•Si
r"
EPA/600/4-85/054
Method 531. Measurement of N-Methyl Carbamoyloximes
and N-Methyl Carbamates in Drinking Water
by Direct Aqueous Injection HPLC with
Post Column Derivatization
Prepared for
Joseph A. Cotruvo
-. Director
Criteria and Standards Division
Office of Drinking Water
Prepared by
Denis L. Foerst
Physical and Chemical Methods Branch
Environmental Monitoring and Support Laboratory
Cincinnati, Ohio 45268
U.S. EnvfimmenM Protection Agency
Region V, tferary
230 South Dearborn Street
Chicago, minots 60604 <
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INDEX
Section
Number
1 .
2
3
4
5
6
7
• 8
9
10
11
12
13
1.
2.
1.
2.
terials
ation and Storage
Subject
Scope and Application
Summary of Method
Definitions
Interferences
Safety
Apparatus and Equipment
Reagents and Consumable
Sample Collection, Prese
Calibration and Standardization
Quality Control
Procedure
Calculations
Precision and Accuracy
References
... TABLES
Single Operator Accuracy and Precision Data in Acid Preserved
Reagent Water
Acceptable Storage Time (for Selected Method 531 Analytes
FIGURES
Block Diagram of HPLC System
HPLC Chromatogram of N-flethyl Carbamoyloximes and
N-Methyl Carbamates
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Method 531. Measurement of N-Methyl
Carbamoyloximes and N-Methyl Carbamates
in Drinking Water by Direct Aqueous
Injection HPLC with Post Column Derivatization
1. SCOPE AND APPLICATION
1.1 This method describes a procedure for the identification and
measurement of N-methyl Carbamoyloximes and N-methyl carbamates in
finished drinking water, raw source water, or drinking water at any
treatment stage.(1,2) Single-laboratory accuracy and precision
data have been determined for the following compounds.
/
Chemical Abstracts
Service Registry Number STORET
Analyte (CASRN) Number
Aldicarb 116-06-3
' Aldicarb sulfone - 1646-88-4
Aldicarb sulfoxide 1646-87-3
Carbaryl 63-25-2 39750
Carbofuran 1563-66-2 81405
3-Hydroxycarbofuran 16655-82-6
Methomyl 16752-77-5 39051
Oxamyl 23135-22-0
Laboratories may use this method to detect and measure additional
analytes after demonstrating obtains acceptable (defined in Section
10) accuracy and precision data for those analytes.
1.2 Method Detection Limits (MDLs) (3) are matrix and compound
dependent. The MDL is that concentration of analyte below which
there is less than a 99% confidence that the concentration is
different from zero. The reagent water MDLs for the analytes given
above vary from 0.5 to 1.6 ug/L. The applicable concentration
range for this procedure is from the MDL to approximately 250 ug/L
of undiluted sample.
1.3 This method is recommended for use by analysts experienced with
high performance liquid chromatography (HPLC) and fluorescence
detection techniques or by experienced technicians under the close
supervision of such qualified analysts.
531-1
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SUMMARY OF METHOD
2.1
The water sample is filtered ahd a 400 yL aliquot is injected into
a reverse phase HPLC column. Separation of the analytes is
achieved using gradient elution chromatography. After elution from
the HPLC column^ the analytes
hydroxide at 95 C. The methyl
reacted with o-phthai aldehyde
derivative whTch is detected
are hydrolyzed with 0.05N sodium
amine formed during hydrolysis is
(OPA) to form a highly fluorescent
sing a fluorescence detector. (4)
DEFINITIONS
External standard — a known amount of a pure analyte that is analyzed
under the same procedures and conditions that are used to analyze
samples containing that compound.
Internal standard — a pure compound added to a sample in a known amount
and used to calibrate concentration measurements of other analytes that
are sample components. The internjal standard must be a compound that is
not a sample component.
Field duplicates — two samples taken at the same time and place under
identical circumstances and treated exactly the same throughout field
and laboratory procedures. Analysis of field duplicates indicates the
precision associated with sample collection, preservation and storage,
as well as with laboratory procedures.
Field reagent blank — reagent water placed in a sample container by the
laboratory, shipped to and from tne sampling site, and treated as a
sample in all respects; including storage, preservation and all
analytical procedures.
Laboratory control standard — a solution of analytes prepared in the
laboratory by dissolving known amounts of pure compounds in a known
amount of reagent water. In this method, the laboratory control
standard is prepared by adding appropriate volumes of the secondary
dilution standard solution to reagent water.
