Determination of the urinary concentrations of six bisphenols in public servants by online solid-phase extraction-liquid chromatography tandem mass spectrometry
Abstract
Bisphenols are widely used as monomers and additives in plastic production. Thus, bisphenol A (BPA) and its most prominent substitutes have been detected in many environmental and human samples. This study proposes an online solid-phase extraction analytical methodology coupled to liquid chromatography with tandem mass spectrometry for the determination of six bisphenols (BPA and bisphenols F (BPF), S (BPS), AF (BPAF), B (BPB), and E (BPE)) in urine samples as an efficient and automated methodology. The method was developed and validated for all bisphenols with good recoveries (92–112%) and repeatability (RSD ≤ 10%) despite the variable matrix effects, except BPAF (which would require a dedicated internal standard), achieving method quantification limits in the 0.05–2.2 ng mL−1 range. The methodology was subsequently applied to 435 urine samples from a non-occupational exposure population (civil servants for the regional government) from Santiago de Compostela (Galicia, Spain). Only BPA, BPF, and BPS were positively detected; the last two presented higher detection frequencies than BPA. When the urinary concentrations are extrapolated to human intake and compared to the European Food Safety Agency (EFSA) tolerable daily intake (TDI) of 2 × 10−4 µg kg−1 day−1 (TDI), all BPA positively identified samples would surpass this threshold. Although no TDI exists currently for the other two identified bisphenols, it is evident that human exposure to bisphenols should be limited. Finally, the results stratification by gender revealed higher levels of exposure to BPF in the women group.
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For Peer Review 1 Supplementary information Determination of the urinary concentrations of six bisphenols in public servants by online solid-phase extraction-liquid chromatography-tandem mass spectrometry Andrea Estévez-Danta*, Rosario Rodil, José Benito Quintana, Rosa Montes* Aquatic One Health Research Center (ARCUS) & Department of Analytical Chemistry, Nutrition and Food Chemistry. R. Constantino Candeira S/N, IIAA building, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain Analytical & Bioanalytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
For Peer Review 2 Text S1. Enzymatic hydrolysis optimization P.3 Text S2. Analytical methodology for creatinine determination in urine. P.4 Table S1. Chemical structure of target bisphenols P.5 Table S2. Summary of sociodemographic characteristics and creatinine levels for the studied population. P.6 Table S3. Linearity parameters, intercept and slope estimates and standard error P.7 Table S4. Comparison of the performance of the method proposed in this work with other online SPE-LC-MS/MS methods published in the literature. P. 8-9 Table S5. Comparison of the urinary concentrations with other recent published studies. P.10-11 Fig. S1 Standardized pareto charts for (a) BPA, (b) BPS and (c) BPF, obtained during enzymatic deconjugation DOE optimization. P.12 Fig. S2 Estimated response surface for (a) BPF and (b) BPS, obtained during enzymatic deconjugation DOE optimization. P.13 Fig. S3 Relative response in urine for the target compounds employing different concentrations of NH4F, as modifier for both LC mobile phases (n=2). P.14 Fig. S4 Chromatogram of a 100 ng mL-1 spiked urine sample under final conditions. P.15 Fig. S5 Effect of online SPE aqueous phase modifiers (B1) in the peak shape and signal intensity for BPS in urine P.16 Fig. S6 Stability of bisphenol in urine stored at room temperature. RSD (n=3) < 10% P.17 Fig. S7 Comparison of creatinine corrected concentrations (µg g-1) according to tobacco use: (a) BPF and (b) BPS P.18 Fig. S8 Comparison of creatinine corrected concentrations (µg g-1) according to the residence environment: (a) BPF and (b) BPS P.19 Page 35 of 52 Analytical & Bioanalytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
