Amino acid analysis column

Free amino acids in serum samples were measured by a HPLC-FLD method using an AccQ·Tag precolumn derivatization technology with waters AccQ·Tag chemistry package (Waters Chemical Co., USA). 70 μL borate extraction agent was added to 10 μL serum, and then 20 μL AccQ-Flour derived reagent was added to the mixture. After incubating at 55°C for 10 min, supernatants were collected for the analysis. The amino acid concentration was measured with high-performance liquid chromatography using an amino acid analysis column (3.9 mm × 150 mm, 4 μm, Waters, USA) and a mobile phase consisting of (A) water containing 10% AccQ·Tag Eluent A and (B) water containing 40% acetonitrile at a flow rate of 1 mL/min at 37°C with an excitation wavelength at 250 nm and an emission wavelength at 395 nm. The mobile phase gradient elution was programmed as follows: 5–98% A (0–0.5 min), 98–93% A (0.5–15 min), 93–90% A (15–19 min), 90–67% A (19–32 min), 67% A (32-33 min), 67–0% A (33-34 min), 0% A (34–37 min), 0–100% A (37-38 min), and 100% A (38–64 min). The sample injection volume was set at 10 μL.

Serum amino acid calibration standards were prepared with AccQ•Tag™ Ultra Amino Acid Analysis Solution (AA) kit from Waters according to instructions with slight modifications for detection on a mass spectrometer [21 (link)]. A 5-point standard concentration curve was made from the calibration standard solution to calculate amino acid concentrations in serum samples. Serum samples of 10 μl were spiked with an internal standard, norvaline then derivatized according to AccQ•Tag instructions. High-resolution separation was done using an ACQUITY UPLC system and injecting 1 μl, with an Amino Acid Analysis column from Waters. Mass detection was completed on a XEVO TQ-S Mass Spectrometry, Waters in ESI positive mode.