Development and validation of LC-MS/MS method for determination of branched chain amino acids and α-keto acids in human plasma

Abstract

Branched chain amino acids (BCAAs) and branched chain keto acids (BCKAs) are involved in various metabolic processes including glucose intake regulation, protein homeostasis in muscles etc. Regulation of these molecules by circadian clock determines importance of food intake, sleep and exercise for maintaining healthy metabolism. A Liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for the determination of BCAAs and BCKAs circadian fluctuations in human plasma. For sample preparation, plasma was deproteinized with a precipitation solution then diluted before introduction into the chromatographic system. LC was operated in gradient elution mode to facilitate accurate determination of each analayte. C8 column was used as stationary phase and formic acid with water and with methanol was used as mobile phase. MS was operated in switching polarity mode. Linear range for BCAAs was 2–50 μg/mL and for BCKAs 0.5–12.5 μg/mL (R2 0.997–0.999). Method performance was checked for recovery (>80%), Matrix effect, Accuracy and precision (inter day accuracy 93.6–103.1%, inter day CV 1.5–13.0%) and stability. Method developed has reduced run time (6 min) and fast sample preparation compared with exiting methods.