Laboratory duplicates — two aliiuots of the same sample that are
treated exactly the same throughout laboratory analytical procedures.
Analysis of laboratory duplicated provides a measure of the precision
associated with laboratory procedures and excludes the precision
associated with sample collectiop, preservation or storage procedures.
Laboratory reagent blank — a SOf-mL portion of acid preserved reagent
water filtered, and analyzed as if it were a sample.
Performance evaluation sample -4 a water soluble solution of method
analytes distributed by the Quality Assurance Branch (QAB), Ohio, to
Environmental Monitoring and Support Laboratory, USEPA, Cincinnati,
multiple laboratories for analysis. A small measured volume of the
solution is added to a known volume of reagent water and analyzed using
procedures identical to those used for samples. Results of analyses are
531-2
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used by the QAB to determine the accuracy and precision that can be
expected when a method is performed (by competent analysts. Analyte true
values are unknown to the analyst.
Quality control check sample — aw
known concentrations of analytes pr
laboratory performing the analysis.
solution to demonstrate that it can
and measurements with a method. A
is added to a known volume of reage
identical to those used for samples
by the analyst.
Stock standard solution — a concentrated solution containing a
certified standard that is a method
solution of an analyte prepared in
reference compound.
ter soluble solution containing
pared by a laboratory other than the
The performing laboratory uses this
obtain acceptable identifications
mall measured volume of the solution
t water and analyzed with procedures
True values of analytes are known
analyte, or a concentrated organic
the laboratory with an assayed
Secondary dilution standard —- an drganic solution of analytes prepared
in the laboratory from stock standard solutions and diluted as needed to
prepare aqueous calibration solutions and laboratory control standards.
4. INTERFERENCES
4.1
Method interferences may be c
reagents, glassware and-other
to discrete artifacts or elev
chromatograms. All reagents
demonstrated to be free from
the analysis by running labor
Section 10.1.4.
.used by contaminants in solvents,
sample processing apparatus that lead
.ted baselines in liquid
d apparatus must be routinely
nterferences under the conditions of
tory reagent blanks as described in
4.2
4.1.1 Glassware must be scrupulously cleaned.(5) Clean all
glassware as soon as possible after use by washing with hot
water and detergent then rinsing with tap and reagent
water. Drain dry. Seal and store by inverting or capping
with aluminum foil in a clean environment to prevent any
accumulation of dust or other contaminants.
4.1.2 The use of high puritl reagents and solvents helps to
minimize interference]problems. Purification of solvents by
distillation in all-glass systems may be required.
Samples may become contaminated during shipment or storage. Field
reagent blanks must be analyzed to determine that sampling and
storage procedures have prevented contamination.
4.3 During analysis, major contajninant sources are impurities in the
mobile phase. Analyses of fneld reagent blanks and laboratory
reagent blanks provide information about the presence of contami-
nants.
531-3
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4.4 Interfering contamination may occur when a sample containing low
concentrations of analytes is analyzed immediately following a
sample containing relatively high concentrations of analytes. A
preventive technique is between-sample rinsing of the sample
syringe and filter holder with two portions of reagent water.
After analysis of a sample containing high concentrations of
analytes, one or more laboratory reagent blanks should be analyzed
to ensure that accurate values are obtained for the next sample.
5. SAFETY
5.1 The toxicity or carcinogenicitjy of chemicals used in this method
has not been precisely defined); each chemical should be treated as
a potential health hazard, and exposure to these chemicals should
be minimized. Each laboratory^ is responsible for maintaining
awareness of OSHA regulations regarding safe handling of chemicals
used in this method. Additional references to laboratory safety
are cited (6-8).
6. APPARATUS AND EQUIPMENT
6.1 SAMPLE CONTAINERS — 100-fflL of larger glass or plastic bottles,
each equipped with a screw cap.
6.2 BALANCE — Analytical,, capabljb of accurately weighing to the
nearest 0.1 mg.
6.3 FILTRATION APPARATUS
6.3.1 Macrofiltration — to
mobile phases used in
filter derivatization solutions and
HPLC. Recommend using 47 inn filters
(Mill i pore Type HA, OJ45 ufn for water and Mi Hi pore Type FH,
0.5 uro for organics on equivalent).
6.3.2 Microfiltration — to filter samples prior to HPLC
analysis. Use 25 mm filter holder (Nuclepore, polycarbonate
420200 or equivalent) and 25 mm diameter 0.4 ym
polycarbonate filters
(Nuclepore 110607 or equivalent).
