Environ. Sci. Technol. 2009, 43, 8283–8289
Perfluorooctane Sulfonate (PFOS) Precursors Can Be Metabolized Enantioselectively: Principle for a New PFOS Source Tracking Tool YUAN WANG,† GILLES ARSENAULT,‡ NICOLE RIDDELL,‡ ROBERT MCCRINDLE,§ ALAN MCALEES,‡ A N D J O N A T H A N W . M A R T I N * ,† Division of Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada, T6G 2G3, Wellington Laboratories Inc., Research Division, Guelph, Ontario, Canada, N1G 3M5, and Department of Chemistry, University of Guelph, Guelph, Ontario, Canada, N1G 2W1
Received July 8, 2009. Revised manuscript received September 15, 2009. Accepted September 16, 2009.
Perfluorooctane sulfonate (PFOS) is the most prominent perfluoroalkyl substance found in the serum of humans and wildlife, yet the major routes by which exposure occurs are not clear. An important issue facing both the scientific and chemical regulatory communities is the extent to which PFOS concentrations in biota are attributable to direct exposure versus metabolism of PFOS-precursors (higher molecular weight derivatives that can be biotransformed to PFOS). Given that certain branched PFOS-precursors are chiral, we hypothesized that nonracemic proportions of PFOS isomers in biological samples could be used as a marker of significant exposure to PFOS-precursors. In this proof-of-principle study we examined the enantiomer-specific biotransformation of a high-purity model PFOS-precursor isomer: C6F13C*F(CF3)SO2N(H)CH2(C6H4)OCH3 (named 1m-PreFOS hereafter, and whereby * indicates the chiral carbon center). A method for the enantiospecific separation of a compound with a long perfluoroalkyl chain and a chiral center was developed and applied to evaluate the enantioselectivity of 1m-PreFOS biotransformation in human liver microsomes. Gradient elution in reversed-phase mode on a Chiralpak IC column permitted the near-baseline separation of the two enantiomers (E1 and E2, nomenclature based on retention order) in 65 min. Microsome incubations demonstrated that E1 and E2 were metabolized at significantly different rates; kE1 ) 6.5((0.3) × 10-2 min-1 (half-life ) 10.6 min) and kE2 ) 5.2((0.3) × 10-2 min-1 (half-life ) 13.3 min), respectively. These results suggest that tracking of PFOS exposure sources by enantiomeric fractionation is feasible, and that new analytical methods for the enantioselective analysis of PFOS isomers in human and environmental samples should be developed.
* Corresponding author phone: 1-780-492-1190; fax: 1-780-4927800; e-mail:
[email protected]. † University of Alberta. ‡ Wellington Laboratories Inc. § University of Guelph. 10.1021/es902041s CCC: $40.75
Published on Web 10/01/2009
2009 American Chemical Society
Introduction Perfluorinated substances consist of a wide range of ionic and neutral compounds containing varying perfluoroalkyl chain lengths (e.g., C4-C18). Perfluorooctane sulfonate (PFOS) is generally the most prominent perfluorinated contaminant in wildlife and human samples around the world (1-4), and is also regarded to be among the most concentrated xenobiotics in human serum in some areas (5). PFOS has a long half-life in human serum (5.4 years) (6), bioaccumulates in foodwebs (7-10), and is extremely persistent in the environment. It is also a developmental toxicant in animal models (11, 12) and has been demonstrated to disrupt the endocrine system (13). After the voluntary phase-out by its primary manufacturer in 2000-2002 (14), PFOS concentrations in human blood (15, 16) and Arctic wildlife (17, 18) have declined. However, in Greenland the concentrations in ringed seals (19) and polar bears (20) have risen since 2000. Furthermore, PFOS concentrations in Guillemot eggs from the Baltic Sea have not shown any increase or decline over the same time period (21). Due to poorly characterized pathways of global exposure, these seemingly divergent historical trends are difficult to explain and future trends or (eco-)toxicological risks are difficult to predict, thereby making effective chemical regulation difficult. PFOS has been (22), and continues to be (23), manufactured and emitted directly to the environment. PFOS was recently listed as a “persistent organic pollutant”, under the Stockholm Convention, but many exemptions were made which allow its continued production and widespread use (24). Because of its lipophilic properties and high thermal and chemical stability, PFOS and its derivatives have been widely used for coatings of textiles, papers, carpets, as well as food packaging to achieve oil, stain, and water repellant properties. PFOS has also been used as a performance chemical in fire-fighting foams, and in the manufacture of paints, adhesives, waxes, polishes, metals, electronics, and caulks (25, 26). Many related perfluorooctanesulfonamides (i.e., C8F17SO2N(R)(R)) have also been manufactured for decades for use as surfactants or incorporation into soil and water repellant polymer formulations. These sulfonamide substances are also known as “PFOS-precursors” because they can be biotransformed to PFOS (27-30) or, to some extent, be oxidized to PFOS in the atmosphere (31). Various PFOS precursor substances are widely detected in the ambient atmosphere (32-34), indoor air, and dust (35, 36), and even in human blood (37). The biodegradation of PFOS precursors, and their possible atmospheric oxidation (31, 38), proceeds through perfluorooctanesulfonamide (FOSA, C8F17SO2NH2) which is also widely detected in human serum, wildlife samples, and the open oceans (18, 39, 40). These data are suggestive that PFOS precursors could contribute significantly to the body burden of PFOS in humans or wildlife, but there are currently no analytical tools to differentiate this from direct PFOS exposure. One model predicted that a subgroup of humans (who have high daily exposures from ingestion of house dust and inhalation of ambient and indoor air) could receive a substantial quantity of PFOS from PFOS precursors (41), but to date no empirical evidence exists. Historical emissions of PFOS and its precursors have always contained a mixture of linear and branched isomers because they were manufactured by electrochemical fluorination (42). Several of the branched isomers contain a chiral carbon center at the perfluoroalkyl branching point, as VOL. 43, NO. 21, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Structure of 1m-PreFOS. The asterisk shows the chiral center carbon.
evidenced by NMR for a technical PFOS standard (43). As has been shown for other chiral pollutants (44), we hypothesized that the enantiomers of branched PFOS precursors isomers will biotransform at different rates, leading to nonracemic proportions of PFOS which could be a novel source-tracking tool for PFOS in biological samples. If this hypothesis holds true, for example, nonracemic signatures of any PFOS isomer in biological samples could be strongly indicative of substantial exposure to PFOS precursors since directly manufactured PFOS is racemic. There are currently two major obstacles to testing this hypothesis. The first difficulty is analytical in nature. There are currently no methods for enantiomer specific analysis of any perfluoroalkyl substance, including PFOS or its precursors. Although there are now methods for the chromatographic and mass spectrometric resolution of the various branched isomers of PFOS precursors (45) and PFOS (45-49), there are currently no methods published for the enantioselective separation of any isomer. This was anticipated to be a significant challenge because PFOS and its precursors do not contain the usual functional groups that are known to interact with enantioselectors on chiral stationary phases (CSPs) or chiral ion pair additives. Second, because all of the metabolic reactions of any PFOS-precursor occur on the SO2N(R)(R) moiety (27), it cannot be assumed that a chiral center on the perfluorooctyl chain could influence an enzymatic reaction rate enough to significantly influence the resulting enantiomeric fraction (EF; the ratio of the concentration of one enantiomer to that of the sum of both enantiomers) of the substrate or its products. We recently showed that perfluorooctyl branching patterns of PFOS can significantly influence uptake, tissue distribution, and elimination rates in male Sprague-Dawley rats (48, 49), but the influence of PFOS isomer chirality has never before been examined. In this study we developed a new HPLC-MS/MS method for enantioselective separation of a model branched PFOSprecursor (Figure 1) and applied it to a proof-of-principle study to examine its enantioselective biotransformation in human liver microsomes.
