31
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Evaluating Commercially
Available Dermal Wipes,
Cotton Suits, and Alternative
Urinary Collection Materials
for Pesticide Sampling
From Infants
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EPA/600/R-04/087
August 2004
Evaluating Commercially Available
Dermal Wipes, Cotton Suits, and
Alternative Urinary Collection Materials
for Pesticide Sampling From Infants
Prepared for
Brian A. Schumacher, Ph.D.
U.S. Environmental Protection Agency
National Exposure Research Laboratory
Environmental Sciences Division, Las Vegas
Characterization and Monitoring Branch
P.O. Office Box 93478
Las Vegas, NV 89193-3478
and
Gary L. Robertson
U.S. Environmental Protection Agency
National Exposure Research Laboratory
Human Exposure and Atmospheric Sciences Division, Las Vegas
Exposure & Dose Research Branch
P.O. Box 93478
Las Vegas, NV 89193-3478
Prepared by
Ye Hu and James Beach
Analytical and Chemical Sciences
Research Triangle Institute
P.O. Box12194
Research Triangle Park, NC 27709
Contract Number: 68-D-99-012
Task Order Number: 003
045EDRB05.COV * 1/5/05
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NOTICE
The information in this document has been funded wholly by the U.S. Environmental
Protection Agency (EPA) under Contract No. 68-D-99-012, Task Order Number 0003, to the
Research Triangle Institute. It has been subjected to the Agency's peer and administrative review
and has been approved for publication as an EPA document. Mention of trade names or
commercial products does not constitute endorsement or recommendation by EPA for use.
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ABSTRACT
This report is a combination report from three separate research efforts each with the
common theme of collecting representative samples for determining pesticide exposure in young
children. The first study examined whether commercially available dermal wipes could be used
for pesticide sampling from infants without eliciting an allergic or toxic reaction from the child.
The second study investigated the use of cotton suits to estimate pesticide exposure to children as
they moved about the home and play areas. Both studies examined the potential analytical
problems and interferences, the adequacy of adsorption, the recoveries of pesticides, and the
uniformity/consistency/capability of the selected brand dermal wipes and cotton suits. The final
study explored improved methods to collect urine samples from infants and to determine the
effect of the collection material on the extraction and quantification of urinary metabolites.
11
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TABLE OF CONTENTS
Section Page
1.0 INTRODUCTION 1-1
2.0 SUMMARY AND CONCLUSIONS 2-1
Part I. Commercial Wipes 2-1
Part E. Baby Suits 2-2
Part m. Urine Collection Materials 2-3
3.0 RECOMMENDATIONS 3-1
3.1 Commercial Wipes 3-1
3.2 Body Suits 3-1
3.3 Urine Collection Materials 3-2
4.0 PART I-THE WIPE STUDY 4-1
4.1 Experimental 4-1
4.1.1 Selection of Wipes : 4-1
4.1.2 Target Pesticides 4-2
4.1.3 Analytical Methods 4-2
4.1.4 Initial Evaluation of Background and Interferences 4-5
4.1.5 Further Evaluation of Interference in Triplicate Spikes 4-5
4.1.6 Surface Interference 4-6
4.1.7 Uniformity/Consistency 4-6
4.1.8 Modified Extraction for Carbamates Only 4-7
4.2 Results and Discussion 4-8
4.2.1 Evaluation of Extraction Methods 4-8
4.2.2 Initial Evaluation of Background and Interferences 4-9
4.2.3 Further Evaluation of Interference in Triplicate Spikes 4-9
4.2.4 Surface Interference Tests '. 4-12
4.2.5 Uniformity/Consistency 4-14
4.2.6 Carbamate Results 4-16
4.2.7 Laboratory Observations 4-19
4.3 Quality Assurance 4-20
4.3.1 Data Quality Goals '. 4-20
4.3.2 Quality Control Results 4-21
111
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TABLE OF CONTENTS
(continued)
Section Page
5.0 PART H-THE COTTON SUIT STUDIES ,5-1
5.1 Study #1: Evaluation of Whole Body Dosimeters for Measuring Dermal
Exposures of Small Children 5-1
5.2 Study #2: Use of Cotton Suits for Pesticide Sampling from Infants 5-8
5.2.1 Introduction 5-8
5.2.2 Experimental 5-9
5.2.3 Results ' 5-10
5.2.4 Conclusion 5-11
5.2.5 Recommendations ....: 5-13
6.0 PART m-ALTERNATIVE URINE COLLECTION METHODS 6-1
6.1 Study #1: Collecting Urine Samples from Young Children Using Cotton
Gauze for Pesticide Studies 6-1
6.2 Study #2: Alternatives for Collecting Urine Samples from Infants 6-9
6.2.1 Experimental 6-9
6.2.2 Results 6-19
6.2.3 Quality Control 6-27
6.2.4 Discussion 6-29
6.3 Study #3: Disposable Diaper to Collect Urine Samples from Young
Children for Pyrethroid Pesticide Studies 6-30
7.0 REFERENCES 7-1
APPENDICES
A WIPES DESCRIPTION AND LIST OF INGREDIENTS A-l
B ANALYTICAL PROTOCOLS B-l
IV
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LIST OF TABLES
Pae
4-1 Commercially Available Dermal Wipes Tested 4-2
4-2 List of Target Compounds 4-3
4-3 Initial Evaluation of Seven Brands of WipesOne Spiked, One Unspiked for
Background Interference Determination 4-10
4-4 Spike RecoverySeven Brands of Wipes Spiked in Triplicate to Identify Other
Background Interferences 4-11
4-5 Determination of Potential Interferences of Wipes from Different Contact Surfaces .4-13
4-6 Uniformity/ConsistencyAmount Detected on Exposed Wipes 4-17
4-7 Carbamate Results: Spiked3 and Unspiked Wipes Analyzed Using Solvent
Exchange Method 4-18
_4-8 Carbamate Results: Spiked3 and Unspiked Wipes Analyzed by the Modified
Extraction Procedure 4-19
4-9 Summary of Quality Control (QC) Goals 4-20
5-1 Recoveries of Pesticides from Whole Body Dosimeters by GC/MS 5-6
5-2 Recoveries of Target Analytes from Field Control Samples (n=3) 5-6
5-3 Concentrations of Analytes Measured in Whole Body Dosimeters and Other Media .. 5-7
5-4 Pesticides in Tested Baby Suits 5-12
6-1 Potential Materials for Urine Collection 6-10
6-2 Materials Selected for Testing 6-13
6-3 Urine Fortification Levels 6-16
6-4 Saturation Water Capacity of Materials 6-20
6-5 Initial TestsMaterial Background Levels in |ig/L and Percent Recovery of High
Level Spike 6-21
6-6 Material Background for 50 mL Urine Test 6-23
6-7 Percent Recovery of Metabolites from Materials Spiked with 50 mL
of Fortified Urine 6-24
6-8 Material Background Expressed as |ig/L Assuming 50 mL of Urine 6-25
6-9 Percent Recovery of Metabolites from Materials Spiked with 100 mL
of Fortified Urine 6-26
6-10 Percent Recovery of Metabolites from Materials Spiked with 200 mL
of Fortified Urine 6-26
6-11 Acceptable Calibration Range by Experiment 6-28
6-12 Percent Recovery of Analytes from Method Controls 6-29
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LIST OF FIGURES
Figure Page
5-1 Reconstructed ion chromatograms from Whole Body Dosimeter field blank 5-8
VI
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ACRONYMS
ABZ 2-Aminobenzamidazole
ALDS Aldicarb sulfoxide
APCI Atmospheric pressure chemical ionization interface
BDMC 4-Bromo-3,5-dimethylphenyl N-methylcarbamate
DAMP . 2-Diethylamino-6-methyl-pyrimidin-4-ol
DEDTP O,O-diethyldithiophosphoric acid
DEP Diethylphosphate
DETP O,O-diethylthiophosphoric acid, potassium salt; diethylphosphorothioate (DEPT)
DMDTP O,O-dimethyldithiophosphoric acid; dimethylphosphorodithioate (DMPDT)
DMP O,O-dimethylphosphoric acid
DMTP O,O-Dimethylthiophosphoric acid; dimethylphosphorothioate CDMPT)
DSS Disulfoton sulfone
EPA U.S. Environmental Protection Agency
ESI , Electrospray interface
FENS Fenamiphos sulfone
GC Gas chromatography
GC/MS Gas chromatography/mass spectrometry
HPLC High performance liquid chromatography
KD Kuderna-Danish
LC Liquid chromatography
LFB .Lab fortified blank
LFM Lab fortified matrix
LMB Lab matrix blank
LOD Limit of detection
LRB Lab reagent blank
MB Method blank
MC Method control
MQL Method quantitation limit
NHEXAS National Human Exposure Assessment Survey
MS Mass spectrometry
NEC N-Ethylcyclohexamine
OP Organophosphate pesticide
PCNB Pentachloronitrobenzene
PERC Perchloroethylene
PFPD Pulsed flame photometric detection
QAPP Quality assurance project plan
QC Quality control
QL Quantitation limit
RTI Research Triangle Institute
SD Specially denatured
WBD Whole body dosimeter
Vll
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SECTION 1.0
INTRODUCTION
As the Human Exposure Program focuses on the exposure of children to pesticides, there
are concerns about the effect, or perceived effect, of components of the sampling procedure on
the health and well-being of the infant and the ability to collect pesticide residues.
One concern involves the materials in wipes used to collect.pesticide residues or other
contact materials on the skin. In recent studies (e.g., the National Human Exposure Assessment
Survey (NHEXAS)), isopropyl alcohol has been used as a solvent in conjunction with a cloth
wipe to obtain samples from the hands of adults and children. Although isopropyl alcohol is
generally considered innocuous, the use of commercially available products could eliminate
concerns about exposure to alcohol. A few studies have evaluated the potential of commercially
available baby wipes to collect personal exposure samples for metals research, but not for the
area of pesticide research (Millson et al., 1994; Campbell et al., 1993; Lichtenwalner et al.,
1993). Therefore, there is a need to evaluate the potential for .using commercially available baby
wipes for collecting pesticide samples from skin and other surfaces.
Another concern involves establishing a convenient and safe method for assessing overall
dermal exposure for children, especially for those in the crawling stage. One route that the U.S.
Environmental Protection Agency (EPA) would like to investigate is the use of cotton body suits
(infant sleepers) as an indicator of potential dermal exposure. As a first step, it is important to
determine effective cleaning procedures to remove background materials on the baby suits so that
minimal interferences will be encountered when the suits are analyzed.
A final concern involves determining updated procedures to collect urine samples from
infants and other pre-toilet trained children. Many of the currently established methods are
designed for clinical settings and are difficult for parents to use at home. For example, the
adhesive collection bag method involves applying glue to a young child's skin for bag
attachment. This procedure is not only technically difficult, but it is also problematic for children
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who are allergic to adhesives. Because the diaper or diaper-insert method is less invasive and
convenient for field sampling, the EPA is interested in developing a diaper liner or insert that
will absorb the urine and will allow for subsequent extraction and analysis of urinary metabolites
of pollutants.
The purpose of this document is to summarize the results of studies conducted by RTI
over the past several years and to provide recommendations to address the above concerns.
1-2
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SECTION 2.0
SUMMARY AND CONCLUSIONS
This report combines results from various studies conducted by RTI. It is organized in
three parts.
PARTI. COMMERCIAL WIPES
Part 1 of this report contains results from a study to evaluate the potential for using
commercially available baby wipes for collecting pesticide samples from skin and other surfaces
(Contract no. 68-D-99-012, Task Order 0003). The pesticides of interest were organophosphorus
pesticides, carbamate pesticides, and other gas chromatography (GC)-suitable pesticides.
Seven brands of wipes were initially selected, including four brands of baby wipes
(Huggies, Food Lion, Pampers, and Playtex Chubs), one brand of isopropanol hospital prep pads
(Kendall Webcol), and two consumer brands of denatured ethanol wipes (Wet Ones, Wash-ups).
No significant background interferences were found for five of the seven brands. Recovery
experiments for analytes of interest indicated significant interferences from surfactants or
emulsifiers in Huggies and Pampers baby wipes. For background recovery from surface
experiments, Wet Ones exhibited interferences for ions of isofenfos, lindane, and simazine,
whereas Food Lion, Playtex Chubs, and Wet Ones all exhibited satisfactory recoveries (70% to
120%) for most of the target organophosphorus pesticides. A uniformity/consistency test was
performed on Playtex Chubs by wiping pesticide-loaded tiles with wipes from different lots.
With the exception of a few compounds, the recoveries of Playtex Chubs were comparable with
the recoveries for the currently used isopropyl alcohol wipes.
RTI developed a relatively simple extraction method to extract most of the target
compounds from the many ingredients in consumer baby wipes. The new method allows good
recovery of most of the target organophosphorus pesticides. Given the relatively simple
extraction procedure and the satisfactory recoveries in background, recovery, and uniformity tests
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for most of the target compounds, we conclude that the Playtex Chubs brand has promise for use
in pesticide field studies.
PART II. BABY SUITS
Part n of this report combines results from two studies using cotton infant body suits.
The first study investigated the choice of baby suit, the cleaning procedures required, and the
recoveries of the selected pesticides. Baby suits were then used in a study of children's exposure
to pesticides in ]S/ttnnesota, and the recovered compounds were compared to measurements of the
same analytes in dust, surface wipes, and air (EPA STAR Grant no. R827444-01-0). Li the
second study, additional cleaning procedures and pesticides were tested (Contract no. 68-D-99-
012, Task Order 0003).
Four procedures used to clean the body suits were evaluated for background removal and
reduction. In the first procedure, suits were home-laundered using detergent that was free of
fragrance and dyes. In the second procedure, suits were cleaned using a CO2-based dry-cleaning
process. For the third procedure, suits were cleaned using a traditional perchloroethylene
(PERC)-based dry-cleaning process, and for the fourth and final procedure, extraction of the suit
using solvents (hexane, acetone) was evaluated. Five types of body suits were evaluated,
including a BabyGap suit (100% cotton, multicolor), two Carters suits (100% cotton pink, 85%
cotton and 15% polyester white), an Ecobaby union suit (100% cotton, natural), and a Dharma
suit (100% cotton, white). All suits had metal snaps.
From these two studies, we concluded:
Only white or natural-colored suits are suitable for sample collection. Colored body
suits (multicolor BabyGap suits, pink Carter suits) produced colored extracts with
solid precipitate in the final solvent volume for analysis;
Regular home laundering, CO2-based dry cleaning, and traditional PERC-based dry-
cleaning methods are not sufficient to prepare a body suit for use as a whole body
dosimeter (WBD);
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Color-free, 100% cotton body suits that have been subjected to extended solvent
extraction provide the lowest background. The recoveries for most tested
organophosphates, atrazine, pyrethroids, and carbamates are in the range of 70% to
130%, with satisfactory precision; and,
Color-free, 100% cotton body suits were able to collect compounds measured in other
environmental media.
PART m. URINE COLLECTION MATERIALS
Part TTT of this report combines results from three studies to determine urine collection
materials. The first study tested the validity of using cotton gauze pads as a medium for
collecting urine samples from young children and examined the stability of the recoveries for
creatinine and pesticide metabolites over a 24-hour period. Metabolites of interest were
2,4-dichlorophenoxyacetic acid (which is mainly eliminated unchanged from urine),
3-phenoxybenzoic acid (metabolite for synthetic pyrethroids), atrazine mercapturate (metabolite
for atrazine), malathion dicarboxylic acid (metabolite for malathion), and
2-isopropyl-4-methyl-6-hydroxypyrimidine (metabolite for diazinon) (EPA STAR Grant no.
R827444-01-0 and Contract no. 68-C5-0011). The second study evaluated materials that can be
readily amendable to extraction for the urinary metabolite of pollutants. Materials investigated
included whole cloth diapers, feminine sanitary pads, and clay (cat litter and diatomaceous earth).
The pesticide metabolites of interest were alkyl phosphate, compound-specific metabolites of
organophosphorus pesticides, and compound-specific metabolites of carbamate pesticides
(Contract no. 68-D-99-012, Task Order 0003). The third study evaluated whether disposable
diapers that contain polyacrylate granules can be extracted using salt solutions, as well as
whether they can be used for collection and quantitative measurement of selected urinary
pyrethroid pesticide metabolites and creatinine. The storage stability of the metabolites and
creatinine in a wet diaper at body temperature and at refrigeration temperature was also evaluated
(EPA STAR Grant no. R829397-01-0).
2-3
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From these three studies, we concluded:
The recoveries for pesticides and creatinine in urine expressed from the cotton gauze
insert were within a range of 70% to 130%. The recoveries were stable at 37 °C for
at least 24 hours;
Among the four alternative materials evaluated, three of the materials showed
promise for collecting urine from children. These three materials are cloth diaper,
after hand washing and drying; Hydromatrix diatomaceous earth, when cleaned by
heating in an oven; and the feminine pad (Maxipad), used as is. Each was free of
interferences, and all gave acceptable recovery when spiked with urine fortified with
target analytes. Cat litter samples showed poor recoveries; and
Disposable diapers that contain polyacrylate granules can be extracted using salt
solutions. The recoveries for the three tested pyrethroid metabolites were mostly in
the range of 65% to 130%. The recoveries for creatinine were in the range of 71% to
133%. The pyrethroid metabolites and creatinine were stable on the diaper for at least
72 hours.
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SECTION 3.0
RECOMMENDATIONS
3.1 COMMERCIAL WIPES
RTI's study (Contract no. 68-D-99-012, Task Order 0003) demonstrated the potential for
the use of commercially available baby wipes for collecting samples for pesticide studies.
Consumer baby wipes contain many ingredients, some of which are high molecular weight
compounds, that are expected to pose analytical problems. Severalcommercially available
wipes, baby wipes, and other wipes have been identified that yield a low background and
acceptable recoveries; however, because of the considerations of national availability and the
task order's request for baby wipes, RTI only tested Playtex Chubs baby wipes in the uniformity
test. In case of production change or brand discontinuation of Playtex Chubs, the other two
selected brands of wipes (Food Lion and Wet Ones) that were not tested in the uniformity test
have excellent potential to be acceptable for use in the field.
3.2 BODYSUITS
Our two studies of cotton body suits (Contract no. 68-D-99-012, Task Order 0003; EPA
STAR Grant no. R827444-01-0) indicated that color-free body suits extracted extensively with
hexane and acetone can be used as a whole body dosimeter for pesticides studies. Among the
color-free body suits tested, the Dharma body suits performed the best as WBDs for the sampling
of pesticide residues. As a textile craft supplier, Dharma provides pure white clothes to artists,
craftspeople, and industry; therefore, supply is relatively stable and the price is reasonable.
We also recommend using the extensive solvent-extraction procedure RTI developed to
clean the body suits before field sampling. Regular home laundering, CO2-based dry cleaning,
and traditional PERC-based dry-cleaning methods are not sufficient to prepare a body suit for use
as a WBD.
A fundamental problem with any alternative method used to clean body suits is that a
body suit will be worn by a child in the field. Therefore, no matter how clean the body suit is
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before it is put on the child, it will inevitably pick up substances (e.g., foods, soil) that may cause
a problem for GC/mass spectrometry (MS) analysis. We recommend further investigation of
procedures to clean the body suit samples before they are submitted to GC/MS analysis, rather ,
than cleaning the body suits before they are sampled. An even better alternative is to investigate
a robust method, such as liquid chromatography (LC)/MS, that does not need much sample
purification prior to analysis. Preliminary test results of the LC/MS method in our lab suggest
that this method can be a very promising candidate as an alternative to GC/MS analysis.
3.3 URINE COLLECTION MATERIALS
Several tested urine-collection materials can be used, or have the potential to be used, for
pesticide studies. Each of these materials have advantages and disadvantages; therefore,
selection of the urine collection material should be determined by the needs of the field study.
The commercial, disposable diaper is the most convenient choice for urine collection.
Because the whole diaper is extracted, this method maximizes the chance for metabolite
detection. Nonetheless, commercial disposable diapers contain polyacrylate polymers, so the
traditional extraction method fails to extract urine and metabolites from the diaper. We found
calcium chloride dihydrate to be satisfactory in releasing, urine and metabolites from the
polymers, and the extract can be concentrated and analyzed using LC/MS/MS. Although it is
highly likely that other metabolites in the extract can be analyzed, we only examined the
recoveries for pyrethroid metabolites in our study. RTI recommends pesticide metabolites that
have not been examined here be evaluated in a similar manner prior to any field study.
Cotton cloth diaper or cotton diaper insert collection, followed by expression, also
demonstrated strong potential for field use. The greatest advantage of a cotton cloth diaper or a
cotton diaper insert is that urine can be expressed instead of extracted.' When urine is expressed,
existing methods for regular urine analysis can be applied directly. The disadvantage of urine
expression is that only a fraction of urine will be analyzed. The volume limitation of expressed
urine can impact both limit of detection (LOD) and the number of analyses possible for a given
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sample. Therefore, we recommend the cotton cloth diaper or the cotton diaper insert only when a
fraction of a urine sample is needed to satisfy the need for a detection limit.
Other alternative collection methods, followed by whole material extraction, also showed
great promise. These materials include the cloth diaper, after hand washing and drying; clay
(Hydromatrix diatomaceous earth), when cleaned by heating in an oven; and the feminine pad
(Maxipad), used as is. These three materials were all free of interferences, and all gave
acceptable recovery when spiked with urine fortified with target analytes.
Finally, we recommend further development of the LC/MS/MS method in analyzing urine
metabolites. The advantage of using LC/MS/MS is that the urine/extract can be analyzed directly
by using a guard column (changed every 50-100 samples) without cleanup. In studies presented
here, RTI used LC/MS/MS to analyze all the tested pesticide metabolites, using either
atmospheric pressure chemical ionization interface (APCI) mode or electrospray interface (ESI).
The LC/MS/MS method showed satisfactory results for most of the tested metabolites. However,
we experienced inconsistencies in diakylphosphate metabolites, especially at the lower spiking
levels. Although the commonly used GC method was reported as being more stable (Moate et
al., 1999), this method analyzes diakylphosphate metabolites only and requires time-consuming
steps, such as pre-cleanup of urine followed by derivatization. This disadvantage makes the GC
method cumbersome when analyzing a list of diverse urinary metabolites, especially in
complicated matrices such as urine in unconventional materials. Given the increasing need for
analysis of polar pesticides and their metabolites in complicated matrices, we recommend fprther
investigation of new and improved instrumental methods such as LC/MS/MS.
3-3
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SECTION 4.0
PART I - THE WIPE STUDY
4.1 EXPERIMENTAL
4.1.1 Selection of Wipes
A preliminary survey of commercially available wipes was conducted to determine the
availability of candidate wipe products, as well as the variation of ingredients used in the
products. The four wipe product categories were foil-wrapped clinical prep pads containing a
solution of 70% isopropanol in water; multipurpose wipes containing specially denatured (SD)
ethanol alcohol as the chief ingredient; a large number and variety of water-based baby wipes,
most containing propylene glycol or glycerine, surfactants, and emulsifiers; and makeup remover
pads. It was determined that many samples in the baby wipe category contained polyethylene
glycol, lanolin, or aloe vera gel, and that the makeup remover pads may be too harsh on the skin
to consider for use on infants.
Seven brands from the first three categories were identified for evaluation and are shown
in Table 4.1, including Web Ones and Wash-ups (multipurpose wipes containing SD); Food
Lion, Playtex Chubs, Pampers, and Huggies (baby wipes); and Webcol (clinical prep pad
containing 70% IP A). Webcol was included so that results from other wipes could be compared
with those from EPA wipes. A list of ingredients of each brand sample is given in Appendix A.
All of the products chosen are marketed to the consumer, and all but one (Food Lion) are readily
available throughout the United States. Of the numerous wipes found through hospital supply
distributors, all contained isopropyl alcohol; however, only one such wipe was included in this
study. The baby wipes, with one exception, were selected on the basis of the popularity of the
brand and product. Such products are presumed to be available for a long period in the future,
and therefore, available for future work. The Food Lion product was chosen because the label
bore a much shorter list of ingredients than the brand-name products. The Food Lion brand is a
generic product and may be available nationally, but bearing the name of a different retailer.
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Table 4-1. Commercially Available Dermal Wipes Tested.
UPC
3600018500
1915491025
7682804721
51107
3582602594
3700032105
7830005006
7830005004
-7830005004
7830005004
Brand
Huggies
Wash-ups
Wet Ones
Kendall
Food Lion
Pampers
Playtex
Playtex
Playtex
Playtex
Style
Original unscented
Anti-bacterial with moisturizer
Original scent, singles
Webcol 2 ply, large
Thick and soft baby wipes
Premium new big wipe
Chubs unscented
Chubs unscented
Chubs unscented
Chubs unscented
. Lot
ML914903A
303659
9274S9
AG0133xX
J169-F
9089E38
9173CS1517
0017AS2343*
9179CS1749*
9180AS0335*
Size (cm)
19x19
15.8 x 12.5
12.5 x 15.0
8.5x4.0
18x20
17.7 x 25.9
19x19
19x19
19x19
19x19
Weight (g)
9.1
2.3
4.1
1.2
6.7
8.6
7.1
7.1
7.1
7.1
* These lots used only for uniformity/consistency tests.
4.1.2 Target Pesticides
The original task order required the following pesticides to be included in the study:
diazinon, chlorpyrifos, dichlorvos, acephate, disulfoton, malathion, EPN, carbaryl, and
methomyl. Among these nine compounds, acephate was dropped because it degraded rapidly in
the solvents used"for wipe extraction. The task order also required the selection of several other
commonly used pesticides (up to three), in addition to the required list. Cis- and trans-
permethrin and propoxur were added. Upon the EPA's request, we also added 12 other GC-
suitable pesticides to the original list, shown in Table 4-2.
4.1.3 Analytical Methods
4,1.3.1 Extraction of Wipes
The extraction method proposed in the quality assurance project plan (QAPP) for this
task order was modified in order to minimize the co-extractives encountered with the consumer
baby wipes. The following method was used for all data presented in this report.
Using methylene chloride-rinsed tweezers or forceps, the wipe was folded and placed into
the extractor. Surrogate compounds were added to 200 mL of acetone in the boiling flask.
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Table 4-2. List of Target Compounds.
Class
Organophosphate Pesticides
Organochlorine Pesticides
Other, Polar Pesticides
Carbamates
Compound
Diazinon
Dichlorvos
Disulfoton
Chlorpyrifos
Fonofos
EPN
Malathion
Parathion
Terbufos
a-Chlordane
y-Chlordane
Dieldrin
Endosulfan I
Endrin
Heptachlor
cis-Permethrin
trans-Permethrin
Acetochlor
Alachlor
Atrazine
Dichloran
Dacthal
Metolachor
Methomyl
Propoxur
Carbaryl
Samples were extracted with methylene chloride overnight (16-20 hours). The extract was
concentrated to 5 mL, using a Kuderna-Danish (KD) concentrator on a hot water bath. Solids
were then precipitated by again adding 100 mL of hexane concentrating extract to 5 mL. The
extract was transferred to a culture tube with two rinses of hexane. Water was removed from the
extract by shaking with 5 g of anhydrous sodium sulfate. The extract was then transferred to a
concentrator tube and evaporated on a warm water bath -0.5 mL. The concentrator tube was
rinsed down with hexane to a volume of 1 mL, and the internal standards added. For the initial
experiments, up through the triplicate-spike experiment, the extract was split into two
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approximately equal portions: one portion for GC/MS analysis and the other for high
performance liquid chromatography (HPLC) (carbamate) analysis. In the surface interference
tests and the uniformity/consistency tests, the extract was not split, since work on the carbamates
had ceased.
The GC/MS portion of the extract split was simply concentrated to 0.1 mL under a gentle
stream of nitrogen and filtered using a 0.2-um pore size nylon syringe filter. The carbamate
analysis required an extract that was primarily aqueous; therefore, a solvent-exchange step was
required. The portion of the extract split (in iso-octane) intended for carbamate analysis was
transferred to a conical vial, and 1 mL acetonitrile was added. The vial was placed in a heated
block (65 °C) on a blow-down apparatus, and the iso-octane layer evaporated with a stream of
N2. The extract was evaporated to 0.2 mL, then it was mixed with 0.8 mL water. A heavy
precipitate resulted. Samples were filtered through a 0.2-u.m nylon syringe filter and analyzed by
HPLC/fluorescence with post-column derivitization.
4.1.3.2 Gas Chrbmatography/Mass Spectrometry (GC/MS)
The method (RTI/ACS-AP-216-004) proposed in the QAPP was used. The sample
extract was injected into a GC/MS system, where analytes were separated with a fused silica
capillary column DB-5. The compounds were identified based on a chromatographic retention
time of at least two representative mass fragment ions, as compared to standard solutions
analyzed under identical conditions. One ion, a primary ion, was used for the quantitation of a
given compound. The secondary ion was utilized as a confirmation ion for a given compound.
Quantitation was carried out by the method of internal standards. Seven calibration solutions,
spanning the target concentration range from 5-750 ng/mL, were analyzed. A second order
regression curve was fitted for each analyte ion. All peak integrations were inspected or drawn
manually. Secondary ion results were checked for agreement with primary ions. Where the
secondary ion differed by more than 50% of the primary ion, the data is flagged in this report
with an asterisk.
4-4
-------
4.1.3.3 Carbamate Analysis
Carbamate analysis was performed according to RTI/ACS-AP-216-005 using
mercaptoethanol in place of N,N-dimethyl-2-mercaptoethylamine hydrochloride. The excitation
wavelength was set to 330, and the emission wavelength was set to 465, with 16 nm slits on both
monochromators. Six calibration standards were analyzed, each containing the internal standard,
4-Bromo-3,5-dimethylphenyl N-methylcarbamate (BDMC) at the same level (100 ng/mL) that
was added to the samples and covering the range of pesticide concentrations: 5-250 ng/mL. The
instrument was calibrated by the internal standard method, fitting straight line curves to the data
from the calibration solutions. The calibration analyses were then quantified using the resulting
curves and checked for fit (i.e., within ±25% of known values).
4.1.4 Initial Evaluation of Background and Interferences
This first step of the evaluation involved an initial evaluation of background and
interference. The purpose of this step was to examine possible interferences with the pesticide
analysis from components of the wipe. One of each of the seven brands of wipes in Table 4-1
was analyzed as is and spiked. Wipes were placed directly from the package into the extractor.
Wet Ones, Wash-ups, and Webcol wipes were transferred from their individual foil packages
using tweezers. The baby wipes Huggies, Pampers, Chubs, and Food Lion were taken
from the middle of their packs, folded with tongs, and placed in the extractor. One of each brand
was spiked with 50 ng of each target compound by applying 50 \iL of a mixture of 32 pesticides
in iso-octane. All samples were spiked with 50 ng of each surrogate compound by adding 50 uL
of a spiking solution to the acetone in the extractor flask. Two laboratory reagent blanks (LRBs)
and two laboratory fortified blanks (LFBs) were prepared and analyzed. LFBs were prepared as
spiked solvent added to empty extractors. Extracts were concentrated to 1 mL and analyzed by
GC/MSandHPLC.
4.1.5 Further Evaluation of Interference in Triplicate Spikes
Whereas all of the seven brands showed no or low background and inferences for most of
the compounds, all seven brands were further evaluated for interferences in triplicate. Three of
each brand were spiked with 50 ng of each target compound and analyzed, as above. In addition
4-5
-------
to the three spiked wipes, one of each brand was analyzed unspiked. Furthermore, three LRBs
and three LFBs were analyzed in this experiment. Surrogate compounds (50 ng each) were
added to the solvent before extraction. Extracts were concentrated to a final volume of 1 mL and
analyzed by GC/MS and HPLC.
4.1.6 Surface Interference
The purpose of this step was to test for interferences that the wipes may pick up from
surfaces similar to those in field use. Three of each brand of wipe were analyzed after exposure
to a new plastic child's toy. Three types of articles were obtained, in lots of five:
Bowl, colored plastic (Gerber),
Teething ring, water filled soft plastic (Kiddie Products), and
Bib, vinyl-backed terrycloth with printed decoration (Hamco).
Each brand of wipe was rubbed across the entire inner surface of a new bowl, the whole
surface of the teething ring, or the front and back of the bib, for a total of 15 exposed wipes and
15 wiped items. One of each wipe was analyzed unexposed as a laboratory matrix blank (LMB).
In addition, two LRBs and two LFBs were analyzed in this experiment.
4.1.7 Uniformity/Consistency
The purpose of this step was to test the uniformity and consistency of wipe production in
terms of a given brand's ability to recover pesticides from a surface to which pesticides have
been applied. Using the results of the preceding background and interference tests, and with
consideration of the intent of the Statement of Work, Playtex Chubs was selected for further
testing. The Food Lion baby wipe gave excellent results in the previous interference tests, and
the identical product may be available nationally in different packaging. Nevertheless, Chubs
was selected on the basis that it is a major brand product, widely available, and easily traceable.
