METHOD 526. DETERMINATION OF SELECTED SEMIVOLATILE ORGANIC
COMPOUNDS IN DRINKING WATER BY SOLID PHASE EXTRACTION
AND CAPILLARY COLUMN GAS CHROMATOGRAPHY/ MASS
SPECTROMETRY (GC/MS)
Revision 1.0
S.D. Winslow, B. Prakash, M.M. Domino, and B.V. Pepich (IT Corporation) - June 2000
D. J. Munch (U.S. EPA, Office of Ground Water and Drinking Water)
NATIONAL EXPOSURE RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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METHOD 526
DETERMINATION OF SELECTED SEMIVOLATILE ORGANIC COMPOUNDS IN
DRINKING WATER BY SOLID PHASE EXTRACTION AND CAPILLARY COLUMN GAS
CHROMATOGRAPHY/ MASS SPECTROMETRY (GC/MS)
1. SCOPE AND APPLICATION
1.1 This is a gas chromatography/mass spectrometry (GC/MS) method for the determination
of selected semivolatile organic compounds in raw and finished drinking waters. This
method is applicable to the organic compounds listed below, which are efficiently
extracted from water using a polystyrene divinylbenzene solid phase sorbent, and are
sufficiently volatile and thermally stable for gas chromatography. Accuracy, precision,
and method detection limit (MDL) data have been generated in reagent water, finished
ground and surface water for the following compounds:
Chemical Abstract Services
Analyte Registry Number
Acetochlor 34256-82-1
Cyanazine 21725-46-2
Diazinon 61790-53-2
2,4-Dichlorophenol 120-83-2
1,2-Diphenylhydrazine 122-66-7
Disulfoton 298-04-4
Fonofos 944-22-9
Nitrobenzene 98-95-3
Prometon 1610-18-0
Terbufos 13071-79-9
2,4,6-Trichlorophenol 88-06-2
1.2 MDLs are compound, instrument, and matrix dependent. The MDL is defined as the
statistically calculated minimum concentration that can be measured with 99%
confidence that the reported value is greater than zero.(1) Experimentally determined
MDLs for the above listed analytes are provided in Section 17, Table 3. The MDL differs
from, and is lower than, the minimum reporting limit (MRL) (Sect. 3.17). Precision and
accuracy were evaluated at 0.5 and 20 ug/L. Precision and accuracy data and sample
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holding time data are presented Section 17, Tables 4 through 8. Analyte retention times
are in Section 17, Table 2.
1.3 This method is restricted to use by or under the supervision of analysts skilled in solid-
phase extractions (SPE) and GC/MS analysis.
2. SUMMARY OF METHOD
2.1 A 1 liter water sample is passed through a SPE disk or cartridge containing
polystyrenedivinylbenzene (SDVB) to extract the target analytes and surrogate
compounds. The extract is dried by passing through a column of anhydrous sodium
sulfate and is concentrated by blowdown with nitrogen to a volume of about 0.7 mL.
Internal standards are added and the extract is diluted to a final volume of 1 mL.
Components are separated chromatographically by injecting an aliquot of the extract onto
a gas chromatograph equipped with a high resolution fused silica capillary column. The
analytes pass from the capillary column into a mass spectrometer where the they are
identified by comparing their measured mass spectra and retention times to reference
spectra and retention times collected for the same compounds. Instrument specific
reference spectra and retention times for analytes are obtained by the analyses of
calibration standards under the same GC/MS conditions used for samples. The
concentration of each identified component is measured by relating the MS response of
the compound's quantitation ion to the internal standard's quantitation ion MS response.
3. DEFINITIONS
3.1 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 solid
phase extraction devices, solvents, surrogate solution, and fortifying solutions. Required
QC samples include Laboratory Reagent Blank, Laboratory Fortified Blank, Laboratory
Fortified Matrix, and either a Field Duplicate or Laboratory Fortified Matrix Duplicate.
3.2 ANALYSIS BATCH - A set of samples that is analyzed on the same instrument during a
24-hour period that begins and ends with the analysis of the appropriate Continuing
Calibration Check standards (CCC). Additional CCCs may be required depending on the
length of the analysis batch and/or the number of Field Samples.
3.3 INTERNAL STANDARD (IS) - A pure analyte added to an extract or standard solution
in a known amount and used to measure the relative responses of other method analytes
and surrogates. The internal standard must be an analyte that is not a sample component.
3.4 SURROGATE ANALYTE (SUR) - A pure analyte, which is extremely unlikely to be
found in any sample, and which is added to a sample aliquot in a known amount before
extraction or other processing, and is measured with the same procedures used to measure
other sample components. The purpose of the SUR is to monitor method performance
with each sample.
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3.5 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, reagents, sample preservatives, internal standards, and surrogates
that are used in the extraction batch. The LRB is analyzed is used to determine if method
analytes or other interferences are present in the laboratory environment, the reagents, or
the apparatus.
3.6 LABORATORY FORTIFIED BLANK (LFB) - An aliquot of reagent water or other
blank matrix to which known quantities of the method analytes and all the preservation
compounds are added. 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.
3.7 LABORATORY FORTIFIED SAMPLE MATRIX (LFM) - An aliquot of an
environmental sample to which known quantities of the method analytes and all the
preservation compounds are added in the laboratory. The LFM is analyzed exactly like a
sample, and its purpose is to determine whether the sample matrix contributes bias to the
analytical results. The background concentrations of the analytes in the sample matrix
must be determined in a separate aliquot and the measured values in the LFM corrected
for background concentrations.
3.8 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFMD) - A second
aliquot of the Field Sample used to prepare the LFM which is fortified, extracted and
analyzed identically. The LFMD is used instead of the Field Duplicate to access method
precision and accuracy when the occurrence of target analytes is low.
3.9 LABORATORY DUPLICATES (LD1 and LD2) - Two aliquots of the same sample
taken in the laboratory and analyzed separately with identical procedures. Analyses of
LD1 and LD2 indicate precision associated with laboratory procedures, but not with
sample collection, preservation, and storage procedures.
3.10 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 with
laboratory procedures.
3.11 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.
3.12 PRIMARY DILUTION STANDARD SOLUTION (PDS) - A solution containing
method analytes prepared in the laboratory from stock standard solutions and diluted as
needed to prepare calibration solutions and other needed analyte solutions.
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3.13 CALIBRATION STANDARD (CAL) - A solution prepared from the primary dilution
standard solution or stock standard solutions and the internal standards and surrogate
analytes. The CAL solutions are used to calibrate the instrument response with respect to
analyte concentration.
3.14 CONTINUING CALIBRATION CHECK (CCC) - A calibration standard containing one
or more method analytes, which is analyzed periodically to verify the accuracy of the
existing calibration for those analytes.
3.15 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. It is used to check standard integrity.
3.16 METHOD DETECTION LIMIT (MDL) - The minimum concentration of an analyte that
can be identified, measured and reported with 99% confidence that the analyte
concentration is greater than zero (Section 9.2.4). This is a statistical determination of
precision. Accurate quantitation is not expected at this level.(1)
3.17 MINIMUM REPORTING LEVEL (MRL) - The minimum concentration that can be
reported as a quantitated value for a target analyte in a sample following analysis. This
defined concentration can be no lower than the concentration of the lowest continuing
calibration check standard for that analyte, and can only be used if acceptable quality
control criteria for this standard are met.
3.18 MATERIAL SAFETY DATA SHEET (MSDS) - Written information provided by
vendors concerning a chemical's toxicity, health hazards, physical properties, fire, and
reactivity data including storage, spill, and handling precautions.
4. INTERFERENCES
4.1 All glassware must be meticulously cleaned. Wash glassware with detergent and tap
water, rinse with tap water, followed by reagent water. Non-volumetric glassware can be
heated in a muffle furnace at 400 °C for 2 hours as a substitute for a solvent rinse.
Volumetric glassware should 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 the chromatograms. All items such as
these must be routinely demonstrated to be free from interferences (less than V3 the MRL
for each target analyte) under the conditions of the analysis by analyzing laboratory
reagent blanks as described in Section 9.4. 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,
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depending upon the nature of the water. Water samples high in total organic carbon may
have elevated baseline or interfering peaks.
4.4 Relatively large quantities of the buffer and preservatives (Sect. 8.1) are added to sample
bottles. The potential for trace-level organic contaminants in these reagents exist.
Interferences from these sources should be monitored by analysis of laboratory reagent
blanks, particularly when new lots of reagents are acquired.
4.5 Benzophenone was an interferent recovered at low level from one manufacturer's lot of
tris(hydroxymethyl)aminomethane hydrochloride. The spectra of benzophenone and 1,2-
diphenylhydrazine are very similar, sharing the major m/z ions of 51, 77, 105, 152, and
182. At first glance, the benzophenone may appear to be a contaminant in the LRB.
Given that the two compounds share common ions, their chromatographic peaks must be
resolved.
4.6 During method development, one lot of anhydrous sodium sulfate was found to add an
agent to the extract that, when injected, caused complete loss of prometon recovery and
caused severe chromatographic tailing of the phenols. It is very important to check a new
sodium sulfate lot before general use. When only one or two samples with the interfering
agent were injected, recovery of good chromatographic performance was possible by
removing the first meter of the capillary column and replacing the deactivated glass inlet
liner.
4.7 Solid phase cartridges and disks and their associated extraction devices have been
observed to be a source of interferences. The analysis of field and laboratory reagent
blanks can provide important information regarding the presence or absence of such
interferences. Brands and lots of solid phase extraction devices should be tested to ensure
that contamination will not preclude analyte identification and quantitation.
4.8 Analyte carryover may occur when a relatively "clean" sample is analyzed immediately
after a sample containing relatively high concentrations of compounds. Syringes and
splitless injection port liners must be cleaned carefully or replaced as needed. After
analysis of a sample, containing high concentrations of compounds, a laboratory reagent
blank should be analyzed to ensure that accurate values are obtained for the next sample.
4.9 Silicone compounds may be leached from punctured autosampler vial septa, particularly
when particles of the septa sit in the vial. These silicone compounds should have no
effect on the analysis, but the analyst should be aware of this potential problem.
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
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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.(2"4)
5.2 Pure standard materials and stock standard solutions of these compounds should be
handled with suitable protection to skin and eyes, and care should be taken not to breath
the vapors or ingest the materials.
6. EQUIPMENT AND SUPPLIES (All specifications are suggested. Catalog numbers are
included for illustration only.)
6.1 SAMPLE CONTAINERS - 1 liter glass bottles fitted with polytetrafluoroethylene
(PTFE) lined screw caps.
6.2 VIALS - Various sizes of amber glass vials with PTFE lined screw caps for storing
standard solutions and extracts. Crimp-top glass autosampler vials, 2 mL, with PTFE
faced septa.
6.3 VOLUMETRIC FLASKS - Class A, suggested sizes include 1, 5, and 10 mL, for
preparation of standards and dilution of extract to final volume.
6.4 GRADUATED CYLINDERS - Suggested sizes include 5, 10, and 250 mL.
6.5 MICRO SYRINGES - Suggested sizes include 25, 50, 100, 250, 500, and 1000 mL.
6.6 DRYING COLUMN - The drying column must be able to contain 5 to 7 g of anhydrous
sodium sulfate. The drying column should not leach interfering compounds or
irreversibly adsorb target analytes. Any small column may be used, such as a glass pipet
with glass wool plug.
6.7 CONICAL CENTRIFUGE TUBES - 15 mL, or other glassware suitable for collection of
the eluate from the cartridge or disk after extraction.
6.8 COLLECTION TUBES OR VIALS - 25 mL or larger, or other glassware suitable for
collecting extract from drying tube. Conical centrifuge tubes, 50 mL, with graduations
(VWR #: 21049-063) were used to develop this method.