Materials and Methods Chemicals. HPLC-grade methanol, water, acetic acid, and analytical grade potassium dihydrogen phosphate and dipotassium hydrogen phosphate were purchased from Fisher Scientific (Ottawa, ON, Canada). The racemic model PFOS precursor in this study was synthesized at Wellington Laboratories Inc. (Guelph, ON, Canada). It is a benzyl derivative of an R-branched PFOS isomer (C6F13C*F(CF3)SO2N(H)CH2(C6H4)OCH3, where * denotes the chiral carbon center) which will subsequently be referred to as 1m-PreFOS (Figure 1). Pooled human liver microsomes (protein content 20 mg/ mL in 250 mM sucrose) and NADPH regenerating solutions (solution A: 31 mM NADP+, 66 mM glucose-6-phosphate and 66 mM MgCl2 in H2O, NADP+ reductase activity: 0.21 µmol/min/mL; solution B: 40 U/mL glucose-6-phosphate dehydrogenase in 5 mM sodium citrate, NADP+ reductase activity: 0.50 umol/min/mL) were purchased from BD Biosciences (Woburn, MA). 8284
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In Vitro Biotransformation and Sample Pretreatment. The substrate, 20 µL of stock 1m-PreFOS dissolved in methanol at concentrations ranging from 0.5 to 10 µg/mL, was spiked into the reaction mixture containing 900 µL of potassium phosphate buffer (100 mM, pH 7.4) and 20 µL of microsomes. After 2 min of preincubation, the biotransformation reaction was started by adding 60 µL NADPH (50 µL of solution A and 10 µL of solution B, prepared following supplied instructions) at 37 °C in a water bath. After a given incubation time, portions of reaction mixture were transferred into equal volumes of cold methanol (0 °C) to quench the reaction, followed by centrifugation at 3000 rpm for 5 min. The supernatant was collected for HPLC-MS/MS analysis. Enantioseparation of 1m-PreFOS by HPLC-MS/MS. The enantioseparation of 1m-PreFOS was conducted on a Chiralpak IC HPLC column (4.6 mm i.d. × 250 mm, 5 µm particles, Chiral technologies Inc. PA) with an HPLC system consisting of an Agilent 1100 binary gradient pump, autosampler, and column oven. The effects of chromatographic conditions on the enantioseparation of 1m-PreFOS were evaluated for various solvent modifiers. Conditions were optimized in reversed-phase mode using mobile phase A, 0.1% aqueous acetic acid, and mobile phase B, methanol with 0.1% acetic acid (v/v). Both isocratic and gradient elution modes were capable of near-baseline enantioseparation of the analyte. Isocratic mode was optimal with 60% mobile phase B, and gradient mode was optimal while ramping mobile phase B from 60 to 63% in 60 min with a 4 min hold. With either elution mode, on-column flow rate was set at 0.5 and 0.21 mL/min of this was split into the MS electrospray ion source. Column temperature was kept at 35 °C and injection volume was always 10 µL. Only the gradient elution method was ultimately applied to all experimental samples reported in the results because of its higher throughput. Identification and quantification of 1m-PreFOS enantiomers was performed on an API 5000 triple quadrupole mass spectrometer equipped with a Turbo V ion spray source (Applied Biosystems/MDS SCIEX, U.S.) in the negative ion mode using multiple reaction monitoring (MRM). The ion transitions were selected by enhanced product ion (EPI) scans by directly infusing the standard with a syringe pump. The precursor ion ([M-H]-, m/z 618) and product ion with the highest abundance ([C8F17]-, m/z 419) was the primary transition monitored. The transition with the second highest abundance ([C4F9]-, m/z 219) was used for confirmation. For the primary and confirmatory transitions, respectively, the declustering potentials were -60 and -65 V, the collision energies were -29 and -36 eV, the collision cell exit potentials were -9 and -15 V, and the entrance potentials were -10 and -9 V. Dwell time was 300 ms for each transition. The ion spray voltage was set at -4000 V. Nitrogen was used as nebulizer, auxiliary, curtain, and collision gas. The source temperature was 450 °C. Mass spectral data were collected and processed by Analyst 1.5 software, followed by integration using Peakfit software (v4.06, Aspire software International, Ashburn, VA). The concentration of each enantiomer was calculated based on its fitted peak area against the corresponding quantification curve. When elution order is unknown, as in the current work, EF is defined as the quotient of the first-eluting enantiomer (E1) concentration divided by the sum concentration of both enantiomers (E1+E2) (50). Quantification of 1m-PreFOS and Biotransformation Kinetics. Dilutions of stock 1m-PreFOS were made in 50% (v/v) incubation solvent (potassium phosphate buffer) and methanol to minimize any matrix effects; this matched the composition of the supernatant taken from the microsome incubation experiments. Standard curve quantification was conducted by plotting the average fitted peak area from triplicate injections at each concentration (range 0.5-25 ng/
