Acquity 1.7 m c18 column

Urine samples were prepared by diluting each sample 1:4 in a 50% acetonitrile solution containing the internal standards, 30 µM of 4-nitrobenzoic acid and 2 µM of debrisoquine. The samples were centrifuged at 18,000 rcf to precipitate out the proteins. A 5 µL aliquot of the recovered supernatant was then injected into a reverse-phase 50 × 2.1 mm ACQUITY^® 1.7 µm C18 column (Waters^® Corp., Milford, MA) coupled to a time-of-flight mass spectrometry (TOFMS). The 13 min long mobile phase gradient started with aqueous solvent (98% water: 2% acetonitrile with 0.1% formic acid) and switched to 100% organic (acetonitrile) at a flow rate of 0.5 mL/min. The Q-TOF Premier™ (Waters Corp.) mass spectrometer was operated in positive (ESI⁺) and negative (ESI⁻) electrospray ionization modes. The acquired centroid mass spectrometer data was then processed using MassLynx™ software (Waters Corp.). Furthermore, twofold dilutions were performed using the internal standards at the initial concentration of 150 µg/mL to the final concentration of 0.59 µg/mL. The internal standards were also spiked into pooled control urine samples and processed every 7 injection intervals. The calculated standard curve for each internal standard was used to determine the relative abundance of different metabolites in each ionization mode.