Determination of bisphenol A, triclosan and their metabolites in human urine using isotope-dilution liquid chromatography–tandem mass spectrometry
Introduction
Bisphenol A (BPA) and triclosan (TCS) are two widely used phenolic compounds with numerous industrial and commercial applications. BPA is the monomer in the production of polycarbonate plastics and is also a component of epoxy resins that are used to produce food and beverage packaging materials and dental sealants among others [1]. Trace amounts of BPA have been shown to leach from polycarbonate containers [2], [3], [4], which led to the ban of polycarbonate baby bottles in Canada in 2010. BPA has been found in foodstuffs [5], [6], and diet is most likely the major source of exposure to this compound [1], [7], [8]. TCS is a bactericidal agent added to soap and toothpaste [9], [10]. Dermal absorption and ingestion of TCS in these personal care products are thought to contribute the most to human exposure [9].
Both BPA and TCS are endocrine disrupting chemicals: BPA is an estrogen receptor agonist and an androgen receptor antagonist [11], [12]. TCS interacts with constitutive androstane and pregnane-X receptors [13] and exhibits antagonistic activity in both estrogen receptor- and androgen receptor-responsive bioassays [14]. Human exposure to these compounds have been associated with various adverse health effects including reproductive and endocrine disorders, cardiovascular disease, obesity and metabolic syndrome, cancer, immune system dysfunction and neurobehavioral defects [1], [15], [16], [17]. However, whether or not these associations indicate an important public health risk is highly controversial.
An argument raised by those supporting a trivial health risk from exposure to these phenolic compounds is their rapid biotransformation to biologically-inactive metabolites [18]. Indeed, following their absorption by the gastro-intestinal tract, free BPA and TCS are rapidly and efficiently conjugated by hepatic uridine diphosphate-glucuronyltransferases to their glucuronidated forms and eliminated in urine [19], [20]. However, the recent demonstration in the dog model of an efficient and rapid absorption of BPA through the oral mucosa by the sublingual route (avoiding the first-pass hepatic metabolism) could indicate a greater systemic exposure to free BPA by the oral route than previously thought [21]. Also, BPAG can be deconjugated by β-glucuronidase found in placenta, liver, kidney, and intestine [18]. The development of an analytical method allowing the determination of unconjugated (“free”) and conjugated forms of BPA and TCS is central to an improved assessment of the health risk associated with exposure to these ubiquitous compounds.
Both liquid chromatography–tandem mass spectrometry (LC–MS/MS) and gas chromatography–mass spectrometry (GC–MS) techniques have been used for the measurement of BPA and its metabolites in biological specimens [22], [23], [24]. Because standards of BPA conjugates were not commercially available, most methods measured only total BPA and TCS concentrations following enzymatic hydrolysis of conjugates [25], [26], [27], [28], [29]. Some researchers measured both free BPA concentration (BPA analysis without enzymatic deconjugation) and total BPA concentration (BPA analysis following deconjugation with β-glucuronidase/sulfatase) [30], [31]. Whereas glucuronides are major conjugated metabolites of BPA and TCS in urine, other minor conjugates include sulfates and glucuronide/sulfate derivatives [32]. To our knowledge, only Völkel et al. [33] developed an isotope dilution (ID)-LC–MS/MS method to specifically measure free BPA and BPAG in serum and urine samples, but their method lacked sensitivity due to interfering peaks. Using this method, they could not detect free BPA in any urine samples collected from 19 human subjects without known exposure to BPA. Most of these samples did contain detectable amounts BPAG but concentrations were always below the limit of quantification of 65 nmol L−1 (26.3 μg L−1) [33]. Recently, Liao and Kannan [34] published a more sensitive LC–MS/MS method to analyze serum and urine samples for free BPA, BPA chlorides and BPA conjugates (BPAG, BPADS). Analysis of urine samples from 31 volunteers in Albany, New York, revealed that 96.8% and 87.1% of samples contained detectable amounts of free BPA and BPAG, respectively; corresponding geometric mean (GM) concentrations were 0.71 and 2.2 μg L−1. BPADS was also found above the LOQ in 35% of the samples (GM = 0.11 μg L−1). A significant drawback of this method is the reliance on only one internal standard (13C12-BPA) for the quantification of all analytes.