mL for each enantiomer, six concentrations); the response of each enantiomer in the standard was assumed to correspond to 50% of the total concentration of 1m-PreFOS. Analyte recovery and matrix effects were determined to confirm the reliability of the method using spike-recovery and standard addition experiments, respectively. Aliquots of 20 µL of 4 µg/mL stock 1m-PreFOS in methanol were incubated in triplicate with human liver microsomes. At six time points (2, 5, 10, 18, 30, and 50 min), 150 µL of reaction mixture was sampled and mixed with an equal volume of cold methanol. Thus, the initial nominal concentration of 1m-PreFOS after pretreatment was 40 ng/mL, 20 ng/mL for each enantiomer, before metabolism. To monitor contamination, blank matrix was prepared by adding 20 µL of pure methanol to the microsome reaction mixture and by subsequently performing the entire incubation and pretreatment procedure as above. Samples were kept at room temperature and were always analyzed by HPLC-MS/MS within 18 h of the experiments. The biotransformation rate constant for each enantiomer was determined by fitting the data to a first-order elimination equation; ln C ) -kt + ln C0, whereby C was the concentration of each 1m-PreFOS enantiomer at reaction time t, C0 was the initial concentration, and k was the biotransformation rate constant. Accordingly, the half-life (t1/2) of each enantiomer was calculated by 0.693/k. The EF of 1m-PreFOS was also calculated at each time point during the biotransformation experiments. Statistical analyses were conducted using SAS (version 9.1, SAS Institute Inc., Cary, NC). Linear regression was performed to determine if the biotransformation rate constants of the two enantiomers were significantly different from each other. We tested the significance of the interaction between enantiomer concentration and time in a model that also included the main effects for these parameters as well as the intercept. Within each batch, we assumed the two enantiomers were independent from each other.
Results and Discussion Selection of the Model PFOS Precursor, 1m-PreFOS. To develop a new enantiospecific analytical method and to provide unambiguous proof-of-principle enantiospecific biotransformation data, a relatively pure model PFOS precursor was needed. Most commercially available branched PFOS-precursors can only be obtained as gifts from the fluorochemical manufacturing industry, and all of these will contain incompletely characterized mixtures of isomers, chain-length homologues, and other minor impurities. Such a mixture would not only introduce analytical complexity, but would also complicate the interpretation of microsome biotransformation kinetic experiments due to potential competitive inhibition. In addition to requiring a relatively pure compound, we also wanted to select a PFOS precursor with an R moiety that might increase interaction with the enantioselectors on the CSP. Finally, we wished to have the chiral center on the perfluorooctyl chain as close to the sulfonamide moiety as possible, to increase the chances that it may influence the biodegradation of each enantiomer. The model compound, 1m-PreFOS (Figure 1), was the only synthesized compound we could obtain to meet all the above demands. It was relatively pure (>89%), based on peak area monitored in a full scan mode, and the only impurities were other isomers which could be well separated from 1mPreFOS. It also had a methoxy substituted aromatic ring to aid interaction with the CSP, and the chiral carbon was immediately adjacent to the sulfonamide moiety. Enantioseparation of 1m-PreFOS. Achieving the appropriate interaction between the racemate and a CSP is the critical objective when developing a new enantioseparation. These may consist of hydrogen bonding, dipole-dipole
