The need to analyze perfluorinated-acid chains of various
Challenges in Perfluorocarb lengths drives the demand for new analytical methods.
Barbara S. Larsen Mary A. Kaiser Dupont’s Corporate Center for Analytical Sciences
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luorinated materials are found in a wide range of applications, because of their unique stability toward redox agents, acids, bases, and heat, as well as for their inert and nonadhering surface properties. They are used in many commercial products, such as paints, polishes, packaging, lubricants, firefighting foams, cookware, and stain repellents (1). During the past 6 years, scientists and consumers became more aware of these materials when 3M, a longtime major manufacturer of these compounds, declared that it was stopping production of some perfluorinated compounds, including an eight-carbon perfluorooctane sulfonate (PFOS) product and perfluorooctanoic acid (PFOA; 2). The primary reason for withdrawing PFOS from the marketplace was the discovery that it is persistent, bioaccu-
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© 2007 AMERICAN CHEMICAL SOCIETY
oxylic Acid Measurements
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mulative, and toxic in animal studies. The U.S. Environmental Protection Agency (EPA) subsequently requested more information on PFOA to ascertain the sources of human exposure and to determine any environmental effects (3). Because of this heightened awareness, there has been a substantial increase in the number of publications about the determination of perfluorooctanoate (PFO) and other fluorinated compounds in various matrices (4 ). Perfluorinated sulfonates and carboxylates have been identified at low levels in human serum and in the environment, indicating their widespread presence (5 –10). Of particular interest to the regulatory agencies are the fully fluorinated eight-carbon compounds with an anionic end group, such as PFOS or PFOA. However, for a
more complete understanding, the entire suite of perfluorinated sulfonates and carboxylates from C5 to C13 should be analyzed. This article will concentrate on recent developments and challenges in the analytical chemistry for a broader range of analytes, namely C5 to C13 perfluorocarboxylic acids.
Background PFOA and perfluorononanoic acid (PFNA) salts are industrial chemicals principally used to aid in the manufacture of fluoropolymers. These salts are surfactants manufactured by either an electrochemical fluorination (ECF) or telomerization process; both processes have perfluorocarboxylate (PFCA) impurities present. The ECF process fully fluorinates an organic J U N E 1 , 2 0 0 7 / A N A LY T I C A L C H E M I S T R Y
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FIGURE 1. Full-scan MS/MS spectra of isomers of perfluorooctanoate: (a) linear perfluorooctanoate, (b) [1,2-13C]-labeled perfluorooctanoate, (c) 3-trifluoromethyl perfluoroheptanoate, and (d) 6-trifluoromethyl perfluoroheptanoate.
compound dissolved in hydrogen fluoride when an electric current is applied (1). The telomerization process is used to manufacture perfluorooctyl iodide with high purity; however, during that process, a linear PFOA is created, which also results in small quantities of 6- and 10-carbon impurities (1). PFOA made by the ECF process usually contains branched materials; PFNA usually contains other PFCAs, ranging from 4 to 13 carbons at various levels (11). Ammonium perfluorooctanoate (APFO) has historically been produced by ECF, which yields a mixture of linear and branched eight-carbon chain isomers with 2–30% branched isomers (1). About 90% of PFCAs released into the environment originate from fluoropolymer manufacturing and use (11). Historical industry-wide emissions of all PFCAs were estimated at 3200– 7300 t. An exposure assessment and risk characterization were funded by industry and performed by a private consulting group with expertise in exposure and risk assessment to better understand the impact on health of PFO in consumer goods such as carpet, apparel, cookware, and paint (12, 13). The assessment concluded that trace levels of PFO in consumer items are not expected to cause adverse health effects, even in sensitive individuals, and would not result in quantifiable levels of PFO in human serum. The study used a combination of an analytical testing program, product formulation information, and the EPA margin-of-exposure calculation (14). Fluorinated compounds have chemical properties that are quite different from their hydrocarbon counterparts, because of the unusually high strength of the C–F bond and the high electronegativity and small size of the fluorine atom. The perfluoroalkyl moiety is both hydrophobic and oleophobic—that is, it is not miscible in either aqueous or hydrocarbon solvents. Perfluoroalkanes are more hydrophobic than hydrocarbons. For example, octane and perfluorooctane are not miscible because octane is more polar than perfluorooctane. Perfluorinated compounds should be considered a third class of compounds—distinct from the hydrophobic and oleophobic classes—because, on the solvatochromic Π* scale (an index of solvent dipolarity/polarizability), water is 1.09, cyclohexane is 0.00, and perfluorooctane is –0.41 (15). Placing perfluorinated compounds in a separate class requires rethinking the analogies that compare perfluorocompounds (especially) to hydrocarbons 3968
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and will help to rationalize the unusual and often unexpected properties of these compounds. The stability of the C–F bond probably accounts for the high stability of PFOA and PFOS as well as their low boiling points relative to their chain lengths. Because the intermolecular interactions are weak, PFOA also sublimes at room temperature (16); it is a relatively strong acid with an apparent pKa of 2.8 (compared with 6.17 for octanoic acid; 17).
