Novapak c18 guardpak

Assay conditions for 4-MU can be found under Inhibition by Aprepitant of UGTs. Analytical conditions for M-6-G were based on a method previously used in this laboratory (Innocenti et al., 2001 (link)). For AZT-G analyses, we used a previously published method (Court et al., 2003 (link)) with slight modification. Briefly, incubations were terminated with addition of 100 µl ACN (with 1 mM p-acetomidophenol), vortexed and centrifuged at 20,817 RCF at 4°C for 30 min. The supernatant was transferred to a polypropylene HPLC vial and 10 µl was injected into the HPLC system. Separations were performed with a reversed phase µBondapak C₁₈ column (10µm, 3.9 × 300 mm, Waters Corp.) preceded by a Novapak C₁₈ guardpak (Waters Corp.) The mobile phase was a mixture of 20 mM, pH 2.2, potassium phosphate buffer in water (pump ‘A’), and acetonitrile with 5% water (pump ‘B’) at a flow rate of 1.0 ml/min. A gradient was applied as follows: 0 – 4.0 min, 80% A. 4.1 – 8.0 min, 50% A. 8.1 – 12.0 min, 30% A. 12.1 – 20 min 80% A. The respective retention times for AZT-G and the internal standard were 5.5 min and 7.3 min respectively.

MA and its oxidative metabolites were quantified by HPLC (Hitachi High Technologies America, San Jose, CA) with slight modification to a previous method (Matin et al. 2002 (link)). The eluate was monitored with UV detection at 280 nm. The analytes were separated on a C₁₈ reversed-phase column (μBondapak, 3.9 × 300 mm, 10 μm, 125Å; Waters, Milford, MA) preceded by a Nova-Pak C₁₈ Guardpak (Waters, Milford, MA). The mobile phase consisted of acetonitrile/water/acetic acid (60/39/1). The flow rate was 1 ml/min with a run time of 25 min. The retention times were: 11.5 min (MA), 5.2 min (M1), 6.7 min (M2), 4.0 (metabolite 3, M3) and 24 min (internal standard). The ratio of the MA metabolite peak areas to the area of the internal standard peak was calculated. The relative formation of M1 and M2 was estimated using a standard curve generated using MA with a concentration range of 5–1000 μM.