Abstract
Bisphenol S (BPS) has been detected in personal care products, water, food and indoor house dust, demonstrating the potential for human exposure. Due to limited data to characterize the hazard of BPS, the National Toxicology Program (NTP) is investigating the toxicity of BPS in rodent models. Generating systemic exposure data is integral to putting toxicological findings into context. The objective of this work was to develop and validate a method to quantitate free (unconjugated parent) and total (free and all conjugated forms of) BPS in rodent plasma, amniotic fluid and fetal homogenate in support of NTP studies. The method used incubation with (total BPS) and without (free BPS) deconjugating enzyme and then protein precipitation followed by ultra-performance liquid chromatography-tandem mass spectrometry. In Sprague Dawley rat plasma, the method was linear (r ≥ 0.99) over the range 5–1,000 ng/mL, accurate (mean relative error (RE) ≤ ±10.5%) and precise (relative standard deviation (RSD) ≤ 7.7%). Mean recoveries were ≥93.1% for both free and total analyses. The limits of detection were 1.15 ng/mL (free) and 0.862 ng/mL (total) in plasma. The method was evaluated in the following study matrices: (i) male Hsd:Sprague Dawley®SD® (HSD) rat plasma, (ii) female HSD rat plasma, (iii) male B6C3F1 mouse plasma, (iv) female B6C3F1 mouse plasma, (v) HSD rat gestational day (GD) 18 dam plasma, (vi) HSD rat GD 18 amniotic fluid, (vii) HSD rat GD 18 fetal homogenate and (viii) HSD rat postnatal day 4 pup plasma (mean %RE ≤ ±8.2 and %RSD ≤ 8.7). Stability of BPS in extracted samples was demonstrated for up to 7 days at various temperatures, and freeze–thaw stability was demonstrated after three cycles over 7 days. BPS in various matrices stored at −80°C for at least 60 days was within 92.1–115% of Day 0 concentrations, demonstrating its stability in these matrices. These data demonstrate that this simple method is suitable for determination of free and total BPS in plasma, amniotic fluid and fetuses following exposure of rodents to BPS.
Introduction
Bisphenol S (BPS, Figure 1) is a structural analogue of bisphenol A. Annual U.S. production of BPS in 2016 was between 1 and 10 million pounds (1). It is a component of polyethersulfone, which is known for its stability to heat and light, and is consequently used in a variety of consumer products such as thermal paper and plastic microwavable containers (2–4). BPS is also used as a chemical intermediate in preparation of fire retardants, couplers for photography, electroplating chemicals and colorant, and as a modifier for leather, fiber and epoxy curing agents (2, 3, 5). Due to this potential widespread use, BPS has been detected in personal care products, food stuffs and indoor house dust (6, 7). It has also been detected in the urine from the general population (8–18).
Figure 1.
Structure of bisphenol S (BPS, top) and bisphenol A (bottom).
Despite potential human exposure, the data to assess the safety of BPS are limited. Summaries available from studies indicate that exposure to BPS can result in mortality and induce hepatic, renal, thymus, reproductive and hematological changes (19). In rodents, uterine growth following subcutaneous exposure (20), as well as reduced testosterone levels and the potential to reduce spermatogenesis following repeated oral exposure (21), has been reported.
Due to limited data, the National Toxicology Program (NTP) is testing BPS in rodent models. Systemic exposure data are critical for interpreting toxicity data and putting toxicological findings into greater context. We have previously shown that BPS was well-absorbed following oral exposure in rodents and rapidly and extensively metabolized to form glucuronide and sulfate conjugates (22). The objective of this work was to develop and validate a simple method to quantitate both free (parent BPS) and total BPS (parent and conjugated forms) in rodent plasma, amniotic fluid and fetuses in support of NTP studies. Methods for the analysis of BPA and other analogs in urine and plasma have used liquid chromatography--tandem mass spectrometry (LC–MS-MS) (11, 23–28), but these methods often required complex sample extraction, derivatization and/or the inability to measure both free BPS and all of its metabolites.
