vvEPA
United States
Environmental Protection
Agency
Method 625.1 - Base/Neutrals and Acids by
GC/MS
December 2014
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U.S. Environmental Protection Agency
Office of Water
Office of Science and Technology
Engineering and Analysis Division (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
EPA-821-R-14-015
Method 625.1 i December 2014
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METHOD 625.1 - BASE/NEUTRALS AND ACIDS BY GC/MS
1. Scope and Application
1.1 This method is for determination of semivolatile organic pollutants in industrial discharges and
other environmental samples by gas chromatography combined with mass spectrometry (GC/MS),
as provided under 40 CFR 136.1. This revision is based on a previous protocol (Reference 1), on
the basic revision promulgated October 26, 1984 (49 FR 43234), and on an interlaboratory method
validation study (Reference 2). Although this method was validated through an interlaboratory
study conducted more than 29 years ago, the fundamental chemistry principles used in this method
remain sound and continue to apply.
1.2 The analytes that may be qualitatively and quantitatively determined using this method and their
CAS Registry numbers are listed in Tables 1 and 2. The method may be extended to determine the
analytes listed in Table 3; however, extraction or gas chromatography of some of these analytes
may make quantitative determination difficult. For examples, benzidine is subject to oxidative
losses during solvent concentration. Under the alkaline conditions of the extraction, alpha-BHC,
gamma-BHC, endosulfan I and II, and endrin are subject to decomposition. Hexachlorocyclo-
pentadiene is subject to thermal decomposition in the inlet of the gas chromatograph, chemical
reaction in acetone solution, and photochemical decomposition. N-nitrosodiphenylamine and other
nitrosoamines may decompose in the gas chromatographic inlet. EPA has provided other methods
(e.g., Method 607 - Nitrosamines) for determination of some of these analytes.
1.3 The large number of analytes in Tables 1-3 of this method makes testing difficult if all analytes are
determined simultaneously. Therefore, it is necessary to determine and perform quality control
(QC) tests for the "analytes of interest" only. Analytes of interest are those required to be
determined by a regulatory/control authority or in a permit, or by a client. If a list of analytes is not
specified, the analytes in Tables 1 and 2 must be determined, at a minimum, and QC testing must be
performed for these analytes. The analytes in Tables 1 and 2, and some of the analytes in Table 3
have been identified as Toxic Pollutants (40 CFR 401.15), expanded to a list of Priority Pollutants
(40 CFR 423, Appendix A).
1.4 In this revision to Method 625, the pesticides and polychlorinated biphenyls (PCBs) have been
moved from Table 1 to Table 3 (Additional Analytes) to distinguish these analytes from the
analytes required in quality control tests (Tables 1 and 2). QC acceptance criteria for pesticides and
PCBs have been retained in Table 6 and may continue to be applied if desired, or if requested or
required by a regulatory/control authority or in a permit. Method 608 should be used for
determination of pesticides and PCBs. Method 1668C may be useful for determination of PCBs as
individual chlorinated biphenyl congeners, and Method 1699B may be useful for determination of
pesticides. At the time of writing of this revision, Methods 1668C and 1699B had not been
approved for use at 40 CFR part 136. The screening procedure for 2,3,7,8-tetrachlorodibenzo-/?-
dioxin (2,3,7,8-TCDD) contained in the version of Method 625 promulgated October 26, 1984 (49
FR 43234) has been replaced with procedures for selected ion monitoring (SIM), and 2,3,7,8-
TCDD may be determined using the SIM procedures. However, EPA Method 613 or 1613B should
be used for analyte-specific determination of 2,3,7,8-TCDD because of the focus of these methods
on this compound. Methods 613 and 1613B are approved for use at 40 CFR part 136.
1.5 Method detection limits (MDLs; Reference 3) for the analytes in Tables 1, 2, and 3 are listed in
those tables. These MDLs were determined in reagent water (Reference 4). Advances in analytical
technology, particularly the use of capillary (open-tubular) columns, allowed laboratories to
routinely achieve MDLs for the analytes in this method that are 2-10 times lower than those in the
Method 625.1 1 December 2014
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version promulgated in 1984 (40 FR 43234). The MDL for an analyte in a specific wastewater may
differ from those listed, depending upon the nature of interferences in the sample matrix.
1.5.1 EPA has promulgated this method at 40 CFRPart 136 for use in wastewater compliance
monitoring under the National Pollutant Discharge Elimination System (NPDES). The data
reporting practices described in Section 15.2 are focused on such monitoring needs and
may not be relevant to other uses of the method.
1.5.2 This method includes "reporting limits" based on EPA's "minimum level" (ML) concept
(see the glossary in Section 22). Tables 1, 2, and 3 contain MDL values and ML values for
many of the analyte s. The MDL for an analyte in a specific wastewater may differ from
those listed in Tables 1, 2, and 3, depending upon the nature of interferences in the sample
matrix.
1.6 This method is performance-based. It may be modified to improve performance (e.g., to overcome
interferences or improve the accuracy of results) provided all performance requirements are met.
1.6.1 Examples of allowed method modifications are described at 40 CFR 136.6. Other
examples of allowed modifications specific to this method are described in Section 8.1.2.
1.6.2 Any modification beyond those expressly permitted at 40 CFR 136.6 or in Section 8.1.2 of
this method shall be considered a major modification subject to application and approval of
an alternate test procedure under 40 CFR 136.4 and 136.5.
1.6.3 For regulatory compliance, any modification must be demonstrated to produce results
equivalent or superior to results produced by this method when applied to relevant
wastewaters (Section 8.3).
1.7 This method is restricted to use by or under the supervision of analysts experienced in the use of a
gas chromatograph/mass spectrometer and in the interpretation of mass spectra. Each laboratory
that uses this method must demonstrate the ability to generate acceptable results using the
procedure in Section 8.2.
1.8 Terms and units of measure used in this method are given in the glossary at the end of the method.
2. Summary of Method
2.1 A measured volume of sample, sufficient to meet an MDL or reporting limit, is serially extracted
with methylene chloride at pH 11-13 and again at a pH less than 2 using a separatory funnel or
continuous liquid/liquid extractor.
2.2 The extract is concentrated to a volume necessary to meet the required compliance or detection
limit, and analyzed by GC/MS. Qualitative identification of an analyte in the extract is performed
using the retention time and the relative abundance of two or more characteristic masses (m/z's).
Quantitative analysis is performed using the internal standard technique with a single characteristic
m/z.
Method 625.1 2 December 2014
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3. Contamination and Interferences
3.1 Solvents, reagents, glassware, and other sample processing labware may yield artifacts, elevated
baselines, or matrix interferences causing misinterpretation of chromatograms and mass spectra.
All materials used in the analysis must be demonstrated to be free from contamination and
interferences by analyzing blanks initially and with each extraction batch (samples started through
the extraction process in a given 12-hour period, to a maximum of 20 samples - see Glossary for
detailed definition), as described in Section 8.5. Specific selection of reagents and purification of
solvents by distillation in all-glass systems may be required. Where possible, labware is cleaned by
extraction or solvent rinse, or baking in a kiln or oven.
3.2 Glassware must be scrupulously cleaned (Reference 5). Clean all glassware as soon as possible
after use by rinsing with the last solvent used in it. Solvent rinsing should be followed by detergent
washing with hot water, and rinses with tap water and reagent water. The glassware should then be
drained dry, and heated at 400 °C for 15 - 30 minutes. Some thermally stable materials, such as
PCBs, may require higher temperatures and longer baking times for removal. Solvent rinses with
pesticide quality acetone, hexane, or other solvents may be substituted for heating. Volumetric
labware should not be heated above 90 °C. After drying and cooling, glassware should be sealed
and stored in a clean environment to prevent any accumulation of dust or other contaminants. Store
inverted or capped with solvent-rinsed or baked aluminum foil.
3.3 Matrix interferences may be caused by contaminants co-extracted from the sample. The extent of
matrix interferences will vary considerably from source to source, depending upon the nature and
diversity of the industrial complex or municipality being sampled. Interferences extracted from
samples high in total organic carbon (TOC) may result in elevated baselines, or by enhancing or
suppressing a signal at or near the retention time of an analyte of interest. Analyses of the matrix
spike and duplicate (Section 8.3) may be useful in identifying matrix interferences, and gel
permeation chromatography (GPC; Section 11.1) and sulfur removal (Section 11.2) may aid in
eliminating these interferences. EPA has provided guidance that may aid in overcoming matrix
interferences (Reference 6).
3.4 In samples that contain an inordinate number of interferences, the use of chemical ionization (CI)
mass spectrometry may make identification easier. Tables 4 and 5 give characteristic CI m/z's for
many of the analytes covered by this method. The use of CI mass spectrometry to support electron
ionization (El) mass spectrometry is encouraged, but not required.
4. Safety
4.1 Hazards associated with each reagent used in this method have not been precisely defined;
however, each chemical compound should be treated as a potential health hazard. From this
viewpoint, exposure to these chemicals must be reduced to the lowest possible level by whatever
means available. The laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A reference file
of safety data sheets (SDSs, OSHA, 29 CFR 1910.1200[g]) should also be made available to all
personnel involved in sample handling and chemical analysis. Additional references to laboratory
safety are available and have been identified (References 7-9) for the information of the analyst.
4.2 The following analytes covered by this method have been tentatively classified as known or
suspected human or mammalian carcinogens: benzo(a)anthracene, benzidine, 3,3'-dichloro-
benzidine, benzo(a)pyrene, alpha-BHC, beta-BHC, delta-BHC, gamma-BHC, Dibenz(a,h)-
anthracene, N-nitrosodimethylamine, 4,4'-DDT, and PCBs. Other compounds in Table 3 may also
Method 625.1 3 December 2014
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be toxic. Primary standards of toxic compounds should be prepared in a chemical fume hood, and a
NIOSH/MESA approved toxic gas respirator should be worn when handling high concentrations of
these compounds.
4.3 This method allows the use of hydrogen as a carrier gas in place of helium (Section 5.6.1.2). The
laboratory should take the necessary precautions in dealing with hydrogen, and should limit
hydrogen flow at the source to prevent buildup of an explosive mixture of hydrogen in air.
5. Apparatus and Materials
Note: Brand names, suppliers, and part numbers are for illustration purposes only. No
endorsement is implied. Equivalent performance may be achieved using equipment and
materials other than those specified here. Demonstrating that the equipment and supplies
used in the laboratory achieves the required performance is the responsibility of the
laboratory. Suppliers for equipment and materials in this method may be found through an
on-line search. Please do not contact EPA for supplier information.
5.1 Sampling equipment, for discrete or composite sampling.
5.1.1 Grab sample bottle - amber glass bottle large enough to contain the necessary sample
volume, fitted with a fluoropolymer-lined screw cap. Foil may be substituted for
fluoropolymer if the sample is not corrosive. If amber bottles are not available, protect
samples from light. Unless pre-cleaned, the bottle and cap liner must be washed, rinsed
with acetone or methylene chloride, and dried before use to minimize contamination.
5.1.2 Automatic sampler (optional) - the sampler must incorporate a pre-cleaned glass sample
container. Samples must be kept refrigerated at <6 °C and protected from light during
compositing. If the sampler uses a peristaltic pump, a minimum length of compressible
silicone rubber tubing may be used. Before use, however, the compressible tubing should
be thoroughly rinsed with methanol, followed by repeated rinsings with reagent water to
minimize the potential for contamination of the sample. An integrating flow meter is
required to collect flow-proportioned composites.
5.2 Glassware
5.2.1 Separatory funnel - Size appropriate to hold sample volume and extraction solvent volume,
and equipped with fluoropolymer stopcock.
5.2.2 Drying column - Chromatographic column, approximately 400 mm long by 19 mm ID,
with coarse frit, or equivalent, sufficient to hold 15 g of anhydrous sodium sulfate.
5.2.3 Concentrator tube, Kuderna-Danish - 10 mL, graduated (Kontes 570050-1025 or
equivalent). Calibration must be checked at the volumes employed in the test. A ground
glass stopper is used to prevent evaporation of extracts.
5.2.4 Evaporative flask, Kuderna-Danish - 500 mL (Kontes 57001-0500 or equivalent). Attach
to concentrator tube with springs.
Note: Use of a solvent recovery system with the K-D or other solvent evaporation
apparatus is strongly recommended.
Method 625.1 4 December 2014
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5.2.5 Snyder column, Kuderna-Danish - Three ball macro (Kontes 503000-0121 or equivalent).
5.2.6 Snyder column, Kuderna-Danish - Two-ball micro (Kontes 569001-0219 or equivalent).
5.2.7 Vials - 10-15 mL, amber glass, with Teflon-lined screw cap.
5.2.8 Continuous liquid-liquid extractor - Equipped with fluoropolymer or glass connecting
joints and stopcocks requiring no lubrication. (Hershberg-Wolf Extractor, Ace Glass
Company, Vineland, N.J., P/N 6848-20, or equivalent.)
5.2.9 In addition to the glassware listed above, the laboratory should be equipped with all
necessary pipets, volumetric flasks, beakers, and other glassware listed in this method and
necessary to perform analyses successfully.
5.3 Boiling chips - Approximately 10/40 mesh, glass, silicon carbide, or equivalent. Heat to 400 °C for
30 minutes, or solvent rinse or Soxhlet extract with methylene chloride.
5.4 Water bath - Heated, with concentric ring cover, capable of temperature control (±2 °C). The bath
should be used in a hood.
5.5 Balances
5.5.1 Analytical, capable of accurately weighing 0.1 mg
5.5.2 Top loading, capable of accurately weighing 10 mg
5.6 GC/MS system
5.6.1 Gas chromatograph (GC) - An analytical system complete with a temperature
programmable gas chromatograph and all required accessories, including syringes and
analytical columns.
5.6.1.1 Injection port - Can be split, splitless, temperature programmable split/splitless
(PTV), solvent-purge, large-volume, on-column, backflushed, or other. An
autosampler is highly recommended because it injects volumes more precisely
than volumes injected manually.
5.6.1.2 Carrier gas - Helium or hydrogen. Data in the tables in this method were
obtained using helium carrier gas. If hydrogen is used, analytical conditions may
need to be adjusted for optimum performance, and calibration and all QC tests
must be performed with hydrogen carrier gas. See Section 4.3 for precautions
regarding the use of hydrogen as a carrier gas.
5.6.2 GC column - See the footnotes to Tables 4 and 5. Other columns or column systems may
be used provided all requirements in this method are met.
5.6.3 Mass spectrometer - Capable of repetitively scanning from 35-450 Daltons (amu) every
two seconds or less, utilizing a 70 eV (nominal) electron energy in the electron impact
ionization mode, and producing a mass spectrum which meets all the criteria in Table 9A or
9B when 50 ng or less of decafluorotriphenyl phosphine (DFTPP; CAS 5074-71-5;
bis(pentafluorophenyl) phenyl phosphine) is injected into the GC.
Method 625.1 5 December 2014
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5.6.4 GC/MS interface - Any GC to MS interface that meets all performance requirements in this
method may be used.
5.6.5 Data system - A computer system must be interfaced to the mass spectrometer that allows
the continuous acquisition and storage of mass spectra acquired throughout the
chromatographic program. The computer must have software that allows searching any
GC/MS data file for specific m/z's (masses) and plotting m/z abundances versus time or
scan number. This type of plot is defined as an extracted ion current profile (EICP).
Software must also be available that allows integrating the abundance at any EICP between
specified time or scan number limits.
5.7 Automated gel permeation chromatograph (GPC)
5.7.1 GPC column - 150 - 700 mm long x 21 - 25 mm ID, packed with 70 g of SX-3 Biobeads;
Bio-Rad Labs, or equivalent
5.7.2 Pump, injection valve, UV detector, and other apparatus necessary to meet the requirements
in this method.
5.8 Nitrogen evaporation device - Equipped with a water bath than can be maintained at 30 - 45 °C;
N-Evap, Organomation Associates, or equivalent.
6. Reagents
6.1 Reagent water - Reagent water is defined as water in which the analytes of interest and interfering
compounds are not detected at the MDLs of the analytes of interest.
6.2 Sodium hydroxide solution (ION)- Dissolve 40 g of NaOH (ACS) in reagent water and dilute to
100 mL.
6.3 Sodium thiosulfate - (ACS) granular.
6.4 Sulfuric acid (1+1) - Slowly add 50 mL of H2SO4 (ACS, sp. gr. 1.84) to 50 mL of reagent water.
6.5 Acetone, methanol, methylene chloride, 2-propanol - High purity pesticide quality, or equivalent,
demonstrated to be free of the analytes of interest and interferences (Section 3). Purification of
solvents by distillation in all-glass systems may be required.
6.6 Sodium sulfate - (ACS) granular, anhydrous, rinsed or Soxhlet extracted with methylene chloride
(20 mL/g), baked at in a shallow tray at 450°C for one hour minimum, cooled in a desiccator, and
stored in a pre-cleaned glass bottle with screw cap that prevents moisture from entering.
6.7 Stock standard solutions (1.00 ug/uL) - Stock standard solutions may be prepared from pure
materials, or purchased as certified solutions. Traceability must be to the National Institute of
Standards and Technology (NIST) or other national standard, when available. Stock solution
concentrations alternate to those below may be used. Because of the toxicity of some of the
compounds, primary dilutions should be prepared in a hood, and a NIOSH/MESA approved toxic
gas respirator should be worn when high concentrations of neat materials are handled. The
following procedure may be used to prepare standards from neat materials.
