2.9. Amino acids quantification by gas chromatography mass spectrometry (GC/MS)

Briefly, amino acids were extracted by mixing 2 μl of humor sample with 8 μl of cold methanol and 1 μl internal standard (Myristic acid-d27). The extracts were dried under vacuum and derivatized by methoxyamine and N-tertbutyldimethylsilyl-N-methyltrifluoroacetamide as described in detail (Du et al., 2015)(Chao et al., 2017). An Agilent 7890B/5977B GC/MS system with an Agilent DB-5MS column (30 m × 0.25 mm × 0.25 μm film) was used for GC separation and analysis of metabolites. Ultra–high-purity helium was the carrier gas at a constant flow rate of 1 mL/min. One microliter of sample was injected in split-less mode by the auto sampler. The temperature gradient started at 95 °C with a hold time of 2 min and then increased at a rate of 10 °C/min to 300 °C, where it was held for 6 min. The temperatures were set as follows: inlet 250 °C, transfer line 280 °C, ion source 230 °C, and quadrupole 150 °C. Mass spectra were collected from m/z 80–600 under selective ion monitoring mode. The data was analyzed by Agilent MassHunter Quantitative Analysis Software.