We developed methods for the determination of free BPA and TCS as well as their metabolites BPAS, BPADS, BPAG, TCSS, TCSG in urine samples, using ID-LC–MS/MS methods that comprised isotope-labeled standards for all analytes. The samples were also analyzed for BPA and TCS (free and total) using a method combining enzymatic hydrolysis, derivatization with pentafluorobenzyl bromide, liquid–liquid extraction and GC–MS/MS, which was previously used in our laboratory. We also assessed possible losses of analytes during collection and storage of urine samples.
Section snippets
Chemicals and reagents
We purchased dansyl chloride, formic acid (>95%), ammonium formate (LC–MS Ultra), ß-glucuronidase from Helix pomatia and 2,3,4,5,6-pentafluorobenzyl bromide from Sigma-Aldrich (St. Louis, MO), acetone (pesticide grade), ammonium hydroxide (ACS – Pur reactive), hexane (Optima), sodium carbonate (ACS grade), anhydrous potassium carbonate (powder), acetic acid (glacial) and sodium acetate (ACS grade) from Fisher Scientific (Fairlawn, NJ), acetonitrile, methanol and dichloromethane (Omnisolv grade)
Development and validation of the methods
Other research groups have used a single ESI− LC–MS/MS method to analyze free BPA and conjugated metabolites [23], [33], [34]. We used a separate method with dansylation and ESI+ LC–MS/MS for the analysis of free BPA and TCS for the following reasons. First and foremost, dansyl derivatization at the beginning of the procedure prevents contamination of the sample by these ubiquitous environmental contaminants along the subsequent steps of sample preparation. During the development of the method,
Conclusions
We have developed isotope-dilution LC–MS/MS methods for the specific quantification of BPA, TCS and their major sulfated and glucuronidated metabolites in urine samples. These methods were fully validated according to the protocol of our laboratory accredited ISO/CEI 17025 and display extended calibration ranges as well as high accuracy, precision and recovery values. The low LOD value for free BPA indicates proper control of contamination during the analytical process. The sensitive methods
Acknowledgments
We acknowledge the contribution of David Bisson and Pierre-Yves Tremblay (CTQ, INSPQ), who respectively collaborated to the development of LC–MS/MS methods and performed LC–QTOF MS experiments to tentatively identify resveratrol-glucuronide as a possible interference during BPA-glucuronide quantification. The technical assistance of Isabelle P. Côté, Nathalie Morissette, Yves Simard and Jean-Sébastien Trudelle (CTQ, INSPQ) is gratefully acknowledged. We thank Dr. Tye Arbuckle (Healthy
References (37)
- et al.
Food Chem. Toxicol.
(2012) J. Steroid Biochem. Mol. Biol.
(2011)- et al.
Chem. Biol. Interact.
(2013) - et al.
Toxicol. In Vitro
(2013) Reprod. Toxicol.
(2013)- et al.
Toxicol. Appl. Pharmacol.
(2008) - et al.
Talanta
(2011) - et al.
Int. J. Hyg. Environ. Health
(2013) - et al.
Environ. Res.
(2011) - et al.
Crit. Rev. Toxicol.
(2011)
Food Addit. Contam. Part A: Chem. Anal. Control Expo. Risk Assess.
J. Toxicol. Environ. Health A
Crit. Rev. Food Sci. Nutr.
Environ. Sci. Technol.
J. Agric. Food Chem.
J. Toxicol. Environ. Health B Crit. Rev.
Crit. Rev. Toxicol.
Environ. Sci. Pollut. Res. Int.
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