Catechol-O-methyltransferase (COMT) is a major drug metabolizing enzyme in humans. COMT expression is directedly associated with various mental diseases and cancers due to its essential role in catalyzing metabolic inactivation of endogenous catecholamines and catechol estrogens. However, a practical method to precisely measure COMT activities in biological samples is lacking. In the current study, we established a liquid chromatography-fluorescence detection (LC-FD) method based on fluorometric detection of the methylated product of 3-BTD, a fluorescent probe for COMT, to sensitively quantify COMT activities in human erythrocytes and cell homogenates. Assay validation of the established LC-FD based method was conducted for selectivity and sensitivity, range of linearity, precision and accuracy, recovery, biological matrices effect and stability. The limit of quantification for 3-BTMD (the methylated product of 3-BTD by COMT) of this method was 0.0083 nM, which is nearly 10-fold lower than that for previously published methods. The method was precise with intra- and inter-day relative standard deviation (RSD) lower than 5%. In addition, this method showed an excellent anti-interference ability with no effects of the endogenous substances on the fluorometric detection of 3-BTMD. The practical use of this method was established by its successful application for the measurement of COMT activities in individual human erythrocytes (n = 13), and in cell homogenates generated from four different human cell lines. Our results suggest that this method will be of great value in accurately determining the native activity of COMT in biological samples, which is beneficial for a complete understand of the role of COMT both in physiological and pathological conditions.

儿茶酚-O-甲基转移酶（COMT）是人类主要的药物代谢酶。由于COMT在催化内源性儿茶酚胺和儿茶酚雌激素的代谢失活中起重要作用，因此它与多种精神疾病和癌症直接相关。但是，缺少一种精确测量生物样品中COMT活性的实用方法。在当前的研究中，我们建立了一种基于荧光检测3-BTD甲基化产物的液相色谱-荧光检测（LC-FD）方法，一种用于COMT的荧光探针，可以灵敏地定量人类红细胞和细胞匀浆中的COMT活性。对建立的基于LC-FD的方法进行选择性，灵敏度，线性范围，精密度和准确性，回收率，生物基质效应和稳定性的测定验证。此方法的3-BTMD（COMT 3-BTD的甲基化产物）的定量限为0.0083 nM，比以前公开的方法低了近10倍。该方法精确，日内和日间相对标准偏差（RSD）低于5％。此外，该方法显示出优异的抗干扰能力，而内源性物质对3-BTMD的荧光检测没有影响。该方法的实际应用是通过成功用于测量单个人红细胞（n = 13）以及从四种不同人细胞系产生的细胞匀浆中的COMT活性而建立的。我们的结果表明，该方法在准确测定生物样品中COMT的天然活性方面将具有重要价值，这对于全面了解COMT在生理和病理条件下的作用是有益的。