6.9 ANALYTICAL BALANCE - Capable of weighing to the nearest 0.0001 g.
6.10 SOLID PHASE EXTRACTION (SPE) APPARATUS USING CARTRIDGES
6.10.1 SOLID PHASE EXTRACTION CARTRIDGES - 6 mL, packed with 500 mg
(125 um dp) of polystyrene divinylbenzene (SDVB) sorbent phase (Varian Bond
Elut ENV phase; cat.#: 1225-5011 or equivalent).
6.10.2 SAMPLE RESERVOIR AND TRANSFER TUBE- Sample reservoirs (VWR
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cat.#: JT7120-3 or equivalent) with a volume of about 75 mL are attached to the
cartridges to hold the water sample. An alternative method is using transfer tubes
(Supelco "Visiprep"; cat.#: 57275 or equivalent) which transfer the sample
directly from the sample container to the SPE cartridge.
6.10.3 VACUUM EXTRACTION MANIFOLD - With flow/vacuum control (Supelco
cat.#: 57044 or equivalent). The use of replaceable needles or valve liners may be
used to prevent cross contamination.
6.10.4 REMOTE VACUUM GAUGE/BLEED VALVE ASSEMBLY - To monitor and
adjust vacuum pressure delivered to the vacuum manifold (Supelco cat.#: 57161-
U or equivalent)
6.10.5 An automatic or robotic system, designed for use with SPE cartridges, may be
used as long as all quality control requirements discussed in Section 9 are met.
Automated systems may use either vacuum or positive pressure to process
samples and solvents through the cartridge. All extraction and elution steps must
be the same as in the manual procedure. Extraction or elution steps may not be
changed or omitted to accommodate the use of an automated system.
6.11 SOLID PHASE EXTRACTION (SPE) APPARATUS USING DISKS
6.11.1 SOLID PHASE EXTRACTION DISKS - 47 mm diameter and 0.5 mm thick,
manufactured with a polystyrene divinylbenzene (SDVB) sorbent phase (Varian
cat. #: 1214-5010 or equivalent). Larger disks may be used as long as the QC
performance criteria outlined in Section 9 are met.
6.11.2 SPE DISK EXTRACTION GLASSWARE - funnel, PTFE coated support screen,
PTFE gasket, base, and clamp used to support SPE disks and contain samples
during extraction. May be purchased as a set (Fisher cat. #:K971100-0047 or
equivalent) or separately.
6.11.3 VACUUM EXTRACTION MANIFOLD - Designed to accommodate extraction
glassware and disks (Varian cat.#: 1214-6001 or equivalent).
6.11.4 An automatic robotic system for disks as described in Section 6.10.5.
6.12 EXTRACT CONCENTRATION SYSTEM - Extracts are concentrated by blowdown
with nitrogen using water bath set to 40°C (Meyer N-Evap, Model 111, Organomation
Associates, Inc. or equivalent).
6.13 LABORATORY OR ASPIRATOR VACUUM SYSTEM - Sufficient capacity to
maintain a vacuum of about 10 inches of mercury for cartridges. A greater vacuum of 15
to 25 inches of mercury may be used with disks.
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6.14 GAS CHROMATOGRAPH/MASS SPECTROMETER (GC/MS)
INSTRUMENTATION
6.14.1 FUSED SILICA CAPILLARY GC COLUMN - 30 m x 0.25 mm i.d. fused silica
capillary column coated with a 0.25 um bonded film of
poly(dimethylsiloxy)poly(l,4-bis(dimethylsiloxy)phenylene)siloxane(J&WDB-
5MS or equivalent). Any capillary column that provides adequate resolution,
capacity, accuracy, and precision as summarized in Section 17 may be used. A
nonpolar, low-bleed column is recommended for use with this method to provide
adequate chromatography and minimize column bleed.
6.14.2 GC INJECTOR AND OVEN - Capable of temperature programming and
equipped for split/splitless injection. Target compounds included in this method
are subject to thermal breakdown in the injector port, which increases when the
injector is not properly deactivated or at excessive temperatures. The injection
system must not allow analytes to contact hot stainless steel or other metal
surfaces that promote decomposition. The performance data in Section 17 was
obtained by hot, splitless injection using a 4 mm i.d. glass, deactivated liner
(Restek cat.#: 20772). Other injection techniques such as temperature
programmed injections, cold on-column injections and large volume injections
may be used if the QC criteria in Sections 9 and 10 are met. Equipment designed
appropriately for these alternate types of injections must be used if these options
are selected.
6.14.3 GC/MS INTERFACE - Interface should allow the capillary column or transfer
line exit to be placed within a few mm of the ion source. Other interfaces, like jet
separators, are acceptable as long as the system has adequate sensitivity and QC
performance criteria are met.
6.14.4 MASS SPECTROMETER - The MS must be capable of electron ionization at a
nominal electron energy of 70 eV to produce positive ions. The spectrometer
must be capable of scanning at a minimum from 45 to 450 amu with a complete
scan cycle time (including scan overhead) of 1.0 second or less. (Scan cycle time
= total MS data acquisition time in seconds divided by number of scans in the
chromatogram). The spectrometer must produce a mass spectrum that meets all
criteria in Table 1 when a solution containing approximately 5 ng of DFTPP is
injected into the GC/MS. Use a single spectrum at the apex of the DFTPP peak,
an average spectrum of the three highest points of the peak, or an average
spectrum across the entire peak to evaluate the performance of the system. The
scan time should be set so that all analytes have a minimum of five scans across
the chromatographic peak. Seven to ten scans across chromatographic peaks are
preferred.
6.14.5 DATA SYSTEM - An interfaced data system is required to acquire, store, and
output mass spectral data. The computer software should have the capability of
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processing stored GC/MS data by recognizing a GC peak within a given retention
time window. The software must allow integration of the ion abundance of any
specific ion between specified time or scan number limits. The software must be
able to calculate relative response factors, construct a linear regression or
quadratic calibration curve, and calculate analyte concentrations.
7. REAGENTS AND STANDARDS
7.1 REAGENTS AND SOLVENTS - Reagent grade or better chemicals should be used in all
tests. Unless otherwise indicated, it is intended that all reagents will conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society (ACS), 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 HELIUM - 99.999% or better, GC carrier gas.
7.1.2 REAGENT WATER - Purified water which does not contain any measurable
quantities of any target analytes or interfering compounds at or above 1/3 the
MRL for each compound of interest.
7.1.3 METHANOL (CAS# 67-56-1) - High purity, demonstrated to be free of analytes
and interferences (B&J Brand GC2®, Capillary GC/GC-MS Grade or equivalent).
7.1.4 ETHYL ACETATE (CAS# 141 -78-6) - High purity, demonstrated to be free of
analytes and interferences (B&J Brand GC2®, Capillary GC/GC-MS Grade or
equivalent).
7.1.5 METHYLENE CHLORIDE (CAS# 75-09-02) - High purity, demonstrated to be
free of analytes and interferences (B&J Brand GC2®, Capillary GC/GC-MS Grade
or equivalent).
7.1.6 SODIUM SULFATE, ANHYDROUS (CAS# 7757-82-6) - Soxhlet extracted
with methylene chloride for a minimum of four hours or heated to 400°C for two
hours in a muffle furnace. One lot of "ACS grade" anhydrous sodium sulfate had
a contaminant that degraded the capillary column so that analyte recoveries were
unacceptable. An "ACS grade, suitable for pesticide residue analysis," or
equivalent, of anhydrous sodium sulfate is recommended.
7.1.7 SAMPLE PRESERVATION REAGENTS - These preservatives are solids at
room temperature and may be added to the sample bottle before shipment to the
field.
7.1.7.1 BUFFER SALT MIX, pH 7 - The sample must be buffered to pH 7 with
two components: 1) tris(hydroxymethyl)aminomethane, also called Tris,
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0.47 g (CAS# 77-86-1, ACS Reagent Grade or equivalent); and 2)
tris(hydroxymethyl)aminomethane hydrochloride, also called Tris HC1,
7.28 g (CAS# 1185-53-1, ACS Reagent Grade or equivalent). Alternately,
7.75 g of a commercial buffer crystal mixture, that is blended in proportion
to the amounts given above, can be used.
7.1.7.2 L-ASCORBIC ACID (CAS# 50-81-7) - Ascorbic acid reduces "free
chlorine" at the time of sample collection (ACS Reagent Grade or
equivalent).
7.1.7.3 ETHYLENEDIAMINETETRAACETIC ACID, TRISODIUM SALT
(Trisodium EDTA, CAS# 10378-22-0) - Trisodium EDTA is added to
inhibit metal-catalyzed hydrolysis of analytes. The trisodium salt is used
instead of the disodium salt because the trisodium salt solution pH is
closer to the desired pH of 7 (Sigma cat.#: ED3SS or equivalent).
7.1.7.4 DIAZOLIDINYL UREA (DZU) (CAS# 78491-02-8) - DZU is added to
inhibit microbial growth. DZU (Sigma cat.#: D-5146) is commonly used
as a preservative in cosmetics such as skin lotion.
STANDARD SOLUTIONS - When a compound purity is assayed to be 96% or greater,
the weight can be used without correction to calculate the concentration of the stock
standard. Solution concentrations listed in this section were used to develop this method
and are included as an example. Standards for sample fortification generally should be
prepared in the smallest volume that can be accurately measured to minimize the addition
of excess organic solvent to aqueous samples. Even though stability times for
standard solutions are suggested in the following sections, laboratories should used
standard QC practices to determine when their standards need to be replaced.
7.2.1 INTERNAL STANDARD SOLUTIONS - This method uses three internal
standard compounds listed in the table below.
Internal Standards
acenaphthene-t/10
phenanthrene-6?10
chrysene-t/12
CAS#
15067-26-2
1517-22-2
1719-03-5
FW
164.3
188.3
240.4
7.2.1.1 INTERNAL STANDARD PRIMARY DILUTION STANDARD (500
ug/mL) - Prepare or purchase commercially the Internal Standard Primary
Dilution Standard (PDS) at a concentration of 500 ug/mL. If prepared
from neat or solid standards, this solution requires the preparation of a
more concentrated stock standard similar to the procedure followed for the
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analyte stock (Sect. 7.2.3.1). The solvent for the Internal Standard PDS
may be acetone or ethyl acetate. The Internal Standard PDS has been
shown to be stable for 1 year in amber glass screw cap vials when stored at
• 10°Corless.
7.2.1.2 INTERNAL STANDARD EXTRACT FORTIFICATION SOLUTION (50
ug/mL) - Dilute a portion of the Internal Standard PDS (500 ug/mL)
(Sect. 7.2.1.1) to a concentration of 50 ug/mL in ethyl acetate and use this
solution to fortify the final 1 mL extracts (Sect. 11.6). The Internal Extract
Fortification Solution has been shown to be stable in amber glass screw
cap vials for 6 months when stored at • 10°C or less.
SURROGATE (SUR) ANALYTE STANDARD SOLUTIONS - The two
surrogates for this method are listed in the table below.
Surrogates
l,3-dimethyl-2-nitrobenzene
triphenylphosphate
CAS#
81-20-9
115-86-6
FW
151.2
326.3
7.2.3
7.2.2.1 SUR STOCK SOLUTION (~ 4 to 10 mg/mL) - Surrogate Stock Solutions
may be purchased commercially or prepared from neat materials. The
solvent for the SUR Stock Solution may be acetone or ethyl acetate.
These solutions have been shown to be stable for one year when stored in
amber glass containers at -10°C or less.