For Peer Review 3 Text S1. Enzymatic hydrolysis optimization Design of experiments (DOE) A central composite design (2^2 + star) with 4 center points was created to make an efficient optimization of the enzyme concentration (experimental domain: 250- 850 units) and incubation time (experimental domain: 1.5 – 4.5 h) variables. The obtained experiments were: Experiment 1. Enzyme concentration = 550 units, incubation time = 0.88 h Experiment 2. Enzyme concentration = 250 units, incubation time = 1.5 h Experiment 3. Enzyme concentration = 850 units, incubation time = 1.5 h Experiment 4. Enzyme concentration = 126 units, incubation time = 3 h Experiment 5. Enzyme concentration = 550 units, incubation time = 3 h Experiment 6. Enzyme concentration = 550 units, incubation time = 3 h Experiment 7. Enzyme concentration = 550 units, incubation time = 3 h Experiment 8. Enzyme concentration = 550 units, incubation time = 3 h Experiment 9. Enzyme concentration = 974 units, incubation time = 3 h Experiment 10. Enzyme concentration = 250 units, incubation time = 4.5 h Experiment 11. Enzyme concentration = 850 units, incubation time = 4.5 h Experiment 12. Enzyme concentration = 550 units, incubation time = 5.12 h Sample preparation and injection 375 µL of filtered urine was adjusted at pH 5 with sodium acetate buffer and spiked with 100 ng mL-1 of available sulfate and glucuronide metabolites mixture (BPA-S, BPA-DS, BPS-S, BPF-S, BPA- G, BPA-DG, BPS-G and BPF-G) + 20 ng mL-1 of ISs BPA-d6 and BPS-d8. Then, each experiment was randomly performed according to the conditions specified in the DOE table and injected into the online SPE-LC-MS/MS system following the optimized protocol. Page 36 of 52Analytical & Bioanalytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
For Peer Review 4 Text S2. Analytical methodology for creatinine determination in urine. Sample preparation Urine samples were filtered through 0.45 µm PVDF syringe-driven filters. Then, each aliquot was diluted 10,000 times in ultrapure water, spiked with the internal standard (creatinine-d3) at 10 ng mL-1 and transferred to an insert for injection in the LC-MS/MS system. Liquid chromatography-tandem mass spectrometry parameters Instrumental analyses were performed with a Waters Acquity UPLC® H class system (Milford, MA, USA) equipped with a quaternary solvent pump, a thermostated LC column compartment, and a sample manager. The UPLC® system was interfaced to a triple quadrupole mass spectrometer Xevo TQD from Waters. The chromatographic separation was performed at 30 ºC on a Luna C18 column (50 x 2.0 mm, I.D., 3 µm particle size) from Phenomenex. A dual eluent system consisting of (A) 0.1% formic acid in ultrapure water and (B) 0.1% formic acid in MeOH was used at a flow rate of 0.3 mL min- 1 in isocratic mode (50:50) for 3 minutes. Injection volume was set at 3 µL. The interface between the UPLC® system and the Xevo TQD mass spectrometer was an electrospray ionization (ESI) source operating in positive mode at a fixed capillary voltage of 3 kV and a temperature of 150 ºC. Nitrogen, provided by a nitrogen generator from Peak Scientific Spain (Barcelona, Spain), was used as desolvation gas at 600 L h-1 and 450 ºC (desolvation temperature), and as cone gas at 10 L h-1. Analyses were performed by MS/MS in Selected Reaction Monitoring (SRM) mode, where: creatinine SRM transitions 114 > 44 (quantification), 114 > 86 (qualification) and creatinine-d3 SRM transitions 117 > 47 (quantification), 117 > 89 (qualification). Validation Calibration curves were prepared in ultrapure water and ranged from the method quantification limit (MQL) to 250 ng mL-1, with IS level of 10 ng mL-1, being the MQL 0.02 ng mL-1 and the obtained R2 was 0.9995. Method repeatability was evaluated at 1 and 50 ng mL-1 and the RSD were 7 and 5 %, respectively for 5 consecutive injections. Method accuracy was evaluated through spiking 6 different real urine samples at 2 mg mL-1 (which corresponds to 200 mg dL-1 in sample and 200 ng mL-1 in the diluted urine) and the obtained values were 102 ± 6 %. Page 37 of 52 Analytical & Bioanalytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
