EPA Document #: EPA/600/R-14/098
METHOD 543. DETERMINATION OF SELECTED ORGANIC CHEMICALS IN
DRINKING WATER BY ON-LINE SOLID PHASE EXTRACTION-
LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY
(ON-LINE SPE-LC/MS/MS)
Version 1.0
March 2015
J.A. Shoemaker 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 543
DETERMINATION OF SELECTED ORGANIC CHEMICALS IN DRINKING
WATER BY ON-LINE SOLID PHASE EXTRACTION-LIQUID
CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY
(ON-LINE SPE-LC/MS/MS)
1. SCOPE AND APPLICATION
1.1 This is an on-line solid phase extraction liquid chromatography/tandem mass
spectrometry (on-line SPE-LC/MS/MS) method for determination of organic
chemicals 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. This method cannot be used for manual (off-line) SPE extractions.
EPA Method 5401 is available for analysis of these analytes by off-line SPE-
LC/MS/MS analysis, if desired.
Chemical Abstract Services
Analvte Registry Number (CASRN)
3-Hydroxycarbofuran 16655-82-6
Bensulide 741-58-2
Fenamiphos 22224-92-6
Fenamiphos sulfone 31972-44-8
Fenamiphos sulfoxide 31972-43-7
Tebuconazole 107534-96-3
Tebufenozide 112410-23-8
1.2 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 0.27-1.7 ng/L, and are listed in Table 6. The procedure used
to determine the LCMRL is described elsewhere.2
1.3 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.
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.3
The DL is compound dependent and is dependent on extraction efficiency, sample
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matrix, fortification concentration, and instrument performance. DLs for analytes in
this method range from 0.13-0.99 ng/L, and are listed in Table 6.
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 analytical LC
column, LC gradient and MS and MS/MS conditions (Sect. 6.5.8, 9.1.1, 10.2, and
12.1). Changes may not be made to sample collection and preservation (Sect. 8),
sample extraction steps (Sect. 11.3), 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. Analvtes 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. 9.3.5).
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 10-mL preserved water sample is fortified with internal standards and analyzed by
automated on-line SPE-LC/MS/MS. A 2-mL aliquot of the sample is loaded onto the in-line
SPE cartridge. The SPE cartridge is washed with 20 mM ammonium acetate. Analytes are
back-eluted from the solid phase with analytical LC gradient. 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.
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.
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3.2 CALIBRATION STANDARD (CAL) - A solution prepared from the primary dilution
standard solution and/or stock standard solution, and the internal standard(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 and internal standard(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.3
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, 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 a sample or standard
solution in a known amount(s) and used to measure the relative response of other
method analytes that are components of the same solution. The internal 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.
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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, and internal standards 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.2
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.
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
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.
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3.19 QUALITY CONTROL SAMPLE (QCS) - 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.
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
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 (ESI) source or low recoveries on the SPE sorbent.4"5 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.
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5. SAFETY
5.1 The toxicity or carcinogen!city 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.6"8
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 - 100-mL amber glass bottles fitted with
polytetrafluoroethylene (PTFE)-lined screw caps. Smaller amber bottles may be used
provided the corresponding amount of preservatives can be accurately measured at
smaller volumes.
6.2 AUTOSAMPLER VIALS - Amber glass 20-mL autosampler vials (Kimble Chase
#60960A-4 or equivalent) with caps containing pre-slit PTFE-silicone septa (Waters
#186006322 or equivalent). Smaller volume autosampler vials are also acceptable.
6.3 MICRO SYRINGES - Suggested sizes include 5, 10, 25, 500, 100, 250, 50, 1000, and
10,000-jiL syringes.
6.4 ANALYTICAL BALANCE - Capable of weighing to the nearest 0.0001 g.
6.5 ON-LINE SOLID PHASE EXTRACTION (SPE) LIQUID CHROMATOGRAPHY
(LC)/TANDEM MASS SPECTROMETER (MS/MS) WITH DATA SYSTEM (See
Figures 2 and 3 for system schematic.)
6.5.1 SPE CARTRIDGES - Waters Oasis HLB, Direct Connect, 20 |im, 2.1x30 mm
cartridges (Waters #186005231) - divinylbenzene N-vinylpyrrolidone copolymer.
6.5.2 LOADING (SPE) PUMP - A binary or quaternary LC pump capable of flow rates
up to 2 mL/min used to deliver sample loading, cartridge washing and cartridge
conditioning solvents.
6.5.3 AUTOSAMPLER - Autosampler must be capable of reproducibly injecting
sample volumes up to 2 mL.
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6.5.4 SWITCHING VALVES - Automatic switching valves capable of switching flows
between the loading and analytical pump through the extraction column and
analytical column. The two parallel cartridge on-line SPE system used during
method development operated with two switching valves. Other configurations
that can perform the procedure in Section 11.3 and meet QC criteria in Section 9
are acceptable.
6.5.5 ANALYTCAL LC PUMP - A binary or quaternary LC pump capable of
performing binary linear gradients at a constant flow rate near the flow rate used
for development of this method (0.4 mL/min). Usage of a cooled autosampler
compartment and a column heater is optional. During method development
samples were not cooled and the analytical LC column was thermostated at 30 °C.
