*2.2. Inhibitory Potential of Mertansine on Human Major UGTs in Human Liver Microsomes*

The inhibitory potential of mertansine on UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, and UGT2B7 activities was evaluated using liquid chromatography–tandem mass spectrometry (LC-MS/MS) with a cocktail of UGT substrates and ultrapooled human liver microsomes [36]. Each incubation mixture was prepared to a final volume of 100 µL as follows: ultrapooled human liver microsomes (0.2 mg/mL), 5 mM UDPGA, 10 mM magnesium chloride, alamethicin (25 µg/mL), 50 mM Tris buffer (pH 7.4), various concentrations of mertansine in methanol (final concentrations of 0.01–50 µM), and the cocktail sets of UGT enzyme-specific substrates. Two cocktail sets were used: set A contained 0.5 µM SN-38 for UGT1A1, 2 µM chenodeoxycholic acid for UGT1A3, and 0.5 µM trifluoperazine for UGT1A4; and set B contained 1 µM N-acetylserotonin for UGT1A6, 0.2 µM mycophenolic acid for UGT1A9, and 1 µM naloxone for UGT2B7. The reactions were initiated by adding UDPGA and incubated in a shaking water bath for 60 min at 37 ◦C. Reactions were terminated by adding 50 µL of ice-cold acetonitrile containing internal standards (IS, propofol glucuronide for set A and meloxicam for set B). Incubation mixtures were centrifuged at 13,000 g for 8 min at 4 ◦C. Next, 50 µL of each supernatant of sets A and B was mixed, and aliquots (5 µL) were analyzed using LC-MS/MS. All assays were performed in triplicate and average values were used in the analysis.

The LC-MS/MS system was comprised of an Agilent 6495 triple quadrupole mass spectrometer coupled with an Agilent 1290 Infinity system (Agilent Technologies, Wilmington, DE, USA). The column and autosampler temperatures were set to 40 ◦C and 4 ◦C, respectively. Six glucuronide metabolites and two ISs were simultaneously separated using an Atlantis dC<sup>18</sup> system (3 µm, 2.1 mm i.d. ×100 mm, Waters Technologies, Milford, MA, USA) with a gradient elution of 5% acetonitrile in 0.1% formic acid (MP A) and 95% acetonitrile in 0.1% formic acid (MP B) at a flow rate of 0.3 mL/min. Separation was achieved using the following sequence: 10% MP B for 1 min, 10% to 60% MP B for 1 min, 60% to 95% MP B for 1 min, 95% MP B for 2 min, 95% to 10% MP B for 0.1 min, and 10% MP B for 2.9 min. The electrospray ionization (ESI) source settings in both positive and negative ion modes were as follows: gas temperature, 200 ◦C; gas flow, 14 L/min; nebulizer, 40 psi; sheath gas temperature, 380 ◦C; sheath gas flow, 11 L/min; capillary voltage, 4500 V; and nozzle voltage, 500 V. Each metabolite was quantified via selected reaction monitoring in the negative ion mode (chenodeoxycholic acid 24-acyl-β-glucuronide, *m*/*z* 567.1 to 391.2; mycophenolic acid β-d-glucuronide, *m*/*z* 495.0 to 319.0; propofol glucuronide (IS), *m*/*z* 353.0 to 177.0) and in the positive ion mode (SN-38 glucuronide, *m*/*z* 568.9 to 392.9; trifluoperazine *N*-β-d-glucuronide, *m*/*z* 583.9 to 407.9; *N*-acetylserotonin β-d-glucuronide, *m*/*z* 394.0 to 219.0; naloxone 3-β-d-glucuronide, *m*/*z* 503.9 to 309.9; meloxicam (IS), *m*/*z* 351.9 to 115.0). Data were processed using MassHunter software (Version B.07.00, Agilent Technologies, Wilmington, DE, USA).
