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.
Highlights
The tear is an extra-cellular uid bathing the cells of the ocular surface
We developed a liquid chromatography high resolution mass spectrometry method for the simultaneous determination of 15 amino acids (AAs) in human tears
The results indicated that most abundant ions of the AAs were their molecular ions [M + H]+ in positive ion mode
Summary
The tear is an extra-cellular uid bathing the cells of the ocular surface. The changes of certain tear metabolites may be associated with ocular surface diseases or potential systemic diseases.[1] For example, dry eye patients showed a lower concentration of carnitine in tear uid.[2] Recently, analysis of amino acids is of particular interest as it involves the pathophysiology of eye diseases.[3,4]. They have long been considered a source of protein synthesis, and play a key role in the regulation of the body metabolism of living organisms. It is widely accepted that changes in amino acid have profound effects on many aspects of cellular functions, such as gene expression, cell signaling, and the transport of amino acids.[5,6,7] Previous reports have pointed out that some amino acids can reduce in ammation by inhibiting NF-kB activation, IL-6 production and expression of the leukocyte adhesion molecule CD62E.8,9 exploring amino acids at a trace level in clinical samples prompted the need for a reliable, sensitive and convenient test method
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