Determination of Enzyme Activity of 4 CYPs in Earthworms by HPLC-MS/MS Based on Probe Substrate Method and Its Application

Based on the probe substrate method, earthworm microsomal suspension was added into the incubation system containing mixed probe substrates of coumarin (50 μmol·L^-1), bupropion (25 μmol·L^-1), amodiquine (50 μmol·L^-1) and dextromethorphan (5 μmol·L^-1), in which the mass concentration of microsomal protein should be controlled at 32-96 mg·L^-1. After incubation at 37 ℃ for 20 min, methanol was added to terminate the reaction. After settling for 10 min, the supernatant was taken and passed through 0.22 μm membrane. The corresponding metabolites of 7-hydroxycoumarin, hydroxy-bupropion, N-deethylamodiquine and dextrophane were determined by HPLC-MS/MS. Then the enzyme activities of CYP2A6, CYP2B6, CYP2C8 and CYP2D6 were evaluated according to the production of corresponding metabolites. An Acquity UPLC HSS T3 chromatographic column was used as separation column, and mixed solutions composed of 0.05% (φ) formic acid solution (containing 1 mmol·L^-1 ammonium acetate) and methanol at different volume ratios were used as mobile phase for gradient elution. Electrospray positive ion source and multiple reaction monitoring mode were used in MS/MS analysis. The matrix matching method was used to prepare mixed standard solution series. It was shown that mass concentrations of 7-hydroxycoumarin, hydroxy-bupropion, N-deethylamodiquine and dextrorphan were linearly related to their corresponding peak areas in the range of 2.5-50 μg·L^-1. Test for recovery by standard addition method was made on blank sample at 3 concentration levels, giving values of recovery in the range of 73.7%-103%, the intra-day RSDs (n=5) ranged from 2.1% to 11%, and the inter-day RSDs (n=5) were in the range of 4.6%-10%. The enzyme activity of CYP2A6, CYP2B6, CYP2C8 and CYP2D6 in earthworms after 21 days of exposure to contaminated soil (exposure groups) with multi-walled carbon nanotubes and general soil (control group) were determined by the proposed method. It was shown that the enzyme activity of CYP2C8 was significantly higher than that of CYP2A6, CYP2B6 and CYP2D6. The enzyme activity of CYP2A6 and CYP2C8 in the exposure groups was significantly higher than that of the control group, while the enzyme activity of CYP2B6 and CYP2D6 was insignificantly different from that of the control group. Therefore, it was concluded that CYP2A6 and CYP2C8 could be used as a set of biomarkers to diagnose soil polluted by multi-walled carbon nanotubes.