6.5.6 TANDEM MASS SPECTROMETER - The mass spectrometer must be capable
of positive ion electrospray ionization (ESI) near the suggested LC flow rate of
0.4 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 are demonstrated in Tables 7-10
using a triple quadrupole mass spectrometer.
6.5.7 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.
6.5.8 ANALYTICAL COLUMN - A UPLC HSS T3 column (2.1 x 50 mm) packed
with 1.8 jam Ci8 solid phase particles (Waters #186003538) was used. Any
equivalent column that provides adequate resolution, peak shape, capacity,
accuracy, and precision (Sect. 9) may be used.
CAUTION: Under the sample, SPE, and mobile phase conditions of this
method, not all columns performed equivalently. Broad peak
shapes were obtained on some Cis columns. A column designed
to more strongly retain polar analytes in highly aqueous mobile
phases is needed for this method.
6.5.9 IN-LINE FILTER UNIT - A UPLC in-line filter holder (Waters #205000343 or
equivalent) with 0.2 |im, 2.1 mm stainless steel filters (Waters #700005698 or
equivalent).
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Note: The in-line filter unit is optional, but highly recommended to extend the
lifetime of the analytical column from particle buildup at the head of the
column.
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).
7.1.3 ACETONITRILE (CH3CN, CAS#: 75-05-8) - High purity, demonstrated to be
free of analytes and interferences (Fisher Optima LC/MS grade or equivalent).
7.1.4 AMMONIUM ACETATE (C2H7NO2, CAS# 631 -61 -8) - High purity,
demonstrated to be free of analytes and interferences (Sigma LC/MS grade or
equivalent).
7.1.5 20 mM AMMONIUM ACETATE - To prepare 1 L, add 1.54 g ammonium
acetate to 1 L of reagent water. This solution is prone to volatility losses and
should be replaced at least every 48 hours.
7.1.6 SAMPLE PRESERVATION REAGENTS - The following preservatives are
solids at room temperature and may be added to the sample bottle before
shipment to the field.
7.1.6.1 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).
7.1.6.2 L-ASCORBIC ACID (CAS# 50-81-7) - Ascorbic acid reduces free chlorine
at the time of sample collection (Sigma-Aldrich #255564 or equivalent).9
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7.1.6.3 2-CHLOROACETAMIDE (CAS# 79-07-2) - Inhibits microbial growth and
analyte degradation (Sigma-Aldrich #C0267 or equivalent).9
7.1.7 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.
7.1.8 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.
7.2 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
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: methomyl-13C2,15N, carbofuran-13Ce, and bensulide-Ji4. 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 (100-1000 |ig/mL) - 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 methomyl-13C2,15Nin methanol
(Cambridge Isotopes #CNLM-7148-1.2) and carbofuran-13Ce in
1,4-dioxane (Cambridge Isotopes # CLM-1911-1.2) were used. Bensulide-
du 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 -15 °C or less in amber glass screw cap
vials.
7.2.1.2 INTERNAL STANDARD PRIMARY DILUTION STANDARD (IS PDS)
(32-80 pg/|iL) - Prepare, or purchase commercially, the IS PDS containing
the three isotopically labeled chemicals at the suggested concentrations 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
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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 32-80 pg/|iL IS PDS
was used to fortify 10-mL samples (Sect. 11.2.2). This will yield a
concentration of 32-80 ng/L of each IS in 10-mL samples.
IS
methomyl-13C2,15N
carbofuran-13Ce
bensulide-Ji4
Cone, of
IS Stock
(ug/mL)
100
100
1000
Vol. Of
IS Stock
(uL)
20
8.0
2.0
Final Vol.
of IS PDS
(mL)
25
Final Cone.
of IS PDS
(pgW
80
32
80
7.2.2 ANALYTE STANDARD SOLUTIONS - Analyte standards may be purchased
commercially as ampulized solutions or prepared from neat materials.
7.2.2.1 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 methanol for a
final concentration of 1000 |ig/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, all the analytes were
purchased from Absolute Standards, Inc as stock standards at 1000 ug/mL.
These stock standards were stable for at least 6 months when stored
at -15 °C or less in amber glass screw cap vials.
7.2.2.2 INTERMEDIATE ANALYTE PRIMARY DILUTION STANDARD (PDS)
SOLUTION (0.40-1.0 ng/nL) - The intermediate 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
intermediate analyte PDS was prepared in acetonitrile at a concentration of
1.0 ng/|aL, except for fenamiphos, tebufenozide and tebuconazole at
0.40 ng/|aL each. The intermediate analyte PDS is prepared by dilution of
the combined Analyte Stock Standard Solutions (Sect.7.2.2.1) and is used to
prepare the analyte SDS (Sect 7.2.2.3). The intermediate analyte PDS has
been shown to be stable for 6 months when stored at -15 °C or less in amber
glass screw cap vials.