Method 625.1 6 December 2014
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6.7.1 Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material.
Dissolve the material in pesticide quality methanol or other suitable solvent and dilute to
volume in a 10 mL volumetric flask. Larger volumes may be used at the convenience of
the laboratory. When compound purity is assayed to be 96% or greater, the weight may be
used without correction to calculate the concentration of the stock standard. Commercially
prepared stock standards may be used at any concentration if they are certified by the
manufacturer or by an independent source.
6.7.2 Transfer the stock standard solutions to fluoropolymer-sealed screw-cap bottles. Store at
<6 °C and protect from light. Stock standard solutions should be checked frequently for
signs of degradation or evaporation, especially just prior to preparing calibration standards
from them.
6.7.3 Replace purchased certified stock standard solutions per the expiration date. Replace stock
standard solutions prepared by the laboratory or mixed with purchased solutions after one
year, or sooner if comparison with QC check samples indicates a problem.
6.8 Surrogate standard spiking solution
6.8.1 Select a minimum of three surrogate compounds from Table 8 that most closely match the
recovery of the analytes of interest. For example, if all analytes tested are considered acids,
use surrogates that have similar chemical attributes. Other compounds may be used as
surrogates so long as they do not interfere in the analysis. The deuterium and carbon-13
labeled compounds in Method 1625 are particularly useful because Method 1625 contains
QC acceptance criteria for recovery of these compounds. If only one or two analytes are
determined, one or two surrogates may be used.
6.8.2 Prepare a solution containing each selected surrogate such that the concentration in the
sample would match the concentration in the mid-point calibration standard. For example,
if the midpoint of the calibration is 100 ug/L, prepare the spiking solution at a
concentration of 100 ug/mL in methanol. Addition of 1.00 mL of this solution to 1000 mL
of sample will produce a concentration of 100 ug/L of the surrogate. Alternate volumes
and concentrations appropriate to the response of the GC/MS instrument or for selective ion
monitoring (SIM) may be used, if desired.
6.8.3 Store the spiking solution at < 6°C in a fluoropolymer-sealed glass container. The solution
should be checked frequently for stability. The solution must be replaced after one year, or
sooner if comparison with quality control check standards indicates a problem.
6.9 Internal standard spiking solution
6.9.1 Select three or more internal standards similar in chromatographic behavior to the analytes
of interest. Internal standards are listed in Table 8. Suggested internal standards are:
l,4-dichlorobenzene-d4; naphthalene-ds; acenaphthene-di0; phenanthrene-di0; chrysene-di2;
and perylene-di2. The laboratory must demonstrate that measurement of the internal
standards is not affected by method or matrix interferences (see also Section 7.3.4).
6.9.2 Prepare the internal standards at a concentration of 10 mg/mL in methylene chloride or
other suitable solvent. When 10 uL of this solution is spiked into a 1-mL extract, the
concentration of the internal standards will be 100 ug/mL. A lower concentration
appropriate to the response of the GC/MS instrument or for SIM may be used, if desired.
Method 625.1 7 December 2014
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6.9.3 To assure accurate analyte identification, particularly when SIM is used, it may be
advantageous to include more internal standards than those suggested in Section 6.9.1. An
analyte will be located most accurately if its retention time relative to an internal standard is
in the range of 0.8 to 1.2.
6.10 DFTPP standard - Prepare a solution of DFTPP in methanol or other suitable solvent such that 50
ng or less will be injected (see Section 13.2). An alternate concentration may be used to
compensate for specific injection volumes or to assure that the operating range of the instrument is
not exceeded, so long as the total injected is 50 ng or less. Include benzidine and pentachloro-
phenol in this solution such that <100 ng of benzidine and <50 ng of pentachlorophenol will be
injected.
6.11 Quality control check sample concentrate - See Section 8.2.1.
6.12 GPC calibration solution
6.12.1 Prepare a methylene chloride solution to contain corn oil, bis(2-ethylhexyl) phthalate
(BEHP), perylene, and sulfur at the concentrations in Section 6.12.2, or at concentrations
appropriate to the response of the detector.
Note: Sulfur does not readily dissolve in methylene chloride, but is soluble in warm corn oil.
The following procedure is suggested for preparation of the solution:
6.12.2 Weigh 8 mg sulfur and 2.5 g corn oil into a 100-mL volumetric flask and warm to dissolve
the sulfur. Separately weigh 100 mg BEHP and 2 mg perylene and add to flask. Bring to
volume with methylene chloride and mix thoroughly.
6.12.3 Store the solution in an amber glass bottle with a fluoropolymer-lined screw cap at 0 - 6 °C.
Protect from light. Refrigeration may cause the corn oil to precipitate. Before use, allow the
solution to stand at room temperature until the corn oil dissolves, or warm slightly to aid in
dissolution. Replace the solution every year, or more frequently if the response of a
component changes.
6.13 Sulfur removal - Copper foil or powder (bright, non-oxidized), or tetrabutylammonium sulfite (TEA
sulfite).
6.13.1 Copper foil, or powder- Fisher, Alfa Aesar 42455-18, 625 mesh, or equivalent. Cut
copper foil into approximately 1-cm squares. Copper must be activated on each day it will
be used, as follows:
6.13.1.1 Place the quantity of copper needed for sulfur removal (Section 11.2.1.3) in a
ground-glass-stoppered Erlenmeyer flask or bottle. Cover the foil or powder
with methanol.
6.13.1.2 AddHCl dropwise (0.5 - 1.0 mL) while swirling, until the copper brightens.
6.13.1.3 Pour off the methanol/HCl and rinse 3 times with reagent water to remove all
traces of acid, then 3 times with acetone, then 3 times with hexane.
6.13.1.4 For copper foil, cover with hexane after the final rinse. Store in a stoppered flask
under nitrogen until used. For the powder, dry on a rotary evaporator or under a
stream of nitrogen. Store in a stoppered flask under nitrogen until used.
Method 625.1 8 December 2014
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6.13.2 Tetrabutylammonium sodium sulfite (TEA sodium sulfite)
6.13.2.1 Tetrabutylammonium hydrogen sulfate, [CH3(CH2)3]4NHSO4
6.13.2.2 Sodium sulfite, Na2SO3
6.13.2.3 Dissolve approximately 3 g tetrabutylammonium hydrogen sulfate in 100 mL of
reagent water in an amber bottle with fluoropolymer-lined screw cap. Extract
with three 20-mL portions of hexane and discard the hexane extracts.
6.13.2.4 Add 25 g sodium sulfite to produce a saturated solution. Store at room
temperature. Replace after 1 month.
7. Calibration
7.1 Establish operating conditions equivalent to those in the footnote to Table 4 or 5 for the
base/neutral or acid fraction, respectively. If a combined base/neutral/acid fraction will be
analyzed, use the conditions in the footnote to Table 4. Alternative temperature program and flow
rate conditions may be used. It is necessary to calibrate the GC/MS for the analytes of interest
(Section 1.3) only.
7.2 Internal standard calibration
7.2.1 Prepare calibration standards for the analytes of interest and surrogates at a minimum of
five concentration levels by adding appropriate volumes of one or more stock standards to
volumetric flasks. One of the calibration standards should be at a concentration near the
ML for the analyte in Table 1, 2, or 3. The ML value may be rounded to a whole number
that is more convenient for preparing the standard, but must not exceed the ML values
listed in Table 1, 2, or 3 for those analytes which list ML values. Alternatively, the
laboratory may establish the ML for each analyte based on the concentration of the lowest
calibration standard in a series of standards obtained from a commercial vendor, again,
provided that the ML values do not exceed the MLs in Tables 1, 2, or 3, and provided that
the resulting calibration meets the acceptance criteria in Section 7.2.3, based on the RSD,
RSE, orR2.
The other concentrations should correspond to the expected range of concentrations found
in real samples or should define the working range of the GC/MS system for full-scan
and/or SIM operation, as appropriate. A minimum of six concentration levels is required
for a second order, non-linear (e.g., quadratic; ax2 + bx + c) calibration. Calibrations higher
than second order are not allowed. To each calibration standard or standard mixture, add a
known constant volume of the internal standard solution (Section 6.9), and dilute to volume
with methylene chloride.
Note: The large number of analytes in Tables 1 through 3 may not be soluble or stable in
a single solution; multiple solutions may be required if a large number of analytes
are to be determined simultaneously.
1.2.1.1 Prior to analysis of the calibration standards, inject the DFTPP standard (Section
6.10) and adjust the scan rate of the mass spectrometer to produce a minimum of
5 mass spectra across the DFTPP GC peak. Adjust instrument conditions until
Method 625.1 9 December 2014
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the DFTPP criteria in Table 9A or 9B are met. Calculate peak tailing factors for
benzidine and pentachlorophenol. Calculation of the tailing factor is illustrated
in Figure 1. The tailing factor for benzidine and pentachlorophenol must be <2;
otherwise, adjust instrument conditions and either replace the column or break
off a short section of the front end of the column, and repeat the test.
Note: The DFTPP spectrum may be evaluated by summing the intensities of the
m/z's across the GC peak, subtracting the background at each m/z in a
region of the chromatogram within 20 scans of but not including any part
of, the DFTPP peak. The DFTPP spectrum may also be evaluated by
fitting a Gaussian to each m/z and using the intensity at the maximum for
each Gaussian or by integrating the area at each m/z and using the
integrated areas. Other means may be used for evaluation of the DFTPP
spectrum so long as the spectrum is not distorted to meet the criteria in
Table 9A or 9B.
7.2.1.2 Analyze the mid-point combined base/neutral and acid calibration standard and
enter or review the retention time, relative retention time, mass spectrum, and
quantitation m/z in the data system for each analyte of interest, surrogate, and
internal standard. If additional analytes (Table 3) are to be quantified, include
these analytes in the standard. The mass spectrum for each analyte must be
comprised of a minimum of 2 m/z's (Tables 4 and 5); 3 to 5 m/z's assure more
reliable analyte identification. Suggested quantitation m/z's are shown in Tables
4 and 5 as the primary m/z. If an interference occurs at the primary m/z, use one
of the secondary m/z's or an alternate m/z. A single m/z only is required for
quantitation.
7.2.1.3 For SIM operation, determine the analytes in each descriptor, the quantitation
and qualifier m/z's for each analyte (the m/z's can be the same as for full-scan
operation; Section 7.2.1.2), the dwell time on each m/z for each analyte, and the
beginning and ending retention time for each descriptor. Analyze the verification
standard in scan mode to verify m/z's and establish the retention times for the
analytes. There must be a minimum of two m/z's for each analyte to assure
analyte identification. To maintain sensitivity and capture enough scans (>5)
across each chromatographic peak, there should be no more than 10 m/z's in a
descriptor. For example, for a descriptor with 10 m/z's and a chromatographic
peak width of 5 sec, a dwell time of 100 ms at each m/z would result in a scan
time of 1 second and provide 5 scans across the GC peak. The quantitation m/z
will usually be the most intense peak in the mass spectrum. The quantitation m/z
and dwell time may be optimized for each analyte. However, if a GC peak spans
two (or more) descriptors, the dwell time and cycle time (scans/sec) should be set
to the same value in both segments in order to maintain equivalent response. The
acquisition table used for SIM must take into account the mass defect (usually
less than 0.2 Daltons) that can occur at each m/z being monitored.
7.2.1.4 For combined scan and SIM operation, set up the scan segments and descriptors
to meet requirements in Sections 7.2.1.1 - 7.2.1.3.
7.2.2 Analyze each calibration standard according to Section 12 and tabulate the area at the
quantitation m/z against concentration for each analyte of interest, surrogate, and internal
standard. If an interference is encountered, use a secondary m/z (Table 4 or 5) for
Method 625.1 10 December 2014
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quantitation. Calculate a response factor (RF) for each analyte of interest at each
concentration using Equation 1.
Equation 1
(AsxCis)
RF = ———
(AisxCs)
where:
As = Area of the characteristic m/z for the analyte of interest or surrogate.
A1S= Area of the characteristic m/z for the internal standard.
Cjs= Concentration of the internal standard (ug/mL).
Cs = Concentration of the analyte of interest or surrogate (ug/mL).
7.2.3 Calculate the mean (average) and relative standard deviation (RSD) of the responses
factors. If the RSD is less than 35%, the RF can be assumed to be invariant and the average
RF can be used for calculations. Alternatively, the results can be used to fit a linear or
quadratic regression of response ratios, AS/A1S, vs. concentration ratios CS/C1S. If used, the
regression must be weighted inversely proportional to concentration. The coefficient of
determination (R2; Reference 10) of the weighted regression must be greater than 0.920.
Alternatively, the relative standard error (Reference 11) may be used as an acceptance
criterion. As with the RSD, the RSE must be less than 35%. If an RSE less than 35%
cannot be achieved for a quadratic regression, system performance is unacceptable and the
system must be adjusted and re-calibrated.
Note: Using capillary columns and current instrumentation, it is quite likely that a laboratory can
calibrate the target analytes in this method and achieve a linearity metric (either RSD or
RSE) well below 35%. Therefore, laboratories are permitted to use more stringent
acceptance criteria for calibration than described here, for example, to harmonize their
application of this method with those from other sources.
7.3 Calibration verification - The RF or calibration curve must be verified immediately after calibration
and at the beginning of each 12-hour shift, by analysis of a mid-point calibration standard (Section
7.2.1). The standard(s) must be obtained from a second manufacturer or a manufacturer's batch
prepared independently from the batch used for calibration. Traceability must be to a national
standard, when available. The concentration of the standard should be near the mid-point of the
calibration. Include the surrogates (Section 6.8) in this solution. It is necessary to verify calibration
for the analytes of interest (Section 1.3) only.
Note: The 12-hour shift begins after the DFTPP (Section 13.1) and DDT/endrin tests (if DDT and
endrin are to be determined), and after analysis of the calibration verification standard.
The 12-hour shift ends 12 hours later. The DFTPP and DDT/endrin tests are outside of the
12-hour shift.
7.3.1 Analyze the calibration verification standard(s) beginning in Section 12. Calculate the
percent recovery of each analyte. Compare the recoveries for the analytes of interest
against the acceptance criteria for recovery (Q) in Table 6, and the recoveries for the
surrogates against the acceptance criteria in Table 8. If recovery of the analytes of interest
and surrogates meet acceptance criteria, system performance is acceptable and analysis of
samples may continue. If any individual recovery is outside its limit, system performance
is unacceptable for that analyte.
Method 625.1 11 December 2014
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Note: The large number ofanalytes in Tables 6 and 8 present a substantial probability
that one or more will fail acceptance criteria when all analytes are tested
simultaneously.
7.3.2 When one or more analytes fail acceptance criteria, analyze a second aliquot of the
calibration verification standard and compare only those analytes that failed the first test
(Section 7.3.1) with their respective acceptance criteria. If these analytes now pass, system
performance is acceptable and analysis of samples may continue. A repeat failure of any
analyte that failed the first test, however, will confirm a general problem with the
measurement system. If this occurs, repair the system (Section 7.2.1.1) and repeat the test
(Section 7.3.1), or prepare a fresh calibration standard and repeat the test. If calibration
cannot be verified after maintenance or injection of the fresh calibration standard, re-
calibrate the instrument.
Note: If it is necessary to perform a repeat verification test frequently; i.e., perform two
tests in order to pass, it may be prudent to perform two injections in succession and
review the results, rather than perform one injection, review the results, then
perform the second injection if results from the first injection fail. To maintain the
validity of the test and re-test, system maintenance and/or adjustment is not
permitted between the injections.
7.3.3 Many of the analytes in Table 3 do not have QC acceptance criteria in Table 6, and some of
the surrogates in Table 8 do not have acceptance criteria. If calibration is to be verified and
other QC tests are to be performed for these analytes, acceptance criteria must be developed
and applied. EPA has provided guidance for development of QC acceptance criteria
(References 12 and 13).
7.3.4 Internal standard responses - Verify that detector sensitivity has not changed by comparing
the response of each internal standard in the calibration verification standard (Section 7.3)
to the response of the respective internal standard in the midpoint calibration standard
(Section 7.2.1). The peak areas or heights of the internal standards in the calibration
verification standard must be within 50% to 200% (1/2 to 2x) of their respective peak areas
or heights in the mid-point calibration standard. If not, repeat the calibration verification
test using a fresh calibration verification standard (7.3), or perform and document system
repair. Subsequent to repair, repeat the calibration verification test (Section 7.3.1). If the
responses are still not within 50% to 200%, re-calibrate the instrument (Section 7.2.2) and
repeat the calibration verification test.
8. Quality Control
8.1 Each laboratory that uses this method is required to operate a formal quality assurance program.
The minimum requirements of this program consist of an initial demonstration of laboratory
capability and ongoing analysis of spiked samples and blanks to evaluate and document data quality
(40 CFR 136.7). The laboratory must maintain records to document the quality of data generated.
Results of ongoing performance tests are compared with established QC acceptance criteria to
determine if the results of analyses meet performance requirements of this method. When results of
spiked samples do not meet the QC acceptance criteria in this method, a quality control check
sample (laboratory control sample; LCS) must be analyzed to confirm that the measurements were
performed in an in-control mode of operation. A laboratory may develop its own performance
criteria (as QC acceptance criteria), provided such criteria are as or more restrictive than the criteria
in this method.
