Govender Katya, Gibhard Liezl, Du Plessis Lissinda, Wiesner Lubbe
Division of Clinical Pharmacology, Department of Medicine, University of Cape Town, Medical School, Cape Town 7925, South Africa.
Division of Clinical Pharmacology, Department of Medicine, University of Cape Town, Medical School, Cape Town 7925, South Africa.
J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Mar 15;985:6-13. doi: 10.1016/j.jchromb.2015.01.015. Epub 2015 Jan 21.
A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantitation of the antimalarial drug, lumefantrine (LF), in mouse whole blood and plasma. The analyte was extracted using a protein precipitation method followed by chromatographic separation on a Phenomenex Luna, PFP (50mm×2.0mm, 5μm) analytical column with a mobile phase consisting of acetonitrile and 0.1% formic acid (formic acid:water, 1:1000, v/v) at a ratio of 3:7 (v/v), delivered at a constant flow rate of 0.5ml/min. Stable isotope labeled lumefantrine (D9-LF) was used as the internal standard. Multiple reaction monitoring was performed using the transitions m/z 530.1→m/z 347.9 and m/z 539.1→m/z 347.9 for the quantification of LF and D9-LF, respectively. Calibration curves were constructed over the concentration range 15.6-4000ng/ml. The mean intra- and inter-assay accuracy values for the analysis of LF in WB was 103% (%CV=5.5) and 99.5% (%CV=5.5), respectively. The mean intra- and inter-assay accuracy values for the analysis of LF in plasma was 93.7% (%CV=3.5) and 93.9% (%CV=5.5), respectively. No significant matrix effect was observed during the method validation. The validated method was applied to an absorption study in mice, to determine and compare LF concentrations in whole blood and plasma samples. Results of the statistical analysis using a linear mixed effects growth curve model concluded that there was no significant difference (p-value=0.668) between WB and plasma LF concentrations. This method utilizes a small sample volume of 20μl, facilitating low blood collection volumes and a short chromatographic run time of 3min which allows for high sample throughput analysis.
建立了一种液相色谱 - 串联质谱（LC-MS/MS）方法，并对其进行了验证，用于定量测定小鼠全血和血浆中的抗疟药物卤泛群（LF）。采用蛋白质沉淀法提取分析物，然后在Phenomenex Luna，PFP（50mm×2.0mm，5μm）分析柱上进行色谱分离，流动相由乙腈和0.1%甲酸（甲酸：水，1:1000，v/v）按3:7（v/v）的比例组成，以0.5ml/min的恒定流速输送。使用稳定同位素标记的卤泛群（D9-LF）作为内标。分别使用m/z 530.1→m/z 347.9和m/z 539.1→m/z 347.9的跃迁进行多反应监测，以定量LF和D9-LF。在15.6 - 4000ng/ml的浓度范围内构建校准曲线。WB中LF分析的平均批内和批间准确度值分别为103%（%CV = 5.5）和99.5%（%CV = 5.5）。血浆中LF分析的平均批内和批间准确度值分别为93.7%（%CV = 3.5）和93.9%（%CV = 5.5）。在方法验证过程中未观察到明显的基质效应。将验证后的方法应用于小鼠吸收研究，以测定和比较全血和血浆样品中的LF浓度。使用线性混合效应生长曲线模型进行统计分析的结果表明，WB和血浆LF浓度之间没有显著差异（p值 = 0.668）。该方法使用20μl的小样本体积，便于采集少量血液，且色谱运行时间短，仅需3分钟，可实现高通量样品分析。
