Method development for the determination of D-and L-isomers of leucine in human plasma by high-performance liquid chromatography tandem mass spectrometry and its application to animal plasma samples

Abstract

We developed a highly sensitive and specific highperformance liquid chromatography tandem mass spectrometry method with an electrospray ionization for the determination of D-and L-isomers of leucine in human plasma. Phosphate-buffered saline was used as the surrogate matrix for preparation of calibration curves and quality control samples. The extraction of D-and L-leucine in plasma samples (100 μL) was performed using cationic exchange solidphase extraction. The enantiomer separation of D-and L-leucine was successfully achieved without derivatization using a CHIRALPAK ZWIX(−) with an isocratic mobile phase comprised of methanol/acetonitrile/1 mol/L ammonium formate/ formic acid (500:500:25:2, v/v/v/v) at a flow rate of 0.5 mL/ min. In addition, the discrimination of DL-leucine from structural isomers DL-isoleucine and DL-allo-isoleucine was performed using the unique precursor and product ion pair transition of DL-leucine (m/z 132.1>43.0) and DL-leucine-d7 (m/z 139.2>93.0) in positive electrospray ionization mode. The standard curves were linear throughout the calibration range from 0.001 to 1 μg/mL for D-leucine and from 1 to 1000 μg/ mL for L-leucine, respectively, with acceptable intra-and inter-day precision and accuracy. The stability of D-and Lleucine in human plasma and solvents was confirmed. The endogenous level of D-and L-leucine in human plasma was 0.00197~0.00591 and 9.63~24.7 μg/mL, respectively. This method was also successfully applied to investigate the species difference in the ratios of D-leucine to total leucine from individual plasma concentrations in humans and various animals. The plasma D-leucine concentrations or their ratio to total leucine in rodents was much higher than that in humans.