Document 5LJwZMwEJypnZ2g8v5m7QX2q5

3 Edition 1.0 Nov 2016 STANDARD TEST METHOD Determination of PFOA in articles of commerce: Disassembly, disjointment and mechanical preparation of solid samples, sample preparation of aqueous polymer systems, sample extraction, and LC/MS/MS quantitative analysis. Table of Content: Introduction Part 1: Disassembly, disjointment and mechanical sample preparation Part 2: Sample Extraction Part 3: Instrumental Analysis Bibliography Page 1 3 11 13 17 INTRODUCTION The use of perfluoroalkyl substances (PFAS) in manufacturing processes and for treatment of articles of commerce has drawn increased attention to their impact on human health and the environment. In many countries this has resulted in the adaptation of regulations affecting the acceptable levels of certain PFAS in articles of commerce. The current EU REACH Annex XV proposal to limit PFOA in products to less than 25 parts per billion, poses particular technical challenges to determine conformance. Careful sample preparation, extraction, and analysis are critical to generating reliable data. The purpose of this document is to provide a test method which will allow analytical laboratories to determine low levels (ppb) of PFOA in articles of commerce on a consistent and reliable basis. This method is a performance based method which emphasizes the use of laboratory matrix spike samples (LMS) fortified with a known quantity of PFOA as the primary quality control (QC) samples used to verify adequate method performance. Other QC components include the use of 13C4-PFOA surrogate recovery standard (SRS), or other isotope labeled analytes that are not used as internal standards (ISs), and fortified into samples prior to preparations, extraction and analysis. Recommended acceptance criteria for reportability of results are also provided and are based on the successful implementation of this method in the laboratory to the analysis of select fluoropolymer substances. The applicability of this method to the analysis of other PFAS substances in articles of commerce should meet the same acceptance criteria as demonstrated by the adequate recovery of the PFAS target analytes from LMS samples and/or recovery of SRSs from samples. The method is outlined in 3 Parts: Part 1 provides the strategies for sampling of articles of commerce along with the mechanical preparation of the samples, Part 2 describes the sample extraction and Part 3 describes the instrumental analysis. WARNING - Persons using this method should be familiar with normal laboratory practices. This standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the laboratory staff and their management to establish appropriate safety and health practices and to ensure compliance with any applicable regulatory requirements. 2 / 17 Part 1: Disassembly, disjointment and mechanical sample preparation 1.1 Scope P a r t 1 o f t h i s m e t h o d provides strategies for sampling of articles of commerce along with the mechanical preparation of t h e samples based on IEC standard methods1 a n d p r e c e d e s Part 2 (Sample Extraction) and Part 3 (Instrumental Analysis) that are based on additional published methods2,3 and are described later in this method. This standard does not provide: - Guidance on each and every product that could be classified as an article of commerce. Since there is a huge variety of commercial products available and they have various structures and processes, along with the continuous innovations in the industry, it is unrealistic to attempt to provide procedures for the disjointment of every type of commercial product available to the general public. - Sampling procedures for packaging and packaging materials - Guidelines for assessment of compliance. 1.2 Terms, definitions and abbreviations 1.2.1 Terms and definitions For the purposes of this document, the following definitions apply. 1.2.1.1 Article The reference to article, or article of commerce, refers to any commercially available product for which the determination of the amount of a substance present is desired to be determined. 1.2.1.2 Composite testing The testing of two or more materials from a single commercial product that is treated as a single sample, but could be mechanically disjointed if necessary 1.2.1.3 Homogenous Sample A homogenous sample can be described as a liquid or sufficiently sized resin or powder or a suspension of fine particles (aqueous, or other). Complex solid articles not of this type will typically require mechanical processing. 1.2.1.4 Certain PFAS substance For this standard method the discussion of certain substances is reserved for PFOA and other PFAS substances that are be quantified in the article of interest. 