College of Pharmacy, Kangwon National University, Chuncheon 200-701, Republic of Korea.
J Pharm Biomed Anal. 2013 Jul-Aug;81-82:151-9. doi: 10.1016/j.jpba.2013.04.018. Epub 2013 Apr 22.
A rapid, simple and sensitive chiral HPLC method was developed and validated for quantification of biologically important alkaloids namely evodiamine enantiomers and rutaecarpine in Evodiae fructus using diphenhydramine as the internal standard (IS). Chromatographic separations were performed on a Chiralpak AD-H column (250 mm × 4.6 mm i.d., 5 μm) with elution of n-hexane-2-propanol-ethanol (70:20:10, v/v/v) in a flow rate of 0.7 ml/min and at λmax 225 nm. To identify the order of elution, small quantities of the each evodiamine enantiomer were isolated by semi preparative HPLC method. Extraction samples were prepared by a simple solid phase extraction (SPE) method. All calibration curves showed good linearity (r(2)≥0.999) within the test ranges. The LOD and LOQ were lower than 0.05 and 0.1 μg/ml, respectively. The RSDs of intra- and interday for relative peak areas of three analytes to IS were less than 3.2 and 2.5%, respectively, and the recoveries were 98.0-103.7%. The validated method was successfully applied to the quantitative analysis of three constituents in 13 batches of samples collected from market. The results showed that S-(+)-evodiamine was the main component while R-(-)-evodiamine was present in low concentration. This study provides a qualitative and quantitative method for analysis of evodiamine enantiomers and rutaecarpine, and should be extendable to pharmacological and toxicological studies of the individual evodiamine enantiomers.
建立并验证了一种快速、简单、灵敏的手性 HPLC 方法，用于定量测定吴茱萸生物中重要的生物碱，即吴茱萸碱对映异构体和吴茱萸次碱，以苯海拉明为内标（IS）。在 Chiralpak AD-H 柱（250mm×4.6mmID，5μm）上进行色谱分离，以 n-己烷-2-丙醇-乙醇（70:20:10，v/v/v）为流动相，流速为 0.7ml/min，在 λmax 225nm 处进行洗脱。为了确定洗脱顺序，通过半制备 HPLC 方法分离出少量的每个吴茱萸碱对映异构体。提取样品通过简单的固相萃取（SPE）方法制备。所有校准曲线在测试范围内均表现出良好的线性（r²≥0.999）。LOD 和 LOQ 均低于 0.05 和 0.1μg/ml。三个分析物相对于 IS 的相对峰面积的日内和日间 RSD 均小于 3.2%和 2.5%，回收率为 98.0-103.7%。验证的方法成功地应用于从市场收集的 13 批样品中三种成分的定量分析。结果表明，S-（+）-吴茱萸碱是主要成分，而 R-（-）-吴茱萸碱的浓度较低。本研究为吴茱萸碱对映异构体和吴茱萸次碱的分析提供了一种定性和定量的方法，并且应该可以扩展到单个吴茱萸碱对映异构体的药理学和毒理学研究。