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7.2.2.3 ANALYTE SECONDARY DILUTION STANDARD (SDS) (4.0-10 pg/jiL) -
The analyte SDS contains all, or a portion, of method analytes at various
concentrations in acetonitrile. The analyte SDS was prepared in acetonitrile
at a concentration of 10 pg/i-iL, except for fenamiphos, tebufenozide and
tebuconazole at 4.0 pg/|oL each. The analyte SDS is prepared by dilution of
the intermediate analyte PDS (Sect.7.2.2.2) and is used to prepare CAL
standards, and fortify LFSMs, LFSMDs and FDs with the method analytes.
The analyte SDS has been shown to be stable for 6 months when stored at
6 °C or less in amber glass screw cap vials.
7.2.3 CALIBRATION STANDARDS (CAL) - Prepare a series of at least five
concentrations of calibration solutions in deionized water, containing all the
preservatives (Sect. 8.1.2), from dilutions of the analyte SDS (Sect 7.2.2.3). 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 0.8-40 ng/L are suggested for
fenamiphos, tebufenozide and tebuconazole, and 2.0-100 ng/L are suggested for
the remaining analytes. Larger concentration ranges will require more calibration
points. The IS is added to CAL standards at a constant concentration. During
method development, the IS concentrations were 32-80 ng/L in the aqueous
samples. 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 and LFBs (Sect. 9.3.2). During method development, CAL standards were
shown to be stable for four weeks when stored at 6 °C or less.
Note: To avoid significant broadening of analyte peak shape, calibration
standards must not contain more than 1.5% acetonitrile.
8. SAMPLE COLLECTION, PRESERVATION AND STORAGE
8.1 SAMPLE BOTTLE PREPARATION
8.1.1 Samples must be collected in amber glass bottles (100-mL or smaller) fitted with
PFTE-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
2-Chloroacetamide
L-Ascorbic acid
Trizma
Amount
2.0 g/L
200 mg/L
7.75 g/L
Purpose
antimicrobial
dechlorinating agent
buffering reagent
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8.2 SAMPLE COLLECTION
8.2.1 Open the cold water 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 HOLDING TIMES - Water samples should be extracted as soon as possible
after collection but must be extracted and analyzed within 28 days of collection.
Samples must not remain at room temperature in the instrument autosampler tray for
more than 29 hours prior to extraction.
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 11 and 12. 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 gradient, internal standards, and MS and MS/MS conditions. Each time such
method modifications are made, the analyst must repeat the procedures of the IDC
(Sect. 9.2). Modifications to LC conditions should still minimize co-elution of
method analytes to reduce the probability of suppression/enhancement
effects. Modifications to the on-line SPE cartridge, the SPE solvents and solvent
volumes are not permitted.
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9.1.2 If an optional on-line dual SPE cartridge system (Sect. 11.1) is used, IDC data
must be obtained on both SPE cartridges and must meet the QC criteria in
Sections 9.2.1-9.2.3 every time a new lot of SPE cartridges is used.
NOTE: Although infrequent, recovery differences were occasionally
observed, during method development, between two on-line SPE
cartridges from the same lot.
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, and disposable pipets are used, it must be
demonstrated that an LRB is reasonably free of contamination and that criteria in
Section 9.3.1 are met.
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
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 (HRpm) using the equation below
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HRpm = 3.963^
where
s = standard deviation
3.963 = a constant value for seven replicates.2
9.2.4.2 Confirm that the upper and lower limits for the Prediction Interval of Result
(PIR = Mean +_ HRpm) meet the upper and lower recovery limits as shown
below
The Upper PIR Limit must be < 150% recovery.
Mean + HRprrf
^ x 100% < 150%
FortifiedConcentration
The Lower PIR Limit must be > 50% recovery.
Mean - HRPIR
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
9.3.7 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.
Replicate analyses for this procedure 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 6 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 11.
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.
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Calculate the DL using the following equation
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. The DL
is a statistical determination of precision only.3 If DL replicates are
fortified at a low enough concentration, it is likely that they will not meet
the precision and accuracy criteria for CCCs. Therefore, no precision
and accuracy criteria are specified.
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. 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. 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. Subtracting blank values from sample results is not permitted.
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) - Because this method utilizes
procedural calibration standards, which are fortified reagent waters, there is no
difference between the LFB and the CCC standards. Consequently, the analysis of
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a separate LFB is not required as part of the ongoing QC; however, the term
"LFB" is used for clarity in the IDC.
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,
analyze a second aliquot of that standard or sample.
9.3.4.1 If analysis of the second aliquot produces acceptable IS responses, report
results for that aliquot.
9.3.4.3 If the IS area counts of the re-analyzed second aliquot still do not meet the IS
criterion, the analyst should check the calibration by analyzing the most recent
CCC. If the IS criterion is met in the CCC but not the sample, report the
sample results as suspect/matrix.
9.3.4.4 If the IS area criterion is not met in both the sample and the CCC, instrument
maintenance such as SPE cartridge replacement or sample cone cleaning may
be necessary. Perform the appropriate instrument maintenance and then re-
analyze the sample in a subsequent analytical batch provided the sample is
still within the holding time. Otherwise, report results obtained from the re-
analyzed sample, but annotate as suspect. Alternatively, collect a new
sample and re-analyze.
9.3.5 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. 9.3.6);
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.
9.3.5.1 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
SDS (Sect. 7.2.2.3). 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.