1.2.2 Abbreviations PFOA: PFAS: LLOQ: Perfluorooctanoic acid and its salts Perfluoroalkyl substances Lower Limit of quantitation 3 / 17 1.3 Introduction to sampling strategy 1.3.1 Introductory remark Obtaining a commercial sample (i.e. sampling) is the first step in analyzing a commercial product for the presence of certain substances. The strategy and process of sampling can be as important as the analytical measurement itself. Hence an effective sampling strategy requires a clear understanding of the product of interest, including its sold state (film, formed article, resin, powder, liquid, etc.), where and how it is sold, reasons for the analysis and the requirements that are to be met. More detailed guidance on effective sampling strategies can be found in IEC 62321-2:2013. 1.4 Sampling Commercial articles (products) for sampling can be acquired via an indirect source (store, online, etc.) or may be acquired directly from the manufacturer. This standard does not provide detailed guidance for how to obtain articles of commerce. 1.4.1 Complete product Sampling a complete product is the first, non-destructive step in the iterative sampling strategy where representative parts of the product can be analyzed in their present form without disassembly or disjointment. However, "complete product" is a relative term. For example, a serge thread on a spindle is a complete product for its manufacturer, but the thread may be just a component of a sewn jacket. Complete products may be evaluated without disassembly if they have a very simple construction, or if the locations of expected certain substances are known and it is possible to test for them without disassembly. 1.4.2 Partial product disassembly The product is disassembled down to its major components and subassemblies and each tested separately, where possible. See Annex E of IEC 62321-2:2013 for more information. 1.4.3 Complete product disassembly Complete disassembly separates all components as far as possible, while still permitting reassembly to give an operational product. See Annex E of IEC 62321-2:2013 for more information. 1.4.4 Partial disjointment Frequently, the detailed analysis of a complete product for certain substances requires disassembled subassemblies and components to be further separated by disjointment. However, it may be impractical for such a product to be disjointed completely into their constituent homogeneous materials. Hence sampling and partial disjointment of subassemblies and components shall typically not be done. Note: After disjointment, the separated subassemblies and/or components would not be able to be reassembled into an operational state. 1.4.5 Complete disjointment The objective of complete disjointment is the complete separation of all components into their homogeneous materials, and is typically not recommended for most commercial products. Generally, this is not practical because "complete disjointment" extends only as far as the separation of components into their homogeneous materials as far as the available tools and techniques permit. 4 / 17 1.4.6 Considerations of sampling and disjointment 1.4.6.1 Special remark Disassembly and disjointment were discussed from the high level down to the detailed level. At the detailed level, certain difficulties become apparent. In theory, sampling to the homogeneous level could continue down to the nano-scale. In practice, however, such sampling is at best very difficult or unacceptably time consuming. Test method detection limits are dependent on sample masses, sizes or volumes. Testing below these masses, sizes, or volumes can render the test method unsuitable to confirm the presence or absence of a given substance below the allowable level. Sampling and analysis of certain substances at the homogeneous material level are often difficult and a proper understanding of the basis of interpretation is crucial, particularly when dealing with composite samples. 1.4.6.2 Sample Size Required Based on practical experience a recommended minimum sample amount is 10 grams of sample for analysis. Samples with high concentrations of certain substances will require dilution which can increase LOQs. In general, every combination of matrix, sample preparation technique and analytical method will have a minimum sample size required to achieve a particular reporting limit. As the size of the sample is reduced, the reporting limit increases. It is important to understand the relationship of sample size and reporting limit for the matrix, sample preparation technique and analytical method used to determine a certain substance. For this method, the minimum sample size needed for a single replicate preparation is 1 gram (1 mL liquids). 1.4.6.3 Determination of Sampling Position for Homogeneous Materials Although commercial homogenous articles such as liquids, powders, resins and milled articles share the same physical or chemical properties, their compositions are not always completely uniform. It is advisable to shake all samples on a tumbling shaker for > 1 hour to homogenize as much as possible prior to sampling. 