Dan A. Markham

Development of a Method for the Determination of Bisphenol A at Trace Concentrations in Human Blood and Urine and Elucidation of Factors Influencing Method Accuracy and Sensitivity

This publication describes a method for the determination of total bisphenol A (BPA and conjugated BPA) following enzyme hydrolysis and is intended as a companion to our previously developed analytical method for the determination of free BPA (the aglycone) in human blood and urine using high-performance liquid chromatography-tandem mass spectrometry ( 1). That free BPA method provided a means to account for and/or eliminate background contamination and demonstrated accuracy and reproducibility in both matrices fortified with BPA or a surrogate analyte ((13)C BPA) at a low method quantitation limit (MQL) of 0.1-0.2 ng/mL. In contrast to the free BPA method results and based on stringent accuracy, precision and confirmation criteria set for the MQLs of the method developed for total BPA, the MQL achieved in blood was 1.020-2.550 and 0.510-1.020 ng/mL in urine. These data showed higher MQLs than the desired MQLs of 0.5 ng/mL (blood) and 0.2 ng/mL (urine) with increased variability between analyses which demonstrates the importance of generating method validation data with each analysis. In contrast, the MQL achieved for (13)C BPA-G (monoglucuronide as a surrogate analyte in blood was 0.2-0.5 and 0.2 ng/mL in urine illustrating that the method is capable of meeting lower MQL requirements if the contribution from exogenous BPA can be well controlled. This method for the determination total BPA in human blood and urine is intended to be used in conjunction with the free BPA method ( 1) to obtain accurate and complete BPA biomonitoring data to support human exposure assessments.

Potential mechanisms of tumorigenic action of diethanolamine in mice

Diethanolamine (DEA), a secondary amine found in a number of consumer products, reportedly induces liver tumors in mice. In an attempt to define the tumorigenic mechanism of DEA, N-nitrosodiethanolamine (NDELA) formation in vivo and development of choline deficiency were examined in mice. DEA was administered with or without supplemental sodium nitrite to B6C3F1 mice via dermal application (with or without access to the application site) or via oral gavage for 2 weeks. Blood levels of DEA reflected the dosing method used; oral greater than dermal with access greater than dermal without access. No NDELA was observed in the urine, blood or gastric contents of any group of treated mice. Choline, phosphocholine and glycerophosphocholine were decreased </=62-84% in an inverse relation to blood DEA levels. These data demonstrated a lack of NDELA formation in vivo at tumorigenic dosages of DEA but revealed a pronounced depletion of choline-containing compounds in mice. It is suggested that the latter effect may underlie DEA tumorigenesis in the mouse.

Quantitative Determination of Bisphenol-A in River Water by Cool On-Column Injection-Gas Chromatography-Mass Spectrometry

Abstract A sensitive and selective method is described for the simultaneous quantitation and confirmation of bisphenol-A (BPA) in river water to support a surface water analysis study. The method employs a cool-on-column injection, gas chromatographic separation and detection by electron impact mass spectrometry (COC-GC-MS). River water samples (40 mL) were fortified with a stable isotope internal standard, deuterium labeled analog of BPA (D8-BPA). The sample was then extracted with toluene and the extract was concentrated (400X) prior to analysis by COC-GC-MS. The analysis involves detection of the M+ ion and the [M-CH3]+ ion (base peak) with quantitation based on the [M-CH3]+ ion response. The quantitation limit, with confirmation, for BPA for this method was determined to be 1 μg BPA/L of river water. Confirmation of BPA was determined by monitoring the ratio of the M+ ion to the [M-CH3]+ ion. Calibration curves for standard concentrations of 1 μg/L to 20 μg/L resulted in correlation coefficients (r2) ...

Determination of 2-butoxyethanol and butoxyacetic acid in rat and human blood by gas chromatography-mass spectrometry

A sensitive and selective gas chromatographic-negative-ion chemical ionization mass spectrometric method was developed to simultaneously quantitate 2-butoxyethanol (BE) and butoxyacetic acid (BAA) in rat and human blood at low ng/g levels as pentafluorobenzoyl and pentafluorobenzyl derivatives, respectively. Analysis of 13C-labeled analogs of BE and BAA were found to improve the limits of quantitation to below 2 ng/g. Deuterium-labeled BE and BAA were used as internal standards. Calibration curves were generally linear over three orders of magnitude, with limits of quantitation of 16-18 ng/g for both BE and BAA, and 1.5 and 0.4 ng/g for [13C2]BE and [13C2]BAA, respectively, in human blood. Linearity in rat blood was similar, with limits of quantitation of 22 ng/g for BE and 5 ng/g for BAA. This method was developed for the support of mammalian metabolism studies and human biomonitoring studies involving exposure to BE or [13C2]BE.