FIGURE 2. Enantioseparation MRM chromatograms of 1m-PreFOS on Chiralpak IC column with (a) isocratic (calculated EF ) 0.500) and (b) gradient (calculated EF ) 0.499) elution. Ten µL of 50 ng/mL standard 1m-PreFOS dissolved in potassium phosphate buffer:methanol (1:1, v/v) was injected on-column. Monitored ion transition was 618/419. Rs ) 1.18(t2 t1)/(w1/2,1 + w1/2,2), in which Rs was resolution of the two enantiomers, t2 and t1 were retention time of the two enantiomers (named E1 and E2 according to elution order), w1/2,1 and w1/2,2 were the half-height peak width, respectively. r ) (t2 - t0)/(t1 - t0), in which r was separation factor of E1 and E2, t0 was dead time of the system. By injection of methanol with the developed method, t0 was detected 6.52 min in this study. The other chromatographic and mass spectrometric conditions are shown in experimental section. interaction, π-π interaction, inclusion and/or steric interaction. The supramolecular structure of the stationary phase, its polarity and electron-donating or withdrawing properties also affect the ability of a CSP to recognize the chirality of an analyte (51). According to their unique chemical and physical properties, we expected that perfluorinated acids and their precursors, which lack moieties capable of interacting with a CSP, would make their enantioseparation very difficult. Thus, choosing an effective CSP for a chiral perfluorinated compound was challenging, since there is no precedent literature on this topic. Several phases were tested, but a chiral stationary phase based on cellulose tris(3,5dichlorophenylcarbamate) (trade name “Chiralpak IC”) was found to be the only one suitable for this application. Due to its immobilized stationary phase (52), this column could be used under both normal-phase and reversed-phase HPLC modes with a wide range of organic modifiers. This aided our optimization and proved useful for successfully separating a wide range of racemates with various structures and properties (53-56). Several chromatographic parameters were optimized on Chiralpak IC: operation mode (normal-phase or reversedphase), type and concentration of organic modifier and acid additives, and separation temperature. We could not accomplish any separation in normal-phase mode, but we did achieve enantioseparation under reversed-phase conditions using methanol as the organic modifier (but not isopropanol or acetronitrile). An acid additive was also essential (without it enantioseparation could not be accomplished) and 0.1% acetic acid in mobile phase was found to be optimal, resulting in near-baseline separation of the enantiomers (Figure 2a). Isocratic elution conditions are most commonly used in chiral HPLC separations. However, because the retention time was relatively long in isocratic mode (∼85 min) the peaks were quite wide (∼3 min peak width at half height). To increase the sensitivity, as well as throughput, we VOL. 43, NO. 21, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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optimized the method by adding a weak gradient elution profile, resulting in 25% less analysis time, negligible loss of resolution, and significantly greater sensitivity (represented by peak height) owing to the sharper peaks (Figure 2b). Thus, the gradient elution method was applied in all subsequent work. After 27 injections at different concentrations (1-150 ng/mL of racemate), the EF value of the racemic 1m-PreFOS was very accurate and precise 0.499 ( 0.002 (average ( SD). It is important to mention that the nitrogen atom of the tertiary amide of 1m-PreFOS could function as a chiral center because of the lone electron pair and the three different substituent groups (sH, sSO2C8F17, and sCH2(C6H4)OCH3 groups, see Figure 1). However, the enantiomers of chiral tertiary amines are known to rapidly interconvert at ambient temperature and, thus, cannot be separated on a chiral column at room temperature; unless the nitrogen is located within a rigid ring structure (57, 58). For 1m-PreFOS, the N-substituent groups are expected to be arranged in a trigonal planar configuration with the benzyl group directed away from the perfluorooctyl group to minimize steric crowding around the SsN bond (59); furthermore, the free energy of activation for hindered rotation about the NsS bond in this type of molecule was estimated to be 60-70 kJ/mol, meaning that rotation should not be hindered at the separation temperature used here (35 °C) (59). Thus, the enantioseparations shown here unambiguously represent the enantiomers of the chiral carbon center. Method Validation. With the developed HPLC-MS/MS method, no carry-over effect was observed when a methanol blank was injected after analysis of the highest concentration standard (150 ng/mL) of 1m-PreFOS. There was also no shift in the baseline, within