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

# U.S. Environmental Protection Agency Office of Water (4303T) 1200 Pennsylvan ia Avenue, NW Washington, DC 20460

EPA

- 821

- R

- 11

- 007Draft PFC Procedure i December 2011 Not approved for either general purpose or regulatory us e 

## 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 pr ecursor 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 b y 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 Engineeri ng 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 Met hod 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. F ollowing 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.govDraft PFC Procedure ii December 2011 Not approved for either general purpose or regulatory us e 

## Table o f 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 Pr eparation, Extraction, and Cleanup of Field Samples and QC Samples

................................ ..... 15

12.0 LC/MS/MS Set Up and Calibration

................................ ................................ ............................... 19

13.0 Instrument Q uality 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 Referenc es

................................ ................................ ................................ ................................ ...... 28

19.0 Glossary

................................ ................................ ................................ ................................ ......... 29

20.0 Tables and Figures

................................ ................................ ................................ ......................... 32

Appendix 1

................................ ................................ ................................ ................................ .................. 46

Appendix 2

................................ ................................ ................................ ................................ .................. 50Draft PFC Procedure 1 D ecember 2011 Not approved for either general purpose or regulatory us e 

## 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 p erfluorinated c arboxylic acids, perfluorinated s ulfonic acids, methyl and ethyl perfluroro

- octanesulfonamide s , and (methyl and ethyl sulfonamido)

- ethanols in se w age 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 T his 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 munic ipal 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 procedure s 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 sample s 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 t his 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 Thi s 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 2 D ecember 2011 Not approved for either general purpose or regulatory us e 

## 2.0 Summary of Procedure

The general steps in this procedure are summarized in Sec s. 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 c ollection and d igestion

– Collect a sample of sewage slu dge or biosolids sufficient to yield at lea s t 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 t ransfer a subsample containing 0.5 g of wet sol ids 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 HCl and

extracted with solvent

.

2.2 Solvent e xtraction and c leanup

– Extract the digested sample twice by shaking and ultrasonic agitation, using 10 mL 50:50/ACN:MeOH (v/v). Dilute the s ample extract

, acidif y it to pH 6.5

, agitate it ultra son i ca lly, and clean up the extract using a weak anion exchange (WAX) solid

- phase extraction ( SPE ) cartridge.

2.3 Analysis

– R econstitute the sample extract with 1 mL 0.3% NH 4 

### OH in m ethanol containing the

labeled injection internal standards . Analyze a 15

- μ L 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 sp iking 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 anal ytical 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 perfluorin ated 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 tes t 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 (HDPE) containers. Storage of aqueous solutions in polypropylene containers has resulted in significant loss of c ertain perfluorocarboxylic acids (C 11 

### and C 12 

### ) 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 b asic methanol have not been observed to degrade when in contact with any of these materials for 6 months

.Draft PFC Procedure 3 D ecember 2011 Not approved for either general purpose or regulatory us e 

### 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 (equ ivalent in volume to the amount used in the procedure) from each lot number of solvent or water should be proofed before use.

C oncentrations of any detected target compounds are compared to the method detection limits ( MDLs ) and minimum levels ( MLs ) to de termine 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, mus t 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 int erferences

– Interferences co

- extracted from samples will vary considerably from source to source. Taurodeoxycholic Acid (TDCA) and some

of its isomers

, including tauroursodeoxycholic acid and tau rochendeoxycholic 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 per fluorohexanesulfonic 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 PFH x S 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 relia bility 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. Diluti on 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 t o diagnose matrix effects, but because it introduces even more material into the ionization chamber, it is unlikely to be useful in resolving those e f fects 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 th ose 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 quantifi cation 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 dilut ed analysis, the sample extract could be subjected to further cleanu p (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 4 D ecember 2011 Not approved for either general purpose or regulatory us e 

## 5.0 Safety

5.1 The toxicity or carcinogenicity of each chemical used in this procedure has not been precise ly 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 h igh 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 specif ied 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 use s this procedure and that the results of this monitoring be made available to the analyst. Additional information on laboratory safety can be found in R efs

. 7 to 9

.

5.3 Spiking solutions or samples suspected to contain high concentrations of these compo unds 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 gl ove 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 n o 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 analyti cal 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, s egregated 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 ot her personnel should be trained in the safe handling of waste. See Sec. 1 7 for additional information on waste handling and disposal.Draft PFC Procedure 5 D ecember 2011 Not approved for either general purpose or regulatory us e 

### 5.4 Sewage sludge and b iosolids samples may contain high concentrations of biohazards, and must be handled with gloves a nd 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 S upplies

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 equivalen t performance that meets the requirements of this procedure is the responsibility of the laboratory.

6.1 Disposable l ab e quipment

6.1.1 Sample bottle s and caps

6.1.1.1 Sample collection bottle

– High density polyethylene (HDPE) sample bottles with pol ypropylene 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 c rimp

- 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. Polypropyle ne 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 (F isher

Scientific Cat. No. 1481728 )

, or equivalent.

6.1.5 Disposable p ipets

– Glass or polypropylene disposable pipets (Fisher Cat. No. 13

- 711

- 17 ) or equivalent.

6.1.6 Miscellaneous l abware

– Glass or polypropylene

, as required.

6.1. 7 pH paper

– Wha tman ® 

### Panpeha ® 

### pH indicator strips pH range 0 to 14 (Sigma

- Aldrich,

Cat. No.: Z134147 )

, or equivalent.

6.1.8 Syringe filters

– Acrodisc 0.45

- μm, 25

- mm filters with nylon membranes (Ann Arbor, MI, USA, Part No.: 4614), or equivalent.Draft PFC Procedure 6 D ecember 2011 Not approved for either general purpose or regulatory us e 

### 6.2 Non

- disposable l ab equipment

6.2.1 Auto

- pipet te s

– Gilson (Middleton, WI, USA) Microman ® 

### positive displacement pipet te s , volume 10

- , 25

- , 100

- , and 1000

- μL with disposable tips, or equivalent.

6.2.2 Balances

– An analytical balance capable of weighing 0.1 mg is used f or 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 oC (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.:

4 15110 ), 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

- p hase e xtraction

6. 3.1 Solid

- p hase e xtraction c artridge

– Oasis weak anion exchange ( WAX ) e xtraction

c artridge, 6

- cc barrel size, 150

- mg sorbent weight, 30

- μ m particle size (Waters Milford, MA, USA)

, or equivalent.

6.3.2 SPE extraction kit

– Vacuum extraction manifold (Al ltech/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 poly propylene 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: A utomated systems may contain parts made of polytetrafluoroethylene ( PTF E

)

. Before use, t he 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 g au ge.

6.4 LC/MS/MS s ystem

– Th e analytical instrumentation used should meet the following requirements :

6.4.1 HPLC s ystem

– 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 opt imization, must be capable of multi

- segment gradient separation, producing the separations for the analytical runs detailed in Table 2 under the instrumentDraft PFC Procedure 7 D ecember 2011 Not approved for either general purpose or regulatory us e 

### conditions detailed in Table 5, and meeting other HPLC requirements in Sec. 12. This system must al so be equipped with a 50

- μ L loop capable of using ‘ partial loop with

needle overfill ’ mode.

6.4.2 Columns

– Injections are introduced into a 50

- μ L loop using ‘ partial loop with needle

overfill ’ mode connected to a C18 guard cartridge

, followed by an analy tical column (PN 186000404, Waters Xterra MS C18 3.5 μm , 2.1 x 100 mm column ) or equivalent.

Alternat iv e columns may be used as long as they provide comparable chromatography to

that described in Table 2.

6.4.3 MS/MS s ystem

– The MS/MS system must be ca pable of negative electrospray ionization (ESI

- ) under the conditions in Table 5, produc ing unique product ions for analytes within specified retention time segments. A minimum of 10 scans across the chromatographic peak of the lowest concentration standa rd 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 s ystem and other s oftware

– 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 acquir e, 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 qu antify 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 b ackground

– Background levels of perf luorinated 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 b e eliminated before analyses pr o c eed. The following instrumentation modifications may be required:

**•** substitution of tubing with PEEK TM 

### tubing,

**•** replacement of PTFE solvent frits with stainl ess steel frits,

**•** inserting a trap column (PN WAT200650, Symmetry C18

, 3.5 μ m, 2.1 x 50 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

**•** inject ion 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

c ontinuing calibration verifications. Prior to daily use, flush the LC column with elution solvents before initiating a sequence. It may also be necessary to flush o ther LC components such as syringes and other system components.Draft PFC Procedure 8 D ecember 2011 Not approved for either general purpose or regulatory us e 

## 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

- g rade w ater (Fishe r 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, Radn o r, PA ) or equivalent. Ar gon gas used should meet or exceed instrument

manufa cture r ’ 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 a erosol generation in ESI liquid spray (Alphagaz 1, Air Liquide Canada

, or Airgas, Radn o r, PA ) or equivalent. Nitrogen also is used to concentrate sample extracts (Ultra High Purity ) or e quivalent. Nitrogen gas used should

meet or exceed instrument manufa cture r ’ s specifications.

7.3 Other reagents

7.3.1 Certified ACS grade sodium hydroxide (Fisher Chemical ) or equivalent.

7.3.2 Ultra p ure hy drochloric acid (Seastar Chemicals Inc.

, Sidney, BC, Canada ) or equivalent.

7.3.3 Glacial acetic acid, HPLC g rade (Fisher Chemical, Fair Lawn, NJ, USA ) or equivalent

.

7.3.4 Ammonium h ydroxide

– (Fisher Chemicals, certified ACS+ grade, 30% in water, Fair Lawn, NJ, USA) is used as received.

7.3.5 Formic a cid

– (Alfa Aesar, greater than 96% purity, Ward Mill, MA, USA,) is used as

received.

7.3.6 Ammonium a cetate

, p urity >98%. ( Sigma Chemicals ) or equivalent.

7.3.7 Reference m atri ces

– 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,

o rganic

- component

- rich

, commercially available top soil may be used as a reference

matrix if testing demonstrates that it does not contain the nativ e PFCs

. Other reference

matrices will be evaluated.Draft PFC Procedure 9 D ecember 2011 Not approved for either general purpose or regulatory us e 

### 7.4 Preparation of r eagents

7.4.1 Pretreatment reagent (1 M NaOH)

– prepared by adding 40 g of NaOH to 1 L of reagent

water.

7.4.2 N eutralization reagent (1M HC l )

– prepared by dilution of 83.5 mL of concentrated hydrochloric acid (HC l ) 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 a cid (3% , v/v )

– prepared by dissolv ing 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% NH 4 

### OH v/v in m ethanol)

– 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%): f ormic 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 a mmonium h ydroxide (0.3% v/v )

– prepared by adding 1 mL, of 30% ammonium hydroxide to 99 mL of reagent water.

7.5 Preparation of LC m obile p hases and w ash s olutions

7.5.1 Aqueous m obile p hase

– 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 m obile p hase

– 90% acetonitrile/10% HPLC water is prepared by adding 400

mL of HPLC

- grade water to 3600 mL of acetonitrile.

7.5.3

. Seal w ash s olution

– 10% acetonitrile/90% HPLC water is prepared by adding 400 mL of

acetonitrile to 3600 mL HPLC

- grade water.

7.5.4 Needle w ash s olution

– m ethanol is used as received.

7.5.5 Purge s olvent

– HPLC

- grade water is used as received

, or aqueous m obile p hase may be

used.



