Bioanalysis
All analytes were quantified with high-performance liquid chromatography (Shimadzu, Kyoto, Japan) tandem mass spectrometry (ABSciex, Ontario, Canada). The substrates and the corresponding metabolites (tizanidine, efavirenz, paclitaxel flurbiprofen, omeprazole, metoprolol, chlorzoxazone, midazolam, hydroxytizanidine, 8’-hydroxyefavirenz, 6α-hydroxypaclitaxel, 4’-hydroxyflurbiprofen, 5’-hydroxyomeprazole, α-hydroxymetoprolol, 6’-hydroxychlorzoxazone and 1’-hydroxymidazolam) were analyzed according to an earlier publication [26]. The main metabolites of metamizole (4-MAA, 4-AA, 4-FAA, 4-AAA) were quantified with a fully validated method [27]. Quantification of ciprofloxacin and fluconazole was performed by HPLC-MS/MS set in positive mode. The following mass transitions were used: ciprofloxacin: m/z 332.3/314.2, ciprofloxacin-d8: m/z 340.3/322.1, fluconazole: m/z 307.1/238.0, fluconazole-d4: m/z 311.2/242.1. As mobile phases, water (mobile phase A and C) and methanol (mobile phase B), both supplemented with 0.1% formic acid, were applied. Mobile phase C was added prior to the analytical column using a T-union. The analytes were separated using a core-shell C18 column (Kinetex C18, 2.6 µM, 50 mm x 2.1 mm, Phenomenex, CA, USA). The following gradient was used (percentage of mobile phase B): 0-0.5 min: 5%, 0.51-1.6 min: 5%-60%, 1.61-2.0 min: 60%-95%, 2.01-2.5 min: 95%, 2.51-3.5 min: 5%. The initial flow rate for pump A and B was 0.1 mL/min. Mobile phase C was added at an initial rate of 0.5 mL/min from the start of the run, decreasing to 0 after 0.5 minutes.
The analysis of the samples met the criteria defined by the US Food and Drug Administration (FDA) guidelines for bioanalytical analysis of study samples [28]. The calibration range was linear from 25-25,000 ng/mL for 4-MAA, 25-10,000 ng/mL for 4-AA, 4-AAA and 4-FAA, and 20-10,000 ng/mL for fluconazole and ciprofloxacin. Intra- and interday accuracy between 85.2%-114.7% with an imprecision of less than 13.6% for all analytes measured.