Development and validation of amisulpride in human plasma by HPLC coupled with tandem mass spectrometry and its application to a pharmacokinetic study

Abstract

In this study, authors developed a simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantification of Amisulpride in human plasma using Amisulpride-d(5) as an internal standard (IS). Chromatographic separation was performed on Zorbax Bonus-RP C18, 4.6 × 75 mm, 3.5 μm column with an isocratic mobile phase composed of 0.2% formic acid:methanol (35:65 v/v), at a flow-rate of 0.5 mL/min. Amisulpride, Amisulpride-d(5) was detected at m/z 370.1→242.1 and 375.1→242.1. The drug and the IS were extracted by a liquid-liquid extraction method. The method was validated over a linear concentration range of 2.0-2500.0 ng/mL for Amisulpride with a correlation coefficient of (r(2)) ≥ 0.9982. This method demonstrated intra- and inter-day precision within 0.9 to 1.7 and 1.5 to 2.8 % and intra- and inter-day accuracy within 98.3 to 101.5 and 96.0 to 101.0 % for Amisulpride. Amisulpride was found to be stable at 3 freeze-thaw cycles, bench top and auto sampler stability studies. The developed method was successfully applied to a pharmacokinetic study.

Keywords: Amisulpride; Liquid chromatography–tandam mass spectrometry; Method Validation; Method development.

Fig. 1

Chemical structures of Amisulpride and Amisulpride-d₅

Fig. 2a.

Mass spectrum of Amisulpride parention

Fig. 2b.

Mass spectrum of Amsulpride production

Fig. 2c.

Mass spectrum of Amsulpride-d₅ parention

Fig. 2d.

Mass spectrum of Amsulpride-d₅ production

Fig. 3a.

MRM Chromatogram of Blank Human Plasma

Fig. 3b.

Chromatogram of LOQ

Fig. 4.

Calibration curve

Fig. 5.

Mean plasma concentrations of test vs. reference after a 50 mg dose (one 50 mg tablet) in 10 healthy volunteers