Early analytical methods
Initial measurements of perfluorinated materials entailed a total organic fluorine determination by using the Wickbold torch combustion procedure (18). The significant component of this method is an oxyhydrogen torch, which reaches a temperature of ~2000 °C in the chamber. That high temperature provides sufficient energy to break the strong C–F bonds. The fluoride ion is captured in water, and total fluoride is determined via a fluoride ion-specific electrode or ion chromatography. This method can attain sensitivity in the low partsper-million range (19). Alternatively, compound-specific methods have been developed using GC with electron capture detection (ECD). The fluorine atom is quite small, which reduces the efficiency of electron capture. Although ECD is selective for compounds that can capture electrons, it is nonspecific. Coeluting analytes will disproportionately impact the amplitude of the detector response. As a result, most GC determinations of fluorinated compounds have migrated to GC/MS. When PFOA is derivatized with diazomethane, a methyl ester is formed; this is the basis for determining PFOA from human plasma and urine using GC/ECD (20, 21) and GC/MS (22). A capillary GC/MS method was reported for the determination of PFOA and other perfluorocarboxylic acids as the methyl ester in aqueous environmental samples, and it achieved a low-nanogram-per-liter detection limit (22). An advantage of this approach is the ability to readily separate isomers (23). In general, GC-based methods require multiple steps and lengthy sample preparation, which makes these methods subject to error. GCsample preparation methods are not easily automated and may also lack sensitivity and selectivity, especially in complex matrices.
LC/MS For nonvolatile or polar fluorinated compounds, LC with ESI MS is now the preferred analytical method (24 ). In negative-ion ESI, the molecular anions for the PFCAs are easily detected without derivitization. For example, PFO and its isomers are detected at – m/z 413 (CF3(CF2)6CO 2). Under collision-induced dissociation conditions, the CO2 is readily lost from the molecular anion, forming the perfluorinated anion CF3(CF2) n– , which can subsequently lose CF2 groups (Figure 1a). Because the CO2 loss is so facile, the instrument must be optimized to minimize collisional fragmentation in the first vacuum region of the ESI source.
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FIGURE 2. The observed background in an LC/MS/MS chromatogram monitoring 413>369 with gradient elution. (a) No guard column. (b) Blank water injection with a guard column. (c) 0.1-ppb and (d) 5-ppb PFOA standard with a guard column. The sharp peak is the injection peak, and the large ill-defined peak is the system peak.
The identification of linear and branched isomers of PFCAs by tandem MS requires standards characterized by NMR because the MS/MS spectra do not have unique, distinguishable transitions (Figures 1c and 1d). A recent report indicates that the various isomers of PFOS have different response factors (25). For purposes of trace quantitation of PFCAs, the chromatographic conditions are selected such that the isomers coelute and the – – [M ]>[M-CO 2 ] transition is used for quantitation. The first LC/MS/MS results reported by Hansen used the transition 413>369 to quantitate PFO and 499>80 for PFOS in human blood at parts-per-billion levels (5). The results aligned with Taves’s early studies in which perfluorinated compounds were identified in blood by the Wickbold torch procedure (26). Although these measurements have been simplified through the use of LC/MS/MS, the analysis is still quite challenging because of matrix and background effects. A particularly challenging issue in the analysis of PFCAs is reducing the background contamination of PFO (Figure 2a). Because APFO and ammonium perfluorononanoate are often used as fluoropolymer processing aids, sampling, sample preparation, and common laboratory equipment and solvents could introduce measurable quantities of PFO that could lead to quantifiable background levels. In our laboratory, we use HPLC-grade water that has been filtered through a C18 LC column for additional cleanup. Risha et al. described a cleanup method in which water that has a low electrical resistance (369). The uptake and clearance of the branched versus linear isomers are very different, suggesting that measuring the concentrations of the various isomers in biota does not necessarily reflect the source of exposure (37, 38). A spike of the analyte or a surrogate is often added to the sample matrix to increase its concentration by a known amount to establish whether an analytical method is performing properly. When the analyte’s or its surrogate’s concentration is measured, percent recovery is usually calculated to assess accuracy. Spike duplicates are sometimes made to determine the measurement’s precision. The surrogate is usually a pure substance with properties that mimic the analyte. The surrogate should be something that is unlikely to be in the sample. In the early era of PFO determinations, the 6-2 fluorotelomer sulfonic acid (6-2 FTS; CF3(CF2)5CH2CH2SO3H) was used as a surrogate. Because many of the PFO methods evolved from PFOS methods, 6-2 FTS might have seemed to be a good surrogate candidate. But 6-2 FTS is too different from PFO in physical and chemical properties, and it is also found in the environment. In general, the telomer-based products containing the ethylene spacer between the functional group and the perfluoro group are probably not good surrogates because the ethylene spacer acts like an electron insulator, making the physical properties (pKa, vapor pressure, solubility) decisively different from perfluoro counterparts. If available, the best surrogate is an isotopically enriched version of the analyte. The level of spiking is an important consideration. Obviously, a high-level spike would make recovery easy, but it would not be
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FIGURE 4. LC/MS/MS chromatograms for the separation of branched isomers of PFOA. (a) Isopropyl PFOA, (b) 5-trifluoromethyl perfluoroheptanoic acid, and (c) linear PFOA.
