EPA # 815-B-01-002
METHOD 531.2. MEASUREMENT OF N-METHYLCARBAMOYLOXIMES AND
N-METHYLCARBAMATES IN WATER BY DIRECT AQUEOUS
INJECTION HPLC WITH POSTCOLUMN DERIVATIZATION
Revision 1.0
September 2001
M.V. Bassett, S.C. Wendelken, and B.V. Pepich (IT Corporation)
D.J. Munch (US EPA, Office of Ground Water and Drinking Water)
L. Henry (American Water Works Service Company)
R.L. Graves - Method 531.1, Revision 3.0 (1989)
T. Engels (Battelle Columbus Laboratories)
D.J. Munch (US EPA, Office of Water) - National Pesticide Survey Method 5, Revision 2.0
(1987)
D.L. Forest - Method 531, Revision 1.0 (1985)
TECHNICAL SUPPORT CENTER
OFFICE OF GROUND WATER AND DRINKING WATER
U. S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
531.2-1
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MEASUREMENT OF N-METHYLCARBAMOYLOXIMES AND
N-METHYLCARBAMATES IN WATER BY DIRECT AQUEOUS INJECTION HPLC
WITH POSTCOLUMN DERIVATIZATION
1. SCOPE AND APPLICATION
1.1 This is a high performance liquid chromatographic (HPLC) method applicable to
the determination of certain N-methylcarbamoyloximes and N-methylcarbamates
in finished drinking waters. The following compounds can be determined using
this method:
Chemical Abstracts Service (CAS)
Analyte Registry Number
Aldicarb 116-06-3
Aldicarb sulfone 1646-88-4
Aldicarb sulfoxide 1646-87-3
Carbaryl 63-25-2
Carbofuran 1563-66-2
3-Hydroxycarbofuran 16655-82-6
Methiocarb 2032-65-7
Methomyl 16752-77-5
1-Naphthol 90-15-3
Oxamyl 23135-22-0
Propoxur 114-26-1
1.2 Detection Limits are compound, instrument, and matrix dependent. The
Detection Limit is defined as the statistically calculated minimum concentration
that can be measured with 99% confidence that the reported value is greater than
zero.(1) Experimentally determined Detection Limits for the above listed analytes
are provided in Section 17, Tables 2-4. The Detection Limit differs from, and is
lower than the Minimum Reporting Level (MRL) (Sect. 3.15). The concentration
range for target analytes in this method was evaluated between 0.2 ug/L and 10
ug/L. Precision and accuracy data and sample holding time data are presented in
Section 17, Tables 5-8.
531.2-2
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1.3 This method is restricted to use by or under the supervision of analysts skilled in
HPLC analysis and the interpretation of HPLC chromatograms.
2. SUMMARY OF METHOD
2.1 A water sample is filtered. Method analytes are chromatographically separated by
injecting an aliquot (up to 1000 uL) into a high performance liquid
chromatographic (HPLC) system equipped with a reversed phase (Clg) column.
After elution from the column, the analytes are hydrolyzed in a postcolumn
reaction with 0.075 N sodium hydroxide (NaOH) at 80 to 100 °C to form methyl
amine. The methyl amine is reacted with o-phthalaldehyde (OPA) and 2-
mercaptoethanol (or N,N-dimethyl-2-mercaptoethylamine) to form a highly
fluorescent isoindole which is detected by a fluorescence detector.(2) Analytes are
quantitated using the external standard technique.
3. DEFINITIONS
3.1 ANALYSIS BATCH - A set of samples prepared and analyzed on the same
instrument during a 24-hour period. An analysis batch begins with a Continuing
Calibration Check (CCC) at or below the MRL. Subsequent CCCs are analyzed
every 10 samples, should alternate between medium and high concentrations, and
must end the analysis batch. An analysis batch is limited to 20 field samples.
Laboratory Reagent Blanks (LRBs), Laboratory Fortified Sample Matrices
(LFSMs), Laboratory Fortified Sample Matrix Duplicates (LFSMDs), Field
Duplicates (FDs), and CCCs are not counted as samples. Required batch QC
samples include: LRB, CCC, LFSM, and either a FD or a LFSMD.
If a sample(s) in an analytical batch needs to be reloaded for analysis within 48
hours of the start of the original analysis batch, it may be considered part of the
original analysis batch. The reanalyzed sample(s) have the same QC requirements
as above. However, reanalysis of the LFSM, and FD or LFMD are not required if
they were already analyzed and passed QC requirements. Any newly prepared
samples would require a full set of QC samples.
3.2 SURROGATE ANALYTE (SUR) - A pure analyte, which chemically resembles
target analytes and is extremely unlikely to be found in any sample. This analyte
is added to a sample aliquot in known amount(s) before filtration or other
processing and is measured with the same procedures used to measure other
sample components. The purpose of the SUR is to monitor method performance
with each sample.
3.3 LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water or
other blank matrix that is treated exactly as a sample including exposure to all
531.2-3
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glassware, equipment, solvents and reagents, sample preservatives, and
surrogates that are used in the analysis batch. The LRB is used to determine if
method analytes or other interferences are present in the laboratory environment,
the reagents, or the apparatus.
3.4 LABORATORY FORTIFIED BLANK (LFB) - An aliquot of reagent water or
other blank matrix to which known quantities of the method analytes and all the
preservation compounds are added in the laboratory. The LFB is analyzed exactly
like a sample, and its purpose is to determine whether the methodology is in
control, and whether the laboratory is capable of making accurate and precise
measurements. For this direct injection method, a LFB is the same as a
Continuing Calibration Check standard (CCC - Sect. 3.12).
3.5 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) - An aliquot of a
preserved field sample to which known quantities of the method analytes are
added in the laboratory. The LFSM is processed and analyzed exactly like a
sample, and its purpose is to determine whether the sample matrix contributes bias
to the analytical results. The background concentrations of the analytes in the
sample matrix must be determined in a separate aliquot and the measured values
in the LFSM corrected for background concentrations.
3.6 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFSMD) - A
second aliquot of the field sample used to prepare the LFSM, fortified, processed,
and analyzed identically to the LFSM. The LFSMD is used instead of the Field
Duplicate to access method precision when the occurrence of target analytes are
low.
3.7 LABORATORY DUPLICATES (LD1 and LD2) - Two aliquots of the same
sample taken in the laboratory and analyzed separately with identical procedures.
Analyses of LD1 and LD2 indicate precision associated with laboratory
procedures, but not with sample collection, preservation, or storage procedures.
3.8 FIELD DUPLICATES (FD1 and FD2) - Two separate samples collected at the
same time and place under identical circumstances, and treated exactly the same
throughout field and laboratory procedures. Analyses of FD1 and FD2 give a
measure of the precision associated with sample collection, preservation, and
storage, as well as laboratory procedures.
3.9 STOCK STANDARD SOLUTION (SSS) - A concentrated solution containing
one or more method analytes prepared in the laboratory using assayed reference
materials or purchased from a reputable commercial source.
3.10 PRIMARY DILUTION STANDARD (PDS) SOLUTION - A solution
containing the analytes prepared in the laboratory from stock standard solutions
531.2-4
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and diluted as needed to prepare calibration solutions and other needed analyte
solutions.
3.11 CALIBRATION STANDARD (CAL) - A solution prepared from the primary
dilution standard solution and/or stock standard solution, and the surrogate
analytes. The CAL solutions are used to calibrate the instrument response with
respect to analyte concentration.
3.12 CONTINUING CALIBRATION CHECK (CCC) - A calibration standard
containing the method analytes and surrogate(s), which is analyzed periodically to
verify the accuracy of the existing calibration for those analytes.
3.13 QUALITY CONTROL SAMPLE (QCS) - A sample prepared using a PDS of
methods analytes that is obtained from a source external to the laboratory and
different from the source of calibration standards. The second source PDS and the
surrogate PDS are used to fortify the QCS at a known concentration. The QCS is
used to check calibration standard integrity.