1.5 Conclusions and Recommendations for Sampling The strategy and process of sampling are critical preliminary steps in the valid analysis of certain substances in commercial products. The quality of analytical results and specifically their representativeness of the analyzed sample are directly affected by the sampling technique. Selection of the sampling strategy depends on the substance(s) to be determined, their allowable limits, and the basis on which the restrictions are applied and possible exemptions. 5 / 17 1.6 Mechanical sample preparation 1.6.1 Overview 1.6.1.1 Field of Application This standard provides strategies on processing samples obtained from an article of commerce for the purpose of determining certain substances by analytical means. Common techniques are covered for mechanical size reduction of articles, their sub- units or portions thereof, prior to the determination of certain substances in the article or article components via extraction and quantitative analysis. Polymer powders, nano-materials, fibers, pastes, etc. may not require size reduction if they are known to be sufficiently homogenous and more than 95% of the material passes a 250 m sieve. Examples of common articles requiring milling prior to extraction and analysis are compacted cakes, fabrics, and agglomerates, melt extruded pellets, fabrics, elastomers and molded articles. The user shall elect to apply one or more of the approaches, or compendium of approaches, to create homogenous samples suitable for extraction and analysis. Selection of the appropriate technique(s) depends on the article. Alternative methods of mechanical sample preparation can be used, provided that the required particle size (avg. particle size approx. 100 m) of the sample is achieved without contaminating or compromising the sample. 1.6.1.2 Quality Assurance Due to the risk of analytical bias resulting from contamination or from loss of material through dust emissions, it is important to select the appropriate equipment and cleaning procedures. The laboratory shall demonstrate by experiment that a mechanical process does not result in contamination by, or loss of, detectable amounts of certain substances. Similarly, that the procedure employed for cleaning the mechanical sample preparation equipment prevents contamination of the sample with certain substances from the previous sample (carry-over). For example, processing and analyzing certified reference materials and blanks before or after processing a material known to contain significant levels of certain substances may be necessary. Use of certified reference materials is not mandatory. However, the materials used shall have a known regulated substance content to determine that the mechanical sample preparation and cleaning processes do not cause contamination or loss of regulated substances. The effectiveness of the mechanical sample preparation technique shall be continuously monitored by using quality control practices, including laboratory matrix spikes (LMS) and/or other quality control (QC) parameters (e.g. use of surrogate recovery standards, like 13C4-PFOA or other isotope labeled analytes not used as internal standards). 1.6.2 Sample Processing Equipment and Materials The following sample processing apparati, equipment and materials are required, subject to the type of material being prepared. Conditions for cryogenic fine milling are provided for the Retsch CryoMill and the SPEX Freezer/Mill, however, alternative cryomilling equipment may be used to generate similar sized particles. a) Scissors or heavy plate shears b) Retsch ZM 200 Grinding Mill (Centrifugal Mill) with 6 or 12 tooth stainless steel (SS) rotor and 1 mm stainless steel bottom sieve c) Retsch CryoMill (Ball Mill), with SS grinding cartridge and 0.25 mm grinding ball (50 mL grinding cartridge, 5 g fill capacity), or equivalent cryogenic mill capable of grinding to < 100 m particles. c) Shaker, end-over-end tumbling shaker or reciprocating shaker are sufficient. d) Analytical balance capable of weighing accurately to 0.0001 g e) Cleaning brushes (different sizes) and Polishing cloths, or cotton balls/swabs g) Assorted glassware (beakers, etc.) h) Liquid nitrogen (LN2) i) HDPE bottles (assorted sizes) j) Various organic solvents for cleaning mill parts (high purity methanol or ethanol, acetone, ASTM 6 / 17 Type I water) k) Ventilated hood or respirator, to minimize exposure to fine particles/dusts/aerosols l) Proper Laboratory Attire: Safety glasses, Laboratory Coat, Nitrile Gloves, etc. m) Ventilated Drying oven capable of > 200C n) Precision sieves and accessories, nickel mesh, 50 m and 150 m sizes 1.6.3 Sample Processing Procedures 1.6.3.1 Manual cutting If possible, manual cutting with shears or scissors is suitable for rough cutting and preparation of samples for further reduction by grinding, etc. Recommended maximum sample sizes is 10 x 10 mm, but will depend on the specification of the equipment used in the subsequent preparation processes: 1.6.3.2 Coarse