Matrix Normalized MALDI-TOF Quantification of a Fluorotelomer- Based Acrylate Polymer
■ INTRODUCTION
Fluorinated polymers are the largest class of commercial fluorochemical products. Despite this fact, there is little known about their environmental fate and potential impact due to difficulties in developing analytical methods to directly measure changes in the formal polymer structure.
Fluorotelomer-based acrylate polymers (FTACPs) are a class of fluorinated polymers widely used as antiwetting and antistaining agents in the textile, upholstery, carpet, and paper industries.1 Specifically, FTACPs are copolymers prepared from fluorotelomer acrylates (FTACs), hydrocarbon acrylates, and often other nonfluorinated monomers.2,3 Similar to other fluorinated polymers, FTACPs benefit from improved repellency, lubricity, and chemical and thermal stability through the replacement of hydrogen with fluorine.4 Consequently, the properties that make FTACPs ideal for industrial applications have also raised concern about their environment fate. Recent studies suggest that the degradation of FTACPs is likely an indirect source of the ubiquitous and persistent perfluoroalkyl carboXylates (PFCAs).5,6 Because long-chain PFCAs (>7 perfluorinated carbons) have been demonstrated to accumulate evaluating the degradation of FTACPs indirectly by measuring the transformation products (i.e., PFCAs) by high performance liquid chromatography tandem mass spectrometry (LC-MS/ MS).12,13 However, PFCAs are also known transformation products of other fluorotelomer-based material such as FTACs and fluorotelomer alcohols (FTOHs),14−20 which have been reported as residuals in the crude FTACP material at levels <5% (w/w).21 Thus, indirect analysis of FTACPs degradation often requires the measurement of transformation products above a high background signal. Alternatively, degradation of FTACP itself could be monitored using a direct analysis method. Our group recently developed a qualitative matriX- assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry method to investigate the degrada- tion of FTACPs as a complementary method to conventional LC-MS/MS.5 Although qualitative, the MALDI-TOF results clearly indicated alterations to the FTACP’s chemical structure caused by microbial degradation. Besides this qualitative MALDI-TOF method, there does not appear to be another direct analysis methods for FTACPs. MALDI-TOF is a technique often used to estimate the weight-average molecular weight (Mw), number-average mo- lecular weight (Mn) and polydispersity index (PDI), as defined in the Supporting Information (SI), and provide structural characterization of synthetic polymers.22−25 Despite having been introduced in the 1980s,26,27 MALDI-TOF has remained primarily a qualitative analytical technique because of poor sample-to-sample reproducibility. Successfully obtaining a ensure intimate contact with the matriX;28 considering the chemical diversity of synthetic polymers, this is not always trivial. Conventional solvent-based sample preparations, such as the dried droplet method,29 rely on proper matriX and solvent selection to increase the likelihood of cocrystallization of the polymer and matriX upon solvent evaporation. To some degree, intensities to minimize the sample-to-sample variability. Calibration curves were generated using a series of poly(8:2 FTAC-co-HDA) standards, which allowed for reliable MALDI- TOF quantification whenever the same matriX solution was used to prepare both the poly(8:2 FTAC-co-HDA) standards and samples. To supplement our understanding of sample knowledge regarding the relative hydrophobicity and polarity of a polymer,30,31 but may require tailoring of unique sample preparation for more problematic polymers.32,33 The choice of a solvent is preferably a single or azeotropic system that allows the polymer, matriX, and cationization agent to be prepared together.34−36 In addition, the rate of solvent evaporation is a contributing factor to polymer and matriX cocrystallization with faster evaporating solvents improving the sample homoge- neity.33,37 Modifications to the deposition method using thin-38,39 and seed-layered,40 and electrospray41−43 have been reported to further enhance sample homogeneity and improve the shot-to-shot reproducibility. However, even with these improvements, MALDI-TOF remains difficult to use for quantification with sufficient precision. A number of studies have recently emerged reporting quantitative MALDI-TOF analysis of microcystins,44 polymer additives,45 saccharides,46 peptides,47,48 proteins49,50 and synthetic polymers.51−53 For synthetic