Experimental
Chemicals and reagents
BPS (>99% purity) was purchased from Ivy Fine Chemicals Inc. (Cherry Hill, NJ), and bis(4-hydroxyphenyl) sulfone-d8 (BPS-d8, internal standard (IS)) and BPS-glucuronide were from Toronto Research Chemicals (Ontario, Canada). All biological matrices used for analytical method validation were purchased from Bioreclamation IVT (Westbury, NY). Those included male and female Hsd:Sprague Dawley®SD® (HSD) rat and B6C3F1 mouse plasma, male Sprague Dawley (SD) rat plasma, HSD rat gestational day (GD) 18 plasma, amniotic fluid, pooled fetuses and HSD rat postnatal day (PND) 4 pup plasma. β-Glucuronidase and sulfatase from Helix Pomatia (at least 300,000 and 10,000 units/g, respectively) were purchased from Sigma-Aldrich (St. Louis, MO). Ultrapure water was generated using the Hydro Picosystem UV Plus purification system (Durham, NC). All other reagents and solvents were procured from commercial suppliers.
Strategy for method development and validation
Ultra-performance liquid chromatography--tandem mass spectrometry (UPLC–MS-MS) conditions were optimized with respect to chromatographic performance, sensitivity, selectivity and dynamic range. Sample preparation with and without enzyme deconjugation (β-glucuronidase and sulfatase) was used prior to extraction with acetonitrile, for analysis of total and free BPS, respectively. The method was fully validated in male SD rat plasma by analyzing calibration standards and quality control (QC) samples in replicates over multiple analysis days to demonstrate linearity, selectivity, sensitivity, recovery, intra- and interday precision and accuracy, and reproducibility over the desired concentration ranges. The concentration range was 5–1,000 ng/mL for both free and total BPS in plasma. Selectivity was assessed by analyzing six matrix blanks (without IS) and six method blanks (matrix blanks with IS) for background interferences at the BPS retention time. Accuracy was evaluated as percent relative error (%RE) and precision as percent relative standard deviation (%RSD). The limit of quantitation (LOQ) was the lowest matrix standard that could be accurately quantitated within 20% of the nominal value and at which six replicates could be reproduced within 20% RSD. The limit of detection (LOD) was defined as three times the standard deviation of the LOQ response expressed as concentration (29). Recovery was expressed as the percentage of the response ratio for BPS in matrix to that in solvent. Intraday precision and accuracy were evaluated at three concentration levels in plasma using three independent QCs prepared on the same day. Interday precision and accuracy were evaluated at the same three concentration levels, using multiple sets of independent QCs prepared and analyzed over multiple analysis days. Carryover was evaluated using three method blanks run immediately following the highest calibration standard. The response in each of these blanks was compared to the mean response for the method blanks run at the beginning of the sample set. Instrument drift (reinjection reproducibility) was assessed by preparing a set of calibration standards and running them at the beginning and end of the sample set, with ∼40 samples run in-between. The first analysis of the matrix calibration standards was used for calibration; the second, re-injected set was analyzed as an independent QC set to assess drift. The method was evaluated in secondary matrices by preparing blanks and QCs in secondary matrix and quantitating using a calibration curve prepared in primary matrix (male SD rat plasma). The secondary matrices were male and female HSD rat plasma, male and female B6C3F1 mouse plasma, HSD rat GD 18 dam plasma, amniotic fluid and fetal homogenate and HSD rat PND 4 pup plasma.
Preparation of stock solutions
Two stock solutions of BPS were prepared in methanol at 100 μg/mL and further diluted in the same solvent to generate spiking solutions over the range 25–5,000 ng/mL, using alternate stocks. A working solution of BPS-d8, IS, was prepared at 2,250 μg/mL in methanol. The deconjugating enzyme solution containing β-glucuronidase and sulfatase was prepared daily in 1 M ammonium acetate buffer (adjusted to pH 5.0 with acetic acid) to yield a final solution of at least 2 units/μL β-glucuronidase and 0.4 units/μL sulfatase.
