Journal of Pharmaceutical and Biomedical Analysis 55 (2011) 1111–1119 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba Simultaneous characterization of bile acids and their sulfate metabolites in mouse liver, plasma, bile, and urine using LC–MS/MS Jiangeng Huang a,1 , Sai Praneeth R. Bathena a , Iván L. Csanaky b , Yazen Alnouti a,∗ a Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198, United States b Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS 66160, United States article info Article history: Received 11 February 2011 Received in revised form 21 March 2011 Accepted 28 March 2011 Available online 6 April 2011 Keywords: LC–MS/MS Bile acids Sulfation Mouse Gender difference abstract Sulfation is a major metabolic pathway involved in the elimination and detoxification of bile acids (BAs). Several lines of evidence are available to support the role of sulfation as a defensive mechanism to attenuate the toxicity of accumulated BAs during hepatobiliary diseases. Individual BAs and their sulfate metabolites vary markedly in their physiological roles as well as their toxicities. Therefore, analytical tech- niques are required for the quantification of individual BAs and BA-sulfates in biological fluids and tissues. Here we report a simple, sensitive, and validated LC–MS/MS method for the simultaneous quantifica- tion of major BAs and BA-sulfates in mouse liver, plasma, bile, and urine. One-step sample preparation using solid-phase extraction (for bile and urine) or protein precipitation (for liver and plasma) was used to extract BAs and BA-sulfates. Base-line separation of all analytes (unsulfated- and sulfated BAs) was achieved in 25 min with a limit of quantification of 1 ng/ml. This LC–MS/MS method was applied to simul- taneously quantify BAs and BA-sulfates in both male and female mouse tissues and fluids. Less than 3% of total BAs are present in the sulfate form in the mouse liver, plasma, and bile, which provides strong evidence that sulfation is a minor metabolic pathway of BA elimination and detoxification in mice. Fur- thermore, we report that the marked female-predominant expression of Sult2a1 is not reflected into a female-predominant pattern of BA-sulfation. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Bile acids (BAs), the major components of bile, are synthesized in hepatocytes from cholesterol, conjugated with glycine or tau- rine, and secreted into the small intestine via bile. Cholic acid (CA) and chenodeoxycholic acid (CDCA) are primary BAs in humans, whereas muricholic acid (MCA) and ursodeoxycholic acid (UDCA) are primary BAs in rodents and bears, respectively. The secondary BAs, deoxycholic acid (DCA) and lithocholic acid (LCA), are pro- duced in the intestine by bacterial metabolism of the primary BAs. Most BAs are reabsorbed in the intestine and they undergo efficient enterohepatic recycling. BAs play important physiological functions in the absorption of fat and fat-soluble vitamins. In addition, BAs are signaling Abbreviations: UPLC, ultra-performance liquid chromatography; LC–MS/MS, liq- uid chromatography–tandem mass spectrometry; BAs, bile acids; G-BAs, glycine conjugated bile acids; T-BAs, taurine conjugated bile acids; MCA, muricholic acid; CA, cholic acid; UDCA, ursodeoxycholic acid; DCA, deoxycholic acid; CDCA, chen- odeoxycholic acid; LCA, lithocholic acid; IS, internal standard; MeOH, methanol; ACN, acetonitrile; QC, quality control. ∗ Corresponding author. Tel.: +1 402 559 4631; fax: +1 402 559 9543. E-mail address: yalnouti@unmc.edu (Y. Alnouti). 1 Both authors contributed equally to this manuscript. molecules with diverse paracrine and endocrine functions [1]. BAs are ligands for the G-protein coupled bile acid receptor (TGR5) [2], and several nuclear receptors, such as the farnesoid-X-receptor (FXR) [3], pregnane-X-receptor (PXR) [4], constitutive androstane receptor (CAR), and vitamin D receptor (VDR) [5]. However, BAs are also cytotoxic and cancer promoters [6]. A variety of pathologic changes induced by BAs, including cholestasis, bile duct infarction, liver fibrosis, liver cirrhosis, liver and colon cancer were demon- strated in previous studies [6,7]. The physiological and pathophysiological activities of individual BAs vary markedly. For example, the monohydroxyl BA (LCA) is the most toxic BA and a potent PXR ligand [4], whereas CDCA is less toxic and is a potent FXR ligand [8]. Therefore, BA homeostasis and the composition of the BA pool must be tightly controlled to prevent the accumulation of toxic levels, and yet maintain physiological levels of BAs in the liver and extrahepatic tissues. Sulfation by sulfotransferase 2A1 (SULT2A1) is the domi- nant metabolic pathway that facilitates BA detoxification and elimination in humans [9], whereas hydroxylation seems to play a major role in BA metabolism in rodents [10]. Sulfation increases BA water solubility, decreases their intestinal absorp- tion, enhances their fecal and urinary excretion, and directly decreases their cytotoxicity by increasing their critical micel- lar concentrations (CMC). Several lines of evidence provided by 0731-7085/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jpba.2011.03.035