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9.3.5.2 Calculate percent recovery (%R) for each analyte using the equation
c
where
A = measured concentration in the fortified sample
B = measured concentration in the unfortified sample
C = fortification concentration.
9.3.5.3 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.
9.3.6 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.
9.3.6. 1 Calculate relative percent difference (RPD) for duplicate measurements
(FD1 and FD2) using the equation
FD1-FD2
RPD = -/ - , — xlOO
FD2)/2
9.3.6.2 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.
9.3.6.3 If an LFSMD is analyzed instead of a FD, calculate the relative percent
difference (RPD) for duplicate LFSMs (LFSM and LFSMD) using the
equation
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\LFSM -LFSMD\
= ^ ^
(LFSM + LFSMD)/2
9.3.6.4 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.
9.3.7 QUALITY CONTROL SAMPLES (QCS) - As part of the IDC (Sect. 9.2), each
time a new Analyte SDS (Sect. 7.2.2.3) 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 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. After the initial calibration is successful, a CCC is required
at the beginning and end of each period in which analyses are performed, and after
every tenth Field Sample.
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 0.4 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
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different optima requiring some compromise between the optima. See
Table 3 for ESI-MS conditions used in method development.
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 0.4 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 5 for MS/MS conditions used in method development.
10.2.2 Establish on-line SPE-LC/MS/MS operating parameters as described in Tables 1,
2 and Section 11. The on-line SPE and LC conditions (solvents, volume of
solvents, solvent modifiers) may not be modified.
10.2.3 Analyze a mid-level CAL standard under on-line SPE-LC/MS conditions to
obtain retention times of each method analyte. Divide the chromatogram into
retention time windows 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 5, although these will be instrument dependent. For
maximum sensitivity, small mass windows of ± 0.5 daltons around the product
ion mass were used for quantitation.
10.2.4 Analyze a mid-level CAL standard under optimized on-line SPE-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 7.2.3. 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 on-line SPE-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
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action is taken to reanalyze the CAL standards, restrict the range of calibration, or
select an alternate method of calibration.
CAUTION: When acquiring MS/MS data, on-line SPE-LC operating
conditions must be carefully reproduced for each analysis to
provide reproducible retention times (Sect. 11.4.1). 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. 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, 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 SDS may be customized to meet
this criteria. Subsequent CCCs should alternate between a medium and high
concentration CAL standard.
10.3.1 Analyze 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 in the CCC. 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,
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and Field Samples show no detection for that method analyte, non-detects
may be reported without re-analysis.
10.3.4 REMEDIAL ACTION - Failure to meet CCC QC performance criteria may
require remedial action. Major maintenance, such as cleaning the ESI probe,
atmospheric pressure ionization source, mass analyzer, replacing the LC column,
replacing the SPE cartridges, etc., requires recalibration (Sect 10.2) and
verification of sensitivity by analyzing a CCC at or below the MRL (Sect 10.3).
CAUTION: The trizma buffer is retained to some degree on the on-line SPE
cartridges and elutes over the whole chromatogram as a broad
band. Thus, fouling of the ESI source cones can occur over
time. It is highly recommended that the ESI probe tip be
positioned as far away from the MS orifice as possible while
maintaining enough sensitivity to meet the MRL. This will aid
in reducing the frequency of the source cleanings.
11. PROCEDURE
11.1 This procedure applies only to fully automated on-line SPE-LC/MS/MS. Data
presented in Tables 6-10 demonstrate data collected by automated on-line SPE-
LC/MS/MS. This method cannot be used for manual (off-line) SPE extractions.
NOTE: SPE cartridges described in this section are designed for multiple use.
During method development, on-line SPE cartridges were shown to be
accurate and precise up to a minimum of 330 injections. Provided QC
criteria in Section 9 are met, laboratories can use the on-line SPE cartridges
for longer periods.
NOTE: During method development, a dual SPE cartridge system was utilized as
displayed in Figures 2 and 3. The parallel two SPE cartridge system
provided the capability to inject on SPE cartridge #1 while conditioning SPE
cartridge #2, thereby increasing sample throughput. This dual on-line SPE
cartridge system is optional. Single on-line cartridge systems may be used
provided the SPE cartridge re-conditioning and re-equilibration steps in
Sections 11.3.6 and 11.3.7 are followed.
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 CCC, 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.
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11.2.2 Transfer a 10-mL aliquot of each sample to 20-mL (or smaller) amber
autosampler vials. Add an aliquot of the IS PDS (Sect. 7.2.1.2) to each sample to
be extracted, cap (caps with pre-slit septa) and invert to mix.
11.2.3 In addition to preservatives, if the sample is a CCC, FD, LFSM, or LFSMD, add
the necessary amount of analyte SDS (Sect. 7.2.2.3). Cap (caps with pre-slit
septa) and invert each sample to mix.
11.3 ON-LINE SPE-LC/MS/MS PROCEDURE
11.3.1 Establish operating conditions equivalent to those summarized in Tables 1-5 of
Section 17. Instrument MS conditions and LC columns should be optimized
prior to initiation of the IDC.
11.3.2 Inj ect a maximum of 2 mL of the preserved water sample. Inj ection volumes
of less than 2 mL may be used provided sufficient sensitivity can be obtained.
