United States
Environmental Protection
Agency
National Exposure
Research Laboratory
Research Triangle Park, NC 27711
Research and Development
EPA/600/SR-97/027
March 1997
SEPA Project Summary
Method Validation for
Measurement of Selected
Semivolatile Phenols in
Dust and Soil
Jane C. Chuang and Donald V. Kenny
The objectives of this study were to
evaluate and validate analytical meth-
ods for analysis of persistent organic
pollutants (POP) in house dust and soil
and to obtain concentration profiles for
the target POP in house dust and soil
samples from the homes of 13 low-
income families.
The analytical method for determin-
ing p-pentylphenol, poctylphenol,
nonylphenols, and bisphenol-A con-
sisted of sequential extraction of the
dust/soil with 5% acetic acid in metha-
nol (MeOH), 100% dichloromethane
(DCM), and 5% acetic acid in water;
liquid-liquid partitioning the resulting
extract with water; and analyzing the
concentrated DCM extract by gas chro-
matography/mass spectrometry (GC/
MS). With this method, quantitative re-
coveries (>80%) of the target phenols
were obtained from the spiked soil
samples. Estimated detection limits for
the target phenols are 0.001 ppm.
The analytical method for 2-
acetylaminofluorene (2AF) and 3-amino-
9-ethylcarbazole (AEC) consisted of ex-
tracting dust/soil with 30% water in
MeOH at pH 10, and analyzing the ex-
tract by liquid chromatography with tan-
dem mass spectrometry (LC/MS/MS).
Recoveries for 2AF and AEC from the
spiked soil samples ranged from 98%
to 110% and from 39% to 110%, re-
spectively. Estimated detection limits
are 0.001 ppm for 2AF and 0.005 ppm
for AEC.
The sums of concentrations of target
phenols ranged from 1.94 to 14.8 ppm
in house dust samples, from 0.047 to
1.51 ppm in entryway dust samples,
and from 0.021 to 0.265 ppm in path-
way soil samples. The observed con-
centration trend was house dust >
entryway dust > pathway soil. There
were no detectable amounts of 2AF and
AEC in any dust or soil samples. Other
compound classes found in dust and
soil samples from one household were
alkanes, aliphatic alcohols, fatty acids,
fatty acid esters, and phthalates.
This Project Summary was developed
by EPA's National Exposure Research
Laboratory, Research Triangle Park, NC,
to announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
Persistent organic pollutants (POP), in-
cluding polycyclic aromatic hydrocarbons
(PAH), polychlorinated biphenyls (PCB),
and other semivolatile organic compounds
(SVOC), nonvolatile organic compounds
(NVOC) and some metals (M) are found
in air, house dust, soil, food, and water.
Many of these compounds are putative
endocrine disrupters and are known mu-
tagens or probable human carcinogens.
Humans can be exposed to these pollut-
ants through inhalation, dietary and non-
dietary ingestion, and dermal adsorption,
and adverse health effects have been
linked to such exposures. The non-dietary
pathway resulting from ingestion of soil
and dust may be more important for young
children because of their play activities.
Children of low-income families or fami-
lies living in urban environments may have
increased exposure to POP and M. This
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may arise because of their proximity to
areas of high traffic, industrial activities, or
lifestyle aspects. Under Cooperative
Agreement CR822073, a preliminary study
to develop and evaluate field methods to
estimate children's exposure to PAH was
conducted. The results from the first two
years of this study indicated that the load-
ings of house dust in several urban low-
income households are more than one
order of magnitude higher than those of
middle-income families. Such high dust
loadings can increase children's exposure
to POP and M through the non-dietary
pathway.
Many POP were not included in the
Cooperative Agreement study. It is desir-
able to include these pollutants in the
evaluation of the field exposure methods
targeted at low-income families. Two ana-
lytical techniques, gas chromatography/
mass spectrometry (GC/MS) and liquid
chromatography with tandem mass spec-
trometry (LC/MS/MS) were evaluated for
analysis of target POP that include puta-
tive endocrine disrupters. The GC/MS
method was evaluated and validated for
analysis of target phenols. The LC/MS/
MS method was evaluated for analysis of
all target POP, but only validated for the
analysis of 2-acetylaminofluorene (2AF)
and 3-amino-9-ethylcarbazole (AEC).
House dust, entryway dust, and pathway
soil samples collected from 13 homes in
other studies were analyzed for target
phenols, 2AF, and AEC using the vali-
dated analytical methods.
The objective of this study was to vali-
date analytical methods for analysis of
target POP in dust and soil, and to deter-
mine target POP in 39 dust/soil samples
collected from the homes of 13 low-in-
come families using the validated analyti-
cal methods.