The spray chamber described in RTI/ACS-AP-216-007 was calibrated to deposit an even
layer of aqueous solution onto the floor of the chamber at a level of 0.005 mL/cm2. It was
4-6
-------
calculated that an aqueous suspension of pesticides at a concentration of 500 ng/mL would result
in the deposition of a residue of 2.5 ng/cm2. First, a stock solution was prepared, containing 20
of the target compounds in iso-octane at a concentration of 0.02 mg/mL. A 12.5 mL aliquot of
this solution was added to 500 mL reagent grade water in a separately funnel, followed by the
addition of 100 uL Triton X-100 surfactant and 100 uL Antifoam A Concentrate (both from
Sigma/Aldrich, St Louis, MO). The funnel was shaken to form a uniform and stable emulsion.
Three polypropylene tiles were prepared by removing the protective wrap and scrubbing
them gently with 5% Isoclean concentrate (Isolab, Akron, OH) in water. Tiles were rinsed
thoroughly with warm tap water, followed by reagent water. After allowing the tiles to air-dry,
they were placed in the spray chamber. The tiles were sprayed, then allowed to dry for two hours
in a fume hood.
Three packages of Chubs were obtained with different lot numbers. Three wipes from
each lot were used to wipe a 10 x 10 cm section of a tile. Three sections of the same size were
wiped with surgical gauze wetted with isopropanol so that the recoveries from Chubs and
isopropanol wipes can be prepared. All nine exposed Chubs, the three IPA wipes, nine
unexposed wipes, two LRBs, and two LFBs were extracted and analyzed by GC/MS. Results
were examined for consistency of recoveries for Chubs and for comparability between the Chubs
and the standard IPA method.
4.1.8 Modified Extraction for Carbamates Only
An additional experiment was conducted to test the hypothesis that the solvent exchange
steps were the cause of poor recovery of spiked carbamates. Wipes were placed inside Soxhlet
extractors and spiked with 50 ng of each target compound. Wipes were extracted overnight with
200 mL acetone. The extracts were concentrated using KD evaporative concentrators, in two
steps, to a final volume of 0.2 mL, or as low as could be achieved on a water bath at 90 °C. The
volumes were adjusted to 1 mL with water. After adding the internal standard BDMC, the
extract was mixed and filtered through a 0.2-[im syringe filter. Extracts were analyzed as given
4-7
-------
in Section 4.1.3.3. Results were compared with those obtained using the extraction procedure
outlined in Section 4.1.3.1.
4.2 RESULTS AND DISCUSSION
4.2.1 Evaluation of Extraction Methods
Before evaluation of the brands, RTI conducted a series of experiments to evaluate the
usefulness of the proposed extraction method (RTI ACS-AP-216-001). In this method, the wipe
is mixed with anhydrous sodium sulfate in a beaker using a mortar and pestle to remove water
from the wipe. The pulverized, dried wipe is transferred to a cellulose thimble with the sodium
sulfate, spiked with pesticides, and extracted overnight with methylene chloride in a Soxhlet
extractor. Two problems emerged from these experiments: 1) the methylene chloride extracted
water from the sodium sulfate, and 2) a large amount of co-extractives dissolved in the
methylene chloride and precipitated at every stage of extract concentration. The result was 1 to 2
rnL of extract, mostly consisting of a white substance resembling raw latex. The filtered extract
presented difficulties for GC/MS, requiring excessive system maintenance. Few target
compounds were detected in the extracts of wipes spiked at the 50 ng level.
A new extraction method (Section 4.3.1) was developed, which deleted the labor-
intensive drying of the wipe and allowed all of the water in the wipe to be displaced. By
extracting the wipe with acetone, one can be sure to recover both polar and nonpolar pesticides
and to avoid the situation where a polar pesticide preferentially partitions into water remaining
on the wipe, rather than the extraction solvent. The water is removed from the extract by a
solvent-exchange step, in which the acetone is replaced with hexane. The hexane extract can
easily be dried by shaking with anhydrous sodium sulfate.
The latex-like co-extractives are removed or reduced during the solvent-exchange step.
This material appears to be more soluble in polar solvents than nonpolar ones. As acetone
distills out of the extract, the material precipitates on the walls of the KD flask. Here the
substance is out of the way, and the refluxing action of the concentrator may recover target
pesticides absorbed in the material. This extraction procedure allows good recovery of most
4-8
-------
target analytes in all of the wipes tested, as shown in the next section. The greatest drawback of
this method may be the cleaning of the glassware at the end of the procedure.
4.2.2 Initial Evaluation of Background and Interferences
Seven brands of wipes (Table 4-3) were tested by analyzing one sample from each brand
spiked with 50 ng of target pesticides. One sample from each brand was also analyzed unspiked,
and two LRBs and two LFBs were analyzed.
Only four compounds were detected in the LRB, none at a level higher than 3 ng total for
the analysis. Recovery of target compounds from the LFB was good, with only five compounds
recovered at less than 70% of the level spiked. Recovery of three of the four surrogate
compounds was acceptable (100%±30%) in the LRBs and LFBs. There appears to be some
interference with the analysis of pentachloronitrobenzene (PCNB).
Initial results for the wipes do not clearly distinguish one brand over the others for
suitability in this task. For the most part, background levels are acceptable (<10 ng) for all of the
unspiked wipes. Recoveries from the fortified wipes are mostly within acceptable limits (100%
±30%). Huggies and Pampers show somewhat poorer performance from the laboratory-fortified
matrix (LFM) in this single analysis test. In addition, disulfoton generally had low recoveries for
all the brands.
4.2.3 Further Evaluation of Interference in Triplicate Spikes
Without sufficient data to narrow the focus of the study to fewer brands, the triplicate
spiking experiment was conducted using all seven of the wipe brands. Three of each brand were
fortified with 50 ng of target compounds and analyzed. In addition, one of each brand was
analyzed without spiking, and three LRBs and three LFBs were analyzed.
The LRBs and LFBs were mostly acceptable, with only disulfoton, terbofus, and EPN not
recovered within quality control (QC) limits (Table 4-4). The wipe results show a more
complete picture of the differences between brands. Huggies and Pampers present significant
4-9
-------
Table 4-3. Initial Evaluation of Seven Brands of WipesOne Spiked, One Unspiked for Background Interference Determination.
>
Dichlorvos
Diazinon
Disulfoton
Malathion
EPN
Permethrin
Terbufos
Fonofos
Parathion
Dichloran
Atrazine
Acetochlor
Heptachlor
Alalchlor
Metolachlor
E>acthal
trans-Chlordane
Endosulfan
cis-Chlordane
Eiieldrin
4,4'-DDE
Endrin
Simazine
Lindane
Chlorpyrifos
Isofenphos
4,4'-DDD
4,4'-DDT
LRB" LFB"
(n=2) (n=2)
ng %recov.
n/de 89 (9)
n/d 96 (5)
n/d n/d
n/d 108 (3)
n/d 50 (3)
1(1) 84(3)
n/d 16 (17)
2 (2) 71 (2)
n/d -67 (4)
n/d 80 (1)
n/d 86 (5)
3(3) 107(2)
n/d 96 (6)
n/d 97 (5)
n/d 100 (2)
n/d 93 (3)
1(1) 98(1)
n/d 102 (0)
n/d 91 (0)
n/d 101 (2)
n/d 89 (5)
n/d 101 (4)
n/d 95(6)
n/d 106 (0)
n/d 89 (5)
n/d 59 (3)
.n/d 87(1)
n/d 89 (4)
\ Huggies ''.
LMBC LFM"
ng %recov
n/d 53
n/d 68
n/d 52
n/d 100
n/d* 379*
n/d 579
n/d* 67
8 52
n/d 82
n/d 48
n/d 54
n/d* 76
n/d 92
n/d 74
n/d 96
3 63
n/d 79
n/d 111
n/d 81
n/d 84
n/d 74
n/d 90
n/d 44*
n/d* 270*
n/d 93
n/d 91
n/d 95*
27* 52*
Pampers
LMB LFM;:
ng %recov
n/d . 57
n/d 98
n/d 73
n/d 95
n/d* 129*
44* 448
3833* -1006*
n/d 881*
n/d 115
. n/d 32*
n/d 37
n/d 90
n/d 99
n/d 105
n/d 100
n/d 84
1 88
n/d 87
n/d 86
n/d 73
n/d 83
n/d 86
n/d 75
n/d 438*
n/d 94
n/d 108
n/d 92
n/d 120
Chubs
LMB LFM
ng %recov
n/d 68
n/d 115
n/d 24
n/d 111
n/d* 122
7 156
n/d 77
n/d 105
n/d 106*
n/d 90
n/d 55
n/d 112
n/d 103
n/d 344
n/d 106
1 85
n/d 96
n/d 106
n/d 93
n/d 84*
n/d 85.
n/d 93
n/d 75
17 65*
n/d 101*
n/d 122
n/d 100
2 120
,Food Lion
LMB ,LFM
ng"~t r%recov'
n/d 81
n/d 103
n/d 13
n/d 156
n/d 98*
n/d 233
n/d 92
4 88
n/d 106
n/d 84
n/d 96
n/d 132
n/d 98
n/d 104
n/d 111
n/d 91
n/d 101
n/d 110
n/d 97
n/d 134
n/d 90
n/d* 101
n/d 97
n/d 96
n/d 106
n/d 112
n/d 113
n/d 116*
! Wet Ones
: LMB . LFM
*' ng %recov
4 59
n/d 89
n/d 11
n/d 83
n/d 79
n/d 213
n/d 81
n/d 82
6 66
n/d 57*
n/d 119
19* 117*
n/d 85
n/d 83
n/d 87
n/d 70
n/d 76
n/d 87
n/d 78
3 72
n/d 73
n/d 78
8 56
n/d 67
n/d 82
n/d 83
9 75*
n/d 103
"Wash-ups < .
LMB « LFM
ng %recov
n/d 10
n/d 98
n/d 58
n/d 0
n/d* 0*
33 192
n/d 89
3 91
n/d 84*
n/d 107
n/d 92
n/d* 107
n/d 103
n/d 96
n/d 103
n/d 87
1 93
n/d 56
n/d 88
n/d 89
n/d 143
n/d 98
5 1.23
n/d 0
n/d 83
n/d 125
n/d 50
n/d 9
WebcolIPA
LMB LFM
ng %recov
n/d 107
n/d 108
n/d 35
n/d 156
n/d 93*
33* 170
n/d 110
2 113
n/d 1 10
n/d 97
n/d 100
n/d 112
n/d 106
n/d 110
n/d 122
n/d 96
1 100
n/d . 107
n/d 101
n/d 51
n/d 93
n/d* 487*
5 96
n/d . 89
n/d 109
n/d 122
5 115
n/d 131
Surrogate .%Recovery'"
Atrazine-d5
PCNB
Parathion-dlO
C13-4,4'-DDT
99(1) 100(1)
116(25) 164(53)
70 (10) 71 (3)
84(3) 91(1)
n/d 60
n/d 105
145 77
101 108
n/d 64
156 182
135 118
122 125
46* 79
256 140
122 117
147 134
89 77
181 130
111 113
131 139
111 73
205 91
110 80
63* 106
167 119
268 85
85 89
44 11
114 105
78 121
23 126
132 138
*(Asterisk) indicates that the quantity reported did not agree with the value calculated from the secondary ion (±50% when 1° ion >10 ng).
LRB-lab reagent blank, %recov = % recovery;b LFB-lab fortified blank;c LMB-lab matrix blank;d LFM-lab fortified matrix
e n/d - not detected
-------
Table 4-4. Spike RecoverySeven Brands of Wipes Spiked Examined in Triplicate to Identify Other Background Interferences.
Dichlorvos
Diazinon
Disulfoton
Malathion
Chlorpyrifos
EPN
Permethrin
Terbufos
Fonofos
Parathion
Dichloran
Atrazine
Acetochlor
Heptachlor
Alalchlor
Metolachlor
Dae thai
trans-Chlordane
Endosulfan
cis-Chlordane
Dieldrin
4,4'-DDE
Endrin
LRB" LFM*
(n=3) (n=3)
5 (4)* 66 (3)
n/d 100 (14)
n/d n/d
n/d 82 (8)
n/d 98 (13)
n/d 39(11)
1 (0) 80 (20)
n/d 30 (14)
0(0) 73(11)
n/d 61 (9)
2 (2) 58 (5)
n/d 101 (18)
3(4) 100(12)
0 (1) 92 (8)
n/d 90 (14)
n/d 93 (15)
n/d 99(11)
1 (0) 103 (10)
n/d 104 (14)
n/d 100(14)
n/d 98 (15)
n/d 122 (22)
n/d 94(15)
Huggies
LMB LFM
(n=l) (n=3)
n/d' 53 (10)
n/d 112(27)
n/d 75 (24)
n/d 236(191)*
n/d 116(26)
n/d* 417 (82)*
n/d 294 (143)
n/d 94 (28)
n/d 94 (24)
n/d 161 (25)*
n/d 63 (18)
n/d 77 (8) .
n/d 105(11)
n/d 85 (30)
n/d 99 (16)
1 105 (14)
n/d 94 (19)
1 87 (22)
n/d 72 (43)
n/d 87(29)
n/d 117(61)
n/d 89 (39)
n/d 103 (29)
hampers
LMB LFM
(n=l) (n=3)
n/d 29 (5)
n/d 105 (5)
n/d 154 (67)
n/d 85 (18)
n/d 115(2)
n/d 141 (30)*
n/d 149 (33)
1137* -538(1210)*
287* 488 (345)*
n/d 128 (19)
n/d 20 (28)
n/d 37 (27)
n/d 104 (17)
n/d 106 (1)
n/d 105 (18)*
n/d 92 (12)
n/d 78 (8)
2 80(4)
n/d 89 (10)
n/d 97(4)
n/d 105 (17)
n/d 129 (1)
n/d 89(5)
Chubs
LMB LFM
(n=l) (n=3)
n/d 62 (14)
n/d 80 (6)
n/d 13 (10)
n/d 85 (12)
n/d 73 (8)
12 55(12)
8 113(22)
n/d 53 (7)
n/d 75 (12)
n/d 74 (9)
12 86(34)
0 73 (19)
5 65 (10)
2 75 (10)
107 52 (22)
5 80 (14)
n/d 82 (9)
1 74 (7)
n/d 74 (12)
0* 79 (9)
19* 35 (9)*
n/d 77 (12)
n/d 81 (10)
Food Lion
LMB LFM
(n=l) (n=3)
1 60 (4)
n/d 91 (8)
n/d 15 (3)
6 106 (10)
n/d 98 (9)
n/d n/d*
31 113(11)
n/d 83 (8)
0 85 (8)
n/d 84 (7)*
2 83 (9)
n/d 85 (9)
15* 94 (7)*
1 86 (6)
n/d 90 (10)
n/d 95 (4)
n/d . 88 (7)
1 91 (5)
n/d 105 (12).
0 92 (7)
37 92 (22)
n/d 91 (9)
n/d 89(11)
Wet Ones
LMB LFM
(n=l) (n=3)
2 66 (8)
n/d 114(1)
n/d 26(2)
n/d 1 10 (7)
n/d 108 (1)
3 84 (2)
44 105 (13)
n/d 97 (5)
n/d 106(2)
n/d 95 (2)
n/d 93 (8)*
n/d 96 (5)
48* 146(15)*
n/d 101 (2)
1 105 (5)
n/d 109 (3)
0* 99 (2)
1 102 (2)
n/d 106 (2)
n/d 101 (3)
1 99 (3)*
n/d 103 (1)
n/d 111 (7)
Wash-ups
LMB LFM
(n=l) (n=3)
n/d 16(15)
n/d 111(9)
n/d 106 (14)
n/d 9 (8)*
n/d 92 (10)
103* -48 (95)*
20 74(19)
n/d 106 (12)
n/d 118(26)
n/d 58 (8)*
6 158(119)
n/d 285 (182)
n/d 128 (23)
n/d 97 (7)
n/d 113(19)
n/d 1 19 (22)
n/d 119(20)
1 128 (24)
n/d 79 (13)
0 115(18)
n/d 120 (24)
n/d 166(24)
n/d 117(20)
Webcol IPA
LMB LFM
(n=l) (n=3)
1 72 (2)
n/d 111(1)
n/d 70(10)
2 109 (3)
n/d 102 (2)
2 40 (8)*
21 80 (6)
n/d 106 (2)
n/d 98 (5)
n/d 60(11)*
n/d 52 (9)
n/d 99 (4)
n/d 1 10 (3)
n/d 97 (3)
n/d 94 (3)
1 101 (2)
n/d 92 (2)
1 98 (3)
n/d 101 (7)
n/d 97 (2)
n/d 382(241)*
n/d 103 (4)
n/d* 572 (16)*
Surrogates (%)
Atrazine-d5
PCNB
Parathion-dlO
C13-4,4'-DDT
100 (5) 103 (10)
88(29) 110(56)
66(11) 64(10)
61(10) 67(11)
54 79 (15)
114 109(38)
141 173 (24)
129 135 (51)
60 53 (7)
166 140 (14)
104 140 (19)
105 133 (6)
230 99 (30)
124 83 (16)
151 85(10)
155 107 (14)
84 92 (9)
87 88 (7)
67 88 (6)
108 121 (10)
89 108(11)
91 107 (4)
81 102 (2)
88* 116(7)*
15 319(186)
33 44(12)
58 63 (10)
57 18 (5)*
107 101 (5)
68 70 (3)
72 67 (14)
95 98 (4)
* (Asterisk) indicates that the quantity reported did not agree with the value calculated from the secondary ion. (±50% when 1° ion >10 ng).
* n/d - not detected; LRB-lab reagent blank; LMB-lab matrix blank; LFM-lab fortified matrix.
-------
interferences to the determination of organophosphate pesticides, atrazine, and permethrin.
Recoveries for dichlorvos were lower than 50% for both Pampers and Huggies. Recoveries for
dichloran and atrazine were also low in Pampers. Recoveries for disulfoton, EPN, permethrin,
terbufos, fonofos, and parathion were high (>130%) for Pampers and/or Huggies. Because of the
high recovery, Pampers and Huggies were not tested in subsequent experiments.
Three brands of baby wipesFood Lion, Chubs, and Wet Ones exhibited recoveries
comparable to those of the LFB. Disulfoton, EPN, and terbufos were poorly recovered from the
LFM for these three brands, probably because of degradation in the GC/MS inlet. Permethrin,
acetochlor, and dieldrin appeared in unspiked wipes (LMBs) at levels greater than the quality
goal (1<10 ng/wipe) in at least two of these three samples. Some of these background
measurements were not confirmed by the secondary ion, and in these cases, the quantity is
flagged with an asterisk. Where the quantity is flagged, it is likely that the measurement reflects
interference from a co-extractive rather than the presence of that compound.
4.2.4 Surface Interference Tests
Using the results of the previous tests, evaluation of two wipe brands, Pampers and
Huggies, was discontinued because of their unsatisfactory recoveries for permethrin, atrazine,
and several organophosphate pesticides. The remaining five brands were evaluated for
background interferences that might be picked up from surfaces after using the wipes on three
objects likely to be encountered in field sampling.
In the previous initial and triplicate tests, urine extract was concentrated to a final volume
of 1 mL before GC/MS analysis. To give a better indication of background levels, the extract
was further concentrated to 0.1 mL in this and next experiments.
As in the initial and triplicate interference tests, the LFB recoveries for disulfoton, EPN,
and terbufos are very low (Table 4-5). In addition, the LFB recoveries for fonofos, parathion,
dichloran, and isofenphos were also low (42% to 55%).
4-12
-------
Table 4-5. Determination of Potential Interferences of Wipes from Different Contact Surfaces.
Dichlorvos
Diazinon
Disulfoton
Malathion
Chlorpyrifos
EPN
Permethrin
Terbufos
Fonofos
Parathion
Dichloran
Atrazine
Acetochlor
Heptachlor
Alalchlor
Metolachlor
Dacthal
trans-Chlordane
Endosulfan
cis-Chlordane
Dieldrin
4,4-DDE
Endrin
Simazine
Lindane
Isofenphos
4,4'-DDD
4,4'-DDT
LRB" LFB"
(n=2) (n=2)
ng %rec
0.4(0.1) 60(3)
n/d 88 (4)
n/d n/d
n/d 82 (6)
n/d 88 (4)
n/d 32 (4)*
n/d 106 (6)
0.2 (0.0) 14 (2)
n/d 55 (2)
n/d 46 (3)*
n/d 42 (1)
n/d 78 (4)
n/d 91 (5)
0.3(0.1) 77(3)
n/d 79 (4)
0.6 (0.4) 95 (5)
n/d 99 (2)
1.1(0.3) 115(3)
n/d 113(6)
0.3 (0.2) 93 (3)
n/d 84 (1)
n/d 81 (1)
n/d 97 (4)
n/d 75 (4)
n/d 70 (4)
n/d 55 (0)
n/d 80 (3)
0.7 (0.2) 96 (1)
Playtex Chubs
LMB" Bowl Ring Bib
0.5 0.9 0.9 0.8
n/d n/d n/d n/d
0.8 n/d n/d n/d
n/d 3.5* 5.2 5.9
n/d n/d n/d n/d
5.2* n/d 13.9* 7.9*
n/d n/d n/d n/d
n/d* n/d* n/d* n/d*
4.8* 6.9 n/d* n/d
n/d n/d n/d n/d
n/d n/d 1.3* n/d
n/d n/d n/d n/d
7.6* n/d n/d n/d
0.3 0.4 0.5 0.6
n/d 1302 1138* 1072*
n/d 0.2 n/d n/d*
0.2 0.5 n/d 0.4
0.7 0.7 0.6 1.4
n/d n/d n/d . n/d
2.3* 3.5* 5.7* 5.6*
n/d n/d n/d n/d
0.4 0.5 n/d 0.4
n/d n/d n/d n/d
n/d* n/d* n/d* n/d*
n/d n/d n/d n/d
3.8 n/d* n/d* n/d
n/d n/d n/d n/d
2.8* 8.4* n/d* 5.6
Food Lion
LMB Bowl Ring Bib
n/d*' 0.4 2.1 1.0
n/d n/d n/d n/d
n/d n/d n/d n/d
n/d 11.2 16.9 12.6
n/d n/d n/d . n/d
n/d n/d n/d n/d
n/d n/d n/d n/d
n/d n/d n/d 0.4
2.0* n/d n/d 0.4
n/d* n/d* 4.7* n/d*
n/d 1.4* n/d n/d
2.8* 1.6* n/d* 1.8*
n/d n/d n/d 6.3*
0.5 0.4 0.2 0.4
0.6 n/d n/d 0.5
' 0.5 1.4 1.3 1.1
n/d n/d n/d n/d
0.7 1.1 0.8 0.8
n/d n/d n/d . n/d
0.4 0.4 0.6 0.3
n/d* n/d n/d n/d
n/d n/d n/d n/d
n/d* n/d* n/d* n/d*
n/d n/d n/d n/d
n/d n/d n/d n/d
n/d n/d n/d n/d
1.0* 1.0* 1.4* 1.1*
4.9* n/d n/d n/d
Wet Ones |
LMB Bowl Ring Bib
n/d* n/d n/d n/d*
n/d n/d n/d n/d
n/d n/d n/d n/d
n/d n/d n/d 11.1
n/d n/d n/d n/d
n/d n/d n/d n/d
n/d n/d n/d n/d
10.3* n/d* n/d* n/d*
2.3* 3.4* 3.1* 3.2*
n/d n/d n/d n/d
n/d n/d n/d n/d
n/d n/d n/d n/d
n/d 2.8 n/d n/d
0.2 0.3 0.3 n/d
1.2 3.0 2.3 2.6
0.6 0.5 0.8 0.6
n/d n/d n/d 0.0
0.7 1.0 0.8 1.0
n/d n/d n/d . n/d
0.3 0.3 0.2 0.3
n/d n/d n/d n/d
n/d n/d n/d n/d
n/d* n/d* n/d* n/d*
66* 818* 769* 791*
15.8* 19.0* 19.5* 18.7*
1970 1570 2515 1791
n/d 2.4* n/d n/d*
n/d n/d n/d n/d
Wash-ups
LMB Bowl Ring Bib
0.6 0.8 0.5 n/d
n/d n/d n/d n/d
n/d n/d n/d n/d
n/d n/d n/d 15.5*
n/d n/d n/d n/d
n/d* n/d n/d* n/d*
n/d n/d n/d n/d
n/d n/d 0.3 n/d*
n/d n/d n/d n/d
n/d n/d n/d n/d
n/d n/d n/d n/d
n/d n/d n/d n/d
n/d n/d n/d n/d
0.5 0.9 0.4 0.5
n/d n/d n/d n/d*
n/d* 0.9 0.7 n/d*
n/d n/d n/d n/d
1.2 1.0 1.3 1.6
n/d* n/d n/d n/d
0.3 0.3 0.6 n/d
n/d* n/d n/d* n/d
n/d n/d n/d n/d
n/d n/d n/d n/d
n/d n/d 0.6 n/d
n/d n/d n/d n/d
n/d n/d* n/d n/d
n/d* n/d 0.6 n/d*
0.9 2.2 1.3 27.9*
Webcol IPA
LMB Bowl Ring Bib
0.7 n/d* 1.3* 1.0
n/d n/d n/d n/d
n/d n/d n/d n/d
n/d 21.3 27.3* 6.9*
n/d n/d n/d n/d
n/d n/d n/d n/d
n/d n/d n/d n/d
n/d n/d n/d n/d
n/d n/d n/d n/d
n/d* n/d n/d n/d*
n/d n/d n/d n/d
n/d n/d n/d n/d
n/d n/d 3.2 7.2*
0.3 0.3 0.8 0.5
n/d n/d n/d n/d
n/d* n/d* n/d 0.7
n/d n/d n/d n/d
0.6 0.5 0.9 1.3
n/d n/d n/d n/d
0.2 0.4 0.6 0.2
n/d n/d* n/d n/d
n/d n/d n/d n/d
n/d n/d* n/d n/d
n/d 3.8* n/d* n/d*
n/d* 12.8 n/d n/d
n/d n/d n/d n/d
n/d* n/d* 2.9 n/d*
n/d* 1.1 n/d n/d*
Surrogates (%)
Atrazine-d5
PCNB
Parathion-dlO
C13-4,4'-DDT
94 (5) 95 (4)
57 (4) 60 (2)
0(0) 121(3)
104(3) 114(2)
37.2 48.2 45.6 51.9
49.3 52.7 50.6 52.3
0.3 0.5 n/d 0.6
68.5 75.3 71.7 71.9
29.9* 29.4 32.8 34.0
39.8 45.1 39.8 41.7
0.1 0.1 0.1 0.0
58.4 69.0 63.2 62.3
30.2 36.6 36.8 38.9
29.1 39.2 40.6 38.5
0.0 n/d n/d n/d
46.2 63.9 58.4 61.7
194.2 103.9* 71.2* 77.0*
20.2 19.6 26.2 26.3
0:0 0.0 n/d n/d
21.8 34.8 11.4 51.8
45,9 44.7 61.2 116.5*
33.2 40.1 43.2 40.4
0.0 n/d 0.1 0.0
68.3 68.4 77.6 80.5
* (Asterisk) indicates that the quantity reported did not agree with the value calculated from the secondary ion. (±50% when 1° ion >1 ng).
' n/d - not detected; LRB-lab reagent blank; LFB-lab fortified blank; LMB-lab matrix blank.
-------
The method quantitation limit (MQL) for this experiment is about 0.5 ng/wipe, or 10
times lower than the previous tests, because of the greater concentration factor. At lower MQL
for background detection, no target compounds were found in the LRB greater than 1.1 ng.
Unexposed (LMB) Wet Ones exhibited high levels (15-2515 ng/wipe) of isofenfos,
lindane, and simazine, or co-extractives that interfered with these ions. None of the other wipes
had values for the LMB greater than 10 ng/wipe. The Webcol prep pad had the lowest LMB
values; Wash-ups also performed very well in this regard. For most of the target compounds, the
Food Lion wipes and the Chub wipes (LMBs) were comparable.
In the Chubs wipes used to wipe all three of the articles, a co-extractive was found that
interfered with the determination of metolachlor. The interferent was not found in the Chubs
LMB. It was deemed unlikely that the compound was metolachlor because of poor agreement
between the primary and secondary ions. Malathion was also found at low levels in most of the
wipes exposed to the test articles, but not in the LMBs. Given the fact that the articles were
straight from the factory, it is likely that the low level was caused by a co-extractive.
4.2.5 Uniformity/Consistency
One brand of wipe was selected for the last round of testing. Results from three
brandsWash-ups, Food Lion, and Chubsindicated that any one of these products might
satisfy the quality goals of this task. However, it is the goal of this study to identify a baby wipe,
if possible, as a substitute for the standard practice of using a surgical sponge saturated with
isopropanol for collecting wipe samples from the skin of children. Therefore, Wash-ups, which
is a multipurpose wipe containing SD ethanol, was dropped for further investigation. At this
point in the study, the Food Lion wipes gave slightly lower background than the Chubs wipes,
but it was thought that some problems might arise for researchers trying to obtain this product.
The Food Lion product has been found in retail stores other than Food Lion outlets, in nearly
identical packaging, but with a different brand name affixed with a sticker. This product may be
available nationally, but the questionable availability of the product precluded the selection of
this product.
4-14
-------
Playtex Chubs were obtained from three different manufacturers' lots for
uniformity/consistency testing. The spray pattern of the pesticide deposition chamber was
adjusted to give an even flow over the tiles. The spray pattern was checked by placing 15
aluminum weigh boats (75 mm diameter, area = 44.2 cm2) in a grid pattern across the floor of the
spray chamber. The sprayer tank was loaded with a water/iso-octane emulsion, and the sprayer
run in the same manner and for the same duration as was done during pesticide deposition. The
wet pans were weighed immediately. Weights ranged from 0.20 to 0.26 g, with an average of
0.224 g and a standard deviation of 0.015 g. The density of the emulsion can be assumed to be
about 1 g/cm3 for the purpose of estimating the level of pesticides applied.
Three clean polypropylene tiles were placed on the floor of the chamber, and the sprayer
was loaded with a suspension containing 500 ng/mL of each target compound. The tiles were
sprayed and removed to a fume hood to dry for 2 hours. The pesticide residue level was
calculated as in Equation 1.
500 ng/mix (0.224 ffiL/44.2 cm2) = 2.54 ng/cm2 (Eq. 1)
Wipes were rubbed against a 10 by 10 cm square of tile before analysis. The level one
would expect to find on the exposed wipes would be about 250 ng, assuming full recovery at
every stage. No target compounds were detected on either of the LRBs (Table 4-6). LFB
recoveries were outside control limits for 5 of the 20 compounds spiked. No peak was observed
for disulfoton, and terbufos could not be determined due to the presence of an interferent.
Malathion was detected in the unexposed (LMB) wipes; however, a co-extractive
interfered with the primary ion (m/z 173), a phenomenon observed previously (see
Section 4.2.4.)
Dichloran was detected in four of the nine LMBs, but the identification could not be
confirmed by the secondary ion. Alachlor was detected in only one unspiked wipe, but that
determination is highly suspect due to the presence of a co-eluting co-extractive that gave high
4-15
-------
values for all nine of the secondary ion measurements. Acetochlor was detected in all of the
unspiked wipes at levels ranging from 31 to 63 ng/wipe, but four of the nine measurements fell
outside of QC criteria for agreement between primary and secondary ions.
As shown in Table 4-6, recovery from the baby wipe was somewhat lower than for the
isopropanol method. In the case of malathion, the yield was much higher and the standard
deviation greater, probably as a result of the high background of the unspiked wipes. The
performance of the aqueous wipes declined with the nonpolar pesticides. The very low recovery
of heptachlor cannot be accounted for. Excluding the results of heptachlor, disulfoton, terbufos,
and fonofos, the ratio of values found for the Chubs wipes to those found by the isopropanol
method was about 88%.