6.4 SYRINGES AND SYRINGE VALVES
6,4.1 One 10-mL glass hypodermic syringe with Luer-Lok tip.
6.4.2 One 3-way syringe valve (Hamilton HV3-3 or equivalent).
6.4.3 One 17 gauge syringe needle, seven to ten cm long, blunt tip.
6.4.4 Micro syringes, various sizes,
6.5 MISCELLANEOUS
6.5.1 Standard solution storage bottles — 10-mL bottles equipped
531-4
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with screw caps and seahed with polytetrafluoroethylene
(PTFE) lined septa.
6.5.2 Helium, for degassing dissolved oxygen,
6.6 HIGH PERFORMANCE LIQUID CHROMATOGRAPH (HPLC)
6.6.1 HPLC system capable of injecting 200 to 400 pL aliquots, and
performing binary lineir gradients at a constant flow rate.
6.6.2 Column — 10 cm long x 8 mm ID radially compressed HPLC
column packed with 10 am y-Bondapak C-18 or equivalent.
This column was used tp generate the method performance
statements in Section 13. Different HPLC columns may be
used in accordance with the provisions in Section 10. Use
of guard columns is highly recommended.
6.6.3 Post Column Reactor —| Capable of mixing reagents into the
mobile phase. Reactor) to be equipped with pumps, to deliver
0.5 mL/min each reagent; mixing tees; two 1.0 mL delay
coils, one thermostatid at 95*C; and constructed using PTFE
tubing. (Kratos URS f)51 and URA 100 or equivalent). See
Figure 1.
6.6.4 Fluorescence Detector — Capable of excitation at 230 nm and
detecting emission energies greater than 419 nm.
Fluorometers should have dispersive optics for excitation
and can utilize eithefr filter or dispersive optics at the
emission detector.
a data system to report retention
is recommended but not required. The
uce a strip chart recording of detector
6.6.5 Data System — Use o
times and peak areas
HPLC system must pro<
response.
7. REAGENTS AND CONSUMABLE MATERIAL|
7.1 HPLC MOBILE PHASE
7.1.1 Reagent water — laboratory grade water in which an
interferent is not observed at the method detection limit.
Filter and degas witjh helium before use.
7.1.2 Organic phase — Methanol and acetonitrile at an 80:20 (v:v)
composition. Prepatre using HPLC grade solvents. Filter and
degas with helium btfore use.
7.2 POST COLUMN DERIVATIZATION SOLUTIONS
7.2.1 Sodium hydroxide (0.05N) — Dissolve 2.0 g of sodium
hydroxide (NaOH) in reagent water. Dilute to 1.0 L with
reagent water. Prepare fresh daily. Filter and degas with
helium just before use.
531-5
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7.2.2 2-Mercaptoethanol (1+1) — Mix 10.0 ml of 2-mercapto-
ethanol and 10,0 ml oif acetonitrile. Cap. Store in hood.
(Caution - stench)
7.2.3 Sodium borate (0.05N)
(N32 8407- 10 H20) in
— Dissolve 19.1 g of sodium borate
reagent water. Dilute to
1.0 L with reagent water. The sodium borate will completely
dissolve at room temperature if prepared a day before use.
7.2.4 OPA Reaction solution!— Dissolve 100 ± 10 mg of
o-phthai aldehyde (mp B5-58*C) in 10 ml of methanol. Add to
T.O L of 0.05N sodium borate. Mix, filter, and degas with
helium. Add 100 yL of 2-niercaptoethanol (1+1) and mix.
Make up fresh solution daily.
7.3 SAMPLE PRESERVATION REAGENTS
7.3.1 Sodium thiosulfate -• granular.
7.3.2 Hydrochloric Acid (ill) — Carefully add 1 volume of
concentrated hydrochloric acid (HC1 sp gr 1.19) to an equal
volume of reagent wajter.
7.4 STOCK STANDARD SOLUTIONS —I These solutions may be purchased as
certified solutions or prepared from pure standard materials using
the following procedures.
7.4.1 Accurately weigh approximately 0.0100 g of pure material.
Dissolve the material in HPLC quality acetonitrile and
dilute to volume in a 10-mL volumetric flask. Larger
volumes may be used at the convenience of the analyst. Mix
by inverting sever a' times.
7.4.2 When the assayed compound purity is certified at 96% or
greater, the weight may be used without correction to
calculate the concentration of the stock standard solution.
7.4.3 Transfer the stock standard solutions into PTFE-sealed screw
cap vials. Store at 4*C and protect from light. Frequently
check stock standard solutions for signs of degradation or
evaporation, especially just prior to preparing a
calibration standarfd from them.
