超高效液相色谱-串联质谱法研究亚硫酸氢钠穿心莲内酯在不同种属血浆中的稳定性

目的: 建立超高效液相色谱-串联质谱的方法,测定血浆中亚硫酸氢钠穿心莲内酯(ASB)的含量,并用于研究ASB在不同种属血浆中的稳定性。 方法: ASB血浆样品用甲醇进行提取,以脱水穿心莲内酯(DAG)为内标进行定量。采用Hypersil Gold C₁₈(50 mm×2.1 mm,1.9 μm)色谱柱,柱温35 ℃;流动相为水-甲醇梯度洗脱,流速0.2 mL·min^-1;分析时间6 min。采用负离子电喷雾离子化电离源和选择反应监测(SRM)模式,进行串联质谱分析;用于定量分析的离子反应分别为m/z 413.2→287.2(ASB)和m/z 331.2→303.3(DAG)。浓度为1 μg·mL^-1 的ASB大鼠、犬、人血浆液37 ℃孵育,于不同时间点测定ASB含量,计算血浆中ASB剩余百分含量。 结果: ASB标准血浆样品的线性范围在10~1000 ng·mL^-1,定量下限为10 ng·mL^-1。方法的日内和日间准确度分别在96.4%~103.2% 和93.7%~98.0%,精密度(RSD)分别小于6.9%和10.8%,基质效应为88.9%~99.9%,提取回收率为71.2%~98.9%。大鼠、犬和人血浆中ASB剩余百分含量均明显下降,在犬血浆中5 min血药浓度降至70%的较高水平,在大鼠和人血浆中5 min降至30%左右,之后趋于平稳。 结论: 本方法适用于血浆中ASB的含量测定,ASB在不同种属血浆中下降均很快,但表现出明显的种属差异。

Objectives: To establish a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the determination of andrographolide sodium bisulphite (ASB) in plasma,and adopt the method to evaluate stability of ASB in plasma of different species. Methods: The analyte from plasma samples extracted by methanol and dehydroandrographolide (DAG) was used as an internal standard for quantitation.The chromatographic separation was achieved within 6 min on a Hypersil Gold C₁₈ column with gradient mobile phase containing a mixture of water and methanol at a flow rate of 0.2 mL·min^-1.Column temperature was 35 ℃.At negative electrospray ionization mode,selected reaction monitoring of the precursor-product ion transitions of m/z 413.2→287.2 for ASB and m/z 331.2→303.3 for DAG was used for quantification.The remaining amount of ASB incubated in rat,dog and human plasma was determined at different time points. Results: The calibration curves were linear in the range of 10-1000 ng·mL^-1 with a lower detection limit of 10 ng·mL^-1.Intra- and inter-day accuracy fell in the ranges of 96.4%-103.2% and 93.7%-98.0%,respectively.Intra- and inter-day precision was less than 6.9% and 10.8%,respectively.The matrix effects ranged from 88.9% to 99.9% and the recovery was between 71.2% and 98.9%.The remaining percentage content of ASB in three species plasma was obviously declined.The ASB remaining content incubated 5 minutes in dog plasma was down to 70%,and in rats and human plasma were down to 30%. Conclusion: The method was suitable for quantification of ASB in plasma.It was showed that ASB remaining content decreased rapidly in plasma of rat,dog and human. But there was difference in species.

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