7.2.2.2 SUR PRIMARY DILUTION STANDARD (SUR PDS) (500 ug/mL) -
The 500 ug/mL SUR PDS may be purchased commercially or prepared by
volumetric dilution of the SUR Stock Solutions (Sect. 7.2.2.1) in acetone
or ethyl acetate The PDS has been shown to be stable for one year when
stored in amber glass screw cap vials at • 10°C or less. This solution is
used to make the 50 ug/mL solution for sample fortification and also to
prepare calibration solutions.
7.2.2.3 SUR SAMPLE FORTIFICATION SOLUTION (50 ug/mL) - Dilute the
500 ug/mL SUR PDS in methanol to make a 50 ug/mL sample
fortification solution. Add 100 uL of this 50 ug/mL solution to each 1 liter
water sample before extraction to give a concentration of 5 ug/L of each
surrogate. This solution has been shown to be stable for six months when
stored in amber glass screw cap vials at • 10°C or less.
ANALYTE STANDARD SOLUTIONS - Obtain the analytes listed in the table in
Section 1.1 as neat or solid standards or as commercially prepared ampulized
solutions from a reputable standard manufacturer. Prepare the Analyte Stock and
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Primary Dilution Standards as described below.
7.2.3.1 ANALYTE STOCK STANDARD SOLUTIONS (1 to 10 mg/mL) -
Analyte standards may be purchased commercially as ampulized solutions
or prepared from neat materials. Stock standards have been shown to be
stable for one year when stored in amber glass screw cap vials at • 10°C or
less.
7.2.3.2 ANALYTE PRIMARY DILUTION STANDARDS (200 ug/mL and 20
ug/mL) - Prepare the 200 ug/mL Analyte PDS by volumetric dilution of
the Analyte Stock Standard Solution (Sect. 7.2.3.1) in ethyl acetate to
make a 200 ug/mL solution. The 20 ug/mL Analyte PDS can be made by a
volumetric dilution of the 200 ug/mL Analyte PDS in ethyl acetate. The
Analyte PDSs are used to prepare calibration and fortification standards.
They have been shown to be stable for six months when stored in an
amber glass screw cap vial at • 10°C or less. Check frequently for signs
of evaporation, especially before preparing calibration solutions.
7.2.4 CALIBRATION SOLUTIONS - Prepare a calibration curve of at least 5 CAL
levels over the concentration range of interest from dilutions of the Analyte PDSs
in ethyl acetate. All calibration solutions should contain at least 80% ethyl acetate
so that gas chromatographic performance is not compromised. The lowest
concentration of calibration standard must be at or below the MRL. The level of
the MRL will depend on system sensitivity. A constant concentration of each
internal standard and surrogate (in the range of 2 to 5 ng/uL) is added to each
CAL. For instance, for method development work, 50 uL of the 500 ug/mL
Internal Standard PDS and 50 uL of the 500 ug/mL SUR PDS were added to each
CAL level standard for final concentrations of 5 ug/mL. The calibration solutions
have been shown to be stable for six months when stored in an amber glass screw
cap vial at • 10°C or less.
7.2.5 ANALYTE FORTIFICATION SOLUTIONS (0.05 to 5.0 ug/mL) - The Analyte
Fortification Solutions contain all method analytes of interest in methanol. They
are prepared by dilution of the Analyte PDSs (200 ug/mL or 20 ug/mL). These
solutions are used to fortify the LFBs, the LFMs and LFMDs with method
analytes. It is recommended that three concentrations be prepared so that the
fortification levels can be rotated. The Analyte Fortification Solutions have been
shown to be stable for six months when stored in an amber glass screw cap vial at
• 10°C or less.
7.2.6 GC/MS TUNE CHECK SOLUTION (5 ug/mL) - Prepare a
Decafluorotriphenylphosphine (DFTPP) solution in methylene chloride. DFTPP
is more stable in methylene chloride than in acetone or ethyl acetate. Store this
solution in an amber glass screw cap vial at • 10°C or less.
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8.
SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 SAMPLE BOTTLE PREPARATION
8.1.1 Grab samples must be collected in accordance with conventional sampling
practices(5) using a 1 liter or 1 quart amber or clear glass bottle fitted with PTFE-
lined screw-caps.
8.1.2 Preservation reagents, listed in the table below, are added to each sample bottle
prior to shipment to the field.
Compound
L- Ascorbic Acid
Ethylenediaminetetraacetic acid
trisodium salt
Diazolidinyl Urea
*Tris(hydroxymethyl)aminomethane
*Tris(hydroxymethyl)aminomethane
hydrochloride
Amount
O.lOg/L
0.35 g/L
1.0 g/L
0.47 g/L
7.28 g/L
Purpose
Dechlorination
Inhibit metal -catalyzed
hydrolysis of targets
Microbial inhibitor
First component of pH 7
buffer mixture
Second component of pH
7 buffer mixture
* Alternately, 7.75 g of a commercial buffer crystal mixture, that is blended in the
proportions given in the table, can be used (Sect. 7.1.7.1).
8.1.2.1 Residual chlorine must be reduced at the time of sample collection with
100 mg of ascorbic acid per liter. Sodium thiosulfate and sodium sulfite
cannot be used because they were found to degrade target analytes. In
addition, while ammonium chloride is effective in converting free chlorine
to chloramines, the chloramines also caused target compound loss.
8.1.2.2 Ethylenediaminetetraacetic acid, trisodium salt (trisodium EDTA) (0.35 g)
must be added to inhibit metal-catalyzed hydrolysis of the target analytes,
principally terbufos, disulfoton, diazinon, fonofos, and cyanazine.
8.1.2.3 Diazolidinyl urea (1.0 g) is added to inhibit microbial degradation of
analytes. Diazolidinyl urea is used in cosmetics such as skin lotion. The
antimicrobial activity of diazolidinyl urea has been proposed as due to
protein alkylation of sulfhydryl groups and the ability to release
formaldehyde.(6) Plate count studies conducted during method
development indicated that it was effective in inhibiting microbial
degradation for extended periods.
8.1.2.4 The sample must be buffered to pH 7 to reduce the acid and base catalyzed
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hydrolysis of target analytes. The pH buffer has two components:
tris(hydroxymethyl)aminomethane (0.47 g) and
tris(hydroxymethyl)aminomethane hydrochloride (7.28 g). A
commercially prepared combination of these two compounds can be
purchased as pre-mixed crystals. When using the pH 7 pre-mixed crystals,
add 7.75 g per liter of water sample.
8.2 SAMPLE COLLECTION
8.2.1 When sampling from a cold water tap, remove the aerator so that no air bubbles
will be trapped in the sample. Open the tap and allow the system to flush until the
water temperature has stabilized (usually about 3-5 minutes). Collect samples
from the flowing system.
8.2.2 When sampling from an open body of water, fill a 1 quart wide-mouth bottle or 1
L beaker with water sampled from a representative area, and carefully fill sample
bottles from the container. Sampling equipment, including automatic samplers,
must be free of plastic tubing, gaskets, and other parts that may leach interfering
analytes into the water sample.
8.2.3 Fill sample bottles, taking care not to flush out the sample preservation reagents.
Samples do not need to be collected headspace free.
8.2.4 After collecting the sample, cap the bottle and agitate by hand until preservatives
are dissolved. 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. Samples should be
confirmed to be at or below 10°C when they are received at the laboratory. Samples
stored in the lab must be held at or below 6°C until extraction, but should not be frozen.
8.4 SAMPLE AND EXTRACT HOLDING TIMES - Results of the sample storage stability
study (Sect. 17, Table 7) indicated that most compounds listed in this method have
adequate stability for 14 days when collected, dechlorinated, preserved, shipped and
stored as described in Sections 8.1, 8.2, and 8.3. At 14 days, even the most unstable
compound, terbufos, was recovered above 60 %. Water samples should be extracted as
soon as possible, but must be extracted within 14 days. The extract storage stability study
must be stored at 0°C or less and analyzed within 28 days after extraction. The extract
storage stability study data are presented in Section 17, Table 8.
9. QUALITY CONTROL
9.1 Quality control (QC) requirements include the Initial Demonstration of Capability (Sect.
17, Table 9), the determination of the MDL, and subsequent analysis in each analysis
batch of a Laboratory Reagent Blank (LRB), Continuing Calibration Check Standards
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(CCC), a Laboratory Fortified Blank (LFB), a Laboratory Fortified Sample Matrix
(LFM), and either a Laboratory Fortified Sample Matrix Duplicate (LFMD) or a Field
Duplicate Sample. This section details the specific requirements for each QC parameter.
The QC criteria discussed in the following sections are summarized in Section 17, Tables
9 and 10. These criteria are considered the minimum acceptable QC criteria, and
laboratories are encouraged to institute additional QC practices to meet their specific
needs.
9.2 INITIAL DEMONSTRATION OF CAPABILITY (IDC) - Requirements for the Initial
Demonstration of Capability are described in the following sections and summarized in
Section 17, Table 9.
9.2.1 INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND - Any time
a new lot of solid phase extraction (SPE) cartridges or disks is used, it must be
demonstrated that a laboratory reagent blank (Sect. 9.4) is reasonably free of
contamination and that the criteria in Section 9.4 are met.
9.2.2 INITIAL DEMONSTRATION OF PRECISION - Prepare, extract, and analyze 4-
7 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 must be added to these samples. The relative standard
deviation (RSD) of the results of the replicate analyses must be less than 20%.
9.2.3 INITIAL DEMONSTRATION OF ACCURACY - Using the same set of replicate
data generated for Section 9.2.2, calculate average recovery. The average
recovery of the replicate values must be within ±30% of the true value.
9.2.4 MDL DETERMINATION - Prepare, extract and analyze at least seven replicate
LFBs at a concentration estimated to be near the MDL, over a period of at least
three days (both extraction and analysis should be conducted over at least three
days) using the procedure described in Section 11. The fortification level may be
estimated by selecting a concentration with a signal of 2 to 5 times the noise level.
The appropriate concentration will be dependent upon the sensitivity of the
GC/MS system being used. Sample preservatives as described in Section 8.1
must be added to these samples. Calculate the MDL using the equation
MDL = St(n. 1; j . ajpj,., = 0 99)
where
V -1, i - alpha = 0.99) = Students t value for the 99% confidence level with n-1
degrees of freedom,
n = number of replicates, and
S = standard deviation of replicate analyses.
NOTE: Do not subtract blank values when performing MDL calculations. The
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MDL is a statistical determination of precision only.(1) If the MDL replicates are
fortified at a low enough concentration, it is likely that they will not meet
precision and accuracy criteria.
9.2.5 METHOD MODIFICATIONS - The analyst is permitted to modify GC columns,
GC conditions, evaporation techniques, internal standards or surrogate standards,
but each time such method modifications are made, the analyst must repeat the
procedures of the IDC (Sect. 9.2).
9.3 MINIMUM REPORTING LEVEL (MRL) - The MRL is a threshold concentration of an
analyte that a laboratory can expect to accurately quantitate in an unknown sample. The
MRL should be established at an analyte concentration that is either greater than three
times the MDL or at an analyte concentration which would yield a response greater than a
signal-to-noise ratio of five. Although the lowest calibration standard for an analyte
may be below the MRL, the MRL must never be established at a concentration
lower than the lowest calibration standard.
9.4 LABORATORY REAGENT BLANK (LRB) - An LRB is required with each extraction
batch (Sect. 3.1) of samples to determine the background system contamination. If the
LRB produces a peak within the retention time window of any analyte that would prevent
the determination of that analyte, determine the source of contamination and eliminate the
interference before processing samples. Background contamination must be reduced to
an acceptable level before proceeding. Background from method analytes or
contaminants that interfere with the measurement of method analytes must be below 1/3
of the MRL. If the target analytes are detected in the LRB at concentrations equal to or
greater than this level, then all data for the problem analyte(s) must be considered invalid
for all samples in the extraction batch.
