A simplified LC−MS/MS method for rapid determination of cycloserine in small-volume human plasma using protein precipitation coupled with dilution techniques to overcome matrix effects and its application to a pharmacokinetic study

Abstract

Matrix effects have been a major concern when developing LC−MS/MS methods for quantitative bioanalysis of cycloserine. Sample handling procedures including solid phase extraction or derivatization have been reported previously by researchers to overcome matrix effects of cycloserine. In the present study, the possibility of reducing matrix effects of cycloserine using protein precipitation coupled with dilution techniques was investigated. Plasma samples were pretreated by protein precipitation with methanol followed by a 40-fold dilution with methanol–water (50:50, v/v). The analyte and the internal standard (mildronate) were chromatographed on a Shim-pack XR-ODS (100 mm × 2.0 mm, 2.2 μm) column using methanol–0.01% formic acid (70:30, v/v) as mobile phase and detected by multiple reaction monitoring mode via positive electrospray ionization. The total run time was only 2 min per sample. The suppression of cycloserine response was reduced with the matrix effects ranging between 80.5 and 87.9%. A lower limit of quantification (LLOQ) of 0.300 μg/mL was achieved using only 10 μL of plasma. The intra- and inter-day precisions were less than 4.8% and the accuracy ranged from −2.6 to 6.6%. The method was successfully applied to a pharmacokinetic study of cycloserine in 30 healthy Chinese male subjects after oral administration of a single dose of cycloserine at 250, 500 and 750 mg under fasting conditions. The newly developed method is simpler, faster, cost-effective, and more robust than previously reported LC-MS/MS methods.