Smaller injection volumes will aid in reducing both matrix effects and fouling
of the ESI source. During method development, a 5-mL autosampler syringe
and 5-mL stainless steel loop were used to deliver the sample to the SPE
cartridge.
Note: During method development, the autosampler syringe was washed
with 10 mL of 50:50 acetonitrile:MeOH followed by 10 mL of
reagent water. Other solvents may be used, if necessary, to prevent
sample carryover, but solvents may not be added to the aqueous
wash as this will cause breakthrough of the analytes.
11.3.3 Load the sample in the loop onto the SPE cartridge with the loading pump
using 20 mM ammonium acetate (Sect. 7.1.5) at a flow rate of 2 mL/min. With
a 5-mL sample loop this takes about 3 minutes.
11.3.4 Wash the SPE cartridge with the loading pump using 20 mM ammonium
acetate (Sect. 7.1.5) at a flow rate of 2 mL/min for 4.1 minutes (Table IB).
11.3.5 Elute the analytes onto the analytical column by back flushing the cartridge
with the analytical pump using the gradient program in Table 1 A. The gradient
steps may be modified, but not the mobile phase constituents: 20 mM
ammonium acetate and acetontrile.
11.3.6 Condition the SPE cartridge (for the next injection) using the loading pump at a
flow rate of 2 mL/min with acetonitrile for 2.9 min. For a dual SPE cartridge
system, this conditioning step occurs concurrently (see Table IB) with the
elution step in Sect. 11.3.5. For a single SPE cartridge system, the conditioning
step is started after completion of the elution step in Section 11.3.5 (see
Table 2).
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11.3.7 Re-equilibrate the SPE cartridge to initial conditions using the loading pump at
a flow rate of 2 mL/min with 20 mM ammonium acetate (Sect. 7.1.5) for 4 min.
The SPE cartridge is now ready for the next injection. For a dual SPE cartridge
system, this re-equilibration step occurs concurrently (see Table IB) with the
elution step in Sect. 11.3.5. For a single SPE cartridge system, the re-
equilibration step is started after completion of the conditioning step in Section
11.3.6 (see Table 2).
NOTE: If different flow rates are used in Sections 11.3.3,11.3.4,11.3.6
and 11.3.7, then the times for each step must be modified to ensure
that the same amount of solvent is used in each step.
11.4 ANALYSIS
11.4.1 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
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.
11.4.2 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 CCC as described in
Section 10.3. If establishing an initial calibration, complete the IDC as described
in Section 9.2.
11.4.3 Begin analyzing Field Samples, including QC samples, at their appropriate
frequency by injecting the same size aliquots (2 mL was used in method
development), under the same conditions used to analyze the CAL standards.
11.4.4 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.
11.4.5 The analyst must not extrapolate beyond the established calibration range. If an
analyte peak area exceeds the range of the initial calibration curve, the sample
may be diluted with reagent water containing the preservatives. Re-analyze the
diluted sample. Incorporate the dilution factor into the final concentration
calculations. The resulting data should be documented as a dilution and MRLs
should be adjusted accordingly.
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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 5. Other
ions may be selected at the discretion of the analyst.
12.2 Calculate analyte concentrations using the multipoint calibration established in
Section 10.2. Do not use daily calibration verification data to quantitate analytes in
samples.
12.3 Prior to reporting data, the chromatogram should be reviewed for any incorrect peak
identification or poor integration.
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 6. Precision and accuracy at two concentration levels are presented
for three water matrices: reagent water (Table 7); chlorinated (finished) ground water
(Table 8); chloraminated/chlorinated (finished) surface water (Table 9).
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 10.
13.3 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: 1) Don
Noot and Ralph Hindle of Vogon Laboratory Services Ltd., Cochrane, AB, Canada
and 2) Karen A. Randazzo, Kevin Durk, and Amanda Comando of Suffolk County
Water Authority, Hauppauge, NY.
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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 April 2014).
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. Shoemaker J.A., Tettenhorst, D.R. U.S. EPA Method 540: Determination of Selected
Organic Chemicals in Drinking Water by Solid Phase Extraction and Liquid
Chromatography/Tandem Mass Spectrometry (LC/MS/MS), Revision 1.0, 2013,
EPA/600/R-13/119.
2. 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.
3. 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.
4. 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." Anal. Chem. 2001, 73, 1461-1471.
5. 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
543-26
-------
Extraction and High-Performance Liquid Chromatography Electrospray lonization Mass
Spectrometry." J. Chromatogr. A, 2004, 1041, 171-180.
6. "Prudent Practices in the Laboratory: Handling and Disposal of Chemicals," National
Academies Press (2011 updated version), ISBN: 9780309138642.
7. "OSHA Safety and Health Standards, General Industry," (29CFR1910), Occupational
Safety and Health Administration, OSHA 2206, (Revised, July 2001).
8. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 7th Edition. Available online at
http ://portal. acs.org/portal/PublicWeb Site/about/governance/committees/chemicalsafety/
publications/WPCP 012294 (accessed April 2014).
9. 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.
