Simultaneous determination of tiopronin and its metabolites in rat blood by LC-ESI-MS-MS using methyl acrylate for stabilization of thiol group.
A sensitive and reproducible HPLC-electrospray tandem mass spectrometric method has been developed for the analysis of tiopronin (TP) and its metabolites, 2-mercaptopropionic acid (2-mpa) and S-methylated TP (SA13), in rat blood using methyl acrylate (MA) for the stabilization of a thiol group. The thiol groups of TP and 2-mpa in rat blood were immediately derivatized by the addition of MA-acetonitrile solution in 0.1 M Tris HCl (pH 9.1). The purification of the derivatives was accomplished by a simple liquid-liquid extraction procedure involving protein precipitation step. The analysis was performed on a Zorbax SB-C18 analytical column by a gradient elution with methanol-0.05 M acetic acid (15:85 and 7:3, v/v). Negative ion electrospray ionization with selected reaction monitoring was employed for the detection of analytes. Linearity of calibration was observed over the range of 0.5-1000 ng/ml for TP and 2-mpa, and 2-1000 ng/ml for SA13. The intra- and inter-assay variability for all analytes at the limit of quantitation (LOQ) level ranged from 5.47 to 16.75% and 4.95 to 7.23%, respectively. The LOQs estimated for TP, 2-mpa and SA13 were 0.5, 0.5 and 2 ng/ml, respectively. This assay method was successively applied to a pharmacokinetics study after an oral administration of TP (10 mg/kg) to rats.
Matsuura
K
K
Developmental Research Division, Santen Pharmaceutical Co. Ltd, Osaka, Japan. kenji.matsuura@santen.co.jp
Murai
K
K
Fukano
Y
Y
Takashina
H
H
D000179:Acrylates / Q000737:chemistry; D000818:Animals; D001711:Biotransformation; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D004563:Electrochemistry; D005260:Female; D013058:Mass Spectrometry; D051381:Rats; D011917:Rats, Inbred Lew; D015203:Reproducibility of Results; D013438:Sulfhydryl Compounds / Q000737:chemistry; D008625:Tiopronin / Q000097:blood* / Q000493:pharmacokinetics
D000179:Acrylates; D013438:Sulfhydryl Compounds; D008625:Tiopronin; C035956:methyl acrylate