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.
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 μL) 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 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 L-leucine 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.
Drug Metabolism and Pharmacokinetics Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan. hiroshi.sugimoto1@takeda.com.
Hiroshi
H
Sugimoto
Drug Metabolism and Pharmacokinetics Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
Masaaki
M
Kakehi
Drug Metabolism and Pharmacokinetics Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa, 251-8555, Japan.
Fumihiro
F
Jinno
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D000882:Haplorhini; D006801:Humans; D007536:Isomerism; D007930:Leucine / Q000032:analysis / Q000097:blood*; D057230:Limit of Detection; D008297:Male; D051379:Mice; D051381:Rats; D053719:Tandem Mass Spectrometry / Q000379:methods*
D007930:Leucine
D- and L-Leucine; Enantiomer separation; LC/MS/MS; Species difference; Surrogate matrix