Preparation of plasma calibration standards
Plasma calibration standards (5, 10, 50, 100, 250, 500 and 1,000 ng/mL) and QC samples (50, 100 and 500 ng/mL) were prepared fresh each day by fortifying 50 µL of male SD rat plasma with 10 µL of the appropriate spiking solution and 10 µL of IS. For method blanks and study samples, 10 µL of methanol were substituted for the spiking solution. Solvent standards (for determination of recovery) were prepared at the same concentrations in the same manner as the matrix standards, except 50 µL of water were used instead of plasma. To each sample, 25 µL of 1 M ammonium acetate buffer (for free analysis) or the β-glucuronidase/sulfatase solution (for total analysis) was added. Samples were briefly mixed and either stored at ∼5°C for 2 hr (for free analysis) or incubated at ∼37°C for 2 hr (for total analysis). Following storage/incubation, 300 µL of acetonitrile were added, and the vials were mixed briefly and centrifuged at 4°C for 10 min at ∼20,000 × g. The supernatant was analyzed by UPLC–MS-MS as described below.
Fetus homogenization
Fetal homogenate was prepared by weighing thawed fetuses into two plastic centrifuge tubes and weighing 2.3 mm stainless steel beads (BioSpec Products, Bartlesville OK), approximately twice the weight of fetus in a 2:1 ratio, into each tube. The fetuses were homogenized using a Geno/Grinder 2010 (SPEX SamplePrep, Metuchen, NY) at ∼1,750 rpm for ∼2 min. Water was added to each tube (1:1 volume ratio of fetus to water, assuming 1 g = 1 mL), and the tissue was homogenized again at ∼1,750 rpm for ∼2 min. The homogenate was combined into one tube, and then homogenized third time at ∼1,750 rpm for ∼2 min. Concentrations in fetal homogenate (ng/mL) can therefore be converted to ng/g fetus using a dilution factor of 2.
Secondary matrix evaluation
The secondary matrices evaluated were (i) male HSD rat plasma, (ii) female HSD rat plasma, (iii) male B6C3F1 mouse plasma, (iv) female B6C3F1 mouse plasma, (v) HSD rat GD 18 dam plasma, (vi) HSD rat GD 18 amniotic fluid, (vii) HSD rat GD 18 fetal homogenate and (viii) HSD rat PND 4 pup plasma. Six replicate matrix samples containing BPS were prepared at 10 ng/mL in each secondary matrix and analyzed using a primary (male SD rat plasma) calibration curve. Six matrix blanks (no IS) and six method blanks (with IS) were prepared in each secondary matrix to evaluate selectivity.
Dilution verification
In anticipation of samples with concentrations higher than the validated range, a dilution verification was conducted to demonstrate that samples with BPS concentrations greater than the upper limit of the validated range could be brought into range by dilution. Triplicate matrix samples were prepared at 20,000 ng/mL, extracted according to the validated method and then diluted by a factor of 200 with extracted plasma (containing IS) prior to analysis.
Stability
Stability of BPS in extracted samples was determined by preparing plasma QCs at three concentration levels (50, 100 and 500 ng/mL), extracting them and then storing the extracts at ambient, refrigerated and freezer (−20°C) temperatures for 7 days. Freeze–thaw stability of BPS in plasma was evaluated by preparing plasma QCs at 50, 100 and 500 ng/mL, storing at −80°C and then subjecting to three freeze–thaw cycles before extraction on the seventh day. To cover the study sample storage conditions and duration, ≥60-day frozen storage stability was also determined by preparing plasma QC samples in all secondary matrices (except PND 4 pup plasma) at two concentration levels (10 and 500 ng/mL) and then storing at −80°C for at least 60 days. The stability samples were analyzed according to the validated method. Determined concentrations were compared to the mean concentration of freshly prepared Day 0 samples, expressed as percent of Day 0.
