R eseaRch a Rticle Tuberculosis, a pervasive and infectious disease of the respiratory system, is one of the most lethal worldwide health threats [1] . Prothiona- mide (PTA; 2-propyl-4-pyridinecabothio- amide) is a thioamide antibiotic, used in the treatment of mycobacterial infections caused by Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium avium complex infec- tions [2] . PTA along with ethionamide (ETA) belongs to the thioamide class and these are considered second-line drugs for the treatment of multidrug-resistant tuberculosis. They are mainly used during resistance to first-line treat- ment with isoniazid and rifampin, and leprosy [3] . They are commonly used in conjunction with other antimycobacterial chemotherapeu- tics. PTA has a bacteriostatic effect and the mode of action is assumed to be similar to that of ETA due to structural similarity. Based on cell activation method, Wang et al. have shown definitive evidence that PTA and ETA form covalent adducts with nicotinamide adenine dinucleotide, which acts as an inhibitor of an M. tuberculosis and M. leprae InhA gene product (a gene encoding a target for PTA and ETA in tuberculosis) [4] . Like ETA, PTA is a prodrug and gets metabolized by mycobacterial enzymes to its active form, prothionamide sulfoxide (PTA-SO), which is then metabolized further to the nicotinamide and nicotinic acid forms, with no antibacterial activity [5] . The bioavailability of PTA is almost 90% after oral administra- tion. It has negligible plasma protein binding and the peak plasma concentration (1–5 μg/ml) is achieved in approximately 1 h [6,101] . Very few reports describe methods for the analysis of PTA and its metabolite PTA-SO in biological fluids [6–10] . Jenner et al . compared the blood levels and rates of urinary excretion of PTA and ETA and their sulfoxide metabo- lites in humans by HPLC–UV [6] . The analytes were determined in plasma, urine and fecal sam- ples. A quantitative thin-layer chromatographic determination of PTA and PTA-SO has been described in human plasma and urine [8] . The linearity was established from 0.75 to 25 μg/ml for both the analytes. Another method based on normal-phase HPLC–UV has been presented for simultaneous determination of PTA and ETA and was used for a PK study in humans [9] . The samples were prepared by liquid–liquid extraction (LLE) in diethyl ether employing 3 ml plasma/serum and urine. A simple, rapid and sensitive HPLC method has been proposed for determination of PTA in human serum [10] . The LLOQ for 300 μl serum was 27 μg/l and the chromatographic run time was 8 min. A comparative summary of reported procedures for PTA and/or PTA-SO in biological samples is shown in Table 1. Ex vivo conversion of prodrug prothionamide to its metabolite prothionamide sulfoxide with different extraction techniques and their estimation in human plasma by LC–MS/MS Background: The present work reports an ex vivo conversion study of prothionamide to its active metabolite prothionamide sulfoxide in human plasma during sample preparation by three conventional extraction techniques. Results: The chromatography was done on a Hypersil™ Gold C18 (50 × 2.1 mm, 3.0 µm) column using 0.1% acetic acid and acetonitrile (20:80, v/v) as the mobile phase. Quantitation of the analytes was done by MS/MS in the positive ionization mode. The method was validated over a wide concentration range of 30 to 6000 ng/ml for prothionamide and 50 to 10,000 ng/ml for prothionamide sulfoxide. The recovery for both the analytes was greater than 89%. Stability was extensively validated under different storage conditions. Conclusion: The extraction protocol was optimized using acetonitrile as protein precipitant for their simultaneous determination in human plasma by LC–MS/MS. The method was applied to a bioequivalence study of 250 mg prothionamide tablet formulation in 14 healthy Indian subjects. Vikas Trivedi 1 , Vivek Upadhyay 1 , Gaurang Shah 1 , Manish Yadav 2 , Pranav S Shrivastav* 2 & Mallika Sanyal 1,3 1 Chemistry Department, Kadi Sarva Vishwavidyalaya, Sarva Vidyalaya Campus, Sector 15/23, Gandhinagar 382015, India 2 Department of Chemistry, School of Sciences, Gujarat University, Navrangpura, Ahmedabad 380009, India 3 Chemistry Department, St Xavier’s College, Navrangpura, Ahmedabad 380009, India *Author for correspondence: Tel.: +91 79 2630 0969 Fax: +91 79 2630 8545 Email: pranav_shrivastav@ yahoo.com 185 ISSN 1757-6180 10.4155/BIO.12.301 © 2013 Future Science Ltd Bioanalysis (2013) 5(2), 185–200 For reprint orders, please contact reprints@future-science.com