1290 uplc system

LC‐MS/MS analysis was performed on an Agilent 1290 UPLC system, SCIEX 4500 tandem mass spectrometer, and ESI source operated in positive ionization mode. Chromatography was performed using a specialized C18 column (SCIEX 4374841, dimensions 5 μm, 4.6 mm × 150 mm) at temperature of 50°C with 0.1% formic acid and 0.01% heptafluorobutyric acid (HFBA) in water (mobile phase A) and 0.1% formic acid and 0.01% HFBA in methanol (mobile phase B), at a rate of 1.0 ml/min using the gradient in Table S1. Sample injection volume was 20 μl. The mass spectrometer was operated in selective reaction monitoring (SRM) mode using the following settings: ion spray voltage 3500 V, entrance potential 10 V, declustering potential 35 V, collision cell exit potential 10 V. The Q1/Q3 transitions (Table S2) for target analytes were provided by the kit manufacturer based on expected fragmentation within the derivatization tag. The Q1 masses for internal standards (stable‐isotope labeled structural analogs) were calculated based on the ¹³C,¹⁵N labeling in native amino acid structure. An additional transition for underivatized stable‐isotope (¹³C,¹⁵N) labeled isoleucine was determined by optimization during direct infusion. Instrument control, data acquisition, and processing were performed using Analyst (SCIEX) software, 1.6.3.

The HPLC system consists of a Waters 2995 controller and 2998 Photodiode Array detector. The separation was performed on an Elite Kromasil C₁₈ column (250 mm × 4.6 mm, 5 μm). The flow rate was set at 1.0 mL/min, with the column temperature set at 25°C, and detective wavelength was set at 254 and 289 nm. The acetonitrile and water containing 0.2% acetic acid were employed as mobile phases A and B, respectively. The binary gradient program was set as follows: 0–10 min, 80% of B; 10–15 min, 80 to 75% of B; 15–25 min, 75% of B; 25–50 min, 75 to 35% of B; and equilibrated for 10 min before the next injection. The injection volume was 10 μL. Data were collected and visualized by Waters Empower Chemstation Software.
The chromatographic peak purity analysis was performed on an Agilent 1290 UPLC system coupled with a SCIEX Triple TOF 4600 mass spectrometer equipped with an ESI interface. The optimized MS conditions were as follows: TOF mass range between 50 and 1700, curtain gas 35 psig, ion spray voltage floating −4500/5000 kV, and ion source temperature 500°C. The collision energy was set at 10 V to obtain more fragment information.