United States Office of Water
Environmental Protection Agency (4303) December 2011
Draft Procedure for Analysis of
Perfluorinated Carboxylic Acids
and Sulfonic Acids in Sewage
Sludge and Biosolids by
HPLC/MS/MS
December 2011
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U.S. Environmental Protection Agency
Office of Water (4303T)
1200 Pennsylvania Avenue, NW
Washington, DC 20460
EPA-821-R-11-007
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Introduction and Disclaimer
This document represents a draft procedure for the analysis of perfluorinated carboxylic acids and
perfluorinated sulfonates in sewage sludge and biosolids, using high performance liquid chromatography
(HPLC) combined with tandem mass spectrometry (MS/MS).
This draft procedure has been tested in one laboratory under contract to EPA's Engineering and Analysis
Division in the Office of Water, but it is incomplete and is undergoing continued development by EPA.
Further method development work and validation must take place including: MDL studies, IPR studies,
and generation of QC acceptance criteria. Following those development activities, the draft procedure
should be validated in multiple laboratories.
However, EPA is publicly releasing this draft procedure because of interest in methods for perfluorinated
compounds as "emerging contaminants."
This procedure does not determine fluorotelomer alcohols and related precursor compounds, which can
degrade to form perfluorinated carboxylic acids and sulfonic acids, and thus may contribute to the overall
concentrations of perfluorinated compounds in sewage sludge and biosolids.
This procedure should be restricted to use by analysts who are experienced in HPLC-MS/MS.
Mention of trade names or commercial products does not constitute endorsement or recommendation for
use.
Acknowledgements
This draft procedure was written by staff in EPA's Engineering and Analysis Division in the Office of
Water, based on extraction, cleanup, and instrumental procedures developed at U.S. EPA's National
Exposure Research Laboratory in Athens, GA (References 1 and 2) and additional supplementary
procedures from EPA Method 537 (Reference 3), developed by the National Exposure Research
Laboratory in Cincinnati, OH.
Initial single-laboratory testing of the procedure was performed by AXYS Analytical Services Ltd.,
Sydney, British Columbia, Canada, under contract to EPA. Following that testing, a version of the
procedure was subjected to a limited internal Agency peer review by two researchers from EPA's
National Risk Management Research Laboratory in Cincinnati, and one researcher from EPA's National
Environmental Research Laboratory in Athens, GA.
EPA gratefully acknowledges the contributions of all of these organizations and individuals.
Contact Information
Questions concerning this draft procedure or its application should be addressed to:
Engineering and Analytical Support Branch
Engineering and Analysis Division (4303T)
Office of Science and Technology, Office of Water
U.S. Environmental Protection Agency
1200 Pennsylvania Avenue NW
Washington, DC 20460
http: //www. epa.gov/waterscience
ostcwamethods@epa.gov
Draft PFC Procedure i December 2011
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Table of Contents
1.0 Scope and Application 1
2.0 Summary of Procedure 2
3.0 Definitions and Units of Measure 2
4.0 Interferences and Contamination 2
5.0 Safety 4
6.0 Equipment and Supplies 5
7.0 Reagents 8
8.0 Standards 9
9.0 Sample Collection, Preservation, Storage, and Holding Times 12
10.0 Quality Control (QC) 12
11.0 Preparation, Extraction, and Cleanup of Field Samples and QC Samples 15
12.0 LC/MS/MS Set Up and Calibration 19
13.0 Instrument Quality Control 21
14.0 Instrumental Analysis 24
15.0 Qualitative Identification and Quantitation 25
16.0 Method Performance 28
17.0 Pollution Prevention and Waste Management 28
18.0 References 28
19.0 Glossary 29
20.0 Tables and Figures 32
Appendix 1 46
Appendix 2 50
Draft PFC Procedure ii December 2011
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Draft Procedure for Analysis of Perfluorinated Carboxylic Acids and
Sulfonic Acids in Sewage Sludge and Biosolids by HPLC/MS/MS
December 2011
1.0 Scope and Application
1.1 This draft procedure is intended for use by EPA in the development of standardized methods for
the determination of perfluorinated carboxylic acids, perfluorinated sulfonic acids, methyl and
ethyl perfluroro-octanesulfonamides, and (methyl and ethyl sulfonamido)-ethanols in sewage
sludge and biosolids. The procedure employs high performance liquid chromatography (HPLC)
combined with tandem mass spectrometry (MS/MS). The list of target analytes is presented in
Table 1.
1.2 This procedure may be applied to various solids from sewage treatment plant operations,
including sewage sludge and biosolids. Sewage sludge is the solid, semisolid, or liquid organic
material that results from the treatment of domestic wastewater by municipal wastewater
treatment plants. Biosolids are defined in EPA regulations at 40 CFR Part 503 as sewage sludge
that has had additional processing for land application. However, EPA often uses the terms
sewage sludge and biosolids interchangeably, and may do so in this procedure as well.
1.3 This draft procedure is based on extraction, cleanup, and instrumental procedures developed at
U.S. EPA's National Exposure Research Laboratory in Athens, GA (References 1 and 2) and
additional supplementary procedures from EPA Method 537 (Reference 3). The quality control
protocols found in this draft procedure are based on existing EPA HPLC/MS/MS methods
(Reference 4).
1.4 Early work on this procedure focused on analysis of the solid portion of sewage sludge samples
and described discarding any supernatant aqueous liquid in the sample. Subsequent efforts
suggest that the extraction procedures are capable of dealing with samples containing large
amounts of water, which may better represent actual sewage sludges and biosolids from
wastewater treatment operations. These capabilities will be the subject of further testing and
refinement of this draft procedure by the EPA Office of Water's Engineering and Analysis
Division.
1.5 This draft procedure does not determine fluorotelomer alcohols and related precursor compounds,
which can degrade to form perfluorinated carboxylic acids and sulfonic acids, and thus may
contribute to the overall concentrations of perfluorinated compounds in sewage sludge and
biosolids.
1.6 This draft procedure should be restricted to use by analysts who are experienced in HPLC
MS/MS.
1.7 The draft procedure has not been published in 40 CFR Part 136 and is not approved for either
general purpose or regulatory use. This draft procedure has not been validated by EPA and is
being used strictly for method development purposes. It is being publicly released because of
interest in methods for perfluorinated compounds as "emerging contaminants."
Draft PFC Procedure 1 December 2011
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2.0 Summary of Procedure
The general steps in this procedure are summarized in Sees. 2.1 to 2.3. A flow chart that
summarizes the procedures for sample preparation, cleanup, and analysis is shown in Figure 1 at
the end of the document.
2.1 Sample collection and digestion - Collect a sample of sewage sludge or biosolids sufficient to
yield at least 0.5 g of wet solids. (Larger samples are recommended to ensure that they are more
representative of the bulk source of the material.) Homogenize the sample and transfer a
subsample containing 0.5 g of wet solids to a centrifuge tube, spike the sample with the labeled
compound spiking solution, and digest the sample for 30 min with 1M NaOH by heating and
ultrasonic agitation, followed by overnight incubation. Samples are neutralized with HC1 and
extracted with solvent.
2.2 Solvent extraction and cleanup - Extract the digested sample twice by shaking and ultrasonic
agitation, using 10 mL 50:50/ACN:MeOH (v/v). Dilute the sample extract, acidify it to pH 6.5,
agitate it ultrasonically, and clean up the extract using a weak anion exchange (WAX) solid-phase
extraction (SPE) cartridge.
2.3 Analysis - Reconstitute the sample extract with 1 mL 0.3% NF^OH in methanol containing the
labeled injection internal standards. Analyze a 15-(iL aliquot on a dedicated HPLC/MS/MS
equipped with a trapping column (if needed), using negative electrospray ionization (ESI-) mode.
Sample concentrations are calculated using either isotope dilution quantitation for those analytes
with exact labeled analogs in the labeled compounds spiking solution, or internal standard
quantitation for those analytes without an exact labeled analog. The recoveries of the labeled
analogs themselves are determined by internal standard quantitation and used as a quality control
check on the overall analytical process.
3.0 Definitions and Units of Measure
Definitions of terms, acronyms, abbreviations, and units of measure are given in the glossary in
Sec. 19 of this document.
4.0 Interferences and Contamination
4.1 Background levels of perfluorinated chemicals must be controlled during this analysis. To
determine if background concentrations of perfluorinated compounds (PFCs) are under control,
analysts must be able to see a significant difference between a blank and a low-level standard.
This test is discussed in Sec. 12.5. If this cannot be achieved, it may be necessary to proof
reagents and equipment to find the source of contamination or to modify the tubing on the
LC/MS/MS system. Modifications to LC/MS/MS systems are discussed in Sec. 6.4.5.
4.2 Containers -Aqueous solutions of PFC compounds should be stable for at least 28 days when
stored in glass or high density polyethylene (FIDPE) containers. Storage of aqueous solutions in
polypropylene containers has resulted in significant loss of certain perfluorocarboxylic acids (Cn
and Ci2) after 7 days, although some researchers have suggested that stability of the
perfluorocarboxylic acids may be improved through the inclusion of a substantial fraction of an
organic solvent. PFC solutions in basic methanol have not been observed to degrade when in
contact with any of these materials for 6 months.
Draft PFC Procedure 2 December 2011
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4.3 All solvents and reagent water used in the analysis must be demonstrated to be free of PFC
contamination and other interferences. A sample (equivalent in volume to the amount used in the
procedure) from each lot number of solvent or water should be proofed before use.
Concentrations of any detected target compounds are compared to the method detection limits
(MDLs) and minimum levels (MLs) to determine whether they are acceptable for use. PFC-free
reagent water is available (see Sec. 7.1.4), but a procedure for "polishing" deionized water is
provided in Appendix 2.
4.4 All materials used in the analysis, and the entire analytical process, must be demonstrated to be
free of PFCs and other interferences by running reference matrix method blanks (Sec. 10.7)
initially, and with each sample batch (samples started through the extraction process on a given
12-hour shift, to a maximum of 20 samples).
4.5 False positives, false negatives, and co-extracted interferences - Interferences co-extracted from
samples will vary considerably from source to source. Taurodeoxycholic Acid (TDCA) and some
of its isomers, including tauroursodeoxycholic acid and taurochendeoxycholic acid, are known
interferences which may overestimate or yield a false positive result for perfluoro-octanesulfonic
acid (PFOS) (Ref 5), while 5-pregnan-3,20-diol-3-sulfate and 34S-3-hydroxy-5-pregnan-20-one
sulfate may interfere with perfluorohexanesulfonic acid (PFHxS) (Ref. 6). Protocols for ensuring
chromatographic separation of PFOS from TDCA and for detecting interferences by monitoring
secondary multiple reaction monitoring (MRM) transitions for both PFOS and PFHxS are
discussed in Sec. 13.5.
4.6 Matrix Effects - Matrix effects can occur during LC/MS/MS analysis when the sample extract
contains material that co-elutes with the target analytes causing enhancement or suppression of
their MS/MS response, potentially impacting the reliability of data obtained for samples with
complex matrices. This type of matrix effect manifests itself as either high or low labeled
compound recovery (indicating that some of the labeled compounds or injection internal
standards are either being enhanced or suppressed).
If the labeled standard recoveries do not meet the acceptance criteria in Table 8, then the
laboratory should determine whether a matrix effect is the cause. One diagnostic test is to dilute
the sample extract and reanalyze it. Dilution decreases the amount of matrix entering the MS/MS
system, diminishing its effect, and therefore dilution should produce a change in labeled standard
recoveries as the effect of matrix is diminished. The "method of standard additions" also can be
used to diagnose matrix effects, but because it introduces even more material into the ionization
chamber, it is unlikely to be useful in resolving those effects in specific samples.
When performing isotope dilution quantification, the labeled compound and its exact analogue
enter the MS/MS system at the same time and are therefore similarly enhanced or suppressed.
Therefore, the quantification of target compounds is not affected, even if the labeled standard
recovery is outside of the specified range. For those target compounds whose concentration is
determined by internal standard quantification (vs. isotope dilution), the matrix enhancement or
suppression may have a different effect on the target compounds compared to the internal
standard used for quantification and therefore, there can be an effect on quantified results.
There are three possible remedies for this situation: the affected target compounds could be
reported from the diluted analysis, the sample extract could be subjected to further cleanup
(repeat the SPE cleanup on the finished extract), or the analysis could be repeated using a smaller
sample size to reduce the matrix effects.
Draft PFC Procedure 3 December 2011
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5.0 Safety
5.1 The toxicity or carcinogenicity of each chemical used in this procedure has not been precisely
determined; however, each compound should be treated as a potential health hazard. Pure
standards of the compounds should be handled only by highly trained personnel thoroughly
familiar with handling and cautionary procedures and the associated risks. It is recommended
that the laboratory purchase dilute standard solutions of the analytes in this method. However, if
primary solutions are prepared, they should be prepared in a hood, and a NIOSH/MESA approved
toxic gas respirator may be necessary when high concentrations are handled.
5.2 This procedure does not address all safety issues associated with its use. The laboratory is
responsible for maintaining a current awareness file of OSHA regulations regarding the safe
handling of the chemicals specified in this procedure. A reference file of material safety data
sheets (MSDSs) should also be made available to all personnel involved in these analyses. It is
also suggested that the laboratory perform personal hygiene monitoring of each analyst who uses
this procedure and that the results of this monitoring be made available to the analyst. Additional
information on laboratory safety can be found in Refs. 7 to 9.
5.3 Spiking solutions or samples suspected to contain high concentrations of these compounds should
be handled with care.
5.3.1 Facility - When finely divided samples (dusts, soils, dry chemicals) are handled, all
operations (including removal of samples from sample containers, weighing, transferring,
and mixing) should be performed in a glove box demonstrated to be leak tight or in a
fume hood demonstrated to have adequate air flow. Gross losses to the laboratory
ventilation system must not be allowed. Handling of the dilute solutions normally used
in analytical and animal work presents no inhalation hazards except in the case of an
accident.
5.3.2 Protective equipment - Disposable plastic gloves (Latex or non-Latex [such as nitrile]),
apron or lab coat, safety glasses or mask, and a glove box or fume hood should be used.
During analytical operations that may give rise to aerosols or dusts, personnel should
wear respirators equipped with activated carbon filters. Eye protection (preferably full
face shields) should be worn while working with exposed samples or pure analytical
standards. Latex or non-Latex (such as nitrile) gloves are commonly used to reduce
exposure of the hands.
5.3.3 Training - Workers must be trained in the proper method of removing contaminated
gloves and clothing without contacting the exterior surfaces.
5.3.4 Personal hygiene - Hands and forearms should be washed thoroughly after each
operation involving high concentrations of the analytes of interest, and before breaks
(coffee, lunch, and shift).
5.3.5 Confinement - Isolated work areas posted with signs, segregated glassware and tools, and
plastic absorbent paper on bench tops will aid in confining contamination.
5.3.6 Waste handling - Good technique includes minimizing contaminated waste. Plastic bag
liners should be used in waste cans. Janitors and other personnel should be trained in the
safe handling of waste. See Sec. 17 for additional information on waste handling and
disposal.
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5.4 Sewage sludge and biosolids samples may contain high concentrations of biohazards, and must be
handled with gloves and opened in a hood or biological safety cabinet to prevent exposure.
Laboratory staff should know and observe the safety procedures required in a microbiology
laboratory that handles pathogenic organisms when handling such samples.
6.0 Equipment and Supplies
Note: Brand names, suppliers, and part numbers are cited for illustration purposes only. No
endorsement is implied. Equivalent performance may be achieved using equipment and
materials other than those specified here. Demonstration of equivalent performance that
meets the requirements of this procedure is the responsibility of the laboratory.
6.1 Disposable lab equipment
6.1.1 Sample bottles and caps
6.1.1.1 Sample collection bottle - High density polyethylene (HDPE) sample bottles
with polypropylene lids, Nalgene, Part No.: 69032, Lima, Ohio, USA, or
equivalent.
6.1.1.2 Extract dilution bottle - HDPE bottles 125-mL Nalgene, Part No.: 332189-
0004, Lima, Ohio, USA, or equivalent.
6.1.2 Centrifuge tubes
6.1.2.1 Polypropylene centrifuge tubes (15 mL) with polypropylene screw caps,
Corning®, Cat. No: 430766, Corning, NY, USA, or equivalent.
6.1.2.2 Glass centrifuge tubes (15 mL), Kimble Chase, Part No.: 7379015, Vineland,
NJ, USA, or equivalent.
6.1.3 Autosampler vials - Polypropylene 0.3-mL autosampler crimp-top vials (Canadian Life
Sciences, Peterborough, ON, Canada, Cat. No.: 30300P-1232), or equivalent, with
polypropylene snap caps (Canadian Life Sciences, Peterborough, ON, Canada. Cat. No.:
30300P-1232), or equivalent. Polypropylene vials and caps are required, but do not
adequately reseal after use. Therefore, due to potential evaporation, multiple injections
from the same vial are not recommended.
6.1.4 Syringes - Polypropylene 5-mL NORM-JECT syringes with Luer-slip fitting (Fisher
Scientific Cat. No. 1481728), or equivalent.
6.1.5 Disposable pipets - Glass or polypropylene disposable pipets (Fisher Cat. No. 13-711-17)
or equivalent.
