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.

中文翻译：

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LC-MS/MS 测定人血浆中支链氨基酸和α-酮酸的方法的开发和验证

摘要 支链氨基酸 (BCAAs) 和支链酮酸 (BCKAs) 参与各种代谢过程，包括葡萄糖摄入调节、肌肉蛋白质稳态等。生物钟对这些分子的调节决定了食物摄入、睡眠和运动的重要性。维持健康的新陈代谢。开发了一种液相色谱串联质谱 (LC-MS/MS) 方法，用于测定人血浆中的 BCAAs 和 BCKAs 昼夜节律波动。对于样品制备，血浆先用沉淀溶液去蛋白质，然后在引入色谱系统之前稀释。LC 以梯度洗脱模式运行，以便于准确测定每种分析物。C8柱作为固定相，甲酸和水和甲醇作为流动相。MS 在切换极性模式下运行。BCAA 的线性范围为 2–50 μg/mL，BCKA 的线性范围为 0.5–12.5 μg/mL (R2 0.997–0.999)。检查方法性能的回收率 (>80%)、基质效应、准确度和精密度（日间准确度 93.6-103.1%，日间 CV 1.5-13.0%）和稳定性。与现有方法相比，开发的方法缩短了运行时间（6 分钟）并加快了样品制备。