9.5 CONTINUING CALIBRATION CHECK (CCC) - A CCC is a standard prepared with all
compounds of interest which is analyzed during the analysis batch to ensure the stability
of the instrument initial calibration. This calibration check is required at the beginning of
each day that samples are analyzed, after every ten injections, and at the end of any group
of sample analyses. See Section 10.3 for concentration requirements and acceptance
criteria.
9.6 LABORATORY FORTIFIED BLANK (LFB) - An LFB is required with each extraction
batch (Sect. 3.6). The fortified concentration of the LFB should be rotated between low,
medium, and high concentrations from day to day. 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 the low-level LFB analyses must be 50-
160% of the true value. Results of the medium and high-level LFB analyses must be 70-
130% of the true value. If the LFB results do not meet these criteria for target analytes,
then all data for the problem analyte(s) must be considered invalid for all samples in the
extraction batch.
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9.7 MS TUNE CHECK - A complete description of the MS tune check is in Section 10.2.1.
This check must be performed each time a major change is made to the mass
spectrometer, and prior to establishing and/or re-establishing an initial calibration (Sect.
10.2). In this method daily DFTPP analysis is not required.
9.8 INTERNAL STANDARDS (IS) - The analyst must monitor the peak area of each
internal standard in all injections during each analysis day. The IS response (as indicated
by peak area) for any chromatographic run must not deviate by more than ±50% from the
average area measured during the initial calibration for that IS. A poor injection could
cause the IS area to exceed these criteria. Inject a second aliquot of the suspect extract to
determine whether the failure is due to poor injection or instrument response drift.
9.8.1 If the reinjected aliquot produces an acceptable internal standard response, report
results for that aliquot.
9.8.2 If the internal standard area for the reinjected extract deviates greater than 50%
from the initial calibration average, the analyst should check the continuing
calibration check standards that ran before and after the sample. If the continuing
calibration check fails the criteria of Section 9.5 and 10.3, recalibration is in order
per Section 10. If the calibration standard is acceptable, extraction of the sample
should be repeated provided the sample is still within holding time. Otherwise,
report results obtained from the reinjected extract, but annotate as suspect.
9.9 SURROGATE RECOVERY - The surrogate standards are fortified into the aqueous
portion of all samples, duplicates, LRBs, LFMs and LFMDs prior to extraction.
Surrogates are also added to the calibration curve and calibration check standards. The
surrogate is a means of assessing method performance from extraction to final
chromatographic measurement.
9.9.1 When surrogate recovery from a sample, blank, or CCC is less than 70% or
greater than 130%, check (1) calculations to locate possible errors, (2) standard
solutions for degradation, (3) contamination, and (4) instrument performance. If
those steps do not reveal the cause of the problem, reanalyze the extract.
9.9.2 If the extract reanalysis meets the surrogate recovery criterion, report only data for
the reanalyzed extract.
9.9.3 If the extract reanalysis fails the 70-130% surrogate recovery criterion, the analyst
should check the surrogate calibration by analyzing the most recently acceptable
calibration standard. If the calibration standard fails the 70-130% surrogate
recovery criteria of Section 9.9.1, recalibration is in order. If the surrogate
recovery of the calibration standard is acceptable, extraction of the sample should
be repeated, provided the sample is still within the holding time. If the sample re-
extract also fails the recovery criterion, report all data for that sample as
suspect/surrogate recovery.
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9.9.4 The surrogate, l,3-dimethyl-2-nitrobenzene, is used to track the recovery of
nitrobenzene. The other surrogate, triphenylphosphate, monitors the recovery of
all the other target analytes. If nitrobenzene is not included on the target analyte
list, then the first surrogate, l,3-dimethyl-2-nitrobenzene, does not need to be
analyzed.
9.10 LABORATORY FORTIFIED SAMPLE MATRIX AND DUPLICATE (LFM AND
LFMD) - Analyses of LFMs (Sect. 3.7) are required in each extraction batch and are used
to determine that the sample matrix does not adversely affect method accuracy. If the
occurrence of target analytes in the samples is infrequent, or if historical trends are
unavailable, a second LFM, or LMFD, must be prepared, extracted, and analyzed from a
duplicate field sample used to prepare the LFM to assess method precision. Extraction
batches that contain LFMDs will not require the analysis of a Field Duplicate (Sect. 9.11).
If a variety of different sample matrices are analyzed regularly, for example, drinking
water from groundwater and surface water sources, method performance should be
established for each. Over time, LFM data should be documented for all routine sample
sources for the laboratory.
9.10.1 Within each extraction batch, a minimum of one field sample is fortified as an
LFM for every 20 samples extracted. The LFM is prepared by spiking a sample
with an appropriate amount of Analyte PDS (Sect. 7.2.5). Select a spiking
concentration that is at least twice the matrix background concentration, if known.
Use historical data or rotate through the designated concentrations to select a
fortifying concentration. Selecting a duplicate bottle of a sample that has already
been analyzed aids in the selection of appropriate spiking levels.
9.10.2 Calculate the percent recovery (R) for each analyte using the equation
(A-B)
R= *100
where: A = measured concentration in the fortified sample,
B = measured concentration in the unfortified sample, and
C = fortification concentration.
9.10.3 Analyte recoveries may exhibit matrix bias. For samples fortified at or above
their native concentration, recoveries should range between 70 and 130%, except
for low-level fortification near or at the MRL where 50 to 160% recoveries are
acceptable. If the accuracy of any analyte falls outside the designated range, and
the laboratory performance for that analyte is shown to be in control in the LFB,
the recovery is judged to be matrix biased. The result for that analyte in the
unfortified sample is labeled suspect/matrix to inform the data user that the results
are suspect due to matrix effects.
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9.10.4 If an LFMD is analyzed instead of a Field Duplicate (Sect. 9.11), calculate the
D „ _ LFM- LFMD , AA
RPD = * 100
(LFM+ LFMD)/2
relative percent difference (RPD) for duplicate LFMs (LFM and LFMD) using the
equation RPDs for duplicate LFMs should fall in the range of ±30% for samples
fortified at or above their native concentration. Greater variability may be
observed when LFMs are spiked near the MRL. At the MRL, RPDs should fall in
the range of ±50% for samples fortified at or above their native concentration. If
the accuracy of any analyte falls outside the designated range, and the laboratory
performance for that analyte is shown to be in control in the LFB, the recovery is
judged to be matrix biased. The result for that analyte in the unfortified sample is
labeled suspect/matrix to inform the data user that the results are suspect due to
matrix effects.
9.11 FIELD DUPLICATES (FD1 AND FD2) - Within each extraction batch, a minimum of
one Field Duplicate (FD) or LFMD (Sect. 9.10) must be analyzed. FDs check the
precision associated with sample collection, preservation, storage, and laboratory
procedures. If target analytes are not routinely observed in field samples, a LFMD (Sect.
9.10) should be analyzed to substitute for this requirement. Extraction batches that
contain LFMDs (Section 9.10) will not require the analysis of a Field Duplicate.
9.11.1 Calculate the relative percent difference (RPD) for duplicate measurements (FD1
and FD2) using the equation
Dnrk FD1-FD2 1AA
RPD = * 100 tr\
(FDl + FD2)/2 l'
9.11.2 RPDs for duplicates should be in the range of ±30%. Greater variability may be
observed when analyte concentrations are near the MRL. At the MRL, RPDs
should fall in the range of ±50%. If the accuracy of any analyte falls outside the
designated range, and the laboratory performance for that analyte is shown to be
in control in the LFB, the recovery is judged to be matrix biased. The result for
that analyte in the unfortified sample is labeled suspect/matrix to inform the data
user that the results are suspect due to matrix effects.
9.12 QUALITY CONTROL SAMPLES (QCS) - Each time that new standards are prepared or
a new calibration curve is run, analyze a QCS from a source different than the source of
the calibration standards. The QCS may be injected as a calibration standard or fortified
into reagent water and analyzed as a LFB. If the QCS is analyzed as a continuing
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calibration, then the acceptance criteria are the same as for the CCC. If the QCS is
analyzed as a LFB, then the acceptance criteria are the same as for an LFB. If measured
analyte concentrations are not of acceptable accuracy, check the entire analytical
procedure to locate and correct the problem source.
10. CALIBRATION AND STANDARDIZATION
10.1 Demonstration and documentation of acceptable mass spectrometer tune and initial
calibration is required before any samples are analyzed. After the initial calibration is
successful, a continuing calibration check is required at the beginning and end of each
period in which analyses are performed, and after every tenth sample. Verification of
mass spectrometer tune must be repeated each time a major instrument modification is
made or maintenance is performed, and prior to analyte calibration.
10.2 INITIAL CALIBRATION
10.2.1 MS TUNE/MS TUNE CHECK- Calibrate the mass and abundance scales of the
MS with calibration compounds and procedures prescribed by the manufacturer
with any modifications necessary to meet tuning requirements. Inject 5 ng or less
of the DFTPP solution (Sect. 7.2.6) into the GC/MS system. Acquire a mass
spectrum that includes data for m/z 45-450. Use a single spectrum at the apex of
the DFTPP peak, an average spectrum of the three highest points of the peak, or
an average spectrum across the entire peak to evaluate the performance of the
system. If the DFTPP mass spectrum does not meet all criteria in Table 1, the MS
must be retuned and adjusted to meet all criteria before proceeding with the initial
calibration.
10.2.2 INSTRUMENT CONDITIONS - Operating conditions are described below.
Conditions different from those described may be used if QC criteria in Section 9
are met. Different conditions include alternate GC columns, temperature
programs, and injection techniques, such as cold on-column and large volume
injections. Equipment designed for alternate types of injections must be used if
these options are selected.
10.2.2.1 Inject 1 uL into a hot, splitless injection port held at 210°C with a split
delay of 1 min. The temperature program is as follows: initially hold at
55°C for one minute, then ramp at 8°C/ min to 320°C. Total run time is
approximately 33 min. Begin data acquisition at 3.5 minutes.
Note: The GC was operated in a constant flow rate mode at a rate of 1.4
mL per minute and an initial head-pressure of 12.2 psi.
10.2.2.2 Many of the target compounds exhibit decreased sensitivity for low-level
injections due to degradation or irreversible adsorption in the injector
port. Deactivated glass or quartz inlet liners are recommended.
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10.2.2.3 MS Detection and Sensitivity - Acquire and store data from m/z 45 to
450 with a total cycle time (including scan overhead time) of 1.0 second
or less. Adjust the cycle time to measure at least five or more spectra
during the elution of each GC peak. Seven to ten scans across each GC
peak are recommended. The GC/MS/DS peak identification software
must be able to recognize a GC peak in the appropriate retention time
window for each of the compounds in the calibration solution, and make
correct qualitative identifications.
10.2.3 CALIBRATION SOLUTIONS - Prepare a set of at least 5 calibration standards
as described in Section 7.2.4. The lowest concentration of the calibration standard
must be at or below the MRL, which will depend on system sensitivity.
Acceptable calibration over a large dynamic range, greater than about 50 fold
range, may require multiple calibration curves.
10.2.4 CALIBRATION - The system is calibrated using the internal standard technique.
Concentrations may be calculated through the use of average relative response
factor (RRF) or through the use of a calibration curve. Calculate the RRFs using
the equation
RRF =
where: Ax = integrated abundance (peak area) of the quantitation ion of
the analyte,
A;s = integrated abundance (peak area) of the quantitation ion
internal standard,
Qx = quantity of analyte injected in ng or concentration units,
and
Qis = quantity of internal standard injected in ng or concentration
units.
Average RRF calibrations may only be used if the RRF values over the calibration
range are relatively constant (<30% RSD). Average RRF is determined by
calculating the mean RRF of a minimum of five calibration concentrations.