grinding/milling Metal components of the articles (i.e. buttons, zippers, bare wire) which would be considered free of certain substances should be removed prior to sample preparation to avoid damage to the centrifugal grinding mill teeth. Processing of metallic samples is not covered by this method. Coarse grinding is suitable for reducing medium to hard samples to ~ 1 mm in diameter for later fine grinding to be more effective. Cool soft samples with the LN2 or dry ice prior to grinding. For organic samples, cryogenic treatment prior to milling is recommended. An example of cryogenic preparation is to put the samples in a polyethylene bottle to cool with LN2. Wait until the LN2 has dissipated and then grind the samples in the grinding mill using a 4 mm stainless steel sieve. During grinding, maintain a sample temperature of < -20 C. Carefully sweep out and collect all particles. Refit the mill with a pre-weighed 1 mm stainless steel bottom sieve and reprocess the 4 mm material in similar fashion. Carefully sweep out the mill and collect all particles. Use a 5 min cooling period between grinding cycles. Reweigh the sieve to determine the quantity of < 1 mm particles captured. Thorough cleaning of the mill components between samples is crucial to avoid cross-contamination of certain substances as described in 1.6.3.4. 1.6.3.3 Fine grinding cryogenic milling Some cryogenic mills are capable of handling materials < 10 mm and can perform both course grinding and fine grinding via use of interchangeable grinding wheels/balls and sieves. Fine grinding via cryogenic milling is suitable for further reducing samples to ~ 100 m in diameter. Be careful not to allow the LN2 to come into direct contact with the sample in order to prevent spattering and sample loss. Cryogenic milling can be accomplished using a Retsch CryoMill (ball mill) with automatic LN2 feed, as described below: Equipment: Retsch CryoMill with 50 mL SS grinding cartridge-Fill quantity: 5g, and 1 SS grinding ball 25mm and LN2 automatic feed with LN2 reservoir. Sample Type Elastomers, Compacted Cake, Agglomerates Melt extruded pellets and molded articles PCT: Precooling time (automatic set) MT: Milling time ICT: In-between cooling time Cycles (No.) 5 7 Frequency (hz) 30 30 PCT MT ICT (minutes) (minutes) (minutes) Auto 2 2 Auto 4 2 7 / 17 Cryogenically mill the sample powder and sweep out the milled material to collect all the powder. The collected material should be precision sieved to obtain a sufficiently homogeneous portion of known particle size range of 50 m to 150 m, by first sieving material through the 150 m sieve, then the 50 m sieve (keep contents captured on 50 m sieve). Size range is 100 + 50 m (i.e. ~ 100 m). Thorough cleaning of the mill components between samples is crucial to avoid cross-contamination of certain substances as described in 1.6.3.4. 1.6.3.4 Cleaning Procedure for Mill Components Thorough cleaning of the mill components between samples is crucial to avoid cross-contamination of certain substances. The following cleaning procedure is recommended: 1) Remove residual polymer thoroughly with DI water and brush 2) If necessary, wipe away last polymer residue with clean polish cloth or cotton ball/swab 3) Rinse all parts with ethanol, use an ultrasonic bath as needed 4) Rinse all parts with DI Water, use an ultrasonic bath as needed 5) Rinse all parts with acetone and allow to dry 6) Repeat process 1-5 as needed. Additionally, SS mill parts can be more thoroughly cleaned of certain substances by heating at > 200C in a ventilated drying oven for 2 hours. 1.7 Sample preparation of aqueous polymer systems/dispersions 1.7.1 Overview 1.7.1.1 Field of Application Aim of this procedure is the preparation of extractable solid polymer samples from aqueous polymer systems like polymer lattices or polymer dispersions used in coating applications under mild drying conditions to prevent loss of (volatile) analytes. . 1.7.1.2 Quality Assurance Due to the risk of analytical bias resulting from contamination or from loss of material through emissions, it is important to select the appropriate equipment and cleaning procedures. The laboratory shall demonstrate by experiment that the freeze drying process does not result in contamination by, or loss of, detectable amounts of certain substances. Similarly, that the procedure employed for cleaning the freeze drying equipment prevents contamination of the sample with certain substances from the previous sample (carry-over). For example, processing and analyzing certified reference materials and/or blanks before or after processing a sample known to contain significant levels of certain substances may be necessary. Use of certified reference materials is not mandatory. However, the materials used shall have a known regulated substance content to determine that the freeze drying processes do not cause contamination or loss of regulated substances. The effectiveness of sample preparation by freeze drying shall be continuously monitored by using quality control practices, including LMS, and/or other QC evaluations (e.g. use of surrogate recovery standards, like 13C4-PFOA or other isotope labeled analytes not used as internal standards). 