LC–MS-MS analysis and analyte quantitation
The UPLC–MS-MS system consisted of a Waters (Milford, MA) Acquity UPLC coupled to a 4000 QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer with a TurboIonSpray (electrospray) source (Sciex, Framingham, MA). Chromatographic separation was performed using a Waters Acquity UPLC BEH C18 column (50 mm × 2.1 mm i.d., 1.7-μm particle size) and a Waters Acquity UPLC BEH C18 guard column (5 mm × 2.1 mm i.d., 1.7 μm particle size). A 5-µL volume of sample was injected onto the column, and elution was achieved at 30°C using a binary gradient and a flow rate of 0.3 mL/min. The mobile phases consisted of (A) water (B) acetonitrile. The gradient was 5% B for 1 min, ramp to 95% B in 5 min, and held at 95% B for 2 min. The electrospray ion source was operated in negative ion mode with an ion spray voltage of −3,000 V and source temperature of 650°C. The curtain gas was 15 psi, the nebulizer gas was 65 psi, the heater gas was 85 psi and the interface heater was on. The selected multiple reaction monitoring (MRM) ion transitions were 248.9 → 107.9 for BPS (248.9 → 155.9 for confirmation) and 257.0 → 112.0 for BPS-d8. The MRM transition for BPS-glucuronide (425.0 → 248.9) was also included in the method for non-quantitative purposes, to demonstrate completeness of the deconjugation reaction. The predicted MRM transitions for BPS-sulfate (329 → 249) and di-conjugates (601 → 249, 409 → 249 and 505 → 249) were monitored as well, for qualitative purposes only. The optimized compound-dependent parameters for BPS and BPS-d8 were declustering potential = −60 V; entrance potential = −10 V; collision energy = −34 V and collision cell exit potential = −9 V. The same parameters were used for BPS-glucuronide and the other metabolites except declustering potential = −45 V and collision energy = −24 V. The Analyst software version 1.6.2 (Sciex) was used for data acquisition and analysis.
Seven-point calibration curves were generated by plotting the peak area ratios of BPS to BPS-d8 as a function of analyte concentration. The regression model for BPS was a linear weighted least squares algorithm with a weighting factor of 1/concentration-squared (1/x2). The concentrations of free and total BPS were calculated using response ratio, the regression equation, initial sample volume and dilution when applicable.
Results
Method validation
Method validation data are summarized in Table I. The LOQ (lowest matrix standard that met the accuracy and precision criteria) for BPS was 5.0 ng/mL for both free and total analyses; the LODs (defined as three times the standard deviation of the lowest concentration, 5 ng/mL) were 1.15 and 0.862 ng/mL, respectively. Matrix and solvent blanks showed detectable levels of BPS, indicating background interference, not unexpected given the ubiquity of BPS in the environment. However, the responses were less than the determined LODs and did not interfere with method performance. Representative chromatograms for BPS in primary matrix (male SD rat plasma) are presented in Figure 2 for free and total analyses.
Table I.
Method Validation Data for Free and Total BPS
| Validation parameter | Free | Total |
|---|---|---|
| Matrix concentration range (ng/mL) | 5.0–1,000 | 5.0–1,000 |
| LOD (ng/mL)a | 1.15 | 0.862 |
| LOQ (ng/mL)b | 5.0 | 5.0 |
| Correlation coefficient (r) | ≥0.99 | ≥0.99 |
| Mean recovery (%) | 97.5 | 93.1 |
| Precision and accuracyc | ||
| Intraday precision (%RSD) | ≤3.7 | ≤2.4 |
| Intraday accuracy (mean %RE) | ≤±6.9 | ≤±10.5 |
| Interday precision (%RSD) | ≤5.1 | ≤7.7 |
| Interday accuracy (mean %RE) | ≤±7.3 | ≤±5.0 |
| Dilution verification (up to 20,000 ng/mL)d | ||
| Precision (%RSD) | 3.3 | 3.8 |
| Accuracy (mean %RE) | 5.7 | −2.3 |
| Secondary matrix evaluatione | ||
| Male HSD plasma | ||
| Precision (%RSD) | 5.5 | 8.7 |
| Accuracy (mean %RE) | −2.3 | 4.1 |
| Female HSD plasma | ||
| Precision (%RSD) | 4.2 | 4.5 |
| Accuracy (mean %RE) | −1.3 | 5.6 |
| Male B6C3F1 plasma | ||
| Precision (%RSD) | 3.2 | 3.3 |
| Accuracy (mean %RE) | 3.5 | −1.7 |
| Female B6C3F1 plasma | ||
| Precision (%RSD) | 3.2 | 4.2 |
| Accuracy (mean %RE) | 2.5 | 0.6 |
| GD 18 plasma | ||
| Precision (%RSD) | 4.8 | 5.8 |
| Accuracy (mean %RE) | −3.7 | 0.2 |
| GD 18 amniotic fluid | ||
| Precision (%RSD) | 6.9 | 4.0 |
| Accuracy (mean %RE) | 1.0 | 5.5 |
| GD 18 fetal homogenate | ||
| Precision (%RSD) | 4.5 | 6.5 |
| Accuracy (mean %RE) | 7.8 | 1.9 |
| PND 4 pup plasma | ||
| Precision (%RSD) | 5.4 | 6.2 |
| Accuracy (mean %RE) | 4.8 | 8.2 |
Defined as three times the standard deviation of the LOQ (5 ng/mL).