10.2.5 As an alternative to calculating average RRFs and applying the RSD test, use the
GC/MS data system software to generate a linear regression or quadratic
calibration curve. The analyst may choose whether or not to force zero to obtain a
curve that best fits the data. Examples of common GC/MS system calibration
curve options are: 1) Ax /Ais vs Qx /Qis and 2) RRF vs Ax /Ajs.
10.2.6 Acceptance criteria for the calibration of each analyte is determined by calculating
the concentration of each analyte and surrogate in each of the analyses used to
generate the calibration curve or average RRF. Each calibration point, except the
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lowest point, for each analyte must calculate to be 70-130 % of its true value. The
lowest point must calculate to be 50-150% of its true value. If this criteria cannot
be met, reanalyze the calibration standards, restrict the range of calibration, or
select an alternate method of calibration. The data presented in this method were
obtained using quadratic fit (RRF vs. amount). Quadratic fit calibrations should
be used with caution, because the non-linear area of the curve may not be
reproducible.
10.3 CONTINUING CALIBRATION CHECK (CCC) - The CCC verifies the initial
calibration at the beginning and end of each group of analyses, and after every 10th
sample during analyses. In this context, a "sample" is considered to be a field sample.
LRBs, LFBs, LFMs, LFMDs and CCCs are not counted as samples. The beginning CCC
for 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
solutions to meet this requirement. Subsequent CCCs should alternate between a
medium and high concentration.
10.3.1 Inject an aliquot of the appropriate concentration calibration solution and analyze
with the same conditions used during the initial calibration.
10.3.2 Determine that the absolute areas of the quantitation ions of the internal standards
have not changed by more than ± 50% from the areas measured during initial
calibration. If any IS area has changed by more this amount, remedial action must
be taken (Sect. 10.3.4). Control charts are useful aids in documenting system
sensitivity changes.
10.3.3 Calculate the concentration of each analyte and surrogate in the check standard.
The calculated amount for each analyte for medium and high level CCCs must be
±30% of the true value. The calculated amount for the lowest calibration level for
each analyte must be within ±50% of the true value. If these conditions do not
exist, then all data for the problem analyte must be considered invalid, and
remedial action (Sect. 10.3.4) should be taken which may require recalibration.
Any field sample extracts 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 continuing calibration fails because
the calculated concentration is greater than 130% (150% for the low-level CCC)
for a particular target compound, and field sample extracts show no detection for
that target compound, 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 an ion source,
cleaning quadrapole rods, replacing filament assemblies, etc., require returning to
the initial calibration step (Sect. 10.2).
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11. PROCEDURE
11.1 Important aspects of this analytical procedure include proper preparation of laboratory
glassware and sample containers (Sect. 4.1), and sample collection and storage (Sect. 8).
This section describes the procedures for sample preparation, solid phase extraction
(SPE) using cartridges or disks, and extract analysis.
11.2 SAMPLE BOTTLE PREPARATION
11.2.1 Samples are preserved, collected and stored as presented in Section 8. All field
and QC samples must contain the preservatives listed in Section 8.1.2, including
the LRB and LFB. Before extraction, mark the level of the sample on the outside
of the sample bottle for later sample volume determination. If using weight to
determine volume (Sect. 11.3.7), weigh the bottle with collected sample before
extraction.
11.2.2 Add an aliquot of the surrogate fortification solution to each sample to be
extracted. For method development work, a 100 uL aliquot of the 50 ug/mL SUR
Sample Fortification Solution (Sect. 7.2.2.3) was added to 1 L for a final
concentration of 5.0 ug/L.
11.2.3 If the sample is an LFB, LFM, or LFMD, add the necessary amount of analyte
fortification solution. Swirl each sample to ensure all components are mixed.
11.2.4 Proceed with sample extraction using either SPE cartridges (Sect. 11.3) or disks
(Sect. 11.4).
11.3 CARTRIDGE SPE PROCEDURE - The cartridge extraction procedure is carried out in a
manual mode or by using a robotic or automatic sample preparation device. This section
describes a SPE manual procedure using the equipment outlined in Section 6.10. The
manual mode of sample addition to cartridges is performed with a large reservoir attached
to the cartridge or with a transfer tube from the sample bottle to the cartridge. Cartridge
extraction data in Section 17 was collected using the transfer tube option described
below.
11.3.1 CARTRIDGE CLEANUP - Attach the extraction cartridges to the vacuum
manifold. Rinse each cartridge with a 5-mL aliquot of ethyl acetate, allowing the
sorbent to soak in the ethyl acetate for about 1 minute by turning off the vacuum
temporarily. Repeat with a 5-mL aliquot of methylene chloride (MeCl2). Let the
cartridge vacuum dry after each flush.
11.3.2 CARTRIDGE CONDITIONING - This conditioning step is critical for recovery
of analytes and can have a marked effect on method precision and accuracy. If the
cartridge goes dry during the conditioning phase, the conditioning must be started
over. Once the conditioning has begun, the cartridge must not go dry until the last
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portion of the sample passes because analyte and surrogate recoveries may be
affected. The analyst should note premature drying of the solid phase, because the
sample may require re-extraction due to low surrogate recoveries.
11.3.2.1 CONDITIONING WITH METHANOL - Rinse each cartridge with a 5-
mL aliquot of methanol (MeOH), allowing the sorbent to soak for about
30 seconds by turning off the vacuum temporarily. From this point on,
do not allow the cartridge to go dry until after extraction is complete.
Drain most of the MeOH without going below the top of the cartridge
packing and rinse again with a 5-mL aliquot of MeOH.
11.3.2.2 CONDITIONING WITH REAGENT WATER - Drain most of the
MeOH and rinse the cartridge with two consecutive 5-mL aliquots of
reagent water, being careful not to allow the water level to go below the
cartridge packing. Turn off the vacuum. Fill the cartridge to the top
with reagent water and attach a reservoir or a transfer tube (Sect. 6.10.2).
11.3.3 CARTRIDGE EXTRACTION - Prepare samples, including the QC samples, as
specified in Section 11.2. The sample may be added to the cartridge using either a
large reservoir attached to the cartridge or using a transfer tube from the sample
bottle to the cartridge.
11.3.3.1 SAMPLE ADDITION USING RESERVOIRS - Attach a reservoir to
the conditioned cartridge from Section 11.3.2. Fill the reservoir with
sample and turn on the vacuum adding additional aliquots of sample
until the entire 1 L sample is processed. Adjust the vacuum so that the
flow rate is about 20 mL/min. Do not let the cartridge packing go dry
before all the sample has been extracted. After all of the sample has
passed through the SPE cartridge, draw air through the cartridge for 10
minutes at full vacuum (minus 10 to 15 inches Hg). If the cartridge is
dried for period much longer than 10 minutes, there may be a loss of
recovery for nitrobenzene and the surrogate l,3-dimethyl-2-
nitrobenzene. After drying, turn off and release vacuum.
11.3.3.2 SAMPLE ADDITION USING TRANSFER TUBES - Fit the PTFE
transfer tube adapter securely to the conditioned cartridge. The screw on
the adapter must be finger-tight, otherwise, air can leak and the cartridge
may go dry. Place the weighted end of the transfer tube inside on the
bottom of the sample bottle. Adjust the flow rate to about 20 mL/min.
If the adapter is securely attached, the water level in the cartridge should
drop only as much as the volume of the transfer tube and no more. Do
not let the SPE sorbent go dry before all the sample has been extracted.
After all the sample has passed through the SPE cartridge, draw air
through the cartridge for 10 minutes at full vacuum (minus 10 to 15
inches Hg). If the cartridge is dried for a much longer period than 10
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minutes, there may be a loss of recovery for nitrobenzene and the
surrogate l,3-dimethyl-2-nitrobenzene. After drying, turn off and
release vacuum.
11.3.4 CARTRIDGE ELUTION - Keep reservoirs or transfer tubes attached. Lift the
manifold top, place collection tubes into the extraction tank, and insert valve
liners into the collection tubes for extract collection. Add 5 mL of ethyl acetate
(EtAc) to the empty sample bottle and rotate the bottle on its side, rinsing the
inside of the bottle. If using the cartridge reservoir method, pour the EtAc from
the bottle into the cartridge reservoir and draw enough of the EtAc through the
cartridge to soak the sorbent. If using the transfer tubes method, pull the EtAc
through the PTFE transfer tubes and draw enough of the EtAc through the tubes
into the cartridge to soak the sorbent. Turn off the vacuum and vent the system
and allow the sorbent to soak in EtAc for approximately 30 seconds. Start a low
vacuum (minus 2-4 in Hg) and pull the ethyl acetate through in a dropwise fashion
into the collection tube. Repeat rinse with 5 mL MeCl2. Take off reservoirs and
transfer tubes and rinse the cartridge body with 2 to 3 mLs of 1:1 mixture of
MeCl2 and EtAc (1:1 MeCl2/EtAc).
11.3.5 DRYING OF THE EXTRACT - Small amounts of residual water from the
sample container and solid phase may form an immiscible layer with the solvent
in the extract. Set up a drying column (Sect. 6.6) packed with about 5 grams of
anhydrous sodium sulfate. Pre-rinse the sodium sulfate column with about 2 mL
of 1:1 EtAc/MeCl2. Place a clean collection tube that can hold at least 20 mL
beneath the drying column. Add the extract to the column and follow with two, 3
mL aliquots of 1:1 EtAc/MeCl2.
11.3.6 EXTRACT CONCENTRATION - Concentrate the extract to about 0.7 mL under
a gentle stream of nitrogen in a warm water bath (at ~ 40°C). Do not blow down
samples to less than 0.5 mL, because the most volatile compounds will show
diminished recovery. Transfer the extract to a 1 mL volumetric flask and add the
internal standard (method development used 100 uL of 50 ug/mL internal standard
solution for an extract concentration of 5 ug/mL). Rinse the collection tube that
held the dried extract with small amounts of EtAc and add to the volumetric flask
to bring the volume up to the 1 mL mark. Transfer to autosampler vial.
11.3.7 SAMPLE VOLUME OR WEIGHT DETERMINATION - Use a graduated
cylinder to measure the volume of water required to fill the original sample bottle
to the mark made prior to extraction (Sect. 11.2.1). Determine volume to the
nearest 10 mL for use in the final calculations of analyte concentration (Sect.
12.2). If using weight to determine volume, reweigh empty sample bottle. From
the weight of the original sample bottle measured in Section 11.2.1, subtract the
empty bottle weight. Use this value for analyte concentration calculations in
Section 12.2.
526-26
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11.4 DISK SPE PROCEDURE - The disk extraction procedure may be carried out in a
manual mode or by using a robotic or automatic sample preparation device. This section
describes the disk SPE procedure using the equipment outlined in Section 6.10 in its
simplest, least expensive mode without the use of a robotics systems. The manual mode
described below was used to collect data presented in Section 17.
11.4.1 SAMPLE PREPARATION - Prepare the sample as given in Section 11.2.
11.4.2 DISK CLEANUP - Assemble the extraction glassware onto the vacuum manifold,
placing disks on a support screen between the funnel and base. Add a 5 mL
aliquot of 1:1 mixture of ethyl acetate (EtAc) and methylene chloride (MeCl2)
(1:1 MeCl2/EtAc), drawing about half through the disk, and allowing the solvent
to soak the disk for about a minute. Draw the remaining solvent through the disk
to waste until the disk is dry of solvent.
11.4.3 DISK CONDITIONING - The conditioning step is critical for recovery of
analytes and can have a marked effect on method precision and accuracy. If the
disk goes dry during the conditioning phase, the conditioning must be started
over. Once the conditioning has begun, the disk must not go dry until the last
portion of the sample passes, because analyte and surrogate recoveries may be
affected. The analyst should note premature drying of the solid phase, because the
sample may require re-extraction due to low surrogate recoveries. During
conditioning, it is not unusual for the middle of the solid phase disk to form a
wrinkle. This typically does not adversely affect extraction.