6.1.6 Miscellaneous labware - Glass or polypropylene, as required.
6.1.7 pH paper - Whatman® Panpeha® pH indicator strips pH range 0 to 14 (Sigma-Aldrich,
Cat. No.: Z134147), or equivalent.
6.1.8 Syringe filters - Acrodisc 0.45-(im, 25-mm filters with nylon membranes (Ann Arbor,
MI, USA, Part No.: 4614), or equivalent.
Draft PFC Procedure 5 December 2011
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6.2 Non-disposable lab equipment
6.2.1 Auto-pipettes - Gilson (Middleton, WI, USA) Microman® positive displacement pipettes,
volume 10-, 25-, 100-, and 1000-(iL with disposable tips, or equivalent.
6.2.2 Balances - An analytical balance capable of weighing 0.1 mg is used for sample weight
measurements. An analytical balance capable of weighing 10 mg is used for measuring
standards.
6.2.3 Equipment for concentration of extracts - Extracts should be concentrated by evaporation
with nitrogen using a water bath set at 40 °C (Meyer N-Evap, Model 111, Organomation
Associates, Inc.) or equivalent.
6.2.4 Ultrasonic bath - Branson Model 5510 ultrasonic bath (Danbury, CT, USA) or
equivalent.
6.2.5 Shaker - Barnstead LabQuake Tube Shaker/Rotators (Waltham, MA, USA, Cat. No.:
415110), or equivalent.
6.2.6 Bench top Centrifuge - Thermo Scientific, Sorvall Legend RT+ (Waltham, MA, USA) or
equivalent.
6.2.7 Vortex mixer - Barnstead (Dubuque, IA, USA Thermolyne Model No: M16715) or
equivalent.
6.3 Solid-phase extraction
6.3.1 Solid-phase extraction cartridge - Oasis weak anion exchange (WAX) extraction
cartridge, 6-cc barrel size, 150-mg sorbent weight, 30-(im particle size (Waters Milford,
MA, USA), or equivalent.
6.3.2 SPE extraction kit - Vacuum extraction manifold (Alltech/Grace Davison, Deerfield, IL,
USA, Part No:. 210016) or equivalent, using 60-mL polypropylene reservoirs (Supelco/
Sigma Aldrich, Part No. 57022) or equivalent, with reservoir adapters (Supelco/Sigma
Aldrich, Part No. 57020-U) or equivalent, and polypropylene needles (Alltech/Grace
Davison, Deerfield, IL, USA, Part No. 210916) or equivalent. Either a manual or
automatic vacuum manifold may be used for SPE extractions.
Caution: Automated systems may contain parts made of polytetrafluoroethylene (PTFE).
Before use, the system should be proofed to ensure it is free from
contamination.
6.3.3 Laboratory vacuum system or aspirator vacuum system - Capable of maintaining 23 in.
Hg, equipped with shutoff valve and vacuum gauge.
6.4 LC/MS/MS system - The analytical instrumentation used should meet the following
requirements:
6.4.1 HPLC system - The HPLC/MS/MS system used must have a high-pressure inlet, must
have a post-column pump for admission of calibrant during mass spectrometry
calibration and optimization, must be capable of multi-segment gradient separation,
producing the separations for the analytical runs detailed in Table 2 under the instrument
Draft PFC Procedure 6 December 2011
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conditions detailed in Table 5, and meeting other HPLC requirements in Sec. 12. This
system must also be equipped with a 50-(iL loop capable of using 'partial loop with
needle overfill' mode.
6.4.2 Columns - Injections are introduced into a 50-(iL loop using 'partial loop with needle
overfill' mode connected to a CIS guard cartridge, followed by an analytical column (PN
186000404, Waters Xterra MS CIS 3.5 (im, 2.1 x 100 mm column) or equivalent.
Alternative columns may be used as long as they provide comparable chromatography to
that described in Table 2.
6.4.3 MS/MS system - The MS/MS system must be capable of negative electrospray ionization
(ESI-) under the conditions in Table 5, producing unique product ions for analytes within
specified retention time segments. A minimum of 10 scans across the chromatographic
peak of the lowest concentration standard is required to ensure adequate precision
(Waters Quattro Ultima tandem mass spectrometer) or equivalent. The system should be
able to resolve native and labeled compounds with a mass difference of two.
6.4.4 Data system and other software - All system operations should be controlled by
appropriate system software (Waters MassLynx 4.1 and QuanLynx 4.1), or equivalent.
The software should be interfaced to the HPLC/MS/MS to control the LC gradient and
other LC and MS/MS operating conditions, and to acquire, store, reduce and output
HPLC/MS/MS data. The software must be able to identify a compound by retention time
and precursor-product m/z, allow integration of an ion abundance of any specific ion
within specified time or scan number limits, be able to quantify the compound using
relative responses and response factors, or linear or quadratic multi-point weighted
regressions, by isotope dilution and internal standard quantitation techniques.
6.4.5 Elimination of PFC background - Background levels of perfluorinated chemicals must be
controlled to ensure that they do not interfere with this analysis. To determine if
background levels of PFCs interfere with this analysis, the background test in Sec. 12.5
must be conducted.
If instrument background levels of PFCs are found (see diagnostic test in Sec. 12.5), they
must be eliminated before analyses proceed. The following instrumentation
modifications may be required:
• substitution of tubing with PEEK™ tubing,
• replacement of PTFE solvent frits with stainless steel frits,
• inserting a trap column (PN WAT200650, Symmetry CIS, 3.5 (im, 2.1x50 mm
column) or equivalent, as part of the solvent manager at the most down-gradient
point in the water-eluent line immediately before the solvent mixing cell, and
• injection of sufficient blanks to cleanse the system (3 to 5 blanks may be required).
Additionally, to minimize buildup of PFCs during mobile phase equilibration and keep
background level constant, the time the system sits at initial conditions should be kept
constant and as short as possible, but should also assure reproducible retention times in
continuing calibration verifications. Prior to daily use, flush the LC column with elution
solvents before initiating a sequence. It may also be necessary to flush other LC components
such as syringes and other system components.
Draft PFC Procedure 7 December 2011
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7.0 Reagents
7.1 Solvents - Each lot of solvent must be demonstrated to be free from contamination on a routine
basis.
7.1.1 Trace-grade methanol (BDH, VWR International, West Chester, PA, USA) or equivalent.
7.1.2 HPLC-grade acetonitrile (VeX Chem, Aurora, ON, Canada) or equivalent.
7.1.3 Plasma-grade reagent water (Fisher Chemicals) or equivalent, for use in the preparation
of standards and samples.
7.1.4 HPLC-grade water (Fisher Chemicals) or equivalent, for use as the LC mobile phase.
7.2 Gases
7.2.1 Argon, used as the collision gas. Ultra high purity (Alphagaz 1, Air Liquide Canada, or
Airgas, Radnor, PA) or equivalent. Argon gas used should meet or exceed instrument
manufacturer's specifications. Nitrogen may be used as the collision gas, provided that
sufficient sensitivity can be achieved.
7.2.2 Nitrogen, ultra high purity or from a nitrogen generator. Nitrogen may be used as a
carrier gas and as a nebulizer gas in aerosol generation in ESI liquid spray (Alphagaz 1,
Air Liquide Canada, or Airgas, Radnor, PA) or equivalent. Nitrogen also is used to
concentrate sample extracts (Ultra High Purity) or equivalent. Nitrogen gas used should
meet or exceed instrument manufacturer's specifications.
7.3 Other reagents
7.3.1 Certified ACS grade sodium hydroxide (Fisher Chemical) or equivalent.
7.3.2 Ultra pure hydrochloric acid (Seastar Chemicals Inc., Sidney, BC, Canada) or equivalent.
7.3.3 Glacial acetic acid, HPLC grade (Fisher Chemical, Fair Lawn, NJ, USA) or equivalent.
7.3.4 Ammonium hydroxide - (Fisher Chemicals, certified ACS+ grade, 30% in water, Fair
Lawn, NJ, USA) is used as received.
7.3.5 Formic acid - (Alfa Aesar, greater than 96% purity, Ward Mill, MA, USA,) is used as
received.
7.3.6 Ammonium acetate, purity >98%. (Sigma Chemicals) or equivalent.
7.3.7 Reference matrices - Reference matrices must be free from contamination. It may be
difficult to obtain a sewage sludge or biosolids sample that does not have detectable
concentrations of one or more of the native PFCs covered by this procedure. Therefore,
organic-component-rich, commercially available top soil may be used as a reference
matrix if testing demonstrates that it does not contain the native PFCs. Other reference
matrices will be evaluated.
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7.4 Preparation of reagents
7.4.1 Pretreatment reagent (1 M NaOH) - prepared by adding 40 g of NaOH to 1 L of reagent
water.
7.4.2 Neutralization reagent (1M HC1) - prepared by dilution of 83.5 mL of concentrated
hydrochloric acid (HC1) to 1 L with plasma-grade reagent water.
7.4.3 Extraction solvent (50:50/ACN:MeOH [v/v]) - prepared by mixing 500 mL of methanol
(MeOH) and 500 mL of Acetonitrile.
7.4.4 Acetic acid (3%, v/v) - prepared by dissolving 30 mL of glacial acetic acid in 1 L of
reagent water.
7.4.5 SPE reagents
7.4.5.1 Basic methanol (0.3% NH4OH v/v in methanol) - prepared by mixing 30 mL
of ammonium hydroxide with 1 L of methanol.
7.4.5.2 Formic acid, 0.1 M - prepared by dissolving 4.8 g formic acid (96%) in 1 L of
reagent water.
7.4.5.3 Methanol (20%): formic acid 0.1M, (80%) - prepared by mixing 200 mL of
methanol with 800 mL of 0.1 M formic acid in reagent water.
7.4.5.4 Aqueous ammonium hydroxide (0.3% v/v) - prepared by adding 1 mL, of 30%
ammonium hydroxide to 99 mL of reagent water.
7.5 Preparation of LC mobile phases and wash solutions
7.5.1 Aqueous mobile phase - 12.1 mM ammonium acetate in 0.1% acetic acid (aqueous) is
prepared by adding 4 g of ammonium acetate and 4 mL of acetic acid to 4 L of HPLC-
grade water.
7.5.2 Organic mobile phase - 90% acetonitrile/10% HPLC water is prepared by adding 400
mL of HPLC-grade water to 3600 mL of acetonitrile.
7.5.3. Seal wash solution - 10% acetonitrile/90% HPLC water is prepared by adding 400 mL of
acetonitrile to 3600 mL HPLC-grade water.
7.5.4 Needle wash solution - methanol is used as received.
7.5.5 Purge solvent - HPLC-grade water is used as received, or aqueous mobile phase may be
used.
8.0 Standards
8.1 Sources of standards
The standards used and suggested suppliers are listed in Table 9. Standards are used as received.
If the stated chemical purity is 98 % or greater, the weight may be used without correction to
Draft PFC Procedure 9 December 2011
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calculate the concentration of the standard. All solution concentrations and calculated results are
reported in terms of the acid form. Where the obtained standards are salts of the analyte
compounds, a salt-to-parent conversion factor must be applied to the concentration.
8.2 Validation
Before preparation of mixed standards, each individual standard is validated by analysis to
confirm its identity and the absence of impurities. A combined working level solution containing
native (unlabeled) standards, labeled standards, and labeled injection standards was prepared and
analyzed to demonstrate accurate quantification against the calibration standards. A combined
solution of labeled standards and labeled injection standards was prepared and analyzed to
demonstrate cleanliness of these solutions.
8.3 Stock solutions
Native (unlabeled) perfluoroheptanesulfonic acid (PFHpS) and all individual labeled compounds,
except perfluoro-n-[l,2-13C9]undecanoic acid, were purchased as solutions in methanol.
Individual native alky 1-octanesulfonamide and (l-octanesulfonamido)-ethanol compounds (N-
Me/Et-FOSA/Es) were purchased as solutions in 90% nonane/10% toluene. Individual natives
for all compounds except PFHpS and perfluoro-n-[l,2-13C9]undecanoic acid were prepared from
solids.
Individual standards stock solutions should be prepared in methanol, except perfluorotetra-
decanoic acid (PFTeDA), which is prepared in acetonitrile (ACN). See Appendix 1 for details on
standards preparation.
Mixed standards stocks and working level solutions should be prepared in 60:40 ACN:H2O or
basic methanol in glass, Class A volumetric flasks. 60:40 ACN:H2O should be prepared from
Optima grade ACN and PFC-free 18-MQ water.
Mixed standards stocks and working level solutions for N-Me/Et-FOSA/Es should be prepared in
methanol.
Prepare the following mixed stock standards. See Appendix 1 for preparation details.
8.3.1 Native stock mix (containing all native perfluorocarboxylic acids, perfluorosulfonic
acids, and PFOSA)
8.3.2 Stock labeled standard mix
8.3.3 Stock labeled injection standard
8.4 Working-level native standards
8.4.1 Working-level native (unlabeled; authentic) compound spiking solution - This solution is
spiked into calibration standards, IPR, OPR, and matrix spike samples. Prepare target
analyte native compounds at the concentrations shown in Table 7 (and detailed in
Appendix 1) in basic methanol. 40 (iL of the native compound spiking solution is added
to each OPR, IPR or matrix spike sample before digestion. See Sec. 11.3.5.
Draft PFC Procedure 10 December 2011
Not approved for either general purpose or regulatory use
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8.4.2 Low-level native standard - This solution is spiked into calibration standards, IPR, OPR,
MDL, and matrix spike samples. Prepare target analytes at the concentrations shown in
Table 7 (and detailed in Appendix 1) in basic methanol.
8.4.3 Working-level native standard for N-Me/Et-FOSA/Es - This solution is spiked into
calibration standards, IPR, OPR, MDL, and matrix spike samples. Prepare target analyte
native compounds at the concentrations shown in Table 7 (and detailed in Appendix 1) in
methanol containing 10% propan-2-ol. A 16-(iL aliquot of this solution is added to each
OPR, IPR, or matrix spike sample before digestion. See Sec. 11.3.5.
Note: Larger volumes of the solutions in Sees. 8.4.1 to 8.4.3 may be used, provided
that the concentrations are adjusted accordingly.
8.5 Labeled internal standards
Labeled internal standard solution (sometimes called the labeled compound spiking solution) -
This solution is spiked directly into samples prior to extraction. Labeled compounds are used to
quantify unlabeled target compounds and perform recovery correction. Prepare the labeled
compounds at the concentrations shown in Table 7 (and detailed in Appendix 1) in basic
methanol. A 100-(iL aliquot of this solution is added to each sample before extraction. See Sec.
11.3.6.
8.6 Labeled injection standards
Labeled injection standard solution - this solution contains the labeled compounds that are used
to quantify all of the other labeled compounds in the labeled internal standard solution (Sec. 8.5),
and is added to the final sample extract prior to instrumental analysis. Prepare the labeled
compounds used as injection standards at the concentrations shown in Table 7 (and detailed in
Appendix 1) in basic methanol. A 12.5-(iL aliquot of this solution is added to each extract in
preparation for LC/MS/MS analysis. See Sec. 11.6.3.
Note: A larger volume of this solution may be used, provided that the concentration is adjusted
accordingly.
8.7 Calibration standards
Combine and dilute the solutions in Sees. 8.4, 8.5, and 8.6 in basic methanol to produce the
calibration solutions at the levels shown in Table 6 or, if available, purchase prepared standards
for calibration solutions. These solutions are used for initial calibration of the analytical system
(Sec. 13.1). The CS-4 standard is used for ongoing calibration verification (Sec. 13.3).
8.8 Storage
Standards are stored in glass in the dark at 4 °C. Longer-term storage stability is to be
determined, but stability for 6 months has been observed. Place a mark on the vial or ampule at
the level of the solution so that solvent loss by evaporation can be detected. Alternatively, weigh
the vial or ampule before storage, record the mass, and reweigh the vial or ampule before the next
use.
Draft PFC Procedure 11 December 2011
Not approved for either general purpose or regulatory use
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8.9 Stability
Shelf life of purchased solutions in methanol is determined by the supplier. Stability of diluted
solutions and working solutions remains to be determined.
Perfluorocarboxylic acid standards in methanol solution may undergo esterification to the methyl
esters. Most purchased perfluorocarboxylic acid standard solutions were received in methanol
containing 4 mole equivalents of NaOH. Basic methanol (Sec. 7.4.5.1), rather than straight
methanol, is used for all standard dilutions to avoid this potential problem.
9.0 Sample Collection, Preservation, Storage, and Holding Times
9.1 Sample collection - Collect samples in amber high density polyethylene (HDPE) containers with
propylene caps/lids, following conventional sampling practices designed to obtain a sample that is
representative of the material of interest. Lids and other materials containing PTFE must be
avoided, due to possible leaching of fluorinated materials.
9.2 Collect a sample of sewage sludge or biosolids sufficient to yield at least 0.5 g of wet solids for
analysis, plus enough sample to allow the determination of % solids determination (Sec. 11.2)
and to provide volume for QC samples (Sec. 10.5). Larger samples are recommended to ensure
that they are more representative of the bulk source of the material.