Method 625.1 12 December 2014
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8.1.1 The laboratory must make an initial demonstration of capability (DOC) to generate
acceptable precision and recovery with this method. This demonstration is detailed in
Section 8.2.
8.1.2 In recognition of advances that are occurring in analytical technology, and to overcome
matrix interferences, the laboratory is permitted certain options (Section 1.6 and 40 CFR
136.6(b)) to improve separations or lower the costs of measurements. These options may
include alternate extraction, concentration, and cleanup procedures (e.g., solid-phase
extraction; rotary-evaporator concentration; column chromatography cleanup), changes in
column and type of mass spectrometer (40 CFR 136.6(b)(4)(xvi)). Alternate determinative
techniques, such as substitution of spectroscopic or immunoassay techniques, and changes
that degrade method performance, are not allowed. If an analytical technique other than
GC/MS is used, that technique must have a specificity equal to or greater than the specificity
of GC/MS for the analytes of interest. The laboratory is also encouraged to participate in
inter-comparison and performance evaluation studies (see Section 8.10).
8.1.2.1 Each time a modification is made to this method, the laboratory is required to
repeat the procedure in Section 8.2. If the detection limit of the method will be
affected by the change, the laboratory must demonstrate that the MDLs (40 CFR
Part 136, Appendix B) are lower than one-third the regulatory compliance limit or
the MDLs in this method, whichever are greater. If calibration will be affected by
the change, the instrument must be recalibrated per Section 7. Once the
modification is demonstrated to produce results equivalent or superior to results
produced by this method, that modification may be used routinely thereafter, so
long as the other requirements in this method are met (e.g., matrix spike/matrix
spike duplicate recovery and relative percent difference).
8.1.2.1.1 If SPE, or another allowed method modification, is to be applied to a
specific discharge, the laboratory must prepare and analyze matrix
spike/matrix spike duplicate (MS/MSD) samples (Section 8.3) and
LCS samples (Section 8.4). The laboratory must include surrogates
(Section 8.7) in each of the samples. The MS/MSD and LCS
samples must be fortified with the analytes of interest (Section 1.3).
If the modification is for nationwide use, MS/MSD samples must be
prepared from a minimum of nine different discharges (See Section
8.1.2.1.2), and all QC acceptance criteria in this method must be
met. This evaluation only needs to be performed once other than for
the routine QC required by this method (for example it could be
performed by the vendor of the SPE materials) but any laboratory
using that specific SPE material must have the results of the study
available. This includes a full data package with the raw data that
will allow an independent reviewer to verify each determination and
calculation performed by the laboratory (see Section 8.1.2.2.5, items
a-q).
8.1.2.1.2 Sample matrices on which MS/MSD tests must be performed for
nationwide use of an allowed modification:
(a) Effluent from a POTW
(b) ASTMD5905 Standard Specification for Substitute Wastewater
(c) Sewage sludge, if sewage sludge will be in the permit
(d) ASTM Dl 141 Standard Specification for Substitute Ocean
Water, if ocean water will be in the permit
Method 625.1 13 December 2014
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(e) Untreated and treated wastewaters up to a total of nine matrix
types (see
http:water.epa.gov/scitech/wastetech/guide/industry.crm) for a
list of industrial categories with existing effluent guidelines).
At least one of the above waste water matrix types must have at
least one of the following characteristics:
(i) Total suspended solids greater than 40 mg/L
(ii) Total dissolved solids greater than 100 mg/L
(iii) Oil and grease greater than 20 mg/L
(iv) NaCl greater than 120 mg/L
(v) CaCO3 greater than 140 mg/L
The interim acceptance criteria for MS, MSB recoveries that do not
have recovery limits specified in Table 6, and recoveries for
surrogates that do not have recovery limits specified in Table 8, must
be no wider than 60-140 %, and the relative percent difference
(RPD) of the concentrations in the MS and MSB that do not have
RPD limits specified in Table 6 must be less than 30%.
Alternatively, the laboratory may use the laboratory's in-house limits
if they are tighter.
(f) A proficiency testing (PT) sample from a recognized provider, in
addition to tests of the nine matrices (Section 8.1.2.1.1).
8.1.2.2 The laboratory is required to maintain records of modifications made to this
method. These records include the following, at a minimum:
8.1.2.2.1 The names, titles, street addresses, telephone numbers, and e-mail
addresses of the analyst(s) that performed the analyses and
modification, and of the quality control officer that witnessed and will
verify the analyses and modifications.
8.1.2.2.2 A list of analytes, by name and CAS Registry Number.
8.1.2.2.3 A narrative stating reason(s) for the modifications.
8.1.2.2.4 Results from all quality control (QC) tests comparing the modified
method to this method, including:
a) Calibration (Section 7).
b) Calibration verification (Section 7).
c) Initial demonstration of capability (Section 8.2).
d) Analysis of blanks (Section 8.5).
e) Matrix spike/matrix spike duplicate analysis (Section 8.3).
f) Laboratory control sample analysis (Section 8.4).
Method 625.1 14 December 2014
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8.1.2.2.5 Data that will allow an independent reviewer to validate each
determination by tracing the instrument output (peak height, area, or
other signal) to the final result. These data are to include:
a) Sample numbers and other identifiers.
b) Extraction dates.
c) Analysis dates and times.
d) Analysis sequence/run chronology.
e) Sample weight or volume (Section 10).
f) Extract volume prior to each cleanup step (Sections 10 and 11).
g) Extract volume after each cleanup step (Section 11).
h) Final extract volume prior to injection (Sections 10 and 12).
i) Injection volume (Section 12.2.3).
j) Sample or extract dilution (Section 12.2.3.2).
k) Instrument and operating conditions.
1) Column (dimensions, material, etc).
m) Operating conditions (temperature program, flow rate, etc).
n) Detector (type, operating conditions, etc).
o) Chromatograms, mass spectra, and other recordings of raw data.
p) Quantitation reports, data system outputs, and other data to link
the raw data to the results reported.
(q) A written Standard Operating Procedure (SOP)
8.1.2.2.6 Each individual laboratory wishing to use a given modification must
perform the start-up tests in Section 8.1.2 (e.g., DOC, MDL), with
the modification as an integral part of this method prior to applying
the modification to specific discharges. Results of the DOC must
meet the QC acceptance criteria in Table 6 for the analytes of interest
(Section 1.3), and the MDLs must be equal to or lower than the
MDLs in Tables 4 and 5 for the analytes of interest.
8.1.3 Before analyzing samples, the laboratory must analyze a blank to demonstrate that
interferences from the analytical system, labware, and reagents, are under control. Each
time a batch of samples is extracted or reagents are changed, a blank must be extracted and
analyzed as a safeguard against laboratory contamination. Requirements for the blank are
given in Section 8.5.
8.1.4 The laboratory must, on an ongoing basis, spike and analyze a minimum of one sample, in
duplicate, with the samples in an extraction batch (Section 3.1). The laboratory must also
spike and analyze, in duplicate, a minimum of 5% of all samples from a given site or
discharge to monitor and evaluate method and laboratory performance on the sample
matrix. The batch and site/discharge samples may be the same. The procedure for spiking
and analysis is given in Section 8.3.
8.1.5 The laboratory must, on an ongoing basis, demonstrate through analysis of a quality control
check sample (laboratory control sample, LCS; on-going precision and recovery sample,
OPR) that the measurement system is in control. This procedure is given in Section 8.4.
8.1.6 The laboratory should maintain performance records to document the quality of data that is
generated. This procedure is given in Section 8.9.
Method 625.1 15 December 2014
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8.1.7 The large number of analytes tested in performance tests in this method present a
substantial probability that one or more will fail acceptance criteria when many analytes are
tested simultaneously, and a re-test is allowed if this situation should occur. If, however,
continued re-testing results in further repeated failures, the laboratory should document the
failures (e.g., as qualifiers on results) and either avoid reporting results for analytes that
failed or report the problem and failures with the data. Failure to report does not relieve a
discharger or permittee of reporting timely results.
8.2 Initial demonstration of capability (DOC) - To establish the ability to generate acceptable recovery
and precision, the laboratory must perform the DOC in Sections 8.2.1 through 8.2.6 for the analytes
of interest. The laboratory must also establish MDLs for the analytes of interest using the MDL
procedure at 40 CFR 136, Appendix B. The laboratory's MDLs must be equal to or lower than
those listed in Tables 1, 2, or 3 or lower than one third the regulatory compliance limit, whichever
is greater. For MDLs not listed in Tables 4 and 5, the laboratory must determine the MDLs using
the MDL procedure at 40 CFR 136, Appendix B under the same conditions used to determine the
MDLs for the analytes listed in Tables 1, 2, and 3. All procedures used in the analysis, including
cleanup procedures, must be included in the DOC.
8.2.1 For the DOC, a QC check sample concentrate containing each analyte of interest (Section
1.3) is prepared in a water-miscible solvent. The QC check sample concentrate must be
prepared independently from those used for calibration, but may be from the same source
as the second-source standard used for calibration verification (Section 7.3). The
concentrate should produce concentrations of the analytes of interest in water at the mid-
point of the calibration range, and may be at the same concentration as the LCS (Section
8.4). Multiple solutions may be required.
Note: QC check sample concentrates are no longer available from EPA.
8.2.2 Using a pipet or micro-syringe, prepare four LCSs by adding an appropriate volume of the
concentrate to each of four 1-L aliquots of reagent water, and mix well. The volume of
reagent water must be the same as the volume that will be used for the sample, blank
(Section 8.5), and MS/MSD (Section 8.3). A concentration of 100 ug/L was used to
develop the QC acceptance criteria in Table 6. Also add an aliquot of the surrogate spiking
solution (Section 6.8). Also add an aliquot of the surrogate spiking solution (Section 6.8)
to the reagent-water aliquots.
8.2.3 Extract and analyze the four LCSs according to the method beginning in Section 10.
8.2.4 Calculate the average percent recovery (X) and the standard deviation of the percent
recovery (s) for each analyte using the four results.
8.2.5 For each analyte, compare s and (X) with the corresponding acceptance criteria for
precision and recovery in Table 6. For analytes in Table 3 not listed in Table 6, DOC QC
acceptance criteria must be developed by the laboratory. EPA has provided guidance for
development of QC acceptance criteria (References 12 and 13). If s and (X) for all
analytes of interest meet the acceptance criteria, system performance is acceptable and
analysis of blanks and samples may begin. If any individual s exceeds the precision limit
or any individual (X) falls outside the range for recovery, system performance is
unacceptable for that analyte.
Note: The large number of analytes in Tables 1 - 3 present a substantial probability that
one or more will fail at least one of the acceptance criteria when many or all
Method 625.1 16 December 2014
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analytes are determined simultaneously. Therefore, the analyst is permitted to
conduct a "re-test" as described in Sec. 8.2.6.
8.2.6 When one or more of the analytes tested fail at least one of the acceptance criteria, repeat
the test for only the analytes that failed. If results for these analytes pass, system
performance is acceptable and analysis of samples and blanks may proceed. If one or more
of the analytes again fail, system performance is unacceptable for the analytes that failed
the acceptance criteria. Correct the problem and repeat the test (Section 8.2). See Section
8.1.7 for disposition of repeated failures.
Note: To maintain the validity of the test and re-test, system maintenance and/or
adjustment is not permitted between this pair of tests.
8.3 Matrix spike and matrix spike duplicate (MS/MSD) - The laboratory must, on an ongoing basis,
spike at least 5% of the samples from each sample site being monitored in duplicate to assess
accuracy (recovery and precision). The data user should identify the sample and the analytes of
interest (Section 1.3) to be spiked. If direction cannot be obtained, the laboratory must spike at
least one sample per extraction batch of up to 20 samples with the analytes in Tables 1 and 2.
Spiked sample results should be reported only to the data user whose sample was spiked, or as
requested or required by a regulatory/control authority.
8.3.1 If, as in compliance monitoring, the concentration of a specific analyte will be checked
against a regulatory concentration limit, the concentration of the spike should be at that
limit; otherwise, the concentration of the spike should be one to five times higher than the
background concentration determined in Section 8.3.2, at or near the midpoint of the
calibration range, or at the concentration in the LCS (Section 8.4) whichever concentration
would be larger.
8.3.2 Analyze one sample aliquot to determine the background concentration (B) of the each
analyte of interest. If necessary, prepare a new check sample concentrate (Section 8.2.1)
appropriate for the background concentration. Spike and analyze two additional sample
aliquots, and determine the concentration after spiking (A] and A2) of each analyte.
Calculate the percent recoveries (Pi and P2) as 100 (Ai - B) / T and 100 (A2 - B) / T, where
T is the known true value of the spike. Also calculate the relative percent difference (RPD)
between the concentrations (A] and A2) as 200 |A] - A2| / (A] + A2). If necessary, adjust the
concentrations used to calculate the RPD to account for differences in the volumes of the
spiked aliquots.
8.3.3 Compare the percent recoveries (Pi and P2) and the RPD for each analyte in the MS/MSD
aliquots with the corresponding QC acceptance criteria in Table 6. A laboratory may
develop and apply QC acceptance criteria more restrictive than the criteria in Table 6, if
desired.
8.3.3.1 If any individual P falls outside the designated range for recovery in either
aliquot, or the RPD limit is exceeded, the result for the analyte in the unspiked
sample is suspect and may not be reported or used for permitting or regulatory
compliance purposes.. See Section 8.1.7 for disposition of failures.
8.3.3.2 The acceptance criteria in Table 6 were calculated to include an allowance for
error in measurement of both the background and spike concentrations, assuming
a spike to background ratio of 5:1. This error will be accounted for to the extent
that the spike to background ratio approaches 5:1 (Reference 14). If spiking is
Method 625.1 17 December 2014
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performed at a concentration lower than 100 ug/L, the laboratory must use either
the QC acceptance criteria in Table 6, or optional QC acceptance criteria
calculated for the specific spike concentration. To use the optional acceptance
criteria: (1) Calculate recovery (X') using the equation in Table 7, substituting
the spike concentration (T) for C; (2) Calculate overall precision (S1) using the
equation in Table 7, substituting X' for X ; (3) Calculate the range for recovery at
the spike concentration as (100 X'/T) ± 2.44(100 S'/T)% (Reference 14). For
analytes in Table 3 not listed in Table 6, QC acceptance criteria must be
developed by the laboratory. EPA has provided guidance for development of QC
acceptance criteria (References 12 and 13).
8.3.4 After analysis of a minimum of 20 MS/MSD samples for each target analyte and surrogate,
the laboratory must calculate and apply in-house QC limits for recovery and RPD of future
MS/MSD samples (Section 8.3). The QC limits for recovery are calculated as the mean
observed recovery ± 3 standard deviations, and the upper QC limit for RPD is calculated as
the mean RPD plus 3 standard deviations of the RPDs. The in-house QC limits must be
updated at least every two years and re-established after any major change in the analytical
instrumentation or process. At least 80% of the analytes tested in the MS/MSD must have
in-house QC acceptance criteria that are tighter than those in Table 6. If an in-house QC
limit for the RPD is greater than the limit in Table 6, then the limit in Table 6 must be used.
Similarly, if an in-house lower limit for recovery is below the lower limit in Table 6, then
the lower limit in Table 6 must be used, and if an in-house upper limit for recovery is above
the upper limit in Table 6, then the upper limit in Table 6 must be used. The laboratory
must evaluate surrogate recovery data in each sample against its in-house surrogate
recovery limits. The laboratory may use 60 -140% as interim acceptance criteria for
surrogate recoveries until in-house limits are developed.
8.4 Laboratory control sample (LCS) - A QC check sample (laboratory control sample, LCS; on-going
precision and recovery sample, OPR) containing each analyte of interest (Section 1.3) and surrogate
must be prepared and analyzed with each extraction batch of up to 20 samples to demonstrate
acceptable recovery of the analytes of interest from a clean sample matrix.
8.4.1 Prepare the LCS by adding QC check sample concentrate (Section 8.2.1) to reagent water.
Include all analytes of interest (Section 1.3) in the LCS. The LCS may be the same sample
prepared for the DOC (Section 8.2.1). The volume of reagent water must be the same as
the volume used for the sample, blank (Section 8.5), and MS/MSD (Section 8.3). Also add
an aliquot of the surrogate spiking solution (Section 6.8). The concentration of the analytes
in reagent water should be the same as the concentration in the DOC (Section 8.2.2).
8.4.2 Analyze the LCS prior to analysis of field samples in the extraction batch. Determine the
concentration (A) of each analyte. Calculate the percent recovery (Ps) as 100 (A/T)%,
where T is the true value of the concentration in the LCS.
8.4.3 Compare the percent recovery (Ps) for each analyte with its corresponding QC acceptance
criterion in Table 6. For analytes of interest in Table 3 not listed in Table 6, use the QC
acceptance criteria developed for the MS/MSD (Section 8.3.3.2). If the recoveries for all
analytes of interest fall within their respective QC acceptance criteria, analysis of blanks
and field samples may proceed. If any individual Ps falls outside the range, proceed
according to Section 8.4.4.
