2.6 LC-MS/MS

DTG concentrations were determined using a LC-MS/MS, LCMS-8030 system (Shimadzu, Kyoto, Japan). Analytes were separated isocratically on a reverse phase C₁₈ column (1.5 × 50 mm, 5 μm, Inertsil^® ODS-3, GL Science, Tokyo, Japan) with 5 mM formic acid-25% (v/v) ethanol at a flow rate of 0.2 mL/min. The column temperature was maintained at 40°C and the sample plate was kept at 4°C. The analytes were detected using positive electrospray ionization mode with multiple reaction monitoring (MRM). The injection volume was 1 μL, and the total analysis time for each sample was 6 min. Argon was used as the nebulizer and desolvation gas. The gas temperature, nebulizing gas flow, drying gas flow, capillary voltage, and collision energy were set at 250°C, 3.0 L/min, 15.0 L/min, 6000 V, and −30 V, respectively. Precursor to product mass transitions for DTG and DTG-d5 were 420/127 and 420/132 (m/z), respectively. LabSolutions software (Shimadzu, Tokyo, Japan) was used for system control and data analysis.