9.3 Holding times - EPA has not yet conducted a formal holding time study and will conduct one after
the procedure is finalized. Until that time, default holding times that begin at the time of sample
collection are as follows:
9.3.1 Begin sample extraction within 60 days of collection (to be validated).
9.3.2 Analyze extracts within 30 days of extraction (to be validated).
9.3.3 Store all samples and extracts at less than 4 °C in HDPE containers
10.0 Quality Control (QC)
10.1 Each laboratory that uses this draft procedure is required to operate a formal quality assurance
program. The minimum requirements of this program consist of initial and ongoing quality
control samples. Initial quality control samples include: an initial precision and recovery (IPR)
study described in Sec. 10.2, an MDL study described in Sec. 10.3, and a reporting limit sample
(RLS) described in Sec. 10.4.
Ongoing quality control samples (Sees. 10.5 - 10.8) include calibration verification (CALVER)
standards at the beginning of every shift, analysis of ongoing calibration verification standards
with every sample batch, and analysis of a method blank with every sample batch.
Laboratory performance is compared to the draft performance criteria (Table 8) to determine if
the results of analyses meet the performance characteristics of the procedure. (The performance
criteria will be revised by EPA as the procedure is finalized.)
Additionally, the test for determination of PFC backgrounds in Sec. 12.5 must be performed on
every instrument to determine the modifications to instrument plumbing that are required.
Draft PFC Procedure 12 December 2011
Not approved for either general purpose or regulatory use
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10.2 Initial precision and recovery (IPR) - To establish the ability to generate acceptable precision and
recovery in reference matrices, that the analytical system is performing properly, and that the
laboratory may perform the procedure, the laboratory must perform the following operations.
10.2.1 Spike four aliquots of the reference matrix (Sec. 7.3.7) with 40 (iL of the working level
native standard (Sec. 8.4.1), 16 (iL of working-level native standard solution forN-
Me/Et-FOSA/Es (Sec. 8.4.3), and 100 (iL of labeled internal standard solution (see Sec.
8.5), and analyze each aliquot according to the procedures in Sees. 11 through 14. All
processing steps that are used for samples, including preparation, extraction, and cleanup
(Sec. 11), must be included in this test.
10.2.2 Using results of the set of four analyses, compute the average percent recovery (X) of
each compound in each extract and the relative standard deviation (RSD) of the recovery
for each compound, by isotope dilution for compounds with a labeled analog, and by
injection internal standard for compounds without a labeled analog and for the labeled
compounds.
10.2.3 For each native and labeled compound, compare RSD and X with the corresponding
limits for initial precision and recovery in Table 8. If RSD and X for all compounds meet
the acceptance criteria, then system performance is acceptable and analysis of blanks and
samples may begin.
Note: EPA has not yet developed formal acceptance criteria for this procedure.
Therefore, use the draft criteria in Table 8 as guidance. If more than one target
compound fails the IPR recovery test, examine the system to determine the cause
and repeat the test.
10.3 Method detection limit (MDL) study - Determine the MDL in accordance with the procedures
described in 40 CFR Part 136, prior to the analysis of field samples.
10.4 Reporting limit sample (RLS) - Use 0.5 gram of the soil reference matrix (Sec. 7.3.7) to prepare
the RLS. Spike this aliquot with 10 (iL of the low-level native standard (Sec. 8.4.2) that is
equivalent to the concentration in the lowest (CS-1) calibration standard. Analyze this sample as
an unknown in the sample batch. Analyze the RLS immediately prior to analysis of the OPR, and
samples from the same batch.
Note: The RLS is not required during routine sample analysis after method development has
been completed and single-lab validation has occurred.
10.5 Matrix spike samples are used to assess performance of the procedure on the biosolids being
analyzed. The laboratory must determine the recovery of both labeled and native compounds
spiked into a biosolids matrix. By preparing two such samples, i.e., a matrix spike and a matrix
spike duplicate, the laboratory can also assess precision of the procedure in routine application.
10.5.1 Spike two 0.5-g aliquots of a sewage sludge or biosolids sample with 40 (iL of the
working-level native standard (Sec. 8.4.1), 16 \\L of the working-level native standard
solution for N-Me/Et-FOSA/Es (Sec. 8.4.3), and 100 \\L of labeled internal standard
solution (Sec. 8.5) and mix thoroughly. Analyze both matrix spike samples according to
the procedures in Sees. 11 through 14.
10.5.2 Compute the recovery of the labeled compounds using the internal standard method
(quantify using the injection internal standard).
Draft PFC Procedure 13 December 2011
Not approved for either general purpose or regulatory use
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10.5.3 It may be necessary to spike the native compounds at concentrations that will allow
meaningful recovery data to be obtained, e.g., 3 to 5 times the background levels in the
unspiked sample. Thus, it may be advisable to spike new aliquots of a sample that has
already been analyzed.
10.5.4 Report the background concentrations of native compounds, and recoveries and spiking
concentrations for both the native and labeled compounds. Compare the recoveries to the
draft acceptance criteria in Table 8.
10.5.5 Calculate and report the relative percent difference (RPD) of the recoveries of the native
analytes in the matrix spike and matrix spike duplicate samples. Compare the RPDs to
the draft acceptance criteria in Table 8.
10.6 Calibration verification
Note: Initial calibration is discussed in Sec. 13.1 and must be performed prior to analysis of
samples.
10.6.1 Frequency - Ongoing calibration verification consists of the analysis of bracketing
calibration verification samples, one at the beginning of every 12-hour shift and another
at the end of every batch or 12-hour shift, whichever is more frequent.
10.6.2 In addition, a calibration verification sample is required during the middle of every
sample batch larger than 10 samples. (Sample batches may be no larger than 20 samples.)
10.6.3 The standards used for calibration, calibration verification, and for initial and ongoing
precision and recovery should be identical, so that the most precise results will be
obtained. The CS-4 calibration standard is used for calibration verification samples.
10.6.4 Inject the VER (CS-4) standard (Table 6) using the analysis procedure in Sec. 14.
Note: The requirements in Sec. 15 must be met when analyzing ongoing calibration verification
samples.
10.7 Method blank - A method blank is analyzed with each sample batch to demonstrate freedom
from contamination. Use a 0.5-g aliquot of the reference matrix (Sec. 7.3.7) to prepare a method
blank. Extract the sample following the procedure described in Sec. 11. The method blank
should be analyzed immediately after the OPR sample. If native compounds will be carried from
the OPR into the method blank, analyze one or more aliquots of solvent between the OPR and the
method blank.
Note: If aliquots of solvent must be analyzed, record how many, and update the analytical
sequence in Sec. 14 of this draft procedure to reflect the use of solvent blanks.
Alternatively, analyze the method blank before the OPR sample, and analyze one or more use
solvent blanks after the OPR to prevent carryover from the OPR into the first sample.
10.8 Ongoing precision and recovery (OPR) sample - The laboratory must, on an ongoing basis,
demonstrate through analysis of the ongoing precision and recovery standard that the analytical
system is in control. Use a 0.5-gram aliquot of the soil reference matrix (Sec. 7.3.7) as the matrix
for the OPR. Spike this sample with 40 (iL of the working-level native standard (Sec. 8.4.1), 16
uL of the working-level native standard for N-Me/Et-FOSA/Es (Sec. 8.4.3), and 100 (iL of the
Draft PFC Procedure 14 December 2011
Not approved for either general purpose or regulatory use
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labeled internal standard solution (Sec. 8.5). The aliquot of native standard spiked is equivalent to
the concentration in the CS-3 calibration standard. Extract the sample following the procedure
described in Sec. 11. Analyze the extracts of the OPR aliquot prior to analysis of samples from
the associated batch. Compare the results to the acceptance criteria in Table 8.
Note: EPA has not yet developed formal acceptance criteria for this procedure. Therefore, use
the draft criteria in Table 8 as guidance. If more than one target compound fails the
OPR test, examine the system to determine the cause and repeat the test.
11.0 Preparation, Extraction, and Cleanup of Field Samples and QC
Samples
11.1 Sample preparation
Note: As noted in Sec. 1.4, early work on this procedure focused on analysis of the solid portion
of sewage sludge samples and described discarding any supernatant aqueous liquid in
the sample. EPA believes that this approach was intended to provide a consistent mass
of solids from a bulk sample that could be used for testing the many extraction conditions
studied by Yoo et al, 2009. At present, EPA believes that the extraction procedures are
capable of dealing with samples containing large amounts of water, which may better
represent actual sewage sludges and biosolids from wastewater treatment operations.
Homogenize the entire sample in the original sample container, by shaking samples that are
pourable liquids, or by stirring solids in their original container with a clean spatula, glass stirring
rod, or other suitable implement. Once homogenized, remove an aliquot of the sample to
determine the percent solids content of the sample using the procedure in Sec. 11.2. Remove a
second aliquot of the sample for analysis, as described in Sec. 11.3.
11.2 Determination of solids content
The percent solids of sewage sludge and biosolids will vary depending on the source of the sample and
the treatment processes applied. The solids content of the bulk sample is determined from a subsample
that is used only for the solids determination. Separate procedures are used for the solids determination,
based on the nature of the sample, as described below.
11.2.1 Single-phase solid samples and multi-phase samples in which the main phase is not
aqueous
11.2.1.1 Using a solvent-rinsed spatula, transfer a 1-gram subsample of the
homogenized sample into tared weighing boat. Record weight of the
subsample to three significant figures. (If there is not sufficient mass of the
original sewage sludge and biosolid sample, a smaller subsample may be used
for the solids determination.)
11.2.1.2 Dry the subsample for a minimum of 12 hours in a drying oven set at 110 ± 5 °
C, and cool in a desiccator.
Draft PFC Procedure 15 December 2011
Not approved for either general purpose or regulatory use
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11.2.1.3 Weigh the dried aliquot and calculate percent solids as follows:
Weight of sample aliquot after drying (g)
/o SO11US — X 1UU
Weight of sample aliquot before drying (g)
11.2.2 Multi-phase biosolids samples consisting of mainly an aqueous phase
11.2.2.1 Dry a GF/A filter and weigh to three significant figures. Mix the bulk sample
in the original container (e.g., cap the bottle and shake) and take a 10.0 ± 0.2
mL aliquot. Filter that aliquot through the filter. Dry the filter in an oven for a
minimum of 12 hours at 110 ± 5 °C and cool in a desiccator.
11.2.2.2 Weigh the filter and calculate percent solids as follows:
Weight of sample aliquot after drying (g) - weight of filter (g)
/o Solids — x 100
11.3 Sample digestion
Each sample batch to be digested and extracted during the same 12-hour shift consists of a
maximum of 20 field samples, plus one method blank, and one OPR sample. A reference matrix
(e.g., reference soil) known to be free (below background levels) of the target analytes is used as
the matrix for the method blank and OPR sample (Sec. 7.3.7).
11.3.1 Place a 0.5-g (wet weight) subsample of thoroughly homogenized sample into a 15-mL
polypropylene centrifuge tube.
11.3.2 Place a 0.5-g (wet weight) aliquot of the reference soil into a 15-mL polypropylene
centrifuge tube. This sample is used as the method blank.
11.3.3 Place another 0.5-g (wet weight) aliquot of the reference soil into a 15-mL polypropylene
centrifuge tube. This sample is used as the OPR sample.
11.3.4 If matrix spike samples are to be analyzed, prepare those aliquots as described in Sec.
10.5 and spike them with the native analytes at 3 - 5 times the background
concentrations.
11.3.5 Spike all of the QC samples (OPR, IPR, matrix spike samples) with 40 (iL of the
working-level native standard solution (Sec. 8.4.1) and 16 (iL of the working-level native
standard solution for N-Me/Et-FOSA/Es (Sec. 8.4.3).
Note: Larger volumes of these solutions may be used, provided that the concentrations
are adjusted accordingly.
11.3.6 Spike all samples and QC samples with 100 (iL of the labeled internal standard solution
(Sec. 8.5). The volume spiked will yield a sample extract with labeled compounds
present at a concentration equivalent to the native compounds in the mid-level calibration
standard.
11.3.7 Add 0.5 mL of 1 M NaOH solution to each sample and QC sample. If a sample is very
dry, add enough NaOH to wet and cover the sample. Record volume of NaOH used.
Draft PFC Procedure 16 December 2011
Not approved for either general purpose or regulatory use
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11.3.8 Sonicate the samples in a heated water bath at 40 °C for 30 min, followed by incubation
overnight (12 h) at ambient temperature.
Note: Mixing the sample by sonication is described in Ref. 1. EPA has not tested
whether or not this step is essential, but may do so in the future.
11.3.9 After the incubation period, neutralize the NaOH by adding an equivalent number of
moles of the 1 M HC1 solution (Sec. 7.4.2), allow the mixture to react for 1 min, and
vortex.
Note: Some biosolids are treated with lime and will require additional HCl to
neutralize them. If the samples are known or suspected to involve lime treatment,
then use a clean glass stirring rod to remove a small amount of the supernatant
from the incubated sample and test the pH with a wide-range pHpaper. Do NOT
dip the pH paper into the sample. If the pH of the incubated sample is not 7.0 ±
0.5, add additional HCl, vortex, and retest the pH until the sample is neutral.
11.4 Sample extraction
11.4.1 Add 10 mL of 50:50/ACN:MeOH (v/v) to the sample in the centrifuge tube.
11.4.2 Shake the mixture for 1 h on a tube shaker (Sec. 6.2.5) at a moderate speed.
11.4.3 Centrifuge the sample at a speed of 3000 x g for 20 min.
11.4.4 Decant the 10 mL supernatant into a 250-mL HDPE bottle containing 180 mL of plasma-
grade reagent water (Sec. 7.1.3).
11.4.5 Repeat the extraction (Sees. 11.4.1 to 11.4.4) one more time and add the extract to the
250-mL HDPE bottle.
11.4.6 After the second extraction, check that the pH of the diluted extract is 6.5 ± 0.5. If
required, adjust the pH with 3% (v/v) acetic acid in reagent water or 0.3% (v/v) aqueous
ammonium hydroxide (Sec. 7.4.5.4).
11.4.7 Sonicate the mixture for 30 min. The extract is ready for cleanup.
11.5 Extract cleanup
All sample extracts are subjected to cleanup using an SPE cartridge, as described below.
11.5.1 Prepare the SPE extraction manifold, reservoirs, SPE adapters, and SPE needles. Use
SPE cartridges that pass the SPE cartridge performance check described in Sec. 11.7.
11.5.2 Label each Oasis WAX SPE cartridge with the sample ID and place the cartridge on the
extraction manifold.
11.5.3 Condition each cartridge with 5 mL of 0.3% NH4OH in methanol, followed by 5 mL of
0.1M formic acid in reagent water. Discard eluants.
11.5.4 Equilibrate the cartridge with 5 mL of plasma-grade reagent water (Sec. 7.1.3). Discard
the eluant.
Draft PFC Procedure 17 December 2011
Not approved for either general purpose or regulatory use
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11.5.5 Load the sample onto the cartridge drop-wise (~5 mL/min).
11.5.6 Wash the sample with 5 mL of 20% MeOH in 80% 0.1M formic acid in reagent water,
followed by 2 mL of 0.3% (v/v) NH4OH in reagent water. Discard the eluants.
Note: Other wash solvents have been reported in the literature and may provide
acceptable results.
11.5.7 Dry the cartridge by pulling air under vacuum for 5 min.
11.5.8 Elute the cartridge into a 15-mL clean glass centrifuge tube with 4 mL 0.3% NlrUOH
(v/v) in MeOH.
11.6 Extract concentration
11.6.1 Reduce the extract to about 50 \\L using a gentle stream of nitrogen and a water bath set
at 40 °C.
11.6.2 Reconstitute the extract with 938 (iL of 0.3% NH4OH (v/v) in MeOH and vortex to mix.
11.6.3 Spike the extract with 12.5 \\L of the injection internal standard solution (Sec. 8.6),
vortex to mix, and filter the sample extract through a syringe filter (Sec. 6.1.8) into a
clean centrifuge tube. The final extract volume is 1.0 mL.
Note: Other volumes and solvents may be usedin Sees. 11.6.1 to 11.6.3, provided that
the mass of the injection internal standard is equal to the mass in the calibration
standards and the final extract volume is the same for all sample extracts.
However, avoid taking the sample extract to dryness in Sec. 11.6.1, because it
may result in loss of short-chain analytes such as PFBA.
11.6.4 Vortex the extract and transfer 300 (iL of the final extract to a polypropylene LC/MS/MS
auto-sampler vial (Sec. 6.1.3) for analysis. Cap the centrifuge tube containing the
remaining 700 (iL and store at 4 °C for backup. Place a mark on the tube at the level of
the solution so that solvent loss by evaporation can be detected. Alternatively, weigh the
tube before storage, record the mass, and reweigh the tube before any subsequent analysis
of the stored extract.
11.7 SPE cartridge performance check
In order to be used for cleanup of sample extracts, the performance of the WAX SPE cartridges
must be checked at least once for each manufacturer's lot of cartridges. This performance check
is accomplished by processing a spiked reagent water sample through the extraction procedure
and analyzing the extract. Labeled compounds are not added to these check samples before
extraction because the recovery correction inherent in isotope dilution will mask problems with
the cartridges. Cartridge performance is acceptable if the recoveries of the native analytes are
within the QC acceptance criteria for the OPR in Table 8. Perform this cartridge check as
outlined below.