Preparation, Characterization, and Scale-up of Ketoconazole with Enhanced Dissolution and Bioavailability

ABSTRACT Many new molecular entities targeted for pharmaceutical applications face serious development challenges because of poor water solubility. Although particle engineering technologies such as controlled precipitation have been shown to enhance aqueous dissolution and bioavailability of poorly water soluble active pharmaceutical ingredients, the data available are the results of laboratory-scale experiments. These technologies must be evaluated at larger scale to ensure that the property enhancement is scalable and that the modified drugs can be processed on conventional equipment. In experiments using ketoconazole as the model drug, the controlled precipitation process was shown to produce kg-scale modified drug powder with enhanced dissolution comparable to that of lab-scale powder. Ketoconazole was demonstrated to be stable throughout the controlled precipitation process, with a residual methanol level below the ICH limit. The modified crystalline powder can be formulated, and then compressed using conventional high-speed tableting equipment, and the resulting tablets showed bioavailability more than double that of commercial tablets. When appropriately protected from moisture, both the modified powder and tablets prepared from the modified powder showed no change in dissolution performance for at least 6 months following storage at accelerated conditions and for at least 18 months following storage at room temperature.

Extensive metabolism and route-dependent pharmacokinetics of bisphenol A (BPA) in neonatal mice following oral or subcutaneous administration.

Orally administered bisphenol A (BPA) undergoes efficient first-pass metabolism to produce the inactive conjugates BPA-glucuronide (BPA-G) and BPA-sulfate (BPA-S). This study was conducted to evaluate the pharmacokinetics of BPA, BPA-G and BPA-S in neonatal mice following the administration of a single oral or subcutaneous (SC) dose. This study consisted of 3 phases: (1) mass-balance phase in which effective dose delivery procedures for oral or SC administration of (3)H-BPA to postnatal day three (PND3) mice were developed; (2) pharmacokinetic phase during which systemic exposure to total (3)H-BPA-derived radioactivity in female PND3 mice was established; and (3) metabolite profiling phase in which 50 female PND3 pups received either a single oral or SC dose of (3)H-BPA. Blood was collected from 5 pups/route/time-point at various times post-dosing, the blood plasma samples were pooled by group, and time-point and samples were profiled by HPLC with fraction collection. Fractions were analyzed for total radioactivity and data used to reconstruct radiochromatograms and to integrate individual peaks. The identity of the BPA, BPA-G, and BPA-S peaks was confirmed using authentic standards and LC-MS/MS analysis. The result of this study revealed that female PND3 mice have the capacity to metabolize BPA to BPA-G, BPA-S and other metabolites after both routes of administration. Systemic exposure to free BPA is route-dependent as the plasma concentrations were lower following oral administration compared to SC injection.

Quantitative analysis of unconjugated and total bisphenol A in human urine using solid-phase extraction and UPLC–MS/MS: Method implementation, method qualification and troubleshooting

The aim of the presented investigation was to document challenges encountered during implementation and qualification of a method for bisphenol A (BPA) analysis and to develop and discuss precautions taken to avoid and to monitor contamination with BPA during sample handling and analysis. Previously developed and published HPLC-MS/MS methods for the determination of unconjugated BPA (Markham et al. Journal of Analytical Toxicology, 34 (2010) 293-303) [17] and total BPA (Markham et al. Journal of Analytical Toxicology, 38 (2014) 194-203) [20] in human urine were combined and transferred into another laboratory. The initial method for unconjugated BPA was developed and evaluated in two independent laboratories simultaneously. The second method for total BPA was developed and evaluated in one of these laboratories to conserve resources. Accurate analysis of BPA at sub-ppb levels is a challenging task as BPA is a widely used material and is ubiquitous in the environment at trace concentrations. Propensity for contamination of biological samples with BPA is reported in the literature during sample collection, storage, and/or analysis. Contamination by trace levels of BPA is so pervasive that even with extraordinary care, it is difficult to completely exclude the introduction of BPA into biological samples and, consequently, contamination might have an impact on BPA biomonitoring data. The applied UPLC-MS/MS method was calibrated from 0.05 to 25ng/ml. The limit of quantification was 0.1ng/ml for unconjugated BPA and 0.2ng/ml for total BPA, respectively, in human urine. Finally, the method was applied to urine samples derived from 20 volunteers. Overall, BPA can be analyzed in human urine with acceptable recovery and repeatability if sufficient measures are taken to avoid contamination throughout the procedure from sample collection until UPLC-MS/MS analysis.

