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.

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.

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.

Preparation, characterization, and scale-up of ketoconazole with enhanced dissolution and bioavailability.

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.

Biodegradation and product identification of [14C]hexabromocyclododecane in wastewater sludge and freshwater aquatic sediment.

In a previous study the biodegradation of hexabromocyclododecane (HBCD) was reported to occur under realistic environmental concentrations in soils and freshwater aquatic sediments with biotransformation half-lives ranging from approximately 2 days to 2 months. In this study we extend our knowledge as to the environmental behavior of HBCD with respect to the fate of the three major diastereomers of HBCD (alpha, beta, and gamma) as well as to the identification of major intermediate metabolites formed during degradation. Substantial biological transformation of the alpha-, beta-, and gamma-[14C]HBCD diastereomers was observed in wastewater (i.e., digester) sludge and in freshwater aquatic sediment microcosms prepared under aerobic and anaerobic conditions. Concomitant with the loss of [14C]HBCD in these matrixes there was a concurrent production of three [14C]products. Using a combination of high performance liquid chromatography atmospheric pressure photoionization mass spectrometry and gas chromatography electron impact ionization mass spectrometry these metabolites were identified as tetrabromocyclododecene, dibromocyclododecadiene, and cyclododecatriene. We propose that HBCD is sequentially debrominated via dihaloelimination where at each step there is the loss of two bromines from vicinal carbons with the subsequent formation of a double bond between the adjacent carbon atoms. These results demonstrate that microorganisms naturally occurring in aquatic sediments and anaerobic digester sludge mediate complete debromination of HBCD.

Investigation of the formation of N-nitrosodiethanolamine in B6C3F1 mice following topical administration of triethanolamine.

To determine potential nitrosation of triethanolamine (TEA) to N-nitrosodiethanolamine (NDELA) at different physiological conditions of the GI tract, in vitro NDELA formation was examined in aqueous reaction mixtures at several pHs (2-10) adjusted with acetic, sulphuric or hydrochloric acids or in cultures of mouse cecal microflora incubated. In vivo NDELA formation was also determined in blood, ingesta, and urine of female B6C3F1 mice after repeated dermal, most relevant human route, or single oral exposure to 1000 mg/kg TEA in the presence of high oral dosages of NaNO(2). Appropriate diethanolamine (DEA) controls were included to account for this impurity in the TEA used. Samples were analyzed for NDELA using GC/MS. The highest degree of nitrosation of TEA to NDELA ( approximately 3%) was observed in the in vitro cultures at pH 4 and acetic acid with lower amounts obtained using sulphuric acid ( approximately 1.3%) and hydrochloric acid ( approximately 1.2%). At pH 7, <1% of the TEA was nitrosated to NDELA and at pH 2 (HCl) or pH 10 (NaOH) no NDELA was found above the limit of detection. In incubated cultures containing cecal microflora and nutrient broth, only 0.68% of TEA was nitrosated to NDELA. No NDELA was formed in rats repeatedly dermally dosed with TEA at the limits of detection in blood (0.001 microg/ml, ppm), ingesta (0.006 microg/ml, ppm), and urine (0.47 microg/ml, ppm). Levels of NDELA measured in blood and ingesta after a single oral dose of TEA and NaNO(2) were less than those in DEA controls. These findings in toto confirm the lack of any significant formation of NDELA from TEA in vivo.