11.4.3.1 CONDITIONING WITH METHANOL - Add approximately 10 mL
methanol to each disk. Pull about 1 mL of MeOH through the disk and
turn off the vacuum temporarily to let the disk soak for about one
minute. Draw most of the remaining MeOH through the disk, but leave
a layer of MeOH on the surface of the disk. The disk must not be
allowed to go dry from this point until the end of the sample extraction.
11.4.3.2 CONDITIONING WITH WATER - Follow the MeOH rinse with two,
10 mL aliquots of reagent water, being careful to keep the water level
above the disk surface. Turn off the vacuum.
11.4.4 DISK EXTRACTION - Add sample to the extraction funnel containing the
conditioned disk and turn on the vacuum. Do not let the disk go dry before all
sample has been extracted. Drain as much water from the sample container as
possible. After all of the sample has passed, draw air through the disk by
maintaining full vacuum (minus 10-15 in Hg) for 10 minutes. If the disk is dried
for a period much longer than 10 minutes, there will be a loss of recovery for
nitrobenzene and the surrogate l,3-dimethyl-2-nitrobenzene. After drying, turn
off and release the vacuum.
526-27
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11.4.5 DISK ELUTION - Detach the glassware base from the manifold without
disassembling the funnel from the base. Dry the underside of the base. Insert
collection tubes into the manifold to catch the extracts as they are eluted from the
disk. The collection tube must fit around the drip tip of the base to ensure
collection of all the eluent. Reattach the base to the manifold. Add 5 mL of ethyl
acetate to the empty sample bottle and rinse the inside of the bottle. Transfer the
ethyl acetate to the disk and, with vacuum, pull enough ethyl acetate into the disk
to soak the sorbent, and allow the solvent to soak the disk for about one minute.
Pull the remaining solvent slowly through the disk into the collection tube.
Repeat the rinse with 5 mL MeCl2. Rinse the SPE funnel surface once with a 2-3
mL aliquot of 1:1 EtAc/MeCl2. Repeat this last rinse of the SPE funnel. Detach
glassware from manifold and remove collection tube from the manifold.
11.4.6 DRYINGOF THE EXTRACT - Proceed with drying the extract, Section 11.3.5.
11.4.7 EXTRACT CONCENTRATION - Proceed with extract concentration, Section
11.3.6.
11.4.8 SAMPLE VOLUME OR WEIGHT DETERMINATION - Proceed with sample
volume or weight determination, Section 11.3.7.
11.5 ANALYSIS OF SAMPLE EXTRACTS
11.5.1 Establish operating conditions as described in Section 10.2.2. Confirm that
retention times, compound separation and resolution are similar to those
summarized in Table 2 and Figure 1.
11.5.2 Establish a valid initial calibration following the procedures outlined in Section
10.2 or confirm that the calibration is still valid by running a CCC as described in
Section 10.3. If establishing an initial calibration for the first time, complete the
IDC as described in Section 9.2.
11.5.3 Analyze aliquots of field and QC samples at appropriate frequencies (Sect. 9) with
the GC/MS system using the conditions used for the initial and continuing
calibrations. At the conclusion of data acquisition, use the same software that was
used in the calibration procedure to tentatively identify peaks in predetermined
retention time windows of interest. Use the data system software to examine the
ion abundances of components of the chromatogram.
11.5.4 COMPOUND IDENTIFICATION - Identify a sample component by comparison
of its mass spectrum (after background subtraction) to a reference spectrum in the
user-created data base.
11.5.4.1 Establish an appropriate retention time window for each target analyte,
internal standard and surrogate standard to identify them in QC and Field
526-28
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Samples chromatograms. Ideally, the retention time window should be
based on measurements of actual retention time variation for each
compound in standard solutions collected on each GC/MS over the
course of time. The suggested variation is plus or minus three times the
standard deviation of the retention time for each compound for a series
of injections. The injections from the initial calibration and from the
Initial Demonstration of Capability may be used to calculate a suggested
window size. However, the experience of the analyst should weigh
heavily on the determination of an appropriate retention window size.
11.5.4.2 In general, all ions that are present above 10% relative abundance in the
mass spectrum of the standard should be present in the mass spectrum of
the sample component and should agree within an absolute 20%. For
example, if an ion has a relative abundance of 30% in the standard
spectrum, its abundance in the sample spectrum should be in the range of
10 to 50%. Some ions, particularly the molecular ion, are of special
importance, and should be evaluated even if they are below 10% relative
abundance.
11.5.5 EXCEEDING CALIBRATION RANGE - An analyst must not extrapolate
beyond the established calibration range. If an analyte result exceeds the range of
the initial calibration curve, the extract may be diluted with ethyl acetate, with the
appropriate amount of internal standard added to match the original level, and the
diluted extract injected. Acceptable surrogate performance (Sect. 9.9) should be
determined from the undiluted sample extract. Incorporate the dilution factor into
final concentration calculations. The dilution will also affect analyte MRLs.
12. DATA ANALYSIS AND CALCULATIONS
12.1 Identify method analytes present in the field and QC samples as described in Section
11.8.4. Complete chromatographic resolution is not necessary for accurate and precise
measurements of analyte concentrations if unique ions with adequate intensities are
available for quantitation.
12.1.1 Identification is hampered when sample components are not resolved
chromatographically and produce mass spectra containing ions contributed by
more than one analyte. When GC peaks obviously represent more than one
sample component (i.e., broadened peak with shoulder(s) or valley between two
or more maxima), appropriate analyte spectra and background spectra can be
selected by examining plots of characteristic ions. When analytes coelute (i.e.,
only one GC peak is apparent), the identification criteria can be met but each
analyte spectrum will contain extraneous ions contributed by the coeluting
compound.
12.1.2 In validating this method, concentrations were calculated by measuring the
526-29
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characteristic ions listed in Table 2. Other ions may be selected at the discretion of
the analyst.
12.2 Calculate analyte and surrogate concentrations, using the multipoint calibration
established in Section 10.2. Do not use daily continuing calibration check data to
quantitate analytes in samples. Adjust the final analyte concentrations to reflect the actual
sample volume or weight determined in Section 11.3.7 or 11.4.8. Field Sample extracts
that require dilution should be treated as described in Section 11.5.5.
12.3 Calculations should utilize all available digits of precision, but final reported
concentrations should be rounded to an appropriate number of significant figures.
13. METHOD PERFORMANCE
13.1 PRECISION, ACCURACY, AND MDLs - Method performance data are summarized in
Section 17. Method detection limits (MDLs) are presented in Table 3 and were
calculated using the formula in Section 9.2.4. Single laboratory precision and accuracy
data are presented for reagent water (Sect. 17, Tables 4A and 4B), chlorinated "finished"
ground water (Sect. 17, Tables 5 A and 5B), and chlorinated "finished" surface water
(Sect. 17, Tables 6A and 6B).
13.2 POTENTIAL PROBLEM COMPOUNDS
13.2.1 MATRIX ENHANCED SENSITIVITY - Cyanazine, and to a lesser extent 2,4,6-
trichlorophenol and prometon, tend to exhibit "matrix-induced chromatographic
response enhancement."(7"n) Compounds that exhibit this phenomenon often give
analytical results that exceed 100 % recovery in fortified extracts at low
concentration and in continuing calibration checks. More frequent recalibration is
required. It has been proposed that these compounds are susceptible to GC inlet
absorption or thermal degradation so that analytes degrade more when injected in
a "cleaner" matrix. The injection of a "dirty" sample extract coats surfaces with
matrix components and "protects" the problem compounds from decomposition or
adsorption. As a result, a relatively greater response is observed for analytes in
sample extracts than in calibration solutions. This effect is minimized by using
deactivated injection liners (Sect. 10.2.2.2). The analyst may also choose to
condition the injection port after maintenance by injecting a few aliquots of a field
sample extract prior to establishing an initial calibration. Preparation of
calibration standards in clean extracts is not allowed.
13.2.2 COMPOUND DEGRADATION - Method development work indicated that
several of the target compounds were unstable when stored in water without
preservation. There were various modes of loss. Hydrolysis of 1,2-
diphenylhydrazine, terbufos, diazinon, disulfoton and cyanazine was accelerated
at low and high pH. In addition, transition metal ions further catalyzed hydrolysis
of terbufos, fonofos, and diazinon. Free chlorine and chloramines degraded 2,4-
526-30
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dichlorophenol, terbufos, fonofos, diazinon, and disulfoton. When water samples
were not properly preserved, after three days, there was more than 80% loss of
some targets, initially fortified at 5 ppb. Sample preservation conditions (Sect. 8)
have been carefully chosen to minimize analyte degradation to acceptable levels
during the 14 day sample holding time.
13.2.3 Inlet liners and/or capillary GC columns that develop active sites can cause a
complete loss of prometon and excessive tailing of 2,4-dichlorophenol and 2,4,6-
trichlorophenol peaks in the chromatogram.
13.3 ANALYTE STABILITY STUDIES
13.3.1 FIELD SAMPLES - Chlorinated surface water samples, fortified with method
analytes at 5.0 ug/L, were preserved and stored as required in Section 8. The
average of triplicate analyses, conducted on days 0, 3, 7, and 14, are presented in
Section 17, Table 7. These data document the 14-day sample holding time. It is
advisable to extract as soon as possible because some compounds exhibit
significant losses by 7 days.
13.3.2 EXTRACTS - Extracts from the day 0 extract holding time study described above
were stored below 0 °C and analyzed on days 0, 6, 13, 20, and 32. The data
presented in Section 17, Table 8, document the 28-day extract holding time.
14. POLLUTION PREVENTION
14.1 This method utilizes solid phase extraction technology to remove the analytes from water.
It requires the use of very small volumes of organic solvent and very small quantities of
pure analytes, thereby minimizing the potential hazards to both the analyst and the
environment involved with 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: Laboratory Chemical Management for Waste
Reduction" available form the American Chemical Society's Department of Government
Relations and Science Policy, 1155 16th Street N.W., Washington, D.C. 20036.
15. WASTE MANAGEMENT
15.1 The analytical procedures described in this method generate relatively small amounts of
waste since only small amounts of reagents and solvents are used. The matrices of
concern are finished drinking water or source water. However, the Agency requires that
laboratory waste management practices be conducted consistent with all applicable rules
and regulations, and that laboratories protect the air, water, and land by minimizing and
526-31
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controlling all releases from fume hoods and bench operations. Also, compliance is
required with any sewage discharge permits and regulations, particularly the hazardous
waste identification rules and land disposal restrictions. For further information on waste
management, consult "The Waste Management Manual for Laboratory Personnel" also
available from the American Chemical Society at the address in Section 14.2.
16 REFERENCES
1. Glaser, J.A., Foerst, D.L., McKee, G.D., Quave, S.A., and Budde, W.L., "Trace Analyses for
Wastewaters," Environ. Sci. Technol.. 15 (1981) 1426-1435.
2. "Carcinogens - Working With Carcinogens," Department of Health, Education, and Welfare,
Public Health Service, Center for Disease Control, National Institute for Occupational Safety and
Health, Publication No. 77-206, Aug. 1977.
3. "OSHA Safety and Health Standards, General Industry," (29CFR1910), Occupational Safety and
Health Administration, OSHA 2206, (Revised, January 1976).
4. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication,
Committee on Chemical Safety, 3rd Edition, 1979.
5. ASTM Annual Book of Standards, Part II, Volume 11.01, D3370-82, "Standard Practice for
Sampling Water," American Society for Testing and Materials, Philadelphia, PA, 1986.
