EPA Document #: EPA/600/R-13/119
METHOD 540. DETERMINATION OF SELECTED ORGANIC CHEMICALS IN
DRINKING WATER BY SOLID PHASE EXTRACTION AND
LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY
(LC/MS/MS)
Version 1.0
September 2013
J.A. Shoemaker US EPA, Office of Research and Development, National Exposure
Research Laboratory
D.R. Tettenhorst US EPA, Office of Research and Development, National Exposure
Research Laboratory
NATIONAL EXPOSURE RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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METHOD 540
DETERMINATION OF SELECTED ORGANIC CHEMICALS IN
DRINKING WATER BY SOLID PHASE EXTRACTION AND LIQUID
CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY (LC/MS/MS)
1. SCOPE AND APPLICATION
1.1
1.2
1.3
This is a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for
determination of organic contaminants in drinking water. Accuracy and precision data
have been generated in reagent water, and finished ground and surface waters for
compounds listed in the table below.
Analyte
3 -Hydroxycarbofuran
Bensulide
Chlorpyrifos oxon
Disulfoton sulfoxide
Fenamiphos
Fenamiphos sulfone
Fenamiphos sulfoxide
Methomyl
Phorate sulfone
Phorate sulfoxide
Tebuconazole
Tebufenozide
Chemical Abstract Services
Registry Number (CASRN)
16655-82-6
741-58-2
5598-15-2
2497-07-6
22224-92-6
31972-44-8
31972-43-7
16752-77-5
2588-04-7
2588-03-6
107534-96-3
112410-23-8
The Minimum Reporting Level (MRL) is the lowest analyte concentration that meets
Data Quality Objectives (DQOs) that are developed based on the intended use of this
method. The single laboratory lowest concentration MRL (LCMRL) is the lowest true
concentration for which the future recovery is predicted to fall, with high confidence
(99%), between 50 and 150% recovery. Single laboratory LCMRLs for analytes in
this method range from 0.30-2.7 ng/L, and are listed in Table 5. The procedure used
to determine the LCMRL is described elsewhere.1
Laboratories using this method will not be required to determine the LCMRL for this
method, but will need to demonstrate that their laboratory MRL meets the require-
ments described in Section 9.2.4.
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1.4 Determining the Detection Limit (DL) for analytes in this method is optional (Sect.
9.2.6). 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.2
The DL is compound dependent and is dependent on extraction efficiency, sample
matrix, fortification concentration, and instrument performance. DLs for analytes in
this method range from 0.15-1.1 ng/L, and are listed in Table 5.
1.5 This method is intended for use by analysts skilled in solid phase extractions,
operation of LC/MS/MS instruments, and the interpretation of associated data.
1.6 METHOD FLEXIBILITY - In recognition of technological advances in analytical
systems and techniques, the laboratory is permitted to modify the evaporation
technique, separation technique, LC column, mobile phase composition, LC conditions
and MS and MS/MS conditions (Sect. 6.9, 9.1.1, 10.2, and 12.1). Changes may not be
made to sample collection and preservation (Sect. 8), sample extraction steps (Sect.
11), or to quality control requirements (Sect. 9). Method modifications should be
considered only to improve method performance. Modifications that are introduced in
the interest of reducing cost or sample processing time, but result in poorer method
performance, should not be used. Analytes must be adequately resolved
chromatographically in order to permit the mass spectrometer to dwell on a minimum
number of compounds eluting within a retention time window. Instrumental
sensitivity (or signal-to-noise) will decrease if too many compounds are permitted to
elute within a retention time window. In all cases where method modifications are
proposed, the analyst must perform the procedures outlined in the initial demonstration
of capability (IDC, Sect. 9.2), verify that all Quality Control (QC) acceptance criteria
(Sect. 9) are met, and that acceptable method performance can be verified in a real
sample matrix (Sect. 9.3.6).
NOTE: The above method flexibility section is intended as an abbreviated summation
of method flexibility. Sections 4-12 provide detailed information of specific
portions of the method that may be modified. If there is any perceived
conflict between the general method flexibility statement in Section 1.6 and
specific information in Sections 4-12, Sections 4-12 supersede Section 1.6.
2. SUMMARY OF METHOD
A 250-mL water sample is fortified with surrogates and passed through a solid phase
extraction (SPE) cartridge to extract the method analytes and surrogates. Compounds are
eluted from the solid phase with a small amount of methanol. The extract is concentrated by
evaporation with nitrogen in a heated water bath, and then adjusted to a 1-mL volume with
methanol after adding the internal standards. A 10-|iL injection is made into an LC equipped
with a Cig column that is interfaced to an MS/MS. Analytes are separated and identified by
comparing the acquired mass spectra and retention times to reference spectra and retention
times for calibration standards acquired under identical LC/MS/MS conditions. The
concentration of each analyte is determined by using the internal standard technique.
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Surrogate analytes are added to all Field and QC Samples to monitor the extraction efficiency
of method analytes.
3. DEFINITIONS
3.1 ANALYSIS BATCH - A set of samples that is analyzed on the same instrument
during a 24-hour period, including no more than 20 Field Samples, that begins and
ends with the analysis of the appropriate Continuing Calibration Check (CCC)
standards. Additional CCCs may be required depending on the length of the analysis
batch and/or the number of Field Samples.
3.2 CALTERATION STANDARD (CAL) - A solution prepared from the primary dilution
standard solution and/or stock standard solution, internal standard(s), and the
surrogate(s). The CAL solutions are used to calibrate the instrument response with
respect to analyte concentration.
3.3 COLLISIONALLY ACTIVATED DISSOCIATION (CAD) - The process of
converting the translational energy of the precursor ion into internal energy by
collisions with neutral gas molecules to bring about dissociation into product ions.
3.4 CONTINUING CALIBRATION CHECK (CCC) - A calibration standard containing
the method analytes, internal standard(s) and surrogate(s). The CCC is analyzed
periodically to verify the accuracy of the existing calibration for those analytes.
3.5 DETECTION LIMIT (DL) - 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. 9.2.6), and
accurate quantitation is not expected at this level.2
3.6 EXTRACTION BATCH - A set of up to 20 Field Samples (not including QC
samples) extracted together by the same person(s) during a work day using the same
lot of SPE devices, solvents, surrogate, internal standard and fortifying solutions.
Required QC samples include Laboratory Reagent Blank, Laboratory Fortified Blank,
Laboratory Fortified Sample Matrix, and either a Field Duplicate or Laboratory
Fortified Sample Matrix Duplicate.
3.7 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.
3.8 INTERNAL STANDARD (IS) - A pure chemical added to an extract or standard
solution in a known amount(s) and used to measure the relative response of other
method analytes and surrogates that are components of the same solution. The internal
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standard must be a chemical that is structurally similar to the method analytes, has no
potential to be present in water samples, and is not a method analyte.
3.9 LABORATORY FORTIFIED BLANK (LFB) - A volume 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.
3.10 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) - 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 sample extraction and the measured values in the LFSM corrected for
background concentrations.
3.11 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFSMD) - A
duplicate of the Field Sample used to prepare the LFSM. The LFSMD is fortified,
extracted, and analyzed identically to the LFSM. The LFSMD is used instead of the
Field Duplicate to assess method precision when the occurrence of method analytes is
low.
3.12 LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water or other
blank matrix that is treated exactly as a sample including exposure to all glassware,
equipment, solvents and reagents, sample preservatives, internal standards, 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.
3.13 LOWEST CONCENTRATION MINIMUM REPORTING LEVEL (LCMRL) - The
single laboratory LCMRL is the lowest true concentration for which a future recovery
is expected, with 99% confidence, to be between 50 and 150% recovery.1
3.14 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.
3.15 MINIMUM REPORTING LEVEL (MRL) - The minimum concentration that can be
reported as a quantitated value for a method analyte in a sample following analysis.
This defined concentration can be no lower than the concentration of the lowest
calibration standard for that analyte and can only be used if acceptable QC criteria for
this standard are met. A procedure for verifying a laboratory's MRL is provided in
Section 9.2.4.
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3.16 PRECURSOR ION - For the purpose of this method, the precursor ion is the
protonated molecule ([M+H]+) of the method analyte. In MS/MS, the precursor ion is
mass selected and fragmented by CAD to produce distinctive product ions of smaller
m/z.
3.17 PRIMARY DILUTION STANDARD (PDS) SOLUTION - A solution containing the
analytes prepared in the laboratory from stock standard solutions and diluted as needed
to prepare calibration solutions and other needed analyte solutions.
3.18 PRODUCT ION - For the purpose of this method, a product ion is one of the fragment
ions produced in MS/MS by CAD of the precursor ion.
3.19 QUALITY CONTROL SAMPLE (QC S) - A solution of method analytes of known
concentrations that is obtained from a source external to the laboratory and different
from the source of calibration standards. The second source stock standard solution is
used to fortify the QCS at a known concentration. The QCS is used to check
calibration standard integrity.
3.20 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.
3.21 SURROGATE ANALYTE (SUR) - A pure chemical which chemically resembles
method analytes and is extremely unlikely to be found in any sample. This chemical is
added to a sample aliquot in known amount(s) before processing and is measured with
the same procedures used to measure other method analytes. The purpose of the SUR
is to monitor method performance with each sample.
4. INTERFERENCES
4.1 All glassware must be meticulously cleaned. Wash glassware with detergent and tap
water, rinse with tap water, followed by a reagent water rinse. Non-volumetric
glassware can be heated in a muffle furnace at 400 °C for two hours or solvent rinsed.
Volumetric glassware should be solvent rinsed and not be heated in an oven above
120 °C.
4.2 Method interferences may be caused by contaminants in solvents, reagents (including
reagent water), sample bottles and caps, and other sample processing hardware that
lead to discrete artifacts and/or elevated baselines in chromatograms. All items must
be routinely demonstrated to be free from interferences (less than 1/3 the MRL for
each method analyte) under the conditions of the analysis by analyzing laboratory
reagent blanks as described in Section 9.3.1. Subtracting blank values from sample
results is not permitted.
4.3 Matrix interferences may be caused by contaminants that are co-extracted from the
sample. The extent of matrix interferences will vary considerably from source to
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source, depending upon the nature of the water. Humic and/or fulvic material can be
co-extracted during SPE and high levels can cause enhancement and/or suppression in
the electrospray ionization source or low recoveries on the SPE sorbent.3"4 Total
organic carbon (TOC) is a good indicator of humic content of the sample.