7.5 SECONDARY DILUTION STANDARD - Use stock standard solutions to
prepare secondary dilution standard solutions that contain the
analytes in acetonitrile. The secondary dilution standard should
be prepared at a concentration such that 50 to 200 pL of the
solution can be added to 25, 50 or 100 mL of reagent water to
prepare aqueous calibration solutions that bracket the working
concentration range. Check the secondary dilution standard
solution frequently for signs of deterioration or evaporation,
especially just before preparing aqueous calibration solutions.
531-6
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7.6 INTERNAL STANDARD SPIKING SOLUTION — Prepare an acetonitrile
solution of the internal standard at a level that gives
approximately 20 ug/L when lOjO yL of the solution is added to
100 mL of the sample. No universal compound can be specified for
use as an internal standard. Choice of internal standard is left
to the analyst,
8. SAMPLE COLLECTION, PRESERVATION AfJD HANDLING
8.2
8.1 Collect samples in glass or ilastic containers. Conventional
sampling practices (9) are to be followed; however, the bottle must
not be prerinsed with samplejbefore collection.
8.1.1 When sampling from a tater tap, open the tap and allow the
system to flush until the water temperature stabilizes.
Adjust the flow to afaput 500 mL/minute and collect samples.
8.1.2 When sampling from an open body of water, fill a 1 quart
wide mouth bottle or a 1 liter beaker with the sample from a
representative area. Carefully fill the sample bottle to
within 2 cm of the tap.
SAMPLE PRESERVATION — Ox
can all degrade quickly in
(1,2). This short term deg
samples are being shipped ai
at room temperature in auto
the analysis of these three
the laboratory. Aldicarb q
when residual chlorine is
storage times vary with th
(Table 2).
lyl, 3-hydroxycarbofuran, and carbaryl
latural waters held at room temperature
adation is of concern during the time
d the time processed samples are held
sampler trays. Samples targeted for
analytes must be preserved at pH 3 in
;ickly oxidizes to aldicarb sulfoxide
esent in the sample. Acceptable
analyte and the preservation technique
8.2.1
Residual chlorine (up to 5 ppm) must be destroyed by adding
6 to 7 mg of sodium thiosulfate per 100 mL of sample. U.S.
EPA methods 330.4 and 330.5 may be used to measure residual
chlorine (10).
purpose.
Field test kits are available for this
8.2.2
After addition of preservative, seal the sample bottle, mix
by inverting the sample several times, and store all samples
over ice at 0 to 4*t
8.3 FIELD REAGENT BLANKS
8.3.1 Field reagent blank
must be included in each sample set. A
sample set consists! of the samples collected from the same
general sample site) at approximately the same time. At the
laboratory, fill field reagent blank sample bottles with
reagent water, seal, and ship to the sampling site with the
empty sample bottles. After collection, ship back to the
531-7
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laboratory with the filled sample bottles. Wherever a set
of samples is shipped
appropriate blanks.
and stored, it is accompanied by
8.3.2 If used in the field, the reducing agent is added to the
blanks after receipt (in the laboratory.
9. CALIBRATION
9.1 INITIAL CALIBRATION
9.1,1 CALIBRATION SOLUTION*
9.1.1.1 At least thr
each analyte
should conta
approaching;
limit (Table
solutions sh
that bracke
example, if
particular
expected to
analyzed, a
prepared at
25 yg/L,
calibration solutions, containing
are needed. One calibration solution
n each analyte at a concentration
but greater than, the method detection
1) for that compound. The other two
uld contain analytes at concentrations
the range expected in samples. For
the method detection limit for a
alyte is 1.0 yg/L, and a sample is
contain approximately 10 »g/L is
ueous solutions of standards should be
concentrations of 2.0 ug/L, 10 yg/L, and
9.1.1.2 To prepare calibration solutions, add appropriate
volumes (yL) of the secondary dilution standard
solution to aliquots of reagent water at pH=3. Add
the internal standard to give solutions at constant
concentration in internal standard.
9.1.2 Analyze duplicate iliquots of each calibration solution
using procedures identical to those used to analyze
samples.
9.1.2.1 If the external standard technique is being used,
prepare a concentration calibration curve for each
analyte b>| plotting integrated area or peak height
of the analyte as a function of the aqueous
concentration (yg/L is equivalent to ng/mL). If the
ratio of area to concentration or peak height to
concentration of an analyte is constant throughout
the concentration range (each point on the
calibration curve is between 0.9 to 1.1 times the
average ratio), the average ratio may be used
instead or a calibration curve.