4.2.6 Carbamate Results
Initially, we used the same extraction and concentration steps outlined in Section 4.1.3.1
for carbamate analysis. The initial solvent exchange step (from acetone to hexane) caused the
precipitation of solids in the extracts of all of the wipes tested except the isopropanol pad. The
subsequent solvent exchange from hexane to acetonitrile was achieved without difficulty and
with little precipitation. However, when water was added to the acetonitrile extract, the result
was a heavy precipitate, which was then removed by filtration. When the extract was analyzed
for organophosphate pesticides, the GC/MS results indicated good method performance for most
compounds in four of the brands tested (Tables 4-3 to 4-7). The HPLC results (Table 4-8), on
the other hand, were wildly erratic. Although only low levels, if any, were seen in the unspiked
wipes, and the levels found in the spiked wipes were far from acceptable. An interfering peak
hampered the determination of propoxur, while methomyl recovery was generally around 50% or
less. Carbaryl suffered from both interferences and low recovery (<50%). The area of the
internal standard was much lower in the extracts than in the standards or controls, suggesting that
the final filtration step may have retained the internal standard (IS), resulting in higher than
expected recoveries of the target compounds. Check standards run interspersed with samples
indicated that the instrument was in control throughout the HDLC analysis.
4-16
-------
Table 4-6. Uniformity/ConsistencyAmount Detected on Exposed Wipes.
Dichlorvos
Diazinon
Disulfoton
Malathion
Chlorpyrifos
EPN
Permethrin
Terbufos
Fonofos
Parathion
Dichloran
Atrazine
Acetochlor
Heptachlor
Alalchlor
Metolachlor
Dacthal
trans-Chlordane
Endosulfan
cis-Chlordane
LRBa
n=2
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
0(0)
LFB"
n=2
72 (13)
56 (12)
0(0)
84 (7)
66 (10)
112(0)
120 (2)
0(0)
23(15)
88 (7)*
83(1)
84(5)
89(2)
87(3)
109 (5)
105 (6)
43 (3)*
79(4)
87(7)
78(5)
LMB"
n=9
0(1)
0(0)
1(3)*
90(17)*
3(2)
0(0)
0(0)
intf
intf
1 (3)
11(13)*
0(0)*
41 (9)*
0(0)
n/d" (95)*
0(0)
0(0)
0(0)
0(0)
0(0)
Used on Sprayed Tile
IPA Wipe Chubs
n=3 n=9
2(2)
18(2)
0(0)
103 (0)
. 33(1)
135 (9)
119(11)
1(2)
9(2)
70(5)*
28 (2)*
73(6)
66(2)
150 (4)
43(3)
68(5)
18(1)*
66(4)
48(3)
61(2)
3(2)
12 (2)*
2(4)*
151 (51)
24 (4)*
98 (13)
' 78 (14)
intf
intf
55 (9)*
51 (10)*
76(11)*
67(8)
8(1)
34 (7)*
48(7)
13 (2)*
31(5)
24(3)
31(5)
Surrogates:
d5-Atrazine
Pentachloronitrobenzene
C13-4,4'-DDT
66(2)
86(1)
84(3)
78(6)
88(0)
93(4)
65(9)
85(9)
87 (12)
76 (n=l)
111(4)
144 (21)
155(18)* .
105 (10)
91 (12)
*(Asterisk) indicates that the quantity reported did not agree with the value calculated from the secondary
ion. (±50% when 1° ion >10 ng). LFB spiked with 100 ng of each pesticide.
Tiles sprayed with about 2.5 ng/cm2 of each pesticide, allowed to dry, then 100 cm2 wiped.
a n/d - not detected; LRB-lab reagent blank; LFB-lab fortified blank; LMB-lab matrix blank; intf -
interference present preventing clear quantitation.
4-17
-------
Table 4-7. Carbamate Results: Spiked8 and Unspiked Wipes
Analyzed Using Solvent Exchange Method.
Wipe Brand s
Method Blank 1
Method Blank 2
Method Control 1
Method Control 2
Pampers-unspiked
Pampers-spkl
Pampers-spk2
Pampers-spk3
Huggies-unspiked
Huggies-spkl
Huggies-spk2
Huggies-spk3
Chubs-unspiked
Chubs-spkl
Chubs-spk2
Chubs-spk3
Food Lion (generic)-unspiked
Food Lion-spkl
Food Lion-spk2
Food Lion-spk3
Wet Ones-unspiked
Wet Ones-spkl
Wet Ones-spk2
Wet Ones-spk3
Wash-ups-unspiked
Wash-ups-spkl
Wash-ups-spk2
Wash-ups-spk3
Webcol (clinical IPA pad)-unspiked
Webcol-spkl
Webcol-spk2
Webcol-spk3
Methomyl
ng/mL .
n/db
n/d
23
28
10
21
11
15
n/d
25
14
18
n/d
20
16
37
n/d
14.
n/d
n/d
n/d
18
63
92
n/d
5
26-
25
n/d
157
483
352
Propoxur
ng/mL
n/d
n/d
39
38
19
170
1415
72
15
125
96
345
. n/d
314
118
101
10
65
n/d
n/d
n/d
113
158
393
n/d
n/d
n/d
n/d
n/d
201
798
416
Garbaryl >
ng/mL !
n/d
n/d
14
31
n/d
34
632
75
n/d
46
37
38
n/d
51
652
64
n/d
34
n/d
n/d
n/d
35
58
77
n/d
n/d
n/d
n/d
n/d
89
264
139
"Wipes spiked with 50 ng of each pesticide; 100% recovery would yield an extract concentration
of 50 ng/mL.
b n/d - not detected.
4-18
-------
Table 4-8. Carbamate Results: Spiked8 and Unspiked Wipes Analyzed by
the Modified Extraction Procedure.
Sample
Method Blank
Method Control
Wet Ones-unspiked
Wet Ones-spkl
Wet Ones-spk2
Wet Ones-spk3
Food Lion-unspiked
Food Lion-spkl
Food Lion-spk2
Food Lion-spk3
Methomyl
ng/mL
n/d
44
93
125
154
81
412
281 .
353
489
Propoxur
ng/mL
. - n/d
46
207
337
165
138
608
668
505
119
Carbaryl
ng/mL
n/d
28
70
39
34
n/d
n/d '
n/d
n/d
n/d
"Spiking level = 50 ng; 100% recovery would yield 50 ng/mL in extract.
Initially we suspected that target compounds were partitioning into the precipitate and
resulting in reduced recovery. The modified method was tested on only two brands of wipes
(Wet Ones and Food Lion). Precipitates formed and adhered to the glassware during the
concentration steps. The extract at 1 mL contained very heavy precipitates. In many cases, the
syringe filter became clogged with precipitates, and the filtration step was repeated in most cases.
The chromatograms from this experiment showed greater levels of interferences. The elevated
recovery seen in Table 4-8 is mostly due to the effect of co-extractives.
Carbamate results are reported only to indicate the magnitude of the difficulties
encountered with this analysis. Because of poor QC results by HPLC, and because of the
interferences observed in the chromatograms, these data should not be considered an accurate
reflection of actual pesticide levels present.
4.2.7 Laboratory Observations
Although some of the data presented here may support the application of baby wipes for
collection of samples in a field study, these data should not be presented without a few
4-19
-------
observations on the problems encountered during the analysis of the wipes. The analysis method
is not a simple procedure, and it is considerably more complex, more labor-intensive, and more
prone to mishap than the simple Soxhlet extraction of the standard isopropanol-impregnated
sponges currently used. The chief antagonist of this method is the co-extractives from the baby
wipes. Depending on the solvent used, the co-extractives from a single wipe may appear as a
sticky, latex-like substance, or as a tenacious residue on the walls of the KD flask. At best, the
residue requires vigorous scrubbing of the glassware; at worst, it may cause expensive
maintenance of the GC/MS. Some of our instrument control data suggest that these co-
extractives will necessitate more frequent routine maintenance of the GC/MS. In short, the
analysis of baby wipes is the analysis of a complex matrix, more like a food sample than an
environmental sample.
4.3 QUALITY ASSURANCE
4.3.1 Data Quality Goals
The data quality goals specified in the QAPP are reproduced in Table 4-9 below.
Table 4-9. Summary of Quality Control (QC) Goals.
|/':^.v^.' £ -.' ,-VC-'9;. - i. ,'. Analytical' ';..".: ^.^'vWipel
Parameter
Accuracy
Precision
Detectability
Matrix
LFBb
LFB
LRB
QC Goals
70% to 130%
30% RSD
10 ng/wipe
Matrix
LFMC
LFM
LMBb
- % :;> - \
Performance*1 * |
QC Goals
70% to 130% '
30% RSD"
10 ng/wipe"
"Maximum allowed to meet reasonable wipe requirements (0.1 ng/cm2).
b LRB-lab reagent blank, % = % recovery; LFB-lab fortified blank.
c LMB-lab matrix blank.
d RSD - relative standard deviation.
As stated previously, the carbamate analyses did not meet-QC goals, and the data reported
for methomyl, propoxur, and carbaryl should be considered unreliable. The following discussion
of QC results will apply only to data obtained by GC/MS.
4-20
-------
4.3.2 Quality Control Results
The GC/MS was calibrated by analyzing a set of eight calibration solutions, spanning the
concentration range from 5 to 750 pg/uL. A quadratic curve was fitted to the data by linear
regression. The calibration curve was then used to compute analyte concentrations. Computed
concentrations in the lowest calibration standard (5 pg/uL) were within 30% of known values.
Higher level standards were within 25% of known analyte concentrations.
The GC/MS calibration was checked at the start of every day and after the analysis of no
more than nine samples. A solution containing all of the analytes at a nominal concentration of
_100 pg/uJL was used for this purpose. When a calibration check failed to meet acceptance
criteria, the calibration check was repeated. All GC/MS data presented in this report were taken
from analyses that fell between two acceptable calibration checks. A calibration check was
considered acceptable if the determination of all analytes fell within 25% of the known
concentration in the calibration check standard.
Nine method blanks (LRBs) were analyzed throughout this study. No analytes were
detected at a level above the QC goal of 10 ng/sample. Nine method controls were prepared and
analyzed in this study. Two target compounds, disulfoton and terbufos, did not pass QC criteria
for recovery (100%±30%) in any of the LRBs. EPN was recovered at levels less than 50% in the
first three experiments, but it was fully recovered in the uniformity/consistency experiment.
Other phosphate compounds passed QC criteria in some, but not all, experiments. These
compounds include dichlorvos, fonofos, parathion, and dichloran. A total of 18 LRBs and LFBs
were fortified with surrogate compounds before the start of Soxhlet extraction. D5-Atrazine was
recovered within QC acceptance criteria (100%±30%) in all 18 QC samples. QC criteria were
met for C13-DDT in 14 samples and for pentachloronitrobenzene in 9 of the 18 LRBs and LFBs.
Data for both the LRBs and the LFBs are included with sample data in Tables 5-1 through 5-4.
4-21
-------
SECTION 5.0
PART II - THE COTTON SUIT STUDIES
5.1 STUDY #1: EVALUATION OF WHOLE BODY DOSIMETERS FOR
MEASURING DERMAL EXPOSURES OF SMALL CHILDREN
The purpose of this work was to evaluate the choice of cotton body suits and the
procedures required for sampling pesticides. The selected body suits were subsequently used in a
study of children's exposures to pesticide in Minnesota, with the recovered compounds compared
o
to measurements of the same analytes in dust, surface wipes, and air.
The following text is from the original poster presented at 10th International Society for
Exposure Analysis Meeting, October 24-27,2000, Monterey Peninsula, CA, USA.
EVALUATION OF WHOLE BODY DOSIMETERS FOR MEASURING
DERMAL EXPOSURES OF SMALL CHILDREN
G. G. Aklanda, J. H. Raymera, Y. Hua, E. D. Pellizzaria, D. Whitak'era, J. Beacha, B. Schumacher
b, and E. Cohen-Hubalb
' Research Triangle Institute, Research Triangle Park, NC;
' U.S. Environmental Protect
Research Triangle Park, NC
U.S. Environmental Protection Agency, National Exposure Research Laboratory,
INTRODUCTION
Whole body dosimeters have been recommended for use in estimating potential dermal
exposure to pesticides for about 20 years; however, the literature is noticeably absent of studies
on the accuracy, precision, and limitations of this methodology. The increased interest in
children's exposures to pesticides as a result of the passage of the Food Quality Protection Act
of 1996 has brought about a renewed interest in developing methods that would more
accurately represent children's dermal exposures. One method that has received growing
5-1
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attention is the use of the WBD. Several factors must be considered in anticipation of using the
WBD on a routine basis for the estimation of dermal exposures. The WBD should be readily
available commercially; cleaning procedures should be established that permit the lowest
possible method-detection limits; the analytes of interest should be reliably recovered during
extraction; and the transfer of the pesticide to the suit from a surface should be consistent and
characterized, both with respect to adsorption/adhesion and to contact time. Additional
concerns relate to the systematic handling of the suits in a field setting, such that reliable data
can be obtained. RTI is currently involved in a laboratory study of commercially available
cotton body suits (pajamas or union suits) for use as a WBD for children under 4 years of age.
This poster highlights work conducted to evaluate the choice of WBD, the cleaning procedures
required, and the recoveries of selected pesticides. In addition, WBDs were used in a study of
children's exposures to pesticides in Minnesota, and the recovered compounds are compared to
measurements of the same analytes in dust, surface wipes, and air.
/
EXPERIMENTAL
Three body suits were evaluated in this work. Two suits (BabyGap, non-flame retardant,
and Carters) were available locally in several department stores. The other suit (Dharma) was
available over the Internet. All three were 100% cotton and had metal snaps; however, the
BabyGap suit had color decoration, the Carters suit was pink, and the Dharma suit was
completely white. All three suits were available in a range of sizes to fit children from ages 6
months to 3 years.
Cleaning Procedures. Three cleaning procedures were evaluated. First, suits were
laundered in the home using detergent free of fragrances and dyes. Second, suits were cleaned
using a CO2-based dry cleaning process. Finally, extraction of the suit using solvents (hexane,
acetone) was evaluated. A single extraction using 2 L of 50:50 hexane:acetone (1 hour as for
analyte extraction) and sequential Soxhlet extraction of the suits using first hexane and then
acetone were also evaluated. In order to evaluate the effectiveness of the cleaning process, the
cleaned, dry suits were extracted with 2 L of solvent (50:50 hexane:acetone) and 1500 mL was
concentrated to 10 mL using a Kuderna-Danish (KD) apparatus.
5-2
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Initial Evaluation of Extraction, Recoveries. The recoveries of pesticides were
evaluated initially by spiking 9 ^.g each of acephate, diazinon, chlorpyrifos, and malathion onto
the Dharma suit in duplicate. One mL of solution was used and was applied to the arms, legs,
and torso of each suit so that the pesticides would be spiked onto various regions of the suit.
The suits were extracted sequentially with 2 L and then 1 L acetone/hexane (50:50) by tumbling
for 1 hour in a 1-gallon glass jar with a Teflon-lined lid. Following the first extraction, 1500 mL
was removed and concentrated to 10 mL using KD. Following the second extraction, 1L was
removed and concentrated to 10 mL. In each case, aliquots were analyzed using GC with
pulsed flame photometric detection (PFPD) (HP 6890 GC fitted with O-I Analytical 5380 PFPD).
Upon obtaining the results from the GC/PFPD analyses, an additional recovery experiment
-was performed using a larger suite of pesticides spiked at 400 ng/suit. Analysis was performed
by GC/MS (HP 5988).
Field Application. The WBDs were applied in the field as part of a study in Minnesota
to evaluate exposures of children to pesticides. Target analytes were diazinon, chlorpyrifos,
atrazine, and malathion. Body suits were provided to the parent/caretaker along with
procedure instructions. Basically, the caregiver was to wash their hands, put the suit on the
child, and let the child behave normally. After 6 to 8 hours, the suit was removed (after hands
were washed) and placed into a 1-gallon glass jar with a Teflon-lined lid. The sample was
shipped to RTI for analysis. Field controls were also prepared. Suits were spiked in the field
with diazinon, chlorpyrifos, atrazine, and malathion in 40 p.L of solvent and shipped to RTI.
Each suit was extracted as described above except that 50 mL of extract was reduced in volume
to 1 mL prior to analysis using GC/MS.
Other samples collected in the MN study included personal, indoor, and outdoor air
(XAD), surface wipes (isopropanol-moistened gauze pad), dermal press (C18 disks), hand rinse
(isopropanol), and house dust. Sample analysis from the summer of 2000 is still in process. For
comparison, results of indoor air, personal air, surface wipes, and house dust were compared to
the same analytes from the WBDs.
5-3
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RESULTS
Cleaning Procedures. The initial extractions that followed laundering resulted in large
amounts of precipitate upon volume reduction of the extract. Both of the suits that contained
color produced colored extracts with approximately 5 mL of the 10 mL final volume occupied
by solid precipitate. There was a lower amount of precipitate and no color in the extract from
the white suit. Given these results, only the white suit was evaluated further. After dry cleaning
the white suit with the CO2 process, the extraction yielded a brown extract and approximately 1
g of oily liquid. Following a single hexane/acetone extraction, the subsequent extract did not.
contain a precipitate. Preliminary GC/MS analysis showed a fairly large background (late-
eluting "hump") that could potentially interfere with the low-level determinations of
pesticides. GC/MS analysis of the extracts obtained following the two 48-hour extractions
showed a much lower background. It was decided that this cleaning approach would be used.
Initial Evaluation of Extraction, Recoveries. Analysis of each of the extracts from the
sequential extraction of the duplicate spiked body suits indicated that the pesticides would be
well-recovered following a single extraction. Given the concentrations measured in the first set
of extracts, the concentrations measured in the second set of extracts could be completely
accounted for by unrecovered solvent from the first extraction that was subsequently diluted by
the additional 1 L solvent volume for the second extraction. The GC/PFPD recoveries ranged
from 93% for malathion to 137% for acephate. The recoveries obtained upon GC/MS analysis at
the lower spike level are shown in Table 1. Recoveries range from 21% for disulfoton to 167%
for methomyl isomer 2. The recoveries of the MN targets were between the targeted acceptable
units of 75% and 125%, so the suits were applied in the Minnesota study.
Field Application. Recoveries from the field control samples are shown in Table 2 and,
for the target analytes, ranged from 74% for diazinon to 110% for malathion; atrazine d-5
(surrogate) was recovered at 140%. The reason for the high recovery for the surrogate is
unclear.
Reconstructed ion chromatograms of the WBD field blank and WBD from Home 1 are
shown in Figure 5-1. It is apparent that much more material was extracted from the WBD worn
by the child. Instrument calibration went out of control frequently during the analysis of the
5-4
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sample extracts, suggesting that further sample clean up might be required. It would be
anticipated that skin oils, foods, and other materials would be deposited on the suit while being
worn by the child. Compounds measured in the WBDs are compared to other matrices in
Table 3. Only two of the samples contained any of the four main targets; however, other
compounds were measured. Triphenyl phosphate was measured in each WBD, as well as the
surface wipes and, with the exception of Home 1, the floor dust. The amounts measured in the
wipes and dust relative to the WBD were not consistent. Of course, the relative amounts on the
WBD would depend upon the activity of the child and WBD contact with surfaces in the
microenvironment (e.g., kitchen floor).
-CONCLUSIONS
Color-free, 100% cotton union suits that have been subjected to extended solvent
extraction provide the lowest background. Recoveries of analytes are generally quite good
following a single extraction. The WBD collected many compounds often measured in other
environmental media. The relationship between WBD measurements and those of the same
compounds in other media will depend on the activities of the child and WBD contact with
surfaces in that microenvironment. Additional method development is suggested to improve
calibration stability.
FUTURE WORK
Videotaped activities should be evaluated to determine if activity patterns relate to
measured WBD concentrations. Additional method development should be pursued to
improve calibration stability if the WBD method is to be applied routinely. Transfer of surface
residues to the WBD should be evaluated in controlled laboratory experiments to provide better
estimates of uncertainty due to factors affecting measurement variability.
ACKNOWLEDGMENT
This work was sponsored by EPA through Contract no. 68-D-99-012, Task Orders 0003
and 0007, and through EPA STAR Grant no. R-827444-01-0. This work has not been subject to
EPA review and no endorsement by EPA should be inferred.
5-5
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Table 5-1. Recoveries of Pesticides from Whole Body Dosimeters by GC/MS.
Compound
Dichlorvos
Acephate
Methomyl
Propoxure
Atrazine-d5
Atrazine
Pentachloronitrobenzene
Diazinon
Disulfoton
Carbaryl
Malathion
Parathion-dlO
Chlorpyrifos
13C-4,4'-DDT
EPN
cz's-Permethrin
fnms-Permethrin
Tralomethrin
Mean % Recovery (n=3)
79
62
167
105
77
89
44
99
21
123
117
42
121
141 -
25
111
106
129
%RSD
9
11
4
7
8
8
16
7
33
6
6
17
6
5
28
6
7
5
Table 5-2. Recoveries of Target Analytes from Field Control Samples (n=3).
; Compound
Spike Level
(ng/suit)
Measured Level
(ng/suit)
. Mean '.....-,>;'
%;Recover^ , - X
Diazinon
Chlorpyrifos
Atrazine
Malathion
Atrazine d-5 (surrogate)
0.98
0.72
0.49
1.3
0.92
0.73
0.67
0.45
1.4
1.3
74
93
93
110
140
13
12
10
6
6
5-6
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Table 5-3. Concentrations of Analytes Measured
in Whole Body Dosimeters and Other Media.
Home
1
2
3
Compound
Chlorpyrifos
4,4-DDE
4,4'-DDT
Triphenyl
Phosphate
Permethrin
Diazinon
4,4-DDE
4,4'-DDT
Triphenyl
Phosphate
Permethrin
Triphenyl
Phosphate
WipeT
(ng/cm2)
1.3
0.20*
4.0 -
8.7
Wipe 2b
(ng/cm2)
12
0.46
2.1
3.5
1.5
Floor
Dustc
(M-g/g)
0.65
2.3
120s
23
Indoor Personal
Aird Air4 WBDe
(ng/m3) (ng/m3) (ng/suit) '
2.5 15
0.13
0.27
14
2.2
0.11
0.23
1.2
14
1.4
> 4.7
' Kitchen floor sampled; approximate LOD = 0.27 ng/cm2
b Window sill sampled; approximate LOD = 0.27 ng/cm2
c Approximate LOD = 0.50 jig/g
d Approximate LOD = 2.1 ng/m3
e Approximate LOD = 0.04 ng/suit based on calibration limit
' Below lower limit of calibration .
g Above upper limit of calibration.
5-7
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Figure 1. Reconstructed Ian
from WBD fksld Isank
i
I
umn
MOM
won
nun
WON
Field Blank
JOiL
JL-UJUU
IMC 1MB
HU» aunt
TIME (minutes)
uoo
Figure 5-1. Reconstructed ion chromatograms from Whole Body Dosimeter field bank.
5.2 STUDY #2: USE OF COTTON SUITS FOR PESTICIDE SAMPLING FROM
INFANTS
5.2.1 Introduction
The concept of this task is to determine if a cotton baby suit can absorb pesticide residues
and allow for the subsequent extraction and quantification of the pesticides without interferences
that may originate from the suit. In a previous study, which was partially supported by EPA Task
Order 0003 and 0007 through Contract no. 68-D-99-012 and EPA STAR Grant no. R-827444-'
01-0, we investigated two cleaning methods: CO2 dry cleaning and solvent extraction. Because
5-8
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the CO2 dry cleaning methods resulted in oily residues and the solvent extraction method is time-
consuming and expensive, this task focused on conventional PERC-dry cleaning, laundering, and
the combinations of these two in reducing the levels of interfering compounds.
5.2.2 Experimental
5.2.2.1 Cotton Suits
We started the task order by first identifying three brands of color-free, 100% cotton
infant body suits. Four of each brand of suits were obtained as follows:
Dharma Style US-12, Size: 24 mo. White, all-cotton jersey union suit with metal
snaps down front. No feet of suit. Dharma Trading, 654 Irwin St., San
Rafael, CA 94901. Phone 800-542-5227.
http://www.dharmatrading.com/home.html
Carters Style 015-751, W Size: Med. (25", 12-18 Ibs.) 85% cotton, 15% polyester
white terrycloth with integral feet, metal snaps down front, and three small
(-1.5 cm) embroidered designs on front. Obtained from local JC Penny
retail outlet.
Ecobaby Item #EBO508NAT, Size: 24 mo. 100% cotton jersey "footie" union suit
with integral feet and metal snaps down front. "Natural color" (tan).
Ecobaby Organics, 332 Coogan Way, El Cajori, CA 92020. Phone 800-
596-7450. http://www.ecobaby.com/
5.2.2.2 Cleaning/Laundering Methods
Two of each brand were taken to Carolina Cleaners, a local dry cleaner, with instructions
to dry clean the suits as a separate machine load in freshly distilled PERC. At a laundromat, one
machine was run through a complete cycle using Liberty LD-1492 non-ionic clean room laundry
detergent. The label of this product describes it as, "filtered pure surfactant that contains no
builders or additives." Liberty Industries 133 Commerce St., East Berlin, CT 06023. Phone 860-
828-6361. Obtained from VWR Scientific, Cat no. 21911-082.
5-9
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After running the empty laundry machine through a complete cycle, one of each brand of
suit was added to the machine and washed and rinsed using hot water as one batch with 100 mL
Liberty LD-1492. The suits were then rinsed again by cycling the machine without the addition
of detergent. The suits were then dried in a stainless steel commercial dryer, heated with natural
gas, at the laundromat. Then, using the same washer and dryer, four dry-cleaned suits, one of
each brand, were washed, rinsed, and dried by the same method.
5.2.2.3 Extraction and Analysis
The following procedures were followed to extract and analyze the body suits. A suit was
placed in a glass 3.8 L jar with 1000 mL of a 1:1 mixture of acetone and hexane. Surrogate
compounds were added to the solvent, the jar was sealed with a Teflon-lined cap, tumbled end-
over-end (~ 30 rpm) for 30 minutes, and 500 mL of the extract was removed and concentrated to
1 mL using a KD apparatus. Internal standard compounds were added, the concentrated extract
mixed, filtered through a 0.45-u.m pore size Teflon syringe filter, and stored at 4 °C. The
extracts were allowed to warm to ambient temperature before analysis. All of the suit extracts
contained suspended precipitate. A note was made to indicate the proportion of the extract
occupied by this precipitate. A portion of the supernatant was withdrawn and analyzed by
GC/MS, using the selected ion mode.
5.2.3 Results
The results of the GC/MS analysis are given in Table 5-1, with analytes reported as
nanograms per suit. Column headings give the data file number and a sample code, where the
prefix Dar, Car, or Eco represents the suit brand, and the suffix indicates the pre-cleaning method
(N: not cleaned, W: washed with detergent, DC: dry cleaned, and DCW: dry cleaning followed
by washing). The far left column indicates the analyte name and the ion monitored. Where a
pure analyte is detected, the amount reported for the various ions of that compound should agree
to within ±25%. Where the ions differ greatly, the identification of the analyte is tenuous, and in
most cases, the data indicate high levels of interfering compounds. For example, in analysis
7546 (Dharma, as is), the two ions of lindane indicate values of 26 and 4 ng/suit. Most likely
5-10
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neither value is accurate (there may be no lindane present at all); rather, these data indicate that
one or more interfering compounds are present;
Concurrent with the extraction of the suits, four method controls (MC) and four method
blanks (MB) were prepared and analyzed. The MC was prepared by adding 100 ng of each target
analyte to 1000 mL of solvent (acetone/hexane) in a 3.8 L jar. The mixture was tumbled,
concentrated, filtered, and analyzed. The MB was prepared and analyzed in the same manner,
but without the addition of target compounds. Results of these analyses are given in Table 5-4 as
the average of the four QC replicates. MC results are given as average percent recovery; MB
_results are given as average values in nanograms per sample for direct comparison with the suit
data.
The QAPP for this task gave a maximum permissible suit background at
75 ng/compound/suit. This figure was derived from a study of adults (Krieger et al., 2000). For
infant body suits with much smaller surface area, a more realistic figure might be 7 ng/suit. With
this lower figure in mind, the data in Table 5-1 looks dismal. Even for the better candidates, for
instance Dharma washed or with dry cleaning and washing, the background exceeds this level for
about half the target compounds. In addition, the chromatograms of most of the tests gave
elevated baselines and interfering peaks. Furthermore, the filtered extracts contained large
amounts of dissolved solids, which indicate the need for an extract cleanup step to prevent
damage to the GC/MS.
5.2.4 Conclusion
In conclusion, the pre-cleaning methods tested here are not sufficient to prepare a cotton
body suit for use as a WBD.
5-11
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Table 5-4. Pesticides in Tested Baby Suits (nanograms per suit)
% Precipitate
Dichlorvos 185
Dichlorvos 187
Dichloran 206
Dichloran 176
Simazine 201
Simazine 186
Atrazine215
Atrazine 202
Atrazine2I7
Lindane 183
Lindane217
Terbufos 23 1
Terbufos 288
Fonofos 246
Fonofos 137
Diazinon 304
Diazinon 227
Disulfotoo 274
DisulfotonSS
Acetochlor 223
Acetochlor 162
Heptachlor 274
Heplachlor 272
Alalchlor 160
Alalchlor 188
Malathion 173
Malathion 158
Malalhion 285
Melolachlor 162
Metolachlor 238
Chlorpyrifos314
Chlorpyrifos.197 ...
Parathion 291
Parathion 109
DacthalSOl
Dacthal332
Isofenphos213
Isofenphos 255
trans-Chlordane 373
trans-Chlordane 375
Endosulfan 339
Endosulfan 307
cis-Chlordane 373
cis-Chlordanc 375
Dieldrin 263
Dieldrin 277
Endrin 263
Endrin 345
EPN 157
EPN 323
Permelhrin 183
Permelhrin 184
Permethrin 163
MC MB
%Recov Avg
ng/samp
84 8 :
66 0
82 22
84 27
83 7
102 6
86 11
79 13
97 11
87 0
. 72 -0....
24 6
43 0
44 5 .-
. ,71 . ?.
73 0
9 1
6 7
. ... 77, ... 6
85 4
92 3
96 3
.. .80 ~_ _ 7
78 0
69 0
- 72 3
. '.57' .; 4 -;;'
, 82 -."':' 0 '
78 3
61 3
78 0
61 _._ ."; . ^3
46 3~"
85 0
84 3
33 . 1
29 2
85 0
85 0
.85 ..,.!_,
J02 o"
84 0
84 . 0 '..
96 _ ;0 ^
88 0
91 0
93 : 3
110 _; .0
62 ~ 5 "
72 18
77 5 -,
79 - :0 '
0
7546 7539 7541 7540
DarN Dar W Dar DC Dar DCW
30 5 15 10
0 . * ' 0 ' r, ,; 3 "; 0 ''.-.
j 0 , _|0 -J. __j j O'i. JL,i
' 45 0 85 0
75 50 0 0
. :.;S3 -v.V.:"130 r'y_;'->43..''-?' '' : '78 ''''
'18 '.I --84 - - £, .INT^L'-'-;; 141 ';.
43 0 0 23
27 0 0 20
113 113 334 263
v:':26"i5;>'-' vSMyiS^f: ,-38. ,. tU162;
Til;'. *«>'jj' ,:. -jSQii/SfeS : 0 " '. '.^.Ri'-'ftiC.^
71 """ 2oT """"*202 "' 183 ~"
19 0 0 0
'60 :'Vl33lNT 0 105
483 !.L- i?S J57 16 . - " 126
0 8 16 9
25 58 18 8
27 0 0 T., 0
8 i__ ;26 - _23_ _* t32_
164 37 OHB 21
20 72 OHB 0
"11 H 22 56. \f 35
13 ,_ Is 25 _ 52_ »J;ia«r33_
9 48 0 15
24 0 0 0
:19 Vi;r f :2485 *> - -:^0'*;p;> ^:j689"?i;
."/;. INT v" :*; ">2000 ntr-f;. /r.'"o HB^JS^OOO +<*\
- .135 -'>;;- } ; ,2984-;; 6S l^?'b^yf 'JJ3i?802i :.,.'
"9" ' ~~ 0 fl" 12
12 HB 13 0 0
: 6 - .-^./; '70 ^s.3i₯$i,-.49;i'*-\;.:-i>;:;.129^:v
- 1929 .'.''-. ..0 '.-'-^ ''''^Si'HB'^*- '-'-'.', .NO :^;,.
26 0 0 0
432 0 EB 0 HB 0
*' '4: ..;/: v '';)( .;-l^>ii23.->A;-rtv'v; .... /0'':Vi'
' .:'2 ' J :' .0' ':Ji^'''S''%'^dE^' ii!".-'rj^
118470 0 116983 0
409 0 644 0
.-0 :-'-:J'- , ;17 ':;.;};;' .123 Sr'.*-:f -"'70'.'',
' 1-1 .-!-".- .... I7.; ^"^.IlS^;^ ^1.^66^.'
"53 "~* o o "" """"o
478 73 0 HB 0
! 0 -'^.V. _'- 9 v.-- -.'--"^ ."i-54 . '^' .. ;33 7.
