Journal of Chromatography B, 969 (2014) 19–28 Contents lists available at ScienceDirect Journal of Chromatography B jou rn al hom ep age: www.elsevier.com/locate/chromb Propylthiouracil quantification in human plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry: Application in a bioequivalence study Gustavo D. Mendes a,b,c,∗ , Samara Bittencourt c,d , Celso Francisco Pimentel Vespasiano d , Tainah Babadópulos c , Thiago Gagliano-Jucá g , André Moreira Martins Arruda c , Elisa Perissutti f , Francesco Frecentese f , Gilberto De Nucci c,d,e a Department of Surgery, Faculty of Odontology, University of Sao Francisco (USF), Braganca Paulista, SP, Brazil b Department of Surgery, Faculty of Odontology, University Camilo Castelo Branco (UNICASTELO), São Paulo, SP, Brazil c Galeno Research Unit, Latino Coelho St., 1301, Parque Taquaral, 13087-010 Campinas, SP, Brazil d Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, SP, Brazil e Department of Pharmacology, Institute of Biomedical Sciences, University of Sao Paulo, São Paulo, SP, Brazil f Department of Pharmaceutical and Toxicological Chemistry, Federico II University, Naples, Italy g Institute of Biophysics Carlos Chagas Filho/UFRJ, CCS-Bloco G, 21949-900 Rio de Janeiro, RJ, Brazil a r t i c l e i n f o Article history: Received 2 November 2013 Received in revised form 1 July 2014 Accepted 13 July 2014 Available online 7 August 2014 Keywords: Bioavailability Pharmacokinetics Healthy volunteer a b s t r a c t A rapid, sensitive and specific method for quantifying propylthiouracil in human plasma using methylth- iouracil as the internal standard (IS) is described. The analyte and the IS were extracted from plasma by liquid–liquid extraction using an organic solvent (ethyl acetate). The extracts were analyzed by high per- formance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC–MS/MS) in negative mode (ES-). Chromatography was performed using a Phenomenex Gemini C18 5 m ana- lytical column (4.6 mm × 150 mm i.d.) and a mobile phase consisting of methanol/water/acetonitrile (40/40/20, v/v/v) + 0.1% of formic acid. For propylthiouracil and I.S., the optimized parameters of the declustering potential, collision energy and collision exit potential were -60 (V), -26 (eV) and -5 (V), respectively. The method had a chromatographic run time of 2.5 min and a linear calibration curve over the range 20–5000 ng/mL. The limit of quantification was 20 ng/mL. The stability tests indicated no significant degradation. This HPLC–MS/MS procedure was used to assess the bioequivalence of two propylthiouracil 100 mg tablet formulations in healthy volunteers of both sexes in fasted and fed state. The geometric mean and 90% confidence interval CI of Test/Reference percent ratios were, without and with food, respectively: 109.28% (103.63–115.25%) and 115.60% (109.03–122.58%) for C max , 103.31% (100.74–105.96%) and 103.40% (101.03–105.84) for AUC last . Conclusion: This method offers advantages over those previously reported, in terms of both a simple liquid–liquid extraction without clean-up pro- cedures, as well as a faster run time (2.5 min). The LOQ of 20 ng/mL is well suited for pharmacokinetic studies. The assay performance results indicate that the method is precise and accurate enough for the routine determination of the propylthiouracil in human plasma. The test formulation with and without food was bioequivalent to reference formulation. Food administration increased the T max and decreased the bioavailability (C max and AUC). © 2014 Elsevier B.V. All rights reserved. ∗ Corresponding author at: 415 Jesuıno Marcondes Machado Ave., 13092-320 Campinas, SP, Brazil. Tel.: +55 193251 6923; fax: +55 19 3252 1516. E-mail addresses: mendesgd@yahoo.com.br, gugamendes@terra.com.br (G.D. Mendes). 1. Introduction Propylthiouracil (6-propyl-2-thiouracil), a drug of the thion- amide class, which blocks thyroid hormone synthesis, is used for treating hyperthyroidism (adults, children and adolescents), hyper- thyroidism in pregnancy and thyrotoxicosis [1–4]. After oral administration of 150 mg of propylthiouracil refer- ence formulation in healthy male volunteers, the C max , AUC 0–8 , http://dx.doi.org/10.1016/j.jchromb.2014.07.016 1570-0232/© 2014 Elsevier B.V. All rights reserved.