A reliable LC-MS/MS method for the quantification of natural amino acids in mouse plasma: Method validation and application to a study on amino acid dynamics during hepatocellular carcinoma progression
A reliable LC-MS/MS method for the quantification of natural amino acids in mouse plasma: Method validation and application to a study on amino acid dynamics during hepatocellular carcinoma progression
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DOI：
10.1016/j.jchromb.2019.05.039
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发表时间：
2019-08-15
影响因子：
3
通讯作者：
Yu, Ai-Ming
中科院分区：
文献类型：
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作者：
Liu, Zhenzhen;Tu, Mei-Juan;Yu, Ai-Ming
建立了一种简单快速的LC-MS/MS方法，用于小体积（5 μ L）小鼠血浆中20种蛋白源性L-氨基酸（AAs）的同时定量分析。在Intrada氨基酸色谱柱上，以0.6 mL/min的流速，用含100 mM甲酸铵的水溶液和含乙腈、水和甲酸（v：v：v = 95：5：0.3）的有机移动的相进行梯度洗脱，在13 mM范围内实现色谱分离。在优化条件下，以正离子模式通过多反应监测（MRM）分析单个AA和相应的稳定同位素标记的AA内标物。方法验证包括线性、灵敏度、准确度和精密度、回收率、基质效应和稳定性，结果证明该LC-MS/MS方法是一种专属性、准确度和可靠的测定方法。因此，该LC-MS/MS方法用于比较在相同条件下饲养的健康和原位肝细胞癌（HCC）异种移植小鼠之间个体血浆AA的动力学。我们的研究结果显示，5周后，肝癌肿瘤进展，血浆L-精氨酸浓度显着降低，而L-丙氨酸和L-苏氨酸水平急剧上升。这些发现支持了这种LC-MS/MS方法的实用性和特异性AA作为HCC可能的生物标志物的前景。
A simple and fast LC-MS/MS method was developed and validated for simultaneous quantification of 20 proteinogenic L-amino acids (AAs) in a small volume (5 mu L) of mouse plasma. Chromatographic separation was achieved on an Intrada Amino Acid column within 13 mM via gradient elution with an aqueous solution containing 100 mM ammonium formate and an organic mobile phase containing acetonitrile, water and formic acid (v:v:v = 95:5:0.3), at the flow rate of 0.6 mL/min. Individual AAs and corresponding stable-isotope-labeled AAs internal standards were analyzed by multiple reaction monitoring (MRM) in positive ion mode under optimized conditions. Method validation consisted of linearity, sensitivity, accuracy and precision, recovery, matrix effect, and stability, and the results demonstrated this LC-MS/MS method as a specific, accurate, and reliable assay. This LC-MS/MS method was thus utilized to compare the dynamics of individual plasma AAs between healthy and orthotopic hepatocellular carcinoma (HCC) xenograft mice housed under identical conditions. Our results revealed that, 5 weeks after HCC tumor progression, plasma L-arginine concentrations were significantly decreased in HCC mice while L-alanine and L-threonine levels were sharply increased. These findings support the utilities of this LC-MS/MS method and the promise of specific AAs as possible biomarkers for HCC.