9,1.2.2 If the internal standard technique is being used,
calculate the response to each compound relative to
the internal standard. Calculate the response
factor (RF) with the equation,
531-8
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RF
where:
Ax - the araa or peak height of the analyte
standarjd;
As « the araa or peak height of the internal
standard;
Qs a concentration of internal standard; and
Qx = concentration of analyte standard.
RF is a unit!
concentration
be equivalen
throughout t!
between 0.9
average RF m<
non-linear R
Areax/Areas
determine an
ss number; units used to express
of analyte and internal standard must
If the RF of an analyte is constant
!e concentration range (each RF is
o 1.1 times the average RF), the
y be used. For an analyte with
, a calibration curve of
lotted versus Qx may be used to
analyte concentration.
9.2 DAILY CALIBRATION — Check balibration data each day by measurement
of one or more laboratory control standards or calibration solu-
tions. If the response for any analyte falls outside of 0.85 to
1.15 times the expected response, prepare and analyze a fresh
calibration solution to determine if the problem is being caused by
deterioration of the calibration solution. When the internal
standard technique is being used, verify each day that response
factors have not changed. Tlf the RF falls outside of 0.85 to 1.15
times the expected RF, prepare and analyze new standard solutions
to determine new response factors.
10. QUALITY CONTROL
10.1 Minimum quality control requirements consist of:
10.1.1 Initial demonstration of laboratory analytical capability
(accuracy and precision procedures, Sect. 10.2 and 10.3),
10.1.2 Analysis of a laboratory control standard near the beginning
of each 8-h work period,
10.1.3 Analysis of a field reagent blank along with each sample set,
10.1.4 Analysis of a laboratory reagent blank when the field
reagent blank contains analytes at concentrations above the
method detection ifimits,
10.1.5 Quarterly analysis! of a quality control check sample, (if
available for anafytes of concern), and
531-9
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10.1.6 Continued maintenance bf performance records to define the
quality of generated data.
10.2 ACCURACY — Determine accura
quality control (QC) check s
analytes of concern. QC che
analytes, are currently aval
Protection Agency, Environme
Quality Assurance Branch, Ci
certified standard solutions
vendors.
, by analyzing four aliquots of a
pie containing known amounts of
ik samples for some, but not all listed
able from the U.S. Environmental
tal Monitoring and Support Laboratory,
cinnati, Ohio 45268; alternatively,
may be purchased from commercial
10.2.1 Calculate accuracy usnng either the external or internal
standard procedure, pe concentration measured in the QC
sample solution is expressed as a percentage (P) of the true
value for the QC sample.
QC
QC
. 100
where:
'QC
thle mean concentration of the QC check
sample determined using an independent
ternal or internal standard calibration,
_ J
the true concentration of the QC check
sample.
NOTE: The internal standard concentration and the volume
injected must be constant for calibration solutions and
all samples for Uich the calibration solutions are used.
10.2.2 For each analyte, the mean accuracy should be in the range
of 80 to 110% (1,2). For some listed analytes, this may not
be feasible for low concentration measurements.
10.3 PRECISION
10.3.1 Calculate the method precision of each analyte as the
standard deviation (s, in yg/L) of the four measured values
obtained in the accuracy calculations:
*•>-
i= 1
S =
where n = number of measurements for each analyte, and
XT = individual measured value.
10.3.2 Calculate the dispersion of the measured values for each
analyte as the percent relative standard deviation (RSD):
531-10
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RSO
s
C"
100
where s = standard deviation, and
C" s mean observed) concentration.
10.3.3 Adequate precision is obtained if the relative standard
deviation is < 15%. (1,2)
10.4 LABORATORY CONTROL STANDARD
calibration curve is valid,
at the beginning of each 8-h
To demonstrate that the current
alyze a laboratory control standard
ork period.
10.4.1 For each analyte to be measured, select a concentration
representative of its (occurrence in drinking water samples,
10.4.2 Prepare the laboratory control standard by adding 50 to
500 pL of the secondary dilution standard to 50 mL of
reagent water at pH
3.
10.5
10.4.3 Add an appropriate volume of the internal standard spiking
solution and analyze (using the same procedures (Sect. 11)
used for samples.
10.4.4 Determine calibration acceptability and appropriate remedial
actions, if needed. (For the external standard technique,
see Sect. 9.1.2.1; for the internal standard technique, see
Sect. 9.1.2.2.)