4.4 Relatively large quantities of the preservatives (Sect. 8.1.2) are added to sample
bottles. The potential exists for trace-level organic contaminants in these reagents.
Interferences from these sources should be monitored by analysis of laboratory reagent
blanks (Sect. 9.3.1), particularly when new lots of reagents are acquired.
4.5 SPE cartridges can be a source of interferences. Analysis of field and laboratory
reagent blanks can provide important information regarding the presence or absence of
such interferences. Brands and lots of SPE devices should be tested to ensure that
contamination does not preclude analyte identification and quantitation.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent 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 an awareness of OSHA regulations regarding safe handling of chemicals
used in this method. A reference file of MSDSs should be made available to all
personnel involved in the chemical analysis. Additional references to laboratory
safety are available.5"7
5.2 Pure standard materials and stock standard solutions of these method analytes should
be handled with suitable protection to skin and eyes, and care should be taken not to
breathe the vapors or ingest the materials.
6. EQUIPMENT AND SUPPLIES (Brand names and/or catalog numbers are included for
illustration only, and do not imply endorsement of the product.)
6.1 SAMPLE CONTAINERS - 250-mL amber glass bottles fitted with
polytetrafluoroethylene (PTFE)-lined screw caps.
6.2 CENTRIFUGE TUBES - 15-mL conical glass centrifuge tubes (Corning #8082-15) or
other glassware suitable for collection of the eluent from the solid phase after
extraction.
6.3 AUTOSAMPLER VIALS - Amber glass 2.0-mL autosampler vials (National
Scientific #C4000-2W or equivalent) with caps containing PTFE-faced septa (National
Scientific #C4000-53 or equivalent).
6.4 MICRO SYRINGES - Suggested sizes include 5, 10, 25, 50, 100, 250, 500 and
1000-jiL syringes.
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6.5 ANALYTICAL BALANCE - Capable of weighing to the nearest 0.0001 g.
6.6 SOLID PHASE EXTRACTION (SPE) APPARATUS FOR USING CARTRIDGES
6.6.1 SPE CARTRIDGES
6.6.1.1 Waters Oasis HLB, 150 mg, 6cc (Waters # 186003365) - divinylbenzene N-
vinylpyrrolidone copolymer.
6.6.1.2 J.T. Baker Speedisk Column H2O-Philic DVB, 200 mg, 6cc (Baker
# 8108-09) - modified divinylbenzene polymer.
6.6.2 VACUUM EXTRACTION MANIFOLD
6.6.2.1 Manual Extraction - A manual vacuum manifold with Visiprep™ large
volume sampler (Supelco #57030 and #57275 or equivalent) for cartridge
extractions.
6.6.2.2 Automated Extraction - An automatic/robotic sample preparation system,
designed for use with SPE cartridges, may be used if all QC requirements
discussed in Section 9 are met. All sorbent washing, conditioning, sample
loading, rinsing, drying and elution steps must be performed as closely as
possible to the manual procedure. Solvents used for washing, conditioning,
and sample elution must be the same as those used in the manual procedure:
however, the amount used may be increased as necessary to achieve the
required data quality. Solvent amounts may not be decreased. Sorbent
drying times prior to elution may be modified to achieve the required data
quality. Caution should be exercised when increasing solvent volumes.
Increased extract volume will likely necessitate the need for extended
evaporation times which may compromise data quality. Caution should also
be exercised when modifying sorbent drying times. Excessive drying may
cause losses due to analyte volatility, and excessive contact with room air
may oxidize some method analytes.
6.6.3 SAMPLE DELIVERY SYSTEM - Use of a transfer tube system (Supelco
"Visiprep," #57275 or equivalent), which transfers the sample directly from the
sample container to the SPE cartridge is recommended.
6.7 EXTRACT CONCENTRATION SYSTEM - Extracts are concentrated by
evaporation with nitrogen using a water bath set no higher than 40 °C (Meyer N-Evap,
Model 111, Organomation Associates, Inc. or equivalent).
6.8 LABORATORY OR ASPIRATOR VACUUM SYSTEM - Sufficient capacity to
maintain a vacuum of approximately 10 to 15 inches of mercury for extracting
cartridges.
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6.9 LIQUID CHROMATOGRAPHY (LC)/TANDEM MASS SPECTROMETER
(MS/MS) WITH DATA SYSTEM
6.9.1 LC SYSTEM - Instrument capable of reproducibly injecting up to 10-|aL aliquots,
and performing binary linear gradients at a constant flow rate near the flow rate
used for development of this method (0.3 mL/min). Usage of a cooled
autosampler compartment and a column heater is optional. Method performance
data were collected using an autosampler thermostated to 4 °C during analyses.
6.9.2 TANDEM MASS SPECTROMETER - The mass spectrometer must be capable
of positive ion electrospray ionization (ESI) near the suggested LC flow rate of
0.3 mL/min. The system must be capable of performing MS/MS to produce
unique product ions (Sect. 3.18) for method analytes within specified retention
time segments. A minimum of 10 scans across the chromatographic peak is
required to ensure adequate precision. Data demonstrated in Section 17 were
collected using a triple quadrupole mass spectrometer.
6.9.3 DATA SYSTEM - An interfaced data system is required to acquire, store, reduce,
and output mass spectral data. The computer software should have the capability
of processing stored LC/MS/MS data by recognizing an LC peak within any given
retention time window. The software must allow integration of the ion abundance
of any specific ion within specified time or scan number limits. The software
must be able to calculate relative response factors, construct linear regressions or
quadratic calibration curves, and calculate analyte concentrations.
6.9.4 ANALYTICAL COLUMN - An LC Cig column (2.1 x 100 mm) packed with
5 jam Cig solid phase particles (Restek Ultra Aqueous #9178512) was used. Any
equivalent column that provides adequate resolution, peak shape, capacity,
accuracy, and precision (Sect. 9) may be used.
7. REAGENTS AND STANDARDS
7.1 GASES, 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 may be 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.
7.1.1 REAGENT WATER - Purified water which does not contain any measurable
quantities of any method analytes or interfering compounds greater than 1/3 the
MRL for each method analyte of interest.
7.1.2 METHANOL (CH3OH, CAS#: 67-56-1) - High purity, demonstrated to be free of
analytes and interferences (Fisher Optima LC/MS grade or equivalent).
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7.1.3 ACETONITRILE (CH3CN, CAS#: 75-05-8) - High purity, demonstrated to be
free of analytes and interferences (Tedia Absolv grade or equivalent).
7.1.4 ACETONE [(CH3)2CO, CAS#: 67-64-1] - High purity, demonstrated to be free of
analytes and interferences (Tedia Absolv grade or equivalent).
7.1.5 FORMIC ACID (CH2O2; CAS# 64-18-6) - High purity, demonstrated to be free
of analytes and interferences (Sigma-Aldrich ACS grade or equivalent).
7.1.6 AMMONIUM FORMATE (CH5O2N, CAS# 540-69-2) - High purity,
demonstrated to be free of analytes and interferences (Fluka LC/MS grade or
equivalent).
7.1.7 10 mM FORMATE BUFFER - To prepare 1 L, add 0.63 g ammonium formate
and 0.5 mL formic acid to 1 L of reagent water. This solution is prone to
volatility losses and should be replaced at least every 48 hours.
7.1.8 SAMPLE PRESERVATION REAGENTS - The following preservatives are
solids at room temperature and may be added to the sample bottle before
shipment to the field.
7.1.8.1 TRIZMA PRESET CRYSTALS, pH 7.0 (Sigma-Aldrich #T-7193 or
equivalent) - Reagent grade. A premixed blend of Tris [Tris(hydroxy-
methyl)aminomethane] and Tris HCL [Tris(hydroxymethyl)aminomethane
hydrochloride]. Alternatively, a mix of the two components with a weight
ratio of 15.5/1 Tris HCL/Tris may be used. These blends are targeted to
produce a pH near 7.0 at 25 °C in reagent water. Trizma functions as a
buffer (Sect. 8.1.2).
7.1.8.2 L-ASCORBIC ACID (CAS# 50-81-7) - Ascorbic acid reduces free chlorine
at the time of sample collection (Sigma-Aldrich #255564 or equivalent).8
7.1.8.3 2-CHLOROACETAMIDE (CAS# 79-07-2) - Inhibits microbial growth and
analyte degradation (Sigma-Aldrich #C0267 or equivalent).8
7.1.9 NITROGEN - Aids in aerosol generation of the ESI liquid spray and is used as
collision gas in some MS/MS instruments. Nitrogen used should meet or exceed
instrument manufacturer's specifications.
7.1.10 ARGON - Used as collision gas during MS/MS experiments. Argon should meet
or exceed instrument manufacturer's specifications. Nitrogen gas may be used as
collision gas provided sufficient sensitivity (product ion formation) is achieved.
STANDARD SOLUTIONS - When the purity of a compound is assayed to be 96% or
greater, the weight can be used without correction to calculate concentration of the
stock standard. The suggested concentrations are a description of concentrations used
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during method development, and may be modified to conform to instrument
sensitivity. Standards for sample fortification generally should be prepared in the
smallest volume that can be accurately measured to minimize addition of excess
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 their standards need to be replaced.
7.2.1 INTERNAL (IS) STOCK STANDARD SOLUTIONS - This method uses three
IS compounds: carbofuran-13Ce, bensulide-t/i4 and phorate-^io. These IS
compounds were carefully chosen during method development because they
encompass some of the functional groups of method analytes. Although alternate
IS compounds may be used provided they are isotopically labeled compounds
with similar functional groups as method analytes, the analyst must have
documented reasons for using alternate IS compounds. Alternate IS compounds
must meet the QC requirements in Section 9.3.4.
7.2.1.1 IS STOCK STANDARD SOLUTIONS - These IS stocks can be obtained
as individual certified stock standard solutions or neat materials. During
development of this method, commercially obtained 100 |ig/mL stock
standard solutions of carbofuran-13Ce in 1,4-dioxane (Cambridge Isotopes #
CLM-1911-1.2) and phorate-dio in acetone (Crescent Chemical #
XA16080100AC) were used. Bensulide-t/i4 was prepared from neat
material (Cambridge Isotopes #DLM-7152) at 1000 |ig/mL in acetonitrile.
IS stock standard solutions were stable for at least six months when stored at
-5 °C or less in amber glass screw cap vials.