polymers, use of an internal standard polymer is possible if it has similar chemical properties to the target polymer to ensure no discrimination during sample preparation. In addition, the mass spectrum of the internal standard polymer cannot overlap with that of the target polymer. The signal intensities or peak areas of the target polymer are then normalized to those of the internal standard polymer. The result minimizes sample-to-sample variability caused by differences in desorption, ionization and crystal- lization. Although studies have shown this to be an effective approach of quantifying synthetic polymers,51−53 processing two overlaid polymer mass spectra can be rather tedious and time-consuming. This makes the work of Ahn et al. on the MALDI-TOF quantification of peptides using the matriX itself as an internal standard an appealing alternative.54 The authors demonstrated that a linear calibration curve can be generated by taking the ratio of peptide to matriX signal intensities if the to provide surface distribution images following crystallization. Future application of this quantitative MALDI-TOF method would provide direct evidence regarding the environmental fate of FTACP and related fluorinated polymers. EXPERIMENTAL SECTION Materials. Dithranol (98.5%), methyl tert-butyl ether (MTBE, ≥99%), sodium trifluoroacetate (NaTFA, 98%), and tetrahydrofuran (THF, ≥99%) were all purchased from Sigma- Aldrich (St. Louis, MO). Graphitic pencils (HB, 2B, 4B, and 8B) were purchased from Faber-Castell USA Inc. (Cleveland, OH). The model FTACP, poly(8:2 FTAC-co-HDA), was synthesized in-house as outlined in a previous study,33 and details are provided in the SI. Sample Preparation. Poly(8:2 FTAC-co-HDA) prepared as standards and samples were dissolved in THF at concentrations of 1, 2, 5, 10, 15, 20, or 25 mg mL−1. Dithranol served as the matriX and was prepared in THF at concentrations of 5, 10, or 20 mg mL−1. NaTFA served as the cationization agent and was prepared in THF at concentrations of 1, 5, or 10 mg mL−1. Each sample was then prepared with a FTACP:matriX:cationization agent (A:M:Cat) miXing ratio of 5:10:1 by volume. A 1 mL aliquot of the prepared poly(8:2 FTAC-co-HDA) standard or sample was then deposited onto a MALDI target plate using the dried droplet method,29 and allowed to dry under ambient conditions. Once crystallized on the MALDI plate, the dithranol molecules absorb the photons emitted from the laser transferring poly(8:2 FTAC-co-HDA) molecules into the gas-phase, at which point they are likely cationized by sodium. For graphite support preparation, a layer of graphitic pencil lead (8B, 4B, 2B, and HB) was applied to the MALDI plate by hand. The poly(8:2 FTAC-co-HDA) standard or sample was then deposited onto the modified MALDI plate. Figure 2. SEM images of (A and B) dithranol and (C and D) poly(8:2 FTAC-co-HDA) at a magnification of 250×. (A) And (C) show the dried droplet crystallization on an unmodified MALDI plate. (B) And (D) show the dried droplet crystallization after coating a MALDI plate with graphitic lead from an 8B pencil. Extraction Method. Poly(8:2 FTAC-co-HDA) samples (n = 3) were prepared in THF at concentrations of 25 mg mL−1, and 250 μL aliquots spiked into 2.5 mL aqueous media. Poly(8:2 FTAC-co-HDA) was then extracted from the aqueous solutions by vortexing for 1 min and sonicating for 15 min with 5 mL of MTBE. The extraction was repeated three times, the MTBE extracts combined and blown to dryness under a gentle stream of nitrogen, and then reconstituted in 250 μL of THF. ■ RESULTS AND DISCUSSION Graphite Support Preparation. Prior to developing the quantitative MALDI-TOF method, the application of a graphite support was investigated as a means to improve sample homogeneity, shot−shot reproducibility, and enhanced signal intensity. Gorka et al. previously reported an 8-fold enhance- ment in signal intensity for peptides and proteins when graphite flakes were applied to the surface of the MALDI target plate.55 Graphite flakes were first investigated as a graphite support in this study; however, the application of graphitic pencil lead (HB, 2B, 4B, and 8B) to the MALDI target plate were observed to produce a more even coating compared to graphite flakes. The relative percentage of graphite in the graphitic pencil lead is defined by their degree of Blackness = B and Hardness = H with 8B pencils having a high percentage and HB having a low homogeneous layer of small dithranol crystals (SI Figure S1), 8B lead was used exclusively because it was the least abrasive. Crystallization of dithranol and poly(8:2 FTAC-co-HDA) in the absence