Integrated PRM and DIA via HILIC‐Orbitrap HRMS for Simultaneous Quantification and Interference Screening of Amino Acids in Human Saliva

ABSTRACT

Free amino acids in human saliva are increasingly recognized as valuable biomarkers for noninvasive health monitoring and disease diagnosis owing to their close association with systemic metabolic status. However, their accurate quantification remains challenging because of low physiological concentrations and high matrix complexity. In this study, a robust method based on hydrophilic interaction liquid chromatography coupled with Orbitrap high‐resolution mass spectrometry (HILIC‐Orbitrap HRMS) was developed for the simultaneous quantification of 20 amino acids in human saliva. Chromatographic separation was achieved using a BEH Amide column (150 mm × 2.1 mm, 1.7 μm) with a gradient elution program. Mass spectrometric detection was performed in positive‐ion mode using a Full MS plus scheduled parallel reaction monitoring (PRM) acquisition scheme (hereafter referred to as PRM). The method exhibited excellent linearity ( R ²  > 0.991), accuracy (80.01%–119.60%), and precision (RSD < 10%). The limits of detection and quantification were 0.030–0.100 and 0.125–0.500 μmol/L, respectively. In addition, data‐independent acquisition (DIA) was used to screen for co‐eluting isobaric interferences; potential interferents such as 5‐aminovaleric acid (for valine) and N , N ‐dimethylglycine (for 4‐aminobutyric acid) were flagged and subsequently resolved with authentic standards. This validated HILIC‐HRMS method provides a reliable platform for targeted quantification of polar amino acids in saliva, with DIA serving as a complementary tool for interference screening to ensure analytical specificity.