Lowest standard at which RE ≤ ±20% and RSD ≤ 20% for n = 6.
Precision and accuracy determined for triplicate QCs at three levels in plasma: 50, 100 and 500 ng/mL. n = 3 for intraday; n = 9 for interday.
Precision and accuracy determined for triplicate QCs at 20,000 ng/mL plasma, diluted into range.
Precision and accuracy for secondary matrices determined for six replicate QCs at 10 ng/mL for male and female HSD rat plasma, male and female B6C3F1 mouse plasma, HSD rat GD 18 dam plasma, amniotic fluid and fetal homogenate, and HSD rat PND 4 pup plasma.
Figure 2.
Representative UPLC–MS-MS chromatograms for BPS in male SD rat plasma: blank rat plasma, blank rat plasma with IS, 5 ng/mL BPS in plasma and 1,000 ng/mL BPS in plasma. Panes on the left are for free analysis; panes on the right are for total analysis.
The calibration curves in plasma were linear with correlation coefficients r ≥ 0.99. The intra- and interday precisions for QCs prepared at 50, 100 and 500 ng/mL were ≤7.7% for both free and total analyses, and accuracies were within ±10.5%RE. Recovery was determined at all calibration levels in each matrix. The mean recoveries were 97.5% for free and 93.1% for total analyses. Carryover was evaluated by assessing the response in three method blanks run immediately following the highest calibration standard of 1,000 ng/mL and comparing them to the mean response for the method blanks run at the beginning of the sample set. No carryover was present for either free or total BPS. To assess instrument drift, a set of matrix calibration standards was prepared in plasma and injected at the start and the end of sample analysis. The relative difference between the determined concentrations for the second set compared to the calibration set were within ±11.3%, indicating minimal instrument drift. Plasma QCs prepared at 20,000 ng/mL and diluted into the validated range using extracted plasma had RE ≤ ±5.7% RE and RSD ≤ 3.8% for both free and total analyses, demonstrating the ability to dilute samples into the range of the calibration curve. Study samples were diluted in this same manner, using extracted plasma.
Matrix evaluation in secondary matrices
Successful matrix evaluations were achieved for BPS in male and female HSD rat plasma, male and female B6C3F1 mouse plasma, HSD rat GD 18 dam plasma, amniotic fluid and fetal homogenate and HSD rat PND 4 pup plasma. Representative chromatograms in one secondary matrix (male HSD rat plasma) are presented in Figure 3. Secondary matrix samples were prepared at 10 ng/mL and quantitated against the male SD rat plasma calibration curve; RE and RSD were ≤±8.2 and ≤8.7%, respectively. All secondary matrix control blanks showed a very minor BPS background peak but responses in the blanks were below the determined LODs and did not interfere with the precision and accuracy of the assay. See Figure 3 (top panes) for a representative secondary matrix blank.
Figure 3.
Representative UPLC–MS-MS chromatograms for BPS in male HSD rat plasma: blank rat plasma, blank rat plasma with IS, 10 ng/mL BPS in plasma and plasma following single gavage administration of BPS (see reference 30). Panes on the left are for free analysis; panes on the right are for total analysis.
