Journal of Pharmaceutical and Biomedical Analysis 81–82 (2013) 151–159 Contents lists available at SciVerse ScienceDirect Journal of Pharmaceutical and Biomedical Analysis jou rn al hom e page: www.elsevier.com/locate/jpba Chiral high-performance liquid chromatographic separation of evodiamine enantiomers and rutaecarpine, isolated from Evodiae fructus Ngoc Van Thi Nguyen a , Kyung Ran Lee a , Yong Jae Lee a , Seungho Choi a , Jong Seong Kang b , Woongchon Mar c , Kyeong Ho Kim a,∗ a College of Pharmacy, Kangwon National University, Chuncheon 200-701, Republic of Korea b College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea c College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea a r t i c l e i n f o Article history: Received 3 December 2012 Received in revised form 31 March 2013 Accepted 6 April 2013 Available online xxx Keywords: Evodiamine Rutaecarpine Evodiae fructus Enantiomer separation Liquid chromatography a b s t r a c t 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 m) 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  max 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 g/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. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Evodiae fructus (Wuzhuyu in China) is the dried, unripe fruit of Evodia rutaecarpa (Juss.) Benth. or E. rutaecarpa (Juss.) Benth. var. officinalis (Dode) Huang, and E. rutaecarpa (Juss.) Benth. var. bodinieri (Dode) Huang belonging to the family Rutaceae. It has been used as one of the traditional Chinese medicines (TCMs) for more than 2000 years and is officially listed in the Chinese Pharma- copeia [1]. Additionally, it also has traditionally been used as folk medicine in Korea for treatment of gastrointestinal disorders, post- partum hemorrhage and amenorrhea [2]. The two major bioactive alkaloids are evodiamine and rutaecarpine (Fig. 1). Modern phar- macological studies have proved their various activities, such as inhibit corticosterone production, anti-inflammation, anti-obesity, cardiotonic, center stimulative, vasodilatatory, antithrombotic and bronchoconstrictive activities [3–6]. Recent studies have shown that rutaecarpine can induce CYP1A1 expression [7], modulate drug metabolizing enzymes [8], ∗ Corresponding author. Tel.: +82 33 250 6918; fax: +82 33 255 7865. E-mail address: kyeong@kangwon.ac.kr (K.H. Kim). and prevent ultraviolet A-induced reactive oxygen species gener- ation [9]. Several studies demonstrated that evodiamine induces tumor cell death through two pathways: apoptosis and necrosis [10], became lead structure of anticancer agents [11]. Further studies demonstrate that evodiamine has anti-tumor potential by inhibiting proliferation, inducing apoptosis and reducing invasion and metastasis of a wide variety of tumor cells, including breast cancer cells [12], prostate cancer cells lines DU145 and PC3 [13], leukemic T-lymphocyte cells [14], melanoma cells [15], cervical cancer cells [16], colon cancer cells [17] and lung cancer cells [18]. Evodiamine posses one chiral center and exists two enan- tiomers, naturally it presents as S-(+)-form in plant [19]. S-(+)-evodiamine and its R-(-)-form can be obtained accord- ing to the chemical synthesis methods, though this alkaloid can be extracted from plants, they are industrially produced by stereos- elective total synthesis [20]. Although much literature devoted to study of the pharmacology of evodiamine can be found, pharmaco- logical studies has usually been performed on racemic evodiamine [20,21]. Significant differences between the pharmacology and toxicology of the individual enantiomers and the racemate have not been demonstrated so far. Many studies have indicated that drug enantiomers may have different pharmacodynamic and 0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.04.018