3.14 DETECTION LIMIT - The minimum concentration of an analyte that can be
identified, measured and reported with 99% confidence that the analyte
concentration is greater than zero. This is a statistical determination of precision
(Sect. 9.2 A), and accurate quantitation is not expected at this level.(1)
3.15 MINIMUM REPORTING LEVEL (MRL) - The minimum concentration that can
be reported as a quantitated value for a target analyte in a sample following
analysis. This defined concentration can be no lower than the concentration of the
lowest continuing calibration standard for that analyte and can only be used if
acceptable quality control criteria for this standard are met.
3.16 MATERIAL SAFETY DATA SHEET (MSDS) - Written information provided
by vendors concerning a chemical's toxicity, health hazards, physical properties,
fire, and reactivity data including storage, spill, and handling precautions.
4. INTERFERENCES
4.1 All glassware must be meticulously cleaned. Wash glassware with detergent and
tap water, rinse with tap water, followed by reagent water. A final rinse with
solvents may be needed. In place of a solvent rinse, non-volumetric glassware can
be heated in a muffle furnace at 400 °C for 2 hours. Volumetric glassware should
not be heated above 120 °C.
4.2 Method interferences may be caused by contaminants, especially amines and
ammonia, in solvents, reagents (including reagent water), sample bottles and caps,
and other sample processing hardware that lead to discrete artifacts and/or
531.2-5
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elevated baselines in the chromatograms. The samples or analytical system may
be contaminated from being handled with bare fingers. All items such as these
must be routinely demonstrated to be free from interferences (less than V3 the
MRL for each target) under the conditions of the analysis by analyzing laboratory
reagent blanks as described in Section 9. Subtracting blank values from sample
results is not permitted.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method has not been
precisely defined; each chemical compound should be treated as a potential health
hazard, and exposure to these chemicals should be minimized. The laboratory is
responsible for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this method. A reference
file of MSDSs should also be made available to all personnel involved in the
chemical analysis. Additional references to laboratory safety are available/3"5'
5.2 Pure standard materials and stock standards of these compounds should be
handled with suitable protection to skin and eyes. Care should be taken not to
breathe the vapors or ingest the materials.
6. EQUIPMENT AND SUPPLIES (All specifications are suggested. Brand names and/or
catalog numbers are included for illustration only.)
6.1 SAMPLE CONTAINERS - Amber glass bottles fitted with PTFE
(polytetrafiuoroethylene) lined screw caps.
6.2 VIALS - Screw cap or crimp top glass autosampler vials with PTFE faced septa,
amber or clear.
6.3 VOLUMETRIC FLASKS - Class A, various sizes used for preparation of
standards.
6.4 GRADUATED CYLINDERS - Various sizes.
6.5 MICRO SYRINGES - Various sizes.
6.6 BALANCE - Analytical, capable of accurately weighing to 0.0001 g.
6.7 DISPOSABLE SYRINGES - 5 to 30 mL (B-D Cat.#: 309603, 309650 or
equivalent) size, used to filter sample extracts before analysis.
531.2-6
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6.8 FILTERS - Disposable filters used to filter samples before analysis (Millipore
0.22 urn PVDF membrane, Cat. #: SLGV 013 NL or equivalent).
6.9 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
INSTRUMENTATION
6.9.1 HPLC SYSTEM - Capable of reproducibly injecting up to 1000-uL
aliquots, and performing binary or ternary linear gradients at a constant
flow rate near the flow rate used for development of this method, e.g., 1.5
mL/min. A ternary gradient was used to achieve the separation pictured in
Figure 1. However, a binary gradient may be used as long as the minimum
resolution is achieved as specified in Sect. 9.10. The use of a column
heater is strongly recommended. During method development, the column
was heated to 30 °C to help insure stable retention times. Column
temperature may be adjusted as long as column performance is not
compromised.
6.9.2 POSTCOLUMN REACTION SYSTEM - Capable of mixing reagents
into the mobile phase. Reactor should be constructed using
polyetheretherketone (PEEK) or PTFE tubing and equipped with two
pumps capable of delivering 0.5 mL/min of each reagent; mixing tees; and
two reaction coils. Postcolumn system manufacturers recommend
different reaction coil temperatures for the carbamate hydrolysis reaction.
Therefore, the first reaction coil temperature may range from 80 to 100 °C.
Analyte signal can increase with temperature over this temperature range;
however, analysts should be aware that with some systems, baseline noise
can also increase with increasing temperatures. If temperatures greater
than 95 °C are used, a slight backpressure must be maintained on the
system to prevent boiling of the post column eluent. This may be
accomplished by use of a restrictor placed in line after the detector. The
second reaction takes place at ambient temperatures. Method performance
data were collected with two postcolumn reaction modules: the Waters
Postcolumn Carbamate System and the Pickering Model 5200 Carbamate
System, both with a reactor temperature of 80 °C.
6.9.3 HPLC DETECTOR - A fluorescence detector capable of excitation at
approximately 340 nm and detection of emission energy at approximately
465 nm. Optimum excitation and emission wavelengths may vary slightly
for each system. For the development of this method, Waters Model 474
and Model 2475 scanning fluorescence detectors were used.
6.9.4 ANALYTICAL COLUMN - For the development of this method, an
HPLC "carbamate" column (3.9 x 150 mm) packed with 4 um dp Clg solid
phase particles (Waters Cat. # : WAT035577) was used. Any column that
531.2-7
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provides adequate resolution, peak shape, capacity, accuracy, and
precision (Sect. 9) may be used.
6.9.5 HPLC DATA SYSTEM - A computerized data system is recommended
for data acquisition and processing. A Waters Millennium software
system was used to generate all data contained in the Section 17 tables.
7. REAGENTS AND STANDARDS
7.1 REAGENTS AND SOLVENTS - Reagent grade or better chemicals should be
used. Unless otherwise indicated, it is intended that all reagents shall conform to
the specifications of the Committee on Analytical Reagents of the American
Chemical Society, where such specifications are available. Other grades maybe
used, provided it is first determined that the reagent is of sufficiently high purity
to permit its use without lessening the quality of the determination. Reagents of
lesser purity may also affect instrument performance, such as clogging of the post
column reactor.
7.1.1 REAGENT WATER - Purified water which does not contain any
measurable quantities of any target analytes or interfering compounds
greater than 1/3 the MRL for each compound of interest.
7.1.2 PRESERVED REAGENT WATER - Reagent water which has sample
preservation reagents added in the same concentrations as the samples. To
prepare 1 liter of preserved reagent water, add a sufficient amount of
potassium dihydrogen citrate (Sect. 7.1.11.1) to yield a concentration of
9.2 to 9.5 g/L and sodium thiosulfate (Sect. 7.1.11.2) to yield a
concentration in the range of 80 to 320 mg/L to a graduated bottle or
volumetric flask. Fill to the 1 L mark with reagent water.
7.1.3 ACETONITRILE (CH3CN, CAS#: 75-05-8) - High purity, demonstrated
to be free of analytes and interferences (HPLC grade or better).
7.1.4 METHANOL (CH3OH, CAS#: 67-56-1) - High purity, demonstrated to
be free of analytes and interferences (HPLC grade or better).
7.1.5 SODIUM HYDROXIDE (NaOH, CAS#: 1310-73-2), 50% (w/w) solution
(Fisher Cat. #: SS254-500 or equivalent). Due to the hygroscopic nature
of NaOH pellets, a 50% (w/w) solution is used in this method for the
preparation of reagents.
7.1.6 POSTCOLUMN REAGENT #1 (hydrolysis solution) - Sodium
hydroxide, 0.075 N. Dilute 4 mL of 50% w/w sodium hydroxide (NaOH)
531.2-8
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solution to 1 L with reagent water. The concentration of the hydrolysis
solution can dramatically effect the analyte response. Filter and degas
with helium just before use.
7.1.7 PREPARATION OF THE OPA SOLUTION (to be used in preparation of
Postcolumn Reagent #2, Sect. 7.1.10) - Dissolve 100 + 10 mg of o-
phthaldehyde (OPA, CAS#: 643-79-8) in 5 - 10 mL of methanol.
7.1.8 PREPARATION OF THE OPA DILUTENT (to be used in preparation of
Postcolumn Reagent #2, Sect. 7.1.10) - Use one of the following
formulations in the preparation of the preliminary OPA dilutent.
