Paar W D, Frankus P, Dengler H J
Department of General Internal Medicine, University of Bonn, Germany.
J Chromatogr B Biomed Appl. 1996 Nov 15;686(2):221-7. doi: 10.1016/s0378-4347(96)00236-8.
A high-performance liquid chromatographic assay for the quantitative determination of the opioid analgesic tramadol and its metabolites is described. A homologue of tramadol [1-(m-hydroxyphenyl)-2-(N-ethyl-N-methylaminomethyl)cycloheptane-1 -ol hydrochloride] is used as internal standard. The assay allows the determination of tramadol O- and N-demethylation activity in vitro in microsomal fractions of human liver. Tramadol and its in vitro generated Phase I metabolites are extracted by a one-step extraction procedure from microsomal incubation mixtures using methylene chloride. Extraction efficiencies of tramadol, O-demethyltramadol and mono-N-demethyltramadol were 70, 91 and 94% respectively. The isocratic high-performance liquid chromatographic system employs a C18 reversed-phase column. The mobile phase is a mixture of methanol, ammonium hydrogencarbonate solution and ammonium hydroxide solution. Sensitivity of the assay was 0.5, 0.2 and 0.2 microgram/ml for tramadol, O-demethyltramadol and mono-N-demethyltramadol, respectively. Within-run precision of the overall assay was 13, 3.1 and 7.6% for tramadol, O-demethyltramadol and mono-N-demethyltramadol, respectively. Accuracy of the assay was determined as mean differences of concentrations added and found in microsomal fractions. It was -2.4% for tramadol, -0.85% for O-demethyltramadol and 0.32% for mono-N-demethyltramadol.
本文描述了一种用于定量测定阿片类镇痛药曲马多及其代谢物的高效液相色谱分析法。使用曲马多的一种同系物[1-(间羟基苯基)-2-(N-乙基-N-甲基氨基甲基)环庚烷-1-醇盐酸盐]作为内标。该分析方法可用于测定人肝微粒体部分体外的曲马多O-和N-去甲基化活性。曲马多及其体外生成的I相代谢物通过一步萃取程序，使用二氯甲烷从微粒体孵育混合物中萃取。曲马多、O-去甲基曲马多和单-N-去甲基曲马多的萃取效率分别为70%、91%和94%。等度高效液相色谱系统采用C18反相柱。流动相是甲醇、碳酸氢铵溶液和氢氧化铵溶液的混合物。该分析方法对曲马多、O-去甲基曲马多和单-N-去甲基曲马多的灵敏度分别为0.5、0.2和0.2微克/毫升。整个分析方法的批内精密度对于曲马多、O-去甲基曲马多和单-N-去甲基曲马多分别为13%、3.1%和7.6%。分析方法的准确度通过添加浓度与微粒体部分中测得浓度的平均差值来确定。曲马多为-2.4%，O-去甲基曲马多为-0.85%，单-N-去甲基曲马多为0.32%。