543-27
-------
17. TABLES, DIAGRAMS, FLOWCHARTS AND VALIDATION DATA
TABLE 1A. ANALYTICAL PUMP CONDITIONS (for dual cartridge system)'
Time (min)
Initial
3.80
4.10
9.00
9.10
Flow rate
mL/min
0.40
0.010
0.40
0.40
0.40
% 20 mM
Ammonium acetate
90
90
90
5.0
90
% Acetonitrile
10
10
10
95
10
Waters Acquity UPLC HSS T3 2.1 x 50 mm, 1.8 jam
a The events in this table are linear gradients. The reduction in flow rate at 3.8 min is to
eliminate potential pressure spikes.
TABLE IB. LOADING PUMP (SPE) CONDITIONS (for dual cartridge system)"
Time (min)
Initial
4.10
7.00
11.00
Flow rate
mL/min
2.0
2.0
2.0
2.0
% 20 mM
Ammonium acetate
100
0
100
100
% Acetonitrile
0
100
0
0
a The events in this table are step movements, not gradients (Sect, 11.3.6 & 11.3.7).
For example, at 4.1 minutes the pump is immediately stepped to 100% acetonitrile
and is constant for 2.9 min.
543-28
-------
TABLE 2. LOADING AND SPE CONDITIONS (for single cartridge system")
Loading Pumpb
Time
(min)
Initial
4.0
4.1
9.0
9.1
12.1
12.2
16.2
Flow rate
mL/min
2.0
2.0
0.10d
0.10
2.0
2.0
2.0
2.0
% 20 mM
Ammonium
acetate
100
100
100
100
0
0
100
100
% Acetonitrile
0
0
0
0
100
100
0
0
Analytical Pumpc
Time
(min)
Initial
3.8e
4.1
9.0
9.1
Flow rate
mL/min
0.40
0.010
0.40
0.40
0.40
% 20 mM
Ammonium
acetate
90
90
90
5.0
90
%
Acetonitrile
10
10
10
95
10
a These single cartridge parameters were not used during method development but were extrapolated from
the dual cartridge parameters for demonstration purposes.
b The events in these columns are step movements, not gradients (Sect, 11.3.6 & 11.3.7). For example, at
4.1 minutes the pump is immediately stepped to 100% acetonitrile and is constant for 5 min
c The events in these columns are linear gradients.
dThe reduction in flowrate is optional but will minimize solvent consumption during the elution step in the
single cartridge system.
e The reduction in flowrate at 3.8 min is to eliminate potential pressure spikes.
TABLE 3. ESI-MS METHOD CONDITIONS"
ESI Conditions
Polarity
Capillary needle voltage
Cone gas flow
Nitrogen desolvation gas
Desolvation gas temp.
Positive ion
4kV
50 L/hr
800 L/hr
450 °C
a The nomenclature used in this table is instrument specific.
Other instruments may use different nomenclature.
543-29
-------
TABLE 4. METHOD ANALYTE SOURCE, RETENTION
TIMES (RTs), AND SUGGESTED IS REFERENCES
Analyte
3 -hydroxycarbofuran
fenamiphos sulfone
fenamiphos sulfoxide
fenamiphos
tebuconazole
tebufenozide
bensulide
methomyl-13C2,15N (IS#1)
carbofuran-13C6 (IS#2)
bensulide-Ji4(IS#3)
Peak#
(Fig. 1)
2
3
4
6
7
8
10
1
5
9
RT
(min)
5.91
6.31
6.70
7.59
7.75
8.06
8.27
5.58
6.82
8.25
IS# Ref
1
2
2
3
3
3
3
_
_
-
TABLE 5. MS/MS METHOD CONDITIONS
a,b
Segment0
1
1
1
2
2
2
2
1
1
2
Analyte
3 -hydroxycarbofuran
fenamiphos sulfoxide
fenamiphos sulfone
fenamiphos
tebuconazole
tebufenozide
bensulide
methomyl-13C2,15N
carbofuran-13Ce
bensulide-Ji4
Precursor
\onA(m/z)
238
320
336
304
308
353
398
166
228
412
Product
Iond'e (m/z)
181
233
266
217
70
133
314
91
171
316
Cone
Voltage (v)
20
32
28
28
26
12
20
15
20
22
Collision
Energyf (v)
10
26
22
24
20
22
12
10
10
12
a The nomenclature used in this table is instrument specific. Other instruments may use different
nomenclature.
b An LC/MS/MS chromatogram of the analytes is shown in Figure 1.
c 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.
e Ions used for quantitation purposes.
f Argon used as collision gas at a flow rate of 0.15 mL/min.
543-30
-------
TABLE 6. DLs AND LCMRLs IN REAGENT WATER
Analyte
3 -hydroxycarbofuran
fenamiphos sulfoxide
fenamiphos sulfone
fenamiphos
tebuconazole
tebufenozide
bensulide
Fortified
Cone. (ng/L)a
1.0
1.0
1.0
0.4
0.4
0.4
1.0
DLb (ng/L)
0.99
0.60
0.63
0.13
0.47
0.26
0.64
LCMRLC
(ng/L)
1.7
1.2
1.4
0.27
1.3
0.47
1.2
a Spiking concentration used to determine DL.
Detection limits were determined by analyzing seven replicates over three days according to
Section 9.2.6.
c LCMRLs were calculated according to the procedure in reference 2.
