Gas chromatographic–mass spectrometric determination of α-ketoisocaproic acid and [ 2H 7]α-ketoisocaproic acid in plasma after derivatization with N-phenyl-1,2-phenylenediamine

Abstract

A method for determination of α-ketoisocaproic acid (KIC) and [4,5,5,5,6,6,6- 2H 7]α-ketoisocaproic acid ([ 2H 7]KIC) in rat plasma was developed using gas chromatography–mass spectrometry-selected ion monitoring (GC–MS-SIM). [5,5,5- 2H 3]α-Ketoisocaproic acid ([ 2H 3]KIC) was used as an analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. The keto acids were extracted by cation-exchange chromatography using BondElut SCX cartridge and derivatized with N-phenyl-1,2-phenylenediamine to form N-phenylquinoxalinone derivatives. Quantitation was performed by SIM of the respective molecular ions at m/z 278, 281 and 285 for the derivatives of KIC, [ 2H 3]KIC and [ 2H 7]KIC on the electron impact method. The limit of detection was found to be 70 fmol per injection ( S/N=3) and the limit of quantitation for [ 2H 7]KIC was around 50 n M in rat plasma. Endogenous KIC concentrations in 50 μl of rat plasma were measured with relative intra- and inter-day precision of 4.0% and 3.3%, respectively. The intra- and inter-day precision for [ 2H 7]KIC spiked to rat plasma in the range of 0.1 to 10 μ M gave good reproducibility with relative standard deviation (RSD) of 6.5% and 5.4%, respectively. The intra- and inter-day relative errors (RE) for [ 2H 7]KIC were less than 6.4% and 3.8%, respectively. The method was applied to determine the plasma concentration of [ 2H 7]KIC after an intravenous administration of [ 2H 7]KIC in rat.