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本研究建立和验证了测定兔血浆中阿霉素含量的液相色谱串联质谱法(LC-MS/MS)。采用乙酸乙酯萃取血浆样品中的阿霉素和柔红霉素(内标)。以配有C18预柱(2.1 mm×10 mm,2.5μm)的C18色谱柱(2.1 mm×50 mm,2.5μm)进行色谱分离,采用0.1%甲酸水溶液–乙腈二元梯度洗脱:在0–1 min,1–8 min和8–15 min两者比例分别为82:18–82:18(v/v),82:18–50:50(v/v)和50:50–82:18(v/v),流速为0.4 mL/min;色谱柱柱温设定为40 oC,样品进样量为10μL。液相流出液使用电喷雾离子源(ESI,正离子模式),质谱多反应监测模式检测,检测离子:阿霉素m/z 544.07→396.96,内标:m/z 528.06→321.05。阿霉素在2–600 ng/mL范围内,线性关系良好,最低定量限为2 ng/mL。该方法的专属性、基质效应、提取回收率及样品稳定性均符合要求。日内准确度和日间准确度的相对误差范围分别为–2.48%~0.18%和–3.78%~1.94%。日内精密度和日间精密度的相对标准偏差值分别小于8.65%和6.41%。结果显示,该方法为检测家兔血浆中阿霉素的含量提供了一种可靠的手段。
This study established and validated a liquid chromatography tandem mass spectrometry (LC-MS / MS) method for the determination of doxorubicin in rabbit plasma. Adriamycin and daunorubicin (internal standard) were extracted from plasma samples with ethyl acetate. Chromatography was performed on a C18 column (2.1 mm × 50 mm, 2.5 μm) equipped with a C18 precolumn (2.1 mm × 10 mm, 2.5 μm) eluting with a 0.1% formic acid aqueous-acetonitrile binary gradient: The ratios of 1: 1, 1-8 and 8-15 min were 82:18 to 82:18 (v / v), 82:18 to 50:50 (v / v) and 50:50 to 82:18 (v / v) at a flow rate of 0.4 mL / min. The column temperature was set at 40 oC and the sample volume was 10 μL. The liquid effluent was detected using an electrospray ionization source (ESI, positive ion mode) and mass spectrometry multiple reaction monitoring mode, detecting ions: doxorubicin m / z 544.07 → 396.96, internal standard: m / z 528.06 → 321.05. Doxorubicin has a good linearity in the range of 2-600 ng / mL with a minimum limit of quantitation of 2 ng / mL. The specificity, matrix effect, extraction recovery, and sample stability of the method met the requirements. The relative error range between day accuracy and day accuracy was -2.48% -0.18% and -3.78% -1.94%, respectively. The relative standard deviations of intra-day precision and intra-day precision were less than 8.65% and 6.41%, respectively. The results show that this method provides a reliable means for the detection of doxorubicin in rabbit plasma.