Study of Enzymatic Activities of CYP1A2, CYP2C9 and CYP3A4 in Earthworms by HPLC-MS/MS Based on Probe Substrate Method

Earthworm sample was dissected by the given method and the internal organs obtained were homogenized with 4 mL of homogeneous buffer mixture giving a homogenate having a volume of 6.0 mL. Microsomes (i.e., the precipitate settled out in the 2nd centrifugation) were separated from the homogenate by differential centrifugation. The microsomes were re-suspended in 3 mL of preservation buffer mixture to give the microsome protein suspension of earthworm. An aliquot of 100 μL of this suspension was taken and incubated in 650 μL of an incubation system and 250 μL of probe substrate solution (containing 100 μmol·L^-1 of phenacetin, 100 μmol·L^-1 of diclofenac sodium and 25 μmol·L^-1 of testosterone) at 37℃ for 20 min, when 200 μL of methanol were added to stop the incubation. After centrifugation for 10 min, the supernatant was taken and filtered through 0.22 μm filtering membrane. The filtrate was passed through Acquity HPLC C₁₈ chromatographic column (50 mm×2.1 mm, 1.8 μm) with the sample solution introduced and using mixtures of various volumic ratios of (A) 1 mmol·L^-1 NH₄OAc solution containing 0.5 mL of formic acid perliter, and (B) methanol, as mobile phases in the gradient elution to separate the 3 specific metabolites (i.e., acetaminophen, 4'-hydroxy diclofenac and 6β-hydroxyl testosterone) produced respectively by the catalytic actions of the sub-enzymes (i.e., CYP1A2, CYP2C9 and CYP3A4) existing in earthworm body on the respective substracts, and then the metabolites were determined by MS/MS under the condition of ESI⁺ and MRM. Linearity ranges of the 3 metabolites were found within 0.40, 0.20, 10.0 μg·L^-1 with detection limits of 0.008, 0.005, 0.1 μg·L^-1 respectively. Based on the amounts of the 3 metabolites produced, the activity for the 3 sub-enzymes existing in body of the earthworm could be evaluated. Furthermore, a study on the variation of activity of the 3 CPY sub-enzymes of earthworms which were breeded in soil contaminated by DDVP for 3 d and 14 d was made by the above method. Beneficial results were obtained, which can be used as an biological symbol in comprehensive understanding on the pollution of soil.