',-,- 2 .i.,.. ..'. ;'" 10 . 'y^--, ;i9i^i- . 34ft .
0 62 HB 219 0
0 709 2563 411
121 '- -?"";, ..;..;, o '-.;;;. 159 >,-;.-;*-« ,;;0'>i;^
' 32 :.>'.:'... ' .'0 '{T" ',r '-..;'<) ''"'l.fiyjfe'-.-'.'OL-^'if
0 494 1438 402
>2000 >2000 >2000EB INT
: >2000:r! >2000 /V;. >2000... . ;/>2000;.;v^
">2000 *'. :":: S>2000 " '. ; ;: >2000 '1: ' ' ' >2000 S^.
>2000 >2000* .'' - OEB, >200b**--
I
7548 7547 7556 7542 '
CarN CarW Car DC Car DCW
30 30 60 20
:'-_ ->3 - 0 '- -:;..; 31 -;',- 0
0 ". '.:.. 0 . ;" ' 11 ",i '. '. 0
22 '29 219 "~" 0
45 223 182 108
- 140 ; 86 :, 0 ; 33
-.. o;;r-v.; 113 . ...-.:-: ,^-o ' iiv^.^ 70
29 86 17 58
24 0 29 0
101 658 332 1741
191 771 :;'. yr: /. 300 _'.,.- 734';.
,._;^.5l.^ . -i: 460;^jr_!._i:331^ ,. ,_. 363 ,.
6 10 1354 11
17 0 456 0
^82 254 47 : '- . 115
i-', ^482 , , " 1010 - CC13377 >i' 582 '
35 7 0 9
52 15 16915 0
0 43 0 0
_ 12 23 _ _ 0 HB _^^ 11
175 581 " 174 369
48 20 27 0
95 ,, 21 161^ 28
112 '_- _ .27 J,^, 133"^_ . 32
47 ' 32 35 24
7079
V ';-. 64 INT jr".'- ,34 ;«; . '.-': 0 HBJ v: : 36
S^2000'iNT^t-'""-23,nn^fck' ' 'VO'iffl ;; >f:.- 28 --
j?nOi5l42lNT^;fe,^l683Xi'^gk'i .'.::'.0fc- ',' * -^ .... 0 -
27 "" " "3l" 21 "" ~18
234 37 0 85
'.-V '44 /'. r-'.'1 73 -; '' gj': 173i'C ; ."183
' . -.11:., '.-:'.. 38,-.;..^. -769 :; , : :^ '. .57 :
55 19 27 68
357 795 635 658
:> o : ''- .-:o *:. -i**-; 6 '-: -?: .0
,....:, 0 ,.../.:.,:; 10 ;,i..ji.:. 5_S ;_..i,.' > '.-...
OHB 38 56382 665
5 12 3306 76
' ; 49 }:. " -23i, . !'.'. 288 ; : 55
i^S -.L.:1-,V .24,,; .-.'.1^291 .'>,., ;1 50,.
5 " 310 0 132
345 0 217 137
... ' 24 11 .-. .;'.-.- 153' -. 26
* V26 ''.':-.'. .12 : .-. : r 153 --;' , ^.y 24.
723 575 500 HB 651
214 218 379 HB 216
.":-.- O/ ,. 238^;.: : ~ .; .'. 8KB" 165
, ...0 , l,-r 0 _*>_ 365, ., .0
122 "24"" """38 """ ~i~56~~'~
19 HB 1338 >2000 959
v;>2ooo. .:; '>'-'-'''. lose . >20oo ;- S;;i494
#>206b' ':*; "j; -; 973 '':-' >2000 -v "-:.. : 1418,,
0 ' -'>2000 "''' >2000 ' >2000''
7557 7552 7549 7553
EcoN EcoW Eco DC Eco DCW
cloudy 45 35 50
.0 4 5 0
0 0 0 0
0000
74 0 0 0
33 0 0 0
50 0 0 0
" 129 34 0 357
0 0 0 19
619 411 283 140
.OHB 0 0 214
0 HB 0 0 0
0 95 91 283
0 25 0 0
63 76 62 28
OHB .50 .0 114
" " 0 0 15 0
217 0 5334 157
41 0 45 0
; o o 12 o
68 59 22 18
42 36 44 42
20 0 0 0
...;. .8 .. .: n o . o
11 0 0 0
OEB 0 0 0
8744 4059 1076 4790
,>2000 >2000 INT INT >2000
l'_ .1 720 __ ^ 0 HB . 201 0
19 OHB 14 0
37 OHB 18 40
7 166 0 213
17293 8205 0 HB 5015
0 0 HB 0 0
0 0 HB 0 HB 0 HB
10 22 3HB 0
'.,3. ... ...7 0 0
104 9725 0 HB 8723
48 7742 0 HB 1 1494
1 14 0 102
: 2 _ .30 1 97
51 '" OHB 8 23
0 KB 0 0 HB 0
4 0 98 58 HB
. 2 - ' 0 92 72
261 HB 0 0 HB 825 HB
416 HB 0 OHB 710 HB
>2000HB >2000HB 3915 >2000
. -.57 : 176 87 317
' ~ 18 " "" 0 OHB 251
0 0 HB 0 HB INT
; OEB . INT INT >2000 DJT
T OEB INT INT INT INT
' 0 EB INT INT >2000
INT = interference in the ion trace / HB - high background / EB = extra high background / >2000 = concentration is greater than the highest calibration ieve!
Dar = Dharma / Car = Carters / Eco = Ecobaby
N = nol cleaned (as is) / W = washed / DC = dry cleaned / DCW = dry cleaned, then washed
% Precipitate -- see text D~l2,
-------
5.2.5 Recommendations
From this study, we concluded the following recommendations:
Only color-free cotton body suits should be used for further testing. Before
selecting a color-free, 100% cotton body suit, we tested the extraction method
using a BabyGap suit with color decoration and a pink Carter suit. Both of the
0
suits produced colored extracts with approximately 5 mL of solid precipitate in -
the 10 mL final volume.
Solvent extraction method is laborious but yields much cleaner background. In
the study co-funded by EPA Contract no. 68-D-99-012, Task 0007, and EPA
STAR Grant no. R-827444-01-0, our lab tried cleaning the body suits using 3 L of
solvents (50:50 hexane, acetone) in Soxhlet overnight. The procedure was very
laborious, but it yielded much cleaner background and good recoveries from many
pesticides.
Recommendations for further study. A fundamental problem with any alternative
methods to clean the body suit is that the body suit will be worn by a child in the
field. No matter how clean the body suit is before putting on the child, it will
inevitably pick up substances (e.g., foods, soil, etc.) that may cause problems for
GC/MS analysis during sampling period. Therefore, we recommend further
investigation on procedures to clean the samples before submitting them to
GC/MS analysis, rather than cleaning the body suit before it is sampled. An even
better alternative is to investigate a robust method, such as LC/MS, that does not
need much cleaning for analysis.
5-13
-------
SECTION 6.0
PART III - ALTERNATIVE URINE COLLECTION METHODS
6.1 STUDY #1: COLLECTING URINE SAMPLES FROM YOUNG CHILDREN
USING COTTON GAUZE FOR PESTICIDE STUDIES
The purpose of this work was (1) to investigate cotton gauze as a collection material for
quantifiable recoveries of creatinine and pesticide metabolites of interest; and (2) to determine
change of pesticide metabolite recoveries over time when collected on cotton gauze.
This work was supported by EPA Contract no. 68-C5-0011 and EPA STAR Grant no.
R827444-01-0. The study was presented in the Journal of Exposure Analysis and
Environmental Epidemiology (2000) 10:703-709.
6-1
-------
Jaerxal
«& .YWJ, \
vn«v .nature jc
Collecting urine samples from young children using cotton gauze for
pesticide studies
YAHU.Dft. BARR. G.AKLAND, L MELMYK, L NEEDHAM, ED. PELIZZARI, J.H. RAW AND
J.M ROBERDS
'Analytical and Chemical Sciences. Research niangla Institute, Research Wangle Park, North Carolina
bQvisco of Uboratdy Sciences, Centerfor Disease Control Atlanta. Georgia
US. EPA National Exposure tesaardi Laboratoq Cincinnati, Ohio
e, urire> smplas are often nseded to aral>ze pstfcide nKtabolhes. HCWUBYET, this is difficult fbrchldren waring diapers bacausa
sirnpkand feasible rechniquessiiilabb for lie-Id cdlcctira are not available. The objectives of missiudy were to fist ihevalidhy cf usingccttontpir/epnda a
mad'uiifa Lollaciiig mire samples (ran young children and to examine tie sUtilhy of die rscovaies foraealinire ail pesticide nElabolires over 71 li Urine
spited wlh a pesticide ad four netatdites, M-dichlaiopheroxyacetic acid (wHcli is mainty eliminated fictn urine unchanged), J-phsncaybenzofc: acid
(metabolite lor sjnthelt pyrethrrmUhatra/iie rnacaplUKte (metabolite for atrazine), malalr ion dicar bony lie a:id (irelabolile for rnabthian), and 2-
i»pop>1-.l-rretlrcl-&h>idroxtpyiimidire (rretabalite far diarinon) was added to the gauze pads and ktja in jars at 31° C in a water ball Uriie was
enpesrad from the gauze pads immediately and after 1, i 4, & and 24 \\ lien analyzed. The raccr.eiEs, catulated a tie percaitage of cuiKcnlrationii
ejpressad urine divided by thatDfttecontrd urine sample, wwevvilhin arangaof 7D- 130% Themetabaliteandcraatinineconcentratbnscldmtcliange'Ailh
lire in either expressed urine samples or cunlroK The results soggestthit cotton gauze fui isaprcmiiing candidate for colbcling uire aniplesfrom young
chldren wearing diapers forstudes in which these Cr« urinary pesticide rretabolites are to be andy/ed. Journal of Exposure Analysis and
10.703-709.
Keyvrords: cotton gauze, creadnins pesticide nretabolitss, urira ycung children.
Intixxhiction
Recent studies have suggested that young children may
experience much higher pesticide exposures than adults
(National Research Council, 1993). To investigate expo-
sure sources, pathways, and health consequences, an
estimation of total pesticide intake based upon urinary
brioniarker measurements is often necessary. However, for
young children who wear diapers, it is difficult to collect
spot urine samples. Therefore, a practical and simple
metliod for field sample collection is needed
To date, three methods haw been developed by pediatric
clinicians to collect urine samples from young children for
diagnosis or treatment. The first method uses a urine
collection bag or similar device that is attached to the skin
using adhesive solutions or adhesive tapes (Perel et al,
1S8S; Pierroetal., 199J; Kaida andTrebse, 1998; Kenda
etaL 19S3). This metal, however, may create potential
Addr«» nil curtvipiio Jcncc Ui: Dr Ye A. llo. Analytical and Chcnguit
Scbnaa. Re»an.-li Triangle IiutiliUe, Put Office Bui 1! 194. Km-arch
Triangle PaifctVC 27709. TcL: U-9l9-54l-a»9. Fai
E-muil: yhn^rtLarn
\ntvniber I9W: acapicd IT Juh- 2lltU.
problems for use during field studies such as leakage or
allergic reactions to the adhesive solutions in aomechildren;
therefore, it is not readily accepted by children or their
parents (Burke, IS95 ).
The second method measures analytes from diapers
directly (Ballauffand Manz, 1988; Ahmad el al.. l*»lj
Ueeram and Dhanireddy, \9) I; Miffatore and Dhanireddy,
1993; Colien^t al., 1997). This method is easy to use, but
most commercially available diapers nowadays contain a
gel that makes extraction of the pesticide metabolites
difficult. In addition, one study reported that the diaper
material selectively adsorbed creatinine (Mock, 1992).
Creatinine is a substance that is excreted at a relatively
constant proportion to the faU free body mass. Therefore, it
is widely used to standardize urinary excretion rate: of a
given substance and to determine exposure.
The third method utilizes cotton inserts that are placed
inside the diaper. Urine is expressed from the insert with a
syringe then analyzed (Roberts and Lucas. 1985: Wonn
etal. 1993; Fell etal l')97j. Analysis of the inserts
demonstrated that cotton does not change the urinary
creatinine concentration nor cuneentrations of most chemi-
cals used for clinical diagnosis. (Robertsand Lucas, I9S5 ).
This method is appealing because the same analytical
6-2
-------
-------
Collecting urine from young children using cotton gauze
au eta\.
urine samples except witliout adding the gauze pads. The
expressed urine samples and controls were frozen until
analysis.
Sample Analysis
"Hie pesticide metabolite concentrations in the expressed
urine samples were determined using the method of
Beeson et al. (1999). Briefly, a 10-ml aliquot of urine
was spiked with stable isotope-labeled analogs of each of .
Ihe pesticide metabolites, a technique known as isotope
dilution which automatically corrects for extraction
recovery and losses in evaporation for each individual
sample The urine samples were treated with B-glucur-
onidase/sulfatasefor 17 h at 37°C to liberate metabolites
as their respective glucuronide or sulfate ester. The urine
was extracted twice with a dichloromethane:diethyl ether
mixture and the extracts were concentrated to approxi-
mately 50 uL Ten microliters of the concentrated extract
was analyzed using isotope dilution on a Finnigan TSQ-
7000 triple quadruple mass spectrometer (Finnigan MAT,
San Jose, CA) equipped with an atmospheric pressure
chemical ionization interface and interfaced to a' high-
pressure liquid chromatograph. The resultant data were
quantified using an internal standard calibration method
based upon selected mass/charge ratio of the ions
characteristic of each metabolite.
Urinary creatinine concentrations were determined
using the established diagnostic enzymatic method avail-
able on Vitros CREA slides (Ortho Clinical Diagnostics,
Raritan, NJ, USA). Through a series of enzymatic
reactions involving creatinine in 10 uLof untreated urine
in a multilayered analytical element coated on a polymer
support on the slides, a leuko dye was oxidized to form a
colored product. Reflectance measurements were made
using a Kodak Ektochrome250 analyzer (Eastman Kodak
Co., NJ, USA) at 385 and 5 min after adding the urine.
The difference in the two reflectance measurements was
directly proportional to the creatinine concentration of the
urine.
Statistical Analysis
The statistical analyses were performed using SAS for
windows version 6.12(S AS Inc., Gary, NC, USA). Two-
way analysis of variance (A'NOVA) with unequal cell
sample sizes was performed using SAS PROC GLM
procedure. The model used was as follows:
where u was the overall mean Concentration fora
metabolite,
-------
til Huetal.
CoBoodnf mine than yoaef dukkw ttfag cotton graze
saturate the gauze, healthy toddles should produce enough
overnight to wet the gauze uner nonnal circumstances.
Recoveries from Spiked Urine Table 3 shows the crealinine
and metabolite concentrations in the expressed urine
samples and controls. Unknown inferences occurred
around the peak formalathion dicarboxylic acid for several
controls and diese data points were excluded Two-way
ANOVA indicated that die cotton gauze pads did not alter
the concentrations for 2.4-dichlorophenoxyaclic acid,
atrazine mercapturate. 2Msopropyl-4-methyl-6-hydroxy-
pyrimidine, and creatinine. For3-plienoxvbenzoic acid,
the concentrations were higher in the expressed urine as
compared to the controls whereas die malathion dicar-
boxylic acid concentrations were consistently lower in the
expressed urine as compared to the controls. However, the
recoveries were consistent through the time course of 24 h.
This indicates that the metabolite concentrations can be
quantified after adjusting for recoveries if urine is
expressed from the pads and kept frozen within 24 h after
voiding.
The recoveries for all the analytes were calculated as the
percentageofmean analyteconcentration in expressed urine
samplesdivided by that af the control. As shown in Table3,
Table 3. Recoveries and metabolite concentrations in expressed urine simples and controls.
Metabolite
2,4' Dichlorophenoxyacetic acid
3-PhenoxybenzDic acid
Atrazine mercapturate
Malathion diacid
Oxypyrimidine
Geatinine
NA not applicable.
706
Time (h) Concentration in expressed urine ( u g / L)
0
. I
2
4
S
24
0
1
2
4
S
24
0
1
2
4
g
24
0
1
2
4
&
24
0
1
2
4
S
24
0
1
2
4
8
24
N
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
3
3
2
3
2
3
3
3
3
3
3
.3
3
3
3
3
3
Mean
57.7
573
58.0
61.0
62.3
55.7
53.7
53.7
54.7
523
533
51.7
483
50.7
533
51.0
513
51.7
22.5
163
19.7
22.0
203
20.0
483
50.0
51.7
51.0
47.0
49.7
57.7
57.0
57.1
56.7
57.6
562
SD
4.0
5.1
0.0
3.6
3.1
0.6 ,
5.0
3.1
3.1
23
2.1
3.1
4.7
0.6
1.2
1.0
3.2
0.6
3.5
23
4.7
42
0.6
2.8
2.1
2.0
0.6
1.0
3.0
1.5
0.6
0.6
0.7
0.5
0.8
0.5
Journal 0
RSD (%)
6.9
S.9
0.0
5.9
5.0
1.1
93
5.8
5.7
4.S
3.9
6.0
9.7
\2
23
2.0
6.2
\2
15.6
153
23.9
19.1
3.0
14.0
4.3
4.0
1.2
2.0
6A
3.0
1.0
1.1
1.2
05
1.4
0.9
f Exposure />.
Concentration in control (u g/L ) Mean recovery
N
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
1
1
1
3
3
3
3
3
3
3
3
3
3
3
3
l/m/n/s an
Mean
57.3
59.3
56.3
59.3
56.0
52.0
44.7
44.7
45.3
43.3
42.7
43.3
51.7
493
513
47.3
393
4S.O
22.7
21.7
233
31.0
31.0
25.0
50.0
463
493
51.0
49.0
43.7
57.7
55.7
573
5S.7
553
55.4
J Environ
SD
2,1
IS
\5
1.5
4.4
7.9
25
1.5
33
23
5.1
4.0
13
3.S
ZI
2.1
7.2
7.8
1.5
33
1.5
NA
NA
NA
2.6
3.8
0.6
1.0
2.0
9.S .
03
1.7
1.6
1.1
3.7
0.7
tmenlall
RSD(%)
3.7
6.4
2.7
2.5
19
15.2
5.6
3.4
7.7
5.3
11.9
9.2
19
7.7
4.1
4.4
183
16.3
6.6
16.1
6.4
NA
NA
NA
5.2
8.2
1.2
2.0 .
4.1
22.4
0.5
3.1
2.8
1.9
6.7
13
.pidemiolagy (2
100.7
96.6
103.0
102.9
1113
107.1
120.1
Ml
120.8
120.8
124.S
119.4
93.4
102.S
103.9
107.8
1303
107.7
99.1
75.1
843
71.0
65.5
80.0
96.6
10S.O
1049
100.0
95.9
113.7
100.0
1023
99.3
96.6
104.2
101.4
000) 10(6)Part2
6-5
-------
Collecting urine from young children using cotton gauze
HuaoL (f)
I 70.0 -I
2-
s
60.0*
I 50.0-
3
I 40.0-
30.0-
20.0
10.0-
Snn
-:
70.0-1
2
I 60.0
I
§ 50.0
i
S 40-°
30.0
20.0-
|- 10.0
0.0'
70.0-
I 60-°'
|| 50.0-
I 40.0-
:| .
0 30.0-
, |"
[ 20.0-
j 10.0-
_ _
ICQR 1* P
'annul of i
~ 70.0-
8 2 « S 600-
!I« ' : J 1
" « 1 50.0-
* J 40.0-
8 30.0-
1
I 20.0-
1
| 10.0-
- I
j «* nn.
20 24 6 8 10 12 14 18 18 20 22 24 28
Time (hours)
70.0-
§ 60.0-
i * * ^
1 1 | S * 1 'Q
o* . * i
§ 40.0
i
* 1 3a0'
f 20.0
S
1 lo-o
20 2 4 8 8 10 12 14 16 18 20 22 24 28 -4
Tlma (hours)
70.0-
? 80.0-
§.-.
2 1 1 I 8 ^ 50,0-
* * A = 1 | .«*
! . . - :-
t *'8'
| 28.*
1 to*
O '- '. .: -
A*.
20 2 48 8 10 tt 14 18 18 20 22 24 28 : ;,.:
TIM* /k \
;i: i : J
20 2 4 8 8 10 12 14 18 18 20 22 24 26
Tlma(hour»)
I'l * ' ' 8
9 gO 0 f j
f»
! 0 2 4 6 8 10 12 14 18 18 20 22 24 28
Time (hours)
|i 1 J i
* .
2 0 2 4 6 8 W 12 14 16 18 20 22 24 26
Tin* (hours)
.. M7
6-6
-------
Hu el at.
Collecting urine flora young children using cotton gauze
the recoveries lor all theaiudytes were wiihin an acceptable
range of 70-130*;. with one exception, which can lie
ailribiued to the unknown interference around the pea/e ol"
melaihiondicaiboxylicaeid lor several Controls. Because of
the interferences, only one sample was analyzed for this
lime interval. This made the estimation of the mean less
reliable statistically. In general, the relative standard
deviations for recoveries, which measure the precision,
were wiihin 25% for all the analytes.
Decomposition of die Metabolites Figure I shows the plots
of the metabolite concentrations ancl crealinine concentra-
tions over the 24-h lime interval. Two-way unbalanced
ANQVA indicated that there was no interaction between
treatment and lime (all P>0.05): time had no significant
effect on the metabolite concentrations in expressed urine
samples and control samples (P>0.09). The results suggest
that the concentrations did not increase nor decrease with
time in both expressed urine samples and controls. Given
the reproducibility of recoveries at different times .for all the
analytes. the urine metabolites did not decompose in gauze
at 37°C for at least 24 h.
recoveries be investigated hefore using cotton gau/c ixicl as
a medium for urine collection.
Recoveries for crealinine and the tested metabolites did
not change over a 24-h-period at 37°C. Tills is important
because under normal field conditions, urine samples cannot
be collected immediately after voiding forchilclren who are
in diapers. However, it is easy to express and store the urine
samples wiihin 24 h after voiding. Consistent recoveries
make it possible lo quantify pesticide excretion in a given
time period in urine samples collected by gauze pads.
In this study, we only tested whether prolonged contact
lime of gauze pads with urine will change the recoveries at
37°C Oilier factors lhal may change the recoveries, such as
contact: with diaper creams/ointments or skin were not
tested. The use of urine spiked with metabolites may rci
representlhe form or behavioroflhe metaboliiesexcreted in
ihe urine. Furthermore, Uie technique of using disposable
syringe lo express urine on site may pose health risks for
field technicians; therefore, a protective procedure is
needed. Despite the limitations, the study results suggest
that the cotton gauze pad is a promising medium for
pesticide studies that require urine samples from cliildten
who are in diapers.
Discussion
This study evaluates the feasibility of using cotton gauze
pads for the collection of overnight urine samples for
pesticide studies from young children who are not toilet-
trained. Since urine samples can be expressed on site, this
method prevents possible further .interaction between
urinary constituents and the gauze, which might occur
when samples are shipped prior to urine expression. Also.
the gauze is small enough to be expressed using a disposable
syringe, which decreases the field effort. The expressed'
samples can be easily stored, shipped, and analyzed.
. Although Ihe data demonstrated lhal most of the tested
analytes were recovered consistently and with good
efficiency, ihese data verify that recoveries are chemical-
specific. This was previously observed with colion inserts
(Roberts and Lucas. 1985). The concentrations of creali-
nine and metabolites indicated that there were no statistical
differences in expressed urine samples and the controls for
234-dichlorophenoxyaceticacid. atra/ine mercapiurate. 2-
isopropyM-meihyl-hydroxypyrimidine. ancl crealinine.
To quantity these metabolites, direct measurement of the
expressed urine appears to be appropriate. The concentra-
tions for malathion dicarboxylic acid and 3-phenoxybenz-
zoic acid.however, were different in the expressed urine-
samples as compared lo die controls. To quantify urinary
malathion diacid ancl .5-phenoxybenzoic acid using the
cotton gauze collection method determined here, the
concentrations should be adjusted by the recoveries. For
chemicals that are not tested here, it is recommended that Ihe
Acknowledgments
The authors thank Mr. Don Whilaker for helping with tlw
expressionof the urine samples and Mr. Andrew Clayton for
advice regarding statistical analysis. This research -was
suppoiWby U.S. EPA STAR grant (R 827444-01-0) and
U.S. EPA contract no. 68-C5-0011.
The U.S. Environmental Protection Agency, through its
Office of Research and Development, partially fundedand
managed the research described here under contract no. 68-
C5-OOII to Research Triangle Institute. It has been
subjected lo agency review and approved for publication.
Mention of trade names or commercial products does not
constitute an endorsement or recommendation for use.
References
Ahmad T., Vielcers D., CainpbcllS.,CoultlurdM.G.,and Pettier & Urine
collection from disposable nappies. Lancet 1991:335:674-676.
BallaulTA,. and Man/ ['. Scheduled urine collection using disposable
diapers with an acoustic signal emitter. Gin Peillutr I9SS: 200:
414-418.
Basel! R.C. Biological MonitoringMethods Ibr Industrial Clremkals,P3G
Publishing Co.. Inc.. Littleton, Massachusetts. I9SS.
Beeram M.R.. and Dhanireddy R. Urinarysis: direct versus diaper
collection. C/in feJialrWl': .10: 27S-2SO.
Bee»n M.D., Driskell WJ. and BarrD.B. Isotope dilution hiyh-
perfomunce liquid chromalojiraphyYlaiideni mass speciroinetry nvllv
o
-------
Collecting urine from young children using cotton gauw
Hit el al.
Burke N. Alternative methods for newborn urine sample collection.
Potato- ,\un 1995: 21: 546-549.
Cohen HA, Wolocli B., Under N., Viudi A_ and Barziloi A. Urine
sample from disposable diapers: an accurate method for urine
cultures. J Fam Prad 1997: 44: 290-292.
Fell J.M.R, Thakkar H, Newman D.J., and Price C.P. Measurement of
albumin and tow molecular weight proteins in the urine of newborn
infants using a cation wool ball collection method. Ada Paefuar
1997: 86: SI8-S22.
Kemla R.B.. ami Trebse R. New paediatric urine collector. Aaa PaetRatr
.,1998: 87: 98r-99.
Kendo R 8., Accctt M.O., and Lamprehi - Rijavec Z.S. A urine collector for
separating the initial, midstream and final portions of voided urine in
infancy and early dtiUbood.fiu J-PediorMepltml 1983:4: 201 -204.
Mock D-M. Rayon balls and disposable diaper material selectively adsorb
creaiinina Am J Ctiit WWr 1992: 55:326-330.
Muraiore C. ami Ohanireddy R. Urine collection from disposable diapers
in premature infants: biochemical analysis. Cliit feeliair 1993: 5:
314-315, '..-.'.
Notional Research Council (NRC). Pesticide!! in the Diets of Infants and
Children. National Academy Press. Washington. DC. 1993.
Percl y., Sdlin B.. Perei C., Arnold P., and Moucliet F. Use ofihe urine
collector for infants from 0 ID 4 years of age in a mass survey of
urinary schistosomiasis in Niger. A/«/ Tnpp 1985: 45: 429-433.
Picrro A., Jones M.O.. and Uoyd D.A. A method for urine collection in
infants, Arcii Dis Cklltt 1993: 69: 8J-86.
Roberts S.B.. and Lucas A. A nappy collection method for measuring
urinary constituent] and 24-hour urine output in infant*. Arch Bis
Child 1985:60: 1018-1020. , .; -;
Smith G.C., and Taylor CM. Recovery of protein from urine sperimens
collectad in cotton wool Areb Oft Child 1992: 67.
Wong W.W.. Ckarke U_, Uaunmdor Ms Ferlic L, and_Kleia-PJ)..Tho
use of cotton balls to collect infant arine samples for ^H/'H and
IKO/"O isotope ratio measurement; Appt Radial Jsot 1993: 44::
1125-1128.' . ' / " ':- ' /' . ' '-'' ' ''"
Journal of Exposure Analyiis ami Environmental Epidemiology (2000) 10(6) Port 2
709
6-8
-------
6.2 STUDY #2: ALTERNATIVES FOR COLLECTING URINE SAMPLES FROM
INFANTS
The objective of this task order was to investigate and evaluate materials that can be
readily amendable to extraction for urinary metabolite of pollutants. The pesticide metabolites of
interest were alkyl phosphate, compound-specific metabolites of organophosphorus pesticides,
and compound-specific metabolites of carbamate pesticides. This work was supported by EPA
Contract no. 68-D-99-012, Task 0004.
6.2.1 Experimental
6.2.1.1 Materials
1. Literature Review
This task order began with a literature review. The objective of the literature review was
to identify nontoxic, nonirritating materials that could be used as media to collect urine from
infants. The databases searched included HealthStar, Medline, and Toxline. Magazines, such as
Consumer Reports, were also searched.
Three clinical, widely used urine collection methods for young children were identified.
The first method uses a urine collection bag. The second method collects and analyzes analytes
from diapers directly. The third method uses diaper inserts, with urine expressed from the inserts
for analysis. An extensive literature search focused on the second and the third methods. The
materials reported in the published literature included cotton ball inserts, cotton gauzes, rayon
ball inserts, disposable diapers (with and without gel), and sanitary towels. The advantages and
disadvantages of each of these materials are listed in Table 6-1.
2. Selection of Materials
Although the literature review indicated that diaper inserts are most popularly used for
pre-toilet trained children in medical research, we opted for whole material extraction to allow
for assessment of media matrix effects. Five materialscloth diapers, disposable diapers,
6-9
-------
Table 6-1. Potential Materials for Urine Collection.
Material
>: Publications and Brief Description
Brand Name
Advantages'
Disadvantages
Cotton ball 1. Roberts and Lucas (1985)
inserts Cotton balls were placed inside a disposable diaper.
Urine was expressed from the cotton balls and the diaper
using syringe.
2. Wong et al. (1993)
Cotton balls were moistened with urine that contained
either natural abundances or enriched 2H or 18O. Urine
was expressed using syringe.
3. Smith and Taylor (1992)
Cotton balls were moistened with urine from normal
children and children with glomerular or tubular
dysfunction. Urine was expressed using syringe.
4. Fell et al. (1997)
Cotton balls placed inside a disposable diaper. Urine
was expressed using syringe.
5. Mock (1992)
' Urine collected from children and adults was absorbed
by cotton ball. Urine was expressed using syringe.
Unknown brand for cotton
ball
Baxter Scientific
Unknown brand
Smith and Nephew
Medical Ltd, Hull, UK
Q-tips cotton balls,
Cheeseborough Ponds
1. No change of most of the urine
constituents.
2. Urine can be easily expressed
with a syringe.
No change of H and O isotope ratio.
Low recoveries for albumin,
alpha-1 -microglobulin
andretinol-binding protein
Good recoveries for albumin, alpha-
1-microglobulin andretinol-binding
protein with introduction of a
detergent extraction ste'p.
Cotton balls did not selectively
adsorb creatinine.
Cotton 6. Hu et al. (2000)
gauze Urine spiked with pesticide metabolites was added to
inserts cotton gauzes. Urine was expressed using syringe.
Surgipad, Johnson &
Johnson
1. Cotton gauze provided good
recoveries for tested pesticide
metabolites.
2. Can be easily attached to diaper.
Rayon ball 1. Mock (1992)
Urine collected from children and adults was absorbed
by rayon ball. Urine was expressed using syringe.
Kendall Curity Rayon
Balls, Kendell Company
Rayon balls selectively
adsorbed creatinine
-------
Table 6-1. (continued)
Material
Publications and brief description
Brand Name
Advantages
Disadvantages
Disposable 1. Roberts and Lucas (1985)
diaper Cotton balls were placed inside a disposable diaper.
without gel Urine was expressed from the cotton ball and the
diaper using syringe.
2. Mock (1992)
Urine collected from adults and children was
absorbed by diaper material. Urine was expressed
using syringe.
3. Cohen (1997)
Diapers were collected from sick children. Lining
layer of diaper was removed and pushed into the
barrel of a syringe to express the urine.
4. Beeram and Dhanireddy (1991)
Urine collected from children and adults was poured
onto the diaper. The absorbent diaper material was
removed and placed into a syringe to express urine.
S.Ahmadetal. (1991)
Diapers were collected from children and expressed
using syringe.
6. Muratore et al. (1993)
Urine was collected from premature infants using
urine collection bag. Half of the collected volume
was added to the diaper and expressed using syringe
for comparison.
Pampers, Procter and
Gamble
Huggies Thick
Various kinds available in
Israel
Premature Infant Pampers
(Procter and Gamble)
Various brands available
in United Kingdom
Premature Infant Pampers
(Procter and Gamble)
1. No change of most of the urine
constituents.
2. Urine can be easily expressed with
a syringe.