8 / 17 1.7.2 Sample processing equipment Freeze drying equipment usable for drying aqueous polymer samples may strongly vary from lab to lab, so only a general description is given. The freeze drying process normally requires the following equipment (list may not be complete and has to be adjusted to the needs / prerequisites of the laboratory): a) Uncontaminated sample containers providing a large sample surface, e.g. wide mouth glass or plastic vials / bottles, aluminum bowls etc. (preferable single use equipment to avoid carry-over of analytes) b) Freeze dryer (commercial instrument e.g. Labconco Freezone Benchtop Systems or equivalent) or improvisational equipment (e.g. desiccator with vacuum hose, cooling trap and rotary vacuum pump) c) Deep freezer / cooling device to prepare deep frozen aqueous polymer samples d) Optional: cooling medium for cooling trap if used, e.g. acetone/dry ice or liquid nitrogen The equipment also strongly depends on the sample size / amount of polymer that has to be dried. As large samples volumes with small surface areas need very long drying times, splitting of big samples into a higher number of small volume samples may be favorable. 1.7.3 Sample processing procedures 1.7.3.1 Freeze drying process As the freeze drying procedure and conditions also strongly depend on the equipment in use, a generic description of the process is provided. Add surrogate recovery standards (SRS) e.g. 13C4-PFOA or other standards in the desired concentration to the aqueous polymer system and homogenize the sample, for example by gentle shaking. Vigorous shaking and stirring has to be avoided, along with addition of larger amounts of standard solutions in organic solvents (e.g. Methanol), as coagulation of the polymer may occur resulting in inhomogeneous samples and strongly increased polymer particle sizes. Additionally the freezing temperature may significantly decrease by adding organic solvents, making it hard or impossible to freeze the samples. Deep freeze the sample(s) until completely frozen, ideally producing large area to volume ratios, e.g. by rolling of samples to produce a film on the vessel walls (see illustration) or by using flat bowls with large surface areas. Source: "A Guide To Freeze Drying for the Laboratory" Labconco Corporation 8811 Prospect Avenue Kansas City, Missouri 64132-2696 816-333-8811, 800-821-5525 FAX: 816-363-0130 www.labconco.com 2010 by Labconco Corporation Printed in the U.S.A. 353-8/10-James-3M-R8 Once the samples are frozen, they can be attached / inserted into to the freeze drying equipment and vacuum is applied until the water is completely removed from the samples. According to equipment and sample amount this may take several hours or even days. If the freeze drying setup includes a cooling trap, ensure the trap is not blocked by ice and sufficient cooling medium is always present. Note: The content of the cooling trap can be used to evaluate possible loss of compounds of interest by analyzing the water and balancing the total mass and mass of sample and evaporated water after drying. In case the drying process was ended before dryness of the samples, keep the samples frozen, or freeze again if thawed, and continue the drying procedure until the samples are completely dry. 9 / 17 1.7.3.2 Cleaning procedure To avoid contamination of samples and carry-over, use new clean single use equipment wherever possible, otherwise refer to section 1.6.3.4 Cleaning Procedure for Mill Components ("Cleaning Procedure for Mill Components") for possible cleaning methods. Before using organic solvents to clean freeze drying equipment, check the material compatibility to avoid any damage. 10 / 17 2.1 Scope Part 2: Sample Extraction Part 2 of this method provides direction for the extraction of certain substances from the prepared samples generated in Part 1. Part 3 (Instrumental Analysis Parameters) are described later. The extraction procedures are based on the extraction described by Liu et al. 2009. The method does not specify quality control aspects other than that obtained through the use of surrogate recovery standards (SRSs) and calibration standards. QC samples of special types may be prepared by some laboratories as extended quality control elements to further support their analysis conclusions. 