6. Llabres, C.M., Ahearn, D.G. "Antimicrobial Activities of N-Chloramines and Diazolidinyl
Urea," Applied and Environmental Microbiology, 49, (1985), 370-373.
7. Erney, D.R., Gillespie, A.M., Gilvydis, D.M., and Poole, C.F., "Explanation of the Matrix-
Induced Chromatographic Response Enhancement of Organophosphorous Pesticides During
Open Tubular Column Gas Chromatography with Splitless or Hot On-Column Injection and
Flame Photometric Detection." J. Chromatogr.. 638 (1993) 57-63.
8. Mol, H.G.J., Althuizen, M., Janssen, H., and Cramers, C.A., Brinkman, U.A.Th.,
"Environmental Applications of Large Volume Injection in Capillary GC Using PTV Injectors,"
J. High Resol. Chromatogr.. 19 (1996) 69-79.
9. Erney, D.R., Pawlowski, T.M., Poole, C.F., "Matrix Induced Peak Enhancement of Pesticides in
Gas Chromatography," J. High Resol. Chromatogr.. 20 (1997) 375-378.
10. Hajslova, J., Holadova, K. , Kocourek, V., Poustka, J., Godula, M., Cuhra, P., Kempny, M.,
"Matrix Induced Effects: A Critical Point in the Gas Chromatographic Analysis of Pesticide
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Residues," J. Chromatogr.. 800 (1998) 283-295.
11. Wylie, P., Uchiyama, M., "Improved Gas Chromatographic Analysis of Organophosphorous
Pesticides with Pulsed Splitless Injection," J. AOAC International. 79, 2, (1996) 571-577.
526-33
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17. TABLES. DIAGRAMS. FLOWCHARTS. AND VALIDATION DATA
TABLE 1. ION ABUNDANCE CRITERIA FOR BIS(PERFLUOROPHENYL)PHENYL
PHOSPHINE, (DECAFLUOROTRIPHENYL PHOSPHINE, DFTPP)
Mass
(M/z)
51
68
70
127
197
198
199
275
365
441
442
443
Relative Abundance Criteria
10-80% of the base peak
<2% of Mass 69
<2% of Mass 69
10-80% of the base peak
<2% of Mass 98
Base peak or >50% of Mass 442
5-9% of Mass 198
10-60% of the base peak
>1% of the base peak
Present and 50% of Mass 198
15-24% of Mass 442
Purpose of Checkpoint1
Low-mass sensitivity
Low-mass resolution
Low-mass resolution
Low- to mid-mass resolution
Mid-mass resolution
Mid-mass resolution and sensitivity
Mid-mass resolution and isotope ratio
Mid- to high-mass sensitivity
Baseline threshold
High-mass resolution
High-mass resolution and sensitivity
High-mass resolution and isotope ratio
JA11 ions are used primarily to check the mass measuring accuracy of the mass spectrometer and data
system, and this is the most important part of the performance test. The three resolution checks, which
include natural abundance isotope ratios, constitute the next most important part of the performance test.
The correct setting of the baseline threshold, as indicated by the presence of low intensity ions, is the
next most important part of the performance test. Finally, the ion abundance ranges are designed to
encourage some standardization to fragmentation patterns.
526-34
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TABLE 2. RETENTION TIMES (RTs), SUGGESTED QUANTITATION IONS (QIs), AND
INTERNAL STANDARD REFERENCE
Peak #a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Analyte
Nitrobenzene
2,4-Dichlorophenol
2,4,6-Trichlorophenol
1 ,2-Diphenylhydrazine
Prometon
Terbufos
Fonofos
Diazinon
Disulfoton
Acetochlor
Cyanazine
Acenaphthene-6?10 (IS#1)
Phenanthrene-J10 (IS#2)
Chrysene-J12 (IS#3)
l,3-Dimethyl-2-Nitrobenzene (SURR)
Triphenylphosphate (SURR)
Peak
Label in
Figure #1
1
2
4
6
7
8
9
11
12
13
14
5
10
16
O
15
RTb
(min)
6.33
7.70
10.81
15.08
16.64
17.08
17.14
17.29
17.51
18.39
19.73
12.88
17.20
24.98
7.98
24.29
Quanti-
tation
Ion
77
162
196
182
225
231
246
179
88
146
225
164
188
240
151
326
IS#
Ref.
1
1
1
2
2
2
2
2
2
2
2
-
-
-
1
O
a- Number refers to peak number in Figure 1.
b- Column: 30 m X 0.25 mm i.d. DBS-MS (J&W), 0.25 urn film thickness.
526-35
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TABLE 3. METHOD DETECTION LIMITS IN REAGENT WATER FOR SDVB DISK AND
CARTRIDGE EXTRACTION PROCEDURES
Compound
Nitrobenzene
2,4-Dichlorophenol
2,4,6-Trichlorophenol
1 ,2-Diphenylhydr azine
Prometon
Terbufos
Fonofos
Diazinon
Disulfoton
Acetochlor
Cyanazine
Disk Extraction
Spiking Cone.
(ug/L)
0.05
0.05
0.05
0.20
0.10
0.05
0.05
0.10
0.10
0.05
0.05
MDLa
(ug/L)
0.015
0.012
0.012
0.028
0.035
0.017
0.022
0.015
0.024
0.015
0.025
Cartridge Extraction
Spiking Cone.
(ug/L)
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
0.20
MDLa
(ug/L)
0.09
0.04
0.14
0.10
0.14
0.05
0.06
0.03
0.05
0.10
0.09
aMethod detection limits samples were extracted and analyzed over 3 days for 7 replicates following the
procedure outlined in Section 9.2.4.
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TABLE 4A. PRECISION, ACCURACY AND SENSITIVITY DATA FOR METHOD ANALYTES
FORTIFIED AT 0.5 AND 20 UG/L IN REAGENT WATER EXTRACTED WITH SDVB
DISKS
Compound
Nitrobenzene
2,4-Dichlorophenol
2,4,6-Trichlorophenol
1 ,2-Diphenylhydr azine
Prometon
Terbufos
Fonofos
Diazinon
Disulfoton
Acetochlor
Cyanazine
l,3-Dimethyl-2-Nitrobenzene(SUR)c
Triphenylphosphate (SUR)C
Concentration= 0.5 ug/L,
n=7
Mean %
Recovery
106
114
136
121
138
111
104
101
105
124
153
86.2
106
%RSDa
3.8
1.9
2.3
2.5
2.3
2.7
2.0
2.5
2.4
2.9
2.5
2.6
2.6
S/N
Ratio b
159
71
134
25
42
71
138
14
103
67
10
NC
NC
Concentration = 20 ug/L,
n=7
Mean %
Recovery
81.5
97.6
104
103
101
91.4
106
98.3
95.0
98.9
104
84.2
105
%RSDa
5.6
4.3
3.4
3.7
3.8
4.0
4.4
4.7
4.8
4.0
4.6
4.6
5.2
""Relative Standard Deviation = (Standard Deviation/Recovery)* 100.
bSignal-to-noise ratios were calculated for each peak by dividing the peak height for each compound by the peak-
to-peak noise, which was determined for each component from the method blank over a period of time equal to
the full peak width in the target analyte's retention time window.
Surrogate fortification concentration of all samples was 5 ug/L.
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TABLE 4B. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES FORTIFIED
AT 0.5 AND 20 UG/L IN REAGENT WATER EXTRACTED WITH SDVB
CARTRIDGES
Compound
Nitrobenzene
2,4-Dichlorophenol
2,4,6-Trichlorophenol
1 ,2-Diphenylhydr azine
Prometon
Terbufos
Fonofos
Diazinon
Disulfoton
Acetochlor
Cyanazine
l,3-Dimethyl-2-Nitrobenzene(SUR)b
Triphenylphosphate (SUR)b
Concentration = 0.5 ug/L,
n=7
Mean %
Recovery
86.3
104
129
96.9
148
115
101
104
106
125
147
81.7
107
%RSDa
6.9
7.3
3.8
16
3.2
2.8
3.8
3.4
3.0
3.0
5.6
4.1
7.5
Concentration = 20 ug/L,
n=7
Mean %
Recovery
73.4
83.8
92.9
123
100
85.0
90.1
91.1
85.5
91.3
101
80.2
108
%RSDa
3.7
3.3
3.0
3.2
0.8
1.8
1.9
1.4
1.7
1.2
1.2
3.0
3.0
""Relative Standard Deviation = (Standard Deviation/Recovery)* 100.
bSurrogate fortification concentration of all samples was 5 ug/L.
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TABLE 5A. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES FORTIFIED
AT 0.5, AND 20 UG/L IN GROUND WATER EXTRACTED WITH SDVB DISKS
Compound
Nitrobenzene
2,4-Dichlorophenol
2,4,6-Trichlorophenol
1 ,2-Diphenylhydr azine
Prometon
Terbufos
Fonofos
Diazinon
Disulfoton
Acetochlor
Cyanazine
l,3-Dimethyl-2-Nitrobenzene(SUR)b
Triphenylphosphate (SUR)b
Concentration = 0.5 ug/L,
n = 7
Mean %
Recovery
110
113
148
134
152
119
105
110
113
130
163
86.3
105
%RSDa
4.7
3.0
2.0
3.8
2.4
1.6
2.4
1.5
2.5
2.1
2.3
4.8
2.9
Concentration = 20 ug/L,
n = 7
Mean %
Recovery
83.5
96.5
103
102
101
93.4
105
97.6
95.1
98.3
104
83.8
106
%RSDa
5.3
5.0
4.1
4.0
3.8
3.5
3.7
3.7
4.2
3.7
3.8
5.5
4.0
""Relative Standard Deviation = (Standard Deviation/Recovery)* 100.
bSurrogate fortification concentration of all samples was 5 ug/L.
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TABLE 5B. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES FORTIFIED
AT 0.5, 5.0 AND 20 UG/L IN GROUND WATER EXTRACTED WITH SDVB
CARTRIDGES
Compound
Nitrobenzene
2,4-Dichlorophenol
2,4,6-Trichlorophenol
1 ,2-Diphenylhydr azine
Prometon
Terbufos
Fonofos
Diazinon
Disulfoton
Acetochlor
Cyanazine
1 ,3-Dimethyl-2-Nitr obenzene
(SUR)b
Triphenylphosphate (SUR)b
Concentration =
0.5 ug/L, n = 7
Mean %
Recovery
114
110
136
122
144
120
107
105
108
129
160
86.7
119
%RSDa
5.4
3.4
4.1
4.3
3.3
3.0
3.0
2.4
3.8
4.5
3.1
6.3
4.3
Concentration =
5.0 ug/L, n = 7
Mean %
Recovery
87.7
89.8
102
84.8
103
87.1
87.9
88.7
93.3
103
106
86.3
123
%RSDa
3.9
3.9
3.8
3.2
2.9
4.2
3.5
3.1
3.7
3.7
3.6
3.3
3.4
Concentration =
20 ug/L, n = 7
Mean %
Recovery
84.1
89.1
98.3
90.4
100
84.9
90.8
91.6
88.6
94.2
98.6
84.1
111
%RSDa
1.9
2.2
1.4
3.7
2.3
2.4
2.4
2.3
3.1
1.1
3.6
4.6
14
3Relative Standard Deviation = (Standard Deviation/Recovery)* 100.
bSurrogate fortification concentration of all samples was 5 ug/L.