Note: 777/5 check is performed whenever a new lot number of cartridges is purchased.
Draft PFC Procedure 18 December 2011
Not approved for either general purpose or regulatory use
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11.7.1 Spike 100 mL reagent water with 40 (iL of the working-level native standard solution
(Sec. 8.4.1) and 16 (iL of the working-level native standard solution for N-Me/Et-
FOSA/Es (Sec. 8.4.3). Do NOT spike the labeled internal standard compounds.
11.7.2 Process the solution through the SPE cleanup procedure in Sec. 11.5.
11.7.3 After cleanup, spike the solution with the labeled internal standard solution (Sec. 8.5) and
complete the analysis as per Sec. 14.
11.7.4 Recovery of the native compounds must be within the QC acceptance criteria for the
OPR in Table 8. If the compounds are not recovered in this range, adjust the elution
volumes or reject the cartridge batch.
12.0 LC/MS/MS Set Up and Calibration
Samples are analyzed on a high performance liquid chromatograph coupled to a triple quadrupole mass
spectrometer (LC/MS/MS), or equivalent, equipped with an electrospray ionization (ESI) source. The
tandem MS systems operate at a nominal resolution of 1 amu. The LC/MS/MS is run in the negative ion
electrospray (ESI-) mode using multiple reaction monitoring (MRM). Data acquisition and quantification
are performed by recording the peak areas of the applicable parent ion /daughter ion transitions. The
instrument manufacturer's software is used to acquire data and calculate results using isotope dilution and
internal standard quantitation (Sec. 15.2).
Once the mass spectrometer has been optimized and the LC/MS/MS operating conditions for the targeted
compounds have been established, the same conditions must be used for the analysis of all standards,
blanks, IPR and OPR standards, field samples, and QC samples.
12.1 Establishing LC/MS/MS Operating Conditions
Prior to any analyses, optimize the following instrumental conditions: mass calibration, MRM
acquisition parameters, scans per peak, chromatographic resolution, retention time calibration,
sensitivity and instrument background elimination. Example analyte-specific instrumental source
parameters for PFCs analysis are found in Table 3, but actual tuning parameters are instrument-
specific and should be optimized according to manufacturer's specifications.
12.2 Mass calibration
The mass spectrometer system must undergo mass calibration according to manufacturer's
specifications to ensure accurate assignments of m/z values by the instrument. Mass calibration
is performed at least annually, after performing major maintenance, or as required to maintain
routine instrument sensitivity and stability performance. The reference calibrant used is a 50:50
isopropanol:water (IPA:water) solution containing sodium cesium iodide (NaCsI), which is
infused directly into the electrospray source during calibration. Mass calibration is performed at
instrumental settings corresponding to a nominal unit mass resolution, but mass resolution is not
directly measured.
In the absence of manufacturer-specific instructions and acceptance criteria, the following
procedure may be used.
12.2.1 Use a NaCsI calibration solution (in 50:50 IPA:water) containing 2 (ig/(iL sodium iodide
and 50 ng/(iL cesium iodide.
Draft PFC Procedure 19 December 2011
Not approved for either general purpose or regulatory use
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12.2.2 Use a syringe pump to infuse the calibration solution as a stable aerosol spray at 10
(iL/min directly into the ESI-MS/MS source.
12.2.3 Scan the MS/MS over the mass range from 20 to 2000 Daltons. Adjust the source
parameters to optimize peak intensity and shape across the mass range. The exact m/z
values for NaCsI calibration are contained as a reference file on the instrument data
system and are:
Calibration Masses (Daltons - m/z)
22.9898
132.9054
172.8840
322.7782
472.6725
622.5667
772.4610
922.3552
1072.2494
1222.1437
1372.0379
1521.9321
1671.8264
1821.7206
1971.6149
2121.5091
2271.4033
2421.2976
2571.1918
2721.0861
2870.9803
During the mass calibration, examine the instrument parameters to ensure detection of the
specified ions.
Mass calibration is judged on the basis of the presence or absence of the exact calibration
masses, e.g., a limit on the number of masses that are "missed." Repeat the test if more
than two masses are missed.
12.3 MRM acquisition parameters
During method setup, the mass spectrometer response must be separately optimized for each target
compound, using a solution containing only the compound of interest. These parameters are then
used for analysis of all standards and samples.
12.3.1 Using a post-column pump, infuse a solution mixture of methanol and mobile phase
containing approximately 1 ppm of the compound of interest directly into the ESI-MS/MS
source. This solution is prepared by adding mobile phase to a 1 ppm methanol solution
of the compound being tested in the ratio of 1 part mobile phase/2 parts test solution.
12.3.2 For each compound, optimize the sensitivity to the specified parent-daughter transitions
(see Table 3) by adjusting the collision energy and cone voltage.
12.3.3 The optimum parameters are compound specific and a set of single settings cannot be
used for all target compounds in the analysis. Use the optimized settings for the analysis
of all standards and samples.
12.4 Chromatographic separation
Establish liquid chromatography conditions suitable for the separation of the target compounds.
To achieve the retention times in Table 2, the HPLC should be operated according to the
parameters in Table 5. A CIS analytical column is used. The exact gradient is optimized for the
chromatography system in use, but the conditions in Table 5 can be used as guidelines. The
chromatographic separation should ensure that there is adequate resolution of target compounds
from potentially interfering substances (Sec. 13.5). Note that the LC gradient selected will affect
the chromatographic resolution of linear and branched PFC compounds. After the LC column
conditions and gradient have been determined, they should be used for all analyses.
Draft PFC Procedure
20
Not approved for either general purpose or regulatory use
December 2011
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12.5 Instrument background determination
To determine if background concentrations of PFCs significantly impact this analysis, a 40 part
per trillion (ppt) standard containing all of the target analytes in 0.3% NF^OH (v/v) in methanol
must be analyzed three times, with alternating instrument solvent (methanol) blank
measurements. If the peak area from the standards is not greater than that of the solvent blank
using a Student's t-test (95% confidence), then it may be necessary to modify the "plumbing" of
the analytical system as outlined in Sec. 6.4.5. This test should be performed prior to any
analysis, at least annually, and after major instrument maintenance.
12.6 Establishing retention time windows
Analyze individual solutions of the each of the target compounds using the LC gradient and
acquisition parameters determined above. Analyze a mixed solution of all target compounds to
confirm their separation and identification. A total ion chromatogram (TIC) indicating the
separation of the target analytes is shown in Figure 2, as an example.
12.7 Analytical data acquisition program
All of the information is now in place to finalize the analytical acquisition routine to be used for
calibration of instrument response and analysis of all samples. The acquisition program will
monitor each of the MRM ions listed in Table 3 at its optimum cone voltage and collision energy,
as determined in Sec. 12.3, and in the appropriate retention time window established in Table 2.
12.8 Instrument sensitivity
Prior to commencement of any analysis, and at least once every 24 hours during extended runs,
determine that the instrument is meeting the sensitivity specifications. Analyze the lowest
concentration calibration solution (CS-1) using the acquisition program described in Sec. 12.7.
Ensure that all compounds are detected with S/N > 3. If sensitivity is inadequate, perform system
cleaning and maintenance and repeat the test. If calibration verification can not be established
after this maintenance, anew initial calibration (Sec. 13.2) is required.
13.0 Instrument Quality Control
The mass spectrometer is optimized and the LC/MS/MS operating conditions are established for the
target compounds as described in Sec. 12. The same conditions must be used for the analysis of all
standards, blanks, IPR and OPR standards, and samples.
13.1 Initial calibration
Initial calibration (ICAL) must be performed prior to analysis of any field sample or QC sample.
Approaches to quantitation vary depending on the compound. Some compounds are quantified
using isotope dilution (for those target compounds with exact labeled analogs) and others are
quantified using internal standard (those without an exact labeled analog). Additionally, labeled
compounds are quantified using the internal standard approach. These two approaches are
discussed in Sees. 13.2.1 and 13.2.2, respectively. The requirements for initial calibration are
outlined in Sec. 13.2, and Table 8. After the initial calibration, analyze an initial calibration blank
and an initial calibration verification (CALVER) standard.
Draft PFC Procedure 21 December 2011
Not approved for either general purpose or regulatory use
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13.2 Calibration by isotope dilution and internal standard
Calibrations should be constructed using regressions of untransformed data and plotting
normalized relative response versus the absolute concentration of analyte. Normalized relative
response is defined as:
NRR = -^ x C,
where:
A,
NRR = Normalized relative response
An = Area of the peak for the native (unlabeled) analyte
AI = Area of the peak for the specified labeled compound
Ci = Concentration of the specified labeled compound (in pg/mL)
Efforts to date indicate that a linear equation may be used for all of the analytes except:
perfluorobutanesulfonic acid (PFBS), perfluoro-n-heptane sulfonic acid (PFHpS),
perfluorooctane sulfonamide (PFOSA), and those compounds containing more than 10 carbons
(Cn - Ci4). For those analytes, the response is better fit (higher r2) with a quadratic equation.
For either a linear regression or a quadratic fit, weight the equation using the inverse of the
concentration (e.g., 1/x), and do not force the calibration through the origin.
13.2.1 Calibration by isotope dilution
Isotope dilution is used for calibration of each native compound for which an exact
labeled analog is available (Table 3). An 8-point initial calibration is prepared for each
native compound. The calibration solutions are listed in Table 6.
13.2.1.1 To calibrate the analytical system by isotope dilution, inject the CS-1 through
CS-8 calibration solutions (Sec. 8.7 and Table 6). Use an injection volume of
ISjiL.
13.2.1.2 For each compound determined by isotope dilution, compute its normalized
relative response (NRR) over the calibration range. Determine the NRR of
each compound using the area responses of the product m/z values specified in
Table 3. Use the labeled compounds listed in the tables as the quantitation
reference and the product m/z values of these labeled compounds for
quantitation. Determine the calibration equation for each compound by
regressing the NRR against the native compound concentration to produce a
calibration weighted inversely proportional to concentration (i.e., a 1/x
weighted linear regression). Select the calibration equation (linear or
quadratic) that provides the best fits to the data, as indicated by the coefficient
of determination (r2). Examine the residuals of the regression (i.e., the distance
between the observed response for each calibration standard and the response
predicted by the regression) to determine if they are randomly distributed (i.e.,
the regression line passes between the calibration points, and not above or
below all of the points). Whichever calibration is selected, r2 must be at least
0.99. Use the resultant equation to calculate the concentration of analyte in
each sample or CALVER.
Draft PFC Procedure 22 December 2011
Not approved for either general purpose or regulatory use
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13.2.1.3 Some of the labeled compounds may contain native analogs as impurities. If
the labeled compounds are present at a constant concentration in calibration
and other solutions (including sample extracts), a constant background
response will be added to the response from analysis of calibration and other
solutions. A calibration not forced through zero will accommodate any such
residual.
13.2.2 Calibration by internal standard
Internal standard calibration is applied to the determination of the native compounds that
do not have exact labeled analogs, and that are not being quantified by isotope dilution.
Internal standard calibration is also used to quantify the labeled compounds themselves.
The internal standard approach utilizes the injection internal standard (IIS) that is added
to the extract after extraction and cleanup and prior to injection into the instrument as the
quantitation reference. The reference compound for each native and/or labeled
compound is listed in Table 3. For the labeled compounds, calibration is performed at a
single concentration, using data from the 8 points in the calibration (all of which contain
the labeled compounds at the same concentration).
13.2.2.1 To calibrate the system for native compounds for which isotope dilution is not
being performed, use the data from the 8-point calibration and the labeled
injection internal standard.
13.2.2.2 For each compound determined by internal standard quantitation, its NRR is
computed over the calibration range. Determine the NRR of each compound
using the area responses of the product m/z's specified in Table 3. Use the
labeled IIS compounds listed in the tables as the quantitation reference and the
product m/z's of these labeled compounds for quantitation. Determine the
calibration equation for each compound by regressing the NRR against the
native compound concentration to produce a calibration weighted inversely
proportional to concentration (i.e., a 1/x weighted linear regression). Do not
force through zero (Sec. 13.2.1.3). Select the calibration equation (linear or
quadratic) that provides the best fits to the data, as indicated by the coefficient
of determination (r2). Examine the residuals of the regression (i.e., the distance
between the observed response for each calibration standard and the response
predicted by the regression) to determine if they are randomly distributed (i.e.,
the regression line passes between the calibration points, and not above or
below all of the points). Whichever calibration is selected, r2 must be at least
0.99. Use the resultant equation to calculate the concentration of analyte in
each sample or CALVER.
13.2.2.3 For each labeled compound, regress its NRR against the concentration of the
labeled compound using the labeled IIS as the quantitation reference as
indicated in Table 3. The labeled compounds and the labeled injection internal
standards are in each calibration solution at a constant concentration. The
regression will simplify to a single-point calibration because the concentrations
are constant.
Draft PFC Procedure 23 December 2011
Not approved for either general purpose or regulatory use
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13.2.3 Quantification of the labeled injection internal standard concentration
The injection internal standard (IIS) itself is quantified by external standard. Monitoring
of the recovery of the IIS is used as a diagnostic test for matrix effects during the LC
analysis (Sec. 4.6).
13.3 Calibration verification
Calibration verification (VER) requires the analysis of bracketing calibration verification samples,
one at the beginning of every 12-hour shift, and another after the analysis of every 10 samples, or at
the end of each 12-hour shift, whichever is more frequent. The standards used for calibration,
calibration verification, and for initial and ongoing precision and recovery should be identical, so
that the most precise results will be obtained. The CS-4 calibration standard is used for calibration
verification samples. Inject the CS-4 calibration standard (Table 7) using the analysis procedure in
Sec. 14. The requirements in Sec. 10.8, and Table 8 must be met when analyzing calibration
verification samples.
13.4 Quantitation of linear and branched isomers
Some PFCs consist of linear and branched isomers, depending on manufacturing processes (Ref.
5). There are reports that during MS/MS analysis, the linear and branched isomers ionize with
different efficiencies, complicating the quantification of PFC compounds (Ref. 3). The LC
analysis performed by this procedure results in partial chromatographic resolution of the isomers.
Additionally, the composition of standards may differ by vendor, and differ from the distribution
of the PFCs in environmental samples. Therefore, peak integration in samples should ensure that
the PFC target peaks include the linear and branched isomers as a single total response.
13.5 Co-extracted interferences
Interferences co-extracted from samples will vary considerably from source to source.
Taurodeoxychloic Acid (TDCA) is a known interference which may lead to an overestimate or
yield a false positive result for PFOS (Ref. 5) while 5-pregnan-3,20-diol-3-sulfate and 34S-3-
hydroxy-5-pregnan-20-one sulfate may interfere with PFHxS (Ref. 6). The 499 > 80 transition is
prominent in all TDCA isomers and in PFOS. However, the 499 > 99 transition for PFOS is not
affected by the TDCA. In the absence of chromatographic separation of TDCA from PFOS, the
499 > 80 transition will result in significant bias in PFOS concentrations. Therefore, both
transitions must be monitored for PFOS and results must agree within 20%, to ensure accurate
quantification of PFOS. Similarly, analysis for PFHxS can be biased by co-eluting interferences.
In this case, the 399 > 80 and the 399 > 99 transitions may both be affected, and therefore, a third
transition, 399 > 119, also must be monitored to demonstrate that there is not a bias from co-
eluting interferences.
14.0 Instrumental Analysis
Once the operating conditions have been established and the instrument tuned (Sec. 12), inject a
15-(iL aliquot of sample extract into a 50-uL loop, using partial-loop-with-needle-overfill mode
onto a trapping column (if needed). Start the gradient according to the parameters found in Table
5. Start data collection 1 to 2 minutes priorto elution of the first analyte. Monitor the product
m/z's for each native and labeled analyte throughout its retention time window. Stop data
Draft PFC Procedure 24 December 2011
Not approved for either general purpose or regulatory use
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collection after elution of the last analyte in each group. Return the gradient to the initial mixture
for analysis of the next sample extract or standard.
A typical instrument analysis sequence is as follows:
• 1-2 Instrument blanks (more may be included)
• 8 Initial calibration standards
• 2 Instrument blanks
• Opening calibration verification standard
• OPR sample
• 1-2 Instrument blanks
• Method blank
• Up to 6 field samples
• Bracketing calibration verification standard (every 10th injection)
• Up to 10 field samples
• Closing calibration verification standard (every 10th injection, which may serve as the
opening VER for the next cycle)
• OPR sample, etc.
15.0 Qualitative Identification and Quantitation
15.1 Qualitative identification - The following requirements must be met in all samples for a
compound to be identified.
15.1.1 Signal to noise requirements - The LC peak representing the quantitation m/z of each
native compound in the upper 7ICAL standards must be present with a S/N of at least 10,
and with a S/N of at least 3 for the lowest standard (CS-1). The LC peak representing each
labeled compound quantitation m/z in the CALVER standard and in extracts from all other
samples must be 10 or greater. If these requirements are not met in ICAL or CALVER
samples, the LC/MS/MS system must be adjusted or recalibrated until these requirements
can be met. If these requirements are not met in samples, the CALVER test should be
repeated.
