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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Abstract
A simple and fast LC-MS/MS method was developed and validated for the quantification
of 20 proteinogenic L-amino acids (AAs) in a small volume (5 μL) of mouse plasma.
Chromatographic separation was achieved on an Intrada Amino Acid column within 13
min 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.