7.2.1.2 INTERNAL STANDARD PRIMARY DILUTION STANDARD (IS PDS)
(0.40 ng/|iL) - Prepare, or purchase commercially, the IS PDS at a
suggested concentration of 0.40 ng/jiL using the three isotopically labeled
chemicals in the table below. If prepared from individual stock standard
solutions (Sect. 7.2.1.1), the table below can be used as a guideline for
preparing the IS PDS although concentrations may need to be adjusted for
instrument sensitivity. The IS PDS used in these studies was prepared in
acetonitrile. The IS PDS has been shown to be stable for at least six months
when stored at 6 °C or less in amber glass screw cap vials. Ten jiL of this
0.40 ng/|iL IS PDS was used to fortify the final 1-mL extracts (Sect. 11.4).
This will yield a concentration of 4 pg/|iL of each IS in 1-mL extracts.
IS
carbofuran-13Ce
bensulide-t/i4
phorate-t/io
Cone, of
IS Stock
(ug/mL)
100
1000
100
Vol. Of
IS Stock
(uL)
20
2.0
20
Final Vol.
of IS PDS
(mL)
5.0
5.0
5.0
Final Cone.
of IS PDS
(ng/uL)
0.40
0.40
0.40
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7.2.2 SURROGATE (SUR) ANALYTE STANDARD SOLUTIONS - The two SUR(s)
for this method are methomyl-13C2,15N and tebuconazole-de- These isotopically
labeled SUR standards were carefully chosen during method development
because they encompass some of the functional groups, as well as the water
solubility range of method analytes. Although alternate SUR standards may be
used provided they are isotopically labeled compounds with similar functional
groups as method analytes, the analyst must have documented reasons for using
alternate SUR standards. Alternate SUR standards chosen must still span the
water solubility range of method analytes. In addition, alternate SUR standards
must meet the QC requirements in Section 9.3.5.
7.2.2.1 SUR STOCK STANDARD SOLUTIONS - These SUR stocks can be
obtained as individual certified stock standard solutions. During
development of this method, commercially obtained 100 |ig/mL stock
standard solutions of methomyl-13C2,15Nin methanol (Cambridge Isotopes
#CNLM-7148-1.2) and tebuconazole-^4 in acetone (Dr. Ehrenstorfer GmbH
#XA17178710AC) were used. SUR stock standard solutions were stable for
at least one year when stored at -5 °C or less in amber glass screw cap vials.
7.2.2.2 SURROGATE PRIMARY DILUTION STANDARD (SUR PDS)
(0.40 ng/|iL) - Prepare, or purchase commercially, the SUR PDS at a
suggested concentration of 0.40 ng/|iL. If prepared from individual stock
standard solutions (Sect. 7.2.2.1), the table below can be used as a guideline
for preparing the SUR PDS. The SUR PDS used in these studies was
prepared in acetonitrile. This solution is used to fortify all QC and Field
Samples. The PDS has been shown to be stable for at least six months when
stored at 6 °C or less. Use 10 |iL of this 0.40 ng/jiL SUR PDS to fortify the
250 mL aqueous QC and Field Samples prior to extraction (Sect. 11.2.2).
This will yield a concentration of 16 ng/L of each SUR in aqueous QC and
Field Samples.
SUR
methomyl-13C2, 15N
tebuconazole-de
Cone. Of
SUR Stock
(ug/mL)
100
100
Vol. of
SUR Stock
GiL)
20
20
Final Vol. of
SUR PDS
(mL)
5.0
5.0
Final Cone.
of SUR PDS
(ng/uL)
0.40
0.40
7.2.3 ANALYTE STANDARD SOLUTIONS - Analyte standards may be purchased
commercially as ampulized solutions or prepared from neat materials (see Table 3
for analyte sources used during method development).
7.2.3.1 ANALYTE STOCK STANDARD SOLUTION (1000 |ig/mL) - If
preparing from neat material, accurately weigh approximately 5 mg of pure
material to the nearest 0.1 mg and dilute to 5 mL with acetonitrile or
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methanol for a final concentration of 1000 ug/mL. Repeat for each method
analyte prepared from neat material. Alternatively, purchase commercially
available individual stock standards of the analytes, preferably in methanol or
acetonitrile, if available. For development of this method, commercially
available stock standards of 1000 ug/mL were purchased except for phorate
sulfoxide, tebufenozide, disulfoton sulfoxide and chlorpyrifos oxon which
were purchased as neat materials. These stock standards were stable for at
least six months when stored at -5 °C or less in amber glass screw cap vials.
7.2.3.2 ANALYTE PRIMARY DILUTION STANDARD (PDS) SOLUTION
(0.16-0.40 ng/|aL) - The analyte PDS contains all, or a portion, of method
analytes at various concentrations in acetonitrile. ESI and MS/MS response
varies by compound; therefore, a mix of concentrations may be needed in
the analyte PDS. During method development, analyte PDS solutions were
prepared such that approximately the same instrument response was
obtained for all analytes. The analyte PDS was prepared in acetonitrile at
concentrations of 0.4 ng/|aL, except for fenamiphos, tebufenozide and
tebuconazole at 0.16 ng/|aL each. The analyte PDS is prepared by dilution
of the combined Analyte Stock Standard Solutions (Sect.7.2.3.1) and is used
to prepare CAL standards, and fortify LFBs, LFSMs, LFSMDs and FDs
with the method analytes. The analyte PDS has been shown to be stable for
six months when stored at 6 °C or less in amber glass screw cap vials.
7.2.4 CALIBRATION STANDARDS (CAL) - Prepare a series of at least five
concentrations of calibration solutions in methanol (see note below), from
dilutions of the analyte PDS (Sect 7.2.3.2). The suggested concentrations in this
paragraph are a description of the concentrations used during method
development, and may be modified to conform with instrument sensitivity.
Concentrations ranging from 0.16-6.4 ug/L are suggested for fenamiphos,
tebufenozide and tebuconazole, and 0.40-16 ug/L are suggested for the remaining
analytes. Larger concentration ranges will require more calibration points. The
IS and SUR are added to CAL standards at a constant concentration. During
method development, the concentrations of the SUR(s) were 0.40 ng/|aL in the
standard (16 ng/L in the aqueous sample) and the IS(s) concentrations were
4 pg/|-iL. The lowest concentration CAL standard must be at or below the MRL,
which may depend on system sensitivity. CAL standards may also be used as
CCCs (Sect. 9.3.2). During method development, CAL standards were shown to
be stable for two weeks when stored at 6 °C or less. Longer storage times are
acceptable provided appropriate QC measures are documented demonstrating the
CAL stability.
Note: Acetonitrile was not used as the solvent for calibration standards
because injection of calibration standards prepared in acetonitrile
distorted peak shapes for early eluting analytes, such as methomyl and
3-hydroxycarbofuran.
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8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
8.1 SAMPLE BOTTLE PREPARATION
8.1.1 Samples must be collected in 250-mL amber glass bottles fitted with teflon-lined
screw caps.
8.1.2 Preservation reagents, listed in the table below, are added to each sample bottle as
a solid prior to shipment to the field (or prior to sample collection).
Compound
Trizma
2-Chloroacetamide
Ascorbic acid
Amount
7.75 g/L
2g/L
100 mg/L
Purpose
buffering reagent
antimicrobial
dechlorinating agent
8.2 SAMPLE COLLECTION
8.2.1 Open the tap and allow the system to flush until the water temperature has
stabilized (approximately 3 to 5 min). Collect samples from the flowing system.
8.2.2 Fill sample bottles, taking care not to flush out the sample preservation reagents.
Samples do not need to be collected headspace free.
8.2.3 After collecting the sample, cap the bottle and agitate by hand until preservative is
dissolved. Note that 2-chloroacetamide is slow to dissolve especially in cold
water. Keep the sample sealed from time of collection until extraction.
8.3 SAMPLE SHIPMENT AND STORAGE - Samples must be chilled during shipment
and must not exceed 10 °C during the first 48 hours after collection. Sample
temperature must be confirmed to be at or below 10 °C when samples are received at
the laboratory. Samples stored in the lab must be held at or below 6 °C until
extraction, but should not be frozen.
NOTE: Samples that are significantly above 10° C, at the time of collection, may need
to be iced or refrigerated for a period of time, in order to chill them prior to
shipping. This will allow them to be shipped with sufficient ice to meet the
above requirements.
8.4 SAMPLE AND EXTRACT HOLDING TIMES - Water samples should be extracted
as soon as possible after collection but must be extracted within 28 days of collection,
except for chlorpyrifos oxon which must be extracted within 7 days. Extracts must be
stored at < 6 °C and analyzed within 28 days after extraction.
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9. QUALITY CONTROL
9.1 QC requirements include the Initial Demonstration of Capability (IDC) and ongoing
QC requirements that must be met when preparing and analyzing Field Samples. This
section describes QC parameters, their required frequencies, and performance criteria
that must be met in order to meet EPA quality objectives. QC criteria discussed in the
following sections are summarized in Tables 14 and 15. These QC requirements are
considered the minimum acceptable QC criteria. Laboratories are encouraged to
institute additional QC practices to meet their specific needs.
9.1.1 METHOD MODIFICATIONS - The analyst is permitted to modify LC columns,
LC conditions, evaporation techniques, internal standards or surrogate standards,
and MS and MS/MS conditions. Each time such method modifications are made,
the analyst must repeat the procedures of the IDC. Modifications to LC
conditions should still minimize co-elution of method analytes to reduce the
probability of suppression/enhancement effects.
9.2 INITIAL DEMONSTRATION OF CAPABILITY (IDC) - The IDC must be
successfully performed prior to analyzing any Field Samples. Prior to conducting the
IDC, the analyst must first generate an acceptable Initial Calibration following the
procedure outlined in Section 10.2.
9.2.1 INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND - Any time
a new lot of SPE cartridges, solvents, centrifuge tubes, disposable pipets, and
autosampler vials are used, it must be demonstrated that an LRB is reasonably
free of contamination and that criteria in Section 9.3.1 are met. If an automated
extraction system is used, an LRB should be extracted on each port to ensure that
all valves and tubing are free from potential contamination.
9.2.2 INITIAL DEMONSTRATION OF PRECISION (IDP) - Prepare, extract, and
analyze four to seven replicate LFBs fortified near the midrange of the initial
calibration curve according to the procedure described in Section 11. Sample
preservatives as described in Section 8.1.2 must be added to these samples. The
relative standard deviation (RSD) of the results of replicate analyses must be less
than 20%.
