A reliable LC-MS/MS method for the quantification of natural amino acids in human plasma and its application in clinic

Junhuan Lin; Yibo Song; Yangrui Zhang; Tao Ke; Fengting Ou; Kui Zeng; Debo He; Li Li; Lushan Yu

doi:10.1016/j.jpba.2025.116672

A reliable LC-MS/MS method for the quantification of natural amino acids in human plasma and its application in clinic

J Pharm Biomed Anal. 2025 Jan 8:256:116672. doi: 10.1016/j.jpba.2025.116672. Online ahead of print.

Authors

Junhuan Lin¹, Yibo Song², Yangrui Zhang³, Tao Ke⁴, Fengting Ou⁵, Kui Zeng⁵, Debo He⁶, Li Li⁷, Lushan Yu⁸

Affiliations

¹ Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China; Jinhua Institute of Zhejiang University, Jinhua 321036, China; State Key Laboratory of Advanced Drug Delivery and Release Systems, Zhejiang University, Hangzhou 310058, China.
² Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China; Department of Pharmacy, Shaoxing People's Hospital (Shaoxing Hospital, Zhejiang University School of Medicine), Shaoxing 312000, China.
³ Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China; Jinhua Institute of Zhejiang University, Jinhua 321036, China.
⁴ Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, Zhejiang University of Technology, Hangzhou 310014, China.
⁵ Jinhua Institute of Zhejiang University, Jinhua 321036, China.
⁶ Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China; State Key Laboratory of Advanced Drug Delivery and Release Systems, Zhejiang University, Hangzhou 310058, China.
⁷ Department of Pharmacy, Zhejiang Hospital, Hangzhou, Zhejiang 310013, China. Electronic address: zjyylili@163.com.
⁸ Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China; Jinhua Institute of Zhejiang University, Jinhua 321036, China; State Key Laboratory of Advanced Drug Delivery and Release Systems, Zhejiang University, Hangzhou 310058, China. Electronic address: yuls@zju.edu.cn.

PMID: 39813777
DOI: 10.1016/j.jpba.2025.116672

Abstract

A simple and fast LC-MS/MS method was developed and validated for simultaneous quantification of 20 L-amino acids (AAs) in human plasma. Chromatographic separation was achieved on an Agilent AdvanceBio Hilic column within 15 min via gradient elution with an aqueous solution containing 5 mM ammonium formate, 5 mM ammonium acetate and 0.1 % formic acid and an organic mobile phase containing 0.1 % formic acid, 5 mM ammonium formate and 5 mM ammonium acetate acetonitrile-water (90:10, v/v) at the flow rate of 0.25 mL/min. Individual AAs and internal standard 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. The method was thus utilized to compare the dynamics of individual plasma AAs between healthy females and patients with ovarian tumors. Our results revealed that, in cancer group, plasma 3-Methyl-L-Histidine, L-Proline, L-Phenylalanine and L-Lysine concentrations were significantly increased in patients with malignant ovarian tumors while L-Leucine and L-Isoleucine levels were sharply decreased. These findings support the utilities of this LC-MS/MS method and the promise of specific AAs as possible biomarkers for ovarian cancer.

Keywords: Amino acids; Biomarker; Ovarian cancer progression; UHPLC-MS/MS.