Division of Clinical Pharmacology and Toxicology, Department of Medecine, Lausanne, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Switzerland.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Dec 15;878(32):3402-14. doi: 10.1016/j.jchromb.2010.10.027. Epub 2010 Oct 30.
There is increasing evidence that the clinical efficacy of tamoxifen, the first and most widely used targeted therapy for estrogen-sensitive breast cancer, depends on the formation of the active metabolites 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen (endoxifen). Large inter-individual variability in endoxifen plasma concentrations has been observed and related both to genetic and environmental (i.e. drug-induced) factors altering CYP450s metabolizing enzymes activity. In this context, we have developed an ultra performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) requiring 100 μL of plasma for the quantification of tamoxifen and three of its major metabolites in breast cancer patients. Plasma is purified by a combination of protein precipitation, evaporation at room temperature under nitrogen, and reconstitution in methanol/20 mM ammonium formate 1:1 (v/v), adjusted to pH 2.9 with formic acid. Reverse-phase chromatographic separation of tamoxifen, N-desmethyl-tamoxifen, 4-hydroxy-tamoxifen and 4-hydroxy-N-desmethyl-tamoxifen is performed within 13 min using elution with a gradient of 10 mM ammonium formate and acetonitrile, both containing 0.1% formic acid. Analytes quantification, using matrix-matched calibration samples spiked with their respective deuterated internal standards, is performed by electrospray ionization-triple quadrupole mass spectrometry using selected reaction monitoring detection in the positive mode. The method was validated according to FDA recommendations, including assessment of relative matrix effects variability, as well as tamoxifen and metabolites short-term stability in plasma and whole blood. The method is precise (inter-day CV%: 2.5-7.8%), accurate (-1.4 to +5.8%) and sensitive (lower limits of quantification comprised between 0.4 and 2.0 ng/mL). Application of this method to patients' samples has made possible the identification of two further metabolites, 4'-hydroxy-tamoxifen and 4'-hydroxy-N-desmethyl-tamoxifen, described for the first time in breast cancer patients. This UPLC-MS/MS assay is currently applied for monitoring plasma levels of tamoxifen and its metabolites in breast cancer patients within the frame of a clinical trial aiming to assess the impact of dose increase on tamoxifen and endoxifen exposure.
越来越多的证据表明，他莫昔芬（一种用于治疗雌激素敏感型乳腺癌的靶向治疗药物）的临床疗效取决于其活性代谢产物 4-羟基他莫昔芬和 4-羟基-N-去甲基他莫昔芬（依维莫司）的形成。已观察到依维莫司血浆浓度存在较大的个体间变异性，与改变 CYP450s 代谢酶活性的遗传和环境（即药物诱导）因素有关。在此背景下，我们开发了一种超高效液相色谱-串联质谱法（UPLC-MS/MS），该方法仅需 100 μL 血浆即可对乳腺癌患者中他莫昔芬及其三种主要代谢物进行定量分析。通过蛋白沉淀、室温氮气蒸发以及在甲醇/20mM 甲酸铵 1:1（v/v）中复溶相结合的方法对血浆进行净化，并用甲酸调节 pH 值至 2.9。采用洗脱液为 10mM 甲酸铵和乙腈（均含 0.1%甲酸）的梯度洗脱方式，在 13min 内即可实现他莫昔芬、N-去甲基他莫昔芬、4-羟基他莫昔芬和 4-羟基-N-去甲基他莫昔芬的反相色谱分离。采用基质匹配校准样品和各自氘代内标物进行分析物定量，通过电喷雾电离三重四极杆质谱法，在正模式下采用选择反应监测检测进行。该方法按照 FDA 建议进行了验证，包括评估相对基质效应变异性，以及他莫昔芬和代谢物在血浆和全血中的短期稳定性。该方法具有良好的精密度（日间 CV%：2.5-7.8%）、准确性（-1.4 至+5.8%）和灵敏度（定量下限为 0.4-2.0ng/mL）。该方法已应用于患者样本，首次在乳腺癌患者中鉴定出两种新的代谢产物 4'-羟基他莫昔芬和 4'-羟基-N-去甲基他莫昔芬。目前，该 UPLC-MS/MS 测定法正在一项临床试验中用于监测乳腺癌患者的他莫昔芬及其代谢物的血浆水平，该试验旨在评估增加剂量对他莫昔芬和依维莫司暴露的影响。