and presence of a graphite support was investigated by SEM at a magnification of 250×, as shown in Figure 2. When dithranol was prepared with NaTFA at a miXing ratio of 10:1 (respective concentration of 20 and 10 mg mL−1), dithranol crystals were observed with diameters up to 200 μm in the absence of the graphite support (Figure 2A). Whereas, in the presence of the graphite support, the diameter of dithranol crystals were at least four times smaller at <50 μm (Figure 2B). When a poly(8:2 FTAC-co-HDA) sample prepared with dithranol and NaTFA at 5:10:1 (respective concentrations of 25, 20, and 10 mg mL−1) was deposited onto the MALDI target plate in the absence of a graphite, cocrystallization was observed in circular rings upon solvent evaporation (Figure 2C). In the presence of the graphite support, cocrystallization of poly(8:2 FTAC-co-HDA) and dithranol was observed to be more homogeneous, and circular rings were absent (Figure 2D). Instead, there appeared to be a reduction in crystal size with an overall increase in the number of crystals per surface area. The diameter of crystals observed in Figure 2C and D are not reported because they tended to decrease in diameter moving from the perimeter toward the center of the MALDI target plate. It is apparent that the presence of a graphite support produced a more homogeneous distribution and higher number of crystals per surface area, which is consistent with the work of Gorka et al., who reported a thinner and more poly(8:2 FTAC-co-HDA) had a wide polydispersity, develop- ment of the calibration curve focused on 40 of the strongest signals from 911 to 4612 Da (SI Table S2). As shown in Figure 3, the dithranol-sodium cluster at 659 Da signal was observed both in the absence (Figure 3A) and presence of poly(8:2 FTAC-co-HDA) (Figure 3B). For both Figures 3A and 3B, NaTFA served as the cationization agent. In the absence of NaTFA, the signal at 659 Da was no longer observed (SI Figure S3), which suggests it is a dithranol-sodium cluster. This dithranol-sodium cluster was consistently observed in all mass spectra obtained in this study, and was investigated as a means of normalizing the poly(8:2 FTAC-co-HDA) signals to reduce sample-to-sample variability. The intensities (I) of the 40 poly(8:2 FTAC-co-HDA) signals (911−4612 Da) were normalized individually to the intensity of the dithranol-sodium cluster at 659 Da (eq 1). Specifically, the normalized polymer response (PN) shown in eq 1 was calculated from the polymer signal intensity (PI) and matriX signal intensity (MI). Summation of the 40 individually normalized polymer signals was defined as PN, and was determined for each poly(8:2 FTAC-co-HDA) mass spectrum. Our working hypothesis was that using the same dithranol solution to prepare a set of poly(8:2 FTAC-co-HDA) standards a calibration curve of PN vs poly(8:2 FTAC-co-HDA) concentration could be achieved. If confirmed, then the calibration curve would allow for quantification of poly(8:2 FTAC-co-HDA) in a sample. When PN was plotted vs poly(8:2 FTAC-co-HDA) standard concentrations (1, 2, 5, 10, 15, 20, and 25 mg mL−1), a nonlinear behavior was observed (SI Figure S4). The corresponding PN for each poly(8:2 FTAC-co-HDA) standard are presented in Table 1. Ahn et al. previously reported that the degree of matriX signal depends on the analyte concentration, and the degree of matriX suppression moves from normal to anomalous.54 Within this normal region (<70% matriX suppression), reliable quantification of peptides was achieved using the signal intensity ratio of analyte to matriX. These results suggested that the MALDI analysis in this study was also suffering from matriX suppression at higher poly(8:2 FTAC-co- HDA) concentrations. However, when the matriX signal intensities were compared in this study, only a moderate reduction in the 659 Da signal was observed for poly(8:2 FTAC-co-HDA) standards prepared with a 5 mg mL−1 dithranol solution (SI Figure S5). Poly(8:2 FTAC-co-HDA) standards prepared with 20 and 10 mg mL−1 dithranol solutions showed no obvious decrease in the 659 Da signal intensity. The complete list of the matriX signal intensities are presented in Table S3. If the nonlinear behavior between the PN and poly(8:2 FTAC-co-HDA) concentration was caused by matriX suppression, a reduction in the matriX signal intensity would have been expected for all three dithranol concen- trations. When the data was replotted as PN vs ln[Poly(8:2 FTAC-co-HDA) concentration], the results fit a logarithmic regression with acceptable coefficient of determination (R2) >0.97 independent of dithranol concentration (Figure 4).
