High-performance liquid chromatographic analysis of Black Cohosh ( Cimicifuga racemosa) constituents with in-line evaporative light scattering and photodiode array detection

Abstract

A validated and reproducible high-performance liquid chromatography (HPLC) method with in-line evaporative light scattering and photodiode array detection was developed for the analysis of major constituents in Black Cohosh ( Cimicifuga racemosa L.). The method is based on the baseline chromatographic separation of 18 compounds reported to be present in Black Cohosh on a C-18 column (5 μm, 4.6 mm×250 mm) with water (0.05% trifluoroacetic acid, TFA)–acetonitrile–water as the mobile phase, and their in-line detection using photodiode array detector (PDA) and evaporative light scattering detector (ELSD). Sixteen of these (caffeic acid, ferulic acid, isoferulic acid, cimicifugoside H-1, cimiracemoside A, cimicifugoside H-2, (26 R)-actein, 26-deoxycimicifugoside, (26 S)-actein, 23- epi-26-deoxyactein, 23-OAc-shengmanol-3- O-β- d-xyloside, 26-deoxyactein, 25-OAc-cimigenol-3- O-α- l-arabinoside, 25-OAc-cimigenol-3- O-β- d-xyloside, cimigenol-3- O-α- l-arabinoside, cimigenol-3- O-β- d-xyloside) were detected to be present and quantifiable. The reputed two flavonoid constituents (kaempferol and formononetin) were not detectable. The validation of the method included tests for sensitivity, linearity, reproducibility, recovery and stability. The detection limits were found to be in the range of 26–55 ng (10 μl per injection) in ELSD for the 16 constituents of Black Cohosh. The exponential linear calibration curves were observed for all the constituents tested with r 2 being >0.99. The reproducibility of the method was evaluated by analyzing three sets of controls on three consecutive days ( n=3) with R.S.D. (%) and relative error (%) <8.14 and 11.27%, respectively. The observed recovery rates were better than 91.75%. The stability of Black Cohosh methanolic sample solution was assessed by consecutively analyzing the methanolic solutions prepared from liquid extract (Sample A), powered extract (Sample B), milled plant material (Sample C) and one of the commercial product (Sample D1) for the content of 16 major constituents with the relative standard deviations (R.S.D., %) <6.39%, indicating the sample matrix of Black Cohosh is stable. Contrary to literature reports, kaempferol and formononetin were not detected in either our field collected plant materials or commercial products of Black Cohosh in the current assay.