15.1.2 Relative intensity - The ion intensity of each monitored ion transition in the ICAL should
be recorded for use in qualitative identification for all other samples. The monitored ion
transitions are found in Table 3. These parameters may be instrument specific. If
alternate transitions are monitored for diagnostic purposes (called confirmatory
transitions), the following requirements should be met:
1) The molecular ion shall preferably be the precursor of one of the selected diagnostic
transitions (the molecular ion, characteristic adducts of the molecular ion,
characteristic fragment ions and all their isotope ions),
2) Diagnostic transitions preferably should not originate from the same part of the
molecule as that for the quantitation transition, and
3) The signal-to-noise ratio for each diagnostic transition must be greater than or equal to
3.
Draft PFC Procedure 25 December 2011
Not approved for either general purpose or regulatory use
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Comparison of the ratio of the confirmation transition response to the quantitation
transition response in a sample to the ratio in the most recent CALVER can assist in
confirmation of the presence or absence of a target compound in samples. The relative
ion ratio requirements are described below.
15.1.3 Relative ion intensity ratio requirement - Depending on the relative intensity of the
confirmatory m/z to the quantitation m/z, the following requirements must be met to
provide confirmation of the presence of the target analyte:
Relative intensity of confirmation
transition to quantitative transition in
most recent CALVER
>50 %
20 to 50 %
10 to 20 %
<10 %
Agreement of ratio of confirmation transition to
quantitative transition in sample relative to
same ratio in most recent CALVER
±20%
±25%
±30%
±50%
Note: If the results for any of the qualitative identification criteria above are
ambiguous, or if false negatives or false positives occur, the laboratory should
consult EPA to determine corrective action and next steps during method
development.
15.1.4 Retention time window requirement - There are two retention time requirements:
1) the retention times of the native and labeled compounds in the initial calibration must be
stored in the system for verification of the retention time window requirement during
analysis of all subsequent samples. Data acquired for all subsequent samples should be
within the required retention time windows. If this is not the first time an initial
calibration is being performed, retention times from this calibration should be checked
against those from previous calibrations to determine if the separation of target analytes
is being affected.
2) the relative retention times for native compounds and the labeled compound for which
each native is being quantified must fall within a certain range of each other. Typical
retention times, relative retention times and retention time windows are listed in Table 2.
15.1.5 Peak asymmetry factor - During initial calibration and calibration verification, the peak
asymmetry factor must be calculated for all analytes using the following figure and
equation:
As = b/a
Draft PFC Procedure
26
Not approved for either general purpose or regulatory use
December 2011
-------
where:
As = peak asymmetry factor
b = width of the back half of the peak measured (at 10% peak height) from the trailing
edge of the peak to a line dropped perpendicularly from the peak apex, and
a = the width of the front half of the peak measured (at 10% peak height) from the
leading edge of the peak to a line dropped perpendicularly from the apex.
Calculated peak asymmetry factors for the first two eluting peaks in the CS-4 standard of
the ICAL must fall within 0.8 to 1.5.
If this criterion cannot be achieved, the LC mobile phase conditions need to be modified.
This criterion must be met each time a new calibration curve is generated.
15.2 Quantitation
15.2.1 Isotope dilution quantitation
Using the most recent multi-point calibration (Sec. 13.2.1), calculate native and labeled
compound concentration in the extract. Do not use calibration verification data to
quantify analytes.
15.2.2 Internal standard quantitation and labeled compound recovery
Compute the concentration of each native compound in the extract that does not have an
exact labeled analog and each labeled compound by internal standard, using the weighted
regression established in Sees. 13.2.2.2 and 13.2.2.3, respectively.
Using the concentration in the extract determined above, compute the percent recovery of
each labeled compound using the following equation:
Concentration found (ng/mL)
Recovery (%) = -^-= x 100
Concentration spiked (ng/mL)
15.2.3 The concentration of a native compound in the solid sample is computed using the
concentration of the compound in the extract, and the wet weight of the solids,
and the percent solids, as follows:
C V
Concentration in solid sample (ng / kg) = —
Ws (% solids)
where:
Cex = Concentration of the compound in the extract in ng/mL
Vex = Extract volume in mL
Ws = Sample weight (wet weight) in kg
% solids = Percent solids determined in Sec. 11.2
If desired, divide the concentration by 1000 to convert ng/kg (ppt) to (ig/kg (ppb).
15.3 Reporting results
Unless otherwise specified, report results in ng/kg (parts-per-trillion) to three significant figures,
based on the dry weight of the sample. Also report the percent solids so that the result may be
converted to wet-weight units by the end user.
Draft PFC Procedure 27 December 2011
Not approved for either general purpose or regulatory use
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Results for a compound in a sample that has been diluted must be reported at the least dilute level
at which the area at the quantitation m/z is within the calibration range. Results should be flagged
to indicate that they are from a diluted analysis.
Project-specific reporting requirements may apply, including reporting results based on the
volume of the original sample, e.g., ng/L or other weight/volume units
16.0 Method performance
This procedure is still under development. Preliminary method performance information can be found in
Tables 4, 8, and 10. Additional performance data will be added by EPA as they are developed.
17.0 Pollution prevention and waste management
17.1 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 Reference 12.
17.2 Samples at pH <2, or pH >12 are hazardous and must be neutralized before being poured down a
drain, or must be handled as hazardous waste.
17.3 Standards should be prepared in volumes consistent with laboratory use to minimize the disposal
of excess volumes of expired standards. 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.
17.4 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.
18.0 References
1. "Analysis of Perfluorinated Chemicals in Sludge: Method Development and Initial Results." Yoo,
Hoon, Washington, John W., Jenkins, Thomas M., Libelo, and E. Laurence. Journal of
Chromatography A. Volume 1216, Issue 45, 6 November 2009, Pages 7831-7839.
2. "Analysis of Perfluorinated Carboxylic Acids in Soils: Detection and Quantitation Issues at Low
Concentrations." Washington, John, W., Ellington, Jackson J., Jenkins, Thomas M., and John Evans.
J. Journal of Chromatography A. Volume 1154, Issues 1-2, 22 June 2007, Pages 111-120.
3. EPA Method 537. "Determination of Selected Perfluorinated Alkyl Acids in Drinking Water By
Solids Phase Extraction and Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS),"
Revision 1.1, EPA Document Number EPA/600/R-08/092. September 2009.
Draft PFC Procedure 28 December 2011
Not approved for either general purpose or regulatory use
-------
4. EPA Method 1694: Pharmaceuticals and Personal Care Products in Water, Soil, Sediment, and
Biosolids by HPLC/MS/MS. EPA Document Number EPA-821-R-08-002. December 2007.
5. "An analytical method for the determination of perfluorinated compounds in whole blood using
acetonitrile and solid phase extraction methods." Leo W.Y., Yeung, Sachi Taniyasu, Kurunthachalam
Kannan, Delia Z.Y. Xu, Keerthi S. Guruge, Paul K.S. Lam, Nobuyoshi Yamashita. Journal of
Chromatography A, 1216 (2009) 4950-4956.
6. "Simultaneous characterization of perfluoroalkyl carboxylate, sulfonate and sulfonamide isomers by
liquid chromatography - tandem mass spectrometry." Benskin, J. P.; Bataineh, M.; and Martin, J. W.
Anal. Chem. 2007, 79, 6455-6464.
7. "Working with Carcinogens," Department of Health, Education, & Welfare, Public Health Service,
Centers for Disease Control, NIOSH, Publication 77-206, September 1977, NTIS PB-277256.
8. "OSHA Safety and Health Standards, General Industry," OSHA 2206, 29 CFR 1910.
9. "Safety in Academic Chemistry Laboratories," ACS Committee on Chemical Safety, 1979.
10. "Structural Identification of Isomers Present In Technical Perfluorooctane Sulfonate By Tandem
Mass Spectrometry." Langlois, I. and Oehme, M. Rapid Commun. Mass Spectrom. 2006, 20, 844-
850.
11. "Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the
performance of analytical methods and the interpretation of results."
http://eur-lex.europa.eu/LexUriServ/site/en/oj/2002/l_221/l_22120020817en00080036.pdf.
12. "Environmental Management Guide for Small Laboratories" USEPA Office of the Administrator,
Washington, DC, EPA 233-B-00-001, May 2000.
19.0 Glossary
These definitions and purposes are specific to this method but have been conformed to common usage to
the extent possible.
Symbols
0 C degrees Celsius
(iL microliter
(im micrometer
< less than
> greater than
% percent
Abbreviations (in alphabetical order)
cm centimeter
ESI- Negative Electrospray lonization
g gram
h hour
ID inside diameter
Draft PFC Procedure 29 December 2011
Not approved for either general purpose or regulatory use
-------
in. inch
L liter
M molecular ion
m meter
mg milligram
min minute
mL milliliter
mm millimeter
m/z mass-to-charge ratio
N normal; gram molecular weight of solute divided by hydrogen equivalent of solute, per liter of
solution
NRR Normalized relative response
OD outside diameter
pg picogram
ppb part-per-billion
ppm part-per-million
ppq part-per-quadrillion
ppt part-per-trillion
psig pounds-per-square inch gauge
v/v volume per unit volume
w/v weight per unit volume
Definitions and acronyms (in alphabetical order)
Analyte -A perfluorinated compound tested for by this method. The analytes are listed in Table 1.
Calibration standard (CAL) - A solution prepared from a secondary standard and/or stock solution and
used to calibrate the response of the HPLC/MS/MS instrument. Referred to as CS-1, CS-2, CS-3, CS-4,
CS-5, CS-6.
Calibration verification standard (CALVER) - A calibration standard close to the mid-point calibration
standard that is used to verify calibration.
HPLC - High performance liquid chromatograph or high performance liquid chromatography
ICAL - Initial calibration
Internal standard quantitation - A means of determining the concentration of (1) a naturally occurring
(native) compound by reference to a compound other than its labeled analog and (2) a labeled compound
by reference to another labeled compound.
IPR - Initial precision and recovery; four aliquots of a reference matrix spiked with the analytes of
interest and labeled compounds and analyzed to establish the ability of the laboratory to generate
acceptable precision and recovery. An IPR is performed prior to the first time this method is used and
any time the method or instrumentation is modified.
Isotope dilution quantitation - A means of determining a naturally occurring (native) compound by
reference to the same compound in which one or more atoms has been isotopically enriched. In this
method, labeled compounds are enriched with deuterium to produce 2H-labeled analogs or carbon-13 to
produce 13C-labeled analogs. The labeled analogs are spiked into each sample to allow identification and
correction of the concentration of the native compounds in the extraction, cleanup and the analytical
process.
Draft PFC Procedure 30 December 2011
Not approved for either general purpose or regulatory use
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Labeled compound, or labeled analog - A molecule in which one or more of the atoms is isotopically
enriched, thereby increasing the mass of the molecule. As used in this procedure, all isotopic labels are
stable (non-radioactive).
Labeled injection standard - A labeled compound used as a reference for quantitation of other labeled
compounds and for quantitation of a native compound for which there is not an exact labeled analog. This
compound is spiked into the sample extract prior to instrumental analysis.
Labeled internal standard - A labeled compound used as a reference for quantitation of native
compounds. This compound is spiked into the sample prior to extraction.
Method blank - An aliquot of a reference matrix 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 method blank is used to determine if analytes or interferences are present in the laboratory
environment, the reagents, or the apparatus.
Method detection limit (MDL) - The minimum concentration of a substance that can be measured and
reported with 99% confidence that the analyte concentration is greater than zero and is determined from
analysis of a sample in a given matrix containing the analyte (see 40 CFR 136, appendix B).
Minimum level (ML) - The greater of a multiple of the MDL or the lowest calibration point (see 68 FR
11790, March 12,2003).
MS - Mass spectrometer or mass spectrometry
Native compound - A molecule in which the atoms all have naturally occurring isotopic abundances
OPR - Ongoing precision and recovery standard (OPR); an aliquot of a reference matrix spiked with
known quantities of analytes. Also known as a "laboratory control sample" (LCS). The OPR 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.
Reagent water - water demonstrated to be free from the analytes of interest and potentially interfering
substances at the method detection limit for the analyte.
Relative standard deviation (RSD) - The standard deviation times 100, divided by the mean. Also termed
the "coefficient of variation."
Relative percent difference (RPD) - The absolute difference between two values, divided by the mean of
the two values. Used to compare results when there is no true value.
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.
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. SPE is used in this procedure as a cleanup technique.