MONITORING THE INTERNAL STANDARD — All samples and laboratory
control standards are at equal concentrations of the internal
standard. The response of pat compound is used to monitor system
performance. If for any sample, the response varies more than a!5%
from that observed in the previous sample or laboratory control
standard, do not report analyte concentrtations for that sample.
10.6
Take remedial action to so
reanalyze the sample,
FIELD REAGENT BLANKS — An
each sample set. If a fie
concentrations above the
laboratory reagent blank.
detected at concentration
laboratory reagent blank
field blank, sampling or
sample contamination, and
must be discarded.
ve the system performance problem and
lyze a field reagent blank along with
d reagetft blank contains analytes at
thod detection limits, analyze a
If one or more analytes that are not
above method detection limits in the
re detected in significant amounts in the
torage procedures have not prevented
the appropriate analyte measurement(s)
10.7 QUALITY CONTROL CHECK SAMPLES — At least quarterly, analyze a
quality control check sample obtained from the U.S. Environmental
Protection Agency, Environmental Monitoring and Support Laboratory,
Quality Assurance Branch, Cincinnati, Ohio. Quality control check
531-11
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samples currently are available for some but not all listed
analytes. If measured analyse concentrations are not within ±20%
of true values, check the entire analytical procedure to locate
and correct the problem sourae.
10.8 Additional QC procedures may be necessary, depending on the purpose
of the analysis performed with this method.
10.8.1 Laboratory Duplicates — Determine the precision associated
with laboratory techniques by analyzing two aliquots (Sect.
11.1.2) of a sample in which some analytes were detected in
measurable quantities. Calculate the range (R) of
concentrations measured for ea'ch duplicate pair:
R » C\ - C2,
where Ci represents the larger and,
Cg represents the smaller of the two
measurements.
Calculate percent relative range (RR) of duplicate
analyses using the formula:
RR
100
where R = range of concentrations measured, and
(I a me'an concentration measured.
Generally, if RR is greater than 30%, precision is
inadequate, and
10.8.2 Field Duplicates —
which some analytes
to indicate precisi
transport and stora
techniques. If ace
analysis of field d
duplicates is usual
aboratory techniques must be improved.
Analyze in duplicate, 10% of samples in
were detected in measurable quantities
limitations imposed by sampling,
e techniques as well as laboratory
ptable results are obtained from
plicates, analysis of laboratory
y not necessary.
10.8.3 Matrix Effects Det
samples to determi
be present in the
unspiked aliquots
concentrations, wh
concentrations mea
analyte, the aroun
exceed twice the a
Analysis of dosed
storage and preser
matrix.
srmination — Dose and analyze 5% of the
Je matrix effects. Because analytes may
Jnspiked aliquots, analysis of one or more
s necessary to determine the initial
ch are then subtracted from the
lured in spiked aliquots. For each
added to determine matrix effects should
lount measured in unspiked aliquots.
iamples over time will indicate if the
'ation procedures are adequate for the
531-12
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11. PROCEDURE
11.1 ANALYSIS PROCEDURES
11.1.1 Adjust the pH of the sample or standard to pH of 3 ± 0.2 by
the dropwise addition of hydrochloric acid (1+1). Fill a 50
ml volumetric flask to the mark with the sample. Add the
constant amount of internal standard, if internal standards
are used (1 pL per ml of sample). Mix by inverting the
flask several times. If 1-Naphthol begins to appear in the
chromatograms of carbaryl standards, hydrolysis is occurring
and is an indication pat the pH of the standard is not low
enough.
11.1,
2 Sample filtration —
syringe. Place a cl
affix the filter hoi
the syringe valve.
reagent water. Prew
water through the filj
leaks. Draw 10 ml o
through the filter.
syringe, expel 5 ml
remaining 5 ml for a
water. Discard the
ffix the three-way valve to a 10 ml
filter in the filter holder and
r and the 7 to 10 cm syringe needle to
inse the needle and syringe with
It the filter by passing 5 ml of reagent
ter. Empty the syringe and check for
sample into the syringe and expel
Draw another 10 mL of sample into the
hrough the filter and collect the
alysis. Rinse the syringe with reagent
'liter.
11.1,
3 Sample injection" —
being used, be sure
volume (between 200
calibration standan
minute linear gradii
4% acetonitrile, 16!
20:80 mix of aceton
30% water, 14% acetc
and 70% of a 20:80
f a constant volume injection loop is
overfill the loop. Inject a constant
[to 400 uL) of the filtered sample or
into the HPLC system. Begin the 10
Int. Initial conditions are 80% water,
methanol (i.e. 80% water and 20% of a
trile/methanol). Final conditions are
initrile, 56% methanol (i.e. 30% water
fix of acetonitrile/methanol).