2.1.1 Extraction Equipment and Materials The following sample extraction equipment and materials are required, subject to the type of material being prepared. a) Shaker, end-over-end tumbling shaker or reciprocating shaker are sufficient. b) Analytical balance capable of weighing accurately to 0.0001 g c) Assorted glassware (beakers, volumetric flasks, etc.) d) HDPE bottles (assorted sizes) e) Methanol (HPLC-MS grade or better) f) 15 mL conical polypropylene centrifuge tubes (FalconTM, or equivalent) g) Centrifuge capable of 5000 RPM and rotor for 15 mL conical centrifuge tubes h) 22 x 75 mm glass test tubes, or equivalent i) Ammonium acetate (HPLC-MS grade or better) j) Ultrasonic water bath k) Micropipettes (variable volumes, from 0.1 mL to 5 mL), and tips. l) RapidVap VertexTM Nitrogen Evaporator, or equivalent m) Autovials and caps n) Milli-QTM water purification system, or equivalent o) Assorted PFAS reference substances and stable-isotope labeled PFASs (e.g. Wellington Laboratories) prepared as stocks at 1.00 mg/mL in methanol. 2.1.2 Extraction of Liquefied Samples 1) Prepare an aqueous solution of 2 mM ammonium acetate using Milli-QTM pure water. 2) Dilute the aqueous 2 mm ammonium acetate with methanol 3:2 to get a solution of 60% methanol and 40% of aqueous ammonium acetate (60/40 MeOH-AA) 3) Accurately measure out 1 mL aliquots of homogenized liquid sample into each of three pre-weighed 15 mL centrifuge tubes. 4) Weight the tubes to determine the mass of sample in each, record the weights. 5) Fortify one of the three with a known quantity of certain substance(s) as a LMS sample to evaluate recovery. 6) Add known quantity of stable isotope labeled internal standard(s) and/or surrogate recovery standards, as appropriate. 7) Add 9 mL of 60/40 MeOH-AA (if sample is not miscible, use 9 mL methanol) 8) Sonicate 10 minutes 9) Centrifuge 5000 RPM for 10 minutes 10) Aliquot supernatant to labeled autovials and cap for analysis. 11) 2.1.3 Extraction of Solid Samples (i.e. samples prepared in Part 1) 1) Accurately weight out 1 gram aliquots of solid sample prepared in part 1 of this method (i.e. 50 to 150 m particle size powder) into each of three pre-weighed 15 mL centrifuge tubes. 2) Record the weights of each 3) Fortify one of the three with a known quantity of certain substance(s) as a LMS sample to evaluate recovery. The other two will be sample and duplicate sample. 11 / 17 4) Add known quantity of stable-isotope labeled internal standard (IS) and/or surrogate recovery standard (SRS), as appropriate. For example 13C4-PFOA can serve as a SRS for PFOA and 13C8-PFOA can serve as IS for PFOA. SRS and IS should be added within the midrange of the anticipated calibration. 5) Shake for 24 hours 6) Centrifuge 5000 RPM for 10 minutes 7) Aliquot clarified supernatant to labeled autovials and cap for analysis 2.1.4 Concentrating Extracts If concentrating of extracts is desired to achieve lower LLOQs, the following procedure can be performed. This will results in a 5-fold increase in measurement sensitivity for certain substances in the extract. 1) Accurately aliquot 5 mL of supernatant from step 3 of sections 2.1.2 or 2.1.3 to a methanol rinsed 22 x 75 mm glass test tube 2) Add 5 mL of aqueous 1 N NaOH 3) Evaporate the contents in Vertex evaporator at 50C water batch temperature 4) Reconstitute with 1 mL of methanol 5) Add known quantity of stable-isotope labeled internal standard(s) and/or surrogate recovery standards, as appropriate. 6) Transfer to autovial and cap for analysis. 2.1.5 Diluting Extracts If dilution of extracts is desired to achieve results within the range of the calibration, in the event they exceed the calibration range, the following procedure can be performed. This will results in a 10-fold dilution. Successive repeated dilutions can result in 100-fold, 1000-fold, and so on. 1) Prepare a 100 mL methanol solution with IS and SRS at same concentrations as in sample extracts prepared in sections 2.1.2, 2.1.3 or 2.1.4 2) Accurately measure 1.00 mL of extract from 2.1.2, 2.1.3 or 2.1.4 to a conical centrifuge tube. 3) Add 9 mL of prepared methanol solution (with IS and SRS) to each 4) Transfer an aliquot to an autovial and cap for analysis. 2.1.6 Preparation of Calibration Standards 1) Combine 1.00 mL of each stock solution of reference substance and SRS in a clean 100 mL volumetric flask. Bring to the mark with methanol solution (2.1.1.5 step 1) to make a combined stock solution with final concentration of each at 10 g/mL of each. 2) Prepare a second multi-component stock by diluting 1.00 mL of the combined stock (step with methanol solution (2.1.1.5 step 1) in a 100 mL volumetric flask. Final concentration of each substance will be 0.100 g/mL. 3) Add known quantity of stable-isotope labeled internal standard (IS) to each of ten 10-mL volumetric flasks. Then, prepare a set of 10 calibration standards in the 10 mL volumetric flasks from the combined stock solutions in step 2 and diluting with methanol solution (2.1.1.5 step 1), as appropriate. Final calibration standard range of 0.100 ng/mL to 100 ng/mL. 4) Analyze the set of calibration standards near the beginning of an analytical run 5) Analyze mid-range calibration standards during and at the end of an analytical run as continuing calibration verification (CCVs) to verify the maintained calibration during the analysis. 