For Peer Review 5 Table S1. Chemical structure of target bisphenols Name Structure Bisphenol A (BPA) Bisphenol AF (BPAF) Bisphenol B (BPB) Bisphenol E (BPE) Bisphenol F (BPF) Bisphenol S (BPS) HO OH S HO OH O O OHHO HO OH CF3 HO OH F3C HO OH Page 38 of 52Analytical & Bioanalytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
For Peer Review 6 Table S2. Summary of sociodemographic characteristics and creatinine levels for the studied population (full data presented in ZENODO repository (https://doi.org/10.5281/zenodo.10477935) Sociodemographic characteristics Gender % Tobacco use % Residence location % Age Creatinine (g L-1) Female Male Yes No Urban Suburban Rural Mean Median SD Mean Median SD 67.6 32.4 16.1 83.9 56.1 33.8 10.1 51.3 52 7.08 0.94 0.82 0.64 Page 39 of 52 Analytical & Bioanalytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
For Peer Review 7 Table S3. Linearity parameters, intercept and slope estimates and standard error. Intercept estimate Intercept standard error Slope estimate Slope standard error Stardard error of estimate BPS 3.74E-04 7.87E-04 3.80E-03 7.83E-05 3.03E-03 BPF 1.43E-03 1.27E-03 7.82E-03 1.31E-04 5.19E-03 BPE -8.49E-04 1.24E-03 1.38E-02 1.30E-04 5.20E-03 BPA 2.85E-03 6.08E-04 4.42E-03 6.42E-05 2.56E-03 BPAF -1.94E-03 9.84E-02 3.94E-01 1.04E-02 4.10E-01 BPB -3.84E-03 1.87E-03 2.07E-02 1.98E-04 7.87E-03 Calculations performed using 3 independent calibration curves with Statgraphics Centurion 19 software Page 40 of 52Analytical & Bioanalytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
For Peer Review 8 Table S4. Comparison of the performance of the method proposed in this work with other online SPE-LC-MS/MS methods published in the literature. Sample preparation Separation and detection Reference Target Bisphenol Pre-treatment Extraction LC-MS/MS %R MQL (ng mL-1) This study BPA, BPAF, BPB, BPE, BPF and BPS 200 µL of urine Filtration through 0.45 µm PVDF syringe-driven filters Addition of IS + 1mM sodium acetate buffer at pH 5 + 700 units of ßglucuronidase + 392 µL of ultrapure water Incubation for 5 h at 37 °C Online SPE on Strata-X 25 µm cartridges Mobile phases: 15 mM of sodium acetate buffer at pH 5 in ultrapure water- MeOH Inj. Vol.: 500 µL LC-(ESI-)-MS/MS on QqQ (SRM) Luna C18 (150 x 2 mm I.D., 3 µm) Mobile phase: 2 mM of NH4F in ultrapure water – 2 mM of NH4F in MeOH 92-112 % (except BPAF 11- 40%) 0.049-2.2 Ye et al., 2005 BPA 100 µL of urine Addition of IS + 50 µL of enzyme/ ammonium acetate Incubation overnight at 37 °C Online SPE on LiChrosphere RP-18 ADS 25 µm column Mobile phases: ultrapure water- MeOH Inj. Vol.: 1000 µL LC-(APCI-)-MS/MS on QqQ (MRM) Chromolith Performance RP-18 (100 x 4.6 mm I.D.) Mobile phases: ultrapure water- MeOH 100 % 1.3 Koch et al., 2012 BPA 300 µL of urine Addition of IS + 1 mM ammonium acetate buffer at pH 5 + 6 µL of ßglucuronidase Incubation for 4 h at 37 °C Online SPE on LiChrosphere RP-8 ADS 25 µm column Mobile phases: ultrapure water- Acetonitrile Inj. Vol.: 100 µL HPLC-(ESI-)-MS/MS on QTRAP (MRM) Waters Atlantis T3 analytical column (150 x 3 mm I.D., 3 µm) Mobile phases: ultrapure water- Acetonitrile 96.8% 0.1 Zhou et. al., 2014 BPA, BPF and BPS 100 µL of urine Addition of IS + dilution to 1 mL with 0.1 M formic acid + 50 µL of ßglucuronidase/sulfatase Incubation for 4 h at 37 °C Stop solution: 750 µL 0.1 M formic acid in ultrapure water Online SPE on LiChrosphere RP-18 ADS 25 µm column Mobile phases: ultrapure water- MeOH Inj. Vol.: 350 µL LC-(APCI-)-MS/MS on QqQ (MRM) Chromolith High Resolution RP-18e (100 x 4.6 mm I.D.) Mobile phases: ultrapure water- MeOH 77-106% 0.03-0.1 Page 41 of 52 Analytical & Bioanalytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