3. Inserts not needed.
Did not change bacterial counts of
interest.
Blood cell count, protein, glucose,
leukocytes, sodium, potassium,
creatinine, urea, osmolality, pH,
specific gravity did not change.
The diaper material did not change
most of the urinary constituents.
The diaper material did not change
most of the urinary constituents
1. Selectively adsorbed
creatinine.
2. Urine was rapidly
absorbed and dispersed
widely, making recovery
of urine difficult and
unpredictable.
The agreement for uric
acid was poor.
7. Consumer Reports (1998)
12 commercially available
diapers, 6 training pants .
Every 6-8 months, the
features are changed to
stay competitive. Diapers
without gel are not easy to
find.
-------
Table 6-1. (continued)
Material
Publications and brief description ,
Brand Name
.Advantages
Disadvantages
Disposable 1. Mock (1992)
Diaper with gel Urine collected from adults arid children was
absorbed by diaper material. Urine was expressed
using syringe.
3. Cohen (1997)
Diapers were collected from sick children. Lining
layer of diaper was removed and pushed into the
barrel of a syringe to express the urine.
5. Ahmad etal. (1991)
Diapers were collected from children and expressed
using syringe.
Huggies Supertrim,
Kimberly-Clark Co.
Various kinds available in
Israel
Various brands available
in UK
Urine forms a gel on
contact with the absorbent
in the diaper, making
syringe express of urine
impossible.
Tested and excluded from
the study because
"extracting urine from
them is difficult and time-
consuming."
Extraction impossible.
Samples contained gel-
like material, not suitable
for mass spectrometry.
to
Sanitary towel
(also called
urine collection
pads by British
researchers)
l.Vemon etal. (1994)
Urine collected from children was divided. Half was
poured onto a sanitary towel. Various urinary
constitutes were compared with the original half.
7 brands tested (brand
name not specified)
The one from UK Medical Supplies
did not change bacterial counts and
other constituents.
One brand had gel, four
had anti-bacterial
properties and one
absorbed urine poorly.
2. Macfarlane et al. (1999)
Urine collection pads were placed inside the diaper of
88 children. Urine was expressed from the pads
using syringe.
NHS supplies
Bacterial contamination
3. Lewis (1998)
Urine expressed from pads was compared with clean-
catch
No change in terms of bacteria
counting.
Cloth Diaper Web site
(http://geocities.com/Heartland/Bluffs/2900/
absorbancytesting.html) Listed absorbency test for
various cloth diapers
Various brand names
The amount absorbed by some of the
diapers seemed to be large.
-------
Table 6-2. Materials Selected for Testing.
UPC
3600052057
1500082906
3700066103
7203627102
p/n 0019-8003
Brand
Huggies
Gerber
Always
Harris Teeter
Varian
Style
Ultratrim diapers, size 4
Diaper service cloth diapers
Maxi regular
Cat Litter 100% Natural
Chem Elut-Hydromatrix
Lot
BI030309B
55510098
268CA1 13 11928
not given
CE7A159
Size
470 x 280 mm
521 x 362 mm
195 x 75 mm
300 g
100 g
feminine sanitary pads, cat litters, and diatomaceous earth were selected for evaluation. The
brands and lot numbers are shown in Table 6-2.
Cloth Diapers. There are two major types of reusable, cotton diapers currently available:
fitted diapers and the conventional flat swath. Fitted diapers are tailored to shape like
underpants, with buttons or velcro for easy closure. The conventional cloth diaper consists of
multiple layers of fabric quilted together to form a single flat cloth that must be folded and
pinned to fit the infant. We chose the latter type for evaluation in order to avoid potential
chemical interference from the plastic closures used in the fitted diapers. We selected Gerber
Diaper Service brand diapers because of the popularity and stability of the brand.
Disposable Diapers. There are three major types of commercially available disposable
diapers: basic, ultratrim, and supreme (Consumer Reports, 1998). The differences among these
types include diaper thickness, the use of plastic or cloth-like covers, and the type of closure
used, including Velcro or adhesive tape. Ultratrim and supreme have more features than basic
(such as Velcro tape for enclosure). Supreme is the thickest. Despite the differences, all the
major brands of disposable diapers currently available in the United States contain polyacrylate
granules to increase water retention. Huggies Ultratrim was selected for initial testing because it
is the most popular brand (Consumer Reports, 1998). Huggies Ultratrim also has cloth-like
covers and fasteners, which are desirable features for field studies.
6-13
-------
Feminine Sanitary Pads. Always Max! brand feminine sanitary pads was selected for
evaluation because, unlike the majority of other pads manufactured today, it uses a tissue core
instead of polyacrylate polymer.
Cat Litter and Diatomaceous Earth. A generic brand of clay cat litter was selected for
initial testing. The product was labeled "Natural" and "100% Clay." After initial tests of this
product yielded poor performance, another product, Varian Hydromatrix, was substituted.
Hydromatrix is diatomaceous earth, with a particle size of about 10-20 mesh. It is used as a
laboratory aid in liquid/liquid partitions and is manufactured and sold by Varian Associates, Palo
Alto, CA.
3. ^Reagents
The following reagents were used in the task:
Reagent Water. Purified, deionized water was generated on-site using a Picosystem
Ultra (Hydro, RTP, NC) ultrapure water system.
Laundry Detergent. Wisk, labeled as "free of perfumes, clear of dyes," UPC No.
1111187041, was obtained from a local grocery store.
Solvents for Liquid Chromatography. Acetonitrile and methanol were obtained in UV
grade from Burdick and Jackson (Muskegon, ME).
Pesticide Metabolites. Compound-specific pesticide metabolites and diethylphosphate
(DEP) were obtained in neat form from Chem Service (West Chester, PA). Other alkyl
phosphates were obtained in neat form from the following sources: O,O-diethyldithiophosphoric
acid, (DEDTP, TCI America); O,O-dimethylthiophosphoric acid (DMP, Pfaltz and Bauer); O,O-
diethylthiophosphoric acid, potassium salt (DETP, Aldrich); and sodium salts of O,O-
dimethylthiophosphoric acid (DMTP) and O,O-dimethyldithiophosphoric acid (DMDTP) from
Applichem (Darmstadt).
6-14
-------
4. Fortified Urine
Human urine (pooled, mixed gender) was obtained from Biochemed Pharmacologicals,
2400 Valley Avenue, Winchester, VA 22602. Stock solutions of single analytes were prepared
in acetonitrile or acetonitrile and water. Stock solutions were combined in proportions that
reflected the anticipated quantification limit (QL) for the instrumental method and diluted in
acetonitrile to make a spiking solution. An aliquot (10-2000 uL) of the spiking solution was
combined with human urine (100 to 1000 mL) and mixed to make fortified urine. Analyte levels
(as prepared) in the fortified urine are given in Table 6-3. Relative analyte levels were adjusted
midway in the work to better reflect the observed instrument QL, which is the lowest level in the.
calibration curve where analytes can be reliably measured. Dimethyl phosphate was not added to
the urine used in the recovery tests. The QL for DMP was found to be too high for the method to
be useful. It was also thought that impurities in the DMP might interfere with the analysis of
other analytes if added at significantly higher levels.
6.2.1.2 Determination of Saturation Water Capacity of Materials
The following test was used to determine the maximum amount (mass) of water that each
of the materials could retain. To determine the saturation water capacity of each material, a
diaper, pad, or 300 g of clay was placed in a 3.8 L jar and weighed. Two L of water was added to
the jar. After 1 hour, the excess water was poured out, and the jar allowed to drain for an
additional hour. The jar containing the water-saturated material was weighed and the difference
calculated. When it was decided to substitute Hydromatrix for cat litter, 100 g of Hydromatrix
was tested by the same method.
6.2.1.3 Analytical Methods
The analytical methods used in this work are described in RTI/ACS-SOP-500-004, given
in Appendix B. The following method was used for urine extraction. The diaper, pad, or clay
was extracted by tumbling in ajar with 0.5 L water. About 15 mL of extract was decanted to a
polypropylene centrifuge tube and placed on a table top centrifuge at 3000 rpm for 15 minutes.
A 10 mL portion of the supernatant was transferred by pipet to another polypropylene centrifuge
6-15
-------
Table 6-3. Urine Fortification Levels.
ON
h-»
ON
Parent Compound
Benomyl, tiqphanate methyl,
carbendazim
Pirimiphos methyl
Cycloate
Aldicarb
Phorate
Phenamiphos
Disulfoton, phorate
Paraoxon, demeton-oxon, diazinon-
oxon, dichlorfenthion
Diazinon, demethon, parathion,
fenchlorphos
Malathion, dimethoate, azinphos-methyl
Dichlorvos, trichlorfon, mevinphos,
malaoxon, dimethoate, fenchlorphos
Fenitrothion, fenchlorphos, malathion,
dimethoate
Target Compound
2-Aminobenzamidazole
2-Diemylarnino-6-methyl-pyrirnidin-4-ol
N-Ethyl-cyclohexamine
Aldicarb sulfoxide
Disulfoton sulfone
Fenamiphos sulfone
Diethyldithiophosphate
Diethylphosphate
Diethylthiophosphate, potassium salt
Dimethyldithiophosphate, sodium salt
Dimethylphosphate
Dimethylthiophosphate, sodium salt
Initial Tests (ng/L)
Abb8
ABZ
DAMP
NEC
ALDS
DSS
FENS
DEDTP
DEP
DETP
DMDTP
DMP
DMTP
Class
Carbamate
OPb
Carbamate
Carbamate
OP
. OP
OP
OP
OP
OP
OP
OP
10
10
10.
50
10
10
10
10
10
10
100
500
500
500
500
500
2500
500
500
500
500
500
500
5000
25000
25000
.Recovery Tests (lig/L)
. :'-;l
1
1
5
3
1
1
50
10
30
10
0
10
10
10
10
50
30
10
10
500
100
300
100
0
100
100
100
100
500
300
100
100
5000
1000
3000
1000
0
1000
' Abbreviation used in this report.
b Organophosphate pesticides.
-------
tube. Samples were chilled to -70 °C in an ultra-cold freezer. The frozen extract was placed on a
freeze-drying apparatus, and the volume reduced to about 0.4 mL. The sample is allowed to
thaw. An internal standard was added, and the volume adjusted to 0.5 mL with reagent water.
The extract was mixed, transferred to an autosampler vial, and analyzed by LC-MS-MS.
Samples that were not to be analyzed immediately were stored at 4 °C.
6.2.1.4 Initial Testing of Materials
Cloth diapers, Huggies, Maxi feminine pads, cat litter, and Hydromatrix were tested as
received without pre-cleaning for analytical interferences. One diaper or pad, 300 g of cat litter,
or 100 g of Hydromatrix were extracted by tumbling in a 3.8 L jar with reagent water for 1 hour.
An aliquot of the water was concentrated and analyzed.
This experiment was also repeated with materials that had gone through a cleaning step.
Cloth diapers were washed by hand with laundry detergent (Wisk Free), using a stainless steel
sink, chloroprene gloves, and tap water. After thorough rinsing, the diapers were wrung by hand
and dried in a glass carboy using a stream of nitrogen. The Maxi pad was cleaned in a similar
fashion but with gentle handling to avoid damage to the pad, leaving in place the plastic sheet
that covers the adhesive underside. The clay and Hydromatrix were baked in glass trays at 400
°C for 4 hours. The cleaned materials were analyzed as above.
The cloth diaper, Maxi pad, cat litter (300 g), and Hydromatrix (100 g) were then tested
for recovery of target compounds. Human urine was fortified with a high level (250-12500
ng/mL) of target compounds in order to obtain initial assessment of the feasibility of the
materials. The levels were 50 times the levels used in method development (Table 3 in
RTI/ACS-SOP-500-004), in which spiked urine samples that did not contact with the materials
were tested. Fifty mL of the fortified urine was applied to each test material. A previous study
indicated that a normal child, aged 1 to 3, produces 78-167 mL urine overnight. We used 50 mL
in this test to be conservative. The materials were then analyzed, and percent recovery was
calculated. This experiment was performed on both cleaned and as received samples of each
type material.
6-17
-------
6.2.1.5 Recovery at Three Levels Using 50 mL Urine
The purpose of this test was to further study the recoveries of the pesticide metabolites in
the four selected materials at three spiking levels. Three of the materialscloth diapers, Maxi
pads, and Hydromatrixwere tested for recovery of analytes from human urine fortified at three
levels. Prior to testing, the diapers used in this experiment were washed and dried as given in
Section 6.2.1.3. The Hydromatrix was cleaned by baking it in an oven at 400 °C for 4 hours.
The Maxi pads were used as-is, directly from the package and leaving in place the plastic sheet
that covers the adhesive underside.
Urine was spiked with analytes at the levels given in the three right hand columns of
Table 6-3. Each type of material was spiked in triplicate at each of the three spiking
concentrations, and 50 mL of fortified urine was used in each case. The materials were allowed
1 hour to absorb the urine before analysis. In addition, three blanks of each of the materials were
extracted without spiking.
6.2.1.6 Recovery at Three Levels Using 100 and 200 mL Urine
The previous experiment was repeated for the same materials, using the same urine
fortification levels, but with single tests using 100 or 200 mL of fortified urine instead of the
triplicate tests using 50 mL. The purpose of this test was to test whether volume also plays a role
in pesticide recoveries, because the possible larger volume results in better mixing and thorough
metabolites-extraction solvent contact; therefore, allowing for better recoveries.
The three materials were prepared (cleaned) as in Section 6.2.1.4. Two of each of the
materials were extracted without the addition of urine. The analytical results were calculated as
|ig/L in the volume of urine applied. For the matrix blanks, where no urine was used, 50 mL
were imputed as the sample volume. Percent recovery was calculated for each of the target
compounds in the spiked samples.
6-18
-------
6.2.2 Results
6.2.2.1 Water Capacity Experiments
Among four tested materials, cat litter had the smallest water capacity. It retained water
approximately 75% of its weight. Cloth diapers and Hydromatrix retained water approximately 3
times their own weight. Huggies and Maxi pads absorbed more than 20 times their weight in
water (Table 6-4).
Although water capacities were similar for the Maxi pads and the Huggies diaper, these
two materials differ in their water retaining mechanisms. The Maxi pad used in this study
contained a tissue core for absorption. The Huggies diaper contained a super-absorbent material,
polyacrylate polymer, for absorption. At a size of between 0.1 and 0.8 mm, these polymer
granules are barely distinguishable from normal salt. However, when exposed to water, the
polymer absorbs liquid and swells to a great extent. The large water capacity of the Huggies
diaper called into question the feasibility of extracting a urine sample from the diaper using 2 L
of water. A Huggies diaper was placed in a 3.8 L jar with 3 L water, capped, and tumbled for 1
hour. At the end, the diaper had torn and the polyacrylate beads had spilled and swelled to
absorb all but a few milliliters of liquid. The unabsorbed liquid was viscous, apparently
containing dissolved polyacrylate. Several brief experiments were conducted to investigate
whether water could be recovered from the acrylate gel beads, but the results were not promising,
therefore further investigation of polyacrylate polymer-containing diapers was canceled.
6.2.2.2 Background and Recovery With and Without Cleaning the Materials
The results indicate the cleaning methods used for the four materials were effective and
cleaned the background substantially. Cleaning reduced the level of interfering compounds to
below the QL for all tested metabolites. The results of the initial tests are shown in Table 6-5.
Among the four tested materials, the cloth diaper gave good recoveries (79% to 106%)
for 8 of the 12 analytes. Among these eight metabolites, five were compound-specific (DAMP,
NEC, ALDS, DSS, and FENS) and three were alkyl phosphates (DEP, DETP, and DMDTP).
6-19
-------
Table 6-4. Saturation Water Capacity of Materials.
Material
Cloth Diaper
Huggies
Maxi Pad
Cat Litter
Hydromatrix
Dry Mass (g)
105
50
9.9
300
100
Water Retained (g)
306
1400
225
225
300
The same five compound-specific metabolites also had good recoveries (75% to 94%) in
Hydromatrix. However, alkyl phosphates had poorer performance in Hydromatrix. Only one of
the alkyl phosphates (DMDTP) had satisfactory recovery (87% for as is and 94% for cleaned).
The tests on Maxi pads gave puzzling results. Although the recoveries for the pre-
cleaned Maxi pads were comparable to that of cloth diapers, the recoveries for the cleaned Maxi
pads were substantially lower. Except for DETP, which had a recovery of 71%, the recoveries
for all the other metabolites were below 70%, without a plausible explanation.
Among the four tested materials, cat Utter had the poorest recoveries. This is not
surprising because when urine was added to the material, the granules of the cat litter formed into
a clump. We extracted the clump regardless, after breaking it using hammer and blender.
The major ingredients of the cat litter were clumpable bentonite clay and silica gel. We
suspect these substances prevented the release of the metabolites when the clump was extracted.
In general, the recoveries were better for the tested compound-specific metabolites
(except for ABZ). Among the six alkyl phosphates, two metabolites, DETP and DMDTP, had
better recoveries than the rest.
6-20
-------
Table 6-5. Initial TestsMaterial Background Levels in ug/L and
Percent Recovery of High Level Spike
Analyte
QL(nfi/L)
Background (ug/L)
Diaper, as is
Diaper after washing
Maxi Pad, as is
Maxi Pad, washed
Cat litter, as is
Cat Litter, baked
Hydromatrix, as is
Hydromatrix, baked
Recovery
Spiking level (ug/L):
Diaper, as is
Diaper after washing
Maxi Pad, as is
Maxi Pad, washed
Cat litter, as is
Cat Litter, baked
Hydromatrix, as is
Hydromatrix, baked
ABZa.
0.5
2.6
n/d
<0.5
<0.5
n/d
n/d
1.3
n/d
250
50%
37%
57%
40%
n/d
n/d
45%
43%
DAMP
0.5
0.8
n/d
n/d .
n/d
n/d
n/d
3.2
n/d
250
94%
95%
88%
59%
2%
n/d
82%
89%
'NEC
5
3.7
n/d
n/d
1.6
2.6
n/d
12.3
n/d
1250
89%
87%
84%
57%
1%
n/d
76%
75%
ALDS
3
<3
n/d
n/d
n/d
n/d
n/d
<3
n/d
250
100%
101%
84%
53%
13%
10%
83%
83%
DSS
0.5
<0.5
<0.5
n/d
<0.5
<0.5
n/d
<0.5
n/d
250
92%
95%
93%
60%
25%
4%
91%
92%
FENS
0.5
0.6
n/d
n/d
0.5
<0.5
n/d
2.3
<0.5
250
96%
93%
89%
58%
9%
1%
92%
94%
DEDTPb
30
n/dd
n/d
n/d
n/d
n/d
n/d
n/d
n/d
250
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
DEP
10
23.4
n/d
n/d
n/d
n/d
n/d
n/d
n/d
250
84%
81%
61%
51%
20%
48%
48%
66%
DETPb
30
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
250
99%
102%
109%
71%
33%
n/d
133%
144%
DMDTP
10
<10
n/d
n/d
n/d
n/d
n/d
21.1
n/d
2500
106%
79%
92%
64%
26%
19%
87%
94%
DMP"
5000
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
12500
38%
41%
29%
31%
n/d
88%
n/d
25%
DMTPC
50
n/d
n/d
n/d
.n/d
n/d
1052
n/d
n/d
12500
n/d
2%
3%
2%
n/d
2%
n/d
n/d
a Analytes abbreviated as follows: ABZ: 2-aminobenzamidazole; DAMP: 2-diethylamino-6-methyl-pyrimidin-4-ol; NEC: N-ethyl-cyclohexamine;
ALDS: aldicarb sulfoxide; DSS: disulfoton sulfone; FENS: fenamiphos sulfone; DEDTP: diethyldithiophosphate; DMTP: dimethylthiophosphate;
DMDTP: dimethyldithiophosphate; DEP: diethylphosphate; DETP: diethylphosphorothioate; DMP: dimethylphosphate.
b Check standards not detected.
c Check standards not in control.
d n/d - not detected
-------
6.2.2.3 Recovery at Three Levels Using 50 mL Urine
We encountered analytical difficulties in this step, which is reflected in the inconsistent
numbers shown in Table 6-6. Further discussion is provided in the QC section (Section 6.2.3).
Initially, we attempted to determine all the analytes in a single run using LC-MS-MS
with an APCI interface. No problems occurred during the method development phase (see
RTI/ACS-SOP-500-004) and the initial test (Section 6.2.2.2.); however, when analyzing samples
at three spiking levels using 50 mL urine, instrument performance for the alkyl phosphates
degraded and remained poor despite many efforts to correct the problem. We therefore changed
the LC-MS-MS to an electrospray (ESI) interface, instead of the original APCI interface. This
modification improved alkyl phosphate measurement greatly, giving a QL of 1 ng/mL for all of
the alkyl phosphates except DMP, which remained at 5000 ng/mL. Because 5000 ng/mL
exceeded the highest levels anticipated in human urine, we did not spike the remaining samples
with DMP. The spiking levels of the other alkyl phosphates were adjusted to place the lowest
spiking level at or near the QL determined from the previous experiment.
Because LC-ESI-MS was optimized for the six alkyl phosphates rather than the six
compound-specific metabolites, we only used this mode to analyze the alky phosphates. The
data reported in the next two sections contains results from compound-specific metabolites
obtained from the LC-APCI-MS analysis and the results of alkyl phosphates obtained from the
LC-ESI-MS analysis of the same extracts.
Unspiked, cleaned material was first analyzed for background (Table 6-6). No
metabolites were found in cloth diaper. Small interferences were found for target compound
DETP in the Maxi pad and for DEDTP in the Hydromatrix. A relatively high level of
interferences was found for DMDTP for Hydromatrix.
Table 6-7 gives the spiking levels (ng/mL in 50 mL urine) and recoveries of the spiked
compounds in percent. The high level spikes gave fairly good and reproducible recoveries.
Recoveries greater than 130% occur more frequently in the mid and low spiked samples, where
6-22
-------
Table 6-6. Material Background for 50 mL Urine Test.
QL
LVl-DIA-Bb
LV2-DIA-BL
LV1-PAD-BL
LV2-PAD-BL
LV3-PAD-BL
LV1-HYD-BL
LV2-HYD-BL
LV3-HYD-BL
ABZa
0.5
n/dc
n/d
n/d
n/d
n/d
n/d
n/d
n/d
DAMP
0.5
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
NEC
5.0
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
ALDS
1.0
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
DSS
0.5
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
FENS
0.5
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
DEDTP
1
n/d
n/d
n/d
n/d
n/d
2
n/d
n/d
DEP
1
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
DETP
1
n/d
n/d
n/d
1
n/d
n/d
n/d
n/d
DMDTP DMP
1
n/d
n/d
n/d
n/d
n/d
n/d
n/d
10
5000
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
DMTP
1
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
' Analytes abbreviated as follows: ABZ: 2-aminobenzamidazole; DAMP: 2-diethylamino-6-methyl-pyrimidin-4-ol; NEC: N-ethyl-cyclohexamine;
ALDS: aldicarb sulfoxide; DSS: disulfoton sulfone; FENS: fenamiphos sulfone; DMPT: dimethylphosphorothioate; DEDTP: diethyldithiophosphate;
DMDTP: dimethyldithiophosphate; DMP: dimethylphosphate; DEP: diethylphosphate; DETP: diethylphosphorothioate; DMTP:
dimethylthiophosphate.
b LV1, LV2 and LV3 denote matrix blanks associated with high, med, and low spiking.
c n/d - not detected
poor chromatographic peak shape and occasional interfering peaks may cause quantification
errors. Because of inconsistent recoveries from method controls (which will later be discussed in
the QC section, Section 6.2.3), we hesitate to draw many conclusions on the performance of the
three tested materials based upon this table.
6-23
-------
Table 6-7. Percent Recovery of Metabolites from Materials Spiked with 50 mL of Fortified Urine.
Spiking level (jig/L)
': : ABZ"
High 100
Med ,..: - -: 10
Low - '"1
Percent Recovery
LV1-DIA-HI1
LV1-DIA-HI2
LV1-DIA-HI3
LV3-DIA-M2
LV3-DIA-M3
LV3-DIA-M4
LV2-DIA-LO1
LV2-DIA-LO2
LV2-DIA-LO3
LV1-PAD-HI1
LV1-PAD-HI2
LV1-PAD-HI3
LV3-P AD-MI
LV3-PAD-M2
LV3-PAD-M3
LV2-PAD-LO1
LV2-PAD-LO2
LV2-PAD-LO3
LV1-HYD-HI1
LV1-HYD-HI2
LV1-HYD-HI3
LV3-HYD-M1
LV3-HYD-M2
LV3-HYD-M3
LV2-HYD-LO1
LV2-HYD-LO2
LV2-HYD-LO3
43
80
45
106
85
93
75
83
109
61
56
64
117
131
119
62
52
71
57
53
46
0
126
107
95
98
86
DAMP
100
10
;V- i
116
143
119
175
145
147
362
455
508
117
132
145
133
149
138
380
310
409
138
145
. 134
0
150
139
361
470
273
"NEC
500
50
,:s"
97
120
118
125
126
117
0
0
119
112
125
139
120
129
119
171
136
0
127
134
122
0
136
139
114
131
0
ALDS
300
30
3
241
427
136
150
127
134
154
182
228
114
113
126
113
136
117
134
87
142
113
104
84
0
131
137
125
223
119
DSS
100
10
1
96
111
99
123
110
116
112
107
121
101
116
128
109
121
108
86
78
72
121
126
112
130
119
118
85
132
75
FENS DEDTP
100 5000
10 - 500
1 50
113
134
106
160
144
145
133-
144
170
102
109
120
126
144
126
124
103
94
109
109
97
151
137
139
113
149
99
66
71
62
91
85
65
58
53
74
89
103
110
159
173
152
83
59
70
89
89
85
155
105
0
72
98
68
DEP
1000
100
10
106
116
105
203
165
142
101
121
144
91
106
110
162
176
158
113
81
118
115
114
111
168
135
213
105
164
105
DETP DMDTP
3000 1000
300 100.
30 10
91
105
86
143
117
107
91
117
131
78
85
102
115
119
104
88
56
63
100
100
96
125
104
177
110
163
105
34
33
27
20
18
15
205
164
185
73
76
91
146
149
108
414
172
200
79
81
78
140
97
440
192
200
194
DMP DMTP
0 1000
.... :-0- 100
0 "10
n/s"
n/s
n/s
. n/s
n/s
n/s
n/s
n/s
n/s
n/s
n/s
n/s
n/s
n/s
n/s
n/s
n/s
n/s
n/s
n/s
n/s
n/s
n/s
n/s
n/s
n/s
n/s
112
129
110
139
114
90
144
160
168
99
99
116
113
113
99
146
98
146
104
104
108
114
97
167
99
187
99
° n/s - not spiked
Analytes abbreviated as follows: ABZ: 2-aminobenzamidazole; DAMP: 2-diethylamino-6-methyl-pyrimidin-4-ol; NEC: N-ethyl-cyclohexamine;
ALDS: aldicarb sulfoxide; DSS: disulfoton sulfone; FENS: fenamiphos sulfone; DEDTP: diethyldithiophosphate; DMTP: dimethylthiophosphate;
DMDTP: dimethyldithiophosphate; DEP: diethylphosphate; DETP: dietbylphosphorothioate; DMP: dimethylphosphate.
6-24
-------
6.2.2.4 Recovery at Three Levels Using 100 and 200 mL Urine
Three materials were pre-cleaned. Unspiked materials were first analyzed for background
(Table 6-8). No background or interferences were found for the metabolites in three tested
materials.
Table 6-8. Material Background Expressed as ng/L Assuming 50 mL of Urine.
QL
Vl-DIA-BL"
V2-DIA-BL
"Vl-PAD-BL
V2-PAD-BL
Vl-HYD-BL
V2-HYD-BL
ABZ"
0.5
n/d"
n/cc
n/d
n/d
n/d
n/d
DAMP
0.5
n/d
n/c
n/d
n/d
n/d
n/d
NEC
5.0
n/d
n/c
n/d
n/d
n/d
n/d
ALDS
1.0
n/d
n/c
n/d
n/d
n/d
n/d
DSS
0.5
n/d
n/c
n/d
n/d
n/d
n/d
FENS'
0.5
n/d
n/c
n/d
n/d
n/d
n/d
DEDTP
. 1
n/d
n/d
n/d
n/d
n/d
.n/d
DEP
1
n/d
n/d
n/d
n/d
n/d
n/d
DETP
1
n/d
n/d
n/d
n/d
n/d
n/d
DMDTP DMP
1
n/d
n/d
n/d
n/d
n/d
n/d
5000
n/d
n/d
n/d
n/d
n/d
n/d
DMTP
1
n/d
n/d
n/d
n/d
n/d
n/d
The results for the compound-specific metabolites (ABZ, DAMP, NEC, ALDS, DSS, FENS) were obtained from the LC-APCI-MS analysis. The'
results for the other (phosphate) compounds were obtained from a later LC-ESI-MS analysis.
"VI and V2 denote matrix blanks associated with 100 mL and 200 mL tests, respectively.
b n/d - not detected; Analytes abbreviated as follows: ABZ: 2-aminobenzamidazole; DAMP: 2-diethylamino-6-methyl-pyrimidin-4-ol; NEC: N-ethyl-
cyclohexamine; ALDS: aldicarb sulfoxide; DSS: disulfoton sulfone; FENS: fenamiphos sulfone; DEDTP: diethyldithiophosphate; DMPT:
dimethylphosphorotnioate; DMDTP: dimethyldithiophosphate; DEP: diethylphosphate; DETP: diethylthiophosphate; DMP: dimethylphosphate:
DMTP: dimethyithiophosphate.
c n/c - not analyzed
The results from 100 mL and 200 mL spike urine tests were comparable for cloth diaper
and Maxi pad (Table 6-9 and Table 6-10). In both tests, good recoveries (70% to 130%) were
seen for most of the metabolites at the mid and high spiking level for the these two materials.
Good recoveries were also seen at high spiking level in Hydromatrix clay. The recoveries were
somewhat low (50% to 60%) for more than half of the target compounds in Hydromatrix at mid-
level. All three materials gave poorer recoveries at the lower spiking level.
6-25
-------
Table 6-9. Percent Recovery of Metabolites from Materials Spiked with 100 mL
of Fortified Urine.
Vl-DIA-HI'
Vl-DIA-M
Vl-DIArLO
Vl-PAD-HI
Vl-PAD-M
Vl-PAD-LO
Vl-HYD-HI
Vl-HYD-M
Vl-HYD-LO
ABZ"
101
80
74
119
110
46
85
60
0
DAMP
113
122
19
94
96
32
87
78
17
NEC.'.
119
89
191
105
123
68
87
58
0
ALDS
115
103
223
92
116
95
80
63
32
DSS
99
94
94
90
99
47
84
54
29
FENS DEDTP
106
97
121
87
115
48
83
53
26
92
50
15
107
100
44
103
51
. 25
DEP DETP ., DMDTP
148
115
169
133
119
57
119
71
46
99
65
91
92
.77
33
88
49
25
92
53
75
107
85
99
93
53
53
DMP
n/sb
n/s
n/s
n/s
n/s
n/s
n/s
n/s
n/s
DMTP!
72
88
209
61
90
45
54
49
35
'Spiking levels are given in Table 5-1.
Bn/s - not spiked; Analytes abbreviated as follows: ABZ: 2-aminobenzamidazole; DAMP: 2-diethylamino-6-methyl-pyrimidin-4-ol; NEC: N-ethyl-
cyclohexamine; ALDS: aldicarb sulfoxide; DSS: disulfoton sulfone; FENS: fenamiphos sulfone; DEDTP: diethyldithiophosphate; DMPT:
dimethylphosphorothioate; DMDTP: dimethyldithiophosphate; DEP: diethylphosphate; DETP: diethylthiophosphate; DMP: dimethylphosphate;
DMTP: dimethylthiophosphate.
Table 6-10. Percent Recovery of Metabolites from Materials Spiked with
200 mL of Fortified Urine.
1 t-
1
V2-DIA-HI
V2-DIA-M
V2-DIA-LO
V2-PAD-HI
V2-PAD-M
V2-PAD-LO
V2-HYD-HI
V2-HYD-M
V2-HYD-LO
ABZ"
92
90
83
105
0
51
85
98
30
DAMP
94
134
249
87
99
96
73
119
38
NEC
93
89
176
87
139
53
72
89
36
ALDS
98
107
272
88
133
71
66
92
31
DSS
88
99
106
80
106
44
67
90
27
FENS
88
102
124.