9.2.3 INITIAL DEMONSTRATION OF ACCURACY (IDA) - Using the same set of
replicate data generated for Section 9.2.2, calculate average recovery. The
average recovery of replicate values must be within ± 30% of the true value.
9.2.4 MINIMUM REPORTING LEVEL (MRL) CONFIRMATION - Establish a target
concentration for the MRL based on the intended use of the method. The MRL
may be established by a laboratory for their specific purpose or may be set by a
regulatory agency. Establish an Initial Calibration following the procedure
outlined in Section 10.2. The lowest CAL standard used to establish Initial
Calibration (as well as the low-level CCC, Section 10.3) must be at or below the
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concentration of the MRL. Establishing the MRL concentration too low may
cause repeated failure of ongoing QC requirements. Confirm the MRL following
the procedure outlined below.
9.2.4.1 Fortify, extract, and analyze seven replicate LFBs at the proposed MRL
concentration. These LFBs must contain all method preservatives described
in Section 8.1.2. Calculate the mean measured concentration (Mean) and
standard deviation for these replicates. Determine the Half Range for the
prediction interval of results (HRpiR) using the equation below
HRPIR = 3.963s
where
s = standard deviation
3.963 = a constant value for seven replicates.1
9.2.4.2 Confirm that the upper and lower limits for the Prediction Interval of Result
(PIR = Mean j_ HRpiR) meet the upper and lower recovery limits as shown
below
The Upper PIR Limit must be < 150% recovery.
Mean + HRP1R
FortifiedConcentration
The Lower PIR Limit must be > 50% recovery.
Mean-HRP1R
FortifiedConcentration
9.2.4.3 The MRL is validated if both the Upper and Lower PIR Limits meet the
criteria described above (Sect. 9.2.4.2). If these criteria are not met, the
MRL has been set too low and must be determined again at a higher
concentration.
9.2.5 CALIBRATION CONFIRMATION - Analyze a QCS as described in Section
9.3.8 to confirm the accuracy of the standards/calibration curve.
9.2.6 DETECTION LIMIT DETERMINATION (optional) - While DL determination
is not a specific requirement of this method, it may be required by various
regulatory bodies associated with compliance monitoring. It is the responsibility
of the laboratory to determine ifDL determination is required based upon the
intended use of the data.
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Replicate analyses for this procedure should be done over at least three days (i.e.,
both the sample extraction and the LC/MS/MS analyses should be done over at
least three days). Prepare at least seven replicate LFBs at a concentration
estimated to be near the DL. This concentration may be estimated by selecting a
concentration at 2-5 times the noise level. DLs in Table 5 were calculated from
LFBs fortified at various concentrations as indicated in the table. Appropriate
fortification concentrations will be dependent upon the sensitivity of the
LC/MS/MS system used. All preservation reagents listed in Section 8.1.2 must
also be added to these samples. Analyze the seven replicates through all steps of
Section 1 1 .
NOTE: If an MRL confirmation data set meets these requirements, a DL may be
calculated from the MRL confirmation data, and no additional analyses
are necessary.
Calculate the DL using the following equation
— J A, t
/ i i /-. /-\/-\\
(w-l,l-a=0.99)
where
s = standard deviation of replicate analyses
t («-i, i-a=o.99) = Student's t value for the 99% confidence
level with n-1 degrees of freedom
n = number of replicates.
NOTE: Do not subtract blank values when performing DL calculations.
9.3 ONGOING QC REQUIREMENTS - This section summarizes ongoing QC criteria
that must be followed when processing and analyzing Field Samples.
9.3.1 LABORATORY REAGENT BLANK (LRB) - An LRB is required with each
extraction batch (Sect. 3.6) to confirm that potential background contaminants are
not interfering with identification or quantitation of method analytes. If more
than 20 Field Samples are included in a batch, analyze an LRB for every 20
samples. If the LRB produces a peak within the retention time window of any
analyte that would prevent 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 other contaminants that inter-
fere with the measurement of method analytes must be below 1/3 of the MRL.
Blank contamination is estimated by extrapolation, if the concentration is below
the lowest CAL standard. This extrapolation procedure is not allowed for sample
results as it may not meet data quality objectives. If method 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 extraction
batch.
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9.3.2 CONTINUING CALIBRATION CHECK (CCC) - CCC standards are analyzed
at the beginning of each analysis batch, after every 10 Field Samples, and at the
end of the analysis batch. See Section 10.3 for concentration requirements and
acceptance criteria.
9.3.3 LABORATORY FORTIFIED BLANK (LFB) - An LFB is required with each
extraction batch (Sect. 3.6). The fortified concentration of the LFB must be
rotated between low, medium, and high concentrations from batch to batch. The
low concentration LFB must be as near as practical to, but no more than two
times, the MRL. Similarly, the high concentration LFB should be near the high
end of the calibration range established during the initial calibration (Sect. 10.2).
Results of low-level LFB analyses must be 50-150% of the true value. Results of
medium and high-level LFB analyses must be 70-130% of the true value. If LFB
results do not meet these criteria for method analytes, then all data for the
problem analyte(s) must be considered invalid for all samples in the extraction
batch.
9.3.4 INTERNAL STANDARDS (IS) - The analyst must monitor peak areas of the
IS(s) in all injections during each analysis day. Internal standard responses (as
indicated by peak areas) for any chromatographic run must not deviate by more
than ± 50% from average areas measured during the initial calibration for the
internal standards. If IS areas in a chromatographic run do not meet these criteria,
inject a second aliquot of that standard or extract.
9.3.4.1 If the reinjected aliquot produces an acceptable IS response, report results
for that aliquot.
9.3.4.2 If the reinjected extract fails again, the analyst should check the calibration
by reanalyzing the most recently acceptable CAL standard. If the CAL
standard fails the criteria of Section 10.3, recalibration is in order per
Section 10.2. If the CAL standard is acceptable, extraction of the sample
may need to be repeated provided the sample is still within the holding time.
Otherwise, report results obtained from the reinjected extract, but annotate
as suspect. Alternatively, collect a new sample and re-analyze.
9.3.5 SURROGATE RECOVERY - The SUR standard is fortified into all samples,
CCCs, LRBs, LFBs, LFSMs, LFSMDs, and FD prior to extraction. It is also
added to CAL standards. The SUR is a means of assessing method performance
from extraction to final chromatographic measurement. Calculate the recovery
(%R) for the SUR using the following equation
( A\
%R= — xlOO
UJ
where
A = calculated SUR concentration for the QC or Field Sample
540-18
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B = fortified concentration of the SUR.
9.3.5.1 SUR recovery must be in the range of 70-130%. When SUR 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. Correct the problem and
reanalyze the extract.
9.3.5.2 If the extract reanalysis meets the SUR recovery criterion, report only data
for the reanalyzed extract.
9.3.5.3 If the extract reanalysis fails the 70-130% recovery criterion, the analyst
should check the calibration by injecting the last CAL standard that passed.
If the CAL standard fails the criteria of Section 10.3, recalibration is in
order per Section 10.2. If the CAL standard is acceptable, extraction of the
sample should be repeated provided the sample is still within the holding
time. If the re-extracted sample also fails the recovery criterion, report all
data for that sample as suspect/SUR recovery to inform the data user that the
results are suspect due to SUR recovery. Alternatively, collect a new
sample and re-analyze.
9.3.6 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) - Analysis of an
LFSM is required in each extraction 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 (FD) (Sect. 9.3.7);
however, infrequent occurrence of method analytes would hinder this assessment.
If the occurrence of method analytes in samples is infrequent, or if historical
trends are unavailable, a second LFSM, or LFSMD, must be prepared, extracted,
and analyzed from a duplicate of the Field Sample. Extraction batches that
contain LFSMDs will not require extraction of a FD. If a variety of different
sample matrices are analyzed regularly, for example, drinking water from ground
water and surface water sources, method performance should be established for
each. Over time, LFSM data should be documented by the laboratory for all
routine sample sources.
9.3.6.1 Within each extraction batch (Sect. 3.6), a minimum of one Field Sample is
fortified as an LFSM for every 20 Field Samples analyzed. The LFSM is
prepared by spiking a sample with an appropriate amount of the Analyte
PDS (Sect. 7.2.3.2). Select a spiking concentration that is greater than or
equal to the matrix background concentration, if known. Use historical data
and rotate through low, mid and high concentrations when selecting a
fortifying concentration.
9.3.6.2 Calculate percent recovery (%R) for each analyte using the equation
540-19
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c
where
A = measured concentration in the fortified sample
B = measured concentration in the unfortified sample
C = fortification concentration.
9.3.6.3 Analyte recoveries may exhibit matrix bias. For samples fortified at or
above their native concentration, recoveries should range between 70-130%,
except for low-level fortification near or at the MRL (within a factor of
two-times the MRL concentration) where 50-150% recoveries are
acceptable. If the accuracy of any analyte falls outside the designated range,
and laboratory performance for that analyte is shown to be in control in
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.
9.3.7 FIELD DUPLICATE OR LABORATORY FORTIFIED SAMPLE MATRIX
DUPLICATE (FD or LFSMD) - Within each extraction batch (not to exceed 20
Field Samples, Sect. 3.6), a minimum of one FD or LFSMD must be analyzed.
Duplicates check the precision associated with sample collection, preservation,
storage, and laboratory procedures. If method analytes are not routinely observed
in Field Samples, an LFSMD should be analyzed rather than an FD.
9.3.7.1 Calculate relative percent difference (RPD) for duplicate measurements
(FD1 and FD2) using the equation
FDI-FD2
RPD = -/ ,— xlOO
(FDl + FD2)/2
9.3.7.2 RPDs for FDs should be < 30%. Greater variability may be observed when
the matrix is fortified at analyte concentrations at or near the MRL (within a
factor of two times the MRL concentration). At these concentrations, FDs
should have RPDs that are < 50%. If the RPD of any analyte falls outside
the designated range, and laboratory performance for that analyte is shown
to be in control in the CCC, 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.
9.3.7.3 If an LFSMD is analyzed instead of a FD, calculate the relative percent
difference (RPD) for duplicate LFSMs (LFSM and LFSMD) using the
equation
\LFSM-LFSMD\
^ ^
(LFSM+LFSMD} 12
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9.3.7.4 RPDs for duplicate LFSMs should be < 30% for samples fortified at or
above their native concentration. Greater variability may be observed when
the matrix is fortified at analyte concentrations at or near the MRL (within a
factor of two times the MRL concentration). LFSMs fortified at these
concentrations should have RPDs that are < 50% for samples fortified at or
above their native concentration. If the RPD of any analyte falls outside the
designated range, and laboratory performance for that analyte is shown to be
in control in the CCC, 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.