the retention window of the enantiomers, when blank matrix was injected indicating no nonspecific interferences. The relative abundance of primary to secondary ion transitions (m/z 618/419 and m/z 618/219) was stable over the entire calibration range and was the same between the two enantiomers, indicating selectivity of the method for the target enantiomers. “Matrix effects” that can affect the ionization process are always an important consideration in electrospray ionization methods, but are of particular importance for EF analysis. For example, a matrix component may coelute with only one enantiomer, causing a suppression or enhancement of the analyte response, and thus cause a racemic signal to appear nonracemic. In this study, matrix effects were monitored by spiking standard racemic 1m-PreFOS into microsome-incubated samples. Aliquots of 20 µL of 4 µg/mL stock 1m-PreFOS in methanol was incubated with human liver microsomes and at two time points, 10 and 50 min, sufficient samples were taken to allow triplicate standard additions of 5 and 25 ng/mL 1m-PreFOS at both time points (total of 12 spiked samples were analyzed to examine any matrix effects). The additional peak area for each enantiomer was calculated by subtracting the prespike peak area from the postspike peak area, and in all samples the EFs based on additional area was in the range of 0.50-0.52; indicating no significant matrix effect. To determine method recovery, NADPH regenerating solutions were heat-killed by boiling before addition to the incubation mixture to prevent any microsomal activity. Two concentrations of 1m-PreFOS were spiked into the reaction mixture resulting in samples with 1m-PreFOS at a nominal concentration of 20 and 50 ng/mL for each enantiomer. At each concentration the method recovery was evaluated in triplicate by comparing the peak area ratio of E1 and E2 in inactive microsomes to the standard 1m-PreFOS. At 20 ng/ mL, recovery was 99.3 ( 3.3% for E1 and 99.6 ( 3.5% for E2. At 50 ng/mL, recovery was 97.5 ( 2.8 for E1 and 97.0 ( 2.2% for E2. 8286
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FIGURE 3. Enantiomeric profile of 1m-PreFOS at different time points after incubating with human liver microsomes. Due to the negligible matrix effects and good recovery observed in this matrix, the series of calibration samples were also prepared by diluting stock standard 1m-PreFOS (10 µg/mL in methanol) into potassium phosphate buffer: methanol (1:1, v:v). Calibration curves for each enantiomer were constructed from a series of six concentrations (range of 0.5-25 ng/mL), each injected in triplicate, by linear regression of peak area versus nominal concentration. Both enantiomer calibration plots appeared linear over the entire range and had correlation coefficients (r2) greater than 0.9995. Biotransformation of 1m-PreFOS Enantiomers. As shown in Figure 3, both of the enantiomers of 1m-PreFOS could be significantly biodegraded by human liver microsomes in 50 min, and simple visual inspection showed that the enantiomeric profile was not constant at different time points. Under the experimental incubation conditions, both enantiomers showed a similar pattern of exponential decay which could be easily fit to a first order decay model. By linear regression, the slopes of both lines were significantly less than 0 and, more importantly, the slopes of the two enantiomers were significantly different from each other (p < 0.0001). E1 was metabolized more quickly than E2, and the biodegradation rate constants of E1 and E2 were 6.5((0.3) × 10-2 min-1 and 5.2 ((0.3) × 10-2 min-1, respectively (Figure 4a). Accordingly, the half-lives for E1 and E2 were 10.6 and 13.3 min, respectively. Consequently, the EF slowly decreased over time (Figure 4b), and overall the data were a clear indication that the biotransformation of 1m-PreFOS was indeed enantioselective. It is germane to note that similar biotransformation rate constants were obtained (6.7((0.2) × 10-2 min-1 for E1 and 5.4((0.2) × 10-2 min-1 for E2) when the data were analyzed by nonlinear regression in an exponential decay model (i.e., without natural log transformation). In control experiments with heat-killed microsomes no enantiospecific degradation was observed, thus all enantiospecific loss in viable incubations can be attributed to biotransformation. Potential for Source Tracking of PFOS. By establishing a sensitive and specific method for the enantioseparation of a perfluoroalkyl substance, this proof-of-principle study demonstrated