CD-MEKC method to analyze triterpene acids in traditional chinese medicines

Article J. Braz. Chem. Soc., Vol. 20, No. 7, 1268-1274, 2009. Printed in Brazil - ©2009 Sociedade Brasileira de Química 0103 - 5053 $6.00+0.00 CD-MEKC Method to Analyze Triterpene Acids in Traditional Chinese Medicines Hui Dua,b and Xiaoqing Chen*,a College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, People’s Republic of China a College of Medicine, Xi’an Jiaotong University, Xi’an 710061, People’s Republic of China b Um método cromatográfico eletrocinético micelar modificado por ciclodextrina (CD-MEKC) foi desenvolvido para separar e determinar ácido betulínico (BA), ácido oleanólico (OA) e ácido ursólico (UA) usados na medicina tradicional Chinesa (TCMs). Três triterpenos pentacíclicos ácidos foram separados em tampão (pH 9,0) de metanol 6% (v/v), contendo 10 mmol L-1 de tetraborato dissódico, 10 mmol L-1 de hidrogênio fosfato de sódio, 50 mmol L-1 de dodecilsulfato de sódio, 15 mmol L-1 de 2-hidroxipropil-β-ciclodextrina em 15 min. Uma boa relação linear entre a área de pico e a concentração foram obtidos nos intervalos de 16,5-330 μg mL-1, 18-360 μg mL-1 e 19,5-390 μg mL-1 para BA, OA e UA, respectivamente. Comparado com cromatografia líquida de alta eficiência com γ-CD como fase móvel quiral aditiva (CMPA-HPLC), CD-MEKC apresentou melhor eficiência analítica para os três ácidos e o tempo analítico (15 min) foi mais curto do que para CMPA-HPLC (80 min). A cyclodextrin-modified micellar electrokinetic chromatographic (CD-MEKC) method was developed to separate and determine betulinic acid (BA), oleanolic acid (OA) and ursolic acid (UA) in traditional Chinese medicines (TCMs). Three pentacyclic triterpene acids were baseline seperated in the buffer (pH 9.0) of 6% (v/v) methanol containing 10 mmol L-1 disodium tetraborate, 10 mmol L-1 sodium dihydrogen phosphate, 50 mmol L-1 sodium dodecylsulfate, 15 mmol L-1 2-hydroxypropyl-β-cyclodextrin within 15 min. The good linear relationships between the peak area and concentration were obtained in the ranges of 16.5-330 μg mL-1, 18-360 μg mL-1 and 19.5-390 μg mL-1 for BA, OA and UA, respectively. Compared with high performance liquid chromatography with γ-CD as chiral mobile phase additive (CMPA-HPLC), CD-MEKC has better analytical efficiency for three acids, and the analytical time (15 min) was shorter than that of CMPA-HPLC (80 min). Keywords: CD-MEKC, betulinic acid, oleanolic acid, ursolic acid, CMPA-HPLC Introduction Triterpenoids, such as betulinic acid (BA), oleanolic acid (OA) and ursolic acid (UA), existing in traditional Chinese medicines (TCMs) in the form of free acids or aglycones for triterpenoid saponins,1 possess important pharmacological properties. BA, a plant-derived pentacyclic lupane-type triterpene, has been shown to exert anti-inflammatory2 and anti-HIV activities.3 OA and UA are position isomers (Figure 1). The only difference between them is the position of the methyl group on the ring E. They have been ascertained to have anti-diabetogenic,4 hepatoprotective,5 antitumorigenesis6,7 and antioxidant effects.8 *e-mail: The main analytical methods of triterpenoids in TCMs are gas chromatography (GC),9-11 high-performance liquid chromatography (HPLC),12 capillary zone electrophoresis (CZE)13,14 and micellar electrokinetic chromatography (MEKC).15 However, the resolution of OA and UA was not satisfied by common HPLC or MEKC due to their similar molecular structures. Gas chromatography-mass spectrometry (GC-MS) via a time-consuming derivatisation reaction has already been used to identify OA and UA,16 and liquid chromatography-electrospray/atmospheric pressure chemical mass spectrometry (LC-ESI/APCI-MS) can be employed to identify BA17 and UA,18 as the triterpenoids have weak chromophores. Cyclodextrins (CDs, α-, β-, γ-) are torus-shaped, enzymatically synthesized, non-reducing oligosaccharides Vol. 20, No. 7, 2009 Du and Chen 1269 including pH value, buffer concentration, organic modifier composition, SDS and CDs concentration in CD-MEKC were investigated. Experimental Materials and reagents BA, OA and UA were provided by the National Institute for the Control of Pharmaceuticals and Biological Products (Beijing, China). Prunella vulgaris L., Diospyros kaki L. f., and Crataegus pinnatifida Bge., were purchased from Hubei, Shanxi and Hebei province of China, respectively. Sodium dodecylsulfate (SDS), sudan III and β-cyclodextrin (β-CD) were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China), 2-hydroxypropyl-β- cyclodextrin (HP-β-CD), 2,6-di-Omethyl-β-cyclodextrin (DM-β-CD) and γ-cyclodextrin (γ-CD) from Sigma (St. Louis, MO, USA). Methanol was of LC grade (Hanbon Technologies, Jiangsu, China). All other reagents and solvents were of analytical reagent grade and used without further purification unless otherwise noted. All aqueous solutions were prepared using newly double-distilled water. Apparatus and conditions Figure 1. Chemical structures of the three pentacyclic triterpene acids. consisting of D-glucopyranose units bonded through α-l,4-1inkages. The cavities of CDs are relatively hydrophobic while the external faces are hydrophilic. A cyclodextrin-modified MEKC (CD-MEKC) method19 and HPLC with CDs as chiral mobile phase additive (CMPAHPLC) 20 greatly expand the analytical applications. The hydrophobic cavities of CDs can form inclusion complexes with the analytes, which improves the separation of components with hydrophobic, isomerous or chiral properties significantly. In this work, for the first time, a comparative study was carried out, in which a CD-MEKC method was validated for separation and determination of triterpene acids in samples and it was compared with CMPA-HPLC. And the factors affecting the separation of triterpenoids, The CE analysis was carried out on a CL1030 capillary electrophoresis system (Beijing Cailu Scientific Inc., Beijing, China) equipped with a UV-Vis detector that can perform wavelength scanning from 190 to 740 nm. An uncoated fused-silica capillary (75 μm × 58 cm, Ruipu Chromatogram Equipment Co., Ltd, Hebei, China) with an effective length of 50 cm was used to separate samples, which were injected into the capillary by hydrodynamic flow at a height differential of 10 cm for 10 s. The applied voltage was 20 kV, and the detection wavelength was 210 nm. At the beginning of experiment, the capillary was purged with 0.5 mol L-1 NaOH for 5 min, followed by 0.1 mol L-1 NaOH for 5 min, deionized water for 5 min and then running buffer for 5 min. Between runs, the capillary was flushed with 0.1 mol L-1 NaOH for 2 min followed by deionized water for 2 min and then running buffer 2 min. HPLC analysis was performed on a Shimadzu LC2010 apparatus equipped with a Shimadzu SPD-M10A photodiode array detector (Shimadzu Corporation, Kyoto, Japan). Separations were carried out at 30 °C on a Hedera ODS C18 column (5 μm, 250 × 4.6 mm, Hanbon Technologies, Jiangsu, China). The optimum wavelength for determination was 210 n (...truncated)