HPLC-DAD DETERMINATION OF BERBERINE, CHELERYTHRINE AND SANGUINARINE IN THE MEXICAN PRICKLY POPPY (Argemone mexicana L. PAPAVERACEAE), A MEDICINAL PLANT

A sensitive, simple, rapid and reliable HPLC-DAD method for the analysis of the benzylisoquinoline alkaloids (BIA's) content in Argemone mexicana (Papaveraceae) is presented. This method allows the simultaneous separation and quantitation of berberine (Bn), chelerythrine (C) and sanguinarine (S) in extracts from A. mexicana tissues, reducing time of analysis in comparison to previous reports. Alkaloids were separated on a C18 Hypersil Gold column using an acetonitrile gradient (20 to 70%) in 1% acetic acid in water. Alkaloids were identified based on retention times and UV spectra and quantified at 254 nm. Linearity between 0.5-20 µg mL-1 was observed for Bn, C and S, with limits of detection (LOD) and quantitation (LOQ) of 0.11 and 0.33 for Bn, 0.10 and 0.30 for C and 0.05 and 0.15 for S, respectively. Maximal intra- and inter-day variation values were < 0.49% in all cases, with alkaloids' recoveries higher than 95%. System suitability tests (SST), including resolution (Rs), retention factor (K'), selectivity (α), tailing factor and number of theoretical plates were performed according to the United States Pharmacopeia (USP), fulfilling recommended values. The method proved to be efficient and reproducible when analyzing different tissues of field-collected A. mexicana plants.

本研究建立了一种灵敏、简便、快速且可靠的高效液相色谱-二极管阵列检测器（High Performance Liquid Chromatography-Diode Array Detector，HPLC-DAD）分析方法，用于测定蓟罂粟（Argemone mexicana，罂粟科Papaveraceae）组织中苄基异喹啉类生物碱（benzylisoquinoline alkaloids，BIA）的含量。该方法可同时分离并定量分析蓟罂粟组织提取物中的小檗碱（berberine，Bn）、白屈菜红碱（chelerythrine，C）与血根碱（sanguinarine，S），相较于既往报道的分析方法缩短了分析时长。生物碱在C18 Hypersil Gold色谱柱上，以含1%乙酸水溶液为水相的乙腈梯度洗脱液（梯度范围20%~70%）完成分离。通过保留时间与紫外光谱对生物碱进行定性鉴定，并在254 nm波长下开展定量检测。小檗碱、白屈菜红碱和血根碱在0.5~20 µg·mL⁻¹范围内线性关系良好，三者的检测限（limits of detection，LOD）与定量限（quantitation，LOQ）分别为：小檗碱0.11、0.33 µg·mL⁻¹，白屈菜红碱0.10、0.30 µg·mL⁻¹，血根碱0.05、0.15 µg·mL⁻¹。所有测试的日内与日间精密度最大值均低于0.49%，生物碱的加样回收率均高于95%。系统适用性试验（System suitability tests，SST）按照美国药典（United States Pharmacopeia，USP）要求开展，涵盖分离度（resolution，Rs）、容量因子（retention factor，K'）、选择性因子（selectivity，α）、拖尾因子及理论塔板数，各项参数均符合推荐标准。将该方法应用于野外采集的蓟罂粟不同组织样品分析时，展现出优异的高效性与重现性。