6490 series esi triple quadrupole mass spectrometer

Protein-bound homocitrulline and chloro-tyrosine were monitored in plasma after acid hydrolysis as previously described [24 (link)]. Briefly, plasma (20 µL) was placed into the vial and 200 µL of acid mixture (6 M HCl supplemented with 0.05% (m/v) phenol) and internal standards were added. Hydrolysis was carried out for 35 min at 110 °C. 13C9-Tyr and 13C15N-Lys were used as internal standards. Samples were evaporated to dryness under nitrogen flow, labeled with a butanolic HCl solution, dried under nitrogen flow, and finally dissolved in 1.0 mL formic acid 0.1% in water before injection into the LC-MS system. The LC system was a 1290 Infinity series UHPLC system (Agilent Technologies, Palo Alto, CA, USA). Amino acid residues were resolved on a Poroshell 120 EC-C18 column (2.1 × 100 mm, 2.7 µm) (Agilent Technologies, Palo Alto, CA, USA) using a gradient of 0.2% formic acid and methanol. Amino acid residues were quantified by tandem MS on a 6490 series ESI-triple quadrupole mass spectrometer using a JetStream source (Agilent Technologies, Palo Alto, CA, USA). Data were acquired using the MassHunter Acquisition^® software and analyzed by the MassHunter Quantitative Analysis^® software (Version B.07, Agilent Technologies, Santa Clara, United States). Data were expressed as the ratio of homocitrulline to lysine and ratio of chloro-tyrosine to tyrosine.