9.3.8 QUALITY CONTROL SAMPLES (QCS) - As part of the IDC (Sect. 9.2), each
time a new Analyte PDS (Sect. 7.2.3.2) is prepared, and at least quarterly, analyze
a QCS sample from a source different from the source of the CAL standards. If a
second vendor is not available, then a different lot of the standard should be used.
The QCS should be prepared and analyzed just like a CCC. Fortify the QCS near
the midpoint of the calibration range. Acceptance criteria for the QCS are identical
to mid- and high-level CCCs; 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.
If the discrepancy is not resolved, one of the standard materials may be degraded
or otherwise compromised and a third standard must be obtained.
10. CALIBRATION AND STANDARDIZATION
10.1 Demonstration and documentation of acceptable initial calibration is required before
any samples are analyzed. The MS tune check and initial calibration must be repeated
each time a major instrument modification is made, or maintenance is performed.
10.2 INITIAL CALIBRATION
10.2.1 ESI-MS/MS TUNE
10.2.1.1 Calibrate the mass scale of the MS with the calibration compounds and
procedures prescribed by the manufacturer.
10.2.1.2 Optimize the [M+H]+ for each method analyte by infusing approximately
0.5-1.0 ng/mL of each analyte (prepared in the initial mobile phase
conditions) directly into the MS at the chosen LC mobile phase flow rate
(approximately 0.3 mL/min). This tune can be done on a mix of method
analytes. MS parameters (voltages, temperatures, gas flows, etc.) are varied
until optimal analyte responses are determined. Method analytes may have
different optima requiring some compromise between the optima. See
Table 2 for ESI-MS conditions used in method development.
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10.2.1.3 Optimize the product ion (Sect. 3.18) for each analyte by infusing
approximately 0.5-1.0 ng/mL of each analyte (prepared in the initial mobile
phase conditions) directly into the MS at the chosen LC mobile phase flow
rate (approximately 0.3 mL/min). This tune can be done on a mix of
method analytes. MS/MS parameters (collision gas pressure, collision
energy, etc.) are varied until optimal analyte responses are determined. See
Table 4 for MS/MS conditions used in method development.
10.2.2 Establish LC operating parameters that optimize resolution and peak shape.
Suggested LC conditions can be found in Table 1. LC conditions listed in Table 1
may not be optimum for all LC systems and may need to be optimized by the
analyst.
10.2.3 Inject a mid-level CAL standard under LC/MS conditions to obtain retention
times of each method analyte. Divide the chromatogram into retention time
windows (segments) each of which contains one or more chromatographic peaks.
During MS/MS analysis, fragment a small number of selected precursor ions
([M+H]+; Sect. 3.16) for the analytes in each window and choose the most
abundant product ion. Product ions (also quantitation ions) chosen during method
development are in Table 4, although these will be instrument dependent. For
maximum sensitivity in subsequent MS/MS analyses, minimize the number of
transitions that are simultaneously monitored within each segment.
10.2.4 Inject a mid-level CAL standard under optimized LC/MS/MS conditions to ensure
that each method analyte is observed in its MS/MS window and that there are at
least 10 scans across the peak for optimum precision.
10.2.5 Prepare a set of at least five CAL standards as described in Section 7.2.4. The
lowest concentration CAL standard must be at or below the MRL, which may
depend on system sensitivity. It is recommended that at least four of the CAL
standards are at a concentration greater than or equal to the MRL.
10.2.6 The LC/MS/MS system is calibrated using the IS technique. Use the LC/MS/MS
data system software to generate a linear regression or quadratic calibration curve
for each of the analytes. Curves may be concentration weighted, if necessary.
10.2.7 CALIBRATION ACCEPTANCE CRITERIA - Validate the initial calibration by
calculating the concentration of each analyte as an unknown against its regression
equation. For calibration levels that are < MRL, the result for each analyte should
be within ± 50% of the true value. All other calibration points must calculate to be
within ± 30% of their true value. If these criteria cannot be met, the analyst will
have difficulty meeting ongoing QC criteria. It is recommended that corrective
action is taken to reanalyze the CAL standards, restrict the range of calibration, or
select an alternate method of calibration.
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CAUTION: When acquiring MS/MS data, LC operating conditions must be
carefully reproduced for each analysis to provide reproducible
retention times. If this is not done, the correct ions will not be
monitored at appropriate times. As a precautionary measure,
chromatographic peaks in each window must not elute too close to
the edge of the segment time window.
10.3 CONTINUING CALIBRATION CHECK (CCC) - Minimum daily calibration
verification is as follows. Analyze a CCC to verify the initial calibration at the
beginning of each analysis batch, after every tenth Field Sample, and at the end of
each analysis batch. LRBs, CCCs, LFBs, LFSMs, FDs and LFSMDs are not counted
as samples. The beginning CCC of each analysis batch must be at or below the MRL
in order to verify instrument sensitivity prior to any analyses. If standards have been
prepared such that all low CAL points are not in the same CAL solution, it may be
necessary to analyze two CAL standards to meet this requirement. Alternatively,
analyte concentrations in the analyte PDS may be customized to meet this criteria.
Subsequent CCCs should alternate between a medium and high concentration CAL
standard.
10.3.1 Inject an aliquot of the appropriate concentration CAL standard and analyze with
the same conditions used during the initial calibration.
10.3.2 Determine that the absolute areas of the quantitation ions of the IS(s) are within
50-150% of the average areas measured during initial calibration. If any of the IS
areas has changed by more than these amounts, adjustments must be made to
restore system sensitivity. These adjustments may include cleaning of the MS ion
source, or other maintenance as indicated in Section 10.3.4. Major instrument
maintenance requires recalibration (Sect 10.2) and verification of sensitivity by
analyzing a CCC at or below the MRL (Sect 10.3). Control charts are useful aids
in documenting system sensitivity changes.
10.3.3 Calculate the concentration of each analyte and SUR in the CCC. The calculated
amount for the SUR must be within ± 30% of the true value. Each analyte
fortified at a level < MRL must calculate to be within ± 50% of the true value.
The calculated concentration of method analytes in CCCs fortified at all other
levels must be within ± 30%. If these conditions do not exist, then all data for the
problem analyte must be considered invalid, and remedial action should be taken
(Sect. 10.3.4) 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, with the following
exception. If the CCC fails because the calculated concentration is greater
than 130% (150% for the low-level CCC) for a particular method analyte,
and Field Sample extracts show no detection for that method analyte, non-
detects may be reported without re-analysis.
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10.3.4 REMEDIAL ACTION - Failure to meet CCC QC performance criteria may
require remedial action. Major maintenance, such as cleaning the electrospray
probe, atmospheric pressure ionization source, mass analyzer, replacing the LC
column, etc., requires recalibration (Sect 10.2) and verification of sensitivity by
analyzing a CCC at or below the MRL (Sect 10.3).
11. PROCEDURE
11.1 This procedure may be performed manually or in an automated mode using a robotic
or automatic sample preparation device. Data presented in Tables 5-13 demonstrate
data collected by manual extraction. If an automated system is used to prepare
samples, follow the manufacturer's operating instructions, but all extraction and
elution steps must be the same as in the manual procedure. Extraction and/or elution
steps may not be changed or omitted to accommodate the use of an automated system.
If an automated system is used, LRBs should be rotated among the ports to ensure that
all valves and tubing meet LRB requirements (Sect. 9.3.1).
NOTE: SPE cartridges described in this section are designed as single use items and
should be discarded after use. They may not be refurbished for reuse in
subsequent analyses.
11.2 SAMPLE PREPARATION
11.2.1 Samples are preserved, collected and stored as presented in Section 8. All Field
and QC Samples, including the LRB, and LFB, must contain the preservatives
listed in Section 8.1.2. Before extraction, verify that the sample pH is 7 ± 0.5. If
the sample pH does not meet this requirement, discard the sample. If the sample
pH is acceptable, proceed with the analysis. Before extraction, mark the level of
the sample on the outside of the sample bottle for later sample volume
determination (Sect. 11.5). If using weight to determine volume, weigh the bottle
with collected sample before extraction.
11.2.2 Add an aliquot of the SUR PDS (Sect. 7.2.2.2) to each sample to be extracted, cap
and invert to mix. During method development, a 10-|aL aliquot of the
0.40 ng/nL SUR PDS (Sect. 7.2.2.2) was added to 250 mL for a final
concentration of 16 ng/L in the aqueous sample.
11.2.3 In addition to SUR(s) and preservatives, if the sample is an LFB, FD, LFSM, or
LFSMD, add the necessary amount of analyte PDS (Sect. 7.2.3.2). Cap and invert
each sample to mix.
11.3 CARTRIDGE SPE PROCEDURE
11.3.1 CARTRIDGE CLEAN-UP AND CONDITIONING - DO NOT allow cartridge
packing material to go dry during any of the conditioning steps. Rinse each
cartridge with 5 mL of methanol. Next, rinse each cartridge with 10 mL of
540-24
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reagent water, without allowing the water to drop below the top edge of the
packing. If the cartridge goes dry during the conditioning phase, the conditioning
must be started over. Add 4-5 mL of reagent water to each cartridge, attach
sample transfer tubes (Sect. 6.6.3), turn on the vacuum, and begin adding sample
to the cartridge.
11.3.2 SAMPLE EXTRACTON - Adjust the vacuum so that the approximate flow rate
is 10-15 mL/min. Do not allow the cartridge to go dry before all the sample has
passed through.
11.3.3 CARTRIDGE RINSE - After the entire sample has passed through the cartridge,
rinse the cartridge with 5 mL of reagent water. Draw air or nitrogen through the
cartridge for 5 min at high vacuum (10-15 in. Hg).
11.3.4 CARTRIDGE ELUTION - Turn off and release the vacuum. Lift the extraction
manifold top and insert a rack with collection tubes into the extraction tank to
collect the extracts as they are eluted from the cartridges. Elute analytes from the
cartridge by pulling 5 mL of methanol through the cartridge. Use a low vacuum
such that the solvent exits the cartridge in a dropwise fashion.