Draft PFC Procedure 31 December 2011
Not approved for either general purpose or regulatory use
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20.0 Tables and figures
Table 1. Names and CAS Registry numbers for PFCs determined by isotope dilution and internal standard
Compound
Native PFCs
Perfluoorobutanoic acid
Perfluoropentanoic acid
Perfluoro-n-hexanoic acid
Perfluoro-n-heptanoic acid
Perfluoro-n-octanoic acid
Perfluoro-n-nonanoic acid
Perfluoro-n-decanoic acid
Perfluoro-n-undecanoic acid
Perfluoro-n-dodecanoic acid
Perfluoro-n-tridecanoic acid
Perfluoro-n-tetradecanoic acid
Perfluorobutanesulfonic acid
Perfluorohexanesulfonic acid
Perfluoro-n-heptanesulfonic acid
Perfluorooctanesulfonic acid
Perfluorooctane sulfonamide
N-methylperfluoro-1-octanesulfonamide
N-ethylperfluoro- 1 -octanesulfonamide
2-(N-methylperfluoro-l-octanesulfonamido)-
ethanol
2-(N-ethylperfluoro- 1 -octanesulfonamido)-
ethanol
Labeled Internal Standards
Perfluoro-n-[l ,2,3,4-13C4]butanoic acid
Perfluoro-n-[l,2-13C2]hexanoic acid
Perfluoro-n-[l ,2,3,4-13C4]octanoic acid
Perfluoro-n-[l,2,3,4,5-13C5]nonanoic acid
Perfluoro-n-[l,2-13C2]decanoic acid
Perfluoro-n-[l,2,3,4,5,6,7,8,9-13C9]decanoic
acid
Perfluoro-n-[l ,2-13C2]undecanoic acid
Perfluoro-n-[2,3,4,5,6,7,8,9,10-13C9]undecanoic
acid
Perfluoro-n-[l ,2-13C2]dodecanoic acid
Perfluoro-l-[l,2-18O2]-hexanesulfonic acid
Perfluoro-n-[l ,2,3,4-13C4]-octanesulfonic acid
2-(N-deuteriomethylperfluoro- 1 -octane
sulfonamido )-!,!, 2 ,2-tetradeuterioethanol
Injection Internal Standards
Perfluoro-n-[l,2,3,4,5,6,7,8-13C8]octanoicacid
2H-Perfluoro-[l ,2-13C2]-2-decenoic acid
CAS
Number
375-22-4
2706-90-3
307-24-4
375-85-9
335-67-1
375-95-1
335-76-2
2058-94-8
307-55-1
72629-94-8
376-06-7
375-73-5
355-46-4
375-92-8
1763-23-1
754-91-6
31506-32-8
4151-50-2
24448-09-7
1691-99-2
Formula
CF3(CF2)2COOH
CF3(CF2)3COOH
CF3(CF2)4COOH
CF3(CF2)5COOH
CF3(CF2)6COOH
CF3(CF2)7COOH
CF3(CF2)8COOH
CF3(CF2)9COOH
CF3(CF2)10COOH
CF3(CF2)nCOOH
CF3(CF2)12COOH
CF3(CF2)3SO3H
CF3(CF2)5SO3H
CF3(CF2)6S03H
CF3(CF2)7SO3H
CF3(CF2)6S02NH2
CF3(CF2)7S02N.H.CH3
CF3(CF2)7SO2N.H.C2H5
CF3(CF2)7SO2N. CH3.C2H4OH
CF3(CF2)7S02N. C2H5.C2H4OH
13CF3(13CF2)213COOH
CF3(CF2)3(13CF2)13COOH
CF3(CF2)3(13CF2)313COOH
CF3(CF2)3(13CF2)413COOH
CF3(CF2)7(13CF2)13COOH
CF3( 13CF2)813COOH
CF3(CF2)8(13CF2)13COOH
CF3(13CF2)9COOH
CF3(CF2)9(13CF2)13COOH
CF3(CF2)5SO18O2H
CF3(CF2)3(13CF2)4S03H
CF3(CF2)7SO2N. CD3.C2D4OH
13CF3(13CF2)613COOH
CF3(CF2)6CF ' 3CH' 3COOH
Acronym
PFBA
PFPA
PFHxA (C6)
PFHpA (C7)
PFOA (C8)
PFNA (C9)
PFDA(CIO)
PFUnDA(Cll)
PFDoDA(C12)
PFTriDA(C13)
PFTeDA(C14)
PFBS (S4)
PFHxS (S6)
PFHpS (S7)
PFOS (S8)
PFOSA (S8)
N-MeFOSA
N-EtFOSA
N-MeFOSE
N-EtFOSE
[13C4]PFBA-(MPFBA)
[13C2]PFHxA-(MPFHxA)
[13C4]PFOA-(M4PFOA)
[13C5]PFNA-(MPFNA)
[13C2]PFDA-(M2PFDA)
[13C9]PFDA-(M9PFDA)
[13C2]PFUnDA-
(M2PFUnDA)
[13C9]PFUnDA-
(M9PFUnDA)
[13C2]PFDoDA-MPFDoDA)
[18O2]PFOS-(MPFHxS)
[13C4]PFOS-(MPFOS)
d7-N-MeFOSE
[13C8]PFOA-(M8PFOA)
[13C2]PFOUEA-MPFOUEA)
Draft PFC Procedure
32
Not approved for either general purpose or regulatory use
December 2011
-------
Table 2. Typical retention times (RT), relative retention times (RRT), and RT windows
Compound
Native PFCs
Perfluoorobutanoic acid
Perfluoropentanoic acid
Perfluoro-n-hexanoic acid
Perfluoro-n-heptanoic acid
Perfluoro-n-octanoic acid
Perfluoro-n-nonanoic acid
Perfluoro-n-decanoic acid
Perfluoro-n-undecanoic acid
Perfluoro-n-dodecanoic acid
Perfluoro-n-tridecanoic acid
Perfluoro-n-tetradecanoic acid
Perfluorobutanesulfonic acid
Perfluoro-n-hexane sulfonic acid
Perfluoro-n-heptane sulfonic acid
Perfluoro-n-octanesulfonic acid
Perfluorooctane sulfonamide
N-methylperfluoro-1-octanesulfonamide
N-ethylperfluoro- 1 -octanesulfonamide
2-(N-methylperfluoro- 1 -octane
sulfonamido)-ethanol
2-(N-ethylperfluoro- 1 -octane sulfonamido)-
ethanol
Mass-labeled PFCs
Perfluoro-n-[l ,2,3,4-13C4]butanoic acid
Perfluoro-n-[l,2-13C2]hexanoic acid
Perfluoro-n-[l,2,3,4-13C4]octanoic acid
Perfluoro-n-[l,2,3,4,5-13C5]nonanoic acid
Perfluoro-n-[l,2-13C2]decanoic acid
Perfluoro-n-[l,2,3,4,5,6,7,8,9-13C9]decanoic
acid
Perfluoro-n-[l ,2-13C2]undecanoic acid
Mean RT in
Calibration
Standard"
5.22
6.36
7.06
7.81
8.78
10.01
11.50
13.16
15.12
17.48
20.56
7.20
9.25
10.61
12.20
17.56
23.15
24.91
21.53
24.27
5.22
7.05
8.78
9.99
11.49
11.48
13.15
Standard
Deviation
of RT
0.025
0.016
0.016
0.014
0.026
0.036
0.026
0.030
0.032
0.038
0.072
0.018
0.019
0.032
0.047
0.032
0.063
0.039
0.057
0.034
0.028
0.011
0.026
0.038
0.033
0.034
0.029
Mean RT
in
Biosolids12'
5.24
6.37
7.07
7.82
8.80
10.05
11.48
13.02
14.99
17.46
20.64
7.21
9.25
10.65
12.15
17.52
23.07
24.95
21.36
24.22
5.20
7.07
8.80
10.03
11.48
11.48
13.06
Standard
Deviation
ofRT
0.036
0.016
0.015
0.020
0.016
0.048
0.040
0.113
0.168
0.073
0.100
0.016
0.016
0.029
0.073
0.064
0.096
0.031
0.127
0.070
0.084
0.015
0.016
0.036
0.040
0.040
0.069
RT
Window
±0.3
±0.1
±0.1
±0.1
±0.1
±0.5
±0.5
±0.5
±0.5
±0.5
±0.5
±0.1
±0.1
±0.5
±0.5
±0.5
±0.5
±0.5
±0.5
±0.5
±0.3
±0.1
±0.1
±0.1
±0.5
±0.5
±0.5
Mean RRT in
a Calibration
Standard*1'
1.000
0.725
1.001
0.891
1.000
1.001
1.002
1.001
1.000
1.993
2.344
0.821
1.001
1.210
1.002
2.002
2.640
2.841
1.009
2.767
0.595
0.804
1.001
1.139
1.310
1.309
1.499
Standard
Deviation
of RRT
0.0026
0.0018
0.0018
0.0015
0.0000
0.0027
0.0017
0.0013
0.0009
0.0043
0.0082
0.0020
0.0020
0.0037
0.0017
0.0036
0.0072
0.0045
0.0024
0.0039
0.0032
0.0012
0.0029
0.0043
0.0037
0.0039
0.0033
Mean RRT
in
Biosolids12'
1.008
0.723
1.000
0.887
1.000
1.002
1.000
1.001
1.001
1.981
2.343
0.819
1.000
1.209
1.001
1.988
2.619
2.832
1.005
2.749
0.590
0.802
0.999
1.138
1.303
1.303
1.482
Standard
deviation
RRT
0.0116
0.0018
0.0000
0.0022
0.0000
0.0039
0.0000
0.0014
0.0010
0.0083
0.0114
0.0018
0.0000
0.0032
0.0013
0.0073
0.0109
0.0036
0.0022
0.0080
0.0096
0.0018
0.0019
0.0041
0.0045
0.0045
0.0079
RRT
Window
±0.05
±0.05
±0.01
±0.05
±0.01
±0.01
±0.01
±0.01
±0.01
±0.05
±0.05
±0.05
±0.01
±0.05
±0.01
±0.05
±0.05
±0.05
±0.01
±0.05
±0.05
±0.05
±0.01
±0.05
±0.05
±0.05
±0.05
Draft PFC Procedure
33
Not approved for either general purpose or regulatory use
December 2011
-------
Table 2. Typical retention times (RT), relative retention times (RRT), and RT windows
Compound
Perfluoro-n-[2,3,4,5,6,7,8,9,10-
13C9]undecanoic acid
Perfluoro-n-[l ,2-13C2]dodecanoic acid
Perfluoro-l-[l,2-18O2]-hexanesulfonic acid
Perfluoro-n-[l,2,3,4-13C4]-octanesulfonate
2-(N-deuteriomethylperfluoro- 1 -octane
sulfonamido)-l,l,2,2-tetradeuterioethanol
Mean RT in
Calibration
Standard"
13.15
15.11
9.24
12.18
21.34
Standard
Deviation
of RT
0.031
0.025
0.021
0.032
0.071
Mean RT
in
Biosolids12'
13.01
14.98
9.25
12.14
21.26
Standard
Deviation
ofRT
0.124
0.162
0.016
0.083
0.094
RT
Window
±0.5
±0.5
±0.1
±0.5
±0.5
Mean RRT in
a Calibration
Standard*1'
1.499
1.723
1.054
1.389
2.433
Standard
Deviation
of RRT
0.0035
0.0029
0.0024
0.0036
0.0081
Mean RRT
in
Biosolids12'
1.477
1.700
1.050
1.378
2.414
Standard
deviation
RRT
0.0141
0.0184
0.0019
0.0095
0.0106
RRT
Window
±0.05
±0.05
±0.05
±0.05
±0.05
Injection Internal Standards (compound added after extraction, but prior to injection)
Perfluoro-n-[l,2,3,4,5,6,7,8-13C8]octanoic
acid
2H-Perfluoro-[l ,2-13C2]-2-decenoic acid
8.78
9.64
0.026
0.037
8.80
9.65
0.016
0.016
±0.1
±0.1
..
-
..
-
..
-
..
-
..
-
Draft PFC Procedure
34
Not approved for either general purpose or regulatory use
December 2011
-------
Table 3. Analytes, ions, quantification references, and instrument conditions
Compound
Precursor
Ion (ni/z)
Quant
Ion
(nVz)
Precursor
formula
Primary Quant
Ion formula
Cone
Voltage
(V)
Collision
(eV)
2nd Qual
Ion Mass
(m/z)
2nd Qual Ion
formula
Collision
(eV)
Quant
by:
Quantitation Reference
Native PFCs
Perfluoorobutanoic acid
Perfluoropentanoic acid
Perfluoro-n-hexanoic acid
Perfluoro-n-heptanoic acid
Perfluoro-n-octanoic acid
Perfluoro-n-nonanoic acid
Perfluoro-n-decanoic acid
Perfluoro-n-undecanoic
acid
Perfluoro-n-dodecanoic
acid
Perfluoro-n-tridecanoic
acid
Perfluoro-n-tetradecanoic
acid
Perfluorobutanesulfonic
acid
Perfluoro-n-hexane
sulfonic acid '
Perfluoro-n-heptane
sulfonic acid
Perfluoro-n-octanesulfonic
acid
Perfluorooctane
sulfonamide
N-methylperfluoro- 1 -
octanesulfonamide
N-ethylperfluoro- 1 -
octanesulfonamide
2-(N-methylperfluoro- 1 -
octanesulfonamido)-ethanol
2-(N-ethylperfluoro- 1 -
octanesulfonamido)-ethanol
213
263
313
363
413
463
513
563
613
663
713
299
399
449
499
498
512
526
616
630
169
219
269
319
369
419
469
519
569
619
669
80
80
80
80
78
169
169
59
59
[CF3(CF2)2C02]-
[CF3(CF2)3C02]-
[CF3(CF2)4C02]-
[CF3(CF2)5C02]-
[CF3(CF2)6C02]-
[CF3(CF2)7C02]-
[CF3(CF2)8C02]-
[CF3(CF2)9C02]-
[CF3(CF2)10C02]-
[CF3(CF2)nC02]-
[CF3(CF2)12C02]-
[CF3(CF2)3S03]-
[CF3(CF2)5S03]-
[CF3(CF2)6S03]-
[CF3(CF2)7S03]-
[CF3(CF2)7SO2N
H]-
[CF3(CF2)7S02
N(CH3) ]-
[CF3(CF2)7S02
N(C2H5)]-
[CF3(CF2)7SO2N(
CH3)C2H,OH-CH
3coj-
[CF3(CF2)7S02N(
C2H5)C2Fl4OH-C
H3CO2]'
[CF3(CF2)2]-
[CF3(CF2)3]-
[CF3(CF2)4]-
[CF3(CF2)5]-
[CF3(CF2)6]-
[CF3(CF2)7]-
[CF3(CF2)8]-
[CF3(CF2)9]-
[CF3(CF2)10]-
[CF3(CF2)U]-
[CF3(CF2)12]-
[SOj]'
[S03]-
[SOJ-
[S03]-
[S02N]-
[CF3(CF2)2]-
[CF3(CF2)2]-
[CH3CO2]-
[CH3CO2]-
27
27
27
27
19
20
21
21
22
20
27
70
30
50
80
80
27
27
27
27
8
8
20
12
12
13
11
15
15
17
21
40
45
39
45
40
45
45
45
45
119
169
169
219
219
269
319
319
319
99
99
99
99
478
[CF3CF2]'
[CF3(CF2)2]-
[CF3(CF2)2]-
[CF3(CF2)3]-
[CF3(CF2)3]-
[CF3(CF2)4]-
[CF3(CF2)5]-
[CF3(CF2)5]-
[CF3(CF2)5]-
[FSOJ-
[FSOJ-
[FSOJ-
[FSOJ-
[(CF2)8S02N]-
8
8
12
12
13
12
12
13
11
35
40
38
40
16
ID
IS
ID
IS
ID
ID
ID
ID
ID
IS
IS
IS
ID
IS
ID
IS
IS
IS
ID
IS
13CF3(13CF2)213COOH
13CF3(13CF2)613COOH
CF3(CF2)3(13CF2)13COOH
13CF3(13CF2)613COOH
CF3(CF2)3(13CF2)3
"COOH
CF3(CF2)3(13CF2)4
"COOH
CF3( 13CF2)813COOH
CF3( 13CF2)9COOH
CF3(CF2)9( 13CF2)13COOH
13CF3(13CF2)613COOH
13CF3(13CF2)613COOH
13CF3(13CF2)613COOH
CF3(CF2)5S(18O)2OH
13CF3(13CF2)613COOH
CF3(CF2)3(13CF2)4SO3H
13CF3(13CF2)613COOH
13CF3(13CF2)613COOH
13CF3(13CF2)613COOH
CF3(CF2)7SO2N
(CD3)C2D4OH-CH3COOH
13CF3(13CF2)613COOH
Draft PFC Procedure
35
Not approved for either general purpose or regulatory use
December 2011
-------
Table 3. Analytes, ions, quantification references, and instrument conditions
Compound
Precursor
Ion (m/z)
Quant
Ion
(m/z)
Precursor
formula
Primary Quant
Ion formula
Cone
Voltage
(V)
Collision
(eV)
2nd Qual
Ion Mass
(m/z)
2nd Qual Ion
formula
Collision
(eV)
Quant
by:
Quantitation Reference
Mass-labeled PFCs
Perfluoro-n-[l,2,3,4-
13C4]butanoic acid
Perfluoro-n-[l,2-
13C2]hexanoic acid
Perfluoro-n-[l,2,3,4-13
C4]octanoic acid
Perfluoro-n-[l,2,3,4,5-13
C5]nonanoic acid
Perfluoro-n-[l,2-
13C2]decanoic acid
Perfluoro-n-
[1,2,3,4,5,6,7,8,9-
13C9]decanoic acid
Perfluoro-n-[l,2-
13C2]undecanoic acid
Perfluoro-n-
[2,3,4,5,6,7,8,9,10-
13C9]undecanoic acid
Perfluoro-n-[l,2-
13C2]dodecanoic acid
Perfluoro-l-[l,2-18 O2]-
hexanesulfonic acid
Perfluoro-n-[l,2,3,4-13 C4]-
octanesulfonate
2-(N-
deuteriomethylperfluoro- 1 -
octanesulfonamido)-
1 , 1 ,2,2-tetradeuterioethanol
217
315
417
468
515
522
565
572
615
403
503
623
172
270
372
423
470
477
520
528
570
84
80
59
[13CF3(13CF2)2
"coj-
[CF3(CF2)3(13CF2)
13coj-
[CF3(CF2)3
(13CF2)313CO2]'
[CF3(CF2)3
(13CF2)413CO2]-
[CF3(CF2)7(13CF2)
"coj-
[CF3(13CF2)8
13coj-
[CF3(CF2)8(13CF2)
"coj-
[CF3(
13CF2)9C02]-
[CF3(CF2)9
(13CF2)13CO2]-
[CF3(CF2)5
S(18O)2O]-
[CF3(CF2)3
(13CF2)4S03]-
[CF3(CF2)7SO2N
(CD3)C2D4OH-C
H3CO2]'
[13CF3(13CF2)2]-
[CF3(CF2)3(13CF
2)]-
[CF3(CF2)3
(13CF2)3]-
[CF3(CF2)3(13CF
2)4]-
[CF3(CF2)2(13CF
2)]'
[CF3(13CF2)8]-
[CF3(CF2)8(13CF
2)]'
[CF3(13CF2)9]-
[CF3(CF2)9
(13CF2)]-
[S(180)20]-
[S03]-
[CH3CO2]-
27
27
21
20
21
20
20
20
22
30
40
27
8
8
12
12
12
12
12
12
12
45
45
45
103
99
[FS(18O)2O]-
[FSOJ-
45
40
IS
IS
IS
IS
IS
IS
IS
IS
IS
IS
IS
IS
13CF3(13CF2)613COOH
13CF3(13CF2)613COOH
13CF3(13CF2)613COOH
13CF3(13CF2)613COOH
13CF3(13CF2)613COOH
13CF3(13CF2)613COOH
13CF3(13CF2)613COOH
13CF3(13CF2)613COOH
13CF3(13CF2)613COOH
13CF3(13CF2)613COOH
13CF3(13CF2)613COOH
13CF3(13CF2)613COOH
Injection Internal Standards (compound added after extraction, but prior to injection)
Perfluoro-n-
[1,2,3,4,5,6,7,8-13
C8]octanoic acid
2H-Perfluoro-[l,2-13C2]-2-
decenoic acid
421
459
376
394
[13CF3( 13CF2)6
13coj-
[CF3(CF2)6
CF13CH13CO2]-
[13CF3(3CF2)6]-
[CF3(CF2)3
(13CF2)3]-
21
21
12
11
ES
ES
' This analyte has a third ion that can be used for qualitative identification. The m/z of that ion is 119, the formula is CF3CF2-, and the collision energy is 30 eV.