TABLE 7. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES IN
FORTIFIED REAGENT WATER (n=5)
Analyte
3 -hydroxycarbofuran
fenamiphos sulfoxide
fenamiphos sulfone
fenamiphos
tebuconazole
tebufenozide
bensulide
methomyl-13C2,15N
carbofuran-13Ce
bensulide-Ji4
Fortified
Cone. (ng/L)
50
50
50
20
20
20
50
80
32
80
Mean %
Recovery
96.8
96.3
94.6
100
99.9
95.6
92.0
97.2
100
96.1
% RSD
9.1
1.7
5.5
4.5
3.6
5.3
2.0
5.6
2.7
4.3
Fortified
Cone. (ng/L)
5.0
5.0
5.0
2.0
2.0
2.0
5.0
80
32
80
Mean %
Recovery
100
102
101
105
112
101
95.2
97.6
100
94.6
% RSD
4.1
4.0
4.4
3.7
5.4
3.6
2.9
3.4
2.9
2.8
543-31
-------
TABLE 8. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES
FORTIFIED FINISHED DRINKING WATER FROM A GROUND WATER
SOURCE8 (n=5)
Analyte
3 -hydroxycarbofuran
fenamiphos sulfoxide
fenamiphos sulfone
fenamiphos
tebuconazole
tebufenozide
bensulide
methomyl-13C2,15N
carbofuran-13Ce
bensulide-Ji4
Fortified
Cone. (ng/L)
50
50
50
20
20
20
50
80
32
80
Mean %
Recovery
90.1
100
101
107
86.4
98.1
99.8
106
98.7
96.0
% RSD
5.5
4.0
2.2
2.7
2.6
2.5
2.0
7.1
5.1
2.9
Fortified
Cone. (ng/L)
5.0
5.0
5.0
2.0
2.0
2.0
5.0
80
32
80
Mean %
Recovery
98.2
106
97.6
97.9
98.1
105
100
102
99.5
94.6
% RSD
8.5
3.3
3.9
5.0
o o
J.J
9.1
5.3
4.5
2.1
3.2
TOC = 0.71 mg/L and hardness = 342 mg/L as calcium carbonate
TABLE 9. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES
FORTIFIED IN FINISHED DRINKING WATER FROM A SURFACE WATER
SOURCE8 (n=5)
Analyte
3 -hydroxycarbofuran
fenamiphos sulfoxide
fenamiphos sulfone
fenamiphos
tebuconazole
tebufenozide
bensulide
methomyl-13C2,15N
carbofuran-13Ce
bensulide-Ji4
Fortified
Cone. (ng/L)
50
50
50
20
20
20
50
80
32
80
Mean %
Recovery
98.2
95.3
99.5
99.2
99.4
105
106
112
103
103
% RSD
4.7
3.2
3.8
2.1
6.9
1.1
4.7
4.3
1.9
2.2
Fortified
Cone. (ng/L)
5.0
5.0
5.0
2.0
2.0
2.0
5.0
80
32
80
Mean %
Recovery
108
95.4
94.1
91.9
87.6
97.5
89.8
98.3
97.4
98.3
% RSD
14
2.1
6.7
3.8
5.6
5.1
3.8
4.3
2.2
1.7
TOC = 3.20 mg/L and hardness = 68.5 mg/L as calcium carbonate
543-32
-------
TABLE 10. 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=5)b
Analyte
3 -hydroxycarbofuran
fenamiphos sulfoxide
fenamiphos sulfone
fenamiphos
tebuconazole
tebufenozide
bensulide
methomyl-13C2,15N
carbofuran-13Ce
bensulide-Ji4
Fortified
Cone.
(ng/L)
50
50
50
20
20
20
50
80
32
80
DayO
Mean
%Rec
115
108
109
109
96.2
109
105
97.1
97.1
85.9
%
RSD
3.8
3.1
3.6
1.8
4.1
4.4
2.2
2.9
2.6
2.6
Day 7
Mean
%Rec
114
107
107
104
110
116
105
96.4
97.9
101
%
RSD
3.8
2.6
0.9
2.9
4.7
2.1
3.8
3.0
2.2
4.7
Day 14
Mean
%Rec
112
106
104
107
112
113
106
99.1
102
97.8
%
RSD
5.0
2.9
3.2
2.2
4.7
0.9
3.8
2.2
1.7
1.6
Day 22
Mean
%Rec
100
110
109
105
102
112
102
97.9
97.8
94.7
%
RSD
7.3
4.0
4.9
6.9
5.3
4.9
8.0
5.1
5.0
5.0
Day 28
Mean
%Rec
94.1
110
110
99.3
108
105
103
106
99.7
100
%
RSD
3.9
2.9
3.2
3.4
2.2
3.2
3.2
3.5
1.5
1.3
a TOC = 0.72 mg/L and hardness =154 mg/L as calcium carbonate.
b Internal standards were not added to samples until the day of extraction.
543-33
-------
TABLE 11. 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 9.3.7
Sect. 9.2.6
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)
Detection Limit (DL)
Determination (optional)
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.