11.4 EXTRACT CONCENTRATION - Concentrate the extract to less than 1 mL (but not
less than 0.5 mL) under a gentle stream of nitrogen in a heated water bath (40 °C).
Add the appropriate amount of methanol and IS PDS (Sect. 7.2.1.2) to the collection
vial to bring the volume to 1 mL and vortex. (10 |aL of the 0.40 ng/|jL IS PDS for
extract concentrations of 4 pg/|iL were used during method development). Transfer a
small aliquot to an autosampler vial.
11.5 SAMPLE VOLUME DETERMINATION - If the level of the sample was marked on
the sample bottle, use a graduated cylinder to measure the volume of water required to
fill the original sample bottle to the mark made prior to extraction. Determine to the
nearest 10 mL. If using weight to determine volume, weigh the empty bottle to the
nearest 10 g and determine the sample weight by subtraction of the empty bottle
weight from the original sample weight (Sect. 11.2.1). Assume a sample density of
1.0 g/mL. In either case, the sample volume will be used in the final calculations of
the analyte concentration (Sect. 12.2).
11.6 EXTRACT ANALYSIS
11.6.1 Establish operating conditions equivalent to those summarized in Tables 1-4 of
Section 17. Instrument conditions and columns should be optimized prior to
initiation of the IDC.
11.6.2 Establish an appropriate retention time window for each analyte. This should be
based on measurements of actual retention time variation for each method analyte
in CAL standard solutions analyzed on the LC over the course of time. A value
of plus or minus three times the standard deviation of the retention time obtained
540-25
-------
for each method analyte while establishing the initial calibration and 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.
11.6.3 Establish a valid initial calibration following the procedures outlined in Sect. 10.2
or confirm that the calibration is still valid by running a CCC as described in Sect.
10.3. If establishing an initial calibration, complete the IDC as described in
Section 9.2.
11.6.4 Begin analyzing Field Samples, including QC samples, at their appropriate
frequency by injecting the same size aliquots (10 |aL was used in method
development), under the same conditions used to analyze the CAL standards.
11.6.5 At the conclusion of data acquisition, use the same software that was used in the
calibration procedure to identify peaks of interest in predetermined retention time
windows. Use the data system software to examine the ion abundances of the
peaks in the chromatogram. Identify an analyte by comparison of its retention
time with that of the corresponding method analyte peak in a reference standard.
Comparison of MS/MS mass spectra is not particularly useful given the limited
±0.5 dalton mass range around a single product ion for each method analyte.
11.6.6 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 methanol and the appropriate amount of IS added to match
the original concentration. Re-inject the diluted extract. Incorporate the dilution
factor into the final concentration calculations. Acceptable SUR performance
(Sect. 9.3.5.1) should be determined from the undiluted sample extract. The
resulting data should be documented as a dilution and MRLs should be adjusted
accordingly.
12. DATA ANALYSIS AND CALCULATION
12.1. Complete chromatographic resolution is not necessary for accurate and precise
measurements of analyte concentrations using MS/MS. In validating this method,
concentrations were calculated by measuring the product ions listed in Table 4. Other
ions may be selected at the discretion of the analyst.
12.2. Calculate analyte and SUR concentrations using the multipoint calibration established
in Section 10.2. Do not use daily calibration verification data to quantitate analytes in
samples. Adjust final analyte concentrations to reflect the actual sample volume
determined in Section 11.5.
12.3 Prior to reporting data, the chromatogram should be reviewed for any incorrect peak
identification or poor integration.
540-26
-------
12.4 Calculations must utilize all available digits of precision, but final reported
concentrations should be rounded to an appropriate number of significant figures (one
digit of uncertainty), typically two, and not more than three significant figures.
NOTE: Some data in Section 17 of this method are reported with more than two
significant figures. This is done to better illustrate method performance.
13. METHOD PERFORMANCE
13.1 PRECISION, ACCURACY, AND MINIMUM REPORTING LEVELS - Tables for
these data are presented in Section 17. LCMRLs and DLs for each method analyte are
presented in Table 5. Precision and accuracy are presented for three water matrices:
reagent water (Tables 6 and 7); chlorinated (finished) ground water (Tables 8 and 9);
chlorinated (finished) surface water (Tables 10 and 11).
13.2 SAMPLE STORAGE STABILITY STUDIES - An analyte storage stability study was
conducted by fortifying the analytes into chlorinated surface water samples that were
collected, preserved, and stored as described in Section 8. Precision and mean
recovery (n=4) of analyses, conducted on Days 0, 7, 14, 23 and 28 are presented in
Table 12.
13.3 EXTRACT STORAGE STABILITY STUDIES - Extract storage stability studies
were conducted on extracts obtained from a chlorinated surface water fortified with
method analytes. Precision and mean recovery (n=4) of injections conducted on Days
0, 7, 14, 23, and 28 are reported in Table 13.
13.4 SECOND LABORATORY DEMONSTRATION - Performance of this method was
demonstrated by multiple laboratories, with results similar to those reported in Section
17. The authors wish to acknowledge the assistance of the analysts and laboratories
for their participation in the multi-laboratory verification studies: a) Dr. Andrew
Eaton and Mr. Ali Haghani of Eurofms Eaton Analytical (EEA), Monrovia, CA, b) Dr.
Yongtao Li and Mr. Joshua S. Whitaker of UL LLC, South Bend, IN, and c) Dr.
Damon Carl of Heritage Environmental Services, Indianapolis, IN.
14. POLLUTION PREVENTION
14.1 This method utilizes SPE to extract analytes from water. It requires the use of very
small volumes of organic solvent and very small quantities of pure analytes, thereby
minimizing potential hazards to both the analyst and the environment as compared to
the use of large volumes of organic solvents in conventional liquid-liquid extractions.
14.2 For information about pollution prevention that may be applicable to laboratory
operations, consult "Less is Better: Guide to Minimizing Waste in Laboratories"
available from the American Chemical Society's Department of Government Relations
and Science Policy, 1155 16th Street N.W., Washington, D.C., 20036 or on-line at
http ://portal. acs.org/portal/fileFetch/CAVPCP 012290/pdf/WPCP 012290.pdf
(accessed June 2012).
540-27
-------
15. WASTE MANAGEMENT
15.1 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 or source water. However, laboratory waste
management practices must 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.
16. REFERENCES
1. Winslow, S.D., Pepich, B.V., Martin, J.J., Hallberg, G.R., Munch, D.J., Frebis, C.P.,
Hedrick, E. J., Krop, R. A. "Statistical Procedures for Determination and Verification of
Minimum Reporting Levels for Drinking Water Methods." Environ. Sci. Technol. 2004, 40,
281-288.
2. Glaser, J.A., D.L. Foerst, G.D. McKee, S.A. Quave, W.L. Budde, "Trace Analyses for
Wastewaters." Environ. Sci. Technol. 1981, 15, 1426-1435.
3. Leenheer, J.A., Rostad, C.E., Gates, P.M., Furlong, E.T., Ferrer, I. "Molecular Resolution
and Fragmentation of Fulvic Acid by Electrospray lonization/Multistage Tandem Mass
Spectrometry.'Mwa/. Chem. 2001, 73, 1461-1471.
4. Cahill, J.D., Furlong E.T., Burkhardt, M.R., Kolpin, D., Anderson, L.G. "Determination of
Pharmaceutical Compounds in Surface- and Ground-Water Samples by Solid-Phase
Extraction and High-Performance Liquid Chromatography Electrospray lonization Mass
Spectrometry." J. Chromatogr. A, 2004, 1041, 171-180.
5. "Prudent Practices in the Laboratory: Handling and Disposal of Chemicals," National
Academies Press (1995), available at http://www.nap.edu (accessed October 5, 2011).
6. "OSHA Safety and Health Standards, General Industry," (29CFR1910), Occupational Safety
and Health Administration, OSHA 2206, (Revised, July 2001).
7. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 7th Edition. Available online at
http://portal.acs.org/portal/PublicWebSite/about/governance/committees/chemicalsafetv/publ
ications/WPCP 012294 (accessed August 2013).
8. Winslow, S. D. , Pepich, S. D. , Bassett, M. V., Wendelken, S. C., Munch, D. J., Sinclair, J.
L. "Microbial inhibitors for U.S. EPA drinking water methods for the determination of
organic compounds." Environ. Sci. Technol., 2001, 35, 4103-4110.
540-28
-------
17. TABLES, DIAGRAMS, FLOWCHARTS AND VALIDATION DATA
TABLE 1. LC METHOD CONDITIONS
Time (min)
Initial
8.0
9.0
28.0
28.1
30.0
30.1
40.0
% Formate Buffer
90.0
60.0
50.0
17.7
10.0
10.0
90.0
90.0
% Methanol
10.0
40.0
50.0
82.3
90.0
90.0
10.0
10.0
Restek Ultra Aqueous 2. 1 x 100 mm packed with 5 jam Cig stationary phase
Flow rate of 0.3 mL/min
10 |aL partial loop injection into a 20 jiL loop
TABLE 2. ESI-MS/MS METHOD CONDITIONS
ESI Conditions
Polarity
Capillary needle voltage
Cone gas flow
Nitrogen desolvation gas
Desolvation gas temp.
Positive ion
4kV
lOOL/hr
HOOL/hr
350 °C
540-29
-------
TABLE 3. METHOD ANALYTE SOURCE, RETENTION TIMES (RTs), AND
SUGGESTED IS REFERENCES
Analyte
methomyl
3 -hydroxycarbofuran
fenamiphos sulfone
fenamiphos sulfoxide
phorate sulfone
phorate sulfoxide
disulfoton sulfoxide
bensulide
tebufenozide
chlorpyrifos oxon
fenamiphos
tebuconazole
methomyl-13Cz 15N(SUR)
tebuconazole-t4 (SUR)
carbofuran-13C6(IS#l)
bensulide-Ji4(IS#2)
phorate- dw (IS#3)
Method Analyte
Source8
Accu Standard
Absolute Standards
Absolute Standards
Absolute Standards
Spex CertiPrep
Crescent Chemical
Chem Service
Absolute Standards
Accu Standard
Chem Service
Crescent Chemical
Crescent Chemical
Cambridge Isotopes
Dr. Ehrenstorfer
Cambridge Isotopes
Cambridge Isotopes
Crescent Chemical
Peak#
(Fig. 1)
1
3
5
6
7
8
9
11
12
13
14
17
2
16
4
10
15
RT
(mill)
6.14
8.09
12.30
12.56
13.12
13.53
13.91
19.14
19.17
19.62
19.64
20.93
6.13
20.88
11.45
18.96
20.35
IS# Ref
1
1
1
1
3
O
3
2
2
O
2
2
1
2
_
_
-
Data presented in this method were obtained using analytes purchased from these
vendors. Other vendors' materials can be used provided the QC requirements in
Section 9 can be met.