Performance data presented in Section 17 were collected using the sodium
tetraborate formulation. Method performance was checked with the boric
acid formulation and found to be equivalent. OPA dilutent is
commercially available.
7.1.8.1 Prepared using sodium tetraborate : Dissolve 19.1 g of sodium
tetraborate decahydrate (Na^O^lOHA CAS#: 1303-96-4) in a 1
L volumetric flask. Bring the volume up to 1.0 L with reagent
water to make a 0.05 M solution. The sodium borate will dissolve
in less than 2 hours if a stir bar is used. Filter and degas prior to
preparation of Postcolumn Reagent #2 (Sect. 7.1.10).
OR
7.1.8.2 Prepared using boric acid: Dissolve 3.0 g of boric acid (H3BO3,
CAS#: 10043-35-3) in a 1 L volumetric flask in approximately 800
mL of reagent water. Add 1.2 mL of a 50% (w/w) NaOH solution.
Bring the volume up to 1.0 L with reagent water. Filter and degas
prior to preparation of Postcolumn Reagent #2 (Sect. 7.1.10).
7.1.9 PREPARATION OF THE NUCLEOPHILIC SOLUTION (to be used in
preparation of Postcolumn Reagent #2, Sect. 7.1.10) - Use one of the
following formulations in the preparation of the nucleophilic solution.
Either one of these compounds reacts with OPA and the target
methylamine to form the isoindole detected by the fluorescence detector.
Both reagents have characteristic strong odors and should be handled in a
fume hood.
7.1.9.1 Prepared using 2-mercaptoethanol (CAS#: 60-24-2): This
compound is in liquid form and may be used directly in the
preparation of the derivatization solution, Postcolumn Reagent
#2 (Sect 7.1.10). Use 1 mL of 2-mercaptoethanol per liter of
Postcolumn Reagent #2.
OR
7.9.1.2 Prepared using N,N-dimethvl-2-mercaptoethvlamine-
531.2-9
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hvdrochloride (CAS#: 13242-44-9): Use approximately 10 mL of
OPA dilutent (Sect. 7.1.8) to dissolve 2.0 + 0.2 g of N,N-
dimethyl-2-mercaptoethylamine-hydrochloride.
7.1.10 FINAL PREPARATION OF POSTCOLUMN REAGENT #2 (OPA
derivatization solution), - Studies have shown that this reagent made with
either of the two nucleophiles listed above (Sect. 7.1.9) is stable for a
period of at least 36 hours. However, individual laboratory conditions vary
and daily preparation of this solution may be necessary.
7.1.10.1 Add the dissolved OPA (Sect. 7.1.7) to 1 L of either formulation
of the OPA dilutent (Sect. 7.1.8.1 or 7.1.8.2), which has been
filtered and degassed.
7.1.10.2 To the 1 liter flask, add either one of the 2 nucleophiles specified
in Section 7.1.9 (7.1.9.1 or 7.1.9.2).
7.1.11 SAMPLE PRESERVATION REAGENTS ( See also Sect. 8.1.1)
7.1.11.1 Potassium dihydrogen citrate (C6H7KO7, CAS #: 866-83-1) -
Added to adjust sample pH to ~ 3.8, which acts as a biocide to
guard against potential degradation of method analytes by
microorganisms and to prevent chemical degradation. Oxamyl,
3-hydroxycarbofuran, carbaryl, and methiocarb all hydrolyze in
neutral or basic waters even when held at refrigerated
temperatures.
7.1.11.2 Sodium thiosulfate (Na2S2O3 CAS#: 7772-98-7) - Acts as a
dechlorinating agent. Aldicarb and methiocarb are rapidly
degraded in waters that are not dechlorinated.
7.2 STANDARD SOLUTIONS - Standard Solutions may be prepared from certified,
commercially available solutions or from neat compounds. Compounds used to
prepare solutions must be 96% pure or greater and the weight may be used
without correction for purity to calculate the concentration of the stock standard.
Solution concentrations listed in this section were used to develop this method
and are included as an example. Standards for sample fortification generally
should be prepared in the smallest volume that can be accurately measured to
minimize the addition of organic solvent to aqueous samples. Even though
stability times for standard solutions are suggested in the following sections,
laboratories should use standard QC practices to determine when Standard
Solutions described in this section need to be replaced.
531.2 -10
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7.2.1 SURROGATE ANALYTE (SUR) STANDARD SOLUTION, 4-
BROMO-3,5-DIMETHYLPHENYL N-METHYLCARB AMATE
(BDMC, CAS #: 672-99-1)
7.2.1.1 SUR STOCK SOLUTION - May be purchased as certified
standard or prepared from neat material. If preparing from neat
material: accurately weigh approximately 25 to 35 mg of the neat
SUR to the nearest 0.1 mg into a tared, 5-mL volumetric flask.
Dilute to the mark with methanol. Stock solutions have been
shown to be stable for 12 months when stored at -10°C or less.
7.2.1.2 SUR PRIMARY DILUTION STANDARD - Prepare the SUR
Primary Dilution Standard (PDS) by adding enough of the SUR
stock standard to a volumetric flask partially filled with methanol
to make a final concentration near 10 ug/mL when filled to the
mark with methanol. The PDS has been shown to be stable for 6
months when stored at -10 °C or less.
7.2.2 ANALYTE STANDARD SOLUTIONS - Obtain the analytes listed in the
table in Section 1.1 as neat or solid standards or as commercially prepared
ampulized solutions from a reputable standard manufacturer. Prepare the
Analyte Stock and Primary Dilutions Standards as described below.
7.2.2.1 ANALYTE STOCK STANDARD SOLUTION - If preparing
from neat material, accurately weigh approximately 25 to 35 mg of
pure material to the nearest 0.1 mg into a tared, 5-mL volumetric
flask. Dilute to the mark with methanol. Repeat for each target
analyte.
7.2.2.2 ANALYTE PRIMARY DILUTION STANDARD (PDS)
SOLUTION - Prepare the analyte PDS by dilution of the target
analyte stock standards. Add enough of each target stock standard
to a volumetric flask partially filled with methanol to make a final
concentration near 10 ug/mL when filled to the mark with
methanol. A serial dilution of this PDS to make a 1.0 ug/mL
solution is useful for low level spiking. The PDSs have been
shown to be stable for 6 months when stored at -10 °C or less.
7.2.3 CALIBRATION STANDARDS (CAL) - At least 5 calibration
concentrations will be required to prepare the initial calibration curve
(Sect. 10.2). Prepare the calibration standards over the concentration
range of interest from dilutions of the Analyte PDSs in preserved reagent
531.2-11
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water (7.1.2), filtering the CAL standards in the same manner as the
samples (Sect. 11.1.4). The lowest concentration of calibration standard
must be at or below the MRL, which may depend on system sensitivity.
The calibration standards for the development of this method were
prepared as specified below. Calibration standards must be prepared using
preserved reagent water (Sect. 7.1.2). An example of the dilutions used to
prepare the CALs used to collect the data in Section 17 are shown in the
table below. These standards may be also be used as CCCs. If stored, the
aqueous standards must be stored in amber glass and refrigerated in the
same manner as the samples.
PREPARATION OF CALIBRATION (CAL) CURVE STANDARDS
CAL
Level
Analyte
PDS Cone.
(ug/mL)
1.0
1.0
1.0
10.0
10.0
10.0
Vol. of
Analyte PDS
(uL)
5.0
12.5
25.0
5.0
12.5
25.0
Vol. of
10 ug/mL
SURPDS
(uL)
5.0
5.0
5.0
5.0
5.0
5.0
Final Vol.
of
CAL Std,
(mL)
25
25
25
25
25
25
Final Cone,
of CAL
Std.
(ug/L)
0.20
0.50
1.00
2.00
5.00
10.0
Final Cone.
ofSUR
Std.
