UHPLC-MS/MS Quantification of Amino Acids

The specific operation steps for UHPLC-MS/MS could refer to our previous work (20 (link)). Shortly described as follows: Equipped with a Waters ACQUITY UPLC BEH Amide column (100 × 2.1 mm, 1.7 μm; Waters Corporation, USA), an Agilent 1290 Infinity II series UHPLC system (Agilent Technologies, California, USA) was used for the UHPLC separation. Mobile phase A and B were respectively made up of 1% formic acid in water and 1% formic acid in acetonitrile. The column temperature was set to 35°C while the auto-sampler temperature was set to 4°C. For assay development, Agilent 6460 Triple Quadrupole mass spectrometer was connected with an Agilent Jet Stream electrospray ionization interface (Agilent Technologies, California, USA).
Isotope standards used for the quantifications were applied and the optimal Multiple Reaction Monitoring (MRM) parameters of the target metabolites were obtained. Agilent MassHunter Work Station Software (B.08.00, Agilent Technologies, California, USA) was used for the MRM data acquisition and processing. The raw data of amino acids concentration in PNI was in the Supplementary Table 2.