Reverse-phase high performance liquid chromatography for the determination of tiopronin in human plasma after derivatization with p-bromophenacyl bromide

• Published in: Analytica chimica acta Vol. 565; no. 2; pp. 178 - 182
• Main Authors: Huang, Taomin, Yang, Bei, Yu, Yingjia, Zheng, Xiaowei, Duan, Gengli
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 21-04-2006; Elsevier
• Subjects: Analytical chemistry; Chemistry; Chromatographic methods and physical methods associated with chromatography; Derivatization; Exact sciences and technology; Other chromatographic methods; p-Bromophenacyl bromide ( p-BPB); Reverse-phase high performance liquid chromatography (HPLC); Tiopronin (TP); Tiopronin (TP); p-Bromophenacyl bromide ( p-BPB); Derivatization; Reverse-phase high performance liquid chromatography (HPLC); Trace analysis; Water; Human; Elution; Thiol; Liquid liquid extraction; HPLC chromatography; Chemical enrichment; Regression coefficient; Ultraviolet detector; Sample preparation; Detection limit; Linearity; Acetonitrile; Bromides; Standard curve; Dichloromethane; p-Bromophenacyl bromide (p-BPB); Adduct; Variation coefficient
• DOI: 10.1016/j.aca.2006.02.049
• EISSN: 1873-4324
• ISSN: 0003-2670

In the study, we developed a simple, rapid and sensitive method for the determination of tiopronin (TP) in human plasma, which was based on derivatization with
p-bromophenacyl bromide (
p-BPB) followed by liquid–liquid extraction and reverse-phase HPLC-UV detection. For the first time, the
p-BPB was introduced into the derivatization of TP. The thiol group of TP was trapped with
p-BPB to form a TP-
p-BPB adduct, which can be very suitable for UV detection. From acidified plasma samples, the derivatized TP was extracted with 5
mL dichloromethane. Effective chromatographic separation was achieved using a C18 column (DIAMONSIL 150
mm
×
4
mm i.d., 5
μm) based on an acetonitrile–water–trifluoroacetic acid (40:59.88:0.12, v/v/v) elution at a flow-rate of 1
mL/min. The IS and the derivatized TP were detected at 263
nm. No endogenous substances were found to interfere. The limit of quantification for derivatized TP (TP-
p-BPB) in plasma was 40
ng/mL. The calibration curve for the derivatized TP showed linearity in the range 0.04–4
μg/mL with a regression coefficient corresponding to 0.9991 and the coefficient of the variation of the points of the calibration curve being lower than 10%. Extraction recoveries of the derivatized TP in plasma were greater than 72%. The method was suitably validated and successfully applied to determination of TP in human plasma samples.