Draft PFC Procedure
36
Not approved for either general purpose or regulatory use
December 2011
-------
Table 4. Method detection limits, minimum levels of quantitation, and provisional health advisory
Compound
MDL (ng/g)
ML (ng/g)
Perfluoorobutanoic acid
Perfluoropentanoic acid
Perfluoro-n-hexanoic acid
Perfluoro-n-heptanoic acid
Perfluoro-n-octanoic acid
Perfluoro-n-nonanoic acid
Perfluoro-n-decanoic acid
Perfluoro-n-undecanoic acid
Perfluoro-n-dodecanoic acid
Perfluoro-n-tridecanoic acid
Perfluoro-n-tetradecanoic acid
Perfluorobutanesulfonic acid
Perfluorohexanesulfonic acid
Perfluoroheptanesulfonic acid
Perfluorooctanesulfonic acid
Perfluorooctane sulfonamide
N-methylperfluoro-1-octanesulfonamide
N-ethy Iperfluoro - 1 -octanesulfonamide
2-(N-methylperfluoro-l-octanesulfonamido)-ethanol
2-(N-ethylperfluoro- 1 -octanesulfonamido)-ethanol
0.125
0.185
0.136
0.054
0.085
0.127
0.084
0.080
0.067
0.067
0.028
0.115
0.268
0.291
0.208
0.040
3.65
3.05
7.65
1.88
0.250
0.250
0.250
0.250
0.250
0.250
0.250
0.250
0.250
0.250
0.250
0.500
0.500
0.500
0.500
0.250
10.0
10.0
5.00
5.00
OW Provisional Health
Advisory
0.4 ppb
0.2 ppb
Draft PFC Procedure
37
Not approved for either general purpose or regulatory use
December 2011
-------
Table 5. Instrument conditions
Instruments
Trapping cartridge
HPLC Column
lonization
Acquisition
Injection Volume
Final Extract Volume
HPLC Gradient Program
Time
(min)
0.00
1.00
5.00
20.00
23.00
26.00
26.50
30.00
32.00
Flow mixture
A=15%
B=85%
A=15%
B=85%
A=50%
B=50%
A=65%
B=35%
A=100%
B=0%
A=100%
B=0%
A=15%
B=85%
A=15%
B=85%
A=15%
B=85%
Waters Acquity high performance liquid chromatography (HPLC) or equivalent
Waters Quattro Ultima tandem mass spectrometer or equivalent
NA
Waters Xtera C18MS analytical column, 100 mm length, 2. 1 mm ID, 3
particle size, or equivalent
.5 um
Negative Ion Electrospray
MRM mode, unit resolution
15 uL
ImL
HPLC Flow
rate
(mL/min)
0.150
0.150
0.200
0.200
0.200
0.200
0.200
0.200
0.150
Gradient
Curve
1
1
4
4
4
4
2
2
2
HPLC conditions
Column Temp (°C)
Max Pressure (bar)
MS Conditions
Source Temp (°C)
Desolvation Temp (°C)
Capillary voltage (kV)
40
345
120
325
3.50
Solvent A = 90% ACN: 10% water (organic phase)
Solvent B = 12.1 mM ammonium acetate and 0.1% acetic acid in water (aqueous phase)
Draft PFC Procedure
38
Not approved for either general purpose or regulatory use
December 2011
-------
Table 6. Concentrations of calibration standards (CS) in pg/mL
Native Analytes
Perfluoorobutanoic acid
Perfluoropentanoic acid
Perfluoro-n-hexanoic acid
Perfluoro-n-heptanoic acid
Perfluoro-n-octanoic acid
Perfluoro-n-nonanoic acid
Perfluoro-n-decanoic acid
Perfluoro-n-undecanoic acid
Perfluoro-n-dodecanoic acid
Perfluoro-n-tridecanoic acid
Perfluoro-n-tetradecanoic acid
Perfluorobutanesulfonic acid
Perfluorohexanesulfonic acid
Perfluoro-n-heptanesulfonic acid
Perfluorooctanesulfonic acid
Perfluorooctane sulfonamide
N-methylperfluoro-1-octanesulfonamide
N-ethylperfluoro- 1 -octanesulfonamide
2-(N-methylperfluoro-l-octanesulfonamido)-ethanol
2-(N-ethylperfluoro- 1 -octanesulfonamido)-ethanol
Labeled Internal Standards
Perfluoro-n-[l ,2,3,4-13C4]butanoic acid
Perfluoro-n-[l,2-13C2]hexanoic acid
Perfluoro-n-[l,2,3,4-13C4]octanoic acid
Perfluoro-n-[l,2,3,4,5-13C5]nonanoic acid
Perfluoro-n-[l,2-13C2]decanoic acid
Perfluoro-n-[l,2,3,4,5,6,7,8,9-13C9]decanoicacid
Perfluoro-n-[l ,2-13C2]undecanoic acid
Perfluoro-n-[2,3,4,5,6,7,8,9,10-13C9]undecanoicacid
Perfluoro-n-[l,2- C2]dodecanoic acid
Perfluoro-l-[l,2-18O2]-hexanesulfonic acid
Perfluoro-n-[l,2,3,4- C4]-octanesulfonic acid
2-(N-deuteriomethylperfluoro-l-octanesulfonamido )-!,!, 2,2-
tetradeuterioethanol
Internal Injection Standards
Perfluoro-n-[l,2,3,4,5,6,7,8-13C8]octanoicacid
2H-Perfluoro-[l ,2-13C2]-2-decenoic acid
CS-1
125
125
125
125
125
125
125
125
125
125
125
250
250
250
250
125
5,000
5,000
2,500
2,500
3,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
4,270
4,310
20,000
3,000
2,500
CS-2
312.5
312.5
312.5
312.5
312.5
312.5
312.5
312.5
312.5
312.5
312.5
625
625
625
625
312.5
10,000
10,000
5,000
5,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
4,270
4,310
20,000
3,000
2,500
CS-3
1,250
1,250
1,250
1,250
1,250
1,250
1,250
1,250
1,250
1,250
1,250
2,500
2,500
2,500
2,500
1,250
20,000
20,000
10,000
10,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
4,270
4,310
20,000
3,000
2,500
CS-4 (VER)
5,000
5,000
5,000
5,000
5,000
5,000
5,000
5,000
5,000
5,000
5,000
10,000
10,000
10,000
10,000
5,000
40,000
40,000
20,000
20,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
4,270
4,310
20,000
3,000
2,500
CS-5
25,000
25,000
25,000
25,000
25,000
25,000
25,000
25,000
25,000
25,000
25,000
50,000
50,000
50,000
50,000
25,000
80,000
80,000
40,000
40,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
4,270
4,310
20,000
3,000
2,500
CS-6
50,000
50,000
50,000
50,000
50,000
50,000
50,000
50,000
50,000
50,000
50,000
100,000
100,000
100,000
100,000
50,000
160,000
160,000
80,000
80,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
4,270
4,310
20,000
3,000
2,500
CS-7
125,000
125,000
125,000
125,000
125,000
125,000
125,000
125,000
125,000
125,000
125,000
250,000
250,000
250,000
250,000
125,000
320,000
320,000
160,000
160,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
4,270
4,310
20,000
3,000
2,500
CS-8
312,500
312,500
312,500
312,500
312,500
312,500
312,500
312,500
312,500
312,500
312,500
625,000
625,000
625,000
625,000
312,500
—
—
-
—
3,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
3,000
4,270
4,310
3,000
2,500
1 Calibration standards are stored in ~ 92% MeOH, up to 2.75% Nonane, 2% Propan-2-ol, !%MeCN, 1% H2O, up to 0.3% Toluene, 0.3% NH4OH solution.
Draft PFC Procedure
39
Not approved for either general purpose or regulatory use
December 2011
-------
Table 7. Detailed concentrations of actual working level standards (pg/mL)
Working-level Standards (pg/mL)
Native standards
Perfluoorobutanoic acid
Perfluoropentanoic acid
Perfluoro-n-hexanoic acid
Perfluoro-n-heptanoic acid
Perfluoro-n-octanoic acid
Perfluoro-n-nonanoic acid
Perfluoro-n-decanoic acid
Perfluoro-n-undecanoic acid
Perfluoro-n-dodecanoic acid
Perfluoro-n-tridecanoic acid
Perfluoro-n-tetradecanoic acid
Perfluorobutanesulfonic acid
Perfluorohexanesulfonic acid
Perfluoro-n-heptanesulfonic acid
Perfluorooctanesulfonic acid
Perfluorooctane sulfonamide
N-Me/Et-FOSA/Es Natives (NAT2)
N-methylperfluoro- 1 -octanesulfonamide
N-ethylperfluoro- 1 -octanesulfonamide
2 -(N-methylperfluoro- 1 -octanesulfonamido)-ethanol
2-(N-ethylperfluoro-l-octanesulfonamido)-ethanol
Labeled Internal Standard Solution
Mass Labeled PFCs
Perfluoro-n-[ 1 ,2,3 ,4-: 3C4]butanoic acid
Perfluoro-n-[l,2-13C2]hexanoic acid
Perfluoro-n-[l,2,3,4-13 C4]octanoic acid
Perfluoro-n-[l,2,3,4,5-13 C5]nonanoic acid
Perfluoro-n-[l,2-13C2]decanoic acid
Perfluoro-n-[l,2-13C9]decanoic acid
Perfluoro-n-[ 1 ,2- 3C2]undecanoic acid
Perfluoro-n-[ 1 ,2- 3C9]undecanoic acid
Perfluoro-n-[l,2-13C2]dodecanoic acid
Perfluoro-l-[l,2-18 O2]-hexanesulfonic acid
Perfluoro-n-[l,2,3,4-13 C4]-octanesulfonic acid
2-(N-deuteriomethylperfluoro-l-octanesulfonamido)-l,l,2,2-tetradeuterioethanol
Labeled Injection Standards
Perfluoro-n-[l,2,3,4,5,6,7,8-13 C8]octanoic acid
2H-Perfluoro-[l,2-13 C2]-2-decenoic acid
Working level
125,000
125,000
125,000
125,000
125,000
125,000
125,000
125,000
125,000
125,000
125,000
250,000
250,000
250,000
250,000
125,000
2,500,000
2,500,000
1,250,000
1,250,000
30,000
30,000
30,000
30,000
30,000
30,000
30,000
30,000
30,000
42,700
43,100
200,000
12,000
10,000
Low level
12,500
12,500
12,500
12,500
12,500
12,500
12,500
12,500
12,500
12,500
12,500
25,000
25,000
25,000
25,000
12,500
Draft PFC Procedure
40
Not approved for either general purpose or regulatory use
December 2011
-------
Table 8. QC acceptance criteria for analytes, labeled compounds and internal standards in VER, IPR, OPR, matrix spikes, and samples
Compound
Quant
ICAL and CALVER
Recovery (%)
Low
High
RSD
IPR Recovery (%)
Low
High
RSD
OPR
Recovery (%)
Low
High
RLS Recovery
(%)
Low
High
Recovery in Matrix
Spikes and Samples
Low
High
RPD
Native PFCs
Perfluoorobutanoic acid
Perfluoropentanoic acid
Perfluoro-n-hexanoic acid
Perfluoro-n-heptanoic acid
Perfluoro-n-octanoic acid
Perfluoro-n-nonanoic acid
Perfluoro-n-decanoic acid
Perfluoro-n-undecanoic acid
Perfluoro-n-dodecanoic acid
Perfluoro-n-tridecanoic acid
Perfluoro-n-tetradecanoic acid
Perfluorobutanesulfonic acid
Perfluorohexanesulfonic acid
Perfluoroheptanesulfonic acid
Perfluorooctanesulfonic acid
Perfluorooctane sulfonamide
N-methylperfluoro- 1 -octanesulfonamide
N-ethylperfluoro- 1 -octanesulfonamide
2-(N-methylperfluoro-l-octanesulfonamido)-ethanol
2-(N-ethylperfluoro-l-octanesulfonamido)-ethanol
ID
IS
ID
IS
ID
ID
ID
ID
ID
IS
IS
IS
IS
ID
ID
IS
IS
IS
ID
IS
70
70
70
70
70
70
70
70
70
70
70
70
70
70
70
70
70
70
70
70
130
130
130
130
130
130
130
130
130
130
130
130
130
130
130
130
130
130
130
130
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
70
70
70
70
70
50
70
70
70
20
20
60
70
70
70
20
5
5
40
10
130
130
130
130
130
150
130
130
130
130
130
130
130
130
130
130
130
130
130
130
20
20
20
20
20
30
20
20
20
30
30
20
20
20
20
30
20
40
30
40
70
70
70
70
70
50
70
70
70
20
20
60
70
70
70
20
5
5
40
10
130
130
130
130
130
150
130
130
130
130
130
130
130
130
130
130
130
130
130
130
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
10
10
50
25
200
200
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
5
5
50
10
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
40
40
30
40
Labeled Internal Standards: Compounds added before extraction
Perfluoro-n-[l ,2,3,4-13C4]butanoic acid
Perfluoro-n-[l ,2-13C2]hexanoic acid
Perfluoro-n-[l ,2,3,4-13C4]octanoic acid
Perfluoro-n-[l,2,3,4,5-13C5]nonanoic acid
Perfluoro-n-[l ,2,3,4,5,6,7,8,9-13C9]decanoic acid
Perfluoro-n-[l ,2,3,4,5,6,7,8,9-13C9]undecanoic acid
Perfluoro-n-[l ,2-13C2]dodecanoic acid
Perfluoro-n-[l ,2,3,4-13C4]-octanesulfonic acid
Perfluoro - 1 - [ ' 8O2] -hexanesulfonic acid
2-(N-deuteriomethylperfluoro-l -octane sulfonamido)-
1 ,1 ,2,2-tetradeuterioethanol
IS
IS
IS
IS
IS
IS
IS
IS
IS
IS
70
70
70
70
70
70
70
70
70
70
130
130
130
130
130
130
130
130
130
130
20
20
20
20
20
20
20
20
20
20
60
70
60
50
60
60
30
60
60
5
130
130
130
130
130
130
130
130
130
130
20
20
20
25
20
20
20
20
20
70
60
70
60
50
60
60
30
60
60
5
130
130
130
130
130
130
130
130
130
130
60
70
60
50
60
60
30
60
60
5
130
130
130
130
130
130
130
130
130
130
60
70
60
50
60
60
30
60
60
5
130
130
130
130
130
130
130
130
130
130
20
20
20
25
20
20
20
20
20
70
Injection Internal Standard: Compound added after extraction, but prior to injection
Perfluoro-n-[l ,2,3,4,5,6,7,8-13C8]octanoic acid
ES
80
120
10
80
120
10
80
120
80
120
80
120
10
Quantitation methods:
Draft PFC Procedure
ID = Isotope dilution IS = Internal standard ES = External standard
41
Not approved for either general purpose or regulatory use
December 2011
-------
Table 9. Sources used for standards, materials, and equipment during method development1
Native Compounds
Perfluoorobutanoic acid
Perfluoropentanoic acid
Perfluoro-n-hexanoic acid
Perfluoro-n-heptanoic acid
Perfluoro-n-octanoic acid
Perfluoro-n-nonanoic acid
Perfluoro-n-decanoic acid
Perfluoro-n-undecanoic acid
Perfluoro-n-dodecanoic acid
Perfluoro-n-tridecanoic acid
Perfluoro-n-tetradecanoic acid
Perfluorobutanesulfonic acid (Tetrabutylammonium salt)
Perfluorohexanesulfonic acid (Potassium salt)
Perfluoro-n-heptanesulfonic acid (Sodium salt)
Perfluorooctanesulfonic acid (Potassium salt)
Perfluorooctane sulfonamide
N-methylperfluoro- 1 -octanesulfonamide
N-ethylperfluoro- 1 -octanesulfonamide
2-(N-methylperfluoro-l-octanesulfonamido)-ethanol
2-(N-ethylperfluoro-l-octanesulfonamido)-ethanol
Vendor
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Wellington Labs
Sigma-Aldrich
Chiron
Wellington Labs
Wellington Labs
Wellington Labs
Wellington Labs
Part Number
164194
77285
29226
34,204-1
17,146-8
77284
17,774-1
446777
40,644-9
654973
446785
86909
50929
L-PFHpS
77282
2043.8
N-MeFOSA
N-EtFOSA
N-MeFOSE
N-EtFOSE
Shelf Life
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
Labeled Internal Standards
Perfluoro-n-[l ,2,3,4-13C4]butanoic acid
Perfluoro-n-[l ,2-13C2]hexanoic acid
Perfluoro-n-[l ,2,3,4-13C4]octanoic acid
Perfluoro-n-[l ,2,3,4,5-13C5]nonanoic acid
Perfluoro-n-[l ,2-13C2]decanoic acid
Perfluoro-n-[l ,2,3,4,5,6,7,8,9-13C9]decanoic acid
Perfluoro-n-[l ,2-13C2]undecanoic acid
Perfluoro-n-[2,3,4,5,6,7,8,9,10-13C9]undecanoicacid
Perfluoro-n-[l ,2-13C2]dodecanoic acid
Perfluoro-l-[l ,2-18O2]-hexanesulfonic acid
Perfluoro-n-[l ,2,3,4-13C4]-octanesulfonic acid
2-(N-deuteriomethylperfluoro-l-octanesulfonamido )-!,!, 2,2-
tetradeuterioethanol
Wellington Labs
Wellington Labs
Wellington Labs
Wellington Labs
Wellington Labs
CIL
Wellington Labs
CIL
Wellington Labs
Wellington Labs
Wellington Labs
Wellington Labs
MPFBA
MPFHxA
MPFOA
MPFNA
MPFDA
CLM-8172-S
MPFUdA
CLM-8240
MPFDoA
MPFHxS
MPFOS
d7-N-MeFOSE-M
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
To be determined
Injection Internal Standards
Perfluoro-n-[l,2,3,4,5,6,7,8-13 C8]octanoic acid
2H-Perfluoro-[l ,2-13C2]-2-decenoic acid
CIL
Wellington Labs
CLM-8005-S
MFOUEA
To be determined
To be determined
1 Provided for informational purposes only. Part numbers subject to change. Other suppliers may have equivalent materials.