6) Calibration standard ranges may be expanded lower, or higher as needed using appropriate dilutions and similar calibration standard preparation procedures described above. 12 / 17 Part 3: Instrumental Analysis 3.1 Scope Part 3 of this method provides direction for the Instrumental Analysis of prepared extracts generated following the procedures of Part 1 and Part 2. Analysis is performed by high performance liquid chromatography with triple quadrupole mass spectrometric detection. Sample extracts are analyzed against calibration standards in an analytical run, as described below. 3.1.1 Analytical Equipment and Materials The following analytical equipment and materials are required, subject to the type of material being prepared. a) Applied Biosystems API 5000 triple quadrupole mass spectrometer, or equivalent (it is advisable to reduce the PTFE components of the HPLC when possible with non-PTFE replacements like stainless steel or PEEK, when possible) b) Agilent 1200 HPLC system, or equivalent (degasser, autosampler, binary pump, column comp.) b) Analytical balance capable of weighing accurately to 0.0001 g c) Assorted glassware (beakers, mobile phase bottles, etc. d) HDPE bottles (assorted sizes) e) Methanol (HPLC-MS grade, or better) f) Acetonitrile (HPLC-MS grade, or better) f) Ammonium acetate (HPLC-MS grade, or better) g) Milli-QTM water purification system, or equivalent h) Micropipettes (variable volumes, from 0.1 mL to 5 mL), and tips. j) Analytical column: Betasil C8, reverse phase column, 150 x 4.6 mm, 5 m (Thermo part no. 70205-154630). Alternative reverse phase columns and manufacturers may be used if demonstrated to give adequate peak shape and analyte peak resolution. 3.1.2 HPLC Conditions (exemplary) 1) Install the analytical column (e.g. Betasil C8 or C18, reverse phase column, 150 x 4.6 mm, 5 m). Column temperature 25.0 C. 2) Prepare an aqueous solution of 2 mM ammonium acetate using Milli-QTM pure water. Fill the mobile phase 1 bottle with aqueous 2 mM ammonium acetate, purge all lines in the HPLC, as needed. 3) Fill mobile phase 2 bottle with acetonitrile. Purge all lines in the HPLC, as needed. 4) Program the HPLC Gradient conditions as shown below: Time (minutes) Flow Rate (mL/minute) Mobile Phase 1: Aqueous 2 mM ammonium acetate (%) Mobile Phase 2: Acetonitrile (%) 0.00 0.750 95.0 5.0 2.00 0.750 95.0 5.0 10.0 0.750 5.0 95.0 12.0 0.750 5.0 95.0 12.5 0.750 95.0 5.0 15.0 0.750 95.0 5.0 Injection volumes: 1 to 20 L are recommended, depending on instrument sensitivity Autosampler Tray at ambient temperature Special Note: If background interference from the leaching of certain substances from components of the HPLC occurs, a pre-column (Betasil C18 2x50 mm, 5 m) can be placed between the binary pump and the autosampler. This will result in a delayed interference peak that separates chromatographically from the sample analyte peak. However, this will increase back pressure on the system. Additionally, select PTFE components in the instrument may be replaced with non-PTFE 13 / 17 components when available, or alternatively HPLC flows can be diverted around unnecessary components. 3.1.3 Mass Spectrometer (MS/MS) Conditions (exemplary) Mass spectrometer conditions described below are for a properly maintained and operated applied Biosystems API 5000 triple quadrupole mass spectrometer fitted with a TurboVTM electrospray ionization source. Other equivalent instruments from the same and other manufacturers may require optimization of the MS/MS parameters to achieve similar or better performance. The TurboVTM source inlet is coupled to the outlet of the HPLC analytical column. The specific parameters provided are for analysis of PFOA, 13C4-PFOA (SRS) and 13C8-PFOA (IS). Other PFAS substances may require additional optimization of specific MS/MS parameters for adequate sensitivity to detect those analytes in the suggested calibration range. The MS/MS ions measured for each substance (when more than one) are typically summed during quantitation. Specific API 5000 parameter settings are as follows: Special Note: The MS/MS ions measured for each substance (when more than one are acquired) are typically summed during the data analysis phase. 2.1.3.1 MS/MS Settings (API 5000) Substance ID Q1 (m/z) Q3 (m/z) Dwell (msec.) PFOA 1 413 369 20.0 PFOA 2 413 219 20.0 PFOA 3 413 169 20.0 13C4-PFOA (SRS) 417 372 20.0 13C8-PFOA (IS) 421 376 20.0 Start and Stop voltages are identical for each parameter DP [a] -35.0 -30.0 -40.0 -40.0 -40.0 CE [a] -14.0 -14.0 -14.0 -14.0 -14.0 CXP [a] -7.0 -7.0 -7.0 -7.0 -7.0 MS/MS Parameter Scan Type Scheduled MRM Polarity Scan Mode Ion Source Resolution (Q1) Resolution (Q3) Intesity Threshold Settling Time MR Pause MCA Step Size CUR GS1 GS2 TEM ihe CAD API 5000 Setting MRM NO Negative N/A Turbo Spray Unit Unit 0.00 cps 0.00 msec 5.0070 msec NO 0.00 Da 35.0 45.0 45.0 450.0 ON 10.0 14 / 17 IS EP CEM (Detector) DF (Detector) -4500.0 -10.0 2200 300.0 3.2 Examples Example 1: Sample preparation, extraction and LC-MS/MS analysis of a PTFE dispersion 151.83 g PTFE dispersion (solid content ~ 13% m/m) in a clean 250 ml PE bottle was spiked with 0.1 ml 13C4PFOA (SRS) in methanol with a concentration of 5 g/ml = 500 ng 13C4-PFOA, agitated gently and allowed to rest overnight at room temperature. 