(ug/L)
2.00
2.00
2.00
2.00
2.00
2.00
8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
8.1 SAMPLE BOTTLE PREPARATION
8.1.1 Prior to shipment to the field, preservatives listed in Section 7.1.11 must
be added as dry solids to each amber bottle fitted with a PTFE lined screw
cap. A 40 or 60-mL sample bottle is recommended. Add a sufficient
amount of potassium dihydrogen citrate to yield a concentration in the
sample of 9.2 to 9.5 g/L. Potassium dihydrogen citrate buffers sample pH
to -3.8 to prevent hydrolysis of oxamyl, 3-hydroxycarbofuran, carbaryl,
and methiocarb. Add sodium thiosulfate to yield a sample concentration
in the range of 80 to 320 mg/L. The concentration of 80 mg/L of sodium
thiosulfate is adequate for dechlorination, but difficult to measure if a
small (e.g., 40-mL size) sample bottle is chosen. Method performance
was tested using up to 320 mg/L without any adverse effect. Sodium
thiosulfate eliminates the residual free chlorine in the samples, which
531.2 - 12
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rapidly degrades aldicarb and methiocarb.
8.1.2 Grab samples must be collected in accordance with conventional sampling
practices (6). Sample bottles must not be prerinsed with sample before
collection. Doing so will wash out the preservatives added to the bottles
prior to shipment.
8.2 SAMPLE COLLECTION
8.2.1 When sampling from a cold water tap, remove the aerator so that no air
bubbles will be trapped in the sample. Open the tap, and allow the system
to flush until the water temperature has stabilized (usually about 3-5
minutes). Collect samples from the flowing system.
8.2.2 When sampling from an open body of water, fill a wide-mouth bottle or
beaker with sample from a representative area, and carefully fill sample
bottles from the container. Sampling equipment, including automatic
samplers, must be free of plastic tubing, gaskets, and other parts that may
leach interfering analytes into the water sample.
8.2.3 If sampling waters high in colloidal iron, filtration of the sample may be
necessary prior to preservation in the field to help prevent the precipitation
of the iron in the reactor.
8.2.4 Fill sample bottles, taking care not to flush out the sample preservation
reagents. Samples do not need to be collected headspace free.
8.2.5 After collecting the sample, cap carefully to avoid spillage, and agitate by
hand for 1 minute. Keep samples sealed from collection time until
analysis.
8.3 SAMPLE SHIPMENT AND STORAGE - All samples should be iced during
shipment and must not exceed 10 °C during the first 48 hours after collection.
Samples should be confirmed to be at or below 10 °C when they are received at
the laboratory. Samples stored in the lab must be held at or below 6 °C and
protected from light until analysis. Samples should not be frozen.
8.4 SAMPLE AND EXTRACT HOLDING TIMES - Results of the sample storage
stability study of all method analytes indicated that all compounds are stable for
28 days in water samples that are collected, dechlorinated, preserved, shipped and
stored as described in Sections 8.1- 8.3. Samples must be analyzed within 28
days.
9. QUALITY CONTROL
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9.1 Quality control (QC) requirements include the Initial Demonstration of
Capability, the determination of the Detection Limit, and subsequent analysis in
each analysis batch of a Laboratory Reagent Blank (LRB), Continuing Calibration
Check Standards (CCC), a Laboratory Fortified Blank (LFB), a Laboratory
Fortified Sample Matrix (LFSM), and either a Laboratory Fortified Sample Matrix
Duplicate (LFSMD) or a Field Duplicate Sample. This section details the specific
requirements for each QC parameter. The QC criteria discussed in the following
sections are summarized in Section 17, Tables 10 and 11. These criteria are
considered the minimum acceptable QC criteria, and laboratories are encouraged
to institute additional QC practices to meet their specific needs.
9.2 INITIAL DEMONSTRATION OF CAPABILITY (IDC) - Requirements for the
Initial Demonstration of Capability are described in the following sections and
summarized in Section 17, Table 10.
9.2.1 INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND -
Before any field samples are analyzed, and any time a new set of reagents
is used, it must be demonstrated that a laboratory reagent blank is
reasonably free of contamination and that the criteria in Section 9.4 are
met.
9.2.2 INITIAL DEMONSTRATION OF ACCURACY - Prior to the analysis of
the IDC samples, verify calibration accuracy with the preparation and
analysis of a mid-level QCS as defined in Section 9.11. If the analyte
recovery is not + 30% of the true value, the accuracy of the method is
unacceptable. The source of the problem must be identified and corrected.
After the accuracy of the calibration has been verified, prepare and analyze
4-7 replicate LFBs (or CCCs in this method) fortified at 2 ug/L, or near the
mid-range of the initial calibration curve, according to the procedure
described in Section 11. Sample preservatives as described in Section
8.1.1 must also be added to these samples. The average recovery of the
replicate values must be within ± 20% of the true value.
9.2.3 INITIAL DEMONSTRATION OF PRECISION - Using the same set of
replicate data generated for Section 9.2.2, calculate the standard deviation
and percent relative standard deviation of the replicate recoveries. The
relative standard deviation (%RSD) of the results of the replicate analyses
must be less than 20%.
9.2.4 DETECTION LIMIT DETERMINATION - Prepare and analyze at least 7
replicate LFBs at a concentration estimated to be near the Detection Limit
over at least 3 days using the procedure described in Section 11. This
fortification level may be estimated by selecting a concentration with a
531.2 - 14
-------
signal of 2 to 5 times the noise level. The appropriate concentration will
be dependent upon the sensitivity of the HPLC system being used. Sample
preservatives as described in Section 8.1.1 must be added to these
samples. Calculate the Detection Limit using the equation
Detection Limit = St(n. l^ _ alpha = 099)
where
t(n-u-aipha = o.99)= Student's t value for the 99% confidence level
withn-1 degrees of freedom
n = number of replicates, and
S = standard deviation of replicate analyses.
NOTE: Calculated Detection Limits need only be less than V3 of the
laboratory's MRL to be considered acceptable. Do not subtract
blank values when performing Detection Limit calculations. The
Detection Limit is a statistical determination of precision only.(1)
If the Detection Limit replicates are fortified at a low enough
concentration, it is likely that they will not meet the precision and
accuracy criteria for CCCs, and may result in a calculated
Detection Limit that is higher than the fortified concentration.
Therefore, no precision and accuracy criteria are specified.
9.2.5 METHOD MODIFICATIONS - The analyst is permitted to modify HPLC
columns and conditions prior to the postcolumn reaction. The analyst is
also allowed to modify the surrogate standard. Each time such method
modifications are made, the analyst must repeat the procedures of the IDC
(Sect. 9.2).
9.3 Minimum Reporting Level (MRL) - The MRL is the threshold concentration of
an analyte that a laboratory can expect to accurately quantitate in an unknown
sample. The MRL should not be established at an analyte concentration that is
less than either three times the Detection Limit or a concentration which would
yield a response less than a signal-to-noise (S/N) ratio of five. Depending upon
the study's data quality objectives it may be set at a higher concentration.
Although the lowest calibration standard must be at or below the MRL, the
MRL must never be established at a concentration lower than the lowest
calibration standard.
9.4 LABORATORY REAGENT BLANK (LRB) - This is a direct injection method
without a conventional extraction. An LRB is required with each analysis batch
(Sect. 3.1) of samples to determine any background system contamination. If
within the retention time window of any analyte, the LRB produces a peak that
531.2 - 15
-------
would prevent the determination of that analyte, determine the source of
contamination and eliminate the interference before processing samples.
Background contamination must be reduced to an acceptable level before
proceeding. Background from method analytes or contaminants that interfere
with the measurement of method analytes must be below V3 the MRL. If the
target analytes are detected in the LRB at concentrations equal to or greater than
this level, then all data for the problem analyte(s) must be considered invalid for
all samples in the analysis batch.
9.5 CONTINUING CALIBRATION CHECK (CCC) - A CCC is prepared in the
same manner as the initial calibration solutions (Sect. 7.2.3), using preserved
reagent water (Sect. 7.1.2) and filtering in the same manner as the samples
(11.1.4). It is analyzed during an analysis batch at a required frequency to confirm
that the instrument meets initial calibration criteria. See Section 10.3 for
concentration requirements, frequency requirements, and acceptance criteria.
