Curticapean Augustin, Muntean Daniela, Curticapean Manuela, Dogaru Maria, Vari Camil
University of Medicine and Pharmacy Tg-Mures, Faculty of Pharmacy, Gh. Marinescu Str. No.38, Tg-Mures RO-540139, Romania.
J Biochem Biophys Methods. 2008 Apr 24;70(6):1304-12. doi: 10.1016/j.jprot.2008.01.012. Epub 2008 Feb 9.
The optimized method for HPLC determination of tramadol and its metabolite O-desmethyl tramadol in human plasma using sotalol as internal standard has been developed and validated by a new approach. The determination by fluorescence detection was performed on re-eluted solution, obtained after liquid-liquid extraction with ethyl acetate of the three analytes from plasma. The chromatographic separation of tramadol under a gradient elution was achieved at a temperature of 15 degrees C with a RP-18 column, guarded by a C18 precolumn. The mobile phase was a mixed aqueous solution containing ortho-phosphoric acid, triethylamine, acetonitrile and methanol in a complex gradient mode. The quantitative determination of tramadol was performed at different successive pairs of excitation/emission wavelengths (200/300 nm, 200/295 nm, 212/305 nm) with lower limits of quantification: LLOQ=4.078 ng/ml for tramadol, respectively LLOQ=3.271 ng/ml for O-desmethyl tramadol. For the LLOQ limits, were calculated the values of the coefficient of variation and difference between mean and the nominal concentration. For tramadol analyte they were CV%=5.147% and bias%=-7.273% in the intra-days and CV%=4.894% and bias%=0.836% in the between-days assay, respectively for the metabolite O-desmethyl tramadol they were CV%=11.517% and bias%=0.337% in the intra-days and CV%=6.41% and bias%=3.259% in the between-days assay. In addition, the stabilities of the analytes were verified in different conditions. Both, tramadol and its metabolite proved to be stable in plasma for four weeks, frozen at -20 degrees C, but also for 48 h at 15 degrees C in the re-eluted solution after liquid-liquid extraction.
已开发并通过一种新方法验证了以索他洛尔为内标物，采用高效液相色谱法（HPLC）测定人血浆中曲马多及其代谢物O - 去甲基曲马多的优化方法。通过荧光检测进行测定，检测对象为经乙酸乙酯液 - 液萃取血浆中三种分析物后获得的再洗脱溶液。在15℃的温度下，使用C18预柱保护的RP - 18柱，通过梯度洗脱实现曲马多的色谱分离。流动相是一种复杂梯度模式的混合水溶液，含有正磷酸、三乙胺、乙腈和甲醇。在不同的连续激发/发射波长对（200/300 nm、200/295 nm、212/305 nm）下进行曲马多的定量测定，定量下限为：曲马多的LLOQ = 4.078 ng/ml，O - 去甲基曲马多的LLOQ = 3.271 ng/ml。对于LLOQ限值，计算了变异系数以及均值与标称浓度之间的差值。对于曲马多分析物，日内CV% = 5.147%，偏差% = -7.273%；日间CV% = 4.894%，偏差% = 0.836%。对于代谢物O - 去甲基曲马多，日内CV% = 11.517%，偏差% = 0.337%；日间CV% = 6.41%，偏差% = 3.259%。此外，还在不同条件下验证了分析物的稳定性。曲马多及其代谢物在血浆中于-20℃冷冻保存四周时稳定，在液 - 液萃取后的再洗脱溶液中于15℃也可稳定保存48小时。