81
128
49
62
92
. 28
DEDTP
94
92
67
95
112
39
65
67
21
J)EP
115
116
139
HI
145
66
67
78
33
DETP
104
120
63
86
124
36
56
60
22
DMDTP,
75
77
60
88
113
89
52
68
55
DMP
n/s'
n/s
n/s
n/s
n/s
n/s
n/s
n/s
n/s
DMTP
66
81
131
64
86
76
51
69
43
" n/s - not spiked; Analytes abbreviated as follows: ABZ: 2-aminobenzamidazole; DAMP: 2-diethylamino-6-methyl-pvrimidin-4-ol; NEC: N-ethyl-
cyclohexamine; ALDS: aldicarb sulfoxide; DSS: disulfoton sulfone; FENS: fenamiphos sulfone; DEDTP: diethyldithiophosphate; DMPT:
dimethylphosphorothioate; DMDTP: dimethyldithiophosphate; DEP: diethylphosphate; DETP: diethylthiophosphate; DMP: dimethylphosphate;
DMTP: dimethylthiophosphate.
6-26
-------
6.2.3 Quality Control
6.2.3.1 Instrument Calibration
The LC-MS-MS configured in the APCI mode was calibrated with a set of calibration
standards containing the target analytes over a range of concentrations. Calibration solutions
were prepared in human urine and contained the internal standard d5-atrazine at 100 ng/mL. The
instrument was calibrated for the compound-specific metabolites ABZ, DAMP, DSS, and FENS
over.a range from 0.5 to 1000 ng/mL for ALDS from 1.5 to 3000 ng/mL; and for NEC from 5 to
5000 ng/mL. The standards were analyzed and the resulting peak area data fitted to Equation 2
using linear regression. This equation was then solved for X and applied to the area data of the
standards to compute, , the 'predicted' analyte concentration. The calibration was acceptable
over the range of analyte concentrations where the 'predicted' concentration differed by no more
than 25% from the actual concentration. The range of the acceptable calibration is given in Table
6-11. A mid-level calibration standard was analyzed after every 10 samples. The calibration for
a single analyte was considered in control if the response factor varied by no more than 25%
from that obtained during calibration. If more than two analytes were out of control, the
instrument was recalibrated.
Area-
= a*X2 + b*X + c (Eq. 2)
Areais
where x=concentration of target/concentration of internal standard.
For the data presented in Sections 6.2.2.3 and 6.2.2.4, the alkyl phosphate data were
obtained from a separate analysis. The LC/MS/MS was configured in the ESI mode and
calibrated with a set of standards containing all of the alkyl phosphates at a range from 1 to
10000 ng/mL. Such a broad range was necessary because the samples were spiked at high levels
for the APCI method. The lower standards were used to measure background. The resulting
calibration plots followed an acute S-curve. Equation 2 could not be fit to the calibration data
without residuals greater than 50%. Quadratic approximations of the curves resulted in a
parabola whose maximum fell below the highest data point, giving no solution of the equation .
6-27
-------
for samples spiked at the high levels. To solve the problem, we employed the method of
quantifying each unknown with the response factor of the standard nearest it in area response.
Among the drawbacks of this method are that there is no smoothing and no measure of the
goodness of fit or variance. Because of this, we cannot calculate the precision of these
measurements. The acceptable range of the calibration is thus defined as the range of standards
over which the relative response varies by no more than 25% between adjacent levels. Table 6-
11 gives the acceptable range of the calibrations used during the two phases of this work.
Table 6-11. Acceptable Calibration Range by Experiment.
?:4;'d^
r.ABZ'-JDAMlK';
NEC, -ALDS- y
pss ; FENS . PEDTP
DEP DETF DMDTP(
DMP pMTpj
Initial Experiments
QL
max
0.5 0.5
1000 1000
5
5000
3
1000
0.5
1000
0.5
1000
30
1000
10
1000
30
1000
10
10000
5000
50000
50
50000
Recovery Experiments
QL
max
1 1
200 200
10
1000
2
600
1
200
1
200
1
10000
1
10000
1
10000
5.
10000
500
10000
1
10000
" Analytes abbreviated as follows: ABZ: 2-aminobenzamidazole; DAMP: 2-diethylamino-6-methyl-pyrimidin-4-ol; NEC: N-ethyl-
cyclohexamine; ALDS: aldicarb sulfoxide; DSS: disulfoton sulfone; FENS: fenamiphos sulfone; DEDTP: diethyldithiophosphate;
DMPT: dimethylphosphorothioate; DMPDT: dimethylphosphorodithioate; DEP: diethylphosphate; DETP: diethylthiophosphate; DMP:
dimethylphosphate; DMTP: dimethylthiophosphate.
6.2.3.2 Quality Control Samples
Nine method control samples were prepared and analyzed over the course of this work.
Method control samples consisted of 50 mL of urine fortified with target compounds in known
amounts. A control sample was prepared and analyzed in conjunction with each experiment and
was intended to reflect the performance of the analysis method. Table 6-12 gives the results of
these analyses.
The recoveries for method control for the initial test were mostly satisfactory for the
compound-specific metabolites, except for ALDS, which had an interference in one duplicate at
mid-spiking level. The.recoveries for method control were inconsistent for the 50 mL spike
urine study reflecting inconsistent instrument response during testing. The recoveries were
especially inconsistent at lower levels for compound-specific metabolites that were measured
6-28
-------
Table 6-12. Percent Recovery of Analytes from Method Controls.
ABZ" DAMP NEC ALDS
DSS FENS DEDTP
DEP DETP DMDTP
BMP DMTP
Spiking levels in ng/L
H(high)
M (medium)
L (low)
Percent Recovery
BN1-MC-M"
BN2-MC-M
SP1-MC-H
SP2-MC-H
LV1-MC-H
LV3-MC-M
-JLV2-MC-L
V2-MC-M
Vl-MC-M
100
10
1
65
78
65
55
88
126
73
86
104
100
10
1
96
76
72
95
218
165
242
98
88
500
50
5
84
79
70
82
184
153
0
77
125
300
30
3
197
81
76
100
161
140
58
.87
114
100
10
1
83
77
75
103
115
125
. 58
75
100
100
10
1
83
75
69
85
77
148
81
83
117
5000
500
50
n/ab
n/a
n/a
n/a
122
146
67
102
56
1000
100
10
n/a
n/a
n/a
n/a
127
144
91
113.
63
3000
300
30
n/a
n/a
n/a
n/a
107
110
92
73
51
.1000
100
10
n/a
n/a
n/a
n/a
97
138
184
98
85
12500
500
0
n/a
n/a
n/a
n/a
n/sc
n/s
n/s
n/s
n/s
1000
100
10
n/a
n/a
n/a
n/a
115
113
103
91
56
" BN1, BN2, SP1, and SP2 denote control samples associated with the initial experiments; LV1, LV2, LV3, VI, and V2 denote matrix blanks
associated with high, med low spike SO mL, 100 mL, and 200 mL tests, respectively. Analytes abbreviated as follows: ABZ: 2-aminobenzamidazole;
DAMP: 2-diethylamino-6-methyl-pyrimidin-4-ol; NEC: N-ethyl-cyclohexamine; ALDS: aldicarb sulfoxide; DSS: disulfoton sulfone; FENS:
fenamiphos sulfone; DEDTP: diethyldithiophosphate; DMPT: dimethylphosphorothioate; DMDTP: dimethyldithiophosphate; DEP: diethylphosphate;
DETP: diethylthiophosphate; DMP: dimethylphosphate; DMTP: dimethylthiophosphate.
b n/a - not applicable
c n/s - not spiked
using LC/APCI/MS. Inconsistent recoveries for method controls suggest that the results in the
50 mL urine spike test may not be reliable for compound-specific metabolites at mid and low
levels. Nonetheless, recoveries were satisfactory for alkyl phosphates, ABZ, DSS, and FENS at
the high spiking level. The recoveries for method control were good for the 100 mL and 200 mL
urine the tests. Comparisons of the tested materials were thus based on these two tests.
6.2.4 Discussion
6.2.4.1 Selection of Analytical Method
The commonly used GC method to analyze dialkylphosphate urinary metabolites of
organophosphorus pesticides involves pre-cleanup of the urine followed by derivatization, which
requires substantial effort (Moate et ah, 1999). The LC/MS/MS method, on the other hand, does
not require cleanup, but it was only used on several compound-specific metabolites by a few
researchers (Baker et ah, 2000). In this study, we tried to use LC/MS/MS to analyze the required
6-29
-------
compound-specific OP metabolites and dialkylphosphate in one run without cleanup. We first
employed LC/APCI/MS, which worked well for the compound-specific metabolites, but was
unsuitable for the alkyl phosphate analytes. We then used an electrospray interface, which
provided good results for the alkyl phosphates, but it was too destructive for use on the other,
more labile metabolites. With both methods, adsorption, compound degradation, and large
changes in response over 24 hours made accuracy and precision goals elusive at times. Although
the results showed some inconsistencies, given its simplicity, the LC/MS/MS method is deemed
suitable for further investigation.
6.2A.2 Alternative Urine Collection Materials
Among the four tested materials, we the cloth diaper and Maxi pad performed well for
urine collection. Both materials demonstrated low background and gave satisfactory recoveries
for most of the tested compound-specific carbamate metabolites (ABZ, NEC, and ALDS),
compound-specific organophosphate pesticide metabolites (DAMP, DSS and FENS), and
compound-nonspecific organophosphate pesticide metabolites (alkyl phosphates).
6.3 STUDY #3: DISPOSABLE DIAPER TO COLLECT URINE SAMPLES FROM
YOUNG CHILDREN FOR PYRETHROID PESTICIDE STUDIES
The objectives of this study were to evaluate whether disposable diapers that contain
polyacrylate granules can be extracted using salt solutions, as well as whether they can be used
for collection and quantitative measurement of selected urinary pyrethroid pesticide metabolites
and creatinine. The storage stability of the pyrethroid metabolites and creatinine in a wet diaper
was also evaluated.
This study was supported by EPA STAR Grant no. R829397-01-0. The study has been
published in the Journal of Exposure Analysis and Environmental Epidemiology (2004) 14:378-
384.
6-30
-------
Journal of Exposure Analysis and Eamrnmattal Epidemiology (2004) 14,378-384 /fffi&
© 2004 Nature Publishing Croup All rights reserved 1053-4245/04/S30.00 lIlP
wwwjiature.com/jea
Disposable diaper to collect urine samples from young children
for pyrethroid pesticide studies
YE HU, JAMES BEACH, JAMES RAYMER AND MICHEAL GARDNER
Analytical and Chemical Sciences, Research Triangle Institute International, Research Triangle Park, NC. VSA
Disposable diapers are widely used in the US and many other areas in the world; therefore, they are ideal media for urine collection for measurement of
young children's exposure to pesticides. However, disposable diapers normally contain polyacrylate polymers that make the extraction and analysis of
urine very difficult The objectives of this paper were to evaluate whether disposable diapers that contain polyacrylate granules can be extracted using salt
solutions, and whether they can be used for .the collection and quantitative measurements of selected urinary pyrethroid pesticide metabolites and
_creatinine. The storage stability of the metabolites and creatinine in a wet diaper at body temperature and at refrigeration temperature was also evaluated.
Salt solutions including calcium chloride dihydrate, magnesium sulfate, ammonium acetate, and sodium chloride solutions were tested for efficiency of
polymer shrinkage. Pyrethroid metabolites 3-<2^2-dichlorovinyl)-2,2-dimethyl-(l-cyclopropane) carboxylic acid (DCCA), 3-(2,2-dibromovuiyl)-
2,2,dimethyl-(l-cyclopropane) carboxylic acid (DBCA) and 3-phenoxybenzoic acid (3-PBA) were analyzed using LC/MS/MS and evaluated for
recoveries in the urine released from the diapers. The study found caMnm chloride dihydate to be satisfactory in releasing urine and metabolites from the
polymers. The percent recoveries for the three tested pyrethroid metabolites were mostly in the range of 65-130. The percent recoveries for creatinine were
in the range of 71-133. The detection limit for each of the three metabolites was 0.1 ugfl. The pyrethroid metabolites and creatinine were stable on the
diaper for at least 72 h. We concluded from this study that calcium chloride dihydrate can successfully release urine and metabolites from polyacrylate-
containing diapers, and the method is promising for studies of pyrethroid metabolites.
Journal of Exposure Analysis and Environmental Epidemiology (2004) 14, 378-384. doi:10.1038/sj.jea.7S00334
Keywords: polyacrylate polymer, pesticide metabolites, pyrethroid, young children, disposable diaper, urine collection.
Introduction
For large cohort studies that involve young children who
wear diapers, it is highly desirable to develop a convenient
and low-cost method to collect urine arid to analyze urinary
metabolites. Disposable diapers are widely used in the US
and many other areas in the world; therefore, they are ideal
media for urine collection. However, disposable diapers
normally contain polyacrylate polymers, which make the
extraction and analysis of urine very difficult. To circumvent
the problem of extraction, we attached gauze pads to the
diaper to collect urine samples (Hu et al., 2000). The problem
with the gauze method, however, is that only 3-12 ml of
urine can be expressed; up to 170ml of urine is produced
from a normal 1-3-year-old child and could be available for
analysis. This volume limitation of the gauze pad can impact
both the limit of detection and the number of analyses
possible for a given sample.
1. Address all correspondence to: Ye Hu, Analytical and Chemical
Sciences, Research Triangle Institute, Post Office Box 12194, Research
Triangle Park, NC 27709, USA. Tel: + 919-541-8799; Fax: +919-541-7208.
E-mail: yhu@rti.org
Received 3 July 2003; accepted 11 November 2003
A typical commercially available disposable diaper con-
tains several parts: an absorbent pad made of polyacrylate
absorbent powder mixed in fluffy wood cellulose fibers, a
porous polypropylene top and back sheet, two lateral tapes, a
frontal tape, and elastic around the leg area. The absorbent
pad loaded with polyacrylate absorbent powder is the
primary part for urine collection (Mukerjee, 2000).
The polyacrylate absorbents used in diapers are a family of
polymers that have extraordinary absorbency. Figure 1
depicts the water absorption process of sodium polyacrylate
([CH2-CH--y, one of the simplest polymers in
commercial, diapers. When in powdered state, the polymer
chains are coiled. When hydrated, the sodium ion detaches so
that the carboxyl groups become negatively charged and
repel one another to uncoil the polymer chain to allow more
water to associate with more carboxyl groups or sodium
atoms. As the polymer continues to uncoil and absorb water,
it swells into a gel-like material. As the polymer also has
weak cross-link properties, it effectively forms a .three-
dimensional structure (Mukerjee, 2000).
In the presence of some metal cations, however, the
polymer collapses or "shrinks". Figure 2 demonstrates the
collapse of the polymer chain caused by Ca2"1". The Ca2+
ions bind the carboxylate groups of the polymer and
neutralizes the polyanions. Due to the hydrophobic nature
6-31
-------
Using disposable diaper for pyrethroid studies
Hu et at.
Dry
'O'Na*
in Water
H
C=O
1
°~H2O
H*° Na*
H2O
H
w
c=o
1
o-
H2O r
Na*
H-jO
fi 2
c=o c=o
1 1
°- H20 0-Hz0
'2° Na* H2O Na+
H20 H^°
HaO
' H2O H20
Figure 1. Mechanism of absorbency for sodium polyacrylate.
of the backbone, the polyacrylate chain collapses. Combined
static and dynamic light scattering suggests a compact
spherical shape of the collapsed polymer (Schwein and
Huber, 2001). This property of the polymer shrinkage makes
it possible to release the urine metabolites from the gel diaper
again. Once the polymer collapses, much of the absorbed
liquid is released, thus recovering the analytes.
Pyrethroids are a class of synthetic broad-spectrum
insecticides that are widely applied to crops, garden plants,
pets, and also directly to humans. By the mid-1990s,
a
c=o
<>H20
i i
rji
6-
S
c=o
0- H20
H
c=o
°-H20
H
C=O
Q-^HaO
^
c=o
1 °~H2O
H2O
Na+
H2O
H20
pyrethroids use had grown to represent 23% of the US
dollar vahie of the world insecticide market, ranking second
only to organophosphorus compounds (Soderiund et al.,
2002). After EPA's ban of two most commonly used
organophosphorus pesticides, chlorpyrifos, and diazinon,
for indoor use, pyrethroids have been increasingly used
indoors. As a neurotoxin, pennethrin acts on the sodium
channel of the excitatory nerves (Miyamoto et al., 1995). The
most important process in the metabolism of pyrethroids is
the cleavage of the central ester linkage, which occurs in the
liver (Leng et al., 1997). Table 1 lists the parent pyrethroids
and their corresponding metabolites that are widely used as
biomarkers for exposure assessment. The metabolites are
mostly excreted in urine. Depending on the pyrethroids
investigated, the half-life of the metabolites ranges from 7 to
55 h in blood (Leng et al., 1996).
In this paper, we tried several salt solutions to collapse the
polymer gel in the diaper. The objectives of this paper were to
evaluate whether disposable diapers that contain polyacrylate
granules can be extracted using salt solutions and whether
they can be used for collection and quantitative measure-
ments of selected urinary pyrethroid pesticide metabolites
and creatinine. The storage stability of the pyrethroid
metabolites and creatinine in a wet diaper was also evaluated.
Methods
Materials
Three pyrethroid metabolites were selected for this study and
are listed in Table 1. 3-(2,2-Dichlorovinyl)-2,2-dhnethyl-(l-
H20
H2O
H2O
H2O
H H20
Figure 2. Collapse of sodium polyacrylate in the presence of Ca2+.
6-32
journal of Exposure Analysis and Environmental Epidemiology (2004) 14(5)
379
-------
Hu el al.
Using disposable diaper for pyrethroid studies
Table 1. List of target pesticide metabolites.
Metabolite
Parent compounds
3-Phenoxybenzoic acid (3-PBA)
3-{2,2-dichlorovinyl)-2^-dimethylcyclopropane-
1-carboxylic acid (DCCA)
3-{2,2-dibromovinyl)-2,2-
-------
Using disposable diaper for pyrethroid studies
Hu et al.
results were compared to determine creatinine recovery
across the entire method.
(4) Recoveries at four metabolite levels: Fortified urine was
prepared at four metabolite concentrations, O.I, 0.2, 1, and
5/ig/l. At each concentration level, three diapers were spiked
with 75 ml fortified urine. All 12 diapers were extracted using
100ml of CaQ2 water solution with a concentration of
ISOg/l. Aqueous extracts were obtained and analyzed as
described above.
(5) Storage Stability: To simulate overnight urine collec-
tion and storage, 15 diapers were spiked with 75 ml of urine
fortified with DBCA, DCCA, and 3-PBA at 1 //g/1. Three
diapers were extracted and analyzed immediately. Six were
placed in an incubator at 37°C; three of these were analyzed
at 4h, then the last three at 8 h. Nine diapers were stored in a
refrigerator (1°Q; three were extracted at 24 h, three at 48 h,
and three at 72 h. The diaper stored at 37°C would reveal
analyte perturbations during prolonged wearing (overnight).
The diaper stored in the refrigerator would reveal allowable
storage after collection and before extraction. Supematants
were also analyzed for creatinine. To determine the creatinine
concentration in urine that was not exposed to the polymers,
six undiluted samples without going through the diapers were
analyzed for creatinine.
Sample Analysis
The LC/MS/MS analyses of the extracts were performed on
a PE-Sciex API-3000 triple quadrupole mass spectrometer
with Turbo Ion Spray source (Pelkin Elmer Biosystem,
Foster City, CA, USA), which was interfaced with a Pelkin
Elmer Series 200 Micro HPLC. The ion source was operated
in the negative ionization mode, and the mass analyzer was
operated in multiple reaction monitoring (MRM) mode. The
precursor ions for all analytes were measured as deproto-
nated quasimolecular ions (M-H). Table 2 shows the
precursor and product ions used for each analyte.
A high-performance liquid chromatography (HPLC)
separation was utilized to minimize ion suppression from
matrix interference. The column was a Luna 3/nn C18(2)
50x2.0 mm2 (Phenomenex, Torrance, CA, USA) with a
mobile phase flow rate of 300/d/min. The mobile phases
were: A = water with 0.2% acetic acid (v/v); B=methanol
Table 2. Precursor and product ions used for the analytes.
Compound
3-{2,2-dichlorovinyl)-2)2-dimethyl-(l-cydopropane)
carboxylic acid
3-{2,2-dibromovinyl)-2,2-dimethyl-(l -cyclopropane)
carboxylic acid
3-phenoxybenzoic acid
Precursor
(m/r)
207
297
213
Product
(m/z) .
35
79, 81"
93
'Signal from both product ions was summed for quantification.
with 0.2% acetic acid (v/v). A gradient program was used as
follows: hold 5% B for 1.0 min, linear 5-50% B in 2.0 min,
linear 50-95% B in 5.0 min, 95% B for 3.0 min, linear 95-
5% B in 1.0 min, hold 6.0 min for column equilibration. A
measure of 50 /d of extracts were injected. The first 7.5 min of
the analysis was diverted to waste to minimize source fouling,
and a make-up flow of 1:1 methanol/water (v/v) was
supplied to the ion source from an additional pump during
this period. ' .
Aqueous calibration standards with 0.2 N NaOH were
analyzed from 1.0 to 50/jg/l prior to sample analysis. An
external standard calibration curve was constructed from a I/
x2 weighted linear regression, with a minimum of four points.
A 5.0 ^g/1 check standard was analyzed before and after
sample sets, with a maximum of 10 samples per set. The
difference between the measured and theoretical concentra-
tion of analytes in the check standards was required to be less
than 20% for sample data to be acceptable.
Creatinine was analyzed by a local clinical laboratory
(Quest Diagnostics, Inc., Gary, NC, USA) using a standard
optical absorbance method. Samples were stored at 1°C until
picked up by the company.
Results
(1) Optimal salts to shrink the polymer: Only the calcium and
magnesium salt solutions caused observable shrinkage of the
polyacrylate. About 50-100 ml of liquid was recovered when
100ml of the calcium chloride solution with 150 g/1 or higher
concentration was applied. About 10-30 ml was recovered
with 100ml of magnesium sulfate at the same concentration
level. No liquid at all could be recovered from diaper
materials treated with the ammonium acetate or sodium
chloride salt with comparable ionic strength.
(2) Recoveries of metabolites for Mg and Ca salt extraction:
Urine samples added with Ca2 +, but not exposed to a diaper
("controls"), had a recovery of 95% for DCAA, 74% for
DBCA, and 66% for 3-PBA. Despite the low recoveries for
3-PBA, the precisions for all three compounds were good
(< 15%). It was felt that this method would be adequate to
estimate recoveries from diapers.
Percent recoveries were calculated by dividing the mea-
sured concentration by the spiking level, and were corrected
.for dilution and reconcentration. Table 3 shows the percent
recoveries of the metabolites in the supernatant obtained
from magnesium and calcium solutions. Calcium chloride
solutions yielded higher and more consistent recoveries than
magnesium sulfate solution. This may be partially explained
by the fact that more liquid (50-100 ml) was recovered by
calcium solution extraction, compared to magnesium solu-
tion extraction (10-30ml). Larger volumes of liquid might
have enabled better mixing and thorough "washing off' of
the metabolites from the polymer. Percent recoveries for
6-34
Journal of Exposure Analysis and Environmental Epidemiology (2004) 14(5)
381
-------
Hu et al.
Using disposable diaper for pyiethroid studies
Table 3. Recoveries of metabolites for Mg and Ca salt extraction (spiked at 1 /ig/1).
Solution (150 g/1)
Percent Recoveries (RSD)
N
DBCA
DCCA
3-PBA
Ca-extracted diaper
Mg-extracted diaper
Controls (urine added with Ca, without going through diaper)
3
3
5
85(9)
60(76)
95 (14)
78(8)
62 (62)
74(12)
61(8)
54(44)
66 (12)
Table 4. Impact of calcium concentration on percent recoveries
(triplicates spiked at
Calcium (g/1)
Percent recovery (RSD)
DBCA
DCCA
3-PBA
Creatinine
50
150
450
68(9)
67(7)
59 (15)
61 (3)
64(9)
62 (29)
53(0)
53(2)
41 (49)
95(2)
71 (23)
NA"
"NA: not available; interference prevented creatinine determination.
Table 5. Recoveries at four metabolite levels in triplicate.
Percent recoveries (RSD)
Metabolite concentration (/tg/1) DBCA
DCCA
3-PBA
0.1
0.2
1.0
5.0"
'Results from one reolicate wer
69 (13)
44(30)
78(13)
66(5)
90 (7) 122 (2)
65(11) 112.(11)
75 (7) 83 (5)
65 (2) 57 (5)
e excluded, based urxra Dixon Otest.
calcium-extracted samples were 85%, 78%, and 61% for
DBCA, DCCA, and 3-PBA, respectively. These recoveries
compared very well to those from urine controls, which were
95%, 74%, and 66% for DBCA, DCCA, and 3-PBA,
respectively.
(3) Impact of calcium level on the recoveries of pesticide
metabolites and creatinine: The impact of calcium level on the
percent recoveries of the pesticide metabolites and creatinine
is shown in Table 4. The results indicate comparable analyte
recoveries with CaCl2 solutions at 50 or 150 g/1 level.
Recoveries were somewhat lower when CaCl2 concentration
was 450 g/1. We noticed that some extracts (in 0.2 N NaOH)
were very cloudy, while others were not. The local laboratory
that analyzed the creatinine also reported some difficulties for
samples extracted with CaCl2 solution with a concentration
of 450 g/l, presumably due to the high background level of
CaCl2.
(4) Recoveries at four metabolite levels: The percent
recoveries at four levels are shown in Table 5. The percent
recoveries were within 65122% with good precision in all
cases except for DBCA at 0.2 /ig/1 level and 3-PBA at 5.0 /ig/1
level. The recovery for DBCA at 0.2/ig/1 level for one
replicate was much lower than the rest (34%). However,
because it did qualify as an "outlier" by Dixon's Q-test, it
was not excluded for data analysis, which lowered the overall
average recovery for DBCA at 0.2//g/1 and caused a
relatively high RSD (30%). The reason for the-lower percent
recoveries for 3-PBA at 5.0 /ig/1 level, however, is unknown.
(5) Storage stability: The percent recoveries for the target
metabolites and creatinine are shown in Table 6. The results
show stable and consistent percent recoveries for all the target
Table 6. Storage stability at 37°C and 1°C.
Percent recovery (RSD)
Five time points DBCA DCCA 3-PBA
Creatinine
Tnitia^
37° 4h
37° 8h
l°24h
l°48h'
1° 72h
86(8)
73(3)
72 (10)
75(11)
39(3)
65(2)
75 (10)
63(6)
65(6)
72(3)
46(2)
72 (4)
75(15)
74(3)
79(8)
77(6)
50(2)
67(4)
96(25)
134(5)
126(21)
121 (15)
122(9)
98(9)
'Results from one replicate were excluded, based upon Dixon Q-test
metabolites and creatinine for over 72 h. Percent recoveries
were between 63% and 133%, except for the samples
extracted at 48 h time point. We suspect that this might be
caused by the fact that we did not analyze this set of extracts
immediately. The extracts were stored 2 days before analysis.
We also noticed that all the extracts of this set were cloudy
with white precipitates.
The percent recoveries for creatinine were calculated by
dividing the estimated urine creatinine concentration in the
diaper by the concentration in original urine samples. To
determine the creatinine concentration in original urine, six
urine samples that did not contact the diapers were analyzed
for creatinine. The mean of the six samples was
79.2 ± 0.89 /ig/1. Compared to these six urine samples that
did not contact the diapers, the estimated creatinine
concentrations in urine released from, diaper demonstrated
382
6-35
Journal of Exposure Analysis and Environmental Epidemiology (2004) 14(5)
-------
Using disposable diaper for pyrethroid studies
Hu et al.
a larger variation. Nonetheless, the relative standard devia-
tions (RSDs) were within an acceptable range of ±25% for
all samples.
Discussion
This study evaluated the feasibility of using commercial
disposable diapers for the analysis of three pyrethroid
pesticide metabolites and creatinine. Calcium chloride has
proved to be effective in collapsing the polymer matrix of the
diaper and releasing the target metabolites from the
polymers. Collapsing of polyacrylate chains in calcium
solutions has been investigated by many researchers in
polymer sciences (Sasaki et al., 1995; Schwein and Huber,
2001) and our study agreed with their findings. To obtain
enough liquid without causing analytical difficulties for
metabolites and creatinine, calcium chloride solution with a
concentration of 150g/l fared better than the other levels.
The lower level of 50 g/1 released less urine; the higher level of
450 g/1 caused analytical problems for both metabolites and
creatinine.
In this study, we started with two brands of disposable
diapers but the Pampers Premium was dropped after
experiment 1. We observed that after the diapers were
wetted, the absorbent pads in Huggies Supreme could be
easily separated from the porous polypropylene top and back
sheet and there was essentially minimum urine absorbed by
the top and back sheet The separation of the absorbent pads
from top and back sheets for Pampers Premium, however,
was more difficult. A substantial amount of absorbent pad
materials could not be easily separated from the top sheet,
which made the quantification of metabolite concentrations
more difficult.
With a limit of detection of 0.2/
-------
Hu ei at. Using disposable diapei for pymhroid studies
Sasaki S., Fujimoto D., and Maeda H. Effects of salt concentration and Schwein R., and Huber K. Collapse of sodium polyacrylate
degree of ionization on the hydrophobic counterion binding to ionic . chains in eaMimi salt solutions. Ear Phys J E 2001: 3:
gel and the concentration of the gel volumn. Polymer Gels Networks 117-126.
1995: 3: 145-158. . Soderlund D.M., Clark J.M., Sheets L.P., Mullin L.S., Pictirfflo V.J.,
Schetlgcn T., Heudorf U., Drexler H., and Angerer J. Pyrethroid Sargent D., Stevens J.T., and Weiner M.L. Mechanisms of
exposure of the general population-is this due to diet Toxicol Lett pyrcthroid neurotoritity: implications for cumulative risk assessment
. 2002: 134: 141-145. Toxicology 2002: 171: 3-59. .
6-37
384 Journal of Exposure Analysis and Environmental Epidemiology (2004) 14(5)
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SECTION 7.0
REFERENCES
Ahmad T., D. Vickers, S. Campbell, M. Coulthard, and S. Pedter. 1991. Urine collection from
disposable nappies. The Lancet 338:674-76.
Baker S.E., D.B. Barr, WJ. Driskell, M.D. Beeson, and L.L. Needham. 2000. Quantification of
selected pesticide metabolites in human urine using isotope dilution high-performance
liquid chromatography/tandem mass spectrometry. J Expo Anal Environ Epidemiol Nov-
Dec 10 (6 Pt 2):789-98.
Beeram M. and R. Dhanireddy. 1991. Urinalysis: direct versus diaper collection. Clin. Pedia.
50:278-80.
Campbell J.L., C.R. Santerre, P.C. Farina, and L.A. Muse. 1993. Wipe testing for surface
contamination by tritiated compounds. Health Phys. <54(5):540-544.
Cohen H., B. Woloch, N. Linder, A. Vardi, and A. Barzilai. 1997. Urine samples from disposable
diapers: an accurate method for urine cultures. / Fam. Pract. 44:290-92.
Consumer Reports. 1998. Diaper Wars. Issue 8: 53-55.
Fell J., H; Thakkar, D. Newman, and C. Price. 1997. Measurement of albumin and low molecular
weight proteins in the urine of newborn infants using a cotton wool ball collection
method. Acta Paediatr 86:518-22.
Hu Y., D. Barr, L. Needham, E.D. Pellizzari, J.H. Raymer, and J.M. Roberds. Collecting urine
samples from young children using cotton gauze for pesticide studies. Submitted to
Journal of Exposure Analysis and Environmental Epidemiology.
7-1
-------
Krieger R.I., C.E. Bernard, T.M. Dinoff, L. Fell, T.G. Osimitz, J.H. Ross, and T.
Thongsinthusak. 2000. Biomonitoring and whole body cotton dosimetry to estimate
potential human dermal exposure to semivolatile chemicals. Journal of Exposure
Analysis and Environmental Epidemiology 70:50-57.
Lewis J. 1998. Clean-catch versus urine collection pads: a prospective trial. Paediatr Nurs.
10(1): 15-6.
Lichtenwalner C.P. 1992. Evaluation of wipe sampling procedures and elemental surface
contamination. American Industrial Hygiene Association 53(10):657-659.
Macfarlan P.I., C. Houghton, and C. Hughes. 1999. The Lancet. 354(9178):571.
Millson M., Eller P.M., and Ashley K. 1994. Evaluation of wipe sampling materials for lead in
surface dust. American Industrial Hygiene Association 55(4):339-342.
Mock D. 1992. Rayon balls and disposable-diaper material selectively adsorb creatinine. Am J
ClinNutr. 55:326-30.