9.6 LABORATORY FORTIFIED BLANKS - For this direct injection method, a
CCC is the same as an LFB. Consequently, the analysis of an LFB is not required.
9.7 SURROGATE RECOVERY - The surrogate standard is fortified into all samples,
blanks, LRBs, and LFSMs and LFSMDs prior to sample filtration. They are also
added to the calibration curve and calibration check standards. The surrogate is a
means of assessing method performance from preparation and filtration to final
chromato graphic measurement.
9.7.1 When surrogate recovery from a sample, blank, or CCC is less than 70%
or greater than 130%, check (1) calculations to locate possible errors, (2)
standard solutions for degradation, (3) contamination, and (4) instrument
performance. If those steps do not reveal the cause of the problem,
reanalyze the sample.
9.7.2 If the reanalysis meets the surrogate recovery criterion, report only data for
the reanalyzed sample.
9.7.3 If the sample reanalysis fails the 70-130% recovery criterion, the analyst
should check the calibration by reinjecting the most recently acceptable
calibration standard. If the calibration standard fails the criteria of Section
10.3, recalibration is in order per Section 10.2. If the calibration standard
is acceptable, preparation (which includes spiking with surrogate and
filtration) and analysis of the sample should be repeated provided the
sample is still within the holding time. If this sample reanalysis also fails
the recovery criterion, report all data for that sample as suspect due to
surrogate recovery.
531.2 -16
-------
9.8 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) - Analysis of an
LFSM is required in each analysis batch and is used to determine that the sample
matrix does not adversely affect method accuracy. Assessment of method
precision is accomplished by analysis of a Field Duplicate (Sect. 9.9), however,
infrequent occurrence of target analytes would hinder this assessment. If the
occurrence of target analytes in the samples is infrequent, or if historical trends are
unavailable, a second LFSM, or LFMSD, must be prepared and analyzed from a
duplicate of the field sample. Analysis batches that contain LFSMDs will not
require the analysis of a Field Duplicate. If a variety of different sample matrices
are analyzed regularly, for example, drinking water from groundwater and surface
water sources, method performance should be established for each. Over time,
LFSM data should be documented for all routine sample sources for the
laboratory.
9.8.1 Within each analysis batch, a minimum of one field sample is fortified as
an LFSM for every 20 samples processed. The LFSM is prepared by
spiking a sample with an appropriate amount of the appropriate Analyte
PDS (Sect. 7.2.2.2). Select a spiking concentration at least twice the
matrix background concentration, if known. Use historical data or rotate
through a range of concentrations when selecting a fortifying
concentration. Selecting a duplicate bottle of a sample that has already
been analyzed aids in the selection of appropriate spiking levels.
9.8.2 Calculate the percent recovery (R) for each analyte using the equation
where
A = measured concentration in the fortified sample
B = measured concentration in the unfortified sample, and
C = fortification concentration.
9.8.3 Analyte recoveries may exhibit a matrix bias. For samples fortified at or
above their native concentration, recoveries should range between 70 -
130%. For LFSM fortification at the MRL, 50 to 150% recoveries are
acceptable. If the accuracy of any analyte falls outside the designated
range, and the laboratory performance for that analyte is shown to be in
control in the CCCs, the recovery is judged to be matrix biased. The result
for that analyte in the unfortified sample is labeled suspect/matrix to
inform the data user that the results are suspect due to matrix effects.
9.9 FIELD DUPLICATE OR LABORATORY FORTIFIED SAMPLE MATRIX
531.2 - 17
-------
DUPLICATE (FD or LFSMD) - Within each analysis batch, a minimum of one
Field Duplicate (FD) or Laboratory Fortified Sample Matrix Duplicate (LFSMD)
must be analyzed. Duplicates check the precision associated with sample
collection, preservation, storage, and laboratory procedures. If target analytes are
not routinely observed in field samples, an LFSMD must be analyzed rather than a
FD.
9.9.1 Calculate the relative percent difference (RPD) for duplicate
measurements (FD1 and FD2) using the equation
FDI-FD2
RPD = , ,— *100
9.9.2 If an LFSMD is analyzed instead of a Field Duplicate, calculate the
relative percent difference (RPD) for duplicate LFSMs (LFSM and
LFSMD) using the equation
LFSM - LFSMD
RPD=7 <— *100
LFSM + LFSMD)/2
9.9.3 RPDs for FDs and duplicate LFSMs should fall in the range of ± 30% for
samples fortified at or above their native concentration. Greater variability
may be observed when LFSMs are spiked near the MRL. At the MRL,
RPDs should fall in the range of 0 to 50% for samples fortified at or above
their native concentration. If the accuracy of any analyte falls outside the
designated range, and the laboratory performance for that analyte is shown
to be in control in the LFB, the recovery is judged to be matrix biased.
The result for that analyte in the unfortified sample is labeled
suspect/matrix to inform the data user that the results are suspect due to
matrix effects
9.10 RESOLUTION CHECK - The resolution of peaks in a calibration standard or
CCC near the mid level of calibration must be monitored in each analytical batch.
During the development of this method, the 2 ug/L level was monitored. Closely
eluting peaks that are not baseline resolved must have a resolution (Rs) of 1.0 or
greater using the equation (7)
R = -
s w +W
"0.5,1 ''0.5,2
531.2 -18
-------
where
tj and t2 = retention times of the first and second adjacent peaks
W0 5 a and W0 5 2 = widths of the adjacent peaks at half height.
Resolution must be monitored once for every 24-hour analytical batch and may be
monitored at any time during the 24-hour period. It is recommended that
resolution be checked prior to sample analysis, especially if the system in use has
a history of resolution problems. If a resolution check fails, all samples must be
reanalyzed after the problem is corrected, including the QC samples.
9.11 QUALITY CONTROL SAMPLE (QCS) - During the analysis of the IDC (Sect.
9.2), each time that new analyte standard solutions (Sects. 7.2.2.1 and 7.2.2.2) are
prepared, or at least quarterly, analyze a QCS from a source different from the
source of the calibration standards. The QCS is fortified into preserved reagent
water (Sect. 7.1.2) and analyzed as a CCC. The acceptance criteria is the same as
the CCC criteria at mid-level; the calculated amount for each analyte must be +
30% of the true value. If measured analyte concentrations are not of acceptable
accuracy, check the entire analytical procedure to locate and correct the problem
source.
10. CALIBRATION AND STANDARDIZATION
10.1 After initial calibration is successful, a Continuing Calibration Check (CCC) is
required at the beginning and end of each analysis batch, and after every tenth
sample (Sect. 10.3). Initial calibration should be repeated each time a major
instrument modification or maintenance is performed.
10.2 INITIAL CALIBRATION
10.2.1 Establish HPLC operating parameters equivalent to the suggested
conditions in Section 17, Table 1, including the postcolumn reactor. The
system is calibrated using the external standard technique. A fluorescence
detector was used with the excitation and emission wavelengths optimized
at 340 and 465 nm, respectively. Other HPLC conditions may be used as
long as all QC requirements in Section 9 are met.
10.2.2 Prepare a set of at least 5 calibration standards as described in Section
7.2.3. The lowest concentration calibration standard must be at or below
the MRL, which may depend on system sensitivity. It is recommended
that at least four of the Calibration Standards are at a concentration greater
than or equal to the MRL.
10.2.3 Generate a calibration curve for each analyte by plotting the peak response
531.2 -19
-------
(area is recommended) against analyte concentration. The instrument used
during method development yielded linear curves for the target analytes
over the concentration range of interest. However, data may be fit with
either a linear regression (response vs. concentration) or quadratic fit
(response vs. concentration). Alternately, if the ratio of the analyte peak
area to concentration (or response factor) is relatively constant (%RSD <
30%) an average response factor maybe used to calculate analyte
concentration.
10.2.4 When quantitated using the initial calibration curve, each calibration point,
except the lowest point, for each analyte should calculate to be 70-130% of
its true value. The lowest calibration point should calculate to be 50-150%
of its true value. Failure to meet this criteria may indicate future difficulty
in meeting CCC QC requirements during the analysis batch.