The following tasks were carried out in
this study:
1. Conduct GC/MS method evaluation/
validation for analysis of p-pentylphenol,
p-octylphenol, nonylphenols, and bis-
phenol-A
2. Conduct LC/MS/MS method valuation/
validation for 2AF, AEC, 2,4-
dinitrotoluene (DNT), anthraquinone,
vinclozolin, and phenols.
3. Analyze 39 samples and one method
blank for target POP using the ap-
propriate validated methods.
Procedure
Analytical Method for Phenols
Two extraction methods were evaluated
for removing phenols from the dust and
soil sample matrices. Initially, the soil
samples were spiked with known amounts
of target phenols and extracted with
dichloromethane (DCM) in a sonication
bath. This approach did not provide satis-
factory recoveries. Another extraction
method was then evaluated. For spike
recovery, known amounts of target phenols
were spiked into each aliquot of the soil
samples. The spiked sample was extracted
sequentially with 10 ml_ of 5% acetic acid
in methanol, 10 ml_ of DCM, and 10 ml_ of
5% acetic acid in distilled water, in a soni-
cation bath for 15 min with each type of
solvent. The resulting extracts were com-
bined and transferred to a separatory fun-
nel. The DCM extract was transferred to
another separatory funnel and washed with
20 ml_ of distilled water. The DCM extract
was dried with sodium sulfate and con-
centrated to 2 ml_ for GC/MS analysis.
Thirty-nine dust and soil samples col-
lected in previous studies from thirteen
low-income households were analyzed for
target phenols. The house dust samples
were collected using the High Volume
Small Surface Sampler (HVS3, Cascade
Stack Sampling Systems, Bend, OR) in
designated areas where the child's great-
est play activity occurred. The entryway
dust samples were collected from a door-
mat at the primary entrance of the house.
The walkway soil samples were collected
from a primary walkway into the home.
Aliquots of the 39 dust/soil samples and
one method blank were prepared by the
above method except that the target
phenols were not spiked into the samples
prior to extraction. Known amounts of in-
ternal standard, phenanthrene-d10, were
added to each concentrated DCM extract
prior to GC/MS analysis. An aliquot of
each DCM extract was also removed for
residue weight measurement.
The extracts were analyzed by 70 eV
electron impact (El) GC/MS. A Finnigan
TSQ-45 GC/MS/MS instrument, operated
in the GC/MS mode, was used. Data ac-
quisition and processing were performed
with an INCOS 2300 data system. The
GC column was a DB-5 fused silica capil-
lary column (60 m x 0.25 mm, 0.25 |im
film thickness, J&W), and the column out-
let is located in the MS ion source. Helium
was used as the GC carrier gas. Follow-
ing injection, the GC column was held at
70°C for 2 min and temperature pro-
grammed to 120°C at 20°C/min and then
to 300°C at 8°C/min. The MS was oper-
ated in the selected ion monitoring (SIM)
mode. Masses monitored were the mo-
lecular ions and their associated charac-
teristic fragment ions. Identification of tar-
get compounds was based on their GC
retention times relative to those of the
internal standard phenanthrene-d10. Quan-
tification of target compounds was based
on comparisons of the respective inte-
grated ion current responses of the target
ions to those of the corresponding internal
standards using average response factors
of the target compounds generated from
standard calibrations. The dust/soil sample
extracts from Household A were analyzed
by GC/MS in full mass scan mode to
identify major compounds tentatively. The
MS was set to scan from m/e 45 to 450
amu at 1 sec/scan. Tentative identification
of the compounds was accomplished by
manual interpretation of background-cor-
rected spectra together with an on-line
computerized library search. The on-line
library was the most currently available
EPA/NIH mass spectral data base, con-
taining 42,197 unique reference spectra.
LC/MS/MS Method Evaluation
The following compounds were evalu-
ated for analysis by LC/MS/MS using the
Sciex TAGA 6000E with an atmospheric
pressure chemical ionization (APCI)
source: 2AF, AEC, DNT, anthraquinone,
vinclozoline, p-pentylphenol, p-octylphenol,
nonylphenols, and bisphenoi-A.
Each compound was analyzed in the
single MS mode to identify the precursor
ion formed by the APCI process. Once
the precursor ion was identified, a frag-
ment ion spectrum (MS/MS) was obtained
by introducing energy to the collision cell.
Standards of the above chemicals were
introduced into the TAGA ion source as
either vapors or liquids. Standards with
sufficient vapor pressure were introduced
by placing an open vial of the standard at
the inlet of the TAGA sampling stream.
For nonvolatile standards, solutions were
prepared at known concentration levels.
