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

摘要

We developed a highly sensitive and specific high-performance 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 mu L) was performed using cationic exchange solid-phase 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-d (7) (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 mu g/mL for d-leucine and from 1 to 1000 mu g/mL for l-leucine, respectively, with acceptable intra- and inter-day precision and accuracy. The stability of d- and l-leucine in human plasma and solvents was confirmed. The endogenous level of d- and l-leucine in human plasma was 0.00197 similar to 0.00591 and 9.63 similar to 24.7 mu g/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.