Department of Biopharmacy and Pharmacokinetics, University of Ljubljana, Faculty of Pharmacy, Aškerčeva cesta 7, 1000 Ljubljana, Slovenia.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Aug 1;879(23):2323-31. doi: 10.1016/j.jchromb.2011.06.031. Epub 2011 Jun 26.
A selective, sensitive, accurate and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of raloxifene and its three glucuronides: raloxifene-6-β-glucuronide (M1), raloxifene-4'-β-glucuronide (M2), raloxifene-6,4'-diglucuronide (M3) in urine samples is presented in this paper. To our knowledge the developed analytical method is the first fully validated method capable of simultaneous determination of raloxifene and its glucuronides in real urine samples. Moreover, for the first time a method for determination of raloxifene diglucuronide in relevant biological samples was introduced. Metabolites were obtained by a bioconversion process of raloxifene to its glucuronides using the microorganism Streptomyces sp. and were used as standards for validation. Urine samples were introduced to a simple solid phase extraction prior to the analysis by LC-MS/MS. The method was linear in a wide range with high determination coefficient (r(2) > 0.997). The limits of quantification achieved were 1.01, 1.95, 2.83 and 4.69 nM for raloxifene, M1, M2 and M3, respectively. The recoveries were higher than 92.5%, the accuracy was within 100 ± 8.8% and the precision was better than 12% for all compounds. The developed method was successfully applied to the real urine samples and showed to be appropriate for use in further research of still not completely discovered raloxifene pharmacokinetics. Furthermore, the presented method could also serve for a potential application in anti-doping analysis.
本文介绍了一种选择性、灵敏性、准确性和精密度高的液相色谱-串联质谱（LC-MS/MS）法，用于测定尿液样品中的雷洛昔芬及其三种葡萄糖醛酸苷：雷洛昔芬-6-β-葡萄糖醛酸苷（M1）、雷洛昔芬-4'-β-葡萄糖醛酸苷（M2）、雷洛昔芬-6,4'-二葡萄糖醛酸苷（M3）。据我们所知，所开发的分析方法是首个能够同时测定真实尿液样品中雷洛昔芬及其葡萄糖醛酸苷的完全验证方法。此外，首次介绍了一种用于测定相关生物样品中雷洛昔芬二葡萄糖醛酸苷的方法。使用微生物链霉菌进行雷洛昔芬向其葡萄糖醛酸苷的生物转化过程获得代谢物，并将其用作验证的标准。在进行 LC-MS/MS 分析之前，将尿液样品进行简单的固相萃取。该方法具有较宽的线性范围，高测定系数（r²＞0.997）。雷洛昔芬、M1、M2 和 M3 的定量限分别为 1.01、1.95、2.83 和 4.69 nM。所有化合物的回收率均高于 92.5%，准确度在 100±8.8%范围内，精密度均优于 12%。所开发的方法成功应用于真实尿液样品，并显示适用于进一步研究尚未完全发现的雷洛昔芬药代动力学。此外，所提出的方法也可用于潜在的反兴奋剂分析。
