Table 1

Incubation and assay conditions for the substrates investigated

CYP3A4 SubstrateProtein ConcentrationIncubation TimeSubstrate ConcentrationAssay
mg/ml min μM
MDZ1.02.52, 5, 10 (HAL), 50Sapelcosil LC-ABZ, 5 μM, 15 cm × 4.6 mm; 65% 0.025 M ammonium acetate (pH 5):35% acetonitrile, flow 1.0 ml/min; HPLC/UV-240 nm; retention times: 4-OH MDZ, 7 min; 1′-OH MDZ, 8 min; MDZ, 12 min; LOQ = 0.025 nmol, interassay CV < 5%
FEL1.01010, 25, 50Extraction by ethyl acetate; Spherisorb 5 ODS2, 15 cm × 4.6 mm; 0.2% TMED (N,N,N′,N′-tetramethylethylenediamine) in water, pH 7.0):methanol (30:70, v/v); flow 1.0 ml/min, HPLC/UV, 235 nm; retention times: FEL-PYR, 7 min; FEL, 9 min; UK-58,790, 12 min; LOQ = 0.05 nmol, interassay CV < 5%
NIF0.51510, 20, 50, 200Lightning C18 (33 mm × 3 mm, 4 μm); 61% acetonitrile:39% ammonium acetate (10 mM, pH 8); flow 0.7 ml/min; MS:positive ion MRM with Turbo ionspray interface (480°C); transitions: 345 → 284 (OX NIF); 480 → 315 (nicardipine, internal standard); LOQ = 0.0125 nmol, interassay CV < 5%
TST0.51525, 50, 100, 200, 500 (HAL)Waters C18 Novapak (150 × 3.9 mm); 50% methanol:50% distilled water; flow 1.0 ml/min, HPLC/UV, 245 nm; retention times: 6β-HTS, 5 min; TST, 26 min; LOQ, 0.0125 nmol, interassay CV < 5%
SV0.5105, 10, 25, 50ACE 5 C18 (10 cm × 4.6 mm); 85% 50 mM ammonium acetate:15% acetonitrile, gradient column temperature: 40°C, flow 1.0 ml/min; MS, Micromass LCT (time of flight mass spectrometer), positive ion full scan MS; LOQ = 0.01 nmol, interassay CV < 5%