Note: The large number of analytes in Tables 1 - 3 present a substantial probability that
one or more will fail the acceptance criteria when all analytes are tested
Method 625.1 18 December 2014
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simultaneously. Because a re-test is allowed in event of failure (Sections 8.1.7 and
8.4.3), it may be prudent to extract and analyze two LCSs together and evaluate
results of the second analysis against the QC acceptance criteria only if an analyte
fails the first test.
8.4.4 Repeat the test only for those analytes that failed to meet the acceptance criteria (Ps). If
these analytes now pass, system performance is acceptable and analysis of blanks and
samples may proceed. Repeated failure, however, will confirm a general problem with the
measurement system. If this occurs, repeat the test using a fresh LCS (Section 8.2.2) or an
LCS prepared with a fresh QC check sample concentrate (Section 8.2.1), or perform and
document system repair. Subsequent to repair, repeat the LCS test (Section 8.4). If failure
of the LCS indicates a systemic problem with samples in the batch, re-extract and re-
analyze the samples in the batch. See Section 8.1.7 for disposition of repeated failures.
Note: To maintain the validity of the test and re-test, system maintenance and/or
adjustment is not permitted between the pair of tests.
8.4.5 After analysis of 20 LCS samples, the laboratory must calculate and apply in-house QC
limits for recovery to future LCS samples (Section 8.4). Limits for recovery in the LCS are
calculated as the mean recovery ±3 standard deviations. A minimum of 80% of the
analytes tested for in the LCS must have QC acceptance criteria tighter than those in Table
6. Many of the analytes and surrogates may not contain recommended acceptance criteria.
The laboratory should use 60 -140% as interim acceptance criteria for recoveries of spiked
analytes and surrogates that do not have recovery limits specified in Table 8, until in-house
LCS and surrogate limits are developed. If an in-house lower limit for recovery is lower
than the lower limit in Table 6, the lower limit in Table 6 must be used, and if an in-house
upper limit for recovery is higher than the upper limit in Table 6, the upper limit in Table 6
must be used.
8.5 Blank - A blank must be extracted and analyzed with each extraction batch to demonstrate that the
reagents and equipment used for preparation and analysis are free from contamination.
8.5.1 Spike the surrogates into the blank. Extract and concentrate the blank using the same
procedures and reagents used for the samples, LCS, and MS/MSD in the batch. Analyze
the blank immediately after analysis of the LCS (Section 8.4) and prior to analysis of the
MS/MSD and samples to demonstrate freedom from contamination.
8.5.2 If any analyte of interest is found in the blank: 1) at a concentration greater than the MDL
for the analyte, 2) at a concentration greater than one-third the regulatory compliance limit,
or 3) at a concentration greater than one-tenth the concentration in a sample in the
extraction batch, whichever is greater, analysis of samples must be halted and samples
affected by the blank must be re-extracted and the extracts re-analyzed. Samples must be
associated with an uncontaminated blank before they may be reported or used for
permitting or regulatory compliance purposes.
8.6 Internal standards responses
8.6.1 Calibration verification - The responses (GC peak heights or areas) of the internal
standards in the calibration verification must be within 50% to 200% (1/2 to 2x) of their
respective responses in the mid-point calibration standard. If they are not, repeat the
calibration verification (Section 7.4) test or perform and document system repair.
Subsequent to repair, repeat the calibration verification. If the responses are still not within
Method 625.1 19 December 2014
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50% to 200%, re-calibrate the instrument (Section 7) and repeat the calibration
verification/LCS test.
8.6.2 Samples, blanks, LCSs, and MS/MSDs - The responses (GC peak heights or areas) of the
internal standards in each sample, blank, and MS/MSD must be within 50% to 200% (1/2
to 2x) of its respective response in the most recent LCS. If, as a group, all internal
standards are not within this range, perform and document system repair, repeat the
calibration verification/LCS test (Section 8.4), and re-analyze the affected samples. If a
single internal standard is not within the 50% to 200% range, use an alternate internal
standard for quantitation of the analyte referenced to the affected internal standard.
8.7 Surrogate recoveries - Spike the surrogates into all samples, blanks, LCSs, and MS/MSDs.
Compare surrogate recoveries against the QC acceptance criteria in Table 8 and/or those developed
in Section 7.3.3. If any recovery fails its criteria, attempt to find and correct the cause of the
failure. Surrogate recoveries from the blank and LCS may be used as pass/fail criteria by the
laboratory or as required by a regulatory authority, or may be used to diagnose problems with the
analytical system.
8.8 DDT and endrin decomposition (breakdown) - If DDT and/or endrin are to be analyzed using this
method, a DDT/endrin decomposition test must be performed to reliably quantify these two
pesticides. The DDT/endrin decomposition test to be used is in EPA Method 608A or 1656.
8.9 As part of the QC program for the laboratory, control charts or statements of accuracy for
wastewater samples must be assessed and records maintained (40 CFR 136.7(c)(l)(viii)). After
analysis of five or more spiked wastewater samples as in Section 8.3, calculate the average percent
recovery (X) and the standard deviation of the percent recovery (sp). Express the accuracy
assessment as a percent interval from X -2spto X +2sp. For example, if X =90%andsp= 10%,
the accuracy interval is expressed as 70 -110%. Update the accuracy assessment for each analyte
on a regular basis (e.g., after each 5-10 new accuracy measurements).
8.10 It is recommended that the laboratory adopt additional quality assurance practices for use with this
method. The specific practices that are most productive depend upon the needs of the laboratory
and the nature of the samples. Field duplicates may be analyzed to assess the precision of
environmental measurements. Whenever possible, the laboratory should analyze standard reference
materials and participate in relevant performance evaluation studies.
9. Sample Collection, Preservation, and Handling
9.1 Collect samples as grab samples in glass bottles or in refrigerated bottles using automatic sampling
equipment. Collect 1-L of ambient waters, effluents, and other aqueous samples. If the sensitivity of
the analytical system is sufficient, a smaller volume (e.g., 250 mL), but no less than 100 mL, may be
used. Conventional sampling practices (Reference 15) should be followed, except that the bottle
must not be pre-rinsed with sample before collection. Automatic sampling equipment must be as
free as possible of polyvinyl chloride or other tubing or other potential sources of contamination. If
needed, collect additional sample(s) for the MS/MSD (Section 8.3).
9.2 Ice or refrigerate samples at <6 °C from the time of collection until extraction, but do not freeze. If
residual chlorine is present, add 80 mg of sodium thiosulfate per liter of sample and mix well. Any
method suitable for field use may be employed to test for residual chlorine (Reference 16). Do not
add excess sodium thiosulfate. If sodium thiosulfate interferes in the determination of the analytes,
an alternate preservative (e.g., ascorbic acid or sodium sulfite) may be used.
Method 625.1 20 December 2014
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9.3 All samples must be extracted within 7 days of collection and sample extracts must be analyzed
within 40 days of extraction.
10. Extraction
10.1 This section contains procedures for separatory funnel liquid-liquid extraction (SFLLE) and
continuous liquid-liquid extraction (CLLE). SFLLE is faster, but may not be as effective as CLLE
for recovery of polar analytes such as phenol. SFLLE is labor intensive and may result in
formation of emulsions that are difficult to break. CLLE is less labor intensive, avoids emulsion
formation, but requires more time (18-24 hours) and more hood space, and may require more
solvent. The procedures assume base-neutral extraction followed by acid extraction. For some
matrices and analytes of interest, improved results may be obtained by acid-neutral extraction
followed by base extraction. A single acid or base extraction may also be performed. If an
extraction scheme alternate to base-neutral followed by acid extraction is used, all QC tests must be
performed and all QC acceptance criteria must be met with that extraction scheme as an integral
part of this method.
10.2 Separatory funnel liquid-liquid extraction (SFLLE) and extract concentration
10.2.1 The SFLLE procedure below assumes a sample volume of 1 L. When a different sample
volume is extracted, adjust the volume of methylene chloride accordingly.
10.2.2 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Pour the entire sample into the separatory funnel. Pipetthe surrogate standard
spiking solution (Section 6.8) into the separatory funnel. If the sample will be used for the
LCS or MS or MSB, pipetthe appropriate check sample concentrate (Section 8.2.1 or
8.3.2) into the separatory funnel. Mix well. Check the pH of the sample with wide-range
pH paper and adjust to pH 11-13 with sodium hydroxide solution.
10.2.3 Add 60 mL of methylene chloride to the sample bottle, seal, and shake for approximately
30 seconds to rinse the inner surface. Transfer the solvent to the separatory funnel and
extract the sample by shaking the funnel for two minutes with periodic venting to release
excess pressure. Allow the organic layer to separate from the water phase for a minimum
of 10 minutes. If the emulsion interface between layers is more than one-third the volume
of the solvent layer, the analyst must employ mechanical techniques to complete the phase
separation. The optimum technique depends upon the sample, but may include stirring,
filtration of the emulsion through glass wool, centrifugation, or other physical methods.
Collect the methylene chloride extract in a flask. If the emulsion cannot be broken
(recovery of <80% of the methylene chloride), transfer the sample, solvent, and emulsion
into a continuous extractor and proceed as described in Section 10.3.
10.2.4 Add a second 60-mL volume of methylene chloride to the sample bottle and repeat the
extraction procedure a second time, combining the extracts in the Erlenmeyer flask.
Perform a third extraction in the same manner.
10.2.5 Adjust the pH of the aqueous phase to less than 2 using sulfuric acid. Serially extract the
acidified aqueous phase three times with 60 mL aliquots of methylene chloride. Collect
and combine the extracts in a flask in the same manner as the base/neutral extracts.
Note: Base/neutral and acid extracts may be combined for concentration and analysis
provided all QC tests are performed and all QC acceptance criteria met for the
Method 625.1 21 December 2014
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analytes of interest with the combined extract as an integral part of this method, and
provided that the analytes of interest are as reliably identified and quantified as
when the extracts are analyzed separately. If doubt exists as to whether
identification and quantitation will be affected by use of a combined extract, the
fractions must be analyzed separately.
10.2.6 For each fraction or the combined fractions, assemble a Kuderna-Danish (K-D)
concentrator by attaching a 10-mL concentrator tube to a 500-mL evaporative flask. Other
concentration devices or techniques may be used in place of the K-D concentrator so long
as the requirements in Section 8.2 are met.
10.2.7 For each fraction or the combined fractions, pour the extract through a solvent-rinsed
drying column containing about 10 cm of anhydrous sodium sulfate, and collect the extract
in the K-D concentrator. Rinse the Erlenmeyer flask and column with 20-30 mL of
methylene chloride to complete the quantitative transfer.
10.2.8 Add one or two clean boiling chips and attach a three-ball Snyder column to the
evaporative flask for each fraction (Section 10.2.7). Pre-wet the Snyder column by adding
about 1 mL of methylene chloride to the top. Place the K-D apparatus on a hot water bath
(60-65°C) so that the concentrator tube is partially immersed in the hot water, and the entire
lower rounded surface of the flask is bathed with hot vapor. Adjust the vertical position of
the apparatus and the water temperature as required to complete the concentration in 15-20
minutes. At the proper rate of distillation, the balls of the column will actively chatter but
the chambers will not flood with condensed solvent. When the apparent volume of liquid
reaches 1 mL or other determined amount, remove the K-D apparatus from the water bath
and allow to drain and cool for at least 10 minutes. Remove the Snyder column and rinse
the flask and its lower joint into the concentrator tube with 1-2 mL of methylene chloride.
A 5-mL syringe is recommended for this operation. If the sample will be cleaned up,
reserve the K-D apparatus for concentration of the cleaned up extract. Adjust the volume
to 5 mL with methylene chloride and proceed to Section 11 for cleanup; otherwise, further
concentrate the extract for GC/MS analysis per Section 10.2.9 or 10.2.10.
10.2.9 Micro Kuderna-Danish concentration - add another one or two clean boiling chips to the
concentrator tube for each fraction and attach a two-ball micro-Snyder column. Pre-wet the
Snyder column by adding about 0.5 mL of methylene chloride to the top. Place the K-D
apparatus on a hot water bath (60-65 °C) so that the concentrator tube is partially immersed
in hot water. Adjust the vertical position of the apparatus and the water temperature as
required to complete the concentration in 5-10 minutes. At the proper rate of distillation
the balls of the column will actively chatter but the chambers will not flood with condensed
solvent. When the apparent volume of liquid reaches about 1 mL or other determined
amount, remove the K-D apparatus from the water bath and allow it to drain and cool for at
least 10 minutes. Remove the Snyder column and rinse the flask and its lower joint into the
concentrator tube with approximately 0.2 mL of or methylene chloride. Adjust the final
volume to 1.0 mL or a volume appropriate to the sensitivity desired (e.g., to meet lower
MDLs or for selected ion monitoring). Record the volume, stopper the concentrator tube
and store refrigerated if further processing will not be performed immediately. If the
extracts will be stored longer than two days, they should be transferred to fluoropolymer-
lined screw-cap vials and labeled base/neutral or acid fraction as appropriate. Mark the
level of the extract on the vial so that solvent loss can be detected.
10.2.10 Nitrogen evaporation and solvent exchange - Extracts may be concentrated for analysis
using nitrogen evaporation in place of micro K-D concentration (Section 10.2.9). Extracts
Method 625.1 22 December 2014
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that have been cleaned up using sulfur removal (Section 12.2) and are ready for analysis are
exchanged into methylene chloride.
10.2.10.1 Transfer the vial containing the sample extract to the nitrogen evaporation
(blowdown) device (Section 5.8). Lower the vial into the water bath and begin
concentrating. If the more volatile analytes (Section 1.2) are to be concentrated,
use room temperature for concentration; otherwise, a slightly elevated (e.g., 30 -
45 °C) may be used. During the solvent evaporation process, keep the solvent
level below the water level of the bath and do not allow the extract to become
dry. Adjust the flow of nitrogen so that the surface of the solvent is just visibly
disturbed. A large vortex in the solvent may cause analyte loss.
10.2.10.2 Extracts to be solvent exchanged - When the volume of the liquid is
approximately 200 uL, add 2 to 3 mL of methylene chloride and continue
concentrating to approximately 100 uL. Repeat the addition of solvent and
concentrate once more. Adjust the final extract volume to be consistent with the
volume extracted and the sensitivity desired.
10.2.10.3 For extracts that have been cleaned up by GPC and that are to be concentrated to
a nominal volume of 1 mL, adjust the final volume to compensate the GPC loss.
For a 50% GPC loss, concentrate the extract to 1/2000 of the volume extracted.
For example, if the volume extracted is 950 mL, adjust the final volume to 0.48
mL. For extracts that have not been cleaned up by GPC and are to be
concentrated to a nominal volume of 1.0 mL, adjust the final extract volume to
1/1000 of the volume extracted. For example, if the volume extracted is 950 mL,
adjust the final extract volume to 0.95 mL.
Note: The difference in the volume fraction for an extract cleaned up by GPC
accounts for the loss in GPC cleanup. Also, by preserving the ratio
between the volume extracted and the final extract volume, the
concentrations and detection limits do not need to be adjusted for
differences in the volume extracted and the extract volume.
10.2.11 Transfer the concentrated extract to a vial with fluoropolymer-lined cap. Seal the vial and
label with the sample number. Store in the dark at room temperature until ready for GC
analysis. If GC analysis will not be performed on the same day, store the vial in the dark at
<6 °C. Analyze the extract by GC/MS per the procedure in Section 12.
10.2.12 Determine the original sample volume by refilling the sample bottle to the mark and
transferring the liquid to an appropriately sized graduated cylinder. For sample volumes on
the order of 1000 mL, record the sample volume to the nearest 10 mL; for sample volumes
on the order of 100 mL, record the volume to the nearest 1 mL. Sample volumes may also
be determined by weighing the container before and after filling to the mark with water.
10.3 Continuous liquid/liquid extraction (CLLE)
Note: With CLLE, phenol, 2,4-dimethylphenol, and some other analytes may be preferentially
extracted into the base-neutral fraction. Determine an analyte in the fraction in which it is
identified and quantified most reliably. Also, the short-chain phthalate esters (e.g.,
dimethyl phthalate, diethyl phthalate) and some other compounds may hydrolyze during
prolonged exposure to basic conditions required for continuous extraction, resulting in low
Method 625.1 23 December 2014
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recovery of these analytes. When these analytes are of interest, their recovery may be
improved by performing the acid extraction first.
10.3.1 Use CLLE when experience with a sample from a given source indicates an emulsion
problem, or when an emulsion is encountered during SFLLE. CLLE may be used for all
samples, if desired.
10.3.2 Mark the water meniscus on the side of the sample bottle for later determination of sample
volume. Check the pH of the sample with wide-range pH paper and adjust to pH 11-13
with sodium hydroxide solution. Transfer the sample to the continuous extractor. Pipet
surrogate standard spiking solution (Section 6.8) into the sample. If the sample will be
used for the LCS or MS or MSB, pipet the appropriate check sample concentrate (Section
8.2.1 or 8.3.2) into the extractor. Mix well. Add 60 mL of methylene chloride to the
sample bottle, seal, and shake for 30 seconds to rinse the inner surface. Transfer the
solvent to the extractor.
10.3.3 Repeat the sample bottle rinse with an additional 50-100 mL portion of methylene chloride
and add the rinse to the extractor.