Although the concentration of dithranol did not impact the nonlinear behavior, it had a clear impact on the magnitude of PN response observed, as shown in Figure 4A. When corrected for the mass of dithranol, the PN for each poly(8:2 FTAC-co- HDA) standards was observed to increase as a function of dithranol concentration (5 < 10 < 20 mg mL−1, Table 1), which suggests the desorption efficiency of poly(8:2 FTAC-co-HDA) molecules was decreasing. It is known that desorption depends on the rapid sublimation of matriX molecules transferring intact analyte molecules into the gas phases. Because FTACPs have a tendency to aggregate,33 decreasing the molar ratio of dithranol to poly(8:2 FTAC-co-HDA) would favor aggregation, and therefore is not likely the explanation for the observed nonlinear behavior. An alternative explanation relates to the total number of poly(8:2 FTAC-co-HDA) ions entering the TOF mass spectrometer. Once the pulsed photons strike the MALDI target plate, a plume of neutrals and charged species are accelerated and focused by a series of short voltage pulses (i.e., time-lag focusing) prior to entering the TOF field-free region. In this method, these voltages, along with the laser parameters, were unchanged in order to maintain a consistency throughout all analyses. It is therefore conceivable that these operating voltages restricted a maximum number of poly(8:2 FTAC-co- HDA) ions entering the field-free region. If true, this would presumably be reflected in a flattening of the signal response similar to the nonlinear behavior reported herein. Also, the ability to detect the correct number of poly(8:2 FTAC-co- HDA) ions is another possible explanation for the nonlinear behavior, and could result from saturation of the microchannel plate (MCP) detector. The consequence of which can result in mass discrimination caused by a lower detection efficiency.59 Although the mass range in this study was up to 10 kDa, MCP saturation tends to occur when analyzing a mass range over 10s of kDa, such as proteins.60 A comparison of the mass spectra acquired for 1 to 25 mg mL−1 poly(8:2 FTAC-co-HDA) standards showed no obvious differences, and suggests MCP saturation did not occur under these conditions. Based on the observation made in the present study, a decrease in desorption efficiency was the most plausible explanation for the nonlinear behavior observed between PN and poly(8:2 FTAC-co-HDA) standards. The results for all calibration curves obtained at various dithranol and NaTFA concentrations indicated reasonable fitness to the linear regressions (R2 > 0.97; Figure 4). The calculated PN for each poly(8:2 FTAC-co-HDA) standard had relative standard deviations (RSDs) <10% with few exception (Table 1 and SI Table S4). Inter- and Intra-Day Variability. The reproducibility of PN calibration curves were investigated over several weeks (interday) and multiple replicates within a single day (intraday), as shown in Figure 5. For the interday experiment (Figure 5A), three different sets of poly(8:2 FTAC-co-HDA) standards (1, 2, 5, 10, 15, 20, and 25 mg mL−1) were analyzed on separate days over 3 weeks. Each set of standards was prepared separately with dithranol (20 mg mL−1) and NaTFA (10 mg mL−1) at a miXing ratio of 5:10:1. The slopes of the linear regressions were observed to range from 0.902 to 2.36 (Figure 5A). Although there were differences in the regression slopes between weeks 1, 2, and 3, the fitness for each week were consistent with R2 >0.97 (Figure 5B), and RSD for each poly(8:2 FTAC-co-HDA) standard <10% (Table S5).The intraday experiment was performed by analyzing a single set of poly(8:2 FTAC-co-HDA) standards (1, 2, 5, 10, 15, 20, and 25 mg mL−1) using three different dithranol (20 mg mL−1) and NaTFA (10 mg mL−1) solutions within a single day. As shown in Figure 5B, the linear regressions slopes from the three replicates were similar and ranged from 1.18 to 1.30. The magnitude of PN was similar among the three replicates of each standard, and once again all measured PN had RSD <10% (SI Table S6). Similar to the interday experiment, the linearity of the regressions were consistent with R2 >0.98 (Figure 5B). The ability to obtain fairly reproducible calibration curves while using separate dithranol solutions suggests that the PN method can mitigate variable sample preparations and MALDI-TOF analyses within a single day. Because the precision of the individual interday PN calibration curve was deemed acceptable, any variation appears to be systematic, and repeated analyses of a single set of poly(8:2 FTAC-co-HDA) standards within a given day (intraday) yielded similar PN calibration curves. The ability to obtain linear calibration curves between PN vs ln[Poly(8:2 FTAC-co-HDA) concentration] reproducibly suggests the PN method could be used to quantify poly(8:2 FTAC-co-HDA) samples with appropriate calibration on the same day by preparing both the standards and samples with the same dithranol solution.
