Quantification of Amino Acid Derivatives

Calibration standards and serum samples were prepared in the same manner. The following concentrations of calibrators, in water, were used: 5, 12.5, 25, 50, 100, 150, 200, 250 µM for l-arginine, 0.05, 0.13, 0.25, 0.5, 1.0, 1.5, 2, and 2.5 µM for ADMA and SDMA, and 1, 2.5, 5, 10, 20, 30, 40, and 50 µM for l-citrulline, and 0.14, 0.35, 0.7, 1.4, 2.8, 4.2, 5.6, 7.0 µM for DMA. The following procedure was conducted: 100 µL aliquots of calibration standards or serum, 10 µL of internal standard solution (50 µM D6-DMA, 20 µM D7-ADMA, and 100 µM D7-arginine, respectively) and 50 µL of borate buffer (0.025 M Na₂B₄O₇·10H₂O, 1.77 mM NaOH, pH 9.2) were placed into 2.0 mL polypropylene tubes and vortexed (1 min, 25 °C). Derivatization was conducted using 400 µL of acetonitrile (ACN) and 10 µL of 10% BCl in ACN. The solutions were incubated and vortexed (5 min, 25 °C), centrifuged (7 min, 10,000 RPM, 4 °C), and 100 µL of the clear supernatant was transferred into glass vials containing 400 µL of water.