10.3.4 Add a suitable volume of methylene chloride to the distilling flask (generally 200 - 500
mL), add sufficient reagent water to ensure proper operation, and extract for 18-24 hours.
A shorter or longer extraction time may be used if all QC acceptance criteria are met. Test
and, if necessary, adjust the pH of the water during the second or third hour of the
extraction. After extraction, allow the apparatus to cool, then detach the distilling flask.
Dry, concentrate, and seal the extract per Sections 10.2.6 through 10.2.11. See the note at
Section 10.2.5 regarding combining extracts of the base/neutral and acid fractions.
10.3.5 Charge the distilling flask with methylene chloride and attach it to the continuous extractor.
Carefully, while stirring, adjust the pH of the aqueous phase to less than 2 using sulfuric
acid. Extract for 18 - 24 hours. A shorter or longer extraction time may be used if all QC
acceptance criteria are met. Test and, if necessary, adjust the pH of the water during the
second or third hour of the extraction. After extraction, allow the apparatus to cool, then
detach the distilling flask. Dry, concentrate, and seal the extract per Sections 10.2.6
through 10.2.11. Determine the sample volume per Section 10.2.12.
11. Extract Cleanup
Note Cleanup may not be necessary for relatively clean samples (e.g., treated effluents,
groundwater, drinking water). If particular circumstances require the use of a cleanup
procedure, the laboratory may use any or all of the procedures below or any other
appropriate procedure. Before using a cleanup procedure, the laboratory must demonstrate
that the requirements of Section 8.1.2 can be met using the cleanup procedure as an integral
part of this method.
11.1 Gel permeation chromatography (GPC)
11.1.1 Calibration
11.1.1.1 Load the calibration solution (Section 6.12) into the sample loop
Method 625.1 24 December 2014
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11.1.1.2 Inj ect the calibration solution and record the signal from the detector. The
elution pattern will be corn oil, bis(2-ethylhexyl) phthalate, pentachlorophenol,
perylene, and sulfur.
11.1.1.3 Set the "dump time" to allow >85% removal of the corn oil and >85% collection
of the phthalate.
11.1.1.4 Set the "collect time" to the peak minimum between perylene and sulfur.
11.1.1.5 Verify calibration with the calibration solution after every 20 or fewer extracts.
Calibration is verified if the recovery of the pentachlorophenol is greater than
85%. If calibration is not verified, recalibrate using the calibration solution, and
re-extract and clean up the preceding extracts using the calibrated GPC system.
11.1.2 Extract cleanup - GPC requires that the column not be overloaded. The column specified
in this method is designed to handle a maximum of 0.5 g of high molecular weight material
in a 5-mL extract. If the extract is known or expected to contain more than 0.5 g, the
extract is split into fractions for GPC and the fractions are combined after elution from the
column. The solids content of the extract may be obtained gravimetrically by evaporating
the solvent from a 50-|oL aliquot.
11.1.2.1 Filter the extract or load through the filter holder to remove particulates. Load
the extract into the sample loop. The maximum capacity of the column is 0.5 -
1.0 g. If necessary, split the extract into multiple aliquots to prevent column
overload.
11.1.2.2 Elute the extract using the calibration data determined in Section 11.1.1. Collect
the eluate in the K-D apparatus reserved in Section 10.2.8.
11.1.3 Concentrate the cleaned up extract per Sections 10.2.8 and 10.2.9 or 10.2.10.
11.1.4 Rinse the sample loading tube thoroughly with methylene chloride between extracts to
prepare for the next sample.
11.1.5 If a particularly dirty extract is encountered, run a methylene chloride blank through the
system to check for carry-over.
11.2 Sulfur removal
Note: Separate procedures using copper or TEA sulfite are provided in this section for sulfur
removal. They may be used separately or in combination, if desired.
11.2.1 Removal with copper (Reference 17)
Note: If (I) an additional compound (Table 3) is to be determined; (2) sulfur is to be
removed; (3) copper will be used for sulfur removal; and (4) a sulfur matrix is
known or suspected to be present, the laboratory must demonstrate that the
additional compound can be successfully extracted and treated with copper in the
sulfur matrix. Some of the additional compounds (Table 3) are known not to be
amenable to sulfur removal with copper (e.g. Atrazine and Diazinon).
Method 625.1 25 December 2014
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11.2.1.1 Quantitatively transfer the extract from Section 10.2.8 to a 40- to 50-mL flask or
bottle. If there is evidence of water in the concentrator tube after the transfer,
rinse the tube with small portions of hexane: acetone (40:60) and add to the flask
or bottle. Mark and set aside the concentrator tube for use in re-concentrating the
extract.
11.2.1.2 Add 10 - 20 g of granular anhydrous sodium sulfate to the flask. Swirl to dry the
extract.
11.2.1.3 Add activated copper (Section 6.13.1.4) and allow to stand for 30 - 60 minutes,
swirling occasionally. If the copper does not remain bright, add more and swirl
occasionally for another 30-60 minutes.
11.2.1.4 After drying and sulfur removal, quantitatively transfer the extract to a nitrogen-
evaporation vial or tube and proceed to Section 10.2.10 for nitrogen evaporation
and solvent exchange, taking care to leave the sodium sulfate and copper in the
flask.
11.2.2 Removal with TEA sulfite
11.2.2.1 Using small volumes of hexane, quantitatively transfer the extract to a 40- to 50-
mL centrifuge tube with fluoropolymer-lined screw cap.
11.2.2.2 Add 1 - 2 mL of TEA sulfite reagent (Section 6.13.2.4), 2 - 3 mL of 2-propanol,
and approximately 0.7 g of sodium sulfite (Section 6.13.2.2) crystals to the tube.
Cap and shake for 1 - 2 minutes. If the sample is colorless or if the initial color is
unchanged, and if clear crystals (precipitated sodium sulfite) are observed,
sufficient sodium sulfite is present. If the precipitated sodium sulfite disappears,
add more crystalline sodium sulfite in approximately 0.5 g portions until a solid
residue remains after repeated shaking.
11.2.2.3 Add 5-10 mL of reagent water and shake for 1 - 2 minutes. Centrifuge to settle
the solids.
11.2.2.4 Quantitatively transfer the hexane (top) layer through a small funnel containing a
few grams of granular anhydrous sodium sulfate to a nitrogen-evaporation vial or
tube and proceed to Section 10.2.10 for nitrogen evaporation and solvent
exchange.
12. Gas Chromatography/Mass Spectrometry
12.1 Establish the operating conditions in Table 4 or 5 for analysis of a base/neutral or acid extract,
respectively. For analysis of a combined extract (Section 10.2.5, note), use the operating conditions
in Table 4. Included in these tables are retention times and MDLs that can be achieved under these
conditions. Examples of the separations achieved are shown in Figure 2 for the combined extract.
Alternative columns or chromatographic conditions may be used if the requirements of Section 8.2
are met. Verify system performance per Section 13.
Method 625.1 26 December 2014
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12.2 Analysis of a standard or extract
12.2.1 Bring the standard or concentrated extract (Section 10.2.9 or 10.2.11) to room temperature
and verify that any precipitate has redissolved. Verify the level on the extract and bring to
the mark with solvent if required.
12.2.2 Add the internal standard solution (Section 6.9) to the extract. Mix thoroughly.
12.2.3 Inject an appropriate volume of the sample extract or standard solution using split, splitless,
solvent purge, large-volume, or on-column injection. If the sample is injected manually the
solvent-flush technique should be used. The injection volume depends upon the technique
used and the ability to meet MDLs or reporting limits for regulatory compliance. Injected
volumes must be the same for standards and sample extracts. Record the volume injected
to two significant figures.
12.2.3.1 Start the GC column oven program upon injection. Start MS data collection after
the solvent peak elutes. Stop data collection after benzo(ghi)perylene elutes for
the base/neutral or combined fractions, or after pentachlorophenol elutes for the
acid fraction. Return the column to the initial temperature for analysis of the
next standard solution or extract.
12.2.3.2 If the concentration of any analyte of interest exceeds the calibration range, either
extract and analyze a smaller sample volume, or dilute and analyze the diluted
extract after bringing the concentrations of the internal standards to the levels in
the undiluted extract.
12.2.4 Perform all qualitative and quantitative measurements as described in Sections 14 and 15.
When standards and extracts are not being used for analyses, store them refrigerated at
<6°C protected from light in screw-cap vials equipped with un-pierced fluoropolymer-lined
septa.
13. Performance tests
13.1 At the beginning of each 12-hour shift during which standards or extracts will be analyzed, perform
the tests in Sections 13.2 - 13.7 to verify system performance. If DDT and/or endrin are to be
determined, perform the decomposition test in Section 13.8. If an extract is concentrated for greater
sensitivity (e.g., by SIM), all tests must be performed at levels consistent with the reduced extract
volume.
13.2 DFTPP - Inject the DFTPP standard (Section 6.10) and verify that the criteria for DFTPP in
Section 7.2.1.1 and Table 9A (Reference 18) for a quadrupole MS, or Table 9B (Reference 19) fora
time-of-flight MS, are met. It is not necessary to meet DFTPP criteria for SIM operation.
13.3 GC resolution - There must be a valley between benzo(b)fluoranthene and benzo(k)fluoranthene at
m/z 252, and the height of the valley must not exceed 25 percent of the shorter of the two peaks.
13.4 Calibration verification - Verify calibration per Sections 7.3 and Table 6.
13.5 Peak tailing -Verify the tailing factor specifications are met per Section 7.2.1.1.
Method 625.1 27 December 2014
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13.6 Laboratory control sample and blank - Analyze the extracts of the LCS and blank at the beginning
of analyses of samples in the extraction batch (Section 3.1). The LCS must meet the requirements
in Section 8.4, and the blank must meet the requirements in Section 8.5 before sample extracts may
be analyzed.
13.7 Matrix spike/matrix spike duplicate - Analyze the background sample for the MS/MSD and the MS
and MSB after the blank (Section 8.3.2). Results for the MS/MSD must meet the requirements in
Section 8.3 before a result for an analyte in any unspiked sample in the batch may be reported or
used for permitting or regulatory compliance purposes.
13.8 DDT/endrin decomposition test - If DDT and/or endrin analytes of interest, the DDT/endrin test
(Section 8.8) must be performed and the QC acceptance criteria must be met before analyzing
samples for DDT and/or endrin.
14. Qualitative Identification
14.1 Identification is accomplished by comparison of data from analysis of a sample or blank with data
stored in the GC/MS data system (Sections 5.6.5 and 7.2.1.2, and Tables 4 and 5). Identification of
an analyte is confirmed per Sections 14.1.1 through 14.1.4.
14.1.1 The signals for all characteristic m/z's stored in the data system for each analyte of interest
must be present and must maximize within the same two consecutive scans.
14.1.2 Based on the relative retention time (RRT), the RRT for the analyte must be within ±0.06
of the RRT of the analyte in the calibration verification run at the beginning of the shift
(Section 7.3 or 13.4). Relative retention time is used to establish the identification window
because it compensates for small changes in the GC temperature program whereas the
absolute retention time does not (see Section 6.9.3).
Note: RRT is a unitless quantity (see Sec. 20.2), although some procedures refer to "RRT
units" in providing the specification for the agreement between the RRT values in the
sample and the calibration verification or other standard.
14.1.3 Either (1) the background corrected EICP areas, or (2) the corrected relative intensities of
the mass spectral peaks at the GC peak maximum, must agree within 50% to 200% (1/2 to
2 times) for all m/z's in the reference mass spectrum stored in the data system (Section
7.2.1.2), or from a reference library. For example, if a peak has an intensity of 20% relative
to the base peak, the analyte is identified if the intensity of the peak in the sample is in the
range of 10% to 40% of the base peak.
14.1.4 The m/z's present in the acquired mass spectrum for the sample that are not present in the
reference mass spectrum must be accounted for by contaminant or background m/z's. A
reference library may be helpful to identify and account for background or contaminant
m/z's. If the acquired mass spectrum is contaminated, or if identification is ambiguous, an
experienced spectrometrist (Section 1.7) must determine the presence or absence of the
compound.
14.2 Structural isomers that have very similar mass spectra can be identified only if the resolution
between authentic isomers in a standard mix is acceptable. Acceptable resolution is achieved if the
baseline to valley height between the isomers is less than 50% of the height of the shorter of the two
peaks. Otherwise, structural isomers are identified as isomeric pairs.
Method 625.1 28 December 2014
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15. Calculations
15.1 When an analyte has been identified, quantitation of that analyte is based on the integrated
abundance from the EICP of the primary characteristic m/z in Table 4 or 5. Calculate the
concentration in the extract using the response factor (RF) determined in Section 7.2.2 and
Equation 2. If the concentration of an analyte exceeds the calibration range, dilute the extract by
the minimum amount to bring the concentration into the calibration range, and re-analyze the
extract. Determine a dilution factor (DF) from the amount of the dilution. For example, if the
extract is diluted by a factor of 2, DF = 2.
Equation 2
Cex (jig/mL)=
As x
AlsxRF
where:
Cex = Concentration of the analyte in the extract, in (ig/mL, and the other terms are as defined in
Equation 1
Calculate the concentration of the analyte in the sample using the concentration in the extract, the
extract volume, the sample volume, and the dilution factor, per Equation 3:
Equation 3
Cs Og/L) =
Cexx~
vs
where:
Cs = Concentration of the analyte in the sample
Cex = Concentration of the analyte in the extract, in (ig/mL
Vex = Volume of extract (mL)
Vs = Volume of sample (L)
DF = Dilution factor
15.2 Reporting of results
As noted in Section 1.4.1, EPA has promulgated this method at 40 CFR Part 136 for use in
wastewater compliance monitoring under the National Pollutant Discharge Elimination System
(NPDES). The data reporting practices described here are focused on such monitoring needs and
may not be relevant to other uses of the method.
15.2.1 Report results for wastewater samples in ug/L without correction for recovery. (Other units
may be used if required by in a permit.) Report all QC data with the sample results.
15.2.2 Reporting level
Unless otherwise specified in by a regulatory authority or in a discharge permit, results for
analytes that meet the identification criteria are reported down to the concentration of the
ML established by the laboratory through calibration of the instrument (see Section 7.3.2
and the glossary for the derivation of the ML). EPA considers the terms "reporting limit,"
"quantitation limit," and "minimum level" to be synonymous.
15.2.2.1 Report a result for each analyte in each sample, blank, or standard at or above the
ML to 3 significant figures. Report a result for each analyte found in each
Method 625.1 29 December 2014
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sample below the ML as "
-------
utilizes significant quantities of methylene chloride. Laboratories are encouraged to recover and
recycle this and other solvents during extract concentration.
17.3 For information about pollution prevention that may be applied to laboratories and research
institutions, consult Less is Better: Laboratory Chemical Management for Waste Reduction,
available from the American Chemical Society's Department of Governmental Relations and
Science Policy, 1155 16th Street NW, Washington DC 20036, 202/872-4477.
18. Waste Management
18.1 The laboratory is responsible for complying with all Federal, State, and local regulations governing
waste management, particularly the hazardous waste identification rules and land disposal
restrictions, and to protect the air, water, and land by minimizing and controlling all releases from
fume hoods and bench operations. Compliance is also required with any sewage discharge permits
and regulations. An overview of requirements can be found in Environmental Management Guide
for Small Laboratories (EPA 233-B-98-001).
18.2 Samples at pH <2, or pH >12 are hazardous and must be neutralized before being poured down a
drain, or must be handled and disposed of as hazardous waste.
18.3 Many analytes in this method decompose above 500 °C. Low-level waste such as absorbent paper,
tissues, and plastic gloves may be burned in an appropriate incinerator. Gross quantities of neat or
highly concentrated solutions of toxic or hazardous chemicals should be packaged securely and
disposed of through commercial or governmental channels that are capable of handling these types
of wastes.
18.4 For further information on waste management, consult The Waste Management Manual for
Laboratory Personnel and Less is Better-Laboratory Chemical Management for Waste Reduction,
available from the American Chemical Society's Department of Government Relations and Science
Policy, 1155 16th Street NW, Washington, DC 20036, 202/872-4477.
19. References
1. "Sampling and Analysis Procedures for Screening of Industrial Effluents for Priority Pollutants,"
U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory,
Cincinnati, Ohio 45268, March 1977, Revised April 1977.
2. "EPA Method Study 30, Method 625, Base/Neutrals, Acids, and Pesticides," EPA 600/4-84-053,
National Technical Information Service, PB84-206572, Springfield, Virginia 22161, June 1984.
3. 40 Code of Federal Regulations 136, appendix B.
4. Olynyk, P., Budde, W.L. and Eichelberger, J.W. "Method Detection Limit for Methods 624 and
625," Unpublished report, May 14, 1980.
5. Annual Book of ASTM Standards, Volume 11.02, D3694-96, "Standard Practices for Preparation
of Sample Containers and for Preservation of Organic Constituents," American Society for Testing
and Materials, Philadelphia.
Method 625.1 31 December 2014
-------
6. Solutions to Analytical Chemistry Problems with Clean Water Act Methods, EPA 821-R-07-002,
March 2007.
7. "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, August 1977.
8. "OSHA Safety and Health Standards, General Industry," (29 CFR Part 1910), Occupational Safety
and Health Administration, OSHA 2206 (Revised, January 1976).
