Development and validation of an LC-MS/MS method for simultaneous quantitative analysis of free and conjugated bisphenol A in human urine Dilek Battal a *, Ismet Cok b , Irfan Unlusayin c and Bahar Tunctan d ABSTRACT: Bisphenol A (BPA) is an environmental endocrine-disrupting chemicals that is widely used in common consumer products. There is an increasing concern regarding human exposure to BPA owing to the potential adverse effects associated with its estrogenic activity. For assessing environmental exposure to BPA, it is essential to have a sensitive, accurate and selective analytical method, especially one that can detect low BPA levels in complex sample matrices. In this study, we devel- oped and validated an accurate, sensitive, and robust liquid chromatography–tandem mass spectrometry method for simulta- neous quantification of free BPA and BPA β-D-glucuronide (BPA-gluc) concentrations in human urine with only a single injection. Calibration curves were linear over a concentration range of 1–100 ng/mL for BPA and 10–1000 ng/mL for BPA-gluc. The levels of the analytes were determined quantitatively with HPLC/ESI-MS/MS by using negative electrospray ionization in the select ion monitoring mode and a pentaflouraphenyl propyl column. The validated method was applied to the analysis of spot urine specimens collected from randomly selected healthy human subjects. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: bisphenol A; endocrine disruptors; urine; quantification Introduction Bisphenol A (BPA) is a monomer used in the manufacture of commercial plastics and synthetic resins. It is currently one of the highest-volume chemicals in production. Owing to its characteristics and widespread use in many consumer products worldwide (toys, baby bottles, plastic containers, heating containers for food and beverages, inner lining of metal cans, medical equipment, consumer electronics, dental sealants, etc.), BPA has occupied a significant role in our daily lives for the past 30 years (Vandenberg et al., 2007). The annual production of BPA is approximately 800,000 tons (ECB, 2003) in the EU, 1,080,000 tons in the USA (US Environmental Protection Agency, 2010) and over 2,700,000 tons globally (Burridge, 2003). BPA is one of the most ubiquitous endocrine disruptor chemicals (Krishnan et al., 1993; Kuiper et al., 1998; Wozniak et al., 2005; Kandarakis et al., 2009) and the most important route for BPA exposure in humans appears to be food consumption (European Food Safety Authority, 2006). BPA is fully absorbed within the digestive tract after ingestion, and is rapidly biotransformed into a glucuronide metabolite (BPA-gluc; Fig. 1). As the excretion of this glucuronide metabolite occurs rapidly and extensively via urine, BPA exposure in individuals can be assessed from urine samples. In humans, BPA has also been indentified in serum, fetal blood, umbilical cord blood, amniotic fluid, placental tissue (Ikezuki et al., 2002; Schönfelder et al., 2002) and the milk of nursing mothers (Sun et al., 2004). In the past two decades, a number of analytical methods have been developed for the identification and measurement of BPA forms (i.e. total BPA, free BPA and BPA-gluc) in biological samples, and significant improvements have been achieved regarding the detection limits of these methods. The analytical methods that have been developed for the determination of BPA in human bio-specimens include enzyme-linked immuno- sorbent assays, GC with MS and MS/MS, LC-MS/MS and HPLC with electrochemical or fluorescence detection. In recent years, there have been numerous methods and studies in the literature attempting to assess BPA and its major metabolite BPA-gluc with the aid of HPLC/MS-MS (Calafat and Needham, 2008; Hong et al., 2009; Mahalingaiah et al., 2008; Nepomnaschy et al., 2009; Teitelbaum et al., 2008; Yang et al., 2009; Ye et al., 2009). The aim of the present study was to develop a rapid, sensitive and simple procedure for the simultaneous and quantitative analysis of BPA and BPA-gluc conjugate levels (major metabolite of BPA) in human urine for use in investigations of the potential endocrine disrupter effect of BPA. * Correspondence to: D. Battal, Mersin University, Faculty of Pharmacy, Department of Toxicology, 33169 Mersin, Turkey. E-mail: diakunal@yahoo.com a Mersin University, Faculty of Pharmacy, Department of Toxicology, 33169 Mersin, Turkey b Gazi University, Faculty of Pharmacy, Department of Toxicology, 06330 Ankara, Turkey c Acibadem Laboratory Medical Research and Development Laboratory, 34662 Istanbul, Turkey d Mersin University, Faculty of Pharmacy, Department of Pharmacology, 33169 Mersin, Turkey Abbreviations used: BPA, bisphenol A; BPA-gluc, BPA β-D-glucuronide; PFP, pentaflouraphenyl propyl; SPE, solid-phase extraction. Biomed. Chromatogr. 2014; 28: 686–693 Copyright © 2013 John Wiley & Sons, Ltd. Research article Received: 14 August 2013, Revised: 16 October 2013, Accepted: 25 October 2013 Published online in Wiley Online Library: 17 December 2013 (wileyonlinelibrary.com) DOI 10.1002/bmc.3090 686