HPLC-ECD色谱法测定青蒿素的研究与应用
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摘要
目的建立高效液相库仑阵列电化学色谱技术(HPLC-ECD)测定青蒿素的方法。用建立的含量测定方法测定青蒿素的溶解度、油水分配系数及较低浓度的生物样品,对测得数据整理分析,初步推测青蒿素生物利用度低的影响因素,为今后提高青蒿素生物利用度的制剂研究奠定基础。
方法以均匀实验设计法优化青蒿素的柱前衍生条件,内容包括：衍生剂氢氧化钠的用量、醋酸的用量、衍生反应时的水浴温度及水浴时间；通过改变流动相比例、pH值、检测电极电压值及柱温等,选择适宜的含量测定色谱条件；对青蒿素的溶解度、油水分配系数及低浓度的血浆生物样品等进行含量测定分析,由测得数据对青蒿素的一些生物药剂学参数进行初步推测,分析整理了有助于指导提高青蒿素生物利用度制剂研究的有用信息。
结果根据均匀实验设计结果分析得出,青蒿素柱前衍生的最佳条件为：1mL青蒿素无水乙醇液与4 mL 0.01 mol·L-1氢氧化钠在70℃水浴锅中恒温反应50 min,取出在自来水中使之快速冷却至室温,再加5 mL 0.06 mol·L-1醋酸,其中醋酸含量对测定结果的影响不显著,可根据具体实验进行调整。测定生物样品时,采用等比例减半的处理方法,由之前的衍生剂加入比例1：4：5变为0.5：2：2.5,醋酸浓度调整为0.02 mol·L-1。适用于HPLC-ECD测定一般青蒿素样品方法的线性范围为50μg·mL-1～0.1μg·mL-1；适用于HPLC-ECD测定青蒿素生物样品方法的线性范围为16.32 ng·mL-1～2.04μg·mL-1。溶解度测定结果显示,青蒿素在25℃水中的溶解度仅有0.0671mg·mL-1；用三氯甲烷作为油相时,青蒿素的油水分配系数均值为732.03,1gP=2.86；用正丁醇作为油相时,青蒿素的油水分配系数均值为184.09,1gP=2.27；小鼠血浆生物样品的含量很低,30 min血药浓度仅150 ng·mL-1左右；根据平衡研究法原理粗略测得青蒿素小鼠体内吸收率约92%。
结论通过对青蒿素的含量测定方法学考察及样品的测定表明HPLC-ECD色谱法适用于青蒿素低浓度生物样品的定量测定。对测得数据分析整理得出青蒿素具有一定脂溶性,初步推测青蒿素可能属于按生物药剂学分类系统分类中的Ⅱ型药物,且青蒿素可能同时受溶出与肝脏的首过作用影响而导致其生物利用度低。该分析结果对提高青蒿素生物利用度的制剂研究有重要的指导意义。
Objective:To establish HPLC-ECD chromatograpH determined Artemisinin method. Using this method measure the solubility of Artemisinin in different solvents, its oil-water distribution coefficients and low concentration biological samples. Through this data preliminary speculate the influence factors of Artemisinin bioavailability.This study may offer helpful reference for study of the preparations of Artemisinin.
Method:Using the uniform experimental design method to determine a good derivative method. The examine factors include:the NaOH amount, the acetic dosage, the water bath temperature and time. To choose a suitable measuring conditions through investigate the ratio of mobile pHase, the value of pH, the detection voltage and the Column temperature. Using this method to determine the solubility of Artemisinin in different solvents, its oil-water distribution coefficients, the low concentration of plasma biological samples and so on. From all above these data analyze which can preliminarily infers the biopHarmaceutical parameter of Artemisinin. This useful information will direct the preparations which can improve the bioavailability of Artemisinin.
Results:Through to analyze the results of experiments, the best group of derivative method is as follows:1mL Artesunate ethanol liquid added 4mL 0.01mol·L-1 NaOH, at the 70℃thermostatic water bath pot reaction 50min, then remove the sample and rapid cooling to room temperature, at last added 5mL 0.06mol·L-1 acetic. The acetic is not significant for the results, so according to the test conditions the acetic dosage can adjustment. Determine biological samples, the amount of derivative agent is change from 1:4:5 to 0.5:2:2.5, the acetic acid concentration is adjusted for 0.02 mol·L-1. The linear range of ordinary Artemisinin samples by HPLC-ECD is 50μg·mL-1～0.1μg·mL-1. The linear range of Artemisinin biological samples by HPLC-ECD is 16.32 ng·mL-1～2.04μg·mL-1. The result shows that the solubility of Artemisinin in water at 25℃is only 0.0671mg·mL-1. To use the chloroform as oil pHase, Artemisinin oil-water distribution coefficient is 732.03, 1gP=2.86; When using n-butyl alcohol as the oil pHase, the oil-water distribution of Artemisinin is 184.09, 1gP=2.27. In plasma biological samples from mice, the Artemisinin content is about 150ng·mL-1 at 30min, according to principle of balance research, roughly determined the body absorbs of Artemisinin is 92%.
Conclusion:Based on the content of Artemisinin samples and methodology investigation the HPLC-ECD chromatograpHic method suitable for low concentration of biological samples quantitatively determined. According to Analyze on the measurement data, it is preliminarily presumed that Artemisinin may belong to biopHarmaceutical classification systemⅡtype drug. The low bioavailability may caused by dissloution and first pass effect. The conclusion has important directive meaning to research the preparation of improve Artemisinin bioavailability.