9. "Safety in Academic Chemistry Laboratories," American Chemical Society Publication, Committee
on Chemical Safety, 7th Edition, 2003.
10. http://en.wikipedia.org/wiki/Coefficient_of_determination (accessed on 09/10/2013)
11. 40 Code of Federal Regulations 136.6(b)(4)(x)
12. 40 Code of Federal Regulations 136.6(b)(2)(/)
13. Protocol^or EPA Approval of New Methods for Organic and Inorganic Analytes in Wastewater and
Drinking Water (EPA-821-B-98-003) March 1999
14. Provost, L.P. and Elder, R.S. "Interpretation of Percent Recovery Data," American Laboratory, 15,
58-63 (1983). (The value 2.44 used in the equation in Section 8.3.3 is two times the value 1.22
derived in this report.)
15. ASTM Annual Book of Standards, Part 31, D3370-76. "Standard Practices for Sampling Water,"
American Society for Testing and Materials, Philadelphia.
16. 40 Code of Federal Regulations 136.3(a), Table IB, Chlorine - Total Residual
17. "Manual of Analytical Methods for the Analysis of Pesticides in Human and Environmental
Samples," EPA-600/8-80-038, U.S. Environmental Protection Agency, Health Effects Research
Laboratory, Research Triangle Park, North Carolina.
18. Eichelberger, J.W., Harris, L.E., and Budde, W.L. "Reference Compound to Calibrate Ion
Abundance Measurement in Gas Chromatography-Mass Spectrometry," Analytical Chemistry, 47,
995 (1975).
19. Letter of approval of acceptance criteria for DFTPP for time-of-flight mass spectrometers from
William A. Telliard and Herb Brass of EPA to Jack Cochran of LECO Corporation, February 9,
2005.
Method 625.1 32 December 2014
-------
20. Tables
Table 1 - Non Pesticide/PCB Base/Neutral Extractables1
Analyte
Acenaphthene
Acenaphthylene
Anthracene
Benzidine 2
Benzo(a)anthracene
Benzo(a)pyrene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(ghi)perylene
Benzyl butyl phthalate
bis(2-Chloroethoxy)methane
bis(2-Ethylhexyl)phthalate
bis(2-Chloroisopropyl) ether (2,2'-Oxybis(l-chloropropane))
4-Bromophenyl phenyl ether
2-Chloro naphthalene
4-Chlorophenyl phenyl ether
Chrysene
Dibenz(a,h)anthracene
Di-w -butylphthalate
3,3' -Dichlorobenzidine
Diethyl phthalate
Dimethyl phthalate
2,4-Dinitrotoluene
2,6-Dinitrotoluene
Di-w -octylphthalate
Fluoranthene
Fluorene
Hexachlorobenzene
Hexachlorobutadiene
Hexachloroethane
Indeno( 1 ,2,3 -cd)pyrene
Isophorone
Naphthalene
Nitrobenzene
N-Nitrosodi-w-propylamine 3
Phenanthrene
Pyrene
1 ,2,4-Trichlorobenzene
CAS Registry
83-32-9
208-96-8
120-12-7
92-87-5
56-55-3
50-32-8
205-99-2
207-08-9
191-24-2
85-68-7
111-91-1
117-81-7
108-60-1
101-55-3
91-58-7
7005-72-3
218-01-9
53-70-3
84-74-2
91-94-1
84-66-2
131-11-3
121-14-2
606-20-2
117-84-0
206-44-0
86-73-7
118-74-1
87-68-3
67-72-1
193-39-5
78-59-1
91-20-3
98-95-3
621-64-7
85-01-8
129-00-0
120-82-1
MDL4
1.9
3.5
1.9
44
7.8
2.5
4.8
2.5
4.1
2.5
5.3
2.5
5.7
1.9
1.9
4.2
2.5
2.5
2.5
16.5
1.9
1.6
5.7
1.9
2.5
2.2
1.9
1.9
0.9
1.6
3.7
2.2
1.6
1.9
-
5.4
1.9
1.9
ML5
5.7
10.5
5.7
132
23.4
7.5
14.4
7.5
12.3
7.5
15.9
7.5
17.1
5.7
5.7
12.6
7.5
7.5
7.5
49.5
5.7
4.8
17.1
5.7
7.5
6.6
5.7
5.7
2.7
4.8
11.1
6.6
4.8
5.7
~
16.2
5.7
5.7
1 All analytes in this table are Priority Pollutants (40 CFR 423, Appendix A)
2 Included for tailing factor testing
3 See Section 1.2
4 MDL values from the 1984 promulgated version of Method 624
5 ML = Minimum Level - see Glossary for definition and derivation
Method 625.1
33
December 2014
-------
Table 2-Acid Extractables1
Analyte
4-Chloro-3-methylphenol
2-Chlorophenol
2,4-Dichlorophenol
2,4-Dimethylphenol
2,4-Dinitrophenol
2-Methyl-4,6-dinitrophenol
2-Nitrophenol
4-Nitrophenol
Pentachlorophenol 2
Phenol
2,4,6-Trichlorophenol
CAS Registry
59-50-7
95-57-8
120-83-2
105-67-9
51-28-5
534-52-1
88-75-5
100-02-7
87-86-5
108-95-2
88-06-2
MDL3
3.0
o o
5.5
2.7
2.7
42
24
3.6
2.4
3.6
1.5
2.7
ML4
9.0
9.9
8.1
8.1
126
72
10.8
7.2
10.8
4.5
8.1
All analytes in this table are Priority Pollutants (40 CFR 423, Appendix A)
2 See Section 1.2; included for tailing factor testing
3 MDL values from the 1984 promulgated version of Method 624
4 ML = Minimum Level - see Glossary for definition and derivation
Method 625.1
34
December 2014
-------
Table 3 - Additional Extractable Analytes1'2
Analyte
Acetophenone
2-Acetylaminofluorene
1 -Acetyl-2-thiourea
Alachlor
Aldrin3
Ametryn
2-Aminoanthraquinone
Aminoazobenzene
4-Aminobiphenyl
3-Amino-9-ethylcarbazole
Anilazine
Aniline
o-Anisidine
Aramite
Atraton
Atrazine
Azinphos-methyl
Barban
Benzanthrone
Benzenethiol
Benzidine 3'4
Benzoic acid
2,3-Benzofluorene
p-Benzoquinone
Benzyl alcohol
alpha-BKC3'4
beta-BUC 3
gamma-BRC (Lindane) 3'4
delta-BRC 3
Biphenyl
Bromacil
2-Bromochlorobenzene
3 -Bromochlorobenzene
Bromoxynil
Butachlor
Butylate
w-CIO (w-decane)
w-C12 (w-undecane)
w-C14 (w-tetradecane)
w-C16 (w-hexadecane)
w-C18 (n-octadecane)
w-C20 (w-eicosane)
w-C22 (w-docosane)
w-C24 (w-tetracosane)
w-C26 (w-hexacosane)
w-C28 (w-octacosane)
w-C30 (w-triacontane)
Captafol
CAS Registry
98-86-2
53-96-3
591-08-2
15972-60-8
309-00-2
834-12-8
117-79-3
60-09-3
92-67-1
132-32-1
101-05-3
62-53-3
90-04-0
140-57-8
1610-17-9
1912-24-9
86-50-0
101-27-9
82-05-3
108-98-5
92-87-5
65-85-0
243-17-4
106-51-4
100-51-6
319-84-6
319-85-7
58-89-8
319-86-8
92-52-4
314-40-9
694-80-4
108-39-2
1689-84-5
2318-4669
2008-41-5
124-18-5
112-40-2
629-59-4
544-76-3
593-45-3
112-95-8
629-97-0
646-31-1
630-01-3
630-02-4
638-68-6
2425-06-1
MDL6
1.9
44
3.1
4.2
ML7
5.7
132
9.3
12.6
Method 625.1
35
December 2014
-------
Table 3 - Additional Extractable Analytes1'2
Analyte
Captan
Carbaryl
Carbazole
Carbofuran
Carboxin
Carbophenothion
Chlordane 3'5
bis(2-Chloroethyl) ether3'4
Chloroneb
4-Chloroaniline
Chlorobenzilate
Chlorfenvinphos
4 -Chloro -2 -methy laniline
3 -(Chloromethyl)pyridine hydrochloride
4 -Chloro -2 -nitroaniline
Chlorpropham
Chlorothalonil
1 -Chloronaphthalene
3 -Chloronitribenzene
4-Chloro-l,2-phenylenediamine
4-Chloro-l,3-phenylenediamine
2-Chlorobiphenyl
Chlorpyrifos
Coumaphos
m+p-Cresol
o-Cresol
/>-Cresidine
Crotoxyphos
2-Cyclohexyl-4,6-dinitro-phenol
Cyanazine
Cycloate
p-Cymene
Dacthal (DCPA)
4,4 '-ODD 3
4,4 '-DDE 3
4,4 '-DDT 3
Demeton-O
Demeton-S
Diallate (cis or trans)
2,4-Diaminotoluene
Diazinon
Dibenz(a,j )acridine
Dibenzofuran
Dibenzo(a,e)pyrene
Dibenzothiophene
l,2-Dibromo-3-chloropropane
3 , 5 -Dibromo -4-hydroxybenzonitrile
2,6-Di-tert-butyl-p-benzoquinone
CAS Registry
133-06-2
63-25-2
86-74-8
1563-66-2
5234-68-4
786-19-6
57-74-9
111-44-4
2675-77-6
106-47-8
510-15-6
470-90-6
95-69-2
6959-48-4
89-63-4
101-21-3
1897-45-6
90-13-1
121-73-3
95-83-0
5131-60-2
2051-60-7
2921-88-2
56-72-4
65794-96-9
95-48-7
120-71-8
7700-17-6
131-89-5
21725-46-2
1134-23-2
99-87-6
1861-32-1
72-54-8
72-55-9
50-29-3
298-03-3
126-75-0
2303-16-4
95-80-7
333-41-5
224-42-0
132-64-9
192-65-4
132-65-0
96-12-8
1689-84-5
719-22-2
MDL6
5.7
2.8
5.6
4.7
ML7
17.1
8.4
16.8
14.1
Method 625.1
36
December 2014
-------
Table 3 - Additional Extractable Analytes1'2
Analyte
Dichlone
2, 3 -Dichloroaniline
2, 3 -Dichlorobiphenyl
2,6-Dichloro-4-nitroaniline
2, 3 -Die hloro nitrobenzene
1 , 3 -Dichloro -2-propanol
2,6-Dichlorophenol
Dichlorvos
Dicrotophos
Dieldrin 3
1,2:3 ,4-Diepoxybutane
Di(2-ethylhexyl) adipate
Diethylstilbestrol
Diethyl sulfate
Dilantin (5,5-Diphenylhydantoin)
Dimethoate
3,3' -Dimethoxybenzidine
Dimethylaminoazobenzene
7, 12-Dimethylbenz(a)anthracene
3,3' -Dimethylbenzidine
N,N-Dimethylformamide
3,6-Dimethylphenathrene
alpha, a/p/za-Dimethylphenethylamine
Dimethyl sulfone
1,2-Dinitrobenzene
1 , 3 -Dinitrobenzene
1,4-Dinitrobenzene
Dinocap
Dinoseb
Diphenylamine
Diphenyl ether
1,2-Diphenylhydrazine
Diphenamid
Diphenyldisulfide
Disulfoton
Disulfoton sulfoxide
Disurfoton sulfone
Endosulfanl3'4
Endosulfanll3'4
Endosulfan surfate 3
Endrin3'4
Endrin aldehyde 3'4
Endrin ketone 3'4
EPN
EPTC
Ethion
Ethoprop
Ethyl carbamate
CAS Registry
117-80-6
608-27-5
16605-91-7
99-30-9
3209-22-1
96-23-1
120-83-2
62-73-7
141-66-2
60-57-1
1464-53-5
103-23-1
56-53-1
64-67-5
57-41-0
60-51-5
119-90-4
60-11-7
57-97-6
119-93-7
68-12-2
1576-67-6
122-09-8
67-71-0
528-29-0
99-65-0
100-25-4
39300-45-3
88-85-7
122-39-4
101-84-8
122-66-7
957-51-7
882-33-7
298-04-4
2497-07-6
2497-06-5
959-98-8
33213-65-9
1031-07-8
72-20-8
7421-93-4
53494.70-5
2104-64-5
759-94-4
563-12-2
13194-48-4
51-79-6
MDL6
2.5
5.6
ML7
7.5
16.8
Method 625.1
37
December 2014
-------
Table 3 - Additional Extractable Analytes1'2
Analyte
Ethyl methanesulfonate
Ethylenethiourea
Etridiazole
Ethynylestradiol-3 -methyl ether
Famphur
Fenamiphos
Fenarimol
Fensulfothion
Fenthion
Fluchloralin
Fluridone
Heptachlor 3
Heptachlor epoxide 3
2,2',3,3',4,4',6-Heptachlorobiphenyl
2,2',4,4',5',6-Hexachlorobiphenyl
Hexachlorocyclopentadiene 3>4
Hexachlorophene
Hexachloropropene
Hexamethylphosphoramide
Hexanoic acid
Hexazinone
Hydroquinone
Isodrin
2-Isopropylnapthalene
Isosafrole
Kepone
Leptophos
Longifolene
Malachite green
Malathion
Maleic anhydride
Merphos
Mestranol
Methapyrilene
Methoxychlor
2-Methylbenzothioazole
3 -Methy Icholanthrene
4,4 ' -Methylenebis(2-chloroaniline)
4,4 ' -Methylenebis(N,N-dimethylaniline)
4, 5 -Methy lenephenanthrene
1 -Methy Ifluorene
Methyl methanesulfonate
2-Methylnaphthalene
Methylparaoxon
Methyl parathion
1 -Methy Ip henanthrene
2-(Methylthio)benzothiazole
Metolachlor
CAS Registry
65-50-0
96-45-7
2593-15-9
72-33-3
52-85-7
22224-92-6
60168-88-9
115-90-2
55-38-9
33245-39-5
59756-60-4
76-44-8
1024-57-3
52663-71-5
60145-22-4
77-47-4
70-30-4
1888-71-7
680-31-9
142-62-1
51235-04-2
123-31-9
465-73-6
2027-17-0
120-58-1
143-50-0
21609-90-5
475-20-7
569-64-2
121-75-5
108-31-6
150-50-5
72-33-3
91-80-5
72-43-5
120-75-2
56-49-5
101-14-4
101-61-1
203-64-5
1730-37-6
66-27-3
91-57-6
950-35-6
298-00-0
832-69-9
615-22-5
5218-45-2
MDL6
1.9
2.2
ML7
5.7
6.6
Method 625.1
38
December 2014
-------
Table 3 - Additional Extractable Analytes1'2
Analyte
Metribuzin
Mevinphos
Mexacarbate
MGK 264
Mirex
Molinate
Monocrotophos
Naled
Napropamide
1,4-Naphthoquinone
1-Naphthylamine
2-Naphthylamine
1 , 5 -Naphthalenediamine
Nicotine
5 -Nitroacenaphthene
2-Nitroaniline
3-Nitroaniline
4-Nitroaniline
5-Nitro-o-anisidine
4-Nitrobiphenyl
Nitrofen
5-Nitro-o-toluidine
Nitroquinoline-1 -oxide
N-Nitrosodi-w-butylamine 4
N-Nitrosodiethylamine 4
N-Nitrosodimethylamine 3'4
N-Nitrosodiphenylamine 3'4
N-Nitrosomethylethylamine 4
N-Nitrosomethylphenylamine 4
N-Nitrosomorpholine 4
N-Nitrosopiperidine 4
N-Nitrosopyrrolidine 4
/raws-Nonachlor
Norflurazon
2,2',3,3',4,5',6,6'-Octachlorobiphenyl
Octamethyl pyrophosphoramide
4,4'-Oxydianiline
Parathion
PCB-10163'5
PCB-12213'5
PCB-12323'5
PCB-12423'5
PCB-12483'5
PCB-12543'5
PCB-12603'5
PCB-12683'5
Pebulate
Pentachlorobenzene
CAS Registry
21087-64-9
7786-34-7
315-18-4
113-48-4
2385-85-5
2212-67-1
6923-22-4
300-76-5
15299-99-7
130-15-4
134-32-7
91-59-8
2243-62-1
54-11-5
602-87-9
88-74-4
99-09-2
100-01-6
99-59-2
92-93-3
1836-75-5
99-55-8
56-57-5
924-16-3
55-18-5
62-75-9
86-30-6
10595-95-6
614-00-6
59-89-2
100-75-5
930-55-2
39765-80-5
27314-13-2
40186-71-8
152-16-9
101-80-4
56-38-2
12674-11-2
11104-28-2
11141-16-5