## 8.0 Standards

8.1 Source s of s tandards

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 toDraft PFC Procedure 10 D ecember 2011 Not approved for either general purpose or regulatory us e 

### 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 prepara tion 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 standard s was prepared and analyzed to

demonstrate cleanliness of these solutions.

8.3 St ock solutions

Native (unlabeled) perfluoroheptanesulfonic acid ( PFHpS ) and all individual labeled compounds

, except p erfluoro

- n

- [1,2

- 13 

### C 9 

### ]undecanoic acid

, were purchased as solutions in methanol.

Individual native a lky 1

- octanesulfonamide and (1

- octanesu lfonamido)

- ethanol compounds (N

Me/Et

- FOSA/Es) were purchased as solutions in 90% n onane/10% t oluene. Individual natives for all compounds except PFHpS and p erfluoro

- n

- [1,2

- 13 

### C 9 

### ]undecanoic acid were prepared from

solids.

Individual standards stock solut ions 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:H 2 

### O or basic methanol in glass, C lass A volumetric flasks. 60:40 ACN:H 2 

### O should be prepared from

O ptima grade ACN and PFC

- free 18

- MΩ water.

Mixed standards stocks and working level solutions for N

- Me/Et

- FOSA/Es should be prepared in

m ethanol.

Prepare t he following mixed stock standards . S ee Appendix 1 for preparation details

.

8.3.1 Native s tock m ix (containing all native perfluoroc arboxylic acids, perfluoros ulfonic

acids

, and PFOSA)

8.3.2 Stock l abeled standard mix

8.3.3 Stock l abeled injection standard

8.4 Working

- level native standards

8.4.1 Working

- level n ative (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 μL 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 11 D ecember 2011 Not approved for either general purpose or regulatory us e 

### 8.4.2 Low

- level native standard

– This solution is spiked into calibration standards , IPR, OPR, MDL, and matrix spike samples. Prepare target a nalyte s 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 s piked into calibration standards, IPR, OPR, MDL

, and matrix spike samples. Prepare t arget analyte native compounds at the concentrations shown in Table 7 (and detailed in Appendix 1) in

methanol containing 10% propan

- 2

- ol

. A 1 6

- μL 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 Secs. 8.4.1 to 8.4.3 may be used, provided that the concentrations are adjusted accordingly.

8.5 Labeled i nternal s tandards

Labeled i nternal s tandard s olution (sometimes called the labeled compound spiking s olution )

– This solution is spiked directly into samples prior to extraction. Labeled compounds are used to quanti fy unlabeled target compounds and perform recovery correction. Prepare the labeled compounds at the concentrations shown in Table 7 (and det ailed in Appendix 1) in basic

methanol

. A 100

- μL aliquot of this solution is added to each sample before extraction. See Sec.

11.3. 6

.

8.6 Labeled i njection s tandards

L abeled injection standard solution

– this solution contains the labeled compounds tha t are used to quanti fy 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 in jection standards at the conce ntrations shown in Table 7 (and detailed in Appendix 1) in basic methanol. A 12.5

- μL 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 tha t the concentration is adjusted accordingly.

8.7 Calibration standards

Combine and dilute the solutions in Secs. 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 da rk 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. Altern a tively, weigh

the vi al or ampule before storage, record the mass, and reweigh the vial or ampule before the next

use.Draft PFC Procedure 12 D ecember 2011 Not approved for either general purpose or regulatory us e 

### 8.9 Stability

Shelf life of purchased solutions in methanol is determined by the supplier

. S tability of diluted

solutions and working solutions remains to be determined.

Perfluorocarboxylic acid standards in methanol solution may undergo esterification to the m ethyl

e ster s

. Most purchased perfluorocarboxylic acid standard solutions were received in methanol containing 4 mole equivalents of NaOH. Basic met hanol ( 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 polye thylene (HDPE) container s with propylene caps/lids

, following conventional sampling practices designed to obtain a sample that is representative of the material of interest. L ids and other materials containing PTFE must be avoided, due to possible leachin g 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 t imes

– EPA has not yet conducted a formal holding time study and will conduct one after the procedure is f inalized

. Until that time, d efault 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 valida ted).

9.3.3 Stor e all samples and extracts at less than 4 o 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 ( R LS) described in Sec. 10.4.

Ongoing quality control samples ( Secs. 10.5

– 10.8) include calibration verification (CALVER)

s tandards at the beginning of every shift, analysis of ongoing calibration verification standards with every sample batch, and analysis of a met hod 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 t h e modifications to instrument plumbing that are required.Draft PFC Procedure 13 D ecember 2011 Not approved for either general purpose or regulatory us e 

### 10.2 Initial precision and rec overy (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 oper ations.

10.2.1 Spike four aliquots of the reference matrix ( Sec. 7.3.7) with 40 μL of the working level

native standard (Sec. 8.4. 1 ), 16 μL of working

- level native standard solution for N

Me/Et

- FOSA/Es ( Sec. 8.4 .3 )

, and 100 μ L of labeled internal standa rd solution (see Sec. 8.5)

, and analyze each aliquot according to the procedures in Secs. 11 through 14. All processing steps that are used for samples, inc luding preparation

, extraction

, and cleanup ( Sec. 1 1 ), must be included in this test.

10.2.2 Us ing 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 t he corresponding

limits for initial precision and recovery in Table 8 . If RSD and X f or 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 d etection l imit (MDL ) s tudy

– Determin e the MDL in accordance with the procedures

described in 40 CFR P art 136

, prior to the analysis of field samples.

10.4 Reporting limit s ample (RLS)

– Use 0.5 gram of the soil reference matrix ( Sec. 7.3.7) to prepare
the RLS

. Spike this aliquot with 10 μL of the l ow

- 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 R LS immediately prior to analysis of the OPR, and samples from the same batch.

Note: T he RLS is not required during rout ine sample analysis after method development has been completed and single

- lab validation has occurred.

10.5 Matrix s pike sample s are used t o assess performance of the procedure on the biosolids being analyzed . T he 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 μL of the

w orking

- l evel n ative standard (Sec. 8.4.1) , 16 μL of the working

- level native standard

solution for N

- Me/Et

- FOSA/Es ( Sec. 8.4 .3 )

, and 100 μ L of l abeled internal standard solution ( Sec. 8 .5) and mix thoroughly . A nalyze both matrix spike sample s according to the procedures in Secs. 11 through 14.

10. 5 .2 Compute the recover y of the labeled compounds using the in ternal standard method (quantify using the injection internal standard).Draft PFC Procedure 14 D ecember 2011 Not approved for either general purpose or regulatory us e 

### 10. 5

. 3 It may be necessary to s pike 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 tha t has already been analyzed.

10. 5

. 4 Report the background concentrations of native compounds, and recoveries and spik ing

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 v erification

N ote: Initial c alibration is discussed in Sec. 13. 1 and must be performed prior to analysis of samples.

10.6.1 Frequency

– Ongoing c alibration v erification consists of the analysis of bracketing calibration verification samples, one at the beginning of eve ry 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 u sed for calibration verification samples.

10.6.4 Inject the VER (CS

- 4 ) standard (Table 6 ) using the analysis procedure in Sec. 1 4

.

Note: The requirements in Sec. 1 5 must be met when analyzing ongoing calibration verification samples.

10.7 Method b lank

– 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 preve nt carryover from the OPR into the first sample.

10.8 Ongoing p recision and r ecovery (OPR) s ample

– The laboratory must, on an ongoing basis, demonstrate through analysis of the ongoing precision and recovery standard that the analytical system is in con trol. 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 μL 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 μ L of theDraft PFC Procedure 15 D ecember 2011 Not approved for either general purpose or regulatory us e 

### l abeled 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 extr acts 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 i n 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

S amples

11.1 Sample p reparation

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

o f 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.

H omogenize the entire sample in the original sample container, by shaking samples that are pourable liquids, or by stirring solids in their original container wit h a clean spatula, glass stirring rod, or other suitable implement. Once homogenized, remove an aliquot of the sample to

d etermine the percent solids content of the sample using the procedure in Sec. 11.2. Remove a second aliquot of the sample for analy sis, as described i n 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 determ ined 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 signifi cant 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 o C, and cool in a desiccator.Draft PFC Procedure 16 D ecember 2011 Not approved for either general purpose or regulatory us e 

### 11.2.1.3 Weigh the dried aliqu ot and calculate percent solids as follows:

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 bott le 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 oC and cool in a desiccator

.

11.2

. 2 .2 Weigh the filter and calculate percent solids as follows:

1 1.3 Sample d igestion

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 backg round 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 sam ple is used as the OPR sample.

11.3.4 If matrix spike samples are to be analyzed, prepare those aliquots a s 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 (O PR, IPR, matrix spike samples ) with 40 μ L of the

w orking

- l evel n ative s tandard solution (Sec. 8.4.1) and 16 μ L 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 th at the concentrations are adjusted accordingly.

11.3. 6 Spike all samples and QC samples with 100 μ L of the l abeled i nternal s tandard s olution ( Sec. 8.5). The volume spiked will yield a sample extract with labeled compounds present at a concentration equ ivalent to the native compounds in the mid

- level calibration

standard.

11.3. 7 A dd 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. 100
g 10 (g) filter of weight

- (g) drying after aliquot sample of Weight Solids % x **=** 100 (g) drying before aliquot sample of Weight (g) drying after aliquot sample of Weight Solids % x **=**Draft PFC Procedure 17 D ecember 2011 Not approved for either general purpose or regulatory us e 

### 11.3. 8 Sonica te the samples in a heated water bath at 40 o 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 incubatio n period, neutralize the NaOH by adding an equivalent number of

moles of the 1 M HCl 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 pH pape r. 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 e xtraction

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 ( Secs. 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, c heck that the pH of the diluted extract is 6.5 ± 0.5. If

req uired, a djust 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 c leanup

All sample extracts are subjected t o 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% NH 4 

### OH in methanol, followed by 5 mL of 0.1M formic acid in reagent water. Discard eluants

.

11. 5 .4 Equilibrate th e cartridge with 5 mL of plasma

- grade reagent water (Sec. 7.1.3)

. Discard

the elua n t.Draft PFC Procedure 18 D ecember 2011 Not approved for either general purpose or regulatory us e 

### 11. 5 .5 Load the sample onto the cartridge drop

- wise (~5 mL/min).

11. 5 .6 W ash the sample with 5 mL of 20% MeOH in 80% 0.1M formic acid in reagent water, followed by 2 m L of 0.3% (v/v) NH 4 

### OH 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 cartridg e into a 15

- mL clean glass centrifuge tube with 4 mL 0.3% NH 4 

### OH (v/v) in MeOH

.

11. 6 Extract concentration

11. 6 .1 Reduce the extract to about 50 μL using a gentle stream of nitrogen and a water bath set

at 40 oC.

11. 6 .2 Reconstitute the extract with 93 8 μL of 0.3% NH 4 

### OH (v/v) in MeOH and vortex to mix.

11.6.3 S pike the extract with 12.5 μ L of the i njection i nternal s tandard solution ( Sec. 8.6),

vortex to mix

, and filter the sample extract through a s yringe f ilter (Sec. 6. 1.8 ) into a clean centrifuge t ube. The final extract volume is 1.0 mL.