that a model alpha perfluoromethyl-branched PFOS (1m-PFOS) precursor could biotransform enantioselectively. Thus, biomonitoring of enantiomeric fractions of 1m-PFOS in humans and wildlife is a feasible method for evaluating the importance of PFOS precursors to PFOS body burdens. However a few potential limitations are notable. First, it is important to point out that only a 1m-branched PFOS precursor was specifically proven to biotransform enantioselectively here. It cannot be assumed that all other chiral branched isomers will also biotransform enantioselectively. For example, the chiral center of a 5m PFOSprecuror (i.e., CF3CF2C*F(CF3)C4F8SO2N(R)(R) is sufficiently far away from the SO2N(R)(R) moiety that it may have an insignificant effect on any enzymatic reaction rate. Second,
biological samples is a reliable enantioseparation analytical method for this chiral compound. However, because 1mPFOS lacks any moieties known to interact with common enantioselectors, we do expect that this compound will be more difficult to enantioseparate than its related precursor examined here. Nevertheless, experiments with existing CSPs should be attempted, and we also suggest using chiral mobile phase additives, or possibly derivatizing the sSO3 moiety by introducing a functional group (e.g., an aromatic moiety) which could aid interaction with a CSP.
Acknowledgments Alberta Ingenuity supported this research through postdoctoral salary support (Y.W.), and an NSERC Discovery grant (J.W.M.) further supported the work through provision of materials and consumable supplies. Alberta Health and Wellness is thanked for support of daily laboratory activities in the Division of Analytical and Environmental Toxicology. Leah Martin (Department of Public Health Sciences, University of Alberta) is thanked for assistance with statistical analysis.
Note Added after ASAP Publication
FIGURE 4. (a) Scatter plots to determine of first-order rate constants of enantiomers of 1m-PreFOS biodegraded by human liver microsomes. Error bars represent standard deviation (n ) 3). Red line, linear fitting curve of ln(concentration) vs time for E1; Black line, linear fitting curve of ln(concentration) vs time for E2. The slope of each linear fitting curve represent biotransformation rate constant (k) for each enantiomer. Corresponding correlation coefficients (r2) and p-values were indicated as well. (b) Enantiomeric fraction (EF) change of 1m-PreFOS through biodegradation by human liver microsomes. EF ) conc.E1/(conc.E1 + conc.E2), in which EF was enantiomeric fraction of 1m-PreFOS, conc.E1, and conc.E2 were calculated concentrations of each enantiomer against their fitted peak areas by corresponding calibration curve. Error bars represent standard deviation (n ) 3). the model PFOS precursor used here is not environmentally relevant. Although the proof-of-principle has clearly been demonstrated, we cannot say with certainty that a perfluorooctane sulfonyl derivative without a methoxy substituted aromatic ring will also biotransform enantioselectively. Finally, for nonracemic patterns of PFOS in biota samples to be useful as indicators of significant exposure to precursors, it is important that absorption, distribution, and excretion of PFOS is not enantioselective and therefore does not interfere with the signature from biotransformation of PFOSprecursors; this could be tested in the laboratory in vivo with model organisms. Given the current results with a 1m-precursor, and the unique behavior of 1m-PFOS in tandem mass spectrometry, it may be most efficient to concentrate on profiling the enantiomer profiles of 1m-PFOS in biological samples. More specifically, 1m-PFOS is the only PFOS isomer in most biological samples that produces a detectable signal in the m/z 419 product ion (i.e., the m/z 499/419 transition) (45). Thus, an enormous analytical advantage to 1m-PFOS enantiomer profiling is that it could be accomplished without any preseparation (online or off-line) from other PFOS isomers. In other words, no development of a complicated multistep or multidimensional chromatographic separation method would be needed unless coeluting PFOS isomers caused a significant matrix effect that biased the EF of 1m-PFOS. Therefore, all that is needed before application of 1mPFOS enantiomer profiles as a source tracking tool for
References 48, 49, and 57 were modified from the version published on October 1. The corrected version was published on October 8, 2009.
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