526-40
-------
TABLE 6A. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES FORTIFIED
AT 0.5, 5.0 AND 20 UG/L IN SURFACE WATER EXTRACTED WITH SDVB
DISKS
Compound
Nitrobenzene
2,4-Dichlorophenol
2,4,6-Trichlorophenol
1 ,2-Diphenylhydr azine
Prometon
Terbufos
Fonofos
Diazinon
Disulfoton
Acetochlor
Cyanazine
1 ,3-Dimethyl-2-Nitr obenzene
(SUR)b
Triphenylphosphate (SUR)b
Concentration =
0.5 ug/L, n = 7
Mean %
Recovery
105
111
143
118
136
109
98.9
102
105
124
151
82.9
101
%RSDa
3.8
4.0
2.8
2.4
3.6
3.0
3.8
3.6
3.4
3.4
3.2
4.5
4.5
Concentration =
5.0 ug/L, n = 7
Mean %
Recovery
72.4
81.2
90.8
105
102
85.7
83.0
83.7
81.2
89.2
105
77.4
98.2
%RSDa
4.4
4.0
4.2
4.5
3.1
4.6
4.2
3.5
4.2
3.6
3.9
4.4
3.6
Concentration =
20 ug/L, n = 7
Mean %
Recovery
76.7
89.9
92.4
88.3
89.5
80.2
89.1
86.1
86.6
89.4
93.6
83.9
98.8
%RSDa
4.1
4.8
4.0
3.9
3.9
3.4
4.0
4.1
3.7
3.4
3.9
3.9
4.3
3Relative Standard Deviation = (Standard Deviation/Recovery)* 100.
bSurrogate fortification concentration of all samples was 5 ug/L.
526-41
-------
TABLE 6B. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES FORTIFIED
AT 0.5 AND 20 UG/L IN SURFACE WATER EXTRACTED WITH SDVB
CARTRIDGES
Compound
Nitrobenzene
2,4-Dichlorophenol
2,4,6-Trichlorophenol
1 ,2-Diphenylhydr azine
Prometon
Terbufos
Fonofos
Diazinon
Disulfoton
Acetochlor
Cyanazine
l,3-Dimethyl-2-Nitrobenzene(SUR)b
Triphenylphosphate (SUR)b
Concentration = 0.5 ug/L,
n = 7
Mean %
Recovery
91.7
113
135
107
158
128
109
112
109
132
158
83.0
104
%RSDa
6.6
4.9
4.0
9.9
3.3
3.0
2.3
3.7
3.0
2.8
1.7
5.3
1.9
Concentration = 20 ug/L,
n = 7
Mean %
Recovery
84.9
89.7
97.2
93.7
101
89.0
91.6
92.8
90.0
94.6
99.9
85.1
99.1
%RSDa
2.9
2.2
1.8
2.3
1.3
2.1
1.6
1.5
1.6
1.4
1.0
3.8
1.60
3Relative Standard Deviation = (Standard Deviation/Recovery)* 100.
bSurrogate fortification concentration of all samples was 5 ug/L.
526-42
-------
TABLE 7. SAMPLE HOLDING TIME DATA3 FOR SAMPLES FROM A CHLORINATED
SURFACE WATER, FORTIFIED WITH METHOD ANALYTES AT 5 UG/L,
AND PRESERVED ACCORDING TO SECTION 8.
Compound
Nitrobenzene
2,4-Dichlorophenol
2,4,6-Trichlorophenol
1 ,2-Diphenylhydr azine
Prometon
Terbufos
Fonofos
Diazinon
Disulfoton
Acetochlor
Cyanazine
DayO
% Rec.
83.5
97.2
110
98.8
104
96.7
94.1
93.0
90.8
100
126
Day 3
% Rec.
82.4
92.6
102
93.9
97.7
79.3
88.0
86.7
84.7
93.3
119
Day?
% Rec.
76.7
86.9
97.3
87.5
93.1
69.1
84.8
83.4
81.5
90.5
112
Day 14
% Rec.
88.1
98.1
108
97.2
102
65.4
93.1
91.3
89.1
97.6
125
3Storage stability is expressed as a percent recovery value. Each percent recovery value represents the mean of 3
replicate analyses. Relative Standard Deviations ([Standard Deviation/Recovery]* 100) for replicate analyses
were all less than 13.8 %.
526-43
-------
TABLE 8. EXTRACT HOLDING TIME DATA3 FOR SAMPLES FROM A CHLORINATED
SURFACE WATER, FORTIFIED WITH METHOD ANALYTES AT 5 UG/L,
AND PRESERVED ACCORDING TO SECTION 8.
Compound
Nitrobenzene
2,4-Dichlorophenol
2,4,6-Trichlorophenol
1 ,2-Diphenylhydr azine
Prometon
Terbufos
Fonofos
Diazinon
Disulfoton
Acetochlor
Cyanazine
l,3-Dimethyl-2-Nitrobenzene(SUR)b
Triphenylphosphate (SUR)b
DayO
%Rec.
83.5
97.2
110
98.8
104
96.7
94.1
93.0
90.8
100
126
79.3
97.9
Day 6
% Rec.
84.7
97.5
110
97.9
104
97.5
95.0
93.0
91.9
100
125
78.5
96.6
Day 13
% Rec.
85.2
96.2
108
97.9
103
97.9
94.3
93.6
92.9
99.8
124
78.7
96.7
Day 20
%Rec.
85.5
96.7
107
97.0
103
98.6
95.3
94.3
94.6
99.8
122
78.5
97.8
Day 32
% Rec.
80.5
95.9
112
98.3
101
89.5
97.3
93.0
93.5
98.7
123
77.1
93.4
""Extracts were stored at less than 0 °C and reinjected periodically. Each table value represents the mean of 3
replicate analyses. Relative Standard Deviations ([Standard Deviation/Recovery]* 100) for replicate analyses
were all less than 5.7 %.
526-44
-------
TABLE 9. INITIAL DEMONSTRATION OF CAPABILITY (IDC) REQUIREMENTS
Method
Reference
Requirement
Specification and
Frequency
Acceptance Criteria
Section
9.2.1
Initial
Demonstration of
Low Method
Background
Analyze LRB prior to any
other IDC steps
Demonstrate that all target
analytes are below V3 the
reporting limit or lowest CAL
standard, and that possible
interference from extraction
media do not prevent the
identification and quantification
of method analytes.
Section
9.2.2
Initial
Demonstration of
Precision (IDP)
Analyze 4-7 replicate LFBs
fortified at midrange
concentration.
%RSD must be • 20%
Section
9.2.3
Initial
Demonstration of
Accuracy
Calculate average recovery for
replicates used in IDP
Mean recovery 70-130% of true
value.
Section
9.2.4
Method Detection
Limit (MDL)
Determination
Over a period of three days,
prepare a minimum of 7
replicate LFBs fortified at a
concentration estimated to be
near the MDL. Analyze the
replicates through all steps of
the analysis. Calculate the
MDL using the equation in
Section 9.2.4.
Note: Data from MDL replicates
are not required to meet method
precision and accuracy criteria. If
the MDL replicates are fortified at
a low enough concentration, it is
likely that they will not meet
precision and accuracy criteria.
526-45
-------
TABLE 10. QUALITY CONTROL REQUIREMENTS (SUMMARY)
Method
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Section
10.2.1
MS Tune Check
Analyze DFTPP to verify MS tune
each time the instrument is
calibrated.
Criteria are given in Table 1.
Section
10.2
Initial Calibration
Use internal standard calibration
technique to generate an average
RRF or first or second order
calibration curve. Use at least 5
standard concentrations.
When each calibration standard
is calculated as an unknown
using the calibration curve, the
result must be 70-130% of the
true value for all except the
lowest standard, which must be
50-150% of the true value.
Section 9.4
Laboratory Reagent
Blank (LRB)
Daily, or with each extraction
batch of up to 20 samples,
whichever is more frequent.
Demonstrate that all target
analytes are below V3 the
method reporting limit or lowest
CAL standard, and that possible
interferences do not prevent
quantification of method
analytes.
If targets exceed V3 the MRL,
results for all subject analytes in
extraction batch are invalid.
Section
10.3
Continuing
Calibration Check
(CCC)
Verify initial calibration by
analyzing a calibration standard at
the beginning of each analysis
batch prior to analyzing samples,
after every 10 samples, and after
the last sample.
Low CCC - near MRL
Mid CCC - near midpoint in initial
calibration curve
High CCC - near highest
calibration standard
1) The result for each analyte
must be 70-130% of the true
value for all but the lowest
standard. The lowest standard
must be 50-150% of the true
value.
2) The peak area of internal
standards must be 50-150% of
the average peak area calculated
during the initial calibration.
Results for analytes that do not
meet IS criteria or are not
bracketed by acceptable CCCs
are invalid.
526-46
-------
Method
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Section 9.6
Laboratory Fortified
Blank (LFB)
One LFB is required daily or for
each extraction batch of up to 20
field samples. Rotate the fortified
concentration between low,
medium, and high amounts.
Results of LFB analyses at
medium and high fortification
must be 70-130% of the true
value for each analyte and
surrogate. LFB Results of the
low level LFB must be 50-160%
of the true value.
Section 9.8
Internal Standard
Acenaphthene-J10
phenanthrene-6?10 (IS#2), and
chrysene-t/12 (IS#3), are added to
all standards and sample extracts.
Peak area counts for all ISs in
LFBs, LRBs, and sample
extracts must be within 50-
150% of the average peak area
calculated during the initial
calibration. If ISs do not meet
criteria, corresponding target
results are invalid.
Section 9.9
Surrogate Standards
Surrogate standards, 1,3-
dimethyl-2-nitrobenzene and
triphenylphosphate, are added to
all calibration standards and
samples, including QC samples. If
nitrobenzene is not to be included
on the target list, then the surrogate
l,3-dimethyl-2-nitrobenzene is not
required.
Surrogate recovery must be 70-
130% of the true value. If
surrogate fails this criterion,
report all results for sample as
suspect/surrogate recovery.
Section
9.10
Laboratory Fortified
Sample Matrix (LFM)
and Laboratory
Fortified Matrix
Duplicate (LFMD)
Analyze one LFM per analysis
batch (20 samples or less) fortified
with method analytes at a
concentration close to but greater
than the native concentration.
LFMD should be used in place of
Field Duplicate if frequency of
detects for targets is low.
Recoveries at mid and high
levels not within 70-130% or
low-level recoveries not within
50-150% of the fortified amount
may indicate a matrix effect.
Target analyte RPDs for LFMD
should be ±30% at mid and high
levels of fortification and ±50%
near MRL.
Section
9.11
Field Duplicates (FD)
Extract and analyze at least one
FD with each extraction batch (20
samples or less). A LFMD may be
substituted for a FD when the
frequency of detects for target
analytes is low.
Target analyte RPDs for FD
should be ±30% at mid and high
levels of fortification and ±50%
near MRL.
Section
9.12
Quality Control
Sample (QCS)
Analyzed QCS quarterly.
Results must be 70-130% of the
expected value.
526-47
-------
Method
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Section 8.4
Sample Holding Time
14 days with appropriate
preservation and storage
Sample results are valid only if
samples are extracted within
sample hold time.
Section 8.4
Extract Holding Time
28 days with appropriate storage
Sample results are valid only if
extracts are analyzed within
extract hold time.
526-48
-------
FIGURE 1. EXAMPLE CHROMATOGRAM FOR METHOD 526. NUMBERED PEAKS ARE IDENTIFIED IN TABLE 2.
10
*
Jv
)
w
5<
1
kH
I
£
k^MWv-f-
t
It
1
*
I ..
\t ^^_
7 3-^
y.
I
i
-
3
!
4
.1. ,.
1
L
ll
5
TOO s.eo &.to 10.00 11.00 12.00
14.00 15.00 18.00 17,00 18.00
Time (min)
ioo
25.03
Figure 1 • Total ion cliroinalograni of snrf;ice water sample exlracl \\il\\ \;trgei compounds, iiilental slandards, ;uid surrogate slatid-irds fortified at 5 ppm
level in extract, Peaks with asterisk (*) are interference associated w,i(h the use ofdia/oiidiiivl urea.
526-49
------- |