10.3 CONTINUING CALIBRATION CHECK (CCC) - Minimum daily calibration
verification is as follows. Verify the initial calibration at the beginning and end of
each group of analyses, and after every tenth sample during analyses. (In this
context, a "sample" is considered to be a field sample. LRBs, CCCs, LFSMs, and
LFSMDs are not counted as samples. The beginning CCC each day must be at or
below the MRL in order to verify instrument sensitivity prior to any analyses.
Subsequent CCCs should alternate between a medium and high concentration
standard.
10.3.1 Inject an aliquot of the appropriate concentration calibration solution and
analyze with the same conditions used during the initial calibration.
10.3.2 Calculate the concentration of each analyte and surrogate in the check
standard. The calculated amount for each analyte for medium and high
level CCCs must be ± 30% of the true value. The calculated amount for
the lowest calibration point for each analyte must be within ± 50% of the
true value. If these conditions do not exist, then all data for the problem
analyte must be considered invalid, and remedial action should be taken
which may require recalibration. Any field or QC samples that have been
analyzed since the last acceptable calibration verification should be
reanalyzed after adequate calibration has been restored.
11. PROCEDURE
11.1 SAMPLE PREPARATION
11.1.1 Samples are preserved, collected and stored as presented in Section 8. All
field and QC samples must contain the preservatives listed in Section
8.1.1. Measure a 25-mL aliquot of sample into a volumetric flask.
531.2 - 20
-------
Alternate volumes are allowed as long as they are determined using a glass
volumetric flask or pipette. When the volumes of SUR and analyte PDS
added to the field and QC samples are kept to a minimum, as described
below, no volume adjustment is necessary.
11.1.2 Add an aliquot of the SUR PDS (Sect. 7.2.1.2) to all samples and mix by
capping and inverting the sample. If the SUR PDS volume is less than 1%
of the total volume, no volume adjustment is necessary. For the
development of this method, the addition of 5.0 uL of a 10 ug/mL SUR
PDS to a 25-mL sample resulted in a SUR concentration of 2.0 ug/L.
11.1.3 If the sample is an LFSM, LFSMD or LFB/CCC, add the necessary
amount of analyte PDS. If the PDS volume is less than 1% of the total
volume, no volume adjustment is necessary. Cap and invert each sample
to ensure all components are properly mixed.
11.1.4 Filter samples prior to filling appropriate autosampler vials (Filters as
specified in Section 6.8 are recommended.).
11.2 ANALYSIS OF SAMPLES
11.2.1 Establish operating conditions as summarized in Table 1 of Section 17 for
the HPLC system, including the postcolumn reactor. HPLC conditions
and columns should be optimized prior to the initiation of the IDC.
Confirm that resolution of target peaks meets the requirements of Section
9.10. Retention times are given for illustration in Table 2. Specific
retention times will depend on the column and chromatographic conditions
chosen and may be modified by the user as long as adequate sensitivity
and peak resolution are achieved.
11.2.2 Load filled autosampler vials into the HPLC autosampler. Start the
injection sequence. For the development of this method, the injection
volume ranged between 200 and 1000 uL and depended on system
sensitivity.
11.2.3 Determine optimal excitation and emission wavelengths (if necessary).
Complete the IDC requirements described in Section 9.2.
11.2.4 Establish an appropriate retention time window for each target and
surrogate to identify them in the QC and field samples. This should be
based on measurements of actual retention time variation for each
compound in standard solutions analyzed on the HPLC over the course of
time. Plus or minus three times the standard deviation of the retention
time for each compound while establishing the initial calibration and
531.2-21
-------
completing the IDC can be used to calculate a suggested window size;
however, the experience of the analyst should weigh heavily on the
determination of the appropriate retention window size.
11.2.5 Calibrate the system by either the analysis of a calibration curve (Sect. 10.
2) or by confirming the initial calibration is still valid by analyzing a
continuing calibration check as described in Section 10.3. Begin
analyzing field and QC samples at their appropriate frequency by injecting
the same size aliquots under the same conditions used to analyze the initial
calibration. Confirm that the system meets the resolution check criteria as
described in Section 9.10 once for every 24 hour analytical batch.
11.2.6 The analyst must not extrapolate beyond the established calibration range.
If an analyte peak area exceeds the range of the initial calibration curve,
the extract may be diluted with preserved reagent water (Sect. 7.1.2).
Acceptable surrogate performance (Sect. 9.7) should be determined from
the undiluted sample extract. Any dilutions will also affect analyte MRL.
12. DATA ANALYSIS AND CALCULATION
12.1 Identify the method analytes in the sample chromatogram by comparing the
retention time of the suspect peak to the retention time of an analyte peak in a
calibration standard. Surrogate retention times should be confirmed to be within
acceptance limits (Sect. 11.2.3) even if no target compounds are detected.
Surrogate concentrations need to be calculated and determined to be within QC
limits (Sect. 9.7).
12.2 Calculate the analyte concentrations using the initial calibration curve generated
as described in Section 10.2. Quantitate only those values that fall between the
MRL and the highest calibration standard. Samples with target analyte responses
that exceed the highest standard require dilution and reanalysis (Sect. 11.2.5).
12.3 Adjust the calculated concentrations of the detected analytes to reflect the initial
sample volume and any dilutions performed.
12.4 Prior to reporting the data, the chromatogram should be reviewed for any incorrect
peak identification or poor integration.
12.5 Analyte concentrations are reported in ug/L (usually to two significant figures).
13. METHOD PERFORMANCE
13.1 PRECISION, ACCURACY, AND DETECTION LIMITS - Detection Limits are
presented in Section 17, Tables 3-5 and were calculated using the formula present
531.2 - 22
-------
in Section 9.2.4. Detection Limits were evaluated using two commercially
available postcolumn systems and two different fluorescence detectors. Single
laboratory precision and accuracy are presented for three water matrices: reagent
water (Table 6); chlorinated (finished) surface water (Table 7); and chlorinated
(finished) ground water (Table 8).
13.2 SAMPLE STORAGE STABILITY STUDIES - Chlorinated (finished) ground
water samples, fortified with method analytes at 2.0 ug/L, were preserved and
stored as required in Section 8. The average of triplicate analyses, conducted on
days 0, 2, 7, 14, and 28 days are presented in Section 17, Table 9. These data
document the 28-day sample holding time.
14. POLLUTION PREVENTION
14.1 For information about pollution prevention that may be applicable to laboratory
operations, consult "Less is Better: Laboratory Chemical Management for Waste
Reduction" available from the American Chemical Society's Department of
Government Relations and Science Policy, 1155 16th Street NW, Washington,
D.C., 20036.
15. WASTE MANAGEMENT
15.1 The analytical procedures described in this method generate relatively small
amounts of waste since only small amounts of reagents and solvents are used.
The matrices of concern are finished drinking water. However, the Agency
requires that laboratory waste management practices be conducted consistent with
all applicable rules and regulations, and that laboratories protect the air, water, and
land by minimizing and controlling all releases from fume hoods and bench
operations. Also, compliance is required with any sewage discharge permits and
regulations, particularly the hazardous waste identification rules and land disposal
restrictions. For further information on waste management, consult "The Waste
Management Manual for Laboratory Personnel" also available from the American
Chemical Society at the address in Section 14.1.
531.2 - 23
-------
16. REFERENCES
1. Glaser, J.A., D.L. Foerst, G.D. McKee, S.A. Quave, and W.L. Budde, "Trace Analyses for
Wastewaters," Environ. Sci. Technol. 1981, 15, 1426-1435.
2. Moye, H.A., Sherrer, S.J., and St. John, P.A., "Dynamic Labeling of Pesticides for High
Performance Liquid Chromatography: Detection of N-Methylcarbamates and o-
Phthalaldehyde," Anal Lett 1977, 10, 1049.
3. "OSHA Safety and Health Standards, General Industry," (29CRF1910). Occupational
Safety and Health Administration, OSHA 2206, (Revised, Jan. 1976).
4. "Carcinogens-Working with Carcinogens," Publication No. 77-206, Department of Health,
Education, and Welfare, Public Health Service, Center for Disease Control, National
Institute of Occupational Safety and Health, Atlanta, Georgia, August 1977.