方法以均匀实验设计法优化青蒿素的柱前衍生条件,内容包括：衍生剂氢氧化钠的用量、醋酸的用量、衍生反应时的水浴温度及水浴时间；通过改变流动相比例、pH值、检测电极电压值及柱温等,选择适宜的含量测定色谱条件；对青蒿素的溶解度、油水分配系数及低浓度的血浆生物样品等进行含量测定分析,由测得数据对青蒿素的一些生物药剂学参数进行初步推测,分析整理了有助于指导提高青蒿素生物利用度制剂研究的有用信息。
结果根据均匀实验设计结果分析得出,青蒿素柱前衍生的最佳条件为：1mL青蒿素无水乙醇液与4 mL 0.01 mol·L-1氢氧化钠在70℃水浴锅中恒温反应50 min,取出在自来水中使之快速冷却至室温,再加5 mL 0.06 mol·L-1醋酸,其中醋酸含量对测定结果的影响不显著,可根据具体实验进行调整。测定生物样品时,采用等比例减半的处理方法,由之前的衍生剂加入比例1：4：5变为0.5：2：2.5,醋酸浓度调整为0.02 mol·L-1。适用于HPLC-ECD测定一般青蒿素样品方法的线性范围为50μg·mL-1～0.1μg·mL-1；适用于HPLC-ECD测定青蒿素生物样品方法的线性范围为16.32 ng·mL-1～2.04μg·mL-1。溶解度测定结果显示,青蒿素在25℃水中的溶解度仅有0.0671mg·mL-1；用三氯甲烷作为油相时,青蒿素的油水分配系数均值为732.03,1gP=2.86；用正丁醇作为油相时,青蒿素的油水分配系数均值为184.09,1gP=2.27；小鼠血浆生物样品的含量很低,30 min血药浓度仅150 ng·mL-1左右；根据平衡研究法原理粗略测得青蒿素小鼠体内吸收率约92%。
结论通过对青蒿素的含量测定方法学考察及样品的测定表明HPLC-ECD色谱法适用于青蒿素低浓度生物样品的定量测定。对测得数据分析整理得出青蒿素具有一定脂溶性,初步推测青蒿素可能属于按生物药剂学分类系统分类中的Ⅱ型药物,且青蒿素可能同时受溶出与肝脏的首过作用影响而导致其生物利用度低。该分析结果对提高青蒿素生物利用度的制剂研究有重要的指导意义。
Objective:To establish HPLC-ECD chromatograpH determined Artemisinin method. Using this method measure the solubility of Artemisinin in different solvents, its oil-water distribution coefficients and low concentration biological samples. Through this data preliminary speculate the influence factors of Artemisinin bioavailability.This study may offer helpful reference for study of the preparations of Artemisinin.
Method:Using the uniform experimental design method to determine a good derivative method. The examine factors include:the NaOH amount, the acetic dosage, the water bath temperature and time. To choose a suitable measuring conditions through investigate the ratio of mobile pHase, the value of pH, the detection voltage and the Column temperature. Using this method to determine the solubility of Artemisinin in different solvents, its oil-water distribution coefficients, the low concentration of plasma biological samples and so on. From all above these data analyze which can preliminarily infers the biopHarmaceutical parameter of Artemisinin. This useful information will direct the preparations which can improve the bioavailability of Artemisinin.
Results:Through to analyze the results of experiments, the best group of derivative method is as follows:1mL Artesunate ethanol liquid added 4mL 0.01mol·L-1 NaOH, at the 70℃thermostatic water bath pot reaction 50min, then remove the sample and rapid cooling to room temperature, at last added 5mL 0.06mol·L-1 acetic. The acetic is not significant for the results, so according to the test conditions the acetic dosage can adjustment. Determine biological samples, the amount of derivative agent is change from 1:4:5 to 0.5:2:2.5, the acetic acid concentration is adjusted for 0.02 mol·L-1. The linear range of ordinary Artemisinin samples by HPLC-ECD is 50μg·mL-1～0.1μg·mL-1. The linear range of Artemisinin biological samples by HPLC-ECD is 16.32 ng·mL-1～2.04μg·mL-1. The result shows that the solubility of Artemisinin in water at 25℃is only 0.0671mg·mL-1. To use the chloroform as oil pHase, Artemisinin oil-water distribution coefficient is 732.03, 1gP=2.86; When using n-butyl alcohol as the oil pHase, the oil-water distribution of Artemisinin is 184.09, 1gP=2.27. In plasma biological samples from mice, the Artemisinin content is about 150ng·mL-1 at 30min, according to principle of balance research, roughly determined the body absorbs of Artemisinin is 92%.
Conclusion:Based on the content of Artemisinin samples and methodology investigation the HPLC-ECD chromatograpHic method suitable for low concentration of biological samples quantitatively determined. According to Analyze on the measurement data, it is preliminarily presumed that Artemisinin may belong to biopHarmaceutical classification systemⅡtype drug. The low bioavailability may caused by dissloution and first pass effect. The conclusion has important directive meaning to research the preparation of improve Artemisinin bioavailability.
引文
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