77.4 g of the spiked dispersion was deep frozen and then freeze dried in a desiccator equipped with a cooling trap (acetone / dry ice) and a rotary oil pump (p ~0,3 mbar). After 45h 10.3 g of dry PTFE powder was obtained, resulting in a solid content of 13.35% m/m and a theoretical 13C4PFOA concentration of 24.7 ng/g. 1.03 g of dry polymer was extracted with 3 ml Methanol at 50C using a shaker at 250 RPM for 16 h and an aliquot of the extract (theoretical 13C4-PFOA concentration 8.56 ng/ml) was analyzed for 13C4-PFOA and PFOA. The analysis revealed a PFOA concentration of 8.5 ng/ml in extract, which calculates a PFOA content of 24.8 ng/g (g/kg). The 13C4-PFOA concentration was 7.35 ng/ml, resulting in an overall recovery rate of 86.0 % (21.4 ng/g polymer). Example 2: Sample preparation, extraction and LC-MS/MS analysis of a PFA dispersion 52.95 g PFA dispersion (solid content ~ 27.5% m/m) was spiked in a clean 100 ml PE bottle with 0.075 ml 13C4-PFOA in methanol with a concentration of 5 g/ml = 375 ng 13C4-PFOA , agitated gently and allowed to rest overnight at room temperature. The spiked dispersion was deep frozen and then freeze dried in a desiccator equipped with a cooling trap (acetone / dry ice) and a rotary oil pump (p ~0,3 mbar). After 45h 14.63 g of dry fine PFA powder was obtained, resulting in a solid content of 27.6 % m/m and a theoretical 13C4-PFOA concentration of 25.6 ng/g. 0.98 g of dry polymer was extracted with 3 ml Methanol at 50C using a shaker at 250 RPM for 16 h and an aliquot of the extract (theoretical 13C4-PFOA concentration 8.4 ng/ml) was analyzed for 13C4-PFOA and PFOA. The PFOA concentration was 6.4 ng/ml in extract, which results a PFOA content of 19.6 ng/g (g/kg). The 13C4-PFOA quantification revealed a concentration of 7.0 ng/ml, resulting in an overall recovery rate of 83.3 % (21.4 ng/g polymer). Example 3: Sample preparation, extraction and LC-MS/MS analysis of a PFA sintered plate About 5 g of a sintered PFA plate (cut in small pieces) was cryo-milled according to section 1.6.3.3 (7 x 4min) and 0.93 g of ground polymer was spiked with 25.5 ng 13C4-PFOA in methanolic solution, resulting in a theoretical 13C4-PFOA polymer content of 27.4 ng/g. After evaporation of the solvent, the polymer sample was extracted with 3 ml methanol at 50C using a shaker at 250 RPM for 16 h and an aliquot of the extract (theoretical 13C4-PFOA concentration 8.5 ng/ml) was analyzed for 13C4-PFOA and PFOA. PFOA was quantified with 1.8 ng/ml in extract, resulting a PFOA content of 5.8 ng/g (g/kg) in polymer. 8.2 ng/ml 13C4-PFOA was found in the extract, giving a recovery rate of 96.5 % (26.5 ng/g in polymer). 3.2 Acceptance Criteria This method is a performance based method and as such relies on the recovery of LMS and/or SRS recoveries for reportability of results. While results may be reported with poor recoveries, this is not recommended and re-preparation and analysis in effort to achieve acceptable results are highly encouraged. The recommended analytical acceptance criteria are as such: Precision: precision of results is demonstrated adequate when relative percent difference (RPD) of duplicate sample results is less than 30%, or for 3 or more replicate results the relative standard deviation (RSD) is less than 30%. Fitted linear regression of calibration standards results (or quadratic regression when needed) should be performed using five (5) or more calibration standards, and should have a correlation coefficient (R) of 0.995 or better, resulting in a coefficient of determination (R2) of better than 0.990. Back calculation calibration 15 / 17 standards (those included in the fit) should be within 100 + 25% of their actual value. Standards which cannot meet that criterion should be excluded from the calibration curve LMS and SRS recoveries should be within 100 + 30% of their known fortification concentration, and flagged in reported data for samples with associated LMS or SRS are outside of that range. 16 / 17 Bibliography [1] IEC 62321-2:2013, Determination of certain substances in electrotechnical products - Part 2: Disassembly, disjointment and mechanical sample preparation [2] ISO 25101:2009(E), Water quality-Determination of perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA)-Method for unfiltered samples using solid phase extraction and liquid chromatography/mass spectrometry [3] Liu et al. "Method development for liquid chromatographic/triple quadrupole mass spectrometric analysis of trace level perfluorocarboxylic acids in articles of commerce" J. Chromatography A. 1216(2009) 3910-3918 . 17 / 17