Note: Other volumes and solvents may be used in Secs. 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 t he 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 μL 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 μL and store at 4 o C for backup. Place a mark on the tube at the level of the solution so that solvent loss by evaporation can be detected. Alte rnatively, 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 SP E 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 : This check is performed when ever a new lot number of cartridges is purchased.Draft PFC Procedure 19 D ecember 2011 Not approved for either general purpose or regulatory us e 

### 11. 7 .1 Spike 100 mL reagent water with 40 μ L of the w orking

- l evel n ative s tandard solut ion (Sec. 8.4.1) and 16 μ L of the working

- level native standard solution for N

- Me/Et

FOSA/Es ( Sec. 8.4 .3 ). Do NOT spike the l abeled i nternal s tandard compounds.

11. 7 .2 Process the solution through the SPE cleanup procedure in Sec. 11. 5

.

11. 7 .3 After c leanup , spike the solution with the l abeled i nternal s tandard solution ( Sec. 8.5) and complete the analysis as per Sec. 14.

11. 7 .4 R ecovery 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 S et U p and C alibration

Samples are analy zed on a high performance liquid chromatograph coupled to a triple quadrupole mass spectrometer (LC/MS/MS), or equi valent, 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 m ultiple r eaction m onitoring (MRM ). Data acquisition an d quantification

are performed by recording the peak areas of the applicable parent ion /daughter ion transitions. The

i nstrument manufacturer ’ s software is used to acquire data and calculate results using isotope dilution and internal standard quantitati on (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 Establish ing 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 cali bration, 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 manufactu rer ’ 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 ann ually, after performing major maintenance

, or as required to maintain

routine instru ment 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 di rectly into the electrospray source during calibration. Mass calibration is performed at

instrumen tal settings corresponding to a nominal unit mass resolution, but mass resolution is not directly mea su red.

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 μg/μL sodium iodi de and 50 ng/μL cesium iodide.Draft PFC Procedure 20 D ecember 2011 Not approved for either general purpose or regulatory us e 

### 12.2.2 Use a syringe pump to infuse the calibra tion solution as a stable aerosol spray at 10 μL/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 exa ct 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 922.3552 1971.6149

132.9054 1072.2494 2121.5091

172.8840 1222.1437 2271.4033

322.7782 1372.0379 242 1.2976

472.6725 1521.9321 2571.1918

622.5667 1671.8264 2721.0861

772.4610 1821.7206 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 pre sence 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 a cquisition p arameters

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 p arts 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 Th e optimum parameters are compound specific and a set of single setting s can not be used for all target compounds in the analysis. Use the optimized settings for the analysis of all standards and samples.

12.4 Chromatographic s eparation

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 C18 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 21 D ecember 2011 Not approved for either general purpose or regulatory us e 

### 12.5 Instrument b ackground d etermination

To determine if background concentrations of PFCs significa ntly impact this analysis, a 40 part per trillion (ppt) standard containing all of the target analytes in 0.3% NH 4 

### OH (v/v) in methanol must be analyzed three times, with alternating instrument solvent (methanol) blank measurements. If the peak area from th e 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 r etention t ime windows

Analyze individual solutions of the each of the target compounds using the LC gradient and acquisition parameters determined above. Analyze a mixed solu tion of all target compounds to confirm their separation and identification. A total ion chromatogram (TIC ) indicating the separation of the target analyte s is shown in Figure 2 , as an example.

12.7 Analytical d ata a cquisition p rogram

All of the informa tion 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 ion s 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 s ensitivity

Prior to commencement of any analysis, and at least once every 24 hours during extended runs,

determine that th e 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

, p erform system cleaning and maintenance and repeat the test. If calibration verification can not be established

after this maintenance

, a new i nitial c alibration ( Sec. 13.2) is required.



## 13. 0 Instrument Q uality C ontrol

The mass spectrometer is optim ized 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 c alibration

Initial c alibration (ICAL) must be performed prior to analysis of any field sample or QC sample

. Approaches to quan ti tation vary depending on the compound . S ome compounds are quanti fied using isotope dilution (for those target compounds with exact labeled analog s) and others are quantified using internal standard (those without an exact labeled analog). Additionally, labeled compounds are quanti fied using the internal standard approach. These two approaches are

discussed in Sec s

. 13. 2 .1 and 13. 2 .2 , respectively. The requirements for initial calibration are

outlined in Sec. 1 3.2

, and Table 8

. After the i nitial c alibration , analyze an initial calibration blank

and an i nitial calibration verification (CALVER) standard

.Draft PFC Procedure 22 D ecember 2011 Not approved for either general purpose or regulatory us e 

### 13. 2 Calibration by isotope dilution and in ternal 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 :

where:

NRR = Normalized rela tive response

A

n 

### = Area of the peak for the native ( unlabeled ) analyte

A l 

### = Area of the peak for the specified labeled compound

C l 

### = Concentration of the specified labeled compound (in pg /mL )

Ef f o r ts to date indicate that a linear equation may be used for all of the analytes except :

p erfluorobutanesulfonic acid (PFBS), perfluoro

- n

- heptane sulfonic acid (PFHpS)

, perfluorooctane sulfonamide (PFOSA)

, and those compounds containing more than 10 carbons (C 11 

### – C 14 

### ) . For those analytes, the response is better f it (higher r 2 

### ) 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 dil ution

Isotope dilution is used for calibration of each native compound for which a n exact labeled analog is available (Table 3) . A n 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 a n injection volume of

15 μ L.

13. 2 .1.2 For each compound determined by isotope dilution, compute its normalized

rel ative response ( NRR ) over the calibration range. Determine the NRR of each compound using the area responses of the product m/z value s specified in Table 3. Use the labeled compounds listed in the tables as the quantitation reference and the product m/z value 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). Select the calibration equation (linear or quadratic) that provides the best fits to the data

, as indicated by the coefficient

of determination ( r 2 

### ) . Examine the residuals of the regression (i.e., the distance

between the ob served 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). Whicheve r calibration is selected, r 2 

### must be at least

0.99. Use the resultant equation to calculate the concentration of analyte i n each sample or CALVER. l

l n 

### C

x A A NRR **=**Draft PFC Procedure 23 D ecember 2011 Not approved for either general purpose or regulatory us e 

### 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 w ill 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 th at are not being quanti fied by isotope dilutio n

. 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 int o 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 calibr ation (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 inte rnal 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

labele d 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 p roduce 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 in dicated by the coefficient of determination (r 2 

### ). 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 distr ibuted (i.e., the regression line passes between the calibration points, and not above or below all of the points). Whichever calibration is selected, r 2 

### 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

s tandards 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 24 D ecember 2011 Not approved for either general purpose or regulatory us e 

### 13. 2 .3 Quantification of the labeled injection internal standard concentration

T he 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 v erification

Calibration v erifica tion (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 standard s 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. Injec t the CS

- 4 calibration standard (Table 7) using the analysis procedure in

Sec. 1 4 . The requirements in Sec. 1 0

. 8

, and Table 8 must be met when analyzing calibration verification samples.

13.4 Quantitation of linear and branched isomers

Some PFCs consi st 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

, pe ak 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 i nterferences

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 )

. Th e 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 interference s. 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 A nalysis

Once the operating condi tions have been established and the instrument tuned ( Sec. 12), inject a 15

- μ L 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 fou nd in Table

5. Start data collection 1 to 2 minutes prior to elution of the first analyte. Monitor the product

m/z ’ s for each native and labeled analyte throughout its retention time window. Stop dataDraft PFC Procedure 25 D ecember 2011 Not approved for either general purpose or regulatory us e 

### 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 c alibration s tandards

**•** 2 Instrument blank s

**•** Opening c alibration v erification s tandard

**•** OPR sample

**•** 1

- 2 Instrument b lanks

**•** Method b lank

**•** Up to 6 field s amples

**•** Bracketing c alibration v erification s tandard (every 10th injection)

**•** Up to 10 field samples

**•** Closing calibration verification standard (every 10t h injection, which may serve as the opening VER for the next cycle )

**•** OPR sample, etc.



## 15.0 Qualitative I dentification and Q uantitat ion

15.1 Qualitative i dentification

– The following requirements must be met in all samples for a compound to be identifie d.

15.1.1 Signal to noise requirements

– The LC peak representing the quantitati on m/z of each native compound in the upper 7 ICAL standard s 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 pe ak representing each labeled compound quantitati on m/z in the CAL VER 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 u ntil these requirements

can be met. If these requirements are not met in samples, the CALVER test should be repeated.

15.1.2 Relative i ntensity

– The ion intensity of each monitored ion transition in the ICAL should be recorded for use in qualitative id entification 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 requirem ents 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) Diagn ostic 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 26 D ecember 2011 Not approved for either general purpose or regulatory us e 

### Comparison of the ratio of the confirmation transition response to the quantitation

transitio n 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 io n i ntensity ratio r equirement

– 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 i ntensity of confirmation

transition to quantitative transition in

most recent CALVER Agreement of ratio of confirmation transition to quantitative transition in sample relative to

same ratio in most recent CALVER

>50 % ± 20%

20 to 50 % ± 25%

1 0 to 20 % ± 30%

<10 % ± 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 t ime w indow r equirement

– 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 retenti on time window requirement during analysis of all subsequent samples. Data acquired for all subsequent sample s 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 a ffected.

2) the relative retention times for native compounds and the labeled compound for which

each native is be ing 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 a symmetry f actor

– During initial calibration and calibration verification, the peak asymmetry factor must be calculated for all analytes using the following figure and equation:

A

s 

### = b/aDraft PFC Procedure 27 D ecember 2011 Not approved for either general purpose or regulatory us e 

### where : A

s 

### = 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 l ine 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 fi rst 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 Quantitati on

15.2.1 Isotope d ilution q uantitation

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 Int ernal 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 establishe d in Secs. 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:

15.2.3 The concentration of a native compound in the solid sampl e is computed using the concentration of the compound in the extract

, and the wet weight of the solids, and the percent solids, as follows:

w here:

C

ex 

### = Concentration of the compound in the extract in ng/mL

V

ex 

### = Extract volume in mL

W

s 

### = Sample weight ( wet weight) in kg

% solids = Percent solids d etermined in Sec. 11.2

If desired, divide the concentration by 1000 to convert ng/kg (ppt) to μg/kg (ppb).

15.3 Reporting r esults

Unless otherwise specified, r eport results in n g/ k g (parts

- per

- trillion) to three significant figures, based on the dry weight of the sample. Also r eport the percent solids so that the result may be

converted to wet

- weight units by the end user. ) solids (% W V C ) kg / ng ( sample solid in ion Concentrat

s ex ex 

### = 

### 100 x (ng/mL) spiked ion Concentrat (ng/mL) found ion Concentrat (%) Recovery **=**Draft PFC Procedure 28 D ecember 2011 Not approved for either general purpose or regulatory us e 

### Results for a compound in a sample that has been diluted must be reported at the lea st 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

.

P roject

- specific reporting requirements may apply , including reporting results based o n the volume of the original sample, e.g., ng/L or other weight/volume units



## 16.0 Method p erformance

This procedure is still under development. Preliminary method performance information can be found in

Tables 4

, 8, and 1 0 . Additional performance data will be added by EPA as they are developed.



## 1 7 .0 Pollution p revention and w aste m anagement

1 7 .1 The laboratory is responsible for complying with all f ederal, s tate, 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. A n overview of requirements can be found in Reference 12

.

1 7 .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.

1 7 .3 Standards should be prepared in volumes consis tent 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 W aste 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

Lab oratory 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, 2 2 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 29 D ecember 2011 Not approved for either general purpose or regulatory us e 

### 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 perfluo rinated compounds in whole blood using acetonitrile and solid phase extraction methods. ” Leo W.Y., Yeung, Sachi Taniyasu, Kurunthachalam Kannan, Della 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

. “ Wor king 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.