5. "Safety In Academic Chemistry Laboratories," 3rd Edition, American Chemical Society
Publication, Committee on Chemical Safety, Washington, D.C., 1979.
6. ASTM Annual Book of Standards, Part II, Volume 11.01, D3370-82, "Standard Practice for
Sampling Water," American Society for Testing and Materials, Philadelphia, PA, 1986.
7. Snyder, L.R.; Kirkland, J.J.; Glajch, J.L. Practical HPLC Method Development, 2nd
ed.;Wiley: New York, 1997; pp 22-25.
531.2 - 24
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17. TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA*
TABLE 1. INSTRUMENT METHOD CONDITIONS
Time
(min)
initial
5.30
5.40
14.00
16.10
20.00
22.00
30.00
%Water
88.0
88.0
68.0
68.0
50.0
50.0
88.0
88.0
%Methanol
12.0
12.0
16.0
16.0
25.0
25.0
12.0
12.0
%Acetonitrile
0.0
0.0
16.0
16.0
25.0
25.0
0.0
0.0
Instrument Method Conditions
Column:
Postcolumn Reactor:
Fluorescence Detector:
HPLC:
Waters Carbamate 3.9 x 150 mm packed with 4.0 um Clg
stationary phase.
Reaction coil set at 80 °C, flow rate for Postcolumn Reagent #1
and #2 = 0.5 mL/min (each) for Waters unit, 0.3 mL/min for the
Pickering unit.
340 nm excitation, 465 nm emission with a 18 nm band width;
Gain = 100; Attn. = 16; Response = Standard; 16 uL flow cell.
A ternary gradient comprised of water, methanol, and acetonitrile
with a flow of 1.5 mL/min as shown in the table above.
* Instrumentation, when specified, does not constitute endorsement. Brand names are included for illustration only.
531.2 - 25
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TABLE 2. RETENTION TIME DATA*
Analyte
Aldicarb sulfoxide
Aldicarb sulfone
Oxamyl
Methomyl
3 -Hydroxycarbofuran
Aldicarb
Propoxur
Carbofuran
Carbaryl
1-Naphthol
Methiocarb
BDMC (SUR)
Retention
Time (min.)
4.36
5.07
5.74
6.53
9.82
11.5
14.3
14.8
17.0
18.6
21.8
22.3
STD
DEV
0.0092
0.0089
0.0095
0.0077
0.013
0.013
0.020
0.024
0.026
0.019
0.015
0.015
%RSD
0.21
0.17
0.17
0.12
0.13
0.11
0.14
0.16
0.16
0.10
0.07
0.07
*Retention time data is calculated from precision and accuracy data results presented in
Table 6 and the calibration curve used to quantitate the data. Retention times may differ
depending on the chromatographic conditions and columns used.
531.2 - 26
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TABLE 3. DETECTION LIMITS IN REAGENT WATER USING THE WATERS
POSTCOLUMN CARBAMATE SYSTEM AND THE WATERS MODEL
474 DETECTOR
Analyte
Aldicarb sulfoxide
Aldicarb sulfone
Oxamyl
Methomyl
3 -Hydroxycarbofuran
Aldicarb
Propoxur
Carbofuran
Carbaryl
1-Naphthol
Methiocarb
Fortification
Level (ug/L)
0.20
0.10
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
Detection
Limif (ug/L)
0.059
0.057
0.065
0.050
0.029
0.026
0.037
0.043
0.045
0.063
0.061
Signal to Noise
Ratio
8:1
3:1
10:1
10:1
18:1
9:1
6:1
9:1
13:1
10:1
11:1
"Detection Limits were determined by analyzing 7 replicates over 3 days using the
conditions outlined in Table 1 with a 1000-uL injection.
531.2 - 27
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TABLE 4. DETECTION LIMITS IN REAGENT WATER USING THE
PICKERING MODEL PCX5200 POSTCOLUMN SYSTEM AND THE
WATERS MODEL 474 DETECTOR
Analyte
Aldicarb sulfoxide
Aldicarb sulfone
Oxamyl
Methomyl
3 -Hydroxycarbofuran
Aldicarb
Propoxur
Carbofuran
Carbaryl
1-Naphthol
Methiocarb
Fortification
Level (ug/L)
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
Detection Limit"
(ug/L)
0.056
0.026
0.045
0.045
0.041
0.042
0.040
0.058
0.065
0.034
0.036
Signal to
Noise Ratio
13:1
15:1
9:1
11:1
11:1
7:1
11:1
7:1
22:1
9:1
5:1
"Detection Limits were determined by analyzing 7 replicates over 3 days using the
conditions outlined in Table 1 with a 250-uL injection.
531.2 - 28
-------
TABLE 5. DETECTION LIMITS IN REAGENT WATER USING THE WATERS
POSTCOLUMN CARBAMATE ANALYSIS SYSTEM AND THE
WATERS MODEL 2475 DETECTOR
Analyte
Aldicarb sulfoxide
Aldicarb sulfone
Oxamyl
Methomyl
3 -Hydroxycarbofuran
Aldicarb
Propoxur
Carbofuran
Carbaryl
1-Naphthol
Methiocarb
Fortification
Level (ug/L)
0.20
0.20
0.10
0.20
0.20
0.20
0.20
0.20
0.10
0.20
0.20
Detection
Limit311 (ug/L)
0.038
0.033
0.044
0.054
0.038
0.049
0.061
0.050
0.043
0.115
0.055
Signal to
Noise Ratio
14:1
9:1
4:1
24:1
7:1
12:1
10:1
12:1
9:1
3:1
5:1
"Detection Limits were determined by analyzing 7 replicates over 3 days using the
conditions outlined in Table 1 with a 200-uL injection.
bThese data were collected at American Water Works Service Company.
531.2 - 29
-------
TABLE 6. PRECISION AND ACCURACY OF LOW AND HIGH LEVEL
FORTIFIED REAGENT WATER3
Analyte
Aldicarb sulfoxide
Aldicarb sulfone
Oxamyl
Methomyl
3 -Hydroxycarbofuran
Aldicarb
Propoxur
Carbofuran
Carbaryl
1-Naphthol
Methiocarb
BDMC (SUR)b
Concentration = 0.20 ug/L
(n=7)
Mean %
Recovery
112
92
101
101
105
95
109
112
112
113
105
108
Relative
Standard
Deviation
(%)
6.2
9.5
8.6
6.5
6.8
7.4
5.9
6.7
7.0
12.6
5.9
4.3
Concentration = 10 ug/L
(n=7)
Mean %
Recovery
106
106
106
106
108
106
109
110
107
108
107
101
Relative
Standard
Deviation
(%)
1.8
2.6
2.2
2.9
1.2
1.3
2.0
2.2
2.1
3.1
1.5
2.3
"Data obtained using conditions in Table 1 using a 1000-uL injection.
bSurrogate concentration in all samples was 2.0 ug/L.
531.2-30
-------
TABLE 7. PRECISION AND ACCURACY OF LOW AND HIGH LEVEL
FORTIFIED CHLORINATED SURFACE WATER3
Analyte
Aldicarb sulfoxide
Aldicarb sulfone
Oxamyl
Methomyl
3 -Hydroxycarbofuran
Aldicarb
Propoxur
Carbofuran
Carbaryl
1-Naphthol
Methiocarb
BDMC (SUR)b
Concentration = 0.20 ug/L
(n=7)
Mean %
Recovery
113
104
107
110
128
123
128
140
112
113
104
108
Relative
Standard
Deviation
(%)
7.0
5.5
6.4
9.8
3.9
2.7
6.0
5.6
9.7
12.1
13.3
2.1
Concentration = 10 ug/L
(n=7)
Mean %
Recovery
104
106
104
104
107
105
106
105
106
101
107
96
Relative
Standard
Deviation
(%)
2.8
1.4
2.2
1.6
1.1
1.5
2.1
2.5
0.9
1.3
1.1
3.9
T)ata obtained using conditions in Table 1 using a 1000-uL injection.
Surrogate concentration in all samples was 2.0 ug/L.