11.1.4 Data Acquisition — Acquire data until the last analyte of
interest elutes. If any peak overloads the photomultiplier
or exceeds the working range of the calibration solutions,
dilute an aliquot off the sample and'reanalyze beginning at
Section 11.1.2.
11.1.5 Equilibration to Initial Conditions — Return the mobile
phase to initial composition. Pump at initial conditions
until the baseline (has flattened. Proceed with the next
analysis.
11.2 IDENTIFICATION PROCEDURES
the retention times of th
authentic standards.
— Analytes are identified by comparing
unknowns to the retention times of
531-13
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11.2.1 Qualitative Identification is achieved if the HPLC retention
time of the analyte in the sample is within 6 seconds of
that observed for the same analyte in the calibration
standard. (See Table ft.)
11.3 CONFIRMATORY PROCEDURES
11.3.1 Analyze using an HPLC (column of different selectivity.
Trimethyl silyl reverse phase columns invert the elution
order of aldicarb sulfone and aldicarb sulfoxide when the
mobile phase is changed from water/acetonitrile to
water/methanol.(1)
11.3.2 Fortify with the analyte of interest and reanalyze. The
presence of the analyte is not confirmed if the native
compound and fortified analyte do not coelute.
11.3.3 Moving belt liquid chromatography/mass spectrometer,(11)
thermospray LC/MS (la) and capillary column GC/MS (13) can
be used to confirm tqe qualitative and quantitative
identifications.
12. CALCULATIONS
12.1 Determine the concentration
using the following equation:
of individual compounds in the sample
where Cx
Ax
RF
Qs -
13. PRECISION AND ACCURACY
RF
analytg' concentration in micrograms per liter;
response of the sample analyte;
response of the standard (either internal or
external), in units consistent with those used
for the analyte response;
response factor (With an external standard,
RF = H because the standard is the same
compound as the measured analyte.);
concentration of internal standard present or
concentration of external standard that
produqed As, in micrograms per liter.
13.1 The single laboratory accuracy and precision data in Table 1 were
collected using a nominal spike level of 2.5 yg/L, a 400 uL
injection volume and a three point calibration curve at nominal
values of 2.5, 5, and 10 Jig/L. Peak height calculations were used
rather than electronic integration. Similar data have been
obtained in two other independent laboratories.(1,2)
531-14
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13.2 The MDLs were calculated using
MDL
where:
-l, 1-<*=0.99)*S
the following equation.
*(n-l,'l-a.0.99) is the
n=7 replicates and
tudents1 t value for o=0.01, and
s is the standard deviation (in yg/L) for the seven replicate
analyses.
REFERENCES
1. Foerst, D.C. and H.A. Moye, "Aldicarb in Drinking Water via Direct
Aqueous Injection HPLC with Post dolumn Derivatization," Proceedings of
the 12th Annual AWWA Water Quality Technology Conference, in press 1985.
2. Hill, K.M., R.H. Hollowell, and UA. DalCortevo, "Determination of
n-Methylcarbamate Pesticides in will Water by Liquid Chromatography and
Post Column Fluoresence Derivatizption," Anal. Chero, 56, 2465 (1984)
3. Glaser, J.A., D.L. Foerst, G.M. MbKee, S.A. Quave, and W.L. Budde,
"Trace Analyses for Wastewaters," Environ. Sci. Techno!. 15, 1426, 1981.
4. Moye, 'H.A., S.J. Scherrer, and P.A. St. John," Dynamic Labeling of
Pesticides for High Performance uiquid Chroraatography: Detection of
n-Methylcarbamates and o_-Phthal aldehyde," Anal. Lett, 10, 1049, 1977.
5. ASTM Annual Book of Standards, plrt 31, D3694, "Standard Practice for
Preparation of Sample Containers and for Preservation," American Society
for Testing and Materials, Philadelphia, PA, p. 679, 1980.
6. "Carcinogens - Working With Carcrinogens," Department of Health,
Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute for (Occupational Safety and Health ,
Publication No. 77-206, Aug. 19/7.
7. "OSHA Safety and Health Standards, General Industry," (29CFR1910),
Occupational Safety and Health fdministration, OSHA 2206, (Revised,
January 1976).
8. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 3rd Edition, 1979.