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useful for determining the accuracy and precision at the lower for the sample preparation. For example, in the method for deconcentration levels. Spike recovery studies should generally be termining PFO in water, the calibrants and the samples are condone at the LOQ and 103 LOQ to provide the most useful in- centrated eightfold with SPE. With appropriate precautions to prevent laboratory contamination, PFO can be quantitated in formation on the quality of the method. The time interval between spiking and attempted recovery is water readily with LOQ in the low parts-per-trillion (nanogramsoften not addressed in method development but may impact re- per-liter) range. covery experiments, especially in biota, soil, and sludge samples. If spike recovery is attempted immediately after the spike is ad- Round-robin study ministered, the amount recovered is likely to be close to 100%. A measure of the credibility of an analytical method is the ability If the spike is allowed to stay in contact with the matrix for hours, of another laboratory to run it successfully with a blind, split days, or weeks, the probability increases that irreversible adsorp- sample. The first interlaboratory study on perfluorinated comtion, absorption, chemical transformation, volatilization, or in- pounds in human and environmental matrices was held in 2005 tercalation might occur. In this event an isotopically labeled ma- (40). Because most published studies have been done in environmental matrices, water, terial should be used. In fish tissue, and fish liver exany case, the interval be1937 First fluoropolymer, called Teflon, discovered tracts were chosen as repretween spiking and analyti1943 PTFE gasket used in Manhattan Project sentative matrices. Human cal determination should plasma and blood and a be reported. 1947 Manufacture of fluoropolymers begins study standard were also To remove the analyte provided; 38 laboratories from the matrix, a solvent is 1953 Fabric and upholstery protector Scotchgard discovered enrolled in the roundadded in which the analyte 1960 Nonstick cookware commercialized robin. is expected to be more solOf the 33 participating uble than in the matrix. 1968 First publication on fluorinated organic species in humans laboratories that reported The proper solvent and exdata on the 7.8 µg/L PFO traction conditions are crit1980 First GC/MS analysis of PFOA study standard, only 21 ical to efficient removal of 2001 First LC/MS/MS analysis of PFOA obtained a satisfactory the analyte from the matrix. value as determined by z Spike-recovery study results, especially if sufficient contact time is allowed, will provide scores using Cofino model statistics (41). The values from the helpful information on extraction conditions. It is important to other 10 laboratories were considered unsatisfactory, and they determine whether the total analyte content of the sample is re- were advised to critically study their calibration methods. Agreequired or simply the quantity extracted in one or multiple ex- ment among the laboratories decreased as the complexity of the tractions. Typically in environmental matrices only one extrac- matrix increased, with only five satisfactory results from the 20 tion is performed. Even if the spike study demonstrated 100% laboratories reporting on the fish tissue sample. No correlation recovery immediately after spiking, there is no guarantee that all was found between the laboratory’s experience with such samthe analyte has been removed. In samples such as fabricated ma- ples and its ability to get satisfactory scores. The study concludterials or soils, multiple extractions might be required to estimate ed that extraction and cleanup methods had a large effect on the results. The study also recommended that certified reference mathe total analyte content. Because PFO is often present in analytical systems and both terials in different matrices be developed as a complementary PFOA and APFO sublime under ambient conditions, blanks are quality assurance tool. an especially important component in method development. Field, trip, solvent, and method blanks should be considered. Future considerations Care should be taken to avoid making standards in close physi- The results from the first round-robin study suggest that more cal proximity to sample preparations, especially if the levels are work needs to be done to establish appropriate, rugged, and reliable analytical methods for perfluorinated acid determinations. expected to be low. The linearity of the method is established by running a 6–8 In June 2006, 12 countries at an International Organization for calibration standard set of known concentrations, bracketing the Standardization meeting in Japan accepted a work item proposhighest and lowest reporting levels. When the ratio of peak area al to produce a standard method for the determination of both for the PFC and its internal standard is used in a linear calibra- PFOS and PFOA in unfiltered water samples using SPE and tion curve with 1/x weighting, the acceptable limit for the coef- LC/MS. That task is still under way; air is the next probable maficient of determination R2 is >0.985. The 1/x weighting is ap- trix that they will address. Because perfluorinated compounds are so different from their propriate for trace analysis because it gives more weight to lowerlevel standards. Analysts should consider other models, especially hydrocarbon analogs, additional care must be taken to ensure for a new matrix/analyte pair (39). The deviation of the cali- that a valid method is demonstrated. Adequate methods include brants should be