1 0

. “ 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.

1 1

. “ 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/200 2/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 bu t have been conformed to common usage to the extent possible.

Symbols

o C degrees Celsius

μ L microliter

μm micrometer

< less than

> greater than

% percent

Abbreviations (in alphabetical order)

cm centimeter

ESI

- Negative Electrospray Ionization

g gram h hour

ID inside diameterDraft PFC Procedure 30 D ecember 2011 Not approved for either general purpose or regulatory us e 

### in

. inch

L liter

M m olecular 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 N ormalized 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 ac ronyms (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 r esponse of the HPLC/ MS/MS instrument. Referred to as CS

- 1, CS

- 2, CS

- 3, CS

- 4,

CS

- 5, CS

- 6.

Calibration verification standard ( CAL VER)

– A calibration standard close to the mid

- point calibration standard that is used to verify calibration.

HPLC

– High per formance liquid chromatograph or high performance liquid chromatography

ICAL

– Initial c alibration

Internal standard quantitation

– A means of determining the concentration of (1) a naturally occurring (native) compound by reference to a compound other t han 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 natu rally 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 2 

### H

- labeled analogs or carbon

- 13 to produce 13 

### C

- 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 31 D ecember 2011 Not approved for either general purpose or regulatory us e 

### Labeled compound , or labeled analog

– A molecule in which o ne 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 quantitati on 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 i nternal s tandard

– 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, r eagents, 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 minim um 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).

Mi nimum 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); a n 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 meth od 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, di vided 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 proced ure as a cleanup technique.Draft PFC Procedure 32 D ecember 2011 Not approved for either general purpose or regulatory us e 

## 20.0 Tables and f igures

Table 1. Names and CAS Registry numbers for PFCs determined by isotope dilution and internal standard

Compound CAS

N umber Formula Acronym

Native PFCs

Perfluoorobutanoic acid 375

- 22

- 4 CF 3 (CF 2 ) 2 COOH PFBA

Perfluoropentanoic acid 2706

- 90

- 3 CF 3 (CF 2 )3COOH PFPA

Perfluoro

- n

- hexanoic acid 307

- 24

- 4 CF 3 (CF 2 ) 4 COOH PFHxA (C6)

Perfluoro

- n

- heptanoic acid 375

- 85

- 9 CF 3 (CF 2 ) 5 COOH PFHpA (C7)

Perfluoro

- n

- octanoic acid 335

- 67

- 1 CF 3 (CF 2 ) 6 COOH PFOA (C8)

Perfluoro

- n

- nonanoic acid 375

- 95

- 1 CF 3 (CF 2 ) 7 COOH PFNA (C9)

Perfluoro

- n

- decanoic acid 335

- 76

- 2 CF 3 (CF 2 ) 8 COOH PFDA (C10)

Perfluoro

- n

- undecanoic acid 2058

- 94

- 8 CF 3 (CF 2 ) 9 COOH PFUnDA (C11)

Perfluoro

- n

- dodecanoic acid 307

- 55

- 1 CF 3 (CF 2 ) 10 COOH PFDoDA (C12)

Perfluoro

- n

- tri decanoic acid 72629

- 94

- 8 CF 3 (CF 2 ) 11 COOH PFTriDA (C13)

Perfluoro

- n

- tetradecanoic acid 376

- 06

- 7 CF 3 (CF 2 ) 12 COOH PFTeDA (C14)

Perfluorobutanesulfonic acid 375

- 73

- 5 CF 3 (CF 2 ) 3 SO 3 H PFBS (S4)

Perfluorohexanesulfonic acid 355

- 46

- 4 CF 3 (CF 2 ) 5 SO 3 H PFHxS (S6)

Perfl uoro

- n

- heptanesulfonic acid 375

- 92

- 8 CF 3 (CF 2 ) 6 SO 3 H PFHpS (S7)

Perfluorooctanesulfonic acid 1763

- 23

- 1 CF 3 (CF 2 ) 7 SO 3 H PFOS (S8)

Perfluorooctane sulfonamide 754

- 91

- 6 CF 3 (CF 2 ) 6 SO 2 NH 2 PFOSA (S8)

N

- methylperfluoro

- 1

- octanesulfonamide 31506

- 32

- 8 CF 3 (CF 2 ) 7 SO 2 N. H.CH 3 N

- MeFOSA

N

- ethylperfluoro

- 1

- octanesulfonamide 4151

- 50

- 2 CF 3 (CF 2 ) 7 SO 2 N.H.C 2 H 5 N

- EtFOSA

2

- (N

- methylperfluoro

- 1

- octanesulfonamido)

- ethanol 24448

- 09

- 7 CF 3 (CF 2 ) 7 SO 2 N. CH 3 .C 2 H 4 OH N

- MeFOSE

2

- (N

- ethylperfluoro

- 1

- octanesulfonamido)

- ethanol 1691

- 99

- 2 CF 3 (CF 2 ) 7 SO 2 N. C 2 H 5 .C 2 H 4 OH N

- EtFOSE

Labeled Internal Standards

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]butanoic acid 13 CF 3 ( 13 CF 2 ) 2 13 COOH [ 13 C 4 ]PFBA

- (MPFBA)

Perfluoro

- n

- [1,2

- 13 C 2 ]hexanoic acid CF 3 (CF 2 ) 3 ( 13 CF 2 ) 13 COOH [ 13 C 2 ]PFHxA

- (MPFHxA)

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]octano ic acid CF 3 (CF 2 ) 3 ( 13 CF 2 ) 3 13 COOH [ 13 C 4 ]PFOA

- (M4PFOA)

Perfluoro

- n

- [1,2,3,4,5

- 13 C 5 ]nonanoic acid CF 3 (CF 2 ) 3 ( 13 CF 2 ) 4 13 COOH [ 13 C 5 ]PFNA

- (MPFNA)

Perfluoro

- n

- [1,2

- 13 C 2 ]decanoic acid CF 3 (CF 2 ) 7 ( 13 CF 2 ) 13 COOH [ 13 C 2 ]PFDA

- (M2PFDA)

Perfluoro

- n

- [1,2,3,4,5,6,7,8,9

- 13 C 9 ]decanoic

acid CF 3 ( 13 CF 2 ) 8 13 COOH [ 13 C 9 ]PFDA

- (M9PFDA)

Perfluoro

- n

- [1,2

- 13 C 2 ]undecanoic acid CF 3 (CF 2 ) 8 ( 13 CF 2 ) 13 COOH [ 13 C 2 ]PFUnDA

- (M2PFUnDA)

Perfluoro

- n

- [2,3,4,5,6,7,8,9,10

- 13 C 9 ] undecanoic

acid CF 3 ( 13 CF 2 ) 9 COOH [ 13 C 9 ]PFUnDA

- (M9PFUnDA)

Perfluoro

- n

- [1,2

- 13 C 2 ]dodecanoic acid CF 3 (CF 2 ) 9 ( 13 CF 2 ) 13 COOH [ 13 C 2 ]PFDoDA

- MPFDoDA)

Perfluoro

- 1

- [1,2

- 18 O 2 ]

- hexanesulfonic acid CF 3 (CF 2 ) 5 SO 18 O 2 H [ 18 O 2 ]PFOS

- (MPFHxS)

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]

- octanesulfonic acid CF 3 (CF 2 ) 3 ( 13 CF 2 ) 4 SO 3 H [ 13 C 4 ]PFOS

- (MPFOS)

2

- (N

- deu teriomethylperfluoro

- 1

- octane sulfonamido)

- 1,1,2,2

- tetradeuterioethanol CF 3 (CF 2 ) 7 SO 2 N. CD 3 .C 2 D 4 OH d 7 - N

- MeFOSE

Injection Internal Standards

Perfluoro

- n

- [1,2,3,4,5,6,7,8

- 13 C 8 ]octanoic acid 13 CF 3 ( 13 CF 2 ) 6 13 COOH [ 13 C 8 ]PFOA

- (M8PFOA)

2H

- Perfluoro

- [1,2

- 13 C 2 ]

- 2

- decenoic acid CF 3 (CF 2 ) 6 CF 13 CH 13 COOH [ 13 C 2 ]PFOUEA

- MPFOUEA)Draft PFC Procedure 33 December 2011 Not approved for either general purpose or regulatory us e T able 2. Typical retention times (RT), relative retention times (RRT), and RT windows

Compound Mean R T in

C alibration

Standard (1) Standard

Deviation

of R T Mean RT

in

Biosolids (2) Standar d

Deviation

of RT RT

Window Mean RRT in

a Calibration

Standard (1) Standard

Deviation

of R RT Mean R RT

in

Biosolids (2 ) Standard

deviation

R RT RRT

Window

Native PFCs

Perfluoorobutanoic acid 5.22 0.025 5.24 0.036 ± 0.3 1.000 0.0026 1.008 0.0116 ± 0.05

Perfl uoropentanoic acid 6.36 0.016 6.37 0.016 ± 0.1 0.725 0.0018 0.723 0.0018 ± 0.05

Perfluoro

- n

- hexanoic acid 7.06 0.016 7.07 0.015 ± 0.1 1.001 0.0018 1.000 0.0000 ± 0.01

Perfluoro

- n

- heptanoic acid 7.81 0.014 7.82 0.020 ± 0.1 0.891 0.0015 0.887 0.0022 ± 0.05

Perfluoro

- n

- octanoic acid 8.78 0.026 8.80 0.016 ± 0.1 1.000 0.0000 1.000 0.0000 ± 0.01

Perfluoro

- n

- nonanoic acid 10.01 0.036 10.05 0.048 ± 0.5 1.001 0.0027 1.002 0.0039 ± 0.01

Perfluoro

- n

- decanoic acid 11.50 0.026 11.48 0.040 ± 0.5 1.002 0.0017 1.000 0 .0000 ± 0.01

Perfluoro

- n

- undecanoic acid 13.16 0.030 13.02 0.113 ± 0.5 1.001 0.0013 1.001 0.0014 ± 0.01

Perfluoro

- n

- dodecanoic acid 15.12 0.032 14.99 0.168 ± 0.5 1.000 0.0009 1.001 0.0010 ± 0.01

Perfluoro

- n

- tridecanoic acid 17.48 0.038 17.46 0.073 ± 0.5 1.993 0.0043 1.981 0.0083 ± 0.05

Perfluoro

- n

- tetradecanoic acid 20.56 0.072 20.64 0.100 ± 0.5 2.344 0.0082 2.343 0.0114 ± 0.05

Perfluorobutanesulfonic acid 7.20 0.018 7.21 0.016 ± 0.1 0.821 0.0020 0.819 0.0018 ± 0.05

Perfluoro

- n

- hexane sulfonic acid 9. 25 0.019 9.25 0.016 ± 0.1 1.001 0.0020 1.000 0.0000 ± 0.01

Perfluoro

- n

- heptane sulfonic acid 10.61 0.032 10.65 0.029 ± 0.5 1.210 0.0037 1.209 0.0032 ± 0.05

Perfluoro

- n

- octanesulfonic acid 12.20 0.047 12.15 0.073 ± 0.5 1.002 0.0017 1.001 0.0013 ± 0.01

Pe rfluorooctane sulfonamide 17.56 0.032 17.52 0.064 ± 0.5 2.002 0.0036 1.988 0.0073 ± 0.05

