Analysis of amino acids in human tears by hydrophilic interaction liquid chromatography and quadrupole orbitrap mass spectrometry

Abstract

Amino acids in human tears play certain physiological roles and their determination is challenging due to complicated chemical properties. This study described a fast and sensitive method for the simultaneous determination of 15 amino acids (AAs) in tears by hydrophilic interaction liquid chromatography and quadrupole orbitrap mass spectrometry (HILIC-Q-Orbitrap-MS). Amino acids in tears were extracted by methanol, and then cleaned up with a solid phase extraction (SPE) cartridge. Chromatographic separation was performed on a 1.7 μm BEH Amide column within 8 min. Tear samples spiked with free AAs were tested in terms of linearity, sensitivity, repeatability, and recovery. Two stable isotope-labeled amino acids were used as internal standards to improve the method performance. Recoveries for all analytes ranged from 89 to 107%. Intra-day and inter-day precision, expressed as relative standard deviations, were all below 10%, and the method detection limits ranged from 0.02 μmol L^-1 to 0.11 μmol L^-1. The developed method with high throughput and high analyte specificity shows good promise for consistent analysis of free amino acids in tears.

Fig. 1. The work flow of analysis of amino acids in human tears by UPLC-Q-Orbitrap-MS.

Fig. 2. Representative chromatograms of 15 amino acids (spiking level of 20 μmol L⁻¹) in HILIC-Q-Orbitrap with PRM mode.