Stability
Studies were performed to evaluate both matrix and extract stability of BPS. The determined concentrations of the stability samples were compared to those of freshly prepared samples (Day 0), and data are presented in Table II. Extracts were 90.8 to 96.4% (free analysis) and 94.7–102% (total analysis) of Day 0, demonstrating that BPS is stable up to 7 days at ambient, refrigerated and freezer (−20°) temperatures. BPS in plasma stored frozen (−80°C) and undergoing three freeze–thaw cycles over 7 days were within 80.4–88.7% (free) and 87.1 and 91.2% (total) of Day 0. BPS in secondary matrices stored frozen (−80°) for 60–79 days was within 92.1–114% (free) and 93.5–115% (total) of Day 0, demonstrating the stability over these conditions and durations.
Table II.
Stability Data for BPS in Rodent Plasma, Amniotic Fluid and Fetal Homogenate
| Mean % of Day 0 (% RSD) | ||
|---|---|---|
| Stability endpoint | Free | Total |
| Extract stability | ||
| Refrigerated extracts (4°C, 7 days) | 94.5–96.4 (%RSD ≤ 4.6%) | 96.2–102 (%RSD ≤ 2.7%) |
| Ambient extracts (RT, 7 days) | 90.8–93.4 (%RSD ≤ 2.7%) | 95.1–99.2 (%RSD ≤ 3.5%) |
| Frozen extracts (−20°C, 7 days) | 91.1–93.4 (%RSD ≤ 2.9%) | 94.7–100 (%RSD ≤ 4.5%) |
| Matrix stability | ||
| Freeze–thaw (three cycles over 7 days) | 80.4–88.7 (%RSD ≤ 4.3%) | 87.1–91.2 (%RSD ≤ 3.1%) |
| Frozen matrix (−80°C, ≥ 60 days) | ||
| Male HSD plasma | 96.8–103 (%RSD ≤ 2.8%) | 101–102 (%RSD ≤ 1.5%) |
| Female HSD plasma | 92.1–95.1 (%RSD ≤ 3.1%) | 93.5–101 (%RSD ≤ 3.7%) |
| Male B6C3F1 plasma | 97.1–108 (%RSD ≤ 8.9%) | 97.6–99.1 (%RSD ≤ 5.5%) |
| Female B6C3F1 plasma | 96.4–108 (%RSD ≤ 5.6%) | 108–114 (%RSD ≤ 4.7%) |
| GD 18 dam plasma | 111–114 (%RSD ≤ 9.8%) | 109–115 (%RSD ≤ 8.0%) |
| GD 18 amniotic fluid | 110–114 (%RSD ≤ 9.2%) | 111–113 (%RSD ≤ 6.5%) |
| GD 18 fetal homogenate | 105–110 (%RSD ≤ 3.6%) | 105–108 (%RSD ≤ 4.0%) |
Extract and freeze–thaw stability determined for triplicate QCs at three levels: 50, 100 and 500 ng/mL in male SD rat plasma.
Frozen matrix stability determined for triplicate QCs at two levels: 10 and 500 ng/mL (male and female HSD rat plasma, male and female B6C3F1 mouse plasma, HSD rat GD 18 dam plasma, amniotic fluid and fetal homogenate). Male and female HSD rat plasma and male and female B6C3F1 mouse plasma were analyzed on Day 60, HSD rat GD 18 dam plasma and amniotic fluid were analyzed on Day 79 and HSD rat GD 18 fetal homogenate was analyzed on Day 75.
Discussion
We have described the development and validation of a simple method to quantitate both free (parent BPS) and total BPS (parent and conjugated forms) in rodent plasma, amniotic fluid and fetuses. Different column chemistries, mobile phases and gradients were explored to optimize analyte retention and peak shape. A BEH C18 column was selected because it gave stable retention and peak shape. Initially, a 10 mM ammonium acetate solution was used as a mobile phase, but it was found to cause an elevated BPS background and was therefore eliminated; a simple mobile phase system of water and acetonitrile was better. A commercially available stable isotope-labeled standard, BPS-d8 was used as the IS. To demonstrate that BPS-glucuronide was stable and that deconjugation did not occur in the absence of glucuronidase enzyme, a brief stability test was performed. A commercially available standard of BPS-glucuronide was obtained, and the UPLC–MS-MS method was optimized to include it. BPS-glucuronide was spiked into rat plasma, and both BPS and BPS-glucuronide transitions were monitored over time. The glucuronide was stable in plasma for 4 hr at room temperature and 24 hr refrigerated before extraction. Thus, in the absence of deconjugating enzyme, one can be confident that only free BPS is measured, and comparison of free vs. total BPS is straightforward and conclusive. It was also found that deconjugation was complete after incubation with enzyme at 37°C for 2 hr. A simple incubation with and without glucuronidase and sulfatase enzymes was thus incorporated into the method preparation prior to extraction with acetonitrile, for analysis of both free and total BPS.
