Anal Bioanal Chem DOI 10.1007/s00216-014-7829-1
RESEARCH PAPER
Quantitative determination of free and total bisphenol A in human urine using labeled BPA glucuronide and isotope dilution mass spectrometry Cariton Kubwabo & Ivana Kosarac & Kaela Lalonde & Warren G. Foster
Received: 5 February 2014 / Revised: 1 April 2014 / Accepted: 9 April 2014 # Her Majesty the Queen in Right of Canada 2014
Abstract Bisphenol A (BPA) is a widely used industrial chemical in the manufacturing of polycarbonate plastic bottles, food and beverage can linings, thermal receipts, and dental sealants. Animal and human studies suggest that BPA may disrupt normal hormonal function and hence, potentially, have negative effects on the human health. While total BPA is frequently reported, it is recognized that free BPA is the biologically active form and is rarely reported in the literature. The objective of this study was to develop a sensitive and improved method for the measurement of free and total BPA in human urine. Use of a labeled conjugated BPA (bisphenol A-d6 β-D-glucuronide) allowed for the optimization of the enzymatic reaction and permitted an accurate determination of the conjugated BPA concentration in urine samples. In addition, a 13C12-BPA internal standard was used to account for the analytical recoveries and performance of the isotope dilution method. Solid-phase extraction (SPE) combined with derivatization and analysis using a triple quadrupole GC-EI/ MS/MS system achieved very low method detection limit of 0.027 ng/mL. BPA concentrations were measured in urine samples collected during the second and third trimesters of pregnancy in 36 Canadian women. Total maternal BPA concentrations in urine samples ranged from not detected to 9.40 ng/mL (median, 1.21 ng/mL), and free BPA concentrations ranged from not detected to 0.950 ng/mL (median, 0.185 ng/mL). Eighty-six percent of the women had detectable levels of conjugated BPA, whereas only 22 % had
C. Kubwabo (*) : I. Kosarac : K. Lalonde Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, ON K1A 0K9, Canada e-mail:
[email protected] W. G. Foster Department of Obstetrics and Gynecology, McMaster University, Hamilton, ON L8N 3Z5, Canada
detectable levels of free BPA in their urine. BPA levels measured in this study agreed well with data reported internationally. Keywords Bisphenol A . Bisphenol A-d6 β-D-glucuronide . Human maternal urine . Derivatization . GC-EI/MS/MS
Introduction Bisphenol A ((2,2-bis(4-hydroxyphenyl)propane (BPA), CAS no. 80-05-7)] is a high production volume chemical widely used to make polycarbonate (PC) plastic and epoxy resins [1]. PC plastic made with BPA is used in a number of consumer products, including reusable water bottles. Epoxy resins are mainly used as a protective lining on the inside of metal-based food and beverage cans. Humans are exposed to BPA regularly during their everyday life, and evidence of this exposure can be detected in biological samples such as breast milk, serum and urine. For example, in a 2008 study, BPA conjugates were found in the urine of approximately 90 % of the US population [2]. Human exposure to BPA has been associated with a number of human diseases [3, 4], and a review of key evidence on the human health hazards of exposure to BPA has been recently published elsewhere [5]. In the human body, BPA is present in two forms, free BPA and conjugated BPA (BPA glucuronide and BPA sulfate, the conjugated species). BPA glucuronide is the water-soluble metabolite produced in the liver during the first-pass metabolism of BPA. This process occurs rapidly, and early studies have shown that the half-life of BPA in the human body is less than 6 h with nearly complete urinary excretion within 24 h [6]. Although BPA is quickly metabolized to BPA monoglucuronide and rapidly excreted in urine in adults [6, 7], recent studies, however, have shown that free BPA can accumulate in tissues and may have a longer half-life in the
C. Kubwabo et al.
human body than was previously thought [8, 9]. As the major source of BPA exposure in humans is assumed to occur via the ingestion pathway, urine has been identified as the matrix of choice for the biomonitoring of BPA. Exposure to BPA can also occur by transdermal and inhalation; however, these routes of exposure are not subject to the first-pass metabolism of BPA through the liver; thus, these modes of exposure allow for free BPA to linger longer in the bloodstream. Free BPA serves as a biomarker to the potential estrogenic effects as only free BPA shows weak estrogenicity [6]. Therefore, the combination of free BPA and conjugated BPA levels found in both serum and urine, should allow for a more comprehensive depiction of human exposure to this ubiquitous environmental contaminant. Many different methods have been used to extract BPA from urine samples. Some of these methods are used specifically to extract free BPA, whereas other methods have focused on measuring total levels of BPA (free BPA+BPA glucuronide) in urine. The determination of the conjugated species (BPA glucuronide) is performed after enzymatic hydrolysis using a glucuronidase/sulfatase mixture followed by sample enrichment and instrumental analysis. Current sample enrichment methods include liquid–liquid extraction [10–13], solid-phase extraction (SPE) [14–17], stir-bar sorptive extraction [18, 19], and miniaturized hollow fiber-assisted liquidphase microextraction [20]. The analysis of BPA in urine has involved chromatographic separation (either GC or LC) coupled to an array of detection techniques. In a study by Fukata et al. [21], BPA results obtained by liquid chromatography with multichannel coulometric electrochemical detection (LC-ECD) method were compared with those obtained by column-switching LC-MS/MS and enzyme-linked immunosorbent assay (ELISA). There was a high correlation found between the LC-MS/MS method and the LC-ECD method (r=0.998), whereas the correlation between these methods and the ELISA were low (r
