Acquity beh 300 uplc column

UPLC-MS analysis was carried out with a Waters Acquity H-Class system utilizing a Waters Acquity BEH-300 UPLC column packed with a C₄ stationary phase (2.1 × 50 mm, 1.7 µm) in conjunction with HRMS analysis by a Waters Xevo-G2 quadropole-TOF electrospray mass spectrometer to detect and analyze protein-drug adducts. Each enzyme (2 µM) in 25 mM Tris buffer, pH 8, was incubated in the absence or presence of drug (50 µM) at room temperature for 5 hrs or for 1 hr and 24 hrs to assess stability of the adducts. Trifluoroacetic acid (0.1% final) was used to quench the reactions, samples were filtered through a 0.22 um filter and analyzed using UPLC-MS as follows: Mobile phase: 0–1 min 90% water + 10% ACN + 0.1% formic acid, 1–7.5 min gradient up to 20% water + 80% ACN + 0.1% formic acid, 7.5–8.4 min 20% water + 80% ACN + 0.1% formic acid, 8.4–8.5 min gradient up to 90% water + 10% ACN + 0.1% formic acid, 8.5–10 min 90% water + 10% ACN + 0.1% formic acid. Flow rate = 0.3 mL min⁻¹. T = 60 °C.