The quantitation range of the validated method is 5–1,000 ng/mL for both free and total BPS, with LODs of 1.15 ng/mL (free) and 0.862 ng/mL (total). This method was successfully used to quantitate free and total BPS in rats and mice following a single gavage and IV administration of BPS (30), as well as via repeated feed exposure (31). Free and total BPS concentrations were measurable in all exposed groups in these studies; for example, for male rats in the lowest gavage dose group (34 mg/kg), mean BPS concentrations ranged from 11.0 ng/mL (32 hr) to 644 ng/mL (0.25 hr) for free BPS and 101 ng/mL (48 hr) to 6,740 ng/mL (0.5 hr) for total BPS. Likewise, for male mice in the lowest feed exposure group (338 ppm), mean BPS concentrations ranged from 10.4 ng/mL (20 hr) to 155 ng/mL (0 hr) for free BPS and 127 ng/mL (24 hr) to 2,280 ng/mL (0 hr) for total BPS. Sensitivity could certainly be improved for biomonitoring studies using a larger sample size, complex sample extraction and/or a larger injection volume; however, the range has been shown to be sufficient for NTP toxicokinetic studies. In addition, because the MRM transitions were qualitatively monitored for both glucuronide and sulfate conjugates in these studies, we observed that both are formed in vivo (see Figure 3 herein, bottom pane), and our incubation procedure with β-glucuronidase and sulfatase was effective in fully deconjugating BPS.
Conclusion
A method was developed and validated to quantitate BPS in rodent plasma, amniotic fluid and fetuses using a simple UPLC–MS-MS method. The method uses incubation with (total BPS) and without (free BPS) deconjugating enzyme and a simple protein precipitation, with no complex sample extraction and/or derivatization. The quantitation range is 5–1,000 ng/mL for both free BPS (parent) and total BPS (free and all conjugated forms). The LODs are 1.15 ng/mL (free) and 0.862 ng/mL (total). BPS was shown to be stable in frozen matrix for 60 days and in extracts at various temperatures for 7 days and could withstand three freeze–thaw cycles over 7 days. In addition, samples above the upper LOQ can be accurately diluted into the calibration range for analysis. This method was used to quantitate free and total BPS in rats and mice following exposure to BPS in support of NTP toxicokinetic and toxicology studies.
Acknowledgments
The authors are grateful to Mr. Brad Collins and Dr. Jason Stanko for their review of this manuscript.
Contributor Information
Melanie A Rehder Silinski, Discovery Sciences Unit, RTI International, P.O. Box 12194, Research Triangle Park, NC 27709, USA.
Brenda L Fletcher, Discovery Sciences Unit, RTI International, P.O. Box 12194, Research Triangle Park, NC 27709, USA.
Reshan A Fernando, Discovery Sciences Unit, RTI International, P.O. Box 12194, Research Triangle Park, NC 27709, USA.
Veronica G Robinson, Division of the National Toxicology Program, NIEHS, P.O. Box 12233, Research Triangle Park, NC 27709, USA.
Suramya Waidyanatha, Division of the National Toxicology Program, NIEHS, P.O. Box 12233, Research Triangle Park, NC 27709, USA.
Funding
This work was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences and Intramural Research Project ZIA ES103316-04 and performed for the National Toxicology Program, National Institute of Environmental Health Sciences, National Institutes of Health and U.S. Department of Health and Human Services, under contract HHSN273201400022C (RTI International, RTP, NC).
Conflict of Interest
The authors declare that they have no conflict of interest.
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