9. ASTM Annual Book of Standards, Part 31, D3370, "Standard Practice for
Sampling Water," American Society for Testing and Materials,
Philadelphia, PA, p. 76, 1980.
10. "Methods 330.4 (Titrimetric DPiJ>-FAS) and 330.5 (Spectrophotometric, DPD)
for Chlorine, Total Residual," Methods for Chemical Analyses of Water
and Wastes, EPA 600/4-79-020, (JSEPA, EMSL, Cincinnati, Ohio 45268,
March 1979.
531-15
-------
11. Wright, L.H., M.O. Oackson, and R.G. Lewis, "Determination of Aldicarb
Residues in Water by Combined High
Mass Spectrometry," Bull. Environ.
Performance Liquid Chromatography/
Contam. Tox. 28, 740, 1982,
12. Voybsner, R.D., J.T. Bursey, and EJD. Pellizzari, "Postcolumn Addition
of Buffer for Thermospray Liquid Cnromatography/Mass Spectrometry
Identification of Pesticides," Ana|l. Chem., 56, 1507, 1984.
13. Trehy, M.L., R.A. Yost, and J.J. McCreay, "Determination of Aldicarb,
Aldicarb Oxime and Aldicarb Nitritte in Water by Gas Chromatography/Mass
Spectrometry,« Anal. Chem., 56, 1281, 1984.
531-16
-------
Table 1. Single Operator Accuracy and Precision
Data in Acid Preserved Reagent Water
Analyte
Aldicarb sulfoxide
Aldicarb sulfone
Oxamyl
Methomyl
? llyrfmxyrnrhnfurfln
Aldicarb
Carbofuran
Carbaryl
1-Naphthol
Retention*
Time
(min)
5.40
6.05
6.45
7.34
__9.08__
11.43
12.54
13.08
13.57
Retention
Window
(sec)
±0.07
±0.08
±0.06
±0.06
±0.06
±0.06
±0.04
±0.06
±0.09
Spike
Level
(ug/L)
2.40
2.56
2.91
2.78
2.41
2.55
3.20
2.57
—
Observed c
Concentration
2.40
2.61
2.65
2.47
2.39
2^85
2.81
1.98
—
Standard
Deviation
(n9/L)
0.22
0.16
0.50
0.21
0.50
OB
0.28
0.22
—
Relative
Standard
Deviation
(%)
9.4
6.1
18.9
8.5
21
J.C • C,
9.8
11.1
—
Accuracy
(%)
100
102
91
89
99
H2
88
•77
—
Method
Detection
Limit
U<]/L)
0.8d
0.5
1.6
0.7d
1.6
Ljd
•*•»•»
0.9
0.7
—
aSee Section 11.1.3 for chromatographic conditions, dead volume time is 1.84 min.
b99% Confidence Limit of retention time, 20 runs over 16 hours.
C400 uL injection, seven replicates. 5 hours storage time.
dMDL base in pooled value see Reference 1.
-------
Table 2. Acceptable Stdrage Time for Selected
Method 531 Analytes*
Reagent Water Recovery Raw Source Water Recovery
Analyte
Aldicarb Sulforide
Aldicarb Sulfone
Oxarryl
Me thorny!
Time in Days)
pH = 7 pH | 3
70
70
53
70
18
41
65
62
Time
pH » 7
35
19
3
70
in Days
pH m 3
40
70
70
70
a All samples stored at 5*C, Acceptable indicates time to show a
15% loss of analyte. See Reference 1.
-------
GRADIENT
CONTROLER
PUMP
WATER
PUMP
ORGANIC
INJECTOR
HPLC
COLUMN
DETECTOR
DATA
0
PTIO
PUMP'
NaOH
0.5 mL/i
m
PUMP
OPA
0.5 mL/m
1.0 mL DELAY
COIL AT 95°C
1.0 mL DELAY
COIL AT AMBIENT
TEMPERATURE
STRIP CHART
SYSTEM
NAL
MOBILE. PHASE
ELECTRONICS
Figure 1. Block Diagram of HPLC System
5" 31^
-------
COLUMN - 8 mm ID X 10 cm C-18 Rad Pak
GRADIENT - from 80:4:16 (water: acetonitrile:methanol) to
30:14:56 (W:A:M) in 15 minutes
DETECTOR - Fluoresehce, 235 nm/ex 419 nm/enr
-e
c.
fO
5-
i.
o
XT
*<.
5-
03
fC
CO
0.
nj
Ffgure Z> HFLC Chronra-tagrapr of N-MethyT Carb amoy To xf mes
and N-MethyT Carbamates
531-32
------- |