For Peer Review 9 Heffernan et al., 2016 BPA, BPAF, BPB, BPF and BPS 50 µL of urine Addition of IS + 25 µL of ß-glucuronidase (200 units) + 440 µL ultrapure water Incubation for 90 min at 37 °C Stop solution: 400 µL of 0.5 % formic acid in ultrapure water. Online SPE on Strata-X 25 µm cartridges Mobile phase: 0.05 % of acetic acid in ultrapure water: 0.05 % of acetic acid in MeOH Inj. Vol.: 500 µL LC-(ESI-)-MS/MS on QTRAP (MRM) Synergi MAX-RP column (150 x 3 mm I.D., 4 µm) Mobile phase: 0.05% of acetic acid in ultrapure water: 0.05 % of acetic acid in MeOH 101-110% 0.005-0.39 Jo et al., 2020 BPA, BPF and BPS 100 µL of urine Addition of IS + 100 µL of ß-glucuronidase/sulfatase (1000 units) Incubation for 24h at 37 °C Stop solution: 80 µL 1 M formic acid + 670 µL ultrapure water Online SPE on MAYI-ODS column 50 µm Mobile phases: ultrapure water- MeOH Inj. Vol.: 100 µL LC-(APCI-)-MS/MS on QqQ (MRM) ACE 5 C18-pentafluorophenyl column (150 x 2.1 mm I.D., 5 µm) Mobile phases: ultrapure water- MeOH 99.4-108% 0.13-0.24 References: 1. Ye X, Kuklenyik Z, Needham LL, Calafat AM. Automated on-line column-switching HPLC-MS/MS method with peak focusing for the determination of nine environmental phenols in urine. Analytical Chemistry 2005; 77: 5407-5413. 2. Koch HM, Kolossa-Gehring M, Schröter-Kermani C, Angerer J, Brüning T. Bisphenol A in 24 h urine and plasma samples of the German Environmental Specimen Bank from 1995 to 2009: a retrospective exposure evaluation. J Expo Sci Environ Epidemiol 2012; 22: 610-6. 3. Zhou X, Kramer JP, Calafat AM, Ye X. Automated on-line column-switching high performance liquid chromatography isotope dilution tandem mass spectrometry method for the quantification of bisphenol A, bisphenol F, bisphenol S, and 11 other phenols in urine. Journal of Chromatography B 2014; 944: 152-156. 4. Heffernan AL, Thompson K, Eaglesham G, Vijayasarathy S, Mueller JF, Sly PD, et al. Rapid, automated online SPE-LC-QTRAP-MS/MS method for the simultaneous analysis of 14 phthalate metabolites and 5 bisphenol analogues in human urine. Talanta 2016; 151: 224-233. 5. Jo MJ, Park J-H, An K-A, Choi H, Kang Y-s, Hwang M. Quantification of bisphenols in Korean urine using online solid-phase extraction-high-performance liquid chromatography-tandem mass spectrometry. Environmental Toxicology and Pharmacology 2020; 80: 103491. Page 42 of 52Analytical & Bioanalytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
For Peer Review 16 Fig. S5 Effect of online SPE aqueous phase modifiers (B1) in the peak shape and signal intensity for BPS in urine 3 x10 0 0.5 1 1.5 213.191 1 2 x10 0 2 4 6 8 13.154 1 3 x10 0 1 2 3 413.348 1 4 x10 0 0.5 1 2.5 13.245 1 4 x10 0 0.5 1 1.5 13.275 1 Time (min) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 0.1% formic acid 0.1% acetic acid 5mM of sodium acetate buffer at pH 5 15mM of sodium acetate buffer at pH 5 25mM of sodium acetate buffer at pH 5 Page 49 of 52 Analytical & Bioanalytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
For Peer Review 17 Fig. S6 Stability of bisphenol in urine stored at room temperature. RSD (n=3) < 10% Page 50 of 52Analytical & Bioanalytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
For Peer Review 18 Fig. S7 Comparison of creatinine corrected concentrations (µg g-1) according to tobacco use: (a) BPF and (b) BPS (a) (b) Page 51 of 52 Analytical & Bioanalytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
For Peer Review 19 Fig. S8 Comparison of creatinine corrected concentrations (µg g-1) according to the residence environment: (a) BPF and (b) BPS (a) (b) Page 52 of 52Analytical & Bioanalytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60