Over a period of three days, prepare a minimum of
seven replicate LFBs fortified at a concentration
estimated to be near the DL. Analyze the
replicates through all steps of the analysis.
Calculate the DL using the equation in Sect. 9.2.6.
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.
Data from DL replicates are not required to meet method
precision and accuracy criteria. If the DL replicates are
fortified at a low enough concentration, it is likely that they
will not meet precision and accuracy criteria.
NOTE: Table 11 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.
543-34
-------
TABLE 12. ONGOING QUALITY CONTROL REQUIREMENTS (SUMMARY)
Method
Reference
Sect. 8.1-
Sect. 8.4
Sect. 9.3.1
Sect. 9.3.4
Sect. 9.3.5
Sect. 9.3.6
Sect. 9.3.7
Sect. 10.2
Sect. 9.3.2
and Sect.
10.3
Requirement
Sample Holding Time
Laboratory Reagent Blank
(LRB)
Internal Standard (IS)
Laboratory Fortified
Sample Matrix (LFSM)
Laboratory Fortified
Sample Matrix Duplicate
(LFSMD) or
Field Duplicates (FD)
Quality Control Sample
(QCS)
Initial Calibration
Continuing Calibration
Check (CCC)
Specification and Frequency
28 days with appropriate preservation and storage as
described in Sections 8.1-8.4.
Analyze one LRB with each extraction batch of up to
20 field samples
Internal standards, methomyl-13C2,15N, carbofuran-
13Ce, andbensulide-<5?i4, are added to all standards and
sample extracts, including QC samples. Compare IS
areas to the average IS area in the initial calibration.
Analyze one LFSM per extraction batch (20 samples
or less) fortified with method analytes at a
concentration greater than or equal to the native
concentration, if known. Calculate LFSM recoveries.
Extract and analyze at least one FD or LFSMD with
each extraction batch (20 samples or less). A
LFSMD may be substituted for a FD when the
frequency of detects are low. Calculate RPDs.
Analyze at least quarterly or when preparing new
standards, as well as during the IDC.
Use IS calibration technique to generate a first or
second order calibration curve. Use at least five
standard concentrations. Check the calibration curve
as described in Sect. 10.2.7.
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.
Acceptance Criteria
Sample results are valid only if samples are extracted within the
sample holding time.
Demonstrate that all method analytes are below 1/3 the MRL, and
confirm that possible interferences do not prevent quantification of
method analytes. If targets exceed 1/3 the MRL or if interferences
are present, results for these subject analytes in the extraction batch
are invalid.
Peak area counts for all ISs in all injections must be within + 50% of
the average peak area calculated during the initial calibration. If ISs
do not meet this criterion, corresponding target results are invalid.
For LFSMs fortified at concentrations < 2 x MRL, the calculated
recovery must be within ± 50% of the true value. At concentrations
greater than the 2 x MRL, the recovery must be ± 30% of the true
value. If these criteria are not met, results are labeled suspect due to
matrix effects.
For LFSMDs or FDs, the calculated relative percent difference must
be < 30%. (< 50% if concentration < 2 x MRL.) If these criteria are
not met, results are labeled suspect due to matrix effects.
Results must be within 70-130% of true value.
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. 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.
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 12 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.
543-35
-------
FIGURE 1. EXAMPLE CHROMATOGRAM (OVERLAID MS/MS SEGMENTS) OF A CALIBRATION STANDARD WITH
METHOD 543 ANALYTES AT CONCENTRATION LEVELS OF 3.2-8.0 ug/L. NUMBERED PEAKS ARE
IDENTIFIED IN TABLE 4.
100
9,10
u
C
93
•a
e
=
4.00 4.50 5.00 5.50 6,00 6.50 7.00
Retention Time, min
7.50 8,00 8.50 9.00
543-36
-------
FIGURE 2. ON-LINE SPE-LC/MS/MS DIAGRAMS FOR A DUAL CARTRIDGE SPE SYSTEM
SPE
Injection port
Analytical
Injection port
ToLC
Loading
pump
SPE
Injection port
Analytical
pump
Analytical
Injection port
ToLC
Optional
To waste
Loading
pump
Analytical
pump
B
SPE
Injection port
Analytical
Injection port
ToLC
Loading
pump
Analytical
pump
A: Sample injection
B: Sample loading and washing of SPE
cartridge
C: Sample elution while conditioning and
equilibrating optional second SPE
cartridge
Diagrams courtesy of Waters Corp.
543-37
-------
FIGURE 3. ON-LINE SPE EVENTS FOR AN SPE DUAL CARTRIDGE SYSTEM
Start
Sample loading
20 mM NH4OAc
Loading Pump
SPE wash
20 mM NH4OAc
Loading Pump
5 min gradient
elution
20 mM NH4OAc
ACN
Re-equilibration
Analytical Pump
SPE Cartridge #1
Analytical Pump
10
nTime, min
12
Start
>
1 '
1
Analytical column
initial conditions .
20 mM NH.OAc
4
ACN
Analytical Pump
K ^
9 2
Re-condition
J
ACN
Loading
Pump
f \
4 6
Re-equilibration
J
20 mM NH4OAc
Loading
Pump
f >
8 10
SPE Cartridge #2
f
Timp m
12
543-38
------- |