Quantification of Total Thyroxine in Plasma from Xenopus laevis

There is evidence that the endocrine systems of certain fish and wildlife can be affected by chemical contaminants, possibly resulting in developmental and reproductive problems. Perturbations in the hypothalamus-pituitary-thyroid (HPT) axis, in particular, can be detrimental during early development. Because the rate of amphibian metamorphosis is controlled by circulating thyroid hormones, tadpoles undergoing metamorphosis have been selected as relevant test organisms for evaluating the potential effects of a substance on the HPT axis in vertebrates. An indicative measure of HPT functioning in these assays is the concentration of the thyroid hormone, thyroxine (T4), in frog plasma. Therefore, there is a need for a validated method to measure T4 in plasma during amphibian metamorphosis. This study describes a method involving mixed-mode strong cation exchange solid-phase extraction (SPE) with ultrahigh-performance liquid chromatography and isotope dilution tandem mass spectrometry (UPLC-ID-MS-MS) to quantify total T4 in a small volume (10 µL) of plasma from Xenopus laevis (African clawed frog). The SPE procedure, together with MS detection, produced the required selectivity for the analysis of both T4 and the T4 internal standard. The limit of detection of the method was determined to be 0.2 ng/mL, whereas the lower limit of quantification was 0.5 ng/mL. The intra-day and inter-day precision values were less than ± 5 and ±10%, respectively. Concentrations of total T4 in the plasma of X. laevis tadpoles at metamorphic peak were calculated to be 10.7 ± 0.8 ng/mL, which is comparable to the results from radioimmunoassay. This validated UPLC-ID-MS-MS method for the determination of total T4 in plasma has demonstrated good accuracy and precision, with low susceptibility to interferences with the utilization of multiple reaction monitoring and ID.

Analytical methods impact estimates of trichloroethylene’s glutathione conjugation and risk assessment

The glutathione (GSH) conjugates, S-(1,2-dichlorovinyl)-glutathione (DCVG) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC), have been implicated in kidney toxicity and kidney cancer from trichloroethylene (TCE) exposure. Considerable differences in blood and tissue levels of DCVG and DCVC have been reported, depending on whether HPLC/UV (High Performance Liquid Chromatography-Ultraviolet) or HPLC/MS (HPLC-Mass Spectrometry) was used. A side-by-side comparison of analytical results with HPLC/UV and HPLC/MS/MS (High Performance Liquid Chromatography-Tandem Mass Spectrometry) detection was undertaken to quantitatively compare estimates for DCVG and DCVG using rat and human tissues. For the HPLC method, DCVG and DCVC were initially derivatized with fluorodinitrobenzene (DNP). The results from the HPLC/UV method showed that derivatized-DCVC eluted at the solvent front and could not be quantified. Derivatized-DCVG, however, was quantified but significant interference was observed in all four control tissues (rat blood, liver, kidney; and human blood), resulting in average spike recoveries of 222-22,990%. In contrast, direct analysis of spiked tissues by HPLC/MS/MS resulted in recoveries of 82-127% and 89-117% for DCVG and DCVC, respectively. These differences in analytical results were further confirmed in tissues from TCE-treated rats, e.g., DCVG levels in rat liver were 18,000 times higher by HPLC/UV as compared to HPLC/MS/MS. Fraction collection of the derivatized-DCVG peak (obtained with the HPLC-UV method), followed by peak identification via an HPLC/UV/Q-TOF/MS/MS method, identified DNP-derivatized endogenous glutamate as the primary interfering substance that contributed to and exaggerated recoveries of DCVG. Thus, estimates of DCVG based on the HPLC/UV methods are not reliable; they will over-estimate the formation of the GSH conjugates of TCE and will artifactually exaggerate the potential cancer risk in humans from TCE exposure. Therefore, it is recommended that any characterization of cancer risks from TCE exposure attributable to the GSH conjugates of TCE rely on results obtained with the more specific and reliable HPLC/MS/MS method.

Comparative metabolism and pharmacokinetics of diisobutyl ketone and diisobutyl carbinol in male SD rats.

Diisobutyl ketone (DIBK) and diisobutyl carbinol (DIBC) are important organic solvents widely used as industrial intermediates. It was hypothesized that DIBC and DIBK have common metabolic pathways and metabolites, and as such, toxicological data on DIBK could be used to characterize the hazards of DIBC. To confirm or refute this hypothesis a comparative metabolism and pharmacokinetics assessment of DIBK and DIBC was conducted. Dosing was via single oral gavage dosing in male SD rats, followed by blood collection, metabolite identification, major biomarker quantitation, and pharmacokinetics analysis. Overall, the major metabolites of both DIBC and DIBK in blood were their corresponding monohydroxylated metabolites (DIBC alcohol and DIBK alcohol) with the site of hydroxylation at the σ and σ-1 positions, respectively. Quantitative analysis of DIBC, DIBK, DIBC-alcohol, and DIBK-alcohol in blood samples collected from 5min to 120h after single dosing indicated the following: (1) DIBC and DIBK are both well absorbed following oral gavage with substantial evidence of enterohepatic recirculation of DIBK, DIBC, DIBK-alcohol, and DIBC-alcohol; (2) DIBK and DIBC are interconverted metabolically in rats; (3) DIBC and DIBK have similar bioavailability after oral administration; (4) higher systemic exposure was found for DIBK-alcohol than DIBC-alcohol, implying that DIBC-alcohol may be more easily conjugated and eliminated in bile. In summary, the metabolic similarities and the difference in systemic exposure to metabolites between these substances observed in the current study support the hypothesis that DIBC might have a lower potential toxicity than that of DIBK. The current study results support that toxicological data on DIBK could be used to characterize the hazards of DIBC.