540-30
-------
TABLE 4. MS/MS METHOD CONDITIONS
Segment11
1
2
2
2
2
2
2
O
3
O
3
O
1
O
2
O
3
Analyte
methomyl
3 -hydroxycarbofuran
fenamiphos sulfone
fenamiphos sulfoxide
phorate sulfone
phorate sulfoxide
disulfoton sulfoxide
bensulide
tebufenozide
chlorpyrifos oxon
fenamiphos
tebuconazole
methomyl-13C2,15N
tebuconazole-de
carbofuran-13Ce
bensulide-t/i4
phorate-t/io
Precursor
Ion c (m/z)
163
238
336
320
293
211
291
398
353
334
304
308
166
314
228
412
271
Product
Ionc'd (m/z)
88
163
266
233
171
97
185
314
133
278
217
70
91
72
171
316
75
Cone
Voltage (v)
15
20
30
30
20
20
20
20
15
25
30
30
15
30
26
20
15
Collision
Energy6 (v)
10
15
20
25
10
30
15
10
20
20
25
20
10
20
13
10
10
An LC/MS/MS chromatogram of the analytes is shown in Figure 1.
Segments are time durations in which single or multiple scan events occur.
Precursor and product ions listed in this table are nominal masses. During MS and MS/MS
optimization, the analyst should determine the precursor and product ion masses to one
decimal place by locating the apex of the mass spectral peak place (e.g., m/z 398.1^313.9 for
bensulide). These precursor and product ion masses (with one decimal place) should be used
in the MS/MS method for all analyses.
Ions used for quantitation purposes.
Argon used as collision gas at a flow rate of 0.3 mL/min.
540-31
-------
TABLE 5. DLs AND LCMRLs IN REAGENT WATER
Analyte
methomyl
3 -hydroxycarbofuran
fenamiphos sulfone
fenamiphos sulfoxide
phorate sulfone
phorate sulfoxide
disulfoton sulfoxide
bensulide
tebufenozide
chlorpyrifos oxon
fenamiphos
tebuconazole
Fortified
Cone. (ng/Lf
1.60
1.60
1.60
1.60
1.60
1.60
1.60
1.60
0.64
1.60
0.64
0.64
Oasis HLB
DLb(ng/L)
0.39
0.45
0.88
0.39
0.57
0.70
0.68
0.51
0.26
1.0
0.30
0.25
LCMRLC
(ng/L)
1.2
1.3
1.0
0.86
0.99
2.0
2.0
1.2
0.81
2.0
0.64
2.0
Speedisk H2O-Philic DVB
DLb(ng/L)
0.32
0.25
0.42
0.32
0.32
0.46
0.37
1.1
0.15
0.77
0.26
0.30
LCMRLC
(ng/L)
0.61
1.2
1.0
1.6
1.0
1.1
1.1
1.4
0.73
2.7
0.30
0.33
Spiking concentration used to determine DL.
' Detection limits were determined by analyzing seven replicates over three days according to
Section 9.2.6.
LCMRLs were calculated according to the procedure in reference 1.
540-32
-------
TABLE 6. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES IN
REAGENT WATER FORTIFIED AT A HIGH CONCENTRATION (n=4)
Analyte
methomyl
3 -hydroxycarbofuran
fenamiphos sulfone
fenamiphos sulfoxide
phorate sulfone
phorate sulfoxide
disulfoton sulfoxide
bensulide
tebufenozide
chlorpyrifos oxon
fenamiphos
tebuconazole
methomyl-13C2,15N
tebuconazole-de
Fortified
Cone. (ng/L)
32.0
32.0
32.0
32.0
32.0
32.0
32.0
32.0
12.8
32.0
12.8
12.8
16.0
16.0
Oasis HLB
Mean %
Recovery
106
104
104
106
104
111
115
90.0
103
97.1
96.9
100
99.0
98.1
% RSD
6.3
2.6
4.1
4.5
5.1
2.6
4.4
4.3
5.6
5.6
2.7
5.2
4.3
8.6
Speedisk H2O-Philic DVB
Mean %
Recovery
105
102
98.4
102
111
116
117
92.7
95.5
104
96.7
97.6
99.8
92.6
% RSD
2.5
3.5
1.8
3.4
1.5
3.1
3.8
13
11
4.3
11
10
2.4
12
540-33
-------
TABLE 7. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES IN
REAGENT WATER FORTIFIED AT A LOW CONCENTRATION (n=4)
Analyte
methomyl
3 -hydroxycarbofuran
fenamiphos sulfone
fenamiphos sulfoxide
phorate sulfone
phorate sulfoxide
disulfoton sulfoxide
bensulide
tebufenozide
chlorpyrifos oxon
fenamiphos
tebuconazole
methomyl-13C2,15N
tebuconazole-de
Fortified
Cone. (ng/L)
4.00
4.00
4.00
4.00
4.00
4.00
4.00
4.00
1.60
4.00
1.60
1.60
16.0
16.0
Oasis HLB
Mean %
Recovery
102
101
101
108
94.2
106
113
93.6
110
95.5
92.6
115
101
98.9
% RSD
1.9
6.1
3.8
2.0
3.2
3.0
2.9
5.5
5.2
1.3
6.8
4.9
3.4
1.4
Speedisk H2O-Philic DVB
Mean %
Recovery
103
107
99.6
108
108
112
111
93.0
105
112
104
114
98.1
105
% RSD
2.6
4.8
3.1
9.6
7.2
4.3
6.5
3.0
2.7
11
6.6
3.4
2.1
4.8
540-34
-------
TABLE 8. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES
FORTIFIED AT A HIGH CONCENTRATION IN FINISHED DRINKING
WATER FROM A GROUND WATER SOURCE8 (n=4)
Analyte
methomyl
3 -hydroxycarbofuran
fenamiphos sulfone
fenamiphos sulfoxide
phorate sulfone
phorate sulfoxide
disulfoton sulfoxide
bensulide
tebufenozide
chlorpyrifos oxon
fenamiphos
tebuconazole
methomyl-13C2,15N
tebuconazole-de
Fortified
Cone. (ng/L)
32.0
32.0
32.0
32.0
32.0
32.0
32.0
32.0
12.8
32.0
12.8
12.8
16.0
16.0
Oasis HLB
Mean %
Recovery
105
101
103
106
97.8
102
103
97.1
100
91.8
104
100
103
93.5
% RSD
1.3
2.3
4.4
3.2
4.9
4.2
4.3
5.9
4.2
3.5
4.5
5.2
2.3
7.5
Speedisk H2O-Philic DVB
Mean %
Recovery
109
107
99.0
105
103
104
108
97.1
102
95.1
103
101
105
98.6
% RSD
2.0
1.5
1.6
1.3
1.5
2.5
2.1
1.2
4.3
2.2
4.4
4.0
1.0
3.9
TOC = <0.50 mg/L and hardness = 342 mg/L as calcium carbonate.
540-35
-------
TABLE 9. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES
FORTIFIED AT A LOW CONCENTRATION IN FINISHED DRINKING
WATER FROM A GROUND WATER SOURCE8 (n=4)
Analyte
methomyl
3 -hydroxycarbofuran
fenamiphos sulfone
fenamiphos sulfoxide
phorate sulfone
phorate sulfoxide
disulfoton sulfoxide
bensulide
tebufenozide
chlorpyrifos oxon
fenamiphos
tebuconazole
methomyl-13C2,15N
tebuconazole-de
Fortified
Cone. (ng/L)
4.00
4.00
4.00
4.00
4.00
4.00
4.00
4.00
1.60
4.00
1.60
1.60
16.0
16.0
Oasis HLB
Mean %
Recovery
101
103
108
99.4
98.1
109
115
88.8
100
124
96.9
98.4
104
96.3
% RSD
4.3
5.4
6.1
3.2
2.4
4.0
1.8
17
5.1
14
3.7
3.2
3.0
5.5
Speedisk H2O-Philic DVB
Mean %
Recovery
103
107
107
100
100
108
111
85.6
90.6
102
98.4
85.9
101
91.7
% RSD
3.4
4.4
1.2
2.0
2.9
7.6
6.2
6.0
8.9
5.1
9.5
7.0
1.7
4.4
TOC = 0.61 mg/L and hardness = 377 mg/L as calcium carbonate.
540-36
-------
TABLE 10. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES
FORTIFIED AT A HIGH CONCENTRATION IN FINISHED DRINKING
WATER FROM A SURFACE WATER SOURCE8 (n=4)
Analyte
Methomyl
3 -hydroxycarbofuran
fenamiphos sulfone
fenamiphos sulfoxide
phorate sulfone
phorate sulfoxide
disulfoton sulfoxide
Bensulide
Tebufenozide
chlorpyrifos oxon
Fenamiphos
Tebuconazole
methomyl-13C2,15N
tebuconazole-de
Fortified
Cone. (ng/L)
32.0
32.0
32.0
32.0
32.0
32.0
32.0
32.0
12.8
32.0
12.8
12.8
16.0
16.0
Oasis HLB
Mean %
Recovery
106
101
106
108
102
105
110
101
101
102
107
107
100
98.9
% RSD
2.2
1.4
0.6
1.8
2.6
2.1
1.8
7.6
5.3
4.1
5.8
5.4
3.5
6.3
Speedisk H2O-PhiIic DVB
Mean %
Recovery
111
105
104
104
105
105
107
100
96.7
103
99.5
107
107
101
% RSD
2.5
1.3
2.5
0.7
6.0
5.2
6.0
7.5
6.1
8.0
6.8
7.5
1.8
6.4
TOC = 1.22 mg/L and hardness =154 mg/L as calcium carbonate.