Draft PFC Procedure
42
Not approved for either general purpose or regulatory use
December 2011
-------
Table 10. Performance data from single-laboratory validation
Compound
Native PFCs
Perfluorobutanoic acid
Perfluoropentanoic acid
Perfluoro-n-hexanoic acid
Perfluoro-n-heptanoic acid
Perfluoro-n-octanoic acid
Perfluoro-n-octanoic acid
Perfluoro-n-nonanoic acid
Perfluoro-n-decanoic acid
Perfluoro-n-undecanoic acid
Perfluoro-n-dodecanoic acid
Perfluoro-n-tridecanoic acid
Perfluoro-n-tetradecanoic acid
Perfluorobutanesulfonic acid
Perfluorobutanesulfonic acid
Perfluoro-n-hexane sulfonate
Perfluoro-n-hexane sulfonate
Perfluoro-n-heptane sulfonate
Perfluoro-n-heptane sulfonate
Perfluoro-n-octanesulfonate
Perfluoro-n-octanesulfonate
Perfluorooctane sulfonamide
N-methylperfluoro-1-octane sulfonamide
N-ethylperfluoro- 1 -octanesulfonamide
2-(N-methylperfluoro-l -octane sulfonamido)-
ethanol
2-(N-ethylperfluoro- 1 -octane sulfonamido)-
ethanol
Labeled Internal Standards
Perfluoro-n-[l ,2,3,4-13C4]butanoic acid
Perfluoro-n-[l,2-13C2]hexanoic acid
Perfluoro-n-[l,2,3,4-13C4]octanoic acid
Perfluoro-n-[l,2,3,4,5-13C5] nonanoic acid
Perfluoro-n-[l,2,3,4,5,6,7,8,9-13C9] decanoic
acid
Perfluoro-n-[2,3,4,5,6,7,8,9,10-13C9]
undecanoic acid
Perfluoro-n-[l ,2-13C2]dodecanoic acid
Perfluoro-n-[l ,2,3,4-13C4]-octane sulfonic
acid
Perfluoro-l-[l,2-18O2]-hexane sulfonic acid
2-(N-deuteriomethylperfluoro- 1 -octanesulfon
amido)-! ,1 ,2,2-tetradeuterioethanol
Injection Internal Standards
Perfmoro-n-[l,2,3,4,5,6,7,8-13C8]octanoic
acid
Transition
Primary
Primary
Primary
Primary
Primary
Secondary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Secondary
Primary
Secondary
Primary
Secondary
Primary
Secondary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
Primary
S
Mean
%Rec
105
114
116
95.6
108
109
102
101
101
106
41.3
48.6
84.0
86.3
101
105
83.7
93.5
98.1
102
39.5
9.33
10.1
87.6
21.8
95.2
98.9
90.4
85.6
85.2
76.7
53.5
76.9
76.8
18.3
108
.olid IPR, n=
Standard
Deviation
1.61
4.46
2.28
3.25
5.59
8.65
13.1
2.37
2.20
2.64
2.41
4.52
5.09
5.55
2.18
4.18
14.6
4.95
5.64
4.30
3.69
0.542
0.939
10.6
1.37
6.84
2.55
6.62
8.35
4.61
0.853
3.50
4.82
3.33
3.32
11.0
4
%RSD
1.54
3.81
1.98
3.40
5.20
7.96
12.7
2.34
2.18
2.48
5.83
9.30
6.07
6.43
2.16
4.00
17.4
5.30
5.75
4.21
9.33
5.81
9.31
12.1
6.27
7.19
2.58
7.32
9.75
5.41
1.11
6.54
6.27
4.33
18.2
10.2
Mean
%Rec
95.8
56.4
115
91.1
111
119
97.3
111
104
109
126
131
69.0
69.9
105
107
90.2
92.7
97.3
99.6
50.7
48.8
14.6
96.9
50.3
81.6
72.0
96.1
92.5
107
80.3
109
78.8
79.4
45.5
84.5
Sludge, n=6
Standard
Deviation
9.55
31.4
9.83
16.3
7.57
14.9
16.8
8.41
7.35
6.50
29.1
16.1
17.1
18.8
11.0
11.5
7.72
5.28
8.36
7.32
3.63
13.3
3.75
8.20
13.6
37.5
18.2
5.03
8.80
11.1
28.9
13.1
7.97
7.73
13.0
7.42
%RSD
9.97
55.6
8.52
17.9
6.83
12.6
17.2
7.59
7.09
5.96
23.1
12.2
24.7
26.9
10.5
10.8
8.57
5.70
8.59
7.35
7.15
27.2
25.7
8.46
27.0
45.9
25.3
5.23
9.52
10.4
36.0
12.0
10.1
9.74
28.6
8.79
Draft PFC Procedure
43
Not approved for either general purpose or regulatory use
December 2011
-------
Determine % Solids
(Sec. 11.2)
Cleanup
Instrumental
Digestion and
Extraction
11.3.1 Place 0.5 g of wet
sludge in a 16-mL
polypropylene centrifuge
tube
i
r
11.3.5. Spike samples with
labeled compounds
.
.
11.3.6-11.3.7
Digestion with 0. 5 mL 1M
NaOH (1/2 hr sonication
with heat) overnight
incubation.
.
.
1 1.3.8 Neutralize with HC1
.
11.4.1. A
50:50/ACN
.
.
dd 10 mL
MeOH (v/v)
.
11.4.2 Shake moderately
forlh
.
.
11.4.3 Centrifuge at 3000 x
g for 20 min
i
r
11.4.4. Decant 10 mL
supernatant and collect in
250-mL HOPE bottle
containing 1 80 mL of
reagent water
i
r
11. 4.5. Collect 20 mL total
in 250-mL HOPE bottle
containing 180 mL reagent
water
4 1
11.4.5 Repeated once
11.
Adjust pH of
acid or 0.30/
nee
i
4.6
diluted extract
/ith 3% acetic
NH4OH,as
ded
r
11.4.7. Sonication
(30 min)
1
11.5. WAX Ca
1) Condition
with 5 mL
MeOH and
formic ack
2) Equilibrate
reagent wa
3) Load samp
mL/min)
4) Wash with
MeOH in f
formic ack
5) Dry SPE ft
vacuum
6) Elute with
NH4OHin
i
11.6.1 Red
dryness (—50
and water b
r
rtridge Cleanup
SPE cartridge
0.3%NH4OHin
SmLO.lM
1
with 5mL
ter
le drop- wise (~5
5 mL 20%
0%0.1M
1
>r 5 min under
4 mL 0.3%
MeOH
r
uce to near
ath at 40°C
11.6.2. Reconstitute
with 938 uL of 0.3%
NfttOH in MeOH and
vortex mix
1 '
11.6.3 Spike 12.5 uL
of internal standard
and vortex mix
^ '
11.6.3 Filter extract
using 0.45-um nylon
membranes filter
1 '
14.0. LC/MS/MS
analysis of 20 uL of
extract
15.0. Quantify target
PFCs
Figure 1. Flow chart for determination of PFCs by LC/MS/MS
Draft PFC Procedure
44
Not approved for either general purpose or regulatory use
December 2011
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0 ^
<
Q
§
t
Q_
<
LL
Q_
HI
CO
O
HI
8
UJ
^^ Time
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
Figure 2. Example Chromatogram for CS-4 Calibration Standard
Draft PFC Procedure
45
Not approved for either general purpose or regulatory use
December 2011
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APPENDIX 1
Details on Preparation of Standards
Draft PFC Procedure 46 December 2011
Not approved for either general purpose or regulatory use
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Appendix 1. Details on Preparation of Standards
Individual PFCs are used to make standard solutions. From solids - native PFBS, PFHxS, and PFOS were
obtained as sodium, potassium or tetrabutylammonium salts - all others were acids.
[13C9]PFUnDA was obtained as a solid.
Native PFHpS and labeled compounds except [13C9]PFUnDA, were obtained as 50(ig/mL solutions in
methanol; 1.2 mL ampule. The PFHpS was a solution of its sodium salt.
Native N-Me/Et-FOSA/Es were obtained as 50(ig/mL solutions in 90% Nonane/ 10% Toluene.
1. Use Fisher brand, Plasma grade water (has 18 MQ specification and a bottle was proofed for PFC
compounds of interest).
2. Prepare 60/40 (v/v) acetonitrile/ plasma grade water mix and 99% methanol / 0.7% water / 0.3%
NF^OH (basic methanol) mixes and store in dedicated 3X methanol-washed 1L HDPE solvent
bottles.
3. Prepare stock solutions of individual native compounds in methanol at ~ 20-60 mg/mL by weighing
20 - 60 mg into a 10 mL glass grade A volumetric flask using a 4 place balance, then diluting to mark
with methanol. For carboxylic acids, 4 mole equivalents of NaOH in methanol was added to reduce
esterification in accordance with purchased standards from Wellington. The same procedure was used
for [13C9]PFUnDA except 2 mg in a 5 mL flask was used. PFTeDA is much less soluble than the
other compounds so a solution is prepared by weighing 10 mg into a 50 mL volumetric flask and
diluting with acetonitrile without NaOH added.
Note that PFTeDA, as well as being difficult to dissolve may also be much more likely to stick to
glass, so the flask used to prepare it should receive more extensive cleaning after use than other
glassware. Sonication for 20 minutes in basic Methanol followed by regular cleaning is
recommended. Similar precautions for PFTriDA are also recommended.
4. Clean glass syringes by rinsing with 20X Toluene, 20X Hexane then sonicating for 20 minutes in
80:20 Toluene/Acetone then rinsing with 3X basic Methanol, 3X Toluene, 3X Hexane.
Clean class A volumetric flasks rinsing with 3X Toluene, 3X Hexane then sonicating for 20 minutes
in 80:20 Toluene/Acetone, then rinsing with 3X Toluene, 3X Hexane, 3X Dichloromethane, then a
final rinse before use with the dilution solvent being used for the standard.
Prepare Stock Mass-Labeled internal standard mix - does not include injection internal standard
compounds.
Calculate the volumes required of each labeled standard to give a concentration of 600 ng/mL for
each carboxylic acid and 900 ng/mL for each sulfonic acid.
Using a cleaned glass syringe that is reserved for mass labeled standards, transfer and combine the
required measured quantities of each labeled standard into a cleaned volumetric flask. Dilute to the
mark with the prepared 60:40 acetonitrile/water mix.
Prepare Labeled Internal Standard Solution (LINT)
4.1 Objective: prepare a solution of 30-45 pg/(iL for the labeled carboxylic and sulfonic
acids, plus 200 pg/(iL of d7-N-MeFOSE, in basic methanol.
4.2 Using cleaned syringes and volumetric flasks transfer a portion of the Stock Mass-
Labeled internal standard mix so that it will be diluted 20X, plus a portion of the 50
(ig/mL d7-N-MeFOSE so that it will be diluted 200X and dilute to mark with the prepared
basic methanol mix.
Draft PFC Procedure 47 December 2011
Not approved for either general purpose or regulatory use
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Prepare Stock Mass-Labeled Injection Internal Standard.
(Can be used as Injection Internal Standard or to prepare a more diluted INJ.)
Calculate the volumes required of each labeled standard to give a concentration of 240 ng/mL 13C8-
Perfluorooctanoic acid and 200 ng/mL 2H-Perfluoro-[l,2-13C2]-2-decenoic acid.
Using a cleaned glass syringe that is reserved for mass labeled standards, transfer and combine the
required measured quantities of each labeled standard into the cleaned volumetric flask. Dilute to the
mark with the prepared 60:40 acetonitrile/water mix.
Prepare Mass-Labeled injection standard
4.3 Objective: prepare a solution of 10-12 ng/mL in basic methanol.
4.4 Using cleaned syringes and volumetric flasks transfer a portion of the Stock Mass-
Labeled internal standard mix and dilute to mark with the prepared basic methanol mix so
that it is diluted 20X.
Prepare Intermediate Stock Mix of native PF Carboxylic and Sulfonic acids
Calculate the volumes required of each native stock standard to give a concentration of 50 (ig/mL for
each carboxylic acid and 100 (ig/mL for each sulfonic acid.
Using a cleaned glass syringe that is reserved for native standards, transfer and combine the required
measured quantities of each individual native standard into the cleaned volumetric flask.
Dilute to the mark with the prepared 60:40 acetonitrile/water mix.
5. Prepare Full Native Stock Mix containing all native PF Carboxylic and Sulfonic acids
Calculate the volumes required of the intermediate Stock Mix standard and native PFHpS solution to
give a concentration of 2.5 (ig/mL for each carboxylic acid and 5 (ig/mL for each sulfonic acid.
Using a cleaned glass syringe that is reserved for native standards, transfer and combine the required
measured quantities of each standard into the cleaned volumetric flask.
Dilute to the mark with the prepared basic methanol mix.
Prepare working level native standard of PF Carboxylic and Sulfonic acids
5.1. Objective: prepare a solution of 125-250 ng/mL in basic methanol.
5.2. Using appropriate cleaned syringes and volumetric flasks, transfer a portion of the Full
Native Stock Mix and dilute to mark with the prepared basic methanol mix so that it is
diluted 20X.
Prepare working-level native standard of N-Me/Et-FOSA/Es
5.3. Objective: prepare a solution of 1250 ng/mL of the FOSEs and 2500 ng/mL of the
FOSAs in methanol, plus 10% propan-2-ol which is required to allow full mixing of the
nonane and methanol.
5.4. Using appropriate cleaned syringes and volumetric flasks, transfer and combine the
required portion of each individual 50 (ig/mL standard, add propan-2-ol so that it is 10%
of the flask volume and dilute to mark with methanol.
Prepare low level native standard for low level spiking and Calibration standard preparation
5.5. Objective: prepare a solution of 12.5-25 ng/mL in basic methanol.
5.6. Using appropriate cleaned syringes and volumetric flasks, transfer a portion of the Full
Native Stock Mix and dilute to mark with the prepared basic methanol mix so that it is
diluted 200X.
Draft PFC Procedure 48 December 2011
Not approved for either general purpose or regulatory use
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6. Prepare Calibration Standards (see Table 6 in the body of the procedure)
Label baked disposable 12mL glass vials.
Using disposable tip pipettors, transfer a 200(iL portion of the Labeled Internal Standard Solution
and 25 (iL of the Stock Mass-Labeled injection internal standard to each vial.
Using disposable tip pipettors, transfer portion of the full Native Stock Mix, working level native
standard or low level native standard to give levels of native compounds matching those shown in
Table 7.
Using disposable tip pipettors, add basic methanol to dilute each Calibration Standard to 2.0 mL. Cap
and mix thoroughly.
For Matrix Matched Calibration standards, only 1 mL of each CAL was prepared, so volumes of
standards used were halved and 0.5 mL of soil extract (containing the equivalent of 0.5 g of clean soil
in 0.5 mL basic Methanol) was added before diluting each Calibration Standard to 1.0 mL with basic
methanol.
Table of Calibration Standards
STD
ID
CS-1
CS-2
CS-3
CS-4
CS-5
CS-6
CS-7
CS-8
Internal
Standard
Solution
(LINT)
HL
200
200
200
200
200
200
200
200
Stock Mass-
Labeled injection
internal standard
mix (INJ) nL
25
25
25
25
25
25
25
25
Low level
native
standard
HL
20
50
Working
level native
standard
(NAT)
HL
20
80
Full
Native
Stock
Mix (iL
20
40
100
250
N-Me/Et-
FOSA/Es
native standard
(NAT2)
HL
4
8
16
32
64
128
256
Final
Volume
HL
2000
2000
2000
2000
2000
2000
2000
2000
Draft PFC Procedure
49
Not approved for either general purpose or regulatory use
December 2011
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APPENDIX 2
Procedure for Polishing Deionized Water
Draft PFC Procedure 50 December 2011
Not approved for either general purpose or regulatory use
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Appendix 2. Procedure for Polishing Deionized Water
Laboratory produced de-ionized water can be purified to produce PFC-free water for use in this analysis.
Polished water is used for preparation of strong needle wash solutions, seal wash solutions, 60:40
ACN:H2O standards solutions LC aqueous mobile phase and during the extraction and cleanup procedure.
1.0 Equipment for polishing water
1.1 Cartridge for polishing 18MQ water - Waters Oasis 35cc (6g) HLB Extraction Cartridge.
1.2 Glassware for polishing 18MQ water - Fisher Scientific, 2000 mL Kimax Brand Volumetric
Flask (or equivalent), Fisher Scientific: 2000 mL Pyrex Filtering Flask (or equivalent) and Fisher
Scientific Glass Magnetic Stir Bar 1.5" x 3/8" (or equivalent). All glassware should be washed 3
times using PFC free methanol.
2.0 Procedure for Polishing Water
2.1 Use glassware dedicated to water polishing (See Sec. 1.2 for specified glassware).
2.2 Pass 2L of 18MQ (nanopure or equivalent) water through a 60 cc "Oasis HLB" cartridge. This
cartridge should be used no more than 3 times.
Polished nanopure water should be stored in dedicated 1-L HDPE containers.
Figure 1. Setup for Water Polishing
Draft PFC Procedure
51
Not approved for either general purpose or regulatory use
December 2011
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