UPLC‐MS/MS Analysis of Ethoxyresorufin O‐Deethylation (EROD) Kinetics in Rat Kidney Microsomes

Abstract

PurposeFormation of resorufin from ethoxyresorufin has been widely used to assess cytochrome P450 1A1 (CYP1A1) activity. Additionally, the kinetics of CYP1A1 activity has been well characterized in the liver microsomes by spectrophotometric/chromatographic methods. However, studies in kidney microsomes are scarce, most likely due to the lower microsomal content of CYP1A1 and lower production of the resorufin in the kidneys relative to those in the liver. In the present study, we aimed to develop a microscale enzymatic incubation method, coupled with a sensitive UPLC‐MS/MS assay for the characterization of kinetic parameters of 7‐ethoxyresorufin O‐deethylation (EROD) in the kidney microsomes.MethodsKidney microsomes were prepared from adult male Sprague‐Dawley rats (n = 3) by differential centrifugation. Protein (0.1, 0.25, 0.5 mg/mL) and time (2.5, 5.0, 7.5, and 10 min) linearity experiments with regard to the rate of production of resorufin from the substrate were determined (n = 3) using a substrate concentration of 0.5 μM. For determination of the Michaelis‐Menten (MM) kinetic parameters of EROD, the reaction mixtures (50 μL), containing microsomal proteins (0.25 mg/mL) were incubated with different concentrations of ethoxyresorufin (0.1–5.0 μM) in the presence of NADPH (1 mM) for 10 min at 37°C. The reaction was terminated by the addition of 100 μL of acetonitrile, containing internal standard (d6‐resorufin). After centrifugation, the supernatant (5 μL) was injected onto a UPLC column (Phenomenex C18), and sample components were separated using a binary gradient mobile phase (A: 5 mM ammonium acetate in water and B: acetonitrile/methanol, 95:5) at a flow rate of 0.3 mL/min. The UPLC–MS/MS system used the electrospray ionization technique in positive ion mode, and the transitions of m/z 213.9→185.9 and 220→192 were used to quantitate resorufin and the IS, respectively. Analytical method validation was performed as per regulatory guidelines.ResultsThe analytical method met the acceptance criteria for method validation in the linear range of 0.5 nM to 75 nM resorufin, with a lower limit of quantitation of 0.5 nM. The intra‐ and inter‐day precision was in the range of 4.55–12.4% and 6.17–9.82%, respectively. The overall recoveries of resorufin (90%–99%) and IS (85%–103%) were relatively high, suggesting an almost complete recovery from the protein precipitation method and confirming the lack of matrix effect on the samples. Using the optimized incubation and quantitation methods, we were able to reduce the incubation volume (50 μL), microsomal protein amount (12.5 μg), and incubation time (10 min), compared with reported methods. The method was successfully applied to the estimation of the MM kinetic parameters of 7‐ethoxyresorufin O‐deethylase activity in the rat kidney microsomes (Fig 1), which showed a maximum velocity of 1.92 ± 0.25 pmol/min/mg and a MM constant of 1.24 ± 0.2 μM (mean ± SD).ConclusionThe use of a sensitive and validated UPLC‐MS/MS method allows determination of the MM kinetic parameters of CYP1A1 activity in the rat kidney microsomes.Support or Funding InformationFunding: Chapman University School of PharmacyFormation rate of resorufin from ethoxyresorufin in rat kidney microsomal incubations (n = 3)Figure 1