540-37
-------
TABLE 11. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES
FORTIFIED AT A LOW CONCENTRATION IN FINISHED DRINKING
WATER FROM A SURFACE WATER SOURCE (n=4)
Analyte
methomyl
3 -hydroxycarbofuran
fenamiphos sulfone
fenamiphos sulfoxide
phorate sulfone
phorate sulfoxide
disulfoton sulfoxide
bensulide
tebufenozide
chlorpyrifos oxon
fenamiphos
tebuconazole
methomyl-13C2,15N
tebuconazole-de
Fortified
Cone. (ng/L)
4.00
4.00
4.00
4.00
4.00
4.00
4.00
4.00
1.60
4.00
1.60
1.60
16.0
16.0
Oasis HLBa
Mean %
Recovery
104
96.6
97.6
106
90.3
102
111
90.4
98.9
94.0
96.1
111
101
100
% RSD
2.1
4.3
2.2
3.1
3.3
5.5
4.8
7.1
9.5
4.6
3.9
6.6
0.8
2.8
Speedisk H2O-Philic DVBb
Mean %
Recovery
109
106
104
105
98.1
105
107
96.4
105
104
100
120
103
101
% RSD
3.1
3.4
4.3
7.4
6.6
5.7
4.4
12
11
7.6
3.1
5.2
1.9
3.7
TOC = 1.22 mg/L and hardness =154 mg/L as calcium carbonate.
' TOC = 1.58 mg/L and hardness =137 mg/L as calcium carbonate.
540-38
-------
TABLE 12. AQUEOUS SAMPLE HOLDING TIME DATA FOR SAMPLES OF FINISHED DRINKING WATER FROM A
SURFACE WATER SOURCE3, FORTIFIED WITH METHOD ANALYTES AND PRESERVED AND STORED
ACCORDING TO SECTION 8 (n=4)h
Analyte
methomyl
3 -hydroxycarbofuran
fenamiphos sulfone
fenamiphos sulfoxide
phorate sulfone
phorate sulfoxide
disulfoton sulfoxide
bensulide
tebufenozide
chlorpyrifos oxon
fenamiphos
tebuconazole
methomyl-1JC2,i:'Nc
tebuconazole-de0
Fortified
Cone.
(ng/L)
32.0
32.0
32.0
32.0
32.0
32.0
32.0
32.0
12.8
32.0
12.8
12.8
16.0
16.0
DayO
Mean
%Rec
106
101
106
108
102
105
110
101
101
102
107
107
100
98.9
%
RSD
2.2
1.4
0.6
1.8
2.6
2.1
1.8
7.6
5.3
4.1
5.8
5.4
3.5
6.3
Day?
Mean
%Rec
104
95.2
103
103
95.9
100
108
99.2
104
79.6
98.4
104
101
91.7
%
RSD
2.5
2.8
1.5
3.0
5.2
5.2
5.0
5.5
7.4
6.2
4.1
6.9
2.5
8.2
Day 14
Mean
%Rec
109
94.7
100
103
89.1
93.2
101
100
109
62.4
104
110
105
102
%
RSD
1.3
5.1
3.2
3.5
11
11
11
4.1
5.8
13
4.4
7.2
1.7
5.4
Day 23
Mean
%Rec
109
90.5
99.4
105
101
105
112
106
108
59.1
107
108
102
102
%
RSD
3.8
1.4
2.1
2.4
1.2
1.5
3.6
6.0
4.1
3.0
5.5
7.1
1.5
6.8
Day 28
Mean
%Rec
113
93.9
104
108
100
103
110
100
101
56.0
98.7
106
104
95.5
%
RSD
1.6
3.6
4.3
1.4
2.8
3.5
5.1
4.6
5.0
3.7
5.9
2.6
2.6
6.4
TOC = 1.22 mg/L and hardness =154 mg/L as calcium carbonate.
Oasis HLB SPE cartridges used for holding time studies.
0 Surrogates were not added to samples until the day of extraction.
540-39
-------
TABLE 13. EXTRACT HOLDING TIME DATA FOR SAMPLES OF FINISHED DRINKING WATER FROM A
SURFACE WATER SOURCE3, FORTIFIED WITH METHOD ANALYTES AND PRESERVED AND
STORED ACCORDING TO SECTION 8 (n=4)a
Analyte
methomyl
3 -hydroxycarbofuran
fenamiphos sulfone
fenamiphos sulfoxide
phorate sulfone
phorate sulfoxide
disulfoton sulfoxide
bensulide
tebufenozide
chlorpyrifos oxon
fenamiphos
tebuconazole
methomyl-1 JC2,1:>N
tebuconazole-de
Fortified
Cone.
(ng/L)
32.0
32.0
32.0
32.0
32.0
32.0
32.0
32.0
12.8
32.0
12.8
12.8
16.0
16.0
DayO
Mean
%Rec
106
101
106
108
102
105
110
101
101
102
107
107
100
98.9
%
RSD
2.2
1.4
0.6
1.8
2.6
2.1
1.8
7.6
5.3
4.1
5.8
5.4
3.5
6.3
Day 7
Mean
%Rec
105
99.0
101
103
98.8
103
108
102
107
97.7
106
104
103
94.4
%
RSD
0.9
1.5
1.6
1.8
6.7
7.6
6.5
1.6
2.0
8.0
4.0
2.2
0.9
4.0
Day 14
Mean
%Rec
109
106
103
106
90.1
94.1
98.9
99.2
107
86.2
105
106
105
94.4
%
RSD
1.9
2.1
2.5
1.2
2.5
4.1
5.5
2.0
2.1
4.1
1.5
2.3
1.6
2.1
Day 23
Mean
%Rec
104
99.8
101
106
95.5
101
104
105
109
92.1
107
111
106
99.2
%
RSD
4.3
2.0
2.3
2.8
3.5
2.3
3.1
5.1
4.6
3.4
3.5
4.9
3.8
4.1
Day 28
Mean
%Rec
108
105
103
109
101
102
106
102
105
97.7
109
115
107
96.8
%
RSD
1.5
2.3
1.2
0.5
2.8
3.0
3.8
4.3
3.8
5.9
3.9
3.6
2.0
2.7
Oasis HLB SPE cartridges used for holding time studies.
540-40
-------
TABLE 14. INITIAL DEMONSTRATION OF CAPABILITY QUALITY CONTROL REQUIREMENTS
Method
Reference
Sect. 9.2.1
and 9.3.1
Sect. 9.2.2
Sect. 9.2.3
Sect. 9.2.4
Sect. 9.2.5
and 9.3.8
Requirement
Initial Demonstration of
Low System Background
Initial Demonstration of
Precision (IDP)
Initial Demonstration of
Accuracy (IDA)
Minimum Reporting Limit
(MRL) Confirmation
Quality Control Sample
(QCS)
Specification and Frequency
Analyze LRB prior to any other IDC steps.
Analyze four to seven replicate LFBs fortified near
the midrange calibration concentration.
Calculate average recovery for replicates used in
IDP.
Fortify, extract and analyze seven replicate LFBs
at the proposed MRL concentration. Calculate the
Mean and the Half Range (HR). Confirm that the
upper and lower limits for the Prediction Interval
of Result (Upper PIR, and Lower PIR, Sect.
9.2.4.2) meet the recovery criteria.
Analyze a standard from a second source, as
part of IDC.
Acceptance Criteria
Demonstrate that all method analytes are below 1/3 the MRL
and that possible interferences from extraction media do not
prevent the identification and quantification of method
analytes.
%RSDmustbe <20%
Mean recovery + 30% of true value
Upper PIR < 150%
Lower PIR > 50%
Results must be within 70-130% of true value.
NOTE: Table 14 is intended as an abbreviated summary of QC requirements provided as a convenience to the method user. Because the information has been
abbreviated to fit the table format, there may be issues that need additional clarification, or areas where important additional information from the method text
is needed. In all cases, the full text of the QC in Section 9 supersedes any missing or conflicting information in this table.
540-41
-------
TABLE 15. ONGOING QUALITY CONTROL REQUIREMENTS (SUMMARY)
Method
Reference
Sect. 8.1-
Sect. 8.4
Sect. 8.4
Sect. 9.3.1
Sect. 9.3.3
Sect. 9.3.4
Sect. 9.3.5
Sect. 9.3.6
Sect. 9.3.7
Sect. 9.3.8
Requirement
Sample Holding Time
Extract Holding Time
Laboratory Reagent Blank
(LRB)
Laboratory Fortified Blank
(LFB)
Internal Standard (IS)
Surrogate Standards
(SUR)
Laboratory Fortified
Sample Matrix (LFSM)
Laboratory Fortified
Sample Matrix Duplicate
(LFSMD) or
Field Duplicates (FD)
Quality Control Sample
(QCS)
Specification and Frequency
28 days with appropriate preservation and storage as
described in Sections 8.1-8.4 except chlorpyrifos
oxon (7 days).
28 days when stored at < 6 °C and protected from
light.
One LRB with each extraction batch of up to 20 Field
Samples.
One LFB is required for each extraction batch of up
to 20 Field Samples. Rotate the fortified
concentrations between low, medium, and high
amounts.
Internal standards, carboruran-13C6, bensulide-
-------
TABLE 15. (Continued)
Method
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Sect. 10.2
Initial Calibration
Use IS calibration technique to generate a linear or
quadratic calibration curve for each analyte. Use
at least five standard concentrations. Check the
calibration curve as described in Sect. 10.2.7.
When each CAL standard is calculated as an unknown using
the calibration curve, the analyte results must be 70-130% of
the true value for all except CAL standards < MRL, which
must be 50-150% of the true value. If this criterion is not
met reanalyze the CAL standards, restrict the range of
calibration, or select an alternate method of calibration.
Sect. 9.3.2
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
Field Samples and after the last sample, rotating
concentrations to cover the calibrated range of the
instrument.
Recovery for each SUR must be within 70-130% of the true
value in all CCCs. Each analyte fortified at a level < MRL
must calculate to be within ± 50% of the true value. The
calculated concentration of the method analytes in CCCs
fortified at all other levels must be within ± 30%.
NOTE: Table 15 is intended as an abbreviated summary of QC requirements provided as a convenience to the method user. Because the information has been
abbreviated to fit the table format, there may be issues that need additional clarification, or areas where important additional information from the method text
is needed. In all cases, the full text of the QC in Section 8-10 supersedes any missing or conflicting information in this table.
540-43
-------
FIGURE 1. EXAMPLE CHROMATOGRAM (OVERLAID MS/MS SEGMENTS) OF A CALIBRATION STANDARD WITH
METHOD 540 ANALYTES AT CONCENTRATION LEVELS OF 3.2-8.0 jig/L. NUMBERED PEAKS ARE
IDENTIFIED IN TABLE 3.
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540-44
------- |