Moate T.F., C. Lu, R.A. Fenske, R.M. Hahne, and D.A. Kalman. 1999. Improved cleanup and
determination of dialkyl phosphates in the urine of children exposed to organophosphorus
insecticides. Journal of Analytical Toxicology. 24(4):230-236.
Muratore C. and R. Dhanireddy. 1993. Urine collection from disposable diapers in premature
infants: biochemical analysis. Clin Pediatr. 5:314-315.
Roberts S. and A. Lucas. 1985. A nappy collection method for measuring urinary constituents
and 24-hour urine output in infants. Arch Dis Child. 60:1018-20.
7-2
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Smith G. and C. Taylor. 1992. Recovery of protein from urine specimens collected in cotton
wool. Arch Dis Child. 67.
Vernon S., A. Redfearn, S. Pedler, H. Lambert, and M. Coulthard. 1994. Urine collection on
sanitary towels. 344.
Wong W., L. Clarke, M. Llaunrador, L. Ferlic, and P. Klein. 1993. The use of cotton balls to
collect infant urine samples for 2HJ1H and 18O/16O isotope ratio measurement. Appl Radial
Isot. 44:1125-1128.
7-3
-------
APPENDIX A
WIPES DESCRIPTION AND LIST OF INGREDIENTS
A-l
-------
WIPES DESCRIPTION AND LIST OF INGREDIENTS
Brand Name and Description
Listed Ingredients
Huggies Original unscented
UPC: 3600018500; lot: ML914903A 0256
19x19 cm, 9.1 g
Water, potassium laureth phosphate, glycerine,
polysorbate 20, tetrasodium EDTA, DMDM,
hydantoin, methylparaben, malic acid
Playtex Chubs unscented (new)
UPC: 7830005006, lot: 9173cs 1517
19x19, 7.1 g.
Water, propylene glycol, aloe barbadenis gel,
PEG-60 lanolin, disodium cocoamphodiacetate,
potassium sorbate, citric acid, disodium EDTA,
propyl gallate, polyaminppropyl giguanidine
-Wash-ups (by Clinipad)
Anti-bacterial with moisturizer
UPC: 1915491025, lot: 303659
15.8 x 12.5 cm, 2.3 g
Active ingredient: benzalkonium chloride. Other
ingredients: water, SD alcohol 40-B, propylene
glycol, oleth 10, PEG-75, lanolin, fragrance,
propylparaben
Wet ones
Original scent, singles
UPC: 7682804721, lot: 9274S9
12.5 x 15.0, 4.1 g
Water, SD alcohol 40, propylene glycol, aloe
barbadenis gel, sodium nonoxyol-9 phosphate,
sorbic acid, oleth-20, fragrance, PEG-60, lanolin,
citric acid, disodium phosphate
Kendall
Webcol 2 ply, large
cat:5110,lot:AG0133xX
8.5x4.0, 1.2geach
Isopropyl alcohol
Food Lion Thick and Soft Baby Wipes
Refill Pack
UPC: 3582602594, lot: J169-F
18x20 cm, 6.7g
Water, aloe vera, potassium sorbate,
tartaric acid, glycerine
Pampers
Premium new big wipe
UPC: 3700032105, lot: 9089E38
17.7x25.9, 8.6 g
Water, propylene glycol, aloe vera gel, PEG-75,
lanolin, disodium cocoamphodiacetate, polysorbate
20, methylparaben, propylparaben,
2-bromo-2-nitropropane-1,3-diol, fragrance
A-2
-------
APPENDIX B
ANALYTICAL PROTOCOLS
B-l
-------
LABORATORY
OPERATIONS
PROTOCOL
RESEARCH TRIANGLE INSTITUTE
POST OFFICE BOX 12194
RESEARCH TRIANGLE PARK, NC 27709-2194
RTI/ACS-AP-216-005
Page 1 of 15
TITLE:
SOURCE;
AUTHOR(s):
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
ANALYSIS OF CARBAMATES
Research Triangle Institute
Post Office Box 12194 ....
Analytical and Cheihical Sciences
Research Triangle Park, NC 27/^09-2194
.Date:
Date:
Date:
APPROVED BY;
Principal Investigator:
QA Officer:
STATUS:
IN PROGRESS:
DRAFT:
FINAL VERSION:
O
D
REVISIONS;
No, Date
0 J
1 .
2
3
4
5
No. Date
6
7
8
9
10
11
Effective date of this version is the date of the last approval signature;
revision 0 is the original version.
-------
RTI/ACS-AP-216-005
Revision 0
Page 2 of IS
HIGH PERFORMANCE LIQUID (^pTOGRAPHY (HPLC) ANALYSIS
. . ;;OF CARBAMATES
TABLE OF CONTENTS
Section
1,0
2.0
3.0
.4-0: :<
..:'--
6.0
7.0
8.0
9.0
10.0
11.0
12.0 '"''
13.0
14.0
15:Q.
16.0
. -...,..'. . '.
.Scope and Application ..
Summary of the Method .
Definitions
. Interferences .:....:. :. .i i ;
/ Safety ..:;%... . : . . . ... . . .:....'
Equipment and Supplies :.
Reagents and Standards :. v ; ..;:.... ....;...
Sample Homogenization and Storage . ;
Quality Control
Calibration and Standardization ..^
Procedure .
"Calculati6ns:-"-!;^" = = v* -^ '" = --' " '
-..'.-, "': - .'
Method Performance
Pollution Prevention
<
. Waste.Management. .... .,..,..,.;. ,....,..
References ; . . . . .. .-;.;..:-.........;..;........;. ;..........
Page
3
3
4
4
4
4
6
8
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8
11
12.
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13
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1.0 SCOPE AND APPLICATION
RTI/ACS-AP-216-005
Revision 0
Page 3 of 15
1.1 This is a high performance liquid chromatdgraphic method for the determination of N-
i
methylcarbamates in extracts of dermal wipes. The extract is assumed to be relatively free of
coextractive (lipids, proteins/ etc.) prior to the application of this method. The method has been
tested using laboratory fortified blanks and is applicable to the carbamates in Table 1.
1.2 For compounds other than those listed in Table 1 or for other sample types, the analyst
must demonstrate the applicability of the method by collecting precision and accuracy data on
fortified samples and providing qualitative confirmation of results by an acceptable analytical
method.
i
Ii3 .;.. Estimated detection limits (EDL) for the above jpesticides must be experimentally
" ' . . ' . i.
determined for each linked analysis scheme. -.,:- . j .-.--. .
1.4 This method is restricted to use.by or under the supervision of analysts experienced in
i
the use of liquid chrqma.tography and in the interpretation of liquid chromatograms. Each
analyst must demonstrate :the ability to generate acceptable results with this method using the
procedure described in Section 9 of RTI/ACS-AP-216-boi.
1.5 When this.method is used to analyze unfamiliar samples for any or all of the analytes
above, analyte identifications must be confirmed by a: least one additional qualitative
technique.
2.0 SUMMARY OF THE METHOD
This method is for the analysis of carbamates ijn extracts of dermal wipes by HPLC with
post-column derivatization and fluorescence detectioft. The extract is filtered and a 20 uL
aliquot is injected into a reverse phase HPL,C column]: Separation of the ainalytes is achieved
using gradient elution chromatography. After elutioii from the HPLC column, the analytes are
i '
hydrolyzed wjthQ.05 N spdium:hydroxide.(PifaOH) a: 95°C. The methyl amine formed during
hydrolysis is rea.cted with 6-phmalaIdehyde (OPA), and 2-mercaptoethanol (or.N,N-dimethyl-2-
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mercaptoethylamine hydrochloride) to fonn a highly fluorescent derivative which is detected
by a fluorescence detector.
3.0 DEFINITIONS . ..--;,. - - -.
3.1 Refer to RTI/ACS^AP-216-001.
3.2 - High Performance Liquid Chromatography (HPLC)
4.0 ^INTERFERENCES- .'.:., ..-.;
4.1 Carryover contamination may occur when a sample containing low concentrations of
compounds is analyzed immediately after a sample containing relatively high concentrations of
compounds. Syringes arid injector must be cleaned carefully or replaced as needed.
4.2 -Matrix interference may be caused by contaminants that are .present in the sample. The
extent of matrix interference will vary considerably from sample to sample. Positive
identifications must be confirmed. ...'. / .. -; "-. .
5.0 SAFETY A
5.1 Refer to RTI/ACS-AP-216-001.
6-0 EQUIPMENT AND SUPPLIES
6.1 .flajanse'-':: '.''"^:-;-. -:.--=.'-: ,.:-. i :.:...;:.... '-.-..
' ; :Analyticali; capable of accurately Weighing to'ttie nearest 0.0001 g.
6.2 : FiltratioriApparatus -'".: '' ' -..'. , -. .- -. .
6:2.1 MaCrbfilttatiofi to filter
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: 6.2.2.^j.Nfiq'ofUtration^ to ffltCT-samplesprior to HPLC analysis. Use 13 mm filter
holder (Millipore stairdess steel XX300/200 or equivalent), and 13 mm diameter
.....; 0.2. jim polyester filters .{Nudepore 130406 or equivalent).
6.3 .... Syringes-and Svringe Valves . ::,.. .... . ;,. .--,, . . ,: .. :
;, ,6.3.1 ..Hypodermic syringe 50 mL glass, with LuerrLok tip . .
..6;3.2 ,:Mjcro syringes-varioussizes ..; , ;..,.'. ,,.,'..
6.4 Miscellaneous
6.4.1 Solution storage bottles Amber glass, 500 to 4000 mL with Teflon-lined screw
caps
6.4.2 Helium, for degassing solutions and solvents
6.4.3 Volumetric flasks ~ ;
63 High Performance Liquid Chromatography (HPLO
... 6.54.. ,HJPLCsystem.,capablepf injecting 20 uL^llquots, and performing binary
':: gradients at a constant flow rate. A data system is recommended for measuring
'"'"' '"" peaik area's) Table:'l listsretention times^observed for method ahalytes using the
column and analytical conditions described below.
6.5i2 Column - 250 mm x 4.6 mm i.d. Zorbax-C8 (Dupont 880952.706) maintained at a
constant 35°C temper;arure using an rffLC coiumnheater.
6.5.3 Mobile Phases: A = 955 waleriacetoriitrile; B = 30:70 water:acetonitrile.
Gradieiiit program at 1^ inL/rnin as follows: 80:20 A:B for 10 min then to 0:100
A:B m 25 min with a 16 min final hold.
. '-!. <'.,-. . :! >...,-..;
6.5.4 Post column hydrolysis chamber -1 Capable of mixing reagents into the mobile
phase. Reactor should be constructed using stairdess steel tubing (3 mx 0.4 mm
i.d.) and equipped with a solvent delivery system capable of delivering 0.1 to
2.Q mL/rruri of the NaOH reagent whiie maintaining a constant uniform 100°C
temperature.
' ; '.-' .':.-?.,.' ^i: 'f '. ''. -. . ' . ' ':. :
6.55 Fluorescence detector - Capable of excitation at 230 nm and detection of
emission energies greater than 418 run. A McPherson Instrument Model FL7508
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" ;:-'fluorescence detector was used to generate the data presented in Methods PAS-
r< 01 to PAS-07. '' '''* '-' ''
6.5.6 Post column reaction chamber - Capable of mixing the derivatization solution
into the mobile phase plus NaOH reagent flow; constructed of stainless steel
tubing (3 m x 0.4 min i.d.) arid-equipped with a solvent delivery system capable
of delivering 0.1 to 3 mL/min of the'derivatizing solution to the column plus
NaOH flow.
7.0 REAGENTS AND STANDARDS
ri'a'Solvent is p'urified/ stabilisers aidded by the manufacturer are removed, thus
: .potentially .making the solvent hazardous. Also,, when a solvent if purified,
preservatives added by the manufacturer are removed, thus potentially reducing the
''shelf-life/ ;< ""'' '~'; *"'<'[-'*' :-..-....: - -
7.1 Reagent Water ..
;. ':, :'.-.;?>! > : <: : -..- ..-. ': J-?-.'.:>.!. :.-.-.'-.o-. >' :-.'/.. .-.- ,
Reagent water is defined as water that is reasonably free of contamination that would
prevent the determination of any a.nalyte of interest Reagent water used to generate the
validation data for this method was Hydro-Systems Picotech 18 M Ohm. water.
- . ": . '.,-.'-,.-. ; . \ ''.' f'- '. '.."." Ci .. ' '. ' ' ' . . *
7.2 Methanol - HPLC grade.
73 Acetonitrile - demonstrated to be free of analytes.
*' .,; ''. I'-.-'.: ;'-. .;'.'..' ;-'-:.:-.: 'i'.v ' .:*.. .'.' .'"<, ' '/' .- ' '
7.4 HPLC Mobile Phase .
. 7.4.1 Reagent water. .Filter and degas with helium before use
7.4.2 Acetonitrile HPLC grade. Filter and degas with helium before use.
7.5 Post Column Derivatization Solutions
75.1 Sodium hydroxide, 0.05 N Dissolve 2.0 g of sodium hydroxide (NaOH) in
. reagent water. .Dilute tp.1.0 L with reagent water which has been filtered and
degassed with helium just before use.
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7.5.2 N>I-cumethylT2-mercaptoethyiamine hydrochloride, Thiofluor crystals
; ./(Pickering Laboratories 3700-2000).-.Two grams of Thiofluor is equivalent to
-,- 1 mL of 2-niercaptoethanoL :?...?: . '.-, ...
7.53 .2-Phthalaldehyde (OPA). Dissolve 100 mg of OPA (Pickering Laboratories 0120)
into 10 mL of UV grade methanol. >,
7.5.4 p^Phthalaldehyde diluent Add one 950 mL bottle of o/phthalaldehyde diluent
(Pickering Laboratories B910) into a reservoir and thoroughly degas.
75.5 Reaction solution. (To the degassed Q-phthalaldehyde diluent). Add the
dissolved o-phthalaldehyde mixture. Mix well. Add 2 grams of Thiofluor
>. ?..-'. directly ..to. the solution and gently mix. :.-. . . '; .
7.6 ^Bromo-S.S-dieihylphenyl N-methylcarbamate (BDMQ : . . .
. .Ninety-eight percent purity, for use as quantisation standard (available from Aldrich
Chemical Co.).. .-.-. , ..'-.:.:.;?:-.: ': :: -:.:. _': . - . . -.
7.7 :. Stock Standard. Solutions (1.00 pg/uL) :, .- .: . .
. ..Stock standard solutions may be purchased as .certified solutions or prepared from pure
standard material using the following procedure.
7.7.1 Prepare stock standard solutions by accurately weighing approximately 0.0100 g
of pure material. Dissolve the material in HPLC grade acetonitrile and dilute to
volume in a 10 mL volumetric flask. Larger volumes may be used at the
convenience of the analyst. If compound purity is certified at 96% or greater, the
weight may be used without correction to calculate the concentration of the
stock standard. Commercially prepared stock standards may be used at any
:": . . - concentration if they are .certified by the manufacturer ot by an independent
source. -.\.<.;.'=-. -.-:?!:. ^ :-. -: .;.= -.:-.' .-,. ,. ; >.'< . '
7.7.2 Transfer the stock standard solutions into Teflon-lined screw cap vials. Store in
.^refrigerator and protect from light. . : . .
7.8 Ouantitation Standard Solution = .>,..: ,-.;,,;. ?.:, . . ..
. . .. Prepare.a quantitation stock standard solution by accurately weighing approximately
0.010 g of pure BDMC Dissolve the BDMC 'in acetonitrile arid dilute to volume in a 10 mL
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volumetric flask to make' a solution of 1000 ng//*L.' Transfer 100 (J.L of this stock solution into a
second 10 mL volumetric flask to prepare a dilute stahdard/the fortification solution, at 10
ng//iL. Transfer the quantisation standard fortification solution to a Teflon-lined screw cap
bottle and store at room temperature. Add 50 /zL of the fortification solution to a 1 mL extract
to yield a final quantitation standard concentration of 500'p'g/AtL. . '
8rO SAMPLE HOMOGEMZAT1ON AND STORAGE
8.1 cHoldm'g'Time;-
Holding times for sample extracts prior to analysis have not been established. It is
recommended that sample extracts be analyzed within.30 days, of extraction.
8.2 it is: anticipated that extracts Will be analyzed shortly after extraction is complete. It is
the analyst's responsibility to assure that any storage of extracts does not affect analytical
results. All extracts should be protected from light .^Extracts may be stored at 4°C for short
/periods of time:<48 hours. 'Extracts should be stored at >-10°C for longer periods of time.
:9.0 QUALITY CONTROL-
9.1 Refer to RTI/AGS-AP-216-001.; -=
10.0 : CALIBRATION AND STANDARDIZATION ;
10.1" Establish HPLG'operatirig parameters equivalent to those indicated in Section 6.5. The
HPLC system should be calibrated using the internal standard technique. '
10.2 '' Internal StandardgalibrationProcedure^*
The analyst must select one or more qtiarititation standards similar in analytical
behavior to the analytes of interest The analyst must further demonstrate, that the
measurement of the'quarititation standard^is hot affected by method or matrix interferences.
BDMC has beeri identified as a suitable quantitation istandard.
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10.2.1 Prepare calibration standards at a minimum of five concentration levels (10 to
2000 pg/uL) for each analyte of interest by adding volumes of one or more of the
stock standards to a volumetric flask. To each calibration standard, add a
; . known constant amount of one or more quantitation standards, and dilute to
volume with acetonitrile. The lowest standard should represent, analyte
concentrations near, but above, their respective EDLs. The remaining standards
.. should bracket the .analyte concentrations.expected.in the sample extracts, or
should define the working range of.the detector.
10.2.2 Analyze .each calibration standard according to the procedure (Section 6.5),
. , Tabulate peak height or area responses, against concentration for each compound
and quantitation standard. Calculate response factors (RF) for each analyte
iusing Equation 1. . ..:
RF =
: where:
Area response for the analyte to be measured.
. Area'response.for .the quantitation standard.
Concentration of the quantitation standard (ug/L).
. GoncehtratiQn pf:.ti\e analyte to be measured (ug/L).
10.2.3 If the:RF.value over the working range is constant (25% RSD or less), the average
-:.--,: RF can be used for calculations. Alternatively, the individual analytes RF results
::: . j cainbe used to plota linear calibration curve.
10.2.4:' .The.working calibration curve orRF. must be verified on each working shift by
; : the measurement of one or more calibration standards. If tiie response for any
. '< . analyte varies from the predicted, response by more than 25%, the test must be
: : repeated using a fresh calib.ratiori standard. If the repetition also fails, a new
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calibration curve must be generated for that analyte using freshly prepared
standards. -
103 Coritinuirig'Galibratioh -Check ' '
Verify the instrument calibration at the beginning of each day that analyses are
performed using the following procedure/ as well as after the analysis of every nine sample
extracts. :
10.3.1 Inject a 20-uL aliquot of a mid level concentration calibration solution arid
analyze with the same conditions used during the initial calibration.
10.3.2 Determine that the absolute areas of the quantitation standards have not
' -decreased by more than 25% from the areas measured in the most recent
''"';- continuing calibration check, or by more man 50% from the areas measured
during initial calibration. If these areas have decreased by more than these
amounts/ adjustments must be made to restore system sensitivity or other
maintenance as indicated in Section 10.3.5 and recalibration.
10.3.3 Calculate the RF for each analyte and surrogate from the data measured in the
continuing calibration check. The RF for each analyte and surrogate is in control
if its RF is'-withiriJ±:-25% from the RF in the calibration curve. Acceptable
performance for the analytical system is met if all analytes are in control. If the
mstrument'is ih'cohtrol, tiie daily RF values are used for sample quantitation. If
these conditions are iiot met/ remedial action (Section 10.3.7) must be taken
which may require recalibration.
10.3.4: After the analysis of every nine samples, the mid level calibration standard is
' '< 'reanalyzed; If the RF values for this analysis have not varied by more than 25%
from the daily RF values, then the instrument is in control and samples may be
quantitated using the daily RFs. If these conditions are not met, then the
. quantitative data for the sample analyzed prior to the check standard are
: 'considered suspect for the analytes that are out-of-control. Sample extracts must
' be reanalyzed if any of the target pesticides that are out-of-control are detected
or if the RF values for any of the pesticides in the check standard are less than
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- 50% of the calibration value: Check standards may be analyzed more often to
minimize the need to repeatedly reanalyze samples.
- >: ;' 10;3.5 '.-Some possible remedial actions * Major maintenance such as cleaning the HPLC,
. mjector syringe and detector...
-10.3.5:1 : Check arid adjust HPLG operating conditions.
10.35:2 ; Check and adjust the fluorescence detector.
-10.3:5.3 .v Flush the HPLC column .with solvent according to manufacturer's
instructions.
-10.-3.5.4 :-; -Replace HPLG.guard column if utilized.
10.3;55 ;: Prepare fresh CAL solutions,'and repeat the initial calibration step.
10.35.6 :'- Replace the HPLC syringe; .>,
10.35.7;: ivReplace any components that allow analytes to come into contact
.: with hot metal surfaces. :" ;
11.0 PROCEDURE
i
11.1 Analyze 20:pL aliquot of .each sample with the HPLC system under the same conditions
used for the initial and continuing calibrations (Section 10).
11.1.1 Section 6.5 summarizes the recommended operating conditions for HPLC.
Table 1 lists retention times observed vising this method.
11.1.2 Verify system calibration daily as described in Section 10. The standards and
samples must be in acetonitrile.
11.1.3 Inject 20 uL of the sample extract- ;Record the volume injected and the resulting
peak size in area units.
11.1.4 If the response for the peak exceeds the working range of the system, dilute the
sample extract with acetohitrile.and reanalyze.
11.2 Identification of Analytes "»:>; '.-' /. :r.= . ; .. :
11.2.1 Identify a sample component by comparison of its retention time to the retention
. .:: ': :.>: .time of a standard :compound: If the retention time of an unknown compound
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-;;. : . corresponds, within limits, to theiietention time of a standard compound, then
identification is -considered positive. : . .. . . .
11.2:2 The width of the retention time window used to make identifications should be
based upon measurements of actual retention time variations of standards over
the course of .a day; ;Three times the standard deviation of a retention time can
be used to calculate a .suggested window.size for a compound. However, the
.'} .: fixperience of the analyst should weigh heavily in the interpretation of
chromatograms. . -...= :-.
11.2.3 Identification requires expert judgement when sample components are not
: . :..; : - .resolved chromatographically. When peaks obviously represent more than one
sample component (i.e., broadened peak with shoulder(s) or valley between two
. .-.- , - or more maxima), or any time doubt exists over the identification of a peak on a
chromatogram, appropriate alternate techniques to help confirm peak
identification need to be employed. For example, more. positive identification
may be made by the use of an alternate detector which operates on a '
chemical/physical principle different from that originally used; e.g., mass
: spectrdmetry, or the use '.of a second chromatography column.
12.0 CALCULATIONS.. -.,,-
12.1 Calculate Ahalte Concentrations - .. .
.'where:
Cx = concentration of analyte pg/fiL in the food extract.
A, = area response of the analyte in the sample. .
Au : = -area response of the quantitation standard in the sample.
Qu - concentration of quantitation standard in pg/uL in the food extract.
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RF = daily response factor of analyte from the continuing calibration check
standard.
122 Calculations should utilize all available digits of precision, but final reported
concentrations should be rounded to an appropriate number of significant figures (one digit of
uncertainty). Experience indicates that three significant figures may be used for extract
concentrations abpye 99. ^.g/L,; two significant figiires .for (concentrations between 1-99 ug/L,
and one significant figure for lower concentrations.
13.0
13.1 Table 1 shows the target carbamates that can be analyzed by this method. Additional
carbamates may be applicable with the method performance evaluated by the analysis
laboratory. The table also indicates the lowest concentration standard that has been
demonstrated to be in the linea? range of the instrument
13.2 The overall method performance will be dictated by the performance of the sample
extraction in combination with the cleanup and analytical method. Method performance for
linked analysis schemes is provided in RTI/ACS-AP-2i6-001.
13.3 When this methods was used to analyze food extracts, substantial interferences were
found for methomyl; small interferences were found for aldicarb.
14.0 POLLUTION PREVENTION
14.1 Refer to RTI/ACS-AP-211-131.
15.0 WASTE MANAGEMENT
15.1 Refer to RTI/ ACS-AP-216-001.
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16.0 REFERENCES
1 1. Sheldon;'L.S. Manual of Analytical Methbds for Determination of Selected
Envkbnmental Contamination^ uYComposite Food Samples. EPA-68-C2-0103,
" ' U.S. EPA, Ehvirdnmental Monitoring Systems Laboratory, Cincinnati, Ohio,
1995. ' "'' -' ' ' '
2. . Methods for the Determination of Organic Compounds in Drinking Water, U.S.
EPA, Cincinnati, Ohio, PB89-22Q461, Dec. 1988, Method 525 (rev. 2.1).
3. U.S. FDA, Pesticide Analytical Manual, Volume 1,3rd Edition, Section 401,
-. '''' Washington; DC, 1994. ' ' " ' ''r ""'' ''-
4, Sheldon, L., J. Keever,). Beach, M. Rob^rds and L. Ellis. Environmental
' Contaminants iri;Footis: 'Pha^ie 3 ^ Validation of Analytical Methods. Final
Report, U.S. EPA EMSL-Cuidnnati Contract Number 68-C2-0103, Work
'''' :Assighment'2-6i. ' "-'""' '' ' ' ''-"'
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TABLE 1. APPLICABLE CARBAMATES FOR RTI/ACS-AP-216-005
Carbamate
RT
(min)
Lowest Calibration Standard
(pg/W
Pesticide
Methomyl
Aldicarb
Propoxur
Carbofuran
Carboxyl
Quantitation Standard
4-Bromo-3^-DimethylphenyI-
N-Methykarbamate
15.15
28.31
32.32i
32.79
33.95
37.60
10
10
10
10
10
NAb
* Retention time.
b Not applicable.
-------
ANALYTICAL
PROTOCOL
RESEARCH TRIANGLE INSTITUTE
POST OFFICE BOX 12194
RESEARCH TRIANGLE PARK, NC 27709-2194
RTI/ACS-AP-21 6-006
Page 1of5
TITLE:
SOURCE:
COLLECTION OF SURFACE WIPE SAMPLES
Research Triangle Institute
Post Office Box 12194 ... ,,
Analytical ancl Chemical Sciences
Research Triangle Park, NC 27709-2194
AUTHOR(s):
^frm*t^
a.
APPROVED BY;
WA Leader:
QA Officer
REVISIONS:
Date:
Date:
Date:
\3 23
STATUS:
IN PROGRESS:
DRAFT:
FINAL VERSION:
D
Q
m
No. Date
0 4
1
2
3
4
5
No. Date
6
7
8
9
10
11
J: Effective date of this version is the date of the last approval signature;
revision 0 is the original version.
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COLLECTION OF SURFACE WIPE SAMPLES
TABLE OFCONTENTS
Section Page
1-0 Scope and Application 3
2.0 Summary of the Method 3
3.0 ': Materials 3
4.0 Sample Collection 4
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1.0 SCOPE AND APPLICATION
These procedures are for the sample collection of surface wipe samples for residual
pesticides from hard surfaces. Surface .wipe samples are. collected to determine the loading
(ng/cm2) of pesticide residue on the surface of an indoor area where pesticides may be
transferred to food or human skin.
2.0 SUMMARY OF THE METHOD ..;.-... > '. -, ;
Sof-Wick cotton sponges:are saturated .with isppropanol and wiped across the surface
area of interest. Alternatively, commercially available .wipes may be used, but approval for the
use of these products for in-field use is pending QC:validation of such products. The wipe
samples are stored in a sealed glass jar for subsequent extraction.
3.0 MATERIALS
3.1 Wipe Materials . . . ;.,...:., .... .
3.1.1 4" x 4" 6-ply cotton sponge dressing 0phnson & Johnson Sof-Wick or equivalent)
3.1.2 Commercially available pre moistened wipes will be used for the Task in which
. . ;. . these products are evaluated in the laboratory as replacements for 3.1.1.3.2Clean
tray, glass - 6" x 6" -.=.. ,
3.3 ; Forceps- : .. .-., ,.:. . .. .-.,* -. :, ;-.. -. ...
3.4 2-Prppanpl (isopropahol), pesticide residue analysis quality (B&J, UV grade or
equivalent)
3.6 . \:. Dispensing bottle:(250-mL) ..,.-, ...>..-/.
3.7 Disposable nitrile gloves, Best1?, N-DEX brand or equivalent
3.8 Glass sample container w /Teflon-lined lid (250-mL)
3.9 Measuring tape
3.10 Masking tape
3.11 Lab tissue
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4.0 SAMPLE COLLECTION
4.1 ' Selection of Surface :
The surfaces for sampling should be hard and free of obstructions. The surface area
should be 12" X12" (929 cm2) if possible, however smaller surface areas may be used if
necessary.
4.2 Performing Sample Wipe - Isopropanol Saturated Sponge
4.2.1 Measure and record the area and location to be wiped, in a laboratory notebook.
If practical, outline the collection area with masking tape.
: 4.2.2 Wipe the tray with ah isoprbpanbl-wetted laboratory tissue.
'NOTE: '> Gloves should beiVirorn during sample collection.
4.2.3 UnWrap a package of two clean sterile 4" X 4" cotton sponge dressings and place
separately on the tray using forceps. ''-.'
4.2.4 Uniformly saturate one dressing with up to 15 mL of isopropanol from the .
solvent dispenser.
4.2.5 Holding the dressing in a gloved hand, wipe the surface uniformly in one
direction while frequently using a fresh surface of the dressing. Repeat the
. wiping motion perpendicular to me first direction. Place the exposed dressing in
' a sample container. ' -v . ; -. '
'4J2.6 Prepare the second dressing as in 4.2.4. Repeat 42.5 reversing the order of the
direction of the wipe motion.
4.2.7 Place the second exposed dressing in the sample container. If the wipes are not
to be immediately extracted, add -50 mL of isopropanol to the container, then
cap tightly.
4.2.8 Label the sample container with a unique sample identification number and
transfer to a sample cooler then store in the freezer until analysis can be
performed. :
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4.3 Performing Sample Wipe - Commercially Available Wipes
4.3.1 Measure and record the area and location to be wiped, in a laboratory notebook.
If practical, outline the collection area with masking tape.
4.3.2 Wipe the tray with an isopropanol-wetted laboratory tissue.
NOTE: Gloves should be worn during sample collection.
4.3.3 Remove two wipes from their package(s) and place separately on the tray using
forceps. .
4.3.4 Holding the dressing in a gloved hand, wipe the surface with the wipe
uniformly in one direction while frequently using a fresh surface of the wipe.
Repeat the wiping motion perpendicular to the first direction. Place the exposed
wipe in a sample container.
4.3.6 Repeat 4.2.4 reversing the order of the direction of the wipe motion.
4.3.7 Place the second exposed wipe in me sample container. Label the sample
container with a unique sample identification number and transfer to a sample
cooler then store in the freezer until analysis can be performed.
-------
ANALYTICAL
PROTOCOL
RESEARCH TRIANGLE INSTITUTE
POST OFFICE BOX 12194
RESEARCH TRIANGLE PARK, NC 27709-2194
RTl/ACS-AP-216-007
Page 1 of 9
TITLE:
SOURCE:
DEPOSITION OF PESTICIDES ON SURFACES OF MATERIALS
Research Triangle Institute
Post Office Bo* .12194 . : .,
Analytical and Chemical Sciences
Research Triangle Park, NC 27709-2194
AUTHOR(s):
APPROVED BY;
WA Leader:
QA Officer:
STATUS:
IN PROGRESS:
DRAFT:
FINAL VERSION:
REVISIONS:
Date:
/w<
ilDate:
Date:
Date: \
Date:
D
No. Date
0 $
1
2
3
4 .
5
No. Date
6
7
8
9
TO
11 . .
Effective date of this version is the date of the last approval signature;
revision 0 is the original version.
-------
- RTI/ACS-AP-216-Q07
Revision 0
Page 2 of 9
DEPOSITION OF PESTTICIpES .OJ^ SURFACES OF MATERIALS
TABLE OF CONTENTS
.or
1.0 . . Scope and Application ....,................... 3
2.0 Summary of the Method 3
3.0 Materials :..... .3
4.0...;. -Methods ...,;....,................,.,. .v,.: .,,;..;/:.. ,....:,. 4
5.0 Spray Chamber Cleaning 7
6.0 Safety .,. 8
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1.0 SCOPE AND APPLICATION
. , Procedures are described fpr,ur\ifprmly depositing pesticides on the surfaces of
materials. Subsequently, the transfer of pesticides from the surface to food is measured.
