Domínguez-Romero Juan C, García-Reyes Juan F, Beneito-Cambra Miriam, Martínez-Romero Rubén, Martinez-Lara Esther, Del Moral-Leal María L, Molina-Díaz Antonio
Analytical Chemistry Research Group, Department of Physical and Analytical Chemistry, University of Jaén, 23071, Jaén, Spain.
Cellular Stress and Age Research Group. Department of Experimental Biology, University of Jaén, 23071, Jaén, Spain.
Biomed Chromatogr. 2015 Aug;29(8):1220-8. doi: 10.1002/bmc.3411. Epub 2015 Jan 22.
Tamoxifen (TMX) is a nonsteroidal estrogen antagonist drug used for the treatment of breast cancer. It is also included in the list of banned substances of the World Anti Doping Agency (WADA) prohibited in and out of competition. In this work, the excretion of urinary metabolites of TMX after a single therapeutic dose administration in rats has been studied using ultra-high-performance liquid chromatography electrospray time-of-flight mass spectrometry (UHPLC-TOFMS). A systematic strategy based on the search of typical biotransformations that a xenobiotic can undergo in living organisms, based on their corresponding molecular formula modification and accurate mass shifts, was applied for the identification of TMX metabolites. Prior to UHPLC-TOFMS analyses, a solid-phase extraction step with polymeric cartridges was applied to urine samples. Up to 38 TMX metabolites were detected. Additional collision induced dissociation (CID) MS/MS fragmentation was performed using UHPLC-QTOFMS. Compared with recent previous studies in human urine and plasma, new metabolites have been reported for the first time in urine. Metabolites identified in rat urine include the oxygen addition, owing to different possibilities for the hydroxylation of the rings in different positions (m/z 388.2271), the incorporation of two oxygen atoms (m/z 404.2220) (including dihydroxylated derivatives or alternatives such as epoxidation plus hydroxylation or N-oxidation and hydroxylation), epoxide formation or hydroxylation and dehydrogenation [m/z 386.2114 (+O -H2 )], hydroxylation of the ring accompanied by N-desmethylation (m/z 374.2115), combined hydroxylation and methoxylation (m/z 418.2377), desaturated TMX derivate (m/z 370.2165) and its N-desmethylated derivate (m/z 356.2009), the two latter modifications not previously being reported in urine. These findings confirm the usefulness of the proposed approach based on UHPLC-TOFMS.
他莫昔芬（TMX）是一种用于治疗乳腺癌的非甾体类雌激素拮抗剂药物。它也被列入世界反兴奋剂机构（WADA）禁止在比赛期间和非比赛期间使用的违禁物质清单。在这项工作中，使用超高效液相色谱电喷雾飞行时间质谱（UHPLC - TOFMS）研究了大鼠单次给予治疗剂量后他莫昔芬尿液代谢物的排泄情况。基于对异生物在活生物体中可能经历的典型生物转化的搜索，根据其相应的分子式修饰和精确质量变化，应用了一种系统策略来鉴定他莫昔芬代谢物。在进行UHPLC - TOFMS分析之前，对尿液样本采用聚合物柱进行固相萃取步骤。共检测到多达38种他莫昔芬代谢物。使用UHPLC - QTOFMS进行了额外的碰撞诱导解离（CID）MS/MS碎片化分析。与之前关于人尿液和血浆的最新研究相比，首次在尿液中报道了新的代谢物。在大鼠尿液中鉴定出的代谢物包括：由于不同位置的环羟基化有不同可能性而导致的加氧（m/z 388.2271）、两个氧原子的掺入（m/z 404.2220）（包括二羟基化衍生物或诸如环氧化加羟基化或N - 氧化和羟基化等替代情况）、环氧化或羟基化和脱氢反应[m/z 386.2114（+O - H2）]、环羟基化伴随N - 去甲基化（m/z 374.2115）、羟基化和甲氧基化联合反应（m/z 418.2377）、去饱和他莫昔芬衍生物（m/z 370.2165）及其N - 去甲基化衍生物（m/z 356.2009），后两种修饰此前未在尿液中报道过。这些发现证实了基于UHPLC - TOFMS所提出方法的有效性。
