HPLC-ECD与HPLC-MS联用测定人尿样中的罗红霉素及其代谢物

Determination of roxithromycin and its active metabolite in human urine by liquid chromatography electrochemical detection and liquid chromatography mass spectrometry

目的：建立灵敏专一的分析方法，测定人尿样中的罗红霉素（Ｅ－Ｒｏｘ）及其活性代谢产物Ｚ－Ｒｏｘ。方法：采用高效液相色谱－电化学检测法（ＨＰＬＣ－ＥＣＤ）与高效液相色谱－质谱法（ＨＰＬＣ－ＭＳ），样品经液－液萃取后，通过ＨＰＬＣ－ＥＣＤ定量分析Ｅ－Ｒｏｘ浓度；然后，采用ＨＰＬＣ－ＭＳ，通过选择性离子反应监测方式（ＳＲＭ），测定Ｚ－Ｒｏｘ与Ｅ－Ｒｏｘ的尿药浓度比值，ＳＲＭ扫描方式进行定量分析的碎片离子为ｍ／ｚ６７９（母离子ｍ／ｚ８３７）。结果：Ｅ－Ｒｏｘ和Ｚ－Ｒｏｘ的线性范围分别为０．１～１０ｍｇ·Ｌ－１和０．０５～５ｍｇ·Ｌ－１，最低检测限分别为０．１ｍｇ·Ｌ－１和０．０５ｍｇ·Ｌ－１，日内、日间ＲＳＤ均小于１２．５％。两法联用，分析了４名健康受试者单剂量口服１５０ｍｇＥ－Ｒｏｘ后的尿样。结论：为深入研究Ｅ－Ｒｏｘ在人体内的代谢规律提供了可靠的分析方法，首次测定了人尿样中的活性代谢产物Ｚ－Ｒｏｘ。

OBJECTIVE:To establish a sensitive method for the determination of roxithromycin (E Rox) and its active metabolite,(9Z) epimeric isomer (Z Rox).METHOD:E Rox and Z Rox were determined by using HPLC ECD in combination with HPLC MS.Quantitative analyse of E Rox was performed with HPLC ECD after liquid liquid extraction.Afterwards,the isomeric ratios of Z Rox and E Rox were determined by using HPLC MS in the selected reaction monitoring (SRM) mode via electrospray ionization (ESI).The SRM transition m/z 837 to m/z 679 was monitored for detection of Z Rox and E Rox isomers.RESULT:The calibration curves were over the concentration range of 0.1～10mg·L -1 for E Rox and 0.05～5mg·L -1 for Z Rox.The limits of quantification were found to be 0.1mg·L -1 for E Rox and 0.05mg·L -1 for Z Rox.The intra and inter day RSD were within 12.5% for each of analytes.The combined method was applied to the human urine samples from 4 healthy volunteers after oral administration of 150 mg E Rox.CONCLUSION:The combined method proved to be suitable for studies on the metabolism of E Rox in human.With the method,the concentrations of the active metabolite Z Rox in human urine were for the first time investigated.