53469-21-9
12672-29-6
11097-69-1
11098-82-5
11100-14-4
1114-71-2
608-93-5
MDL6
30
36
ML7
90
108
Method 625.1
39
December 2014
-------
Table 3 - Additional Extractable Analytes1'2
Analyte
Pentachloronitrobenzene
2,2',3,4',6-Pentachlorobiphenyl
Pentachloroethane
Pentamethylbenzene
Perylene
Phenacetin
c/'s-Permethrin
/raws-Permethrin
Phenobarbital
Phenothiazene
1,4-Phenylenediamine
1 -Phenylnaphthalene
2-Phenylnaphthalene
Phorate
Phosalone
Phosmet
Phosphamidon
Phthalic anhydride
alpha-Picoline (2-Methylpyridine)
Piperonyl sulfoxide
Prometon
Prometryn
Pronamide
Propachlor
Propazine
Propylthiouracil
Pyridine
Resorcinol (1,3-Benzenediol)
Safrole
Simazine
Simetryn
Squalene
Stirofos
Strychnine
Styrene
Sulfallate
Tebuthiuron
Terbacil
Terbufos
Terbutryn
a/p/za-Terpineol
1,2,4,5-Tetrachlorobenzene
2,2 ' ,4,4' -Tetrachlorobiphenyl
2,3,7,8-Tetrachlorodibenzo-p-dioxin
2,3,4,6-Tetrachlorophenol
Tetrachlorvinphos
Tetraethyl dithiopyrophosphate
Tetraethyl pyrophosphate
CAS Registry
82-68-8
68194-05-8
76-01-7
700-12-9
198-55-0
62-44-2
61949-76-6
61949-77-7
50-06-6
92-84-2
624-18-0
605-02-7
612-94-2
298-02-2
2310-18-0
732-11-6
13171-21-6
85-44-9
109-06-8
120-62-7
1610-18-0
7287-19-6
23950-58-5
1918-16-7
139-40-2
51-52-5
110-86-1
108-46-3
94-59-7
122-34-9
1014-70-6
7683-64-9
22248-79-9
57-24-9
100-42-5
95-06-7
34014-18-1
5902-51-2
13071-79-9
886-50-0
98-55-5
95-94-3
2437-79-8
1746-01-6
58-90-2
22248-79-9
3689-24-5
107-49-3
MDL6
ML7
Method 625.1
40
December 2014
-------
Table 3 - Additional Extractable Analytes1'2
Analyte
Thianaphthene (2,3-Benzothiophene)
Thioacetamide
Thionazin
Thiophenol (Benzenethiol)
Thioxanthone
Toluene- 1 , 3 -diisocyanate
Toluene-2,4-diisocyanate
o-Toluidine
Toxaphene 3'5
Triadimefon
1,2,3 -Trichlorobenzene
2,4,5 -Trichlorobiphenyl
2, 3 ,6 -Trichlorophenol
2,4,5 -Trichlorophenol
Tricyclazole
Trifluralin
1,2,3 -Trimethoxybenzene
2,4,5 -Trimethylaniline
Trimethyl phosphate
Triphenylene
Tripropyleneglycolmethyl ether
1,3,5-Trinitrobenzene
Tris(2,3-dibromopropyl) phosphate
Tri-p-tolyl phosphate
O,O,O-Triethyl phosphorothioate
Trithiane
Vernolate
CAS Registry
95-15-8
62-55-5
297-97-2
108-98-5
492-22-8
26471-62-5
584-84-9
95-53-4
8001-35-2
43121-43-3
87-61-6
15862-07-4
933-75-5
95-95-4
41814-78-2
1582-09-8
634-36-6
137-17-7
512-56-1
217-59-4
20324-33-8
99-35-4
126-72-7
78-32-0
126-68-1
291-29-4
1929-77-7
MDL6
ML7
Compounds that have been demonstrated amenable to extraction and gas chromatography
Determine each analyte in the fraction that gives the most accurate result
Priority Pollutant (40 CFR 423, Appendix A)
See Section 1.2
These compounds are mixtures of various isomers
MDL values from the 1984 promulgated version of Method 624
ML = Minimum Level - see Glossary for definition and derivation
Method 625.1
41
December 2014
-------
Table 4 - Chromatographic Conditions and Characteristic m/z's for Base/Neutral Extractables
Analyte
N-Nitrosodimethylamine
bis(2-Chloroethyl) ether
bis(2-Chloroisopropyl) ether
Hexachloroethane
N-Nitrosodi-w-propylamine
Nitrobenzene
Isophorone
bis(2-Chloroethoxy) methane
1 ,2,4-Trichlorobenzene
Naphthalene
Hexachlorobutadiene
Hexachlorocyclopentadiene
2-Chloro naphthalene
Acenaphthylene
Dimethyl phthalate
2,6-Dinitrotoluene
Acenaphthene
2,4-Dinitrotoluene
Fluorene
4-Chlorophenyl phenyl ether
Diethyl phthalate
N-Nitrosodiphenylamine
4-Bromophenyl phenyl ether
alpha-BHC
Hexachlorobenzene
beta-BHC
gamma-BHC
Phenanthrene
Anthracene
delta-BUC
Heptachlor
Di-w-butyl phthalate
Aldrin
Fluoranthene
Heptachlor epoxide
ga/w/wa-Chlordane
Pyrene
Benzidine 2
a/p/za-Chlordane
Endosulfan I
4,4'-DDE
Dieldrin
Endrin
Endosulfan II
4,4'-DDD
Endrin aldehyde
Retention
Time
(sec) 1
385
704
799
823
830
849
889
939
958
967
1006
1142
1200
1247
1273
1300
1304
1364
1401
1409
1414
1464
1498
1514
1522
1544
1557
1583
1592
1599
1683
1723
1753
1817
1820
1834
1852
1853
1854
1855
1892
1907
1935
2014
2019
2031
Characteristic m/z's
Electron impact ionization
Primary
42
93
45
117
130
77
82
93
180
128
225
237
162
152
163
165
154
165
166
204
149
169
248
183
284
183
181
178
178
183
100
149
66
202
353
373
202
184
373
237
246
79
81
237
235
67
Second
74
63
77
201
42
123
95
95
182
129
223
235
164
151
194
89
153
63
165
206
177
168
250
181
142
181
183
179
179
109
272
150
263
101
355
375
101
92
375
339
248
263
263
339
237
345
Second
44
95
79
199
101
65
138
123
145
127
227
272
127
153
164
121
152
182
167
141
150
167
141
109
249
109
109
176
176
181
274
104
220
100
351
377
100
185
377
341
176
279
82
341
165
250
Chemical ionization
Methane
63
77
199
124
139
65
181
129
223
235
163
152
151
183
154
183
166
177
169
249
284
178
178
149
203
203
185
107
135
201
152
167
107
183
157
225
237
191
153
163
211
155
211
167
223
170
251
286
179
179
205
231
231
213
109
137
203
164
178
137
209
169
227
239
203
181
164
223
183
223
195
251
198
277
288
207
207
279
243
243
225
Method 625.1
42
December 2014
-------
Table 4 - Chromatographic Conditions and Characteristic m/z's for Base/Neutral Extractables
Analyte
Butyl benzyl phthalate
Endosulfan sulfate
4,4'-DDT
Chrysene
3,3' -Dichlorobenzidine
Benzo(a)anthracene
bis(2-Ethylhexyl) phthalate
Di-w-octyl phthalate
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(a)pyrene
Indeno(l,2,3-cd) pyrene
Dibenz(a,h)anthracene
Benzo(ghi)perylene
Toxaphene
PCB 1016
PCB 1221
PCB 1232
PCB 1242
PCB 1248
PCB 1254
PCB 1260
Retention
Time
(sec) 1
2060
2068
2073
2083
2086
2090
2124
2240
2286
2293
2350
2650
2660
2750
Characteristic m/z's
Electron impact ionization
Primary
149
272
235
228
252
228
149
149
252
252
252
276
278
276
159
224
190
190
224
294
294
330
Second
91
387
237
226
254
229
167
43
253
253
253
138
139
138
231
260
224
224
260
330
330
362
Second
206
422
165
229
126
226
279
57
125
125
125
277
279
277
233
294
260
260
294
262
362
394
Chemical ionization
Methane
149
228
228
149
252
252
252
276
278
276
299
229
229
253
253
253
277
279
277
327
257
257
281
281
281
305
307
305
Column: 30 m x 0.25 mm ID; 94% methyl, 5% phenyl, 1% vinyl bonded phase fused silica capillary
Conditions: 5 min at 30°C; 30 - 280 at 8°C per min; isothermal at 280°C until benzo(ghi)perylene elutes
Gas velocity: 30 cm/sec at 30°C (at constant pressure).
See Section 1.2; included for tailing factor testing
Method 625.1
43
December 2014
-------
Table 5 - Chromatographic Conditions and Characteristic m/z's for Acid Extractables
Analyte
2-Chlorophenol
Phenol
2-Nitrophenol
2,4-Dimethylphenol
2,4-Dichlorophenol
4-Chloro-3-methylphenol
2,4,6-Trichlorophenol
2,4-Dinitrophenol
4-Nitrophenol
2-Methyl-4,6-dinitrophenol
Pentachlorophenol
Retention
Time (sec) 1
705
700
900
924
947
1091
1165
1325
1354
1435
1561
Characteristic m/z's
Electron impact ionization
Prime
128
94
139
122
162
142
196
184
65
198
266
Second
64
65
65
107
164
107
198
63
139
182
264
Second
130
66
109
121
98
144
200
154
109
77
268
Chemical ionization
Methane
129
95
140
123
163
143
197
185
140
199
267
131
123
168
151
165
171
199
213
168
227
265
157
135
122
163
167
183
201
225
122
239
269
Column: 30 m x 0.25 mm ID; 94% methyl, 5% phenyl, 1% vinyl bonded phase fused silica capillary
Conditions: 5 min at 30°C; 30 - 250 at 8°C per min; isothermal at 280°C until pentachlorophenol elutes
Gas velocity: 30 cm/sec at 30°C (at constant pressure)
Method 625.1
44
December 2014
-------
Table 6 - QC Acceptance Criteria - Method 625 1
Analyte
Acenaphthene
Acenaphthylene
Aldrin
Anthracene
Benzo(a)anthracene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(a)pyrene
Benzo(ghi)perylene
Benzyl butyl phthalate
beta-BHC
delta-BRC
bis(2 -Chloroethyl)ether
bis(2-Chloroethoxy)methane
bis(2-Chloroisopropyl) ether
bis(2-Ethylhexyl) phthalate
4-Bromophenyl phenyl ether
2-Chloro naphthalene
4-Chlorophenyl phenyl ether
Chrysene
4,4 '-ODD
4,4 '-DDE
4,4 '-DDT
Dibenz(a,h)anthracene
Di-w-butyl phthalate
3,3 '-Dichlorobenzidine
Dieldrin
Diethyl phthalate
Dimethyl phthalate
2,4-Dinitrotoluene
2,6-Dinitrotoluene
Di-w-octyl phthalate
Endosulfan sulfate
Endrin aldehyde
Fluoranthene
Fluorene
Heptachlor
Heptachlor epoxide
Hexachlorobenzene
Hexachlorobutadiene
Hexachloroethane
Indeno( 1 ,2,3 -cd)pyrene
Isophorone
Naphthalene
Nitrobenzene
N-Nitrosodi-w-propylamine
Range for Q
(%) 2
70-130
60-130
7-152
58-130
42-133
42-140
25-146
32-148
13-195
43-140
42-131
D-130
52-130
52-164
63-139
43-137
70-130
70-130
57-145
44-140
D-135
19-130
D-171
13-200
52-130
18-213
70-130
47-130
50-130
53-130
68-137
21-132
D-130
D-189
47-130
70-130
D-172
70-130
38-142
68-130
55-130
13-151
52-180
70-130
54-158
59-170
Limit for s
(%) 3
29
45
39
40
32
43
38
43
61
36
37
77
65
32
46
50
26
15
36
53
56
46
81
75
28
65
38
60
110
25
29
42
42
45
40
23
44
61
33
38
32
60
56
39
37
52
Range for X
(%) 3
60-132
54-126
7-152
43-120
42-133
42-140
25-146
32-148
D-195
D-140
42-131
D-120
43-126
49-165
63-139
29-137
65-120
65-120
38-145
44-140
D-135
19-120
D-171
D-200
8-120
8-213
44-119
D-120
D-120
48-127
68-137
19-132
D-120
D-189
43-121
70-120
D-172
71-120
8-142
38-120
55-120
D-151
47-180
36-120
54-158
14-198
Range for
P,PS(%)3
47-145
33-145
D-166
27-133
33-143
24-159
11-162
17-163
D-219
D-152
24-149
D-120
12-158
33-184
36-166
8-158
53-127
60-120
25-158
17-168
D-145
4-136
D-203
D-227
1-120
D-262
29-136
D-120
D-120
39-139
50-158
4-146
D-120
D-209
26-137
59-121
D-192
26-155
D-152
24-120
40-120
D-171
21-196
21-133
35-180
D-230
Limit for
RPD (%)
48
74
81
66
53
71
63
72
97
60
61
129
108
54
76
82
43
24
61
87
93
77
135
126
47
108
62
100
183
42
48
69
70
75
66
38
74
101
55
62
52
99
93
65
62
87
Method 625.1
45
December 2014
-------
Table 6 - QC Acceptance Criteria - Method 625 1
Analyte
PCB-1260
Phenanthrene
Pyrene
1 ,2,4-Trichlorobenzene
4-Chloro-3-methylphenol
2-Chlorophenol
2,4-Dichlorophenol
2,4-Dimethylphenol
2,4-Dinitrophenol
2-Methyl-4,6-dinitrophenol
2-Nitrophenol
4-Nitrophenol
Pentachlorophenol
Phenol
2,4,6-Trichlorophenol
Range for Q
(%) 2
19-130
67-130
70-130
61-130
68-130
55-130
64-130
58-130
39-173
56-130
61-163
35-130
42-152
48-130
69-130
Limit for s
(%) 3
77
24
30
30
44
37
30
35
79
122
33
79
52
39
35
Range for X
(%) 3
19-130
65-120
70-120
57-130
41-128
36-120
53-122
42-120
D-173
53-130
45-167
13-129
38-152
17-120
52-129
Range for
P,PS(%)3
D-164
54-120
52-120
44-142
22-147
23-134
39-135
32-120
D-191
D-181
29-182
D-132
14-176
5-120
37-144
Limit for
RPD (%)
128
39
49
50
73
61
50
58
132
203
55
131
86
64
58
Acceptance criteria are based upon method performance data in Table 7 and from EPA Method 1625. Where
necessary, limits for recovery have been broadened to assure applicability to concentrations below those used
to develop Table 7.
2 Test concentration = 100 ug/mL
3 Test concentration = 100 ug/L
Q = Calibration verification (Sections 7.3.1 and 13.4)
s = Standard deviation for four recovery measurements in the DOC test (Section 8.2.4).
X = Average recovery for four recovery measurements in the DOC test (Section 8.2.4).
P, Ps = MS/MSD recovery (Section 8.3.2, Section 8.4.2).
RPD = MS/MSD relative percent difference (RPD; Section 8.3.3).
D = Detected; result must be greater than zero.