Figure 5. Calibration curves obtained (A) replicated over a three-week period and (B) replicated three times within a single day. For all experiments, dithranol and NaTFA were prepared at 20 and 10 mg mL−1, respectively.
Testing the PN Method. The PN method was first tested using a set of three poly(8:2 FTAC-co-HDA) samples (5, 10, and 20 mg mL−1) and quantified using four separate calibration curves over several days. The poly(8:2 FTAC-co-HDA) samples (n = 3) were weighed and then dissolved in THF giving theoretical concentrations of 5.0, 10.9, and 20.4 mg mL−1 (SI Table S7). Preparation of the samples and each set of poly(8:2 FTAC-co-HDA) standards was performed using the same dithranol solution on the given day of analysis. Thus, calculation of PN values would be consistent between the samples and standards for each calibration curve. Calibration curves had acceptable linearity with R2 >0.98, and are presented along with their corresponding regression equations in Table S8. With each calibration curve, analysis of individual samples was replicated nine times. The sample concentrations determined from each of the four calibration curves were then combined, and are reported as measured concentration in SI Table S7. The measured concentrations were 4.0 (RSD 11%), 13.2 (RSD 17.3%), and 22.6 mg mL−1 (RSD 6.50%), and were within 20% of the theoretical concentrations.
To further test the PN method, poly(8:2 FTAC-co-HDA) samples (5, 10, and 20 mg mL−1) were spiked into deionized and river (collected from the Etobicoke Creek in Brampton, ON) water, along with a poly(8:2 FTAC-co-HDA) control in the absence of an aqueous media. Following extraction with MTBE, the extracts were prepared for analysis with a set of poly(8:2 FTAC-co-HDA) standards using the same dithranol solution as described above. The calibration curves used to validate the PN method had acceptable linearity with R2 >0.98, and are presented along with their corresponding regression equations in SI Table S9. Each sample was extracted in triplicate (n = 3), and then analyzed three times. The mean poly(8:2 FTAC-co-HDA) concentrations ranged from 2.99 to 4.94, 9.80−13.3, and 16.5−21.1 mg mL−1, and were typically measured within 25% of the theoretical concentrations of 4.67, 10.6, and 22.2 mg mL−1 (Table 2), with recoveries ranging from 55 to 127%.
In addition to the above test samples, a poly(8:2 FTAC-co- HDA) sample (n = 5) from an on going hydrolysis study was measured to be 22.9 ± 2.45 mg mL−1, and was within 9% of the theoretical concentration of 25.1 mg mL−1 (Table 2). The results from the different test samples demonstrated the reasonable effectiveness and reliability of the PN method to quantify poly(8:2 FTAC-co-HDA) samples having different concentrations.
■ ENVIRONMENTAL IMPLICATIONS
The results from this study demonstrated the quantitative MALDI-TOF analysis of poly(8:2 FTAC-co-HDA) using our PN method to develop reproducible calibration curves. By using the same dithranol solution in the sample preparation for samples and standards, PN values were calculated from the ion abundance ratio of poly(8:2 FTAC-co-HDA) to a dithranol- sodium cluster can be directly compared. This approach minimized the sample-to-sample irreproducibility that is often associated with MALDI-TOF analysis by normalizing with the same dithranol solution, and did not require an internal standard polymer. With the PN method, the limit of quantification of poly(8:2 FTAC-co-HDA) was observed to be 1 mg mL−1. Lower molecular weight poly(8:2 FTAC-co- HDA) oligomers (<3000 Da) were detected well below 1 mg mL−1, but could not quantified because the higher molecular weight poly(8:2 FTAC-co-HDA) oligomers (>3000 Da) typically had a signal-to-noise <10. Considering MALDI-TOF is not as accurate as other analytical methods Sodium acrylate such as GC-MS and LC-MS/MS, quantifying a poly(8:2 FTAC-co-HDA) sample within 25% is promising.