N

- methylperfluoro

- 1

- octanesulfonamide 23.15 0.063 23.07 0.096 ± 0.5 2.640 0.0072 2.619 0.0109 ± 0.05

N

- ethylperfluoro

- 1

- octanesulfonamide 24.91 0.039 24.95 0.031 ± 0 .5 2.841 0.0045 2.832 0.0036 ± 0.05

2

- (N

- methylperfluoro

- 1

- octane sulfonamido)

- ethanol 21.53 0.057 21.36 0.127 ± 0.5 1.009 0.0024 1.005 0.0022 ± 0.01

2

- (N

- ethylperfluoro

- 1

- octane sulfonamido)

- ethanol 24.27 0.034 24.22 0.070 ± 0.5 2.767 0.0039 2.749 0.008 0 ± 0.05

Mass

- labeled PFCs

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]butanoic acid 5.22 0.028 5.20 0.084 ±0.3 0.595 0.0032 0.590 0.0096 ± 0.05

Perfluoro

- n

- [1,2

- 13 C 2 ]hexanoic acid 7.05 0.011 7.07 0.015 ± 0.1 0.804 0.0012 0.802 0.0018 ± 0.05

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]oc tanoic acid 8.78 0.026 8.80 0.016 ± 0.1 1.001 0.0029 0.999 0.0019 ± 0.01

Perfluoro

- n

- [1,2,3,4,5

- 13 C 5 ]nonanoic acid 9.99 0.038 10.03 0.036 ± 0.1 1.139 0.0043 1.138 0.0041 ± 0.05

Perfluoro

- n

- [1,2

- 13 C 2 ]decanoic acid 11.49 0.033 11.48 0.040 ± 0.5 1.310 0.003 7 1.303 0.0045 ± 0.05

Perfluoro

- n

- [1,2,3,4,5,6,7,8,9

- 13 C 9 ]decanoic

acid 11.48 0.034 11.48 0.040 ± 0.5 1.309 0.0039 1.303 0.0045 ± 0.05

Perfluoro

- n

- [1,2

- 13 C 2 ]undecanoic acid 13.15 0.029 13.06 0.069 ± 0.5 1.499 0.0033 1.482 0.0079 ± 0.05Draft PFC Procedure 34 December 2011 Not approved for either general purpose or regulatory us e T able 2. Typical retention times (RT), relative retention times (RRT), and RT windows

Compound Mean R T in

C alibration

Standard (1) Standard

Deviation

of R T Mean RT

in

Biosolids (2) Standar d

Deviation

of RT RT

Window Mean RRT in

a Calibration

Standard (1) Standard

Deviation

of R RT Mean R RT

in

Biosolids (2 ) Standard

deviation

R RT RRT

Window

Perfluoro

- n

- [2,3, 4,5,6,7,8,9,10

- 13 C 9 ]undecanoic acid 13.15 0.031 13.01 0.124 ± 0.5 1.499 0.0035 1.477 0.0141 ± 0.05

Perfluoro

- n

- [1,2

- 13 C 2 ]dodecanoic acid 15.11 0.025 14.98 0.162 ± 0.5 1.723 0.0029 1.700 0.0184 ± 0.05

Perfluoro

- 1

- [1,2

- 18 O 2 ]

- hexanesulfonic acid 9.24 0.021 9.25 0.016 ± 0.1 1.054 0.0024 1.050 0.0019 ± 0.05

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]

- o ctanesulfonate 12.18 0.032 12.14 0.083 ± 0.5 1.389 0.0036 1.378 0.0095 ± 0.05

2

- (N

- deuteriomethylperfluoro

- 1

- octane sulfonamido)

- 1,1,2,2

- tetradeuterioethanol 21.34 0.071 21.26 0.094 ± 0.5 2.433 0.0081 2.414 0.0106 ± 0.05

Injection Internal Standards (compound added after extraction, but prior to injection)

Perfluoro

- n

- [1,2,3,4,5,6,7,8

- 13 C 8 ]octanoic

acid 8.78 0.026 8.80 0.016 ± 0.1

-- -- -- -- --

2H

- Perfluoro

- [1,2

- 13 C 2 ]

- 2

- dece noic acid 9.64 0.037 9.65 0.016 ± 0.1

-- -- -- -- --Draft PFC Procedure 35 December 2011 Not approved for either general purpose or regulatory us e Table 3. A nalytes, 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 (e V) 2nd Qual

Ion Mass

(m/z) 2nd Qual Ion

formula Collision (eV) Quant by: Quantitation Reference

Native PFCs

Perfluoorobutanoic acid 213 169 [CF 3 (CF 2 ) 2 CO 2 ]

- [CF 3 (CF 2 ) 2 ]

- 27 8 ID 13 CF 3 ( 13 CF 2 ) 2 13 COOH

Perfluoropentanoic acid 263 219 [CF 3 (CF 2 ) 3 CO 2 ]

- [CF 3 (CF 2 ) 3 ]

- 27 8 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

Perfluoro

- n

- hexanoic acid 313 269 [CF 3 (CF 2 ) 4 CO 2 ]

- [CF 3 (CF 2 ) 4 ]

- 27 20 119 [CF 3 CF 2 ]

- 8 ID CF 3 (CF 2 ) 3 ( 13 CF 2 ) 13 COOH

Perfluoro

- n

- heptanoic acid 363 319 [CF 3 (CF 2 ) 5 CO 2 ]

- [CF 3 (CF 2 ) 5 ]

- 27 12 169 [CF 3 (CF 2 ) 2 ]

- 8 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

Perfluoro

- n

- octanoic acid 413 369 [CF 3 (CF 2 ) 6 CO 2 ]

- [CF 3 (CF 2 ) 6 ]

- 19 12 169 [CF 3 (CF 2 ) 2 ]

- 12 ID CF 3 (CF 2 ) 3 ( 13 CF 2 ) 3 13 COOH

Perfluoro

- n

- nonanoic acid 463 419 [CF 3 (CF 2 ) 7 CO 2 ]

- [CF 3 (CF 2 ) 7 ]

- 20 13 219 [CF 3 (CF 2 ) 3 ]

- 12 ID CF 3 (CF 2 ) 3 ( 13 CF 2 ) 4 13 COOH

Perfluo ro

- n

- decanoic acid 513 469 [CF 3 (CF 2 ) 8 CO 2 ]

- [CF 3 (CF 2 ) 8 ]

- 21 11 219 [CF 3 (CF 2 ) 3 ]

- 13 ID CF 3 ( 13 CF 2 ) 8 13 COOH

Perfluoro

- n

- undecanoic

acid 563 519 [CF 3 (CF 2 ) 9 CO 2 ]

- [CF 3 (CF 2 ) 9 ]

- 21 15 269 [CF 3 (CF 2 ) 4 ]

- 12 ID CF 3 ( 13 CF 2 ) 9 COOH

Perfluoro

- n

- dodecanoic

acid 613 569 [C F 3 (CF 2 ) 10 CO 2 ]

- [CF 3 (CF 2 ) 10 ]

- 22 15 319 [CF 3 (CF 2 ) 5 ]

- 12 ID CF 3 (CF 2 ) 9 ( 13 CF 2 ) 13 COOH

Perfluoro

- n

- tridecanoic

acid 663 619 [CF 3 (CF 2 ) 11 CO 2 ]

- [CF 3 (CF 2 ) 11 ]

- 20 17 319 [CF 3 (CF 2 ) 5 ]

- 13 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

Perfluoro

- n

- tetradecanoic

acid 713 669 [CF 3 (CF 2 ) 12 CO 2 ]

- [CF 3 (CF 2 ) 12 ]

- 27 21 319 [CF 3 (CF 2 ) 5 ]

- 11 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

Perfluorobutanesulfonic

acid 299 80 [CF 3 (CF 2 ) 3 SO 3 ]

- [SO 3 ]

- 70 40 99 [FSO 3 ]

- 35 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

Perfluoro

- n

- hexane

sulfonic acid 1 399 80 [CF 3 (CF 2 ) 5 SO 3 ]

- [SO 3 ]

- 30 45 99 [FSO 3 ]

- 40 ID CF 3 (CF 2 ) 5 S( 18 O) 2 OH

Perfluoro

- n

- heptane

sulfonic acid 449 80 [CF 3 (CF 2 ) 6 SO 3 ]

- [SO 3 ]

- 50 39 99 [FSO 3 ]

- 38 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

Perfluoro

- n

- octanesulfonic

acid 499 80 [CF 3 (CF 2 ) 7 SO 3 ]

- [SO 3 ]

- 80 45 99 [FSO 3 ]

- 40 ID CF 3 (CF 2 ) 3 ( 13 CF 2 ) 4 SO 3 H

Perfluorooctane

sulf onamide 498 78 [CF 3 (CF 2 ) 7 SO 2 N H]

- [SO 2 N]

- 80 40 478 [(CF 2 ) 8 SO 2 N]

- 16 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

N

- methylperfluoro

- 1

- octanesulfonamide 512 169 [CF 3 (CF 2 ) 7 SO 2 N(CH 3 ) ]

- [CF 3 (CF 2 ) 2 ]

- 27 45 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

N

- ethylperfluoro

- 1

- octanesulfonamide 526 169 [ CF 3 (CF 2 ) 7 SO 2 N(C 2 H 5 ) ]

- [CF 3 (CF 2 ) 2 ]

- 27 45 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

2

- (N

- methylperfluoro

- 1

- octanesulfonamido)

- ethanol 616 59 [CF 3 (CF 2 ) 7 SO 2 N(

CH 3 )C 2 H 4 OH

· CH

3 CO 2 ]

- [CH 3 CO 2 ]

- 27 45 ID CF 3 (CF 2 ) 7 SO 2 N (CD 3 )C 2 D 4 OH

· CH 3 COOH

2

- (N

- ethylperfluoro

- 1

- octanesulfonam ido)

- ethanol 630 59 [CF 3 (CF 2 ) 7 SO 2 N(

C 2 H 5 )C 2 H 4 OH

· C

H 3 CO 2 ]

- [CH 3 CO 2 ]

- 27 45 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOHDraft PFC Procedure 36 December 2011 Not approved for either general purpose or regulatory us e Table 3. A nalytes, 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 (e V) 2nd Qual

Ion Mass

(m/z) 2nd Qual Ion

formula Collision (eV) Quant by: Quantitation Reference

Mass

- labeled PFCs

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]butanoic acid 217 172 [ 13 CF 3 ( 13 CF 2 ) 2 13 CO 2 ]

- [ 13 CF 3 ( 13 CF 2 ) 2 ]

- 27 8 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

Perfluoro

- n

- [1,2

- 13 C 2 ]hexanoic acid 315 270 [CF 3 (CF 2 ) 3 ( 13 CF 2 )

13 CO 2 ]

- [CF 3 (CF 2 ) 3 ( 13 CF

2 ) ]

- 27 8 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

Perfluoro

- n

- [1,2,3,4

- 13

C 4 ]octanoic acid 417 372 [CF 3 (CF 2 ) 3 ( 13 CF 2 ) 3 13 CO 2 ]

- [CF 3 (CF 2 ) 3 ( 13 CF 2 ) 3 ]

- 21 12 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

Perfluoro

- n

- [1,2,3 ,4,5

- 13

C 5 ]nonanoic acid 468 423 [CF 3 (CF 2 ) 3 ( 13 CF 2 ) 4 13 CO 2 ]

