Pre column

HPLC was used to analyze the drug biotransformation activity of human hepatic unit cultured in the PCL HF membrane bioreactor and treated with 10 µg/mL of diazepam for the whole culture time. Diazepam and its specific metabolites temazepam, oxazepam, nordiazepam and 4-hydroxy-diazepam were extracted by the samples and detected in HPLC (Agilent Technologies, Cernusco sul Naviglio (MI), Italy) with UV detector set at 236 nm, using a C18-RP Purosphere Star 5 µm, 250 × 4.6 mm column, equipped with a precolumn (Merck KGaA, Darmstadt, Germany) as previously reported [12 (link)]. Before the injection, samples were treated with 20% of 4 M NaOH, 1:10 isopropanol, 5:1 ethyl acetate (5:1). After 10 min under gentle rocking and 15 min of centrifugation at 200 g, the ethyl acetate phase was evaporated and dried under vacuum condition and the resulting pellet resuspended in 96 µL mobile phase consisting of 25/35/40 acetonitrile/methanol/0.04%triethylamine pH 7.04, respectively. The sample injection volume was 20 µL. The mobile phase was delivered at 0.8 mL/min and the column was operated at ambient temperature. For all the metabolites calibration curves ranging between 10 ng/mL and 10 µg/mL were previously run.

The HILIC separation was performed with a SeQuant ZIC-pHILIC column (150 × 4.6 mm, 5 µm) equipped with the corresponding pre-column (Merck KGaA, Darmstadt, Germany). A linear biphasic LC gradient was conducted from 80 % B to 20 % B over 15 min, followed by a 2 min wash with 5 % B, and 7 min re-equilibration with 80 % B, where solvent B is acetonitrile and solvent A is 20 mM ammonium carbonate in water. The flow rate was 300 μL/min, column temperature was maintained at 25 °C, injection volume was 10 μL, and samples were maintained at 4 °C in the autosampler (Creek et al. 2011 (link)).

The samples were analyzed using a Thermo Scientific Ultimate 3000 RSLCnano system (Thermo Scientific, CA, United States). The pHILIC separation was performed with a SeQuant ZIC-pHILIC column (150 × 4.6 mm, 5 μm) equipped with the corresponding pre-column (Merck KGaA, Darmstadt, Germany) – the column temperature was maintained at 25°C. A linear biphasic LC gradient was conducted from 80% B to 20% B over 15 min, followed by a 2 min wash with 5% B, and 8 min re-equilibration with 80% B, where solvent B is acetonitrile and solvent A is 20 mM ammonium carbonate in water. The flow rate was 300 μL/min, column temperature was held at 25°C, injection volume was 10 μL, and samples were maintained at 4°C in the autosampler (Creek et al., 2011 (link)).