Aliquots of the standard solutions were
introduced into the ion source through a
Battelle-developed vapor jet system. Char-
acteristic fragment ions for each standard
were selected from the MS/MS spectrum,
for use in the SIM mode. A series of
standard solutions was analyzed by LC/
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MS/MS to establish calibration curves and
to estimate detection limits. The initial
evaluation results showed that the LC/
MS/MS technique can provide adequate
detection sensitivity for two of the above
standards, namely 2AF and AEC. These
two compounds were selected for further
analysis in dust/soil samples.
Analytical Method for 2-
Acetylaminofluorene and 3-
A mino-9-ethylcarbazole
Extraction recovery experiments were
conducted for 2AF and AEC. Two extrac-
tion methods were evaluated for removal
of the AEC and 2AF from the dust/soil
samples. The first method, sonication with
methanol (MeOH), did not provide satis-
factory recoveries for AEC. The extraction
solvent was then changed to 30% water
in MeOH at pH 10. A spike recovery study
was conducted, where known amounts of
the two target compounds were spiked
into aliquots of selected soil samples. The
spiked sample was extracted with 5 ml_
aliquots of 30% water in MeOH at pH 10
in a sonication bath for 15 min. This step
was repeated four times. The resulting
extracts were combined, filtered, and con-
centrated to 3 ml_ for LC/MS/MS analysis.
The LC gradient elution conditions for
the analysis of the standards and sample
extracts are:
Column: Supelco LC-304
Guard Column
Flow Rate: 1.2 mL/min
Sample Loop: 50 \iL
Gradient Elution Scheme:
0 - 2 min
2-8 min 100%
MeOH - " 25%
100%H2O
H2O/0%
H2O/75% MeOH
25%
H2O/75%
25% H2O/75%
• 100%H2O
8 - 10 min
MeOH
10 - 15 min
MeOH
The mass spectrometer was operated
in the MS/MS (SIM) mode. Vaporized elu-
ent from the LC was introduced into the
APCI ion source, where the samples were
ionized using a corona discharge. Proto-
nated precursor ions were selected with
the first quadrupole mass analyzer (thus
eliminating all other possible interference
ions). The precursor ions were then fo-
cused into the collision cell where they
were fragmented at a collision energy of
35 volts (Elab) with argon as the collision
gas with a target thickness of approxi-
mately 350 x 1012 molecules/cm2. Selected
fragment ions from the isolated precursor
ions were passed through the second MS
and were detected by an electron multi-
plier. For 2AF, two precursor/fragment ion
transitions were monitored, namely m/z
224/182 and 224/43. For AEC, three pre-
cursor/fragment ion transitions were moni-
tored: m/z 211/182, 211/194, and 2117
179. Identification of the target compounds
was based on their correct LC retention
times and their correct relative responses
for each of the precursor/fragment ion tran-
sitions when compared with those from
the standards calibrations. Quantification
of the target compounds was based on
comparisons of the respective integrated
ion current responses of the target com-
pounds in the sample extract to those in
the standard solutions.
Results
GC/MS Analysis of Dust and
Soil Samples
The analytical method for analyzing tar-
get phenols consisted of sequentially ex-
tracting the samples by sonication with
5% acetic acid in methanol, DCM, and
5% acetic acid in water, followed by liq-
uid-liquid partitioning, and analyzing the
concentrated DCM by GC/MS. Quantita-
tive recoveries (>80%) of the spiked
phenols were obtained. The recoveries
ranged from 90% to 104% at 5 ppm spiked
levels, from 84% to 101% at 0.2 ppm
spiked levels and from 84% to 110% at
0.1 ppm spiked levels. The precision for
the phenols for the triplicate spiked
samples was within 13% (relative stan-
dard deviation).
Among the measured phenols, the most
abundant were nonylphenols. The least
abundant phenols were in general, p-
pentylphenol and its isomer. The concen-
trations of phenols ranged from 0.043 ppm
of p-pentylphenol to 11.1 ppm of p-
octylphenol in house dust samples. Rela-
tively lower concentrations were found in
entryway dust samples and ranged from <
0.001 ppm of p-pentylphenol to 0.974 ppm
of nonylphenols. The concentrations of
phenols in pathway soil samples were from
< 0.001 ppm of p-octylphenol to 0.204 ppm
of nonylphenols. The relative concentra-
tion trend within individual households was
house dust > entryway soil > pathway soil.
Levels of nonylphenols found in house
dust samples were greater than 1 ppm,
while those levels found in entryway dust
and pathway soil samples were less than
1 ppm. The concentrations of nonylphenols
ranged from 1.24 to 3.56 ppm in house
dust, from 0.024 to 0.974 ppm in entryway
dust, and from 0.015 to 0.204 ppm in path-
way soil. The concentrations of bisphenol-
A were lower than those of nonylphenols
in the dust/soil samples. The levels of
bisphenol-A ranged from 0.322 to 3.50
ppm in house dust, from 0.019 to 0.335
ppm in entryway dust, and from <0.001 to
0.036 ppm in pathway soil.