- [CF 3 (CF 2 ) 3 ( 13 CF

2 ) 4 ]

- 20 12 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

Perfluoro

- n

- [1,2

- 13 C 2 ]decanoic acid 515 470 [CF 3 (CF 2 ) 7 ( 13 CF 2 )

13 CO 2 ]

- [CF 3 (CF 2 ) 2 ( 13 CF

2 ) ]

- 21 12 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

Perfluoro

- n

- [1,2, 3,4,5,6,7,8,9

- 13 C 9 ]decanoic acid 522 477 [CF 3 ( 13 CF 2 ) 8 13 CO 2 ]

- [CF 3 ( 13 CF 2 ) 8 ]

- 20 12 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

Perfluoro

- n

- [1,2

- 13 C 2 ]undecanoic acid 565 520 [CF 3 (CF 2 ) 8 ( 13 CF 2 )

13 CO 2 ]

- [CF 3 (CF 2 ) 8 ( 13 CF

2 ) ]

- 20 12 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

Perfluoro

- n

- [2,3, 4,5,6,7,8,9,10

- 13 C 9 ]undecanoic acid 572 528 [CF 3 (

13 CF 2 ) 9 CO 2 ]

- [CF 3 ( 13 CF 2 ) 9 ]

- 20 12 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

Perfluoro

- n

- [1,2

- 13 C 2 ]dodecanoic acid 615 570 [CF 3 (CF 2 ) 9 ( 13 CF 2 ) 13 CO 2 ]

- [CF 3 (CF 2 ) 9 ( 13 CF 2 ) ]

- 22 12 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

Perfluoro

- 1

- [ 1,2

- 18 O 2 ]

- hexanesulfonic acid 403 84 [CF 3 (CF 2 ) 5 S( 18 O) 2 O]

- [S( 18 O) 2 O]

- 30 45 103 [FS( 18 O) 2 O]

- 45 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]

- octanesulfonate 503 80 [CF 3 (CF 2 ) 3 ( 13 CF 2 ) 4 SO 3 ]

- [SO 3 ]

- 40 45 99 [FSO 3 ]

- 40 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

2

- (N

deuteriomethylperfluoro

- 1

- octanesulfonamido)

- 1,1,2,2

- tetradeuterioethanol 623 59 [CF 3 (CF 2 ) 7 SO 2 N (CD 3 )C 2 D 4 OH

· C

H 3 CO 2 ]

- [CH 3 CO 2 ]

- 27 45 IS 13 CF 3 ( 13 CF 2 ) 6 13 COOH

Injection Internal Standards (compound added after extraction, but prior to injection)

Perfluor o

- n

- [1,2,3,4,5,6,7,8

- 13

C 8 ]octanoic acid 421 376 [ 13 CF 3 ( 13 CF 2 ) 6 13 CO 2 ]

- [ 13 CF 3 ( 3 CF 2 ) 6 ]

- 21 12 ES

2H

- Perfluoro

- [1,2

- 13 C 2 ]

- 2

- decenoic acid 459 394 [CF 3 (CF 2 ) 6 CF 13 CH 13 CO 2 ]

- [CF 3 (CF 2 ) 3 ( 13 CF 2 ) 3 ]

- 21 11 ES

1 This analyte has a third ion tha t 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 37 December 2011 Not approved for either general purpose or regulatory us e Table 4. Method detection limits, minimum levels of quantitation, and provisional health advisory

Compound MDL (ng/g) ML (ng/g) OW Provisional Health Advisory

Perfluoorobutanoic acid 0.125 0.250 Perfluoropentanoic acid 0.185 0.250

Perfluoro

- n

- hexanoic acid 0.136 0.250

Perfluoro

- n

- heptanoic acid 0.054 0.250

Perfluoro

- n

- octanoic acid 0.085 0.250 0.4 ppb

Perfluo ro

- n

- nonanoic acid 0.127 0.250

Perfluoro

- n

- decanoic acid 0.084 0.250

Perfluoro

- n

- undecanoic acid 0.080 0.250

Perfluoro

- n

- dodecanoic acid 0.067 0.250

Perfluoro

- n

- tridecanoic acid 0.067 0.250

Perfluoro

- n

- tetradecanoic acid 0.028 0.250

Perfluorobu tanesulfonic acid 0.115 0.500

Perfluorohexanesulfonic acid 0.268 0.500 Perfluoroheptanesulfonic acid 0.291 0.500

Perfluorooctanesulfonic acid 0.208 0.500 0.2 ppb

Perfluorooctane sulfonamide 0.040 0.250

N

- methylperfluoro

- 1

- octanesulfonamide 3.65 10.0

N

- ethylperfluoro

- 1

- octanesulfonamide 3.05 10.0

2

- (N

- methylperfluoro

- 1

- octanesulfonamido)

- ethanol 7.65 5.00

2

- (N

- ethylperfluoro

- 1

- octanesulfonamido)

- ethanol 1.88 5.00Draft PFC Procedure 38 December 2011 Not approved for either general purpose or regulatory us e Table 5. Instrument conditions

Instruments Waters Acquity high performance liquid chromatography (HPLC) or equivalent

Waters Quattro Ultima tandem mass spectrometer or equivalent

Trapping cartridge NA

HPLC Column Waters Xtera C18MS analytical column, 100 mm length, 2.1 mm ID , 3.5 μ m particle size, or equivalent

Ionization Ne gative Ion Electrospray

Acquisition MRM mode, unit resolution

Injection V olume 15 μL

Final Extract Volume 1 mL

HPLC Gradient Program HPLC Flow

rate Gradient

Curve

HPLC conditions Time (min) Flow mixture (mL/min)

0.00 A=15%

B=85% 0.150 1 Column Te mp (°C) 40

1.00 A=15%

B=85% 0.150 1 Max Pressure (bar) 345

5.00 A=50%

B=50% 0.200 4

MS Conditions 20.00 A=65%

B=35% 0.200 4

23.00 A=100%

B=0% 0.200 4

26.00 A=100%

B=0% 0.200 4

26.50 A=15%

B=85% 0.200 2 Source Temp (°C) 120

30.00 A=15%

B=85% 0.20 0 2 Desolvation Temp (oC) 325

32.00 A=15%

B=85% 0.150 2 Capillary voltage (kV) 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 39 December 2011 Not approved for either general purpose or regulatory us e Table 6. Concentrations of c alibrat ion s tandards (CS) in pg/mL

CS

- 1 CS

- 2 CS

- 3 CS

- 4 (VER) CS

- 5 CS

- 6 CS

- 7 CS

- 8
Native Analytes

Perfluoorobutanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500
Perfluoropentanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

Perfluo ro

- n

- hexanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

Perfluoro

- n

- heptanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

Perfluoro

- n

- octanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

Perfluoro

- n

- nonanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

Perfluoro

- n

- decanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

Perfluoro

- n

- undecanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

Perfluoro

- n

- dodecanoic acid 125 312.5 1,25 0 5,000 25,000 50,000 125,000 312,500

Perfluoro

- n

- tridecanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

Perfluoro

- n

- tetradecanoic acid 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

Perfluorobutanesulfonic acid 250 625 2,500 10,000 5 0,000 100,000 250,000 625,000

Perfluorohexanesulfonic acid 250 625 2,500 10,000 50,000 100,000 250,000 625,000

Perfluoro

- n

- heptanesulfonic acid 250 625 2,500 10,000 50,000 100,000 250,000 625,000

Perfluorooctanesulfonic acid 250 625 2,500 10,000 50,000 100,000 250,000 625,000

Perfluorooctane sulfonamide 125 312.5 1,250 5,000 25,000 50,000 125,000 312,500

N

- methylperfluoro

- 1

- octanesulfonamide 5

, 000 10

, 000 20,000 40,000 80,000 160,000 320,000

-- N

- ethylperfluoro

- 1

- octanesulfonamide 5

, 000 10

, 000 20,000 40,000 80,000 160,000 320,000

-- 2

- (N

- methylperfluoro

- 1

- octanesulfonamido)

- ethanol 2

, 500 5

, 000 10,000 20,000 40,000 80,000 160,000

-- 2

- (N

- ethylperfluoro

- 1

- octanesulfonamido)

- ethanol 2

, 500 5

, 000 10,000 20,000 40,000 80,000 160,000

-- Labeled Internal St andards

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]butanoic acid 3,000 3,000 3,000 3,000 3,000 3,000 3,000 3,000

Perfluoro

- n

- [1,2

- 13 C 2 ]hexanoic acid 3,000 3,000 3,000 3,000 3,000 3,000 3,000 3,000

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]octanoic acid 3,000 3,000 3,000 3,000 3,000 3,0 00 3,000 3,000

Perfluoro

- n

- [1,2,3,4,5

- 13 C 5 ]nonanoic acid 3,000 3,000 3,000 3,000 3,000 3,000 3,000 3,000

Perfluoro

- n

- [1,2

- 13 C 2 ]decanoic acid 3,000 3,000 3,000 3,000 3,000 3,000 3,000 3,000

Perfluoro

- n

- [1,2,3,4,5,6,7,8,9

- 13 C 9 ]decanoic acid 3,000 3,000 3, 000 3,000 3,000 3,000 3,000 3,000

Perfluoro

- n

- [1,2

- 13 C 2 ]undecanoic acid 3,000 3,000 3,000 3,000 3,000 3,000 3,000 3,000

Perfluoro

- n

- [2,3,4,5,6,7,8,9,10

- 13 C 9 ]undecanoic acid 3,000 3,000 3,000 3,000 3,000 3,000 3,000 3,000

Perfluoro

- n

- [1,2

- 13 C 2 ]dodecanoic acid 3,000 3,000 3,000 3,000 3,000 3,000 3,000 3,000

Perfluoro

- 1

- [1,2

- 18 O 2 ]

- hexanesulfonic acid 4,270 4,270 4,270 4,270 4,270 4,270 4,270 4,270

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]

- octanesulfonic acid 4,310 4,310 4,310 4,310 4,310 4,310 4,310 4,310

2

- (N

- deuterio methylperfluoro

- 1

- octanesulfonamido)

- 1,1,2,2

- tetradeuterioethanol 20,000 20,000 20,000 20,000 20,000 20,000 20,000
Internal Injection Standards

Perfluoro

- n

- [1,2,3,4,5,6,7,8

- 13 C 8 ]octanoic acid 3,000 3,000 3,000 3,000 3,000 3,000 3,000 3,000

2H

- Perfluoro

- [1,2

- 13 C 2 ]

- 2

- decenoic acid 2,500 2,500 2,500 2,500 2,500 2,500 2,500 2,500

1 Calibration standards are stored in ~ 92% MeOH, up to 2.75% Nonane, 2% Propan

- 2

- ol, 1%MeCN, 1% H2O, up to 0.3% Toluene, 0.3% NH 4 OH solution.Draft PFC Procedure 40 December 2011 Not approve d for either general purpose or regulatory us e Table 7. Detailed concentrations of actual working level standards (pg/mL)

Working

- level Standards ( pg/mL )