Method 625.1
46
December 2014
-------
Table 7 - Precision and Recovery as Functions of Concentration - Method 625 1
Analyte
Acenaphthene
Acenaphthylene
Aldrin
Anthracene
Benzo(a)anthracene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(a)pyrene
Benzo(ghi)perylene
Benzyl butyl phthalate
beta-BHC
delta-BRC
bis(2 -Chloroethyl)ether
bis(2-Chloroethoxy)methane
bis(2-Chloroisopropyl)ether
bis(2 -Ethy lhexyl)phthalate
4-Bromophenyl phenyl ether
2-Chloro naphthalene
4-Chlorophenyl phenyl ether
Chrysene
4,4 '-ODD
4,4 '-DDE
4,4 '-DDT
Dibenz(a,h)anthracene
Di-w-butyl phthalate
3,3 '-Dichlorobenzidine
Dieldrin
Diethyl phthalate
Dimethyl phthalate
2,4-Dinitrotoluene
2,6-Dinitrotoluene
Di-w-octyl phthalate
Endosulfan sulfate
Endrin aldehyde
Fluoranthene
Fluorene
Heptachlor
Heptachlor epoxide
Hexachlorobenzene
Hexachlorobutadiene
Hexachloroethane
Recovery, X' (jig/L)
0.96C+0.19
0.89C+0.74
0.78C+1.66
0.80C+0.68
0.88C-0.60
0.93C-1.80
0.87C-1.56
0.90C-0.13
0.98C-0.86
0.66C-1.68
0.87C-0.94
0.29C-1.09
0.86C-1.54
1.12C-5.04
1.03C-2.31
0.84C-1.18
0.91C-1.34
0.89C+0.01
0.91C+0.53
0.93C-1.00
0.56C-0.40
0.70C-0.54
0.79C-3.28
0.88C+4.72
0.59C+0.71
1.23C-12.65
0.82C-0.16
0.43C+1.00
0.20C+1.03
0.92C-4.81
1.06C-3.60
0.76C-0.79
0.39C+0.41
0.76C-3.86
0.81C+1.10
0.90C-0.00
0.87C-2.97
0.92C-1.87
0.74C+0.66
0.71C-1.01
0.73C-0.83
Single analyst
precision, sr' (jig/L)
0.15 X-0.12
0.24 X-1.06
0.27 X-1.28
0.21 X-0.32
0.15 X+0.93
0.22 X+0.43
0.19 X+1.03
0.22 X+0.48
0.29 X+2.40
0.18 X+0.94
0.20 X-0.58
0.34 X+0.86
0.35 X-0.99
0.16 X+1.34
0.24 X+0.28
0.26 X+0.73
0.13 X+0.66
0.07 X+0.52
0.20 X-0.94
0.28 X+0.13
0.29 X-0.32
0.26 X-1.17
0.42 X+0.19
0.30 X+8.51
0.13 X+1.16
0.28 X+7.33
0.20 X-0.16
0.28 X+1.44
0.54 X+0.19
0.12 X+1.06
0.14 X+1.26
0.21 X+1.19
0.12 X+2.47
0.18 X+3.91
0.22 X+0.73
0.12 X+0.26
0.24 X-0.56
0.33 X-0.46
0.18 X-0.10
0.19 X+0.92
0.17 X+0.67
Overall precision,
S' (ng/L)
0.21 X-0.67
0.26 X-0.54
0.43 X+1.13
0.27 X-0.64
0.26 X-0.28
0.29 X+0.96
0.35 X+0.40
0.32 X+1.35
0.51 X-0.44
0.53 X+0.92
0.30 X-1.94
0.93 X-0.17
0.35 X+0.10
0.26 X+2.01
0.25 X+1.04
0.36 X+0.67
0.16 X+0.66
0.13 X+0.34
0.30 X-0.46
0.33 X-0.09
0.66 X-0.96
0.39 X-1.04
0.65 X-0.58
0.59 X+0.25
0.39 X+0.60
0.47 X+3.45
0.26 X-0.07
0.52 X+0.22
1.05 X-0.92
0.21 X+1.50
0.19 X+0.35
0.37 X+1.19
0.63 X-1.03
0.73 X-0.62
0.28 X-0.60
0.13 X+0.61
0.50 X-0.23
0.28 X+0.64
0.43 X-0.52
0.26 X+0.49
0.17 X+0.80
Method 625.1
47
December 2014
-------
Table 7 - Precision and Recovery as Functions of Concentration - Method 625 1
Analyte
Indeno( 1 ,2,3 -cd)pyrene
Isophorone
Naphthalene
Nitrobenzene
N-Nitrosodi-w-propylamine
PCB-1260
Phenanthrene
Pyrene
1 ,2,4-Trichlorobenzene
4-Chloro-3-methylphenol
2-Chlorophenol
2,4-Dichlorophenol
2,4-Dimethylphenol
2,4-Dinitrophenol
2-Methyl-4,6-Dinitrophenol
2-Nitrophenol
4-Nitrophenol
Pentachlorophenol
Phenol
2,4,6-Trichlorophenol
Recovery, X' (jig/L)
0.78C-3.10
1.12C+1.41
0.76C+1.58
1.09C-3.05
1.12C-6.22
0.81C-10.86
0.87C-0.06
0.84C-0.16
0.94C-0.79
0.84C+0.35
0.78C+0.29
0.87C+0.13
0.71C+4.41
0.81C-18.04
1.04C-28.04
1.07C-1.15
0.61C-1.22
0.93C+1.99
0.43C+1.26
0.91C-0.18
Single analyst
precision, sr' (jig/L)
0.29 X+1.46
0.27 X+0.77
0.21 X-0.41
0.19 X+0.92
0.27 X+0.68
0.35 X+3.61
0.12 X+0.57
0.16 X+0.06
0.15 X+0.85
0.23 X+0.75
0.18 X+1.46
0.15 X+1.25
0.16 X+1.21
0.38 X+2.36
0.05 X +42.29
0.16 X+1.94
0.38 X+2.57
0.24 X+3.03
0.26 X+0.73
0.16 X+2.22
Overall precision,
S' (ng/L)
0.50 X+0.44
0.33 X+0.26
0.30 X-0.68
0.27 X+0.21
0.44 X+0.47
0.43 X+1.82
0.15 X+0.25
0.15 X+0.31
0.21 X+0.39
0.29 X+1.31
0.28 X+0.97
0.21 X+1.28
0.22 X+1.31
0.42 X +26.29
0.26 X+23.10
0.27 X+2.60
0.44 X+3.24
0.30 X+4.33
0.35 X+0.58
0.22 X+1.81
Regressions based on data from Reference 2
X' = Expected recovery for one or more measurements of a sample containing a concentration of C, in ug/L.
sr' = Expected single analyst standard deviation of measurements at an average concentration found of X , in ug/L.
S' = Expected interlaboratory standard deviation of measurements at an average concentration found of X , in
C = True value for the concentration, in ug/L.
X = Average recovery found for measurements of samples containing a concentration of C, in ug/L.
Method 625.1
48
December 2014
-------
Table 8 - Suggested Internal and Surrogate Standards
Base/neutral fraction
Acenaphthalene -d8
Acenaphthene-dio
Aniline-ds
Anthracene-d10
Benzo(a)anthracene-d12
Benzo(a)pyrene-di2
4-Chloroaniline-d4
bis(2-Chloroethyl) ether-d8
Chrysene-d12
Decafluorobiphenyl
4,4 '-Dibromobiphenyl
4,4 '-Dibromooctafluorobiphenyl
l,4-Dichlorobenzene-d4
2,2 '-Difluorobiphenyl
Dimethyl phthalate-d6
Fluoranthene-d10
Fluorene-dio
4-Fluoroaniline
1 -Fluoronaphthalene
2-Fluoronaphthalene
2-Methylnaphthalene-d! 0
Naphthalene-d8
Nitrobenzene-ds
2,3,4,5,6-Pentafluorobiphenyl
Perylene-di2
Phenanthrene-d10
Pyrene-d10
Pyridine-d5
Acid fraction
2-Chlorophenol-d4
2,4-Dichlorophenol-d3
4,6-Dinitro-2-methylphenol-d2
2-Fluorophenol
4-Methylphenol-ds
2-Nitrophenol-d4
4-Nitrophenol-d4
Pentafluorophenol
2-Perfluoromethylphenol
Phenol-d5
Range for Surrogate Recovery (%) 1
Calibration verification
66 - 152
71-141
58-171
28-357
32 - 194
1-145
52 - 194
23 - 290
65 - 153
47-211
47-215
61 - 164
50 - 150
71-141
46-219
67 - 149
48-210
55 - 180
64 - 157
56 - 177
25-111
61 - 163
35-287
48 - 208
Recovery from samples
33 - 168
30 - 180
23 - 142
22 - 329
32 - 194
1-145
25 - 222
23 - 290
11-245
1-500
30 - 187
38 - 172
50 - 150
22 - 192
15-314
34 - 168
28 - 196
33 - 180
34 - 182
22 - 307
25-111
37 - 163
6-500
8-424
Recovery from samples is the wider of the criteria in the CLP SOW for organics or in Method 1625
Method 625.1
49
December 2014
-------
Table 9A - DFTPP Key m/z's and Abundance Criteria for Quadrupole Instruments 1
m/z
51
68
70
127
197
198
199
275
365
441
442
443
Abundance criteria
30 - 60 percent of m/z 198
Less than 2 percent of m/z 69
Less than 2 percent of m/z 69
40 - 60 percent of base peak m/z 198
Less than 1 percent of m/z 198
Base peak, 100 percent relative abundance
5 - 9 percent of m/z 198
10 - 30 percent of m/z 198
Greater than 1 percent of m/z 198
Present but less than m/z 443
40 -100 percent of m/z 198
17-23 percent of m/z 442
1 Criteria in these tables are for quadrupole and time-of-flight instruments. Alternative tuning criteria may be used
for other instruments, provided method performance is not adversely affected.
Table 9B - DFTPP Key m/z's and Abundance Criteria for Time-of-flight Instruments 1
m/z
51
68
70
127
197
198
199
275
365
441
442
443
Abundance criteria
10 - 85 percent of the base peak
Less than 2 percent of m/z 69
Less than 2 percent of m/z 69
10 - 80 percent of the base peak
Less than 2 percent of Mass 198
Base peak, or greater than 50% of m/z 442
5-9 percent of m/z 198
10 - 60 percent of the base peak
Greater than 0.5 percent of m/z 198
Less than 150 percent of m/z 443
Base peak or greater than 30 percent of m/z 198
15-24 percent of m/z 442
1 Criteria in these tables are for quadrupole and time-of-flight instruments. Alternative tuning criteria may be used
for other instruments, provided method performance is not adversely affected.
Method 625.1
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21. Figures
TAILING FACTORS —
AB
Example calculation: Peak Height = DE = 100 mm
10% Peak Heights BD =10 mm
Peak Width at 10% Peak Heights AC = 23 mm
AB = 11 mm
BC = 12mm
12
Therefore: Tailing Factors — =1.1
11
Figure 1 Tailing factor calculation
Method 625.1
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100.0-,
j
see
?«35
715776.
Jt
ieee
15s 58
1500
23s 45
3ls48
T*"^""" '»
2586
39s 35
3900
scan
TIME
Figure 2 Chromatogram of combined acid/base/neutral standard
Method 625.1
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22. Glossary
These definitions and purposes are specific to this method but have been conformed to common usage
to the extent possible.
22.1 Units of weight and measure and their abbreviations
22.1.1 Symbols
°C degrees Celsius
(ig microgram
(iL microliter
< less than
> greater than
< less than or equal to
% percent
22.1.2 Abbreviations (in alphabetical order)
cm centimeter
g gram
h hour
ID inside diameter
in. inch
L liter
M Molecular ion
m mass or meter
mg milligram
min minute
mL milliliter
mm millimeter
ms millisecond
m/z mass-to-charge ratio
N normal; gram molecular weight of solute divided by hydrogen equivalent of solute,
per liter of solution
ng nanogram
pg picogram
ppb part-per-billion
ppm part-per-million
ppt part-per-trillion
psig pounds-per-square inch gauge
22.2 Definitions and acronyms (in alphabetical order)
Analyte - A compound or mixture of compounds (e.g., PCBs) tested for by this method. The analytes are
listed in Tables 1-3.
Batch - See Extraction
Blank - An aliquot of reagent water that is treated exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and surrogates that are used with samples. The blank is
Method 625.1 53 December 2014
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used to determine if analytes or interferences are present in the laboratory environment, the reagents, or the
apparatus.
Calibration - The process of determining the relationship between the output or response of a measuring
instrument and the value of an input standard. Historically, EPA has referred to a multi-point calibration
as the "initial calibration," to differentiate it from a single-point calibration verification.
Calibration standard - A solution prepared from stock solutions and/or a secondary standards and containing
the analytes of interest, surrogates, and internal standards. The calibration standard is used to calibrate the
response of the GC/MS instrument against analyte concentration.
Calibration verification standard - The mid-point calibration standard used to verify calibration. See
Sections 7.3 and 13.4.
Descriptor - In SIM, the beginning and ending retention times for the RT window, the m/z's sampled in
the RT window, and the dwell time at each m/z.
Extracted ion current profile (EICP) - The line described by the signal at a given m/z.
Extraction Batch - A set of up to 20 field samples (not including QC samples) started through the
extraction process on a given 12-hour shift (Section 3.1). Each extraction batch must be accompanied by
a blank (Section 8.5), a laboratory control sample (LCS, Section 8.4), and a matrix spike and duplicate
(MS/MSD; Section 8.3), resulting in a minimum of five analyses (1 sample, 1 blank, 1 LCS, 1 MS, and 1
MSB) and a maximum of 24 analyses (20 field samples, 1 blank, 1 LCS, 1 MS, and 1 MSB) for the
batch. If greater than 20 samples are to be extracted in a 12-hour shift, the samples must be separated into
extraction batches of 20 or fewer samples.
Field Buplicates - Two samples collected at the same time and place under identical conditions, and
treated identically throughout field and laboratory procedures. Results of analyses the field duplicates
provide an estimate of the precision associated with sample collection, preservation, and storage, as well
as with laboratory procedures.
Field blank - An aliquot of reagent water or other reference matrix that is placed in a sample container in
the field, and treated as a sample in all respects, including exposure to sampling site conditions, storage,
preservation, and all analytical procedures. The purpose of the field blank is to determine if the field or
sample transporting procedures and environments have contaminated the sample.
GC - Gas chromatograph or gas chromatography
Internal standard - A compound added to an extract or standard solution in a known amount and used as a
reference for quantitation of the analytes of interest and surrogates. In this method the internal standards
are stable isotopically labeled analogs of selected method analytes (Table 8). Also see Internal standard
quantitation.
Internal standard quantitation - A means of determining the concentration of an analyte of interest (Tables
1 - 3) by reference to a compound not expected to be found in a sample.
BOC - Initial demonstration of capability (Section 8.2); four aliquots of reagent water spiked with the
analytes of interest and analyzed to establish the ability of the laboratory to generate acceptable precision
and recovery. An BOC is performed prior to the first time this method is used and any time the method or
instrumentation is modified.
Method 625.1 54 December 2014
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Laboratory Control Sample (LCS; laboratory fortified blank; Section 8.4) - An aliquot of reagent water
spiked with known quantities of the analytes of interest and surrogates. The LCS is analyzed exactly like a
sample. Its purpose is to assure that the results produced by the laboratory remain within the limits specified
in this method for precision and recovery.
Laboratory fortified sample matrix - See Matrix spike
Laboratory reagent blank - A blank run on laboratory reagents; e.g., methylene chloride (Section 11.1.5).
Matrix spike (MS) and matrix spike duplicate (MSB) (laboratory fortified sample matrix and duplicate) -
Two aliquots of an environmental sample to which known quantities of the analytes of interest and
surrogates are added in the laboratory. The MS/MSD are prepared and analyzed exactly like a field
sample. Their purpose is to quantify any additional bias and imprecision caused by the sample matrix.
The background concentrations of the analytes in the sample matrix must be determined in a separate
aliquot and the measured values in the MS/MSD corrected for background concentrations.
May - This action, activity, or procedural step is neither required nor prohibited.
May not - This action, activity, or procedural step is prohibited.
Method blank - See blank.
Method detection limit (MDL) - A detection limit determined by the procedure at 40 CFR 136, Appendix
B. The MDLs determined by EPA in the original version of the method are listed in Tables 1, 2 and 3. As
noted in Sec. 1.5, use the MDLs in Tables 1, 2, and 3 in conjunction with current MDL data from the
laboratory actually analyzing samples to assess the sensitivity of this procedure relative to project objectives
and regulatory requirements (where applicable).
Minimum level (ML) - The term "minimum level" refers to either the sample concentration equivalent to
the lowest calibration point in a method or a multiple of the method detection limit (MDL), whichever is
higher. Minimum levels may be obtained in several ways: They may be published in a method; they may be
based on the lowest acceptable calibration point used by a laboratory; or they may be calculated by
multiplying the MDL in a method, or the MDL determined by a laboratory, by a factor of 3. For the
purposes of NPDES compliance monitoring, EPA considers the following terms to be synonymous:
"quantitation limit," "reporting limit," and "minimum level."
MS - Mass spectrometer or mass spectrometry, or matrix spike (a QC sample type)
MSD - Matrix spike duplicate (a QC sample type)
Must - This action, activity, or procedural step is required.
m/z - The ratio of the mass of an ion (m) detected in the mass spectrometer to the charge (z) of that ion
Preparation blank - See blank
Quality control check sample (QCS) - See Laboratory Control Sample
Reagent water - Water demonstrated to be free from the analytes of interest and potentially interfering
substances at the MDLs for the analytes in this method.
Method 625.1 55 December 2014
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Regulatory compliance limit (or regulatory concentration limit) - A limit on the concentration or amount
of a pollutant or contaminant specified in a nationwide standard, in a permit, or otherwise established by a
regulatory/control authority.
Relative retention time (RRT) - The ratio of the retention time of an analyte to the retention time of its
associated internal standard. RRT compensates for small changes in the GC temperature program that
can affect the absolute retention times of the analyte and internal standard. RRT is a unitless quantity.
Relative standard deviation (RSD) - The standard deviation times 100 divided by the mean. Also termed
"coefficient of variation."
RF - Response factor. See Section 7.2.2.
RSD - See relative standard deviation
Safety Data Sheet (SDS) - Written information on a chemical's toxicity, health hazards, physical properties,
fire, and reactivity, including storage, spill, and handling precautions that meet the requirements of OSHA,
29 CFR 1910.1200(g) and Appendix D. United Nations Globally Harmonized System of Classification and
Labelling of Chemicals (GHS), third revised edition, United Nations, 2009.
Selected Ion Monitoring (SIM) - An MS technique in which a few m/z's are monitored. When used with
gas chromatography, the m/z's monitored are usually changed periodically throughout the
chromatographic run, to correlate with the characteristic m/z's of the analytes, surrogates, and internal
standards as they elute from the chromatographic column. The technique is often used to increase
sensitivity and minimize interferences.
Signal-to-noise ratio (S/N) - The height of the signal as measured from the mean (average) of the noise to
the peak maximum divided by the width of the noise.
Should - This action, activity, or procedural step is suggested but not required.
SPE - Solid-phase extraction; an extraction technique in which an analyte is extracted from an aqueous
solution by passage over or through a material capable of reversibly adsorbing the analyte. Also termed
liquid-solid extraction.
Stock solution - A solution containing an analyte that is prepared using a reference material traceable to
EPA, the National Institute of Science and Technology (NIST), or a source that will attest to the purity,
authenticity, and concentration of the standard.
Surrogate - A compound unlikely to be found in a sample, and which is spiked into sample in a known
amount before extraction or other processing, and is quantitated with the same procedures used to
quantify other sample components. The purpose of the surrogate is to monitor method performance with
each sample.
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