按规定方法解剖蚯蚓取得其内脏，经清洗后转移至盛有4 mL匀浆缓冲液的组织研磨器中均匀破碎，并将匀浆体积定为6.0 mL。将此匀浆液通过差速离心法取其微粒体（即第2次离心后的沉淀物）用3 mL保存缓冲液重新悬浮，制得蚯蚓微粒体蛋白悬浮液。将此悬浮液100 μL加入于含孵育体系650 μL及探针底物混合液（含非那西汀100 μmmol·L^-1、双氯芬酸钠100 μmmol·L^-1和睾酮25 μmmol·L^-1）250 μL中，升温至37℃，孵化20 min，加入甲醇200 μL中止反应。离心10 min，取其上清液，经0.22 μm滤膜过滤。所得滤液流经Acquity HPLC C₁₈色谱柱（50 mm×2.1 mm，1.8 μm），并用不同体积比的（A）每升溶液中含甲酸0.5 mL的1 mmol·L^-1乙酸铵溶液和（B）甲醇组成的混合液作为流动相进行梯度洗脱，使所加入的3种底物被蚯蚓体内的细胞色素P450亚酶的催化作用而相应产生的3种特异代射物分离。然后在电喷雾正离子源和多反应监测模式条件下，用串联质谱法测得其含量。结果表明：所述3种代谢物（对乙酰氨基酚、4'-羟基双氯芬酸、6β-羟基睾酮）的线性范围依次在0.40，0.20，10.0 μg·L^-1以内，检出限（3S/N）依次为0.008，0.005，0.1 μg·L^-1。根据所测得3种代谢物的生成量可分别评价蚯蚓体内3种相应的亚酶CYP1A2、CYP2C9和CYP3A4活力。此外，还对受敌敌畏染毒的土壤中培养3 d和14 d后的蚯蚓样品，按上述方法分析，获得了染毒的土壤对蚯蚓体内上述3种CPY亚酶活力的变化情况。所得结果可作为土壤污染全面诊断的生物标记。

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.