Native standards Working level Low level

Perfluoorobutanoic acid 125,000 12,500

Perfluoropentanoic acid 125,000 12,500

Perfluoro

- n

- hexanoic acid 125,000 12,500

Perfluoro

- n

- hepta noic acid 125,000 12,500

Perfluoro

- n

- octanoic acid 125,000 12,500

Perfluoro

- n

- nonanoic acid 125,000 12,500

Perfluoro

- n

- decanoic acid 125,000 12,500

Perfluoro

- n

- undecanoic acid 125,000 12,500

Perfluoro

- n

- dodecanoic acid 125,000 12,500

Perfluoro

- n

- trid ecanoic acid 125,000 12,500

Perfluoro

- n

- tetradecanoic acid 125,000 12,500

Perfluorobutanesulfonic acid 250,000 25,000

Perfluorohexanesulfonic acid 250,000 25,000

Perfluoro

- n

- heptanesulfonic acid 250,000 25,000

Perfluorooctanesulfonic acid 250,000 25,0 00

Perfluorooctane sulfonamide 125,000 12,500

N

- Me/Et

- FOSA/Es Natives (NAT2)

N

- methylperfluoro

- 1

- octanesulfonamide 2,500,000

N

- ethylperfluoro

- 1

- octanesulfonamide 2,500,000

2

- (N

- methylperfluoro

- 1

- octanesulfonamido)

- ethanol 1,250,000

2

- (N

- ethylperf luoro

- 1

- octanesulfonamido)

- ethanol 1,250,000

Labeled Internal Standard Solution

Mass Labeled PFCs

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]butanoic acid 30,000

Perfluoro

- n

- [1,2

- 13 C 2 ]hexanoic acid 30,000

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]octanoic acid 30,000

Perfluoro

- n

- [1,2,3,4,5

- 13 C 5 ]nonanoic acid 30,000

Perfluoro

- n

- [1,2

- 13 C 2 ]decanoic acid 30,000

Perfluoro

- n

- [1,2

- 13 C 9 ]decanoic acid 30,000

Perfluoro

- n

- [1,2

- 13 C 2 ]undecanoic acid 30,000

Perfluoro

- n

- [1,2

- 13 C 9 ]undecanoic acid 30,000

Perfluoro

- n

- [1,2

- 13 C 2 ]dodecanoic aci d 30,000

Perfluoro

- 1

- [1,2

- 18 O 2 ]

- hexanesulfonic acid 42,700

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]

- octanesulfonic acid 43,100

2

- (N

- deuteriomethylperfluoro

- 1

- octanesulfonamido)

- 1,1,2,2

- tetradeuterioethanol 200,000

Labeled Injection Standards

Perfluoro

- n

- [1,2,3,4 ,5,6,7,8

- 13 C 8 ]octanoic acid 12,000

2H

- Perfluoro

- [1,2

- 13 C 2 ]

- 2

- decenoic acid 10,000Draft PFC Procedure 41 December 2011 Not approved for either general purpose or regulatory us e 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

R ecovery (%) IPR Recovery (%) OPR Recovery (%) RLS Recovery (%) Recovery in Matrix Spikes and Samples

Low High RSD Low High RSD Low High Low High Low High RPD

Native PFCs

Perfluoorobutanoic acid ID 70 130 20 70 130 20 70 130 50 200 50 150 30

Perfluo ropentanoic acid IS 70 130 20 70 130 20 70 130 50 200 50 150 30

Perfluoro

- n

- hexanoic acid ID 70 130 20 70 130 20 70 130 50 150 50 150 30

Perfluoro

- n

- heptanoic acid IS 70 130 20 70 130 20 70 130 50 150 50 150 30

Perfluoro

- n

- octanoic acid ID 70 130 20 70 130 20 70 130 50 150 50 150 30

Perfluoro

- n

- nonanoic acid ID 70 130 20 50 150 30 50 150 50 150 50 150 30

Perfluoro

- n

- decanoic acid ID 70 130 20 70 130 20 70 130 50 150 50 150 30

Perfluoro

- n

- undecanoic acid ID 70 130 20 70 130 20 70 130 50 150 50 150 30

Perfluoro

- n

- dodecanoic acid ID 70 130 20 70 130 20 70 130 50 150 50 150 30

Perfluoro

- n

- tridecanoic acid IS 70 130 20 20 130 30 20 130 50 150 50 150 30

Perfluoro

- n

- tetradecanoic acid IS 70 130 20 20 130 30 20 130 50 150 50 150 30

Perfluorobutanesulfonic acid IS 70 130 20 60 130 20 60 130 50 150 50 150 30

Perfluorohexanesulfonic acid IS 70 130 20 70 130 20 70 130 50 150 50 150 30
Perfluoroheptanesulfonic acid ID 70 130 20 70 130 20 70 130 50 150 50 150 30

Perfluorooctanesulfonic acid ID 70 130 20 70 130 20 70 130 50 150 50 150 30

Perfluorooctane sulfonamide IS 70 130 20 20 130 30 20 130 50 150 50 150 30

N

- methylperfluoro

- 1

- octanesulfonamide IS 70 130 20 5 130 20 5 130 10 150 5 150 40

N

- ethylperfluoro

- 1

- octanesulfonamide IS 70 130 20 5 130 40 5 130 10 150 5 150 40

2

- (N

- methylperfluoro

- 1

- octanesulfonamido)

- ethanol ID 70 130 20 40 130 30 40 130 50 150 50 150 30

2

- (N

- ethylperfluoro

- 1

- octanesulfonamido)

- ethanol IS 70 130 20 10 130 40 10 130 25 150 10 150 40

Labeled Internal Standards: Compounds added bef ore extraction

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]butanoic acid IS 70 130 20 60 130 20 60 130 60 130 60 130 20

Perfluoro

- n

- [1,2

- 13 C 2 ]hexanoic acid IS 70 130 20 70 130 20 70 130 70 130 70 130 20

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]octanoic acid IS 70 130 20 60 130 20 60 13 0 60 130 60 130 20

Perfluoro

- n

- [1,2,3,4,5

- 13 C 5 ]nonanoic acid IS 70 130 20 50 130 25 50 130 50 130 50 130 25

Perfluoro

- n

- [1,2,3,4,5,6,7,8,9

- 13 C 9 ]decanoic acid IS 70 130 20 60 130 20 60 130 60 130 60 130 20

Perfluoro

- n

- [1,2,3,4,5,6,7,8,9

- 13 C 9 ]undecanoic a cid IS 70 130 20 60 130 20 60 130 60 130 60 130 20

Perfluoro

- n

- [1,2

- 13 C 2 ]dodecanoic acid IS 70 130 20 30 130 20 30 130 30 130 30 130 20

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]

- octanesulfonic acid IS 70 130 20 60 130 20 60 130 60 130 60 130 20

Perfluoro

- 1

- [ 18 O 2 ]

- hexa nesulfonic acid IS 70 130 20 60 130 20 60 130 60 130 60 130 20

2

- (N

- deuteriomethylperfluoro

- 1

- octane sulfonamido)

- 1,1,2,2

- tetradeuterioethanol IS 70 130 20 5 130 70 5 130 5 130 5 130 70

Injection Internal Standard: Compound added after extraction, but pr ior to injection

Perfluoro

- n

- [1,2,3,4,5,6,7,8

- 13 C 8 ]octanoic acid ES 80 120 10 80 120 10 80 120 80 120 80 120 10

Quantitation methods: ID = Isotope d ilution IS = Internal s tandard ES = External s tandardDraft PFC Procedure 42 December 2011 Not approved for eith er general purpose or regulatory us e Table 9

. Sources used for standards, materials

, a nd equipment during method development 1

Native Compounds Vendor Part Number Shelf Life

Perfluoorobutanoic acid Sigma

- Aldrich 164194 To be determined

Perfluoropentanoic acid Sigma

- Aldrich 77285 To be determined

Perfluoro

- n

- hexanoic acid Sigma

- Aldrich 29 226 To be determined

Perfluoro

- n

- heptanoic acid Sigma

- Aldrich 34,204

- 1 To be determined

Perfluoro

- n

- octanoic acid Sigma

- Aldrich 17,146

- 8 To be determined

Perfluoro

- n

- nonanoic acid Sigma

- Aldrich 77284 To be determined

Perfluoro

- n

- decanoic acid Sigma

- Ald rich 17,774

- 1 To be determined

Perfluoro

- n

- undecanoic acid Sigma

- Aldrich 446777 To be determined

Perfluoro

- n

- dodecanoic acid Sigma

- Aldrich 40,644

- 9 To be determined

Perfluoro

- n

- tridecanoic acid Sigma

- Aldrich 654973 To be determined

Perfluoro

- n

- tetradec anoic acid Sigma

- Aldrich 446785 To be determined

Perfluorobutanesulfonic acid (Tetrabutylammonium salt) Sigma

- Aldrich 86909 To be determined

Perfluorohexanesulfonic acid (Potassium salt) Sigma

- Aldrich 50929 To be determined

Perfluoro

- n

- heptanesulfonic a cid (Sodium salt) Wellington Labs L

- PFHpS To be determined

Perfluorooctanesulfonic acid (Potassium salt) Sigma

- Aldrich 77282 To be determined

Perfluorooctane sulfonamide Chiron 2043.8 To be determined

N

- methylperfluoro

- 1

- octanesulfonamide Wellington L abs N

- MeFOSA To be determined

N

- ethylperfluoro

- 1

- octanesulfonamide Wellington Labs N

- EtFOSA To be determined

2

- (N

- methylperfluoro

- 1

- octanesulfonamido)

- ethanol Wellington Labs N

- MeFOSE To be determined

2

- (N

- ethylperfluoro

- 1

- octanesulfonamido)

- ethanol Wel lington Labs N

- EtFOSE To be determined

Labeled Internal Standards

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]butanoic acid Wellington Labs MPFBA To be determined

Perfluoro

- n

- [1,2

- 13 C 2 ]hexanoic acid Wellington Labs MPFHxA To be determined

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]octan oic acid Wellington Labs MPFOA To be determined

Perfluoro

- n

- [1,2,3,4,5

- 13 C 5 ]nonanoic acid Wellington Labs MPFNA To be determined

Perfluoro

- n

- [1,2

- 13 C 2 ]decanoic acid Wellington Labs MPFDA To be determined

Perfluoro

- n

- [1,2,3,4,5,6,7,8,9

- 13 C 9 ]decanoic acid CIL CLM

- 8172

- S To be determined

Perfluoro

- n

- [1,2

- 13 C 2 ]undecanoic acid Wellington Labs MPFUdA To be determined

Perfluoro

- n

- [2,3,4,5,6,7,8,9,10

- 13 C 9 ]undecanoic acid CIL CLM

- 8240 To be determined

Perfluoro

- n

- [1,2

- 13 C 2 ]dodecanoic acid Wellington Labs MPFDo A To be determined

Perfluoro

- 1

- [1,2

- 18 O 2 ]

- hexanesulfonic acid Wellington Labs MPFHxS To be determined

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]

- octanesulfonic acid Wellington Labs MPFOS To be determined

2

- (N

- deuteriomethylperfluoro

- 1

- octanesulfonamido)

- 1,1,2,2

- tetrade uterioethanol Wellington Labs d7

- N

- MeFOSE

- M To be determined

Injection Internal Standards

Perfluoro

- n

- [1,2,3,4,5,6,7,8

- 13 C 8 ]octanoic acid CIL CLM

- 8005

- S To be determined

2H

- Perfluoro

- [1,2

- 13 C 2 ]

- 2

- decenoic acid Wellington Labs MFOUEA To be determined

1 Provided for informational purposes only. Part numbers subject to change. Other suppliers may have equivalent materials.Draft PFC Procedure 43 December 2011 Not approved for eith er general purpose or regulatory us e Table 1 0