The sample extracts of dust/soil samples
from Household A were analyzed by GC/
MS in the full mass scan mode to deter-
mine the major components present in
the samples. The major components found
in house dust were alkanes, fatty acids,
fatty acid esters, phthalates, and aliphatic
alcohols. Similar components including al-
kanes, fatty acid esters and phthalates
were found in entryway dust, but at lower
levels. The pathway soil samples showed
the fewest chromatographic peaks of the
three samples. The major components
found in pathway soil samples were ali-
phatic alcohols, alkanes, fatty acid esters,
and phthalates.
LC/MS/MS Analysis of Dust and
Soil Samples
In order for a compound to be ionized
by APCI, its gas phase basicity (for posi-
tive ion mode) or gas phase acidity (for
negative ion mode) should be greater than
the gas phase basicity/acidity of water.
For this reason, the MS/MS spectra of
anthraquinone, phenols, and vinclozolin
could not be obtained. Of all the com-
pounds evaluated, MS/MS spectra were
obtained for only three compounds, namely
2AF, AEC, and DNT. The 2AF and AEC
spectra were obtained under positive ion
APCI conditions and the DNT spectrum
was obtained by negative ion APCI. Stan-
dard solutions of 2AF, AEC and DNT were
prepared in the range from 1 ng/mL to
1200 ng/mL (1 ppb to 12 ppm) and ana-
lyzed using LC/MS/MS. The estimated
detection limits for 2AF, AEC and DNT
were 1 ng/mL, 5 ng/mL, and 500 ng/mL,
respectively. Since an adequate overall
method detection limit for DNT could not
be obtained, only 2AF and AEC were se-
lected as target analytes.
The analytical method for 2AF and AEC
consisted of extracting the sample with
30% water in MeOH at pH 10, concentrat-
ing the extract, and analyzing the concen-
trated extract by LC/MS/MS. Quantitative
recoveries were obtained for 2AF.
Conclusions
An analytical method for determining
phenols in dust and soil samples was
validated. This method consisted of (1)
sequential sonication of the dust/soil
sample with 5% acetic acid in methanol,
100% DCM, and 5% acetic acid in water,
(2) liquid-liquid partitioning the resulting
extract with water, and (3) GC/MS analy-
sis of the concentrated DCM extract. With
this method, quantitative recoveries (>80%)
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of the phenols were obtained from the
spiked soil samples and the estimated
detection limits are 0.001 ppm of target
phenols in dust/soil.
An analytical method consisting of ex-
tracting the sample with 30% water in
methanol at pH 10 and analyzing the ex-
tract by LC/MS/MS was validated for the
determination of 2AF and AEC in dust/
soil. The recoveries of spiked 2AF ranged
from 98% to 110% in the soil samples.
The recoveries of spiked AEC ranged from
39% to 110% and showed more varia-
tions than the recoveries of 2AF. The esti-
mated detection limits for this method were
0.001 ppm for 2AF and 0.005 ppm for
AEC. The LC/MS/MS method was evalu-
ated but not validated for the analysis of
other target POP either because appropri-
ate MS/MS conditions could not be estab-
lished or because detection limits were
inadequate.
The most abundant target phenols were
nonylphenols and the least abundant one
was, in general, p-pentylphenol in the dust
and soil samples. The concentrations of
target phenols ranged from 0.043 to
3.56 ppm in house dust, from < 0.001 to
0.974 ppm in entryway dust, and from <
0.001 to 0.204 ppm in pathway soil. There
were no detectible levels of 2AF and AEC
in these dust and soil samples. Other com-
pound classes found in the dust/soil
samples from one household were al-
kanes, aliphatic alcohols, fatty acids, fatty
acid esters, and phthalates.
The general concentration trend ob-
served for phenols in these samples was
house dust > entryway dust > pathway
soil. Therefore, human exposure to these
compounds, especially exposures of young
children, through non-dietary ingestion or
dermal contact of house dust should not
be overlooked.
Jane C. Chuang and Donald V. Kenny are with Battelle, Columbus, OH 43201-2693.
Nancy K. Wilson is the EPA Project Officer (see below).
The complete report, entitled "Method Validation for Measurement of Selected
Semivolatile Phenols in Dust and Soil,"(Order No. PB97-143150; Cost:
$21.50, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
National Exposure Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
United States
Environmental Protection Agency
Center for Environmental Research Information
Cincinnati, OH 45268
Official Business
Penalty for Private Use $300
BULK RATE
POSTAGE & FEES PAID
EPA
PERMIT No. G-35
EPA/600/SR-97/027
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