Stereoselective analysis of tiopronin enantiomers in rat plasma using high‐performance liquid chromatography‐electrospray ionization mass spectrometry after chiral derivatization

A specific and relatively sensitive high‐performance liquid chromatography‐electrospray ionization mass spectrometry (HPLC‐ESI‐MS) was developed for the quantitative analysis of tiopronin enantiomers in rat plasma. The method is based on the derivatization of (+)‐tiopronin and (−)‐tiopronin with 2,3,4,6‐tetra‐ O ‐acetyl‐β‐glucopyranosyl isothiocyanate (GITC) in acetonitrile. The separation of resulting diastereomic derivatives was performed on C 18 column (150 mm × 2.0 mm ID, packed with 5.0 μm C 18 silica RP particle), using a mobile phase of methanol/water (containing 5.3 mM formic acid) with gradient elution. LC–MS was performed in the selected ion monitoring and positive ion mode using target ions at m/z: 575 for the diastereomic derivatives of tiopronin and m/z: 603 for the derivative of N ‐isobutyryl‐ D ‐cysteine (internal standard). The method was validated in terms of specificity, linearity, sensitivity, precision, accuracy, matrix effect, and stability. The calibration curves were linear over the concentration range of 0.025–5 μg/ml for both enantiomers of tiopronin. For both enantiomers of tiopronin, the interbatch and intrabatch variability values were less than 15%, and the accuracy was within ±17% in terms of relative error. The method was successfully applied to a pharmacokinetic study of rac ‐tiopronin in rat. Chirality, 2009. © 2008 Wiley‐Liss, Inc.