. Performance d ata from single

- laboratory validation

Compound Transition Solid IPR, n=4 Sludge, n=6

Mean

% Rec Standa rd

Deviation %RSD Mean

% Rec Standard

Deviation %RSD

Native PFCs

Perflu oro butanoic acid Primary 105 1.61 1.54 95.8 9.55 9.97
Perfluoropentanoic acid Primary 114 4.46 3.81 56.4 31.4 55.6

Perfluoro

- n

- hexanoic acid Primary 116 2.28 1.98 115 9.83 8.52

Per fluoro

- n

- heptanoic acid Primary 95.6 3.25 3.40 91.1 16.3 17.9

Perfluoro

- n

- octanoic acid Primary 108 5.59 5.20 111 7.57 6.83

Perfluoro

- n

- octanoic acid Secondary 109 8.65 7.96 119 14.9 12.6

Perfluoro

- n

- nonanoic acid Primary 102 13.1 12.7 97.3 16.8 17.2

P erfluoro

- n

- decanoic acid Primary 101 2.37 2.34 111 8.41 7.59

Perfluoro

- n

- undecanoic acid Primary 101 2.20 2.18 104 7.35 7.09

Perfluoro

- n

- dodecanoic acid Primary 106 2.64 2.48 109 6.50 5.96

Perfluoro

- n

- tridecanoic acid Primary 41.3 2.41 5.83 126 29.1 23. 1

Perfluoro

- n

- tetradecanoic acid Primary 48.6 4.52 9.30 131 16.1 12.2

Perfluorobutanesulfonic acid Primary 84.0 5.09 6.07 69.0 17.1 24.7

Perfluorobutanesulfonic acid Secondary 86.3 5.55 6.43 69.9 18.8 26.9

Perfluoro

- n

- hexane sulfonate Primary 101 2.18 2.16 105 11.0 10.5

Perfluoro

- n

- hexane sulfonate Secondary 105 4.18 4.00 107 11.5 10.8

Perfluoro

- n

- heptane sulfonate Primary 83.7 14.6 17.4 90.2 7.72 8.57

Perfluoro

- n

- heptane sulfonate Secondary 93.5 4.95 5.30 92.7 5.28 5.70

Perfluoro

- n

- octanesulfonate Primary 98.1 5.64 5.75 97.3 8.36 8.59

Perfluoro

- n

- octanesulfonate Secondary 102 4.30 4.21 99.6 7.32 7.35

Perfluorooctane sulfonamide Primary 39.5 3.69 9.33 50.7 3.63 7.15

N

- methylperfluoro

- 1

- o ctane sulfonamide Primary 9.33 0.542 5.81 48.8 13.3 27.2

N

- e thylperfluoro

- 1

- octanesulfonamide Primary 10.1 0.939 9.31 14.6 3.75 25.7

2

- (N

- methylperfluoro

- 1

- octane sulfonamido)

- ethanol Primary 87.6 10.6 12.1 96.9 8.20 8.46

2

- (N

- ethylperfluoro

- 1

- octane sulfonamido)

- ethanol Primary 21.8 1.37 6.27 50.3 13.6 27.0

Lab eled Internal Standards

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]butanoic acid Primary 95.2 6.84 7.19 81.6 37.5 45.9

Perfluoro

- n

- [1,2

- 13 C 2 ]hexanoic acid Primary 98.9 2.55 2.58 72.0 18.2 25.3

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]octanoic acid Primary 90.4 6.62 7.32 96.1 5.03 5.23

Perfluoro

- n

- [1,2,3,4,5

- 13 C 5 ] nonanoic acid Primary 85.6 8.35 9.75 92.5 8.80 9.52

Perfluoro

- n

- [1,2,3,4,5,6,7,8,9

- 13 C 9 ] decanoic

acid Primary 85.2 4.61 5.41 107 11.1 10.4

Perfluoro

- n

- [2,3,4,5,6,7,8,9,10

- 13 C 9 ]

undecanoic acid Primary 76.7 0.853 1.11 80.3 28.9 36.0

Perfluoro

- n

- [1,2

- 13 C 2 ]dodecanoic acid Primary 53.5 3.50 6.54 109 13.1 12.0

Perfluoro

- n

- [1,2,3,4

- 13 C 4 ]

- octane sulfonic

acid Primary 76.9 4.82 6.27 78.8 7.97 10.1

Perfluoro

- 1

- [1,2

- 18 O 2 ]

- hexane sulfonic acid Primary 76.8 3.33 4.33 79.4 7.73 9.74

2

- (N

- deuteriomethylperfluoro

- 1

- octanesulfon amido)

- 1,1,2,2

- tetradeuterioethanol Primary 18.3 3.32 18.2 45.5 13.0 28.6

Injection Internal Standards

Perfluoro

- n

- [1,2,3,4,5,6,7,8

- 13 C 8 ] octanoic

acid Primary 108 11.0 10.2 84.5 7.42 8.79Draft PFC Procedure 44 December 2011 Not approved for eith er general purpose or regulatory us e 

### Figure 1. Flow c hart for d etermination of PFCs by LC/MS/MS 11.3.1 Place 0.5 g of w et

s ludge in a 16

- mL

po lypropylene centrifuge

tube

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) o vernight

incubation.

11.3.8 Neutraliz e with HCl

1 1. 4.1 . Add 10 mL

50:50/A C N:MeOH (v/ v)

11. 4.2 Shake moderately

for 1 h

11. 4

. 3 Centrifuge at 3000 x g for 20 min

11. 4.4 . Decant 10 mL supernatant and collect in

250

- mL HDPE bottle containing 180 mL of

r eagent water 

### Cleanup

11.4 .6 Adjust pH of diluted ex tract
to 6.5 ± 0.5 with 3% acetic

acid or 0.3% NH 4 OH

, as needed

11. 4.7 . Sonication (30 min)

11. 5 . WAX Cartridge Cleanup 1) Condition SPE cartridge

with 5 mL 0.3% NH 4 OH in

MeOH and 5 mL 0.1 M

form ic acid 2) Equilibrate with 5mL
reagent water 3) Load sample drop

- wise (~5 mL/min) 4) Wash with 5 mL 20%

MeOH in 80% 0.1 M

formic acid 5) Dry SPE for 5 min under

vacuum 6) Elute with 4 mL 0.3%

NH 4 OH in MeOH

11. 6 .1 Reduce to near dryness (~50 uL) under N 2

and water bath at 40°C 

### Instrumental

11. 6 .2. Reconstitute

with 938 uL of 0.3%

NH 4 OH in MeOH and

vortex mix

11. 6

. 3 Filter extract using 0.45

- μm n ylon

membranes filter

1 4 .0. LC / MS/MS analysis o f 20 μ L of

extract 

### Determine % Solids (Sec. 11.2)11.4.5Repeatedonce 

### Digestion and

Extraction

11. 4.5 . Collect 20 mL total

in 250

- mL HDPE bottle containing 180 mL reagent

water 11. 6 .3 Spike 12.5 μ L

of internal standard

and vortex mix

1 5 .0. Quantify target

PFCsDraft PFC Procedure 45 December 2011 Not approved for either general purpose or regulatory us e 

### Figure 2. Example Chromatogram for CS

- 4 Calibration Standard Time 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00%

0N-EtFOSAN-EtFOSEN-MeFOSAN-MeFOSEPFTeDAPFOSA and PFTriDAPFOSAPFDoDAPFOSPFDAPFUnDAPFHpSPFNAPFHxSPFOAPFHpAPFBS and PFHxAPFPAPFBADraft PFC Procedure 46 December 2011 Not approved for either genera l purpose or regulatory us e 

## APPENDIX 1

Details on Preparation of StandardsDraft PFC Procedure 47 December 2011 Not approved for either genera l purpose or regulatory us e 

### Appendix 1. Details on Preparation of Standards

Individual P FCs 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. [ 13 

### C 9 

### ]PFUnDA was obtained as a solid

.

Native PFHpS and labeled compounds except [ 13 

### C 9 

### ]PFUnDA, were obtained as 50μg/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 50μg/mL solutions in 90% Nonane/ 10% Toluene

.

1. Use Fisher brand, Plasma grade water ( has 18 MΩ 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%

NH 4 

### OH (basic methanol) mixes and store in dedicated 3X methanol

- washed 1L HDPE s olvent

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 mo le equivalents of NaOH in methanol was added to reduce esterification in accordance with purchased standards from Wellington. The same procedure was used for [ 13 

### C 9 

### ]PFUnDA except 2 mg in a 5 mL flask was used. PFTeDA is much less soluble than the other comp ounds 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 rinsi ng with 20X Toluene, 20X H exane 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 stand ard 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/μL for the labeled carboxylic and sulfonic acids, plus 200 pg/μL of d 7 

### - 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 μg/mL d 7 

### - N

- MeFOSE so that it will be diluted 200X and dilute to mark with the prepared

basic methanol mix.Draft PFC Procedure 48 December 2011 Not approved for either genera l purpose or regulatory us e 

### 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 13 

### C 8 

### - Perfluorooctanoic acid and 200 ng/mL 2 

### H

- Perfluoro

- [1,2

- 13 

### C 2 

### ]

- 2

- decenoic acid

. Using a cleaned glass syringe that is reserved for mass labeled standard s, 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 o f 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 μg/mL for each carboxylic acid and 100 μg/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 μg/mL for each carboxylic acid and 5 μg/mL for each sulfonic acid. Using a cleaned g lass 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 o f 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 methan ol 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 μg/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 Mi x and dilute to mark with the prepared basic methanol mix so that it is

diluted 200X.Draft PFC Procedure 49 December 2011 Not approved for either genera l purpose or regulatory us e 

### 6. Prepare Calibration Standards ( s ee Table 6 in the body of the procedure )

Label baked disposable 12mL glass vials. Using disposable tip pipettors, transfer a 200μL port ion of the Labeled Internal Standard Solution and 25 μL 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 sta ndard 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 Internal

Standard

Solution (LINT)

μL Stock Mass

Labeled injection

internal standard

mix (INJ) μL Low level

native

standard μL Working

level native

standard (NAT)

μL Full

Native

Stock Mix μL N

- Me/Et

FOSA/Es

native standard (NAT2 ) μL Final

Volume μL

CS

- 1 200 25 20 4 2000

CS

- 2 200 25 50 8 2000

CS

- 3 200 25 20 16 2000

CS

- 4 200 25 80 32 2000

CS

- 5 200 25 20 64 2000

CS

- 6 200 25 40 128 2000

CS

- 7 200 25 100 256 2000

CS

- 8 200 25 250 2000Draft PFC Procedure 50 December 2011 Not approved for either genera l purpose or regulatory us e 

## APPENDIX 2

Procedure for Polishing Deionized WaterDraft PFC Procedure 51 December 2011 Not approved for either genera l purpose or regulatory us e 

### 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 p reparation of strong needle wash solutions, seal wash solutions, 60:40

ACN:H 2 

### O standards solutions LC aqueous mobile phase and during the extraction and cleanup procedure.

1.0 Equipment for polishing water

1.1 Cartridge for polishing 18MΩ water

– Water s Oasis 35cc (6g) HLB Extraction Cartridge.

1.2 Glassware for polishing 18MΩ water

– Fisher Scientific, 2000 mL Kimax Brand Volumetric Flask (or equivalent), Fisher Scientific: 2000 mL Pyrex Filtering Flask (or equivalent) and Fisher Scientific Glass Magn etic 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 18MΩ (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. Set u p for Water Polishing