Surfaces can be prepared both ;with and without an initial application of household dust.
Examples of surfaces upon, which pesticides ajet deposited include carpet, finished wood,
linoleum or oth.er plastics, doth and formica. These surf aces are used to simulate those surfaces
most likely to come in. contact with food or children's hands when they are eating at home or in
day care environments.
2.0
Up to three 12" x 12" coupons of the selected surface can be prepared at a time using this
method. Prior to use, the surfaces are cleaned by washing with a water and soap solution or
vacuuming. A liquid mixture of the pesticides .of interest is prepared and sprayed uniformly
onto the surface of the material in a chamber. After depositing the pesticide, surfaces are
allowed to air dry and then used in experiments to (determine the extent of pesticide transferred
to food.
3.0 MATERIALS
' V;.- .= ' .:". .'.. .::.i;:x '.
3.1 Pesticide Spray Deposition Chamber
The Pesticide .Spray, peposition Chamber (PDC) is used for uniformly depositing liquid
mixtures of pesticides onto surfaces. The PDC is shown in Figure 1 and consists of the
following components; ,.:,,.., ,.,.--
(1) a 66" X 30 " polypropylene chamber with a removable top, side access door and
exhaust ports (4" in diameter) on each end
(2) a pressurized liquid delivery tank capable of delivering pesticide mixtures at
pressures up to 30 psi
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(3) a spray tip control unit that includes a solenoid controlled spray nozzle tip
; mounted on a rail drive system capable of traversing the length of the chamber
"'' ;: ' at variable speeds ''-''" "'"'" '--'' -'' .''
i(4) a charcoal filtered exhaust system for removing contaminated air from the
'' chamber during arid after pesticide solution application.
3.2 ' Test surf ace materials (i.e.', c arpet, hardwood)(hit'into 12" by 12" coupohs.
3.3 Test pesticide mixtures triade from commercially available pesticide mixtures (with
emulsifiers) or neat pesticide matrices in solvent
3.4 Aluminum weigh boats.
3.5 Reagent grade water. A Millipore Super-Q-Water System or equivalent may be used to
generate reagent water.
3.7 Aluminum foil. -.-.>., .. :
3.8' NitrUe gibves/Best, N-Etex brand or equivalent :
3.9 4 L Plaistic botties, cleaned, certified pesticide grade
3.10 '"Stir plate:- "-: '">''-' ' :<-V>i: =' ." "-:\ '-' '-
; 3.11 ' Anialytical Balance - capable of weighing to 6;01 g.
4.0 METHODS
The methods described in this section are guides for performing spray applications of
pesticides on surfaces. The general procedure is as follows^
(1) prepare me spray solution and.perform the spray application onto surface
'coupons " ' ' ''' :/: .'':
(2) determine the spray application loading rate and resulting surface
''concentrations and adjust as necessary
(3) perform spray applications onto surfaces.
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4.1 Performing Spray Application :?,' .-. ,-,. :: .-:
4.1.1 Prepare a 2 L pesticide spray solution by .diluting individual pesticide mixtures
. . : (with emulsifiers) into reagent grade water. Alternatively, the spray solution cart
;';... be prepared by diluting stock pesticide solvent standards into reagent water.
4.1.2 Transfer the mixture to a 4 L plastic jar which is then placed inside the spray
tank. .
NOTE: During the spray application it may be necessary to continuously stir the
mixture to prevent settling of tine pesticide emulsions/ this is accomplished by placing
: : theI pia&tic^ container^^ WitftHrife pesticide koiutioii dri a'1 Stir plate inside the spray tank.
4.1.3 .Close to seal the/tank lid and apply a pressure of 30 psi to the tank.
4.1.4 Attach the spray tank to the spray tip control assembly.
4.1.5 Place three surface coupons in the middle of the chamber as shown in Figure 1.
The coupons should be placed on a sheet of aluminum foil and arranged across
the length of the chamber as close fo the middle as possible.
4.1.6 : Place at least two. weighed aluminum weigh boats between the surface coupons
to determine the mass liquid loading onto ;the surface. -. .
.... ... 4.1.7 . .-Attach the charcoal filteringexhaustsystem to one end of the chamber and start
the fan. The exhaust port on the opposite end of the chamber should be left open
:.;<.: .! to allow for .adequate air flow through the chamber.
NOTE: The air flow through the chamber must not interfere With the spray
-,(..v: -.-:',-: .: deposition--,If. the .air flow is too'strong the exhaust system can not be used
..:!. ;;..'during spraying. < . .-.. -,.:;.?.. .-...: . -. .
.=.'; ;4,3.;8 Position the spray tip control behind one of the spray baffles and start the spray
control.. Allow the solution to spray .through the tip until all of the air bubbles
are removed and the spray is uniform.
. :-. :'4.1.9 iStart the spray unit drive device. The unit will automatically stop after each
.!>:,. -pass, ::.*::''!';* >-. .. , :::'. ..-- . t. :
4.1.10 Perform as many passes as necessary to spray at the desired surface
. '' '..
concentration. '"\..
4.1.11 Stop the flow of solution to the spray tip.
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4.1.12 Immediately remove the aluminum weigh boats and,weigh to determine the
'.',:.=; :;;:: '..' mass o^quid 'that was -deposited. ''-; =;: .''.. ..
'."' '4.1.13 Remove the sprayed surface coupons and place in a ventilated hood with a
,'. -' '. ','.> ."Charcoal exhaust -to dry. ;?Allowrthe surfaces to dry for a minimum of 2 hours or
: .-;: '*'-.' :. until no visible liquid remains on the "surface. Remove the aluminum foil sheets
and discard.
4.2 Initial Application 'Testing'- ''. ?>' :'" vu^-:- ':!::;-.,:':. ^-';-'-' '/:.!'., ::''. . '
' ."4-2.i ,fe&trj^?:m^
pesticide mixture concentration needed to obtain surface concentrations of 10-50
ng/cm2. 'The application loading-rate (ALH) is calculated as ng/cm2 as
.:s.vv>-,.J6 J-..-/(-.o..-x:v^-:VA^>-: =.= .- ' '-
ALR =
' - : '' - -':-'!- -'1 ' * -' '- - -- =
where
! : < -'>' W ="0 Weight in rig of spray Solution deposited in the aluminum weigh pan
A = Area in cm2- of thfe altuniriuin weigh boat >:--' ; ; '
'-- . '4^2.2 v^Perforrh a Spray depoSition-'ohtd 3 clean test coupons of the desired surface type
4.2.3 Allow the surf aces to dry for a minimum of 2 hovirs or until no visible liquid
v..; .;* ::- remains onithe surface. ;-.--XVT: :/.-.. -!.V.\iv;ro-'.; -. -/;: '''
4^.4 ; J -After the surfaces have dried, collect and extract surface wipe samples (hard
surfaces) or extract the soft surface sample (carpet of doth) and analyze the
.:;.: ;: >:.-.. extracts to 'measure the surface concentration on each of the three test surfaces.
v : ; o -r-./d ' -,', -. (Protocols RTI./ACS-AP-212-q03/;RTI/ACS-AP-212-004/ and
RTI/ACS-AP-212-006). ;-:^-.v.-»: -. v. -. v y.-. :,.:..--..-:
^'-. 4.2.5 - Calculate ^thesurfa'ce loading/' The surface concentration measured on each of
the three coupons should be within ± 20 % Relative Standard Deviation (RSD).
%J?SD = x 100
SD
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4.2.6 If the surface loading is riot Within the desired loading range or RSD, adjust the
pesticide mixture concentration or spray rate and repeat the application test.
4.2.7 After the correct application rate is achieved, proceed with preparation of
surfaces for transfer testing^
: 4.2.8 "Repeat as necessary to demonstrate continued reproducible and uniform
deposition of test pesticide mixtures; ':
4.3 Direct Pesticide Application Onto Surfaces
4.3.1 Up to three 12 'x 12" surface coupons can be prepared at a time using this
method. If a smaller coupon size is used (i.e., 12" x 6") more coupons may be
' '''" : prepared at a time. ''-': > ..-.'.
4.3.2 : Hard surface coupons are cut to the desired dimensions and washed with a
''' water arid soap solution and allowed to dry. Soft surface coupons (carpet) are
cut to size and vacuumed to remove fabric particles.
4.3;3 The spray application is performed as described in Section 4.1. Several batches
of coupons may be sprayed using the same solution.
4.3.4"" Allow the surfaces todry as in Section 4.1. :
4.4 Application with Dust F611bwe
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5.3 . Rinse the entire chamber by spraying all of the surfaces, with water. Dispose of the rinse
water in a safe manner. . , ,. ... ..:.... =-,-
5.4 As necessary,;Clean .the .surfaces of the chamber by wiping with towels and an
appropriate solvent. Dispose of the towels in a safe manner.
5.5 As .necessary, clean the spray chamber, hoses/ and nozzles fay wiping with appropriate
solvent. Discard the rinses in a safe manner;
6.0 SAFETY
In order to achieve surface concentrations in the 10-50 ng/cm2 range as described in this
protocol, pesticide spray solutions are prepared at levels of 1 to. 5% of the recommended levels
for performing applications as specified by the manufacturers on the pesticide mixture
container labels. However, good laboratory safety practices should still be followed when
performing .the pesticide depositions, :.: :>!::-
6.1 The toxicity and carcinogenicity of.pesticides, used .in this protocol have not been
precisely defined; each chemical and chemical mixture should be treated as a potential health
hazard, and exposure to these chemicals should be rninimized.
6.2 . Read and follow all warnings and instructions on the pesticide container labels.
6.3 Always wear gloves, safety glasses, and laboratory coats when handling treated
surfaces or spray chamber parts..
6.4 Dispose all pesticide contaminated materials in a safe manner following institutional
guidelines and the guidelines listed in the individual pesticide container label.
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Exhaust Spray Tip
Speed Adjustment
Exhaust Pan
Exhaust Tubing
Spray Rail
Spray Baffle
TOP VIEW
Figure 1. Pesticide spray deposition chamber.
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Analysis of Freeze-dried Aqiieoiis Diaj^r Extracts
Using a PE Sciex Series 200 LC witti PE Sciex Triple Quadrupole
Autiior: Approved:
f!B.Beach Date .... .;p.I^Snuth.. ,., ...... Date
E. D. Pellizzari Date
ACS Vice-President
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.... : : ... ,:>,.-; .StandardOperating^rpcedurefor \
Using a PE Sciex Series 200 LC with PE Sciex Triple Quadrupole
.:/.,-: v-.-. \ RH/ACS-SOP-500-O04 . >. .
1.0 SCOPE AND APPLICATION
Use of a liquid chromatogyaph witfi a triple quadrupole .mass spectrometer
(LC/MS/MS) is often necessary to meet the increasing deniand for mass spectrpmetry of
organic sample analytes of Mgh molecular we^ initial
selection :of a parent ion which is determmed by the first quadrupple, then fragmented further
using a positively (or negatiyely) charged gas at the second quadrupole, then the resulting
daughter ion is selected usfrg;ffoe third quadrupole,;,
This SOP covers fee.system consisting pf components frpm.Perkin-Elmer (PE) and PE-
Sciex. The liquid chromatograph (LC) is a pair of PE Series 200 mpdular type pumps "with a PE
Series 200 autosampler. The electrospray (E|S) mterface usedt on this system is made by PE-
Sciex. By coupling these units, this system is capable of carrying out LC/MS/MS for singly-
charged ions up to mfz 3000. The system also has an Atmospheric Pressure Chemical
lonization (APCI) spurce which is an altematiye introduction technique to the mass
spectrometer. This technique also allows both positive and negative ions to be detected up to
m/z 3000 and is capable :of .performing lyiS/MS in either positive or negative modes. Operational
conditions for both electrospray and APCI analysis are shown in Table 1.
. The purpose of this SOP ,is to provide guidelines to operate the PE-Sriex 3000
t. i ' * . * .v' A ' .' \ ** .. . v.. *' t t '.'.' W ..-.* ;*%*.*..** T .''*"
LC/MS/MS system for the analysis of pesticide metabolites. A sample freeze-drying step is
provided for the concentration of dilute urine obtained} by aqueous extraction of diapers or
other absorbent material used to collect urine from pre-toiletrtrained children. Table 2 lists the
compounds which may be analyzed using this method. The samples will be analyzed by
MS/MS using APCI in the positive ion mode. Quantitative analysis will be conducted using
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internal standard calibration. Experimeritil results'for the LC/MS/MS analysis and
performance data for tte freeze^lry conceritratibn step Sire shown in Table 3.
'\,^-.^\-:;--^'.^.:.<':. ''; '.':; -^-Y . .' '.< :', v. <-. ' -,.: ' - :' ' .
2.0 INSTRUMENT OPERATION AND ANALYTICAL PROCEDURES
2.1 Analytical System ''''''
Hie LC/MS/MS system consists of the following components. The manufacturers are
Perkin-Elmer Saex/N^tibnal msfrumeniy; {^^U'tioniputer.
G^pufe'sy'steBi;far'&uiss spfectrbmeter and chrbmatogpraphy systeih
:'i ''' '-'' -'i:J^\v^mdii^1^'dp^lKCG)Ci-SQO KiTbr+ (Pentium HI processor, A07
; ; ' :BIOS) personal computer fwi^i 262 Mb RAM, CD-ROM drive, a Smart Serial 6
:.-) '.' ..-.- ; eaf^ ariti^Gb Hard Drivel, Hewlett Packard laser ^ Jet 5L printer, and a
National Instruments GPIB-SCSI-A -external interface. . ' ' ;
:.'.; ."V/ 'Sorrware'usecl ^^is Arualj^t ^^bperating'iiriderWiridbwsNT.
' -;" ;:; PS 266 Series ^Vacuum Deg'as'ser,^hanriel
;, Y;,< > 'E ^.200 Series IsaiiaHcMi^LC 'Pumps'
e ;;;KeyVt6rie Scientific 4^6 x 100 mm Aquasil column with guard column
:^;;^:'^PE^e^^30^^/MS''':V:;' ^V
' J "''' Atmosphere 'Priessur* iln^face^Eiectrbspray Source (SciexP/N 16486,
Amend E)^ : ':/ :";<< '
' ' ^ '
AtrribspKeric ^^ Pressure' Oieiracal Ibnization iSdu^ce (SciexP/N 14989, Amend J)
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PEStiex Mass Spectrometer Standards Kit (P/N 401936)
22 . Freeze-DryingApparatus - . . ..,-. . ;
, Virtis Benchtpp 3.3 Vaccu-Freeze refrigerated freeze dying system, or equivalent
'including: -.- . ;-.; ..= :-, -.-.'. ::. -.-....:;..:. ..-:. . ' .
Vacuum manifold ... . ;
».'.. , Valve ports for manifold .;.., . , ; ...
.-,.; .500.or :100d mL wide-mouth, freeze drying flasks. ...
..;».-; Glass desiccator, (to accommodate a greater number of samples), and vacuum
hose to connect the desiccator to the manifold.
High vacuum grease may be needed to seal joints.
23 Reagent Preparation . ;
. HPLC mobile phases: Mobile phase A - 0.2% Acetic acid in water. Add 2 mL acetic acid
to one liter of HPLC grade water and mix. Mobile phase B = 0.1% Acetic acid in MethanoL Add
2 mL of acetic acid to one liter of UV grade (or equivalent) methanol and mix.
.2.4. . Standard Preparation ,, . .. , . ...
2.4.1 .Stock Solutions .., .-.>,...:
Stock solutions of analytes and internal standards are prepared in 5050
methanol/water using volumetric flasks. Once mixed the solutions are
transferred to amber glass vials and stored in refrigerators at approximately 4°C.
2.4.2 Working Standards
;.. , Working standards will be prepared from title stock solutions listed in Section
2.4.1. Standards will be prepared in background free pooled urine.
2.4.3-..:Calibration Standards .... .. ;
; :. Solutions -varying in cpncentratipn oyer the expected detectable range (i.e. from 1
>, to 2^0 pg/um)wiU be prepared using background free pooled urine. Calibration
,-,-=curve ranges may vary depending^^on the sample type and expected target
compound concentration; The labeled isotopes used as internal standards will be
added at a fixed concentration (i.e., 100 pg/um) for each calibration standard.
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.2.4.4 Internal Standard Solution ' ' ''' '- ' '-' : '"
A solution of dS-atrazine in acetonitrile at a concentration of 10 ng/uL. Another
' compound may 'be used/ provided that the cbmpound used is extremely unlikely
to be found in samples, and can be analyzed free from matrix interferences.
2,5 Instrumental Operational Conditions
The MS/MS analysis will be conducted using positive ion electrospray or APQ in either
positive or negative ion modes. Operational conditions are shown in Table 1. The target
compounds and internal standards along With the MS/MS ion pairs monitored are shown in
Table 2. ':'' >" ":''-'- - - ' '
2.6 Instrument Start-up Procedure' ; ,- ; ., ,.--. ;
When the computer is on, it will prompt Microsoft Windows.
1. ^Choose Analyst" Software "by double clicking on the software icon with tile
2. Tiirn ori'the Mutdsarriipler; purrips, and detectors.
3. Tune the mass spectrometer as specified in the ACS-SOE-184r013 for me tuning
of doe API 3000 mass spectrometer. If performance requirements are met,
: proceed to the analysis. ''- "."' :
4^ Sdect a method frcoh the memod menu.
5. ' :if me method needs modification, edit to include the suitable parameters for
analysis. !. . :
.- jj . ; , . ..-. jf the experiment ^ itedtiires MS/MS analysis 'the following items are necessary.
petenriihe the mode of ibruzati6n(ES or APCI) and mode of operation (positive
or negative ion mode). This may be accomplished by flow injection or infusion
;: 6f ithe target analyte solution. Optimize for the appropriate parent ion (usually
the (M+l]+ or the [M-l]' ion) either manually or using the automated software
program/' Adjust the Operational parameters to create product ions. Optimize
product ion formation ioi the ions of interest The selected product ion will be
'-fnr riiianiNtatinri~ "' : ' '' '''....':' ' ::' ' .
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7. Set up calibration standards for analysis in the sequence table.
8. Analyze samples against the calibration curve constructed in the previous step.
For urine samples, analyze the samples against a calibration curve prepared in
.urine. ^... -, ... . .; .
2.7;.: Performance^ Check .. . .. .. ; ..... . , . ....... ;..,.;.
.-, ; The performance of. the instr^nT,ent.\YUl be verified prior to arty sample analysis. The
system must meet mii^x^ sensitivity and ^ tuning requirements as described below.
2.73 Sensitivity and Tuning Checks ......
, . . , .. - Tuning is conduced using the PI? Sciex mixture of PPGs. The mass spectrometer
. ., , is i tunedip .meet, the specifications for .sensitivity and mass accuracy as described
/jnAC^SpP-18^^ . .-.....- -,.
.,. : .2.7^ Acceptance Criteria . ..._%?..,...%-., ... , .,.......;... .
. .; , .H; any acceptance criterion is not n\et, necessary corrective actions must be
; performed, .to bring the instrument to an acceptable performance before sample
,. ..analysis. Refer to the ACS^SpP^lS^W "Standard Operating Procedure for me
2.8 . .System Suitability
Acceptable chromatographic performance shall be established prior to analyzing the
calibration curve standards and samples. The system shall, provide sufficient : chromatographic
peak symmetry((T) and retentipn of anajyteis..(kp). .A .test solution of methyl anisate should be
used j» evaluate title chromatQgraphic .system. Both k^ and T values are calculated using the
V "'*."'* 'I-1 ..... ' ' * ...... " ' "
formulas described ^in ACS-SpP-185-OQl. Accep.table values are.k' .= 10 to 15, and T=0.5 to 2.0
: for the methyl anisate performance test- Sensitivity shall, be determined by the analysis of the
calibration curve, .previous evaluations have detennmed that the limit of detection of 1 to 5
pg/um levd was obtainable. All target analytes mustdipw >5:1 signal-to-noise for the lowest
calibration curve point used in the generation of the calibratipn curve-
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3.0 CALIBRATION FOR QUANTITATIVE ANALYSIS
3.1 Mass Spectrometer Calibration
The mass spectrometer is calibrated using standards of the target analytes shown in
Table Z For those ahalytes with isotopically labeled analogs, isotope dilution will be used for
preparation of trie calibration''curve. The other analytes will utilize these isotopically labeled
compounds as internal standards. -.-.'-...<;-.= ;.-.-.:. .-
l Calibratibri'bf the instrUmehi is required before any sample analysis by constructing a
new 'calibration curve with reference stahdard(s). A multi- point calibration curve is
constructed by analyzing the calibration solutions described in Section 2.3. Urine samples
should be quantitated using a calibration curve prepared In pooled, background free urine. The
urine calibratioh solutions should b£ prepared and filtered prior to analysis (Section 4.1.1).
Samples may be quantitated using'either average response factors of regression of the
calibration curve. Average response factbrs:tausthave ^25% RSD over the calibration range.
Acceptable calibration curVes'muit have a r2 value > 0.98. For compounds with high LODs, the
calibration curves shown have a limited number of points, which will therefore require the use
of additional higher level calibration points to complete the curves. '
3.2 ' Cbriimi^$:Cri&&iior\Checkq '''=>'''' .'..." ''=. = '"
Calibration verification tfheck standards wfll be analyzed at the beginning of each day
and bracket me samples With a check standard every 10 samples throughout the sample
analysis to monitor the stability of me calibration! The check standard will be composed of a
rriid-levei standard: The check standard should have a're'sp'onse factor within ±25% for the same
level standard from the 'initial calibration oirVe or me calculated amount should not vary by
mciire thari ± 25%^frbrri mit calculated formesame levelstandard frorfi the initial calibration
curve if regression is used for quahtitatibri. ' ' -
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4.0 SAMPLE ANALYSIS
4:1 v- grecohcentration of Aqueous Diaper Extracts.. -..: -.., . ,; .
-: i When this method is used to analyze aqueous extracts of baby diapers, the extract must
be concentrated to achieve a sufficiently low MQL. .The extract container is shaken for 15 .
seconds to homogenize the extract A 10.0 mL aliquot of the extract is then transferred to a 15
mL polypropylene tube, using a graduated pipette. The tube is capped and the sample code
transferred to the tube. The extract is frozen by laying the tube horizontally on a rack inside an
ultra-cold (<-70 °C) freezer for at least: two hours. Freezing the tube horizontally allows a
passageway for vapor to escape when the samples are later placed under vacuum. The freeze
dryer must be started about an hour before use.- The compressor is turned on, arid the system
allowed to cool to below *40 °G. The refrigerator "OK" light should come on before starting the
Vacuum pump;The system should pump down to < 200 millitorr in about 15 minutes, and the
vacuum "OK" light should come on. When the samples have chilled to < -70 °C, the caps are
removed/ and the tubes are each covered with a Kimwipe, held in place by a rubber band. The
frozen samples are then placed inside a freeze drying flask and attached to the manifold of the
freeze dryer.
The samples are kept under vacuum, attached to the freeze-dryer, until only about 0.4
mL of ice remains. The tubes are removed, recapped; and allowed to thaw. Ten, microliters of
thefintemal standard spiking solution is then added, and the volumes adjusted to 0.5 mL with
HPLC grade water. -The extracts are th.en mixed using a vortex mixer and pushed through a 0,2
micron syringe filter into an autosampler.vial. The sample codeisjiansferred to .the vial with a
permanent marker.; >v\; '::,* '-A- ;:':'.'..'... y ;-.>-.- :-..:- -,.-.> ;.'
''-. . (Additional information on-the operation and;maintenance of the Virtis freeze-dryer can
befoundinRTI/AeS-SOPrl74-030. :. v.
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*./ sample Analysis
An appropriate calibration check standard(s) should be analyzed at least every lu
samples in addition to any quality control sample(s) required by the project. The calculated
amount of the check standard- should not vary by mqre than ±25% from that calculated for uu?
same level standard from the initial calibration curve for analysis to continue. All valid data
must be bracketed by calibration check standards with acceptable performance. The sequence of
analyses oh any given day of analysis will include, at a minimum
; ' : Mid level calibration check point ->'>?,:-.... =
" '' '" -' -Method blank " r :':. ;-.- -.; .;;- ,- -. :., .- .: ,.
'' - Up to 10 samples ^including one duplicate :
-:..., ';-Mid level calibration checkpoint .%..,.<:''....
43 Sample Custody ' '' ':''" : ' ' '.- '' ': ..:/,.=':.:- -. = - . . -
' ;: ' : When a batch of sample extracts -is submitted to the analytical lab for analysis, the MS
analyst is responsible'fbr sample custody: Custody records will be maintained which describe
storage, custody, and sample analysis information for the sample once received in the mass
5.0 DATA REPORTING
"'''' Hard cbpies of the method and the calibration tables should generated. The original
hard copies should be filed with necessary information (project No., date of analysis, etc.) in a
: specific location -(file cabinet No., Room. No.) depending on the project requirement.
: Saved data is stored bh'the computer hard drive." Data files and method files should be
archived to CD-ROM disks on a regular basis. All CD-ROM disks should be labeled
appropriately to reflect:-their (content artd stored in a location specified by the project protocols.
All final results are calculated from the raw data using Analyst software. At the end of
the analysis the analyst is responsible for reviewing the data generated to verify that data
quality objectives are met. The task leader or the laboratory manager is responsible for
8
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October 2000
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reviewing the data before they are reported or provided to, the principal investigator. Data
review forms may be utilized by the task leader .or the laboratory manager to record review of
data. ... . ..:; :,.../; . ... t -.-,
6.0. ,..REFERENCES
6.1... . Standard Operating Procedures ,.;. ,-
' \ ' ~"
Standard Operating Procedure (SOP) for the Calibration and Maintenance of
, ,T , :VHPLC Pumps, RTI/A^
Standard Operating Procedure for the Performance Verification and
... .- _ . , ..., Maintenance of HPLQ Autpmjectors, RTI/ACS-SOP-165TOP6, Rev. 0, Jury 1997.
6.2 Instrument Manuals with Performance Check i
., - , .. ..PE.Sciex LC/JylS/IylS System bistallatipn Manual. [Installation checkout and
performance specifications]
6.3 Other Instrument lyjanyia^s . ....
PE Series 20pLCAutosamplerU^
Part No. 0993:89^ Release C, September 1995. :
PE Series 200 LC Pump User's Manual, Perkin Elnver, Norwalk, CT, PE Part No.
0993-8910, Release F, April 1998.
PE Series 200 Vacuum Degasser User's Manual Perkin Elmer, Norwalk, CT, PE
Part No. 0993-6206, Release A, April 1996.
PE Model 785A Programmable Absorbance Detector User's Manual, Perkin
Elmer, Norwalk, CT, PE Part No. 0993-6189, Release A, August 1996.
API 3000 LC/MS/MS Systems Operator's Manual, PE-Stiex, Foster City, CA,
Part No. 017544, Rev. A, April 1998.
API TUNE Manual, PE-Sciex, Foster City, CA, Part No. 015909, Rev. B, May 1998.
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i ' : Sample Corifrol LC/MS/MS Systems User's Manual, PE-Sciex, Foster Qty, CA,
: '' * " ;ParfN6^di5947/ RevVE/April 1998; v; ' >'=-
API 3000 High Sensitivity Procedures Operator's Manual, PE-Sciex, Foster City,
CA, Part No. 017541, Rev. B, September 1998.
Turbo Ion Spray Inlet for the API LC/MS System Operator's Manual, PE-Sciex,
Foster City, CA, Part No. 016601-B, October 1996.
Heated Nebulizer Inlet for the APCI Ion Source Operators Manual, PE-Sciex,
v;"' -::Fosier'eity;cA/P^ ;
PE-Sciex Analyst Software Operator's Manual, PE-Sciex, Foster City, CA, Part
PEi-Sciex'A^iaiyst Software Administratbir's Guide, PE-Sciex, Foster City, CA, Part
No. 0175551, Rev: A'ijuly 1999; ''- K: ; ; - ' ; ;
Dell OpenManage Client Administrator and Dell OpenManage Client 43
^ ''' instail^tibn mstructibns and Features -Deli Computer Corp., Part No. 5252R,
Rev. AOO, March 1999. .-..,,,.. ^.
Dell-Installed Microsoft Windows NT Workstation 4.0 Setup Guidei Dell
t!dinpu»er CbrpC'Pkrt No/ 4760R, Rev. AOi/March 1999.
Dell OptiPlex Systems - System mfoririiu'an Guide, Dell Computer Corp., Part
10
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ETI/ACS-SOP-500-004
October 2000
Revision 0
< ?; '''TABLE 1. OPMATidNAl/CONDITIONS
HfPLC Conditions
lyipbaiePhase
Gradient:
Column:
Flow Rate (mL/min):
Injection Volume:
APCI Conditions
ATCI Temperature:
islebulizer setting: ;
Nebulizer flow setting:
Auxiliary gas setting:
Curtain Gas setting:
Needle Current
''.'.' :. >.-'. '.''.."
lonSpray Vdltager* "
' A=99:$% Water (6.1 % acetic acid), B= 99.8 % Methanol
100% A( 5 min hold) to 95% B .In 23 min ( 7 min hold)
YMC QD&-AQ 4.6 x 150 mm, 5 um particles
1.0 (sptit 3:1 for electrospray)
50tolob.(um)
475°C
8 .
r ^ ,s> ;.
14
13
2uAmps
4200V (+), -3800V ()
Electrospray Conditions
Nebulizer flow setting: 8
Curtain Gas setting: 13
Needle Current: 3,0
Turboionspray Temperature: 350°C
Mass Spectrometer Conditions
Mode:
Scan Type:
CEM
Positive or Negative ionization
MRM
approximately 2000 V
11
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RTI/ACS-SOP-500-004
October 2000
Revision 0
TABLEZ TARGET OOMPOUTsH^JONIZATION MODE
Target and Internal Standards
tWmethyiphosphate (DMP)
Dimethylphosphorodithioate {DMPDT)
Dimethylphosphorodithionate (t)NIPt)
Diethylphosphorothioate (DEFT)
Diethyl phosphate (DEP)
Diethylphosphorodithioate (DEPDT)
N-Ethyl-cyclohexamine
Aldicarb sulfoxide
2-Aminobenzamidazole
Fenamlphos sulfone
Disulf oton sulfone
2-Diethyiairuno-6-methylpyriihidin-4-ol
Atrazfne-D5'
Parent Pesticide
organophosphates
'; . ,,. , ::-. ( ;.:_ ,--:-;'.:'v
organophosphates
.organophosphates
organophosphates
organophosphates
organophosphates
Cydoate (c)
Aldicarb (c)
Benomyl (c)
Fenamiphos (p)
Disulfoton (p) .
Pirimiphos (p)
(IS)
Ion Pairs
195.8/35
158.9/124.6
143.0/110.7
171.0/114:6
155.0/127.0
.186.9/130.8
1282/83.1
207.1/131.0
134.0/92.0
3362/265.8
306.9/153.0
182.0/154.0
221/179.1
lonization
Mode
+APQ
+APC3
*APQ
+APQ
+APQ
+APCI
+Apa
+APQ
+APO
+Apa
+APCI
+Apa
+APO
"Internal standard
(p) Designates organophosphate pesticides.
(c) Designates carbamate pesticides.
12
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RTl/ ACS-SOP-500-004
October 2000
Revision 0
TABLE 3. PERFORMANCE DATA
Target Compound
Dimethyl phosphate (DMP)
DimetiKylphosphorodithioate
(DMPDT)
Dimethylphosphorothionate
(DMPT)
Diethylphosphorothioate
(DEFT)
Diethyl Phosphate (DBF)
Diethylphosphorodithioate
(DEPOT)
Aldicarb sulfoxide
. N-Eihyl-cyclohexamine
2-Aminbbenzamidazole
Fenamiphos sulfone
Phorate sulfone
Disulfoton sulfone
2-Diethylamino-6-meth.yl-
pyrimidin-4-ol
Atrazine-Ds1
Parent
Pesticide
OP
OP
OP
OP
OP
OP
Aldicarb
Cycloate
e
Benomyl
Fenamiphos
Phorate
Disulfoton
Pirimiphos
(IS)
Limit of
Calibration Detection
Fit (R) (ps/ian)"
NC -50
1.0 (3 pts) 46
0.9206 11
'1.000 <1
tt9977 <1
0.9894 105
0.999 <1
0.9994 5-4
0.9999
5
5
50
5
5
5
5
5
5
10
100
From urine matrix calibration with (S/N x 3).
bn>5.
e Three samples of urine were collected from volunteers and spiked at the level given in the column to
the eight 0.5 mL of spiked urine was diluted to 10 mL in water, then concentrated by the freeze-dry
method (Section 4.1) back to 0.5 mL. Recovery was calculated by comparison with the unprocessed
samples.
d Internal standard
13
6-38
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