531.2-31
-------
TABLE 8. PRECISION AND ACCURACY OF LOW AND HIGH LEVEL
FORTIFIED CHLORINATED GROUND WATER
Analyte
Aldicarb sulfoxide
Aldicarb sulfone
Oxamyl
Methomyl
3 -Hydroxycarbofuran
Aldicarb
Propoxur
Carbofuran
Carbaryl
1-Naphthol
Methiocarb
BDMC (SUR)b
Concentration = 0.20 ug/L
(n=7)
Mean %
Recovery
111
98
99
99
107
100
112
112
119
109
105
109
Relative
Standard
Deviation
(%)
7.3
9.2
8.4
10.2
3.0
6.3
6.1
4.1
5.1
8.2
3.9
2.0
Concentration = 10 ug/L
(n=7)
Mean %
Recovery
106
106
105
105
108
105
107
107
108
109
107
97
Relative
Standard
Deviation
(%)
1.1
0.9
1.2
1.4
0.4
0.6
0.8
1.6
1.3
1.2
1.0
4.3
j'Data obtained using conditions in Table 1 using a 1000-uL injection.
Surrogate concentration in all samples was 2.0 ug/L.
531.2 - 32
-------
TABLE 9. SAMPLE HOLDING TIME DATA FOR CHLORINATED GROUND
WATER SAMPLES FORTIFIED WITH METHOD ANALYTES AT 2.0
ug/L
Analyte
Aldicarb sulfoxide
Aldicarb sulfone
Oxamyl
Methomyl
3 -Hydroxycarbofuran
Aldicarb
Propoxur
Carbofuran
Carbaryl
1-Naphthol
Methiocarb
BDMC (SUR)b
% Recovery for Samples Fortified at 2.0 ug/L
DayO
94
93
97
96
96
95
95
96
96
96
95
99
Day 2
97
99
97
95
99
100
98
99
97
97
98
105
Day8
93
97
101
99
96
96
97
97
98
99
96
100
Day 15
93
98
103
98
95
92
98
97
94
95
94
99
Day 28
96
98
101
97
98
93
99
100
100
98
97
99
''Data obtained using conditions in Table 1 using a 500-uL injection.
Surrogate concentration in all samples was 2.0 ug/L.
531.2 - 33
-------
TABLE 10. INITIAL DEMONSTRATION OF CAPABILITY (IDC)
REQUIREMENTS
Method
Reference
Sect. 9.2.1
Sect. 9.2.2
Sect. 9.2.2
Sect. 9.2.3
Sect. 9.2.4
Requirement
Initial
Demonstration of
Low System
Background
Quality Control
Sample (QCS)
Initial
Demonstration of
Accuracy (IDA)
Initial
Demonstration of
Precision (IDP)
Detection Limit
Determination
Specification and
Frequency
Analyze LRB prior to any other
IDC steps.
Second source standard, used to
fortify preserved reagent water
(Sect. 7.1.2). Analyze as a CCC
after initial calibration but prior
to the analysis of the IDA
samples.
Analyze 4-7 replicate
LFBs/CCCs fortified at
midrange concentration.
Calculate average recovery for
replicates used in IDA.
Over a period of three days,
prepare a minimum of 7
replicate LFBs fortified at a
concentration estimated to be
near the Detection Limit.
Analyze the replicates through
all steps of the analysis.
Calculate the Detection Limit
using the equation in Sect. 9.2.4.
Acceptance Criteria
Demonstrate that all target
analytes are below 1/3 the
intended MRL or lowest
CAL standard, and that
possible interference from
reagents and glassware do
not prevent the identification
and quantitation of method
analytes.
Verifies initial calibration
accuracy, recovery must be
within + 30% of true value.
Mean recovery + 20% of true
value.
%RSD must be < 20%.
Data from Detection Limit
replicates are not required to
meet method precision and
accuracy criteria. If the
Detection Limit replicates are
fortified at a low enough
concentration, it is likely that
they will not meet precision
and accuracy criteria.
531.2 - 34
-------
TABLE 11. QUALITY CONTROL REQUIREMENTS (SUMMARY)
Method
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Sect. 8.3
and Sect. 8.4
Sample and
Extract Holding
Times
Properly preserved samples must
be shipped at or below 10 °C and
may be held in the lab at or
below 6 °C for 28 days. Samples
should not be frozen.
Do not report data for samples
that have not been properly
preserved or stored, or that
have exceeded their holding
time.
Sect. 9.4
Laboratory
Reagent Blank
(LRB)
Include a LRB with each analysis
batch (up to 20 samples).
Analyze prior to analyzing
samples and determine to be free
from interferences.
Demonstrate that all target
analytes are below 1/3 the
intended MRL, and that
possible interference from
reagents and glassware do not
prevent the identification and
quantitation of method
analytes.
If targets exceed 1/3 the MRL,
results for all subject analytes
in the analytical batch are
invalid.
Sect. 9.5 and
Sect. 10.3
Continuing
Calibration
Check (CCC)
Verify initial calibration by
analyzing a low level (at the
MRL or below) CCC prior to
analyzing samples. CCCs are
then injected after every 10
samples and after the last sample,
rotating concentrations to cover
the calibrated range of the
instrument.
Recovery for each analyte
must be 70-130% of the true
value for all but the lowest
level of calibration. The
lowest calibration level CCC
must be 50-150% of the true
value.
Results that are not bracketed
by acceptable CCCs are
invalid.
Sect. 9.7
Surrogate
Standards
The surrogate, 4-bromo-3,5-
dimithylphenyl
-methylcarbamate is added to all
field and QC samples.
Surrogate recovery must be
70-130% of the true value.
Samples that fail criteria, must
be reported as suspect due to
surrogate recovery.
531.2-35
-------
Method
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Sect. 9.8
Laboratory
Fortified
Sample Matrix
(LFSM) and
Laboratory
Fortified
Sample Matrix
Duplicate
(LFSMD)
With each analysis batch (Sect.
3.1), a minimum of one LFSM is
extracted and analyzed. A
duplicate LFSM, or LFSMD,
should be extracted when
occurrence of target analytes is
low. Field Duplicate analysis is
not required for analysis batches
containing an LFSMD.
Recoveries not within 70-
130% (50-150% at the MRL)
of the fortified amount may
indicate a matrix effect.
If an LFSMD is analyzed
instead of a Laboratory
Duplicate , target RPDs
should be within + 30%.
If all CCCs meet acceptance
criteria, and LFSM or
LFSMD do not, sample is
designated suspect/matrix.
Sect. 9.9
Field Duplicates
Analyze at least one duplicate
with each analysis batch (20
samples or less). A Laboratory
Fortified Sample Matrix
Duplicate maybe substituted for
a Field Duplicate when the
occurrence of target analytes is
low.
RPDs should be within +
30%.
If all CCCs meet acceptance
criteria, and Field Duplicates
do not, sample is designated
suspect/matrix.
Sect 9.10
Resolution
Check
Monitor once for every 24 hour
analysis period.
Closely eluting peaks that are
not baseline resolved must
have a resolution of > 1.0 or
greater using the equation in
Sect. 9.10
Sect. 9.11
Quality Control
Sample
Analyze at least quarterly or
when preparing new standards, as
well as during the IDC.
Same acceptance criteria as a
mid-level CCC.
Sect. 10.2
Initial
Calibration
Use external standard calibration
technique to generate a
calibration curve with at least
five standards.
When each calibration
standard is calculated using
the calibration curve, the
results should be 70-130% of
the true value for all but the
lowest standard. The lowest
standard should be 50-150%
of the true value. The lowest
CAL standard concentration
must be as low or lower than
the intended MRL.
531.2-36
-------
FIGURE 1. SAMPLE CHROMATOGRAM OF METHOD 531.2 ANALYTES
6.00-
5.00
4.00
3.00:
mV 2.00
1.00:
-1 .00 -
-2.00-
i i i | i i i | i i i | i i i | i i i | i i i | i
4.00 6.00 8.00 10.00 12.00 14.00 16.00
Minutes
Reagent water, sample preservatives added, fortified at 2.0 ug/L.
18.00
' I '
20.00
' I '
22.00
n\
24.00
n I
26.00
531.2-37
-------
531.2-38
------- |