Analytical methods for determination of magnoflorine and saponins from roots of Caulophyllum thalictroides (L.) Michx. Using UPLC, HPLC and HPTLC

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Abstract

Analytical methods including HPLC, UPLC and HPTLC are presented for the determination of major alkaloid and triterpene saponins from the roots of Caulophyllum thalictroides (L.) Michx. (blue cohosh) and dietary supplements claiming to contain blue cohosh. A separation by LC was achieved using a reversed phase column, PDA with ELS detection, and ammonium acetate/acetonitrile gradient as the mobile phase. Owing to their low UV absorption, the triterpene saponins were detected by evaporative light scattering. The eight triterpene saponins (cauloside H, leonticin D, cauloside G, cauloside D, cauloside B, cauloside C, cauloside A and saponin PE) and the alkaloid magnoflorine could be separated within 35 min using HPLC method and within 8.0 min using UPLC method with detection limits of 10 μg/mL for saponins and 1 μg/mL for magnoflorine. The detection wavelength was 320 nm for magnoflorine and ELS detection was used for the eight saponins. The methods were also successfully applied to analyze different dietary supplements. For the products claiming to contain blue cohosh, there was a significant variability in the amounts of triterpene saponins detected. Calculations based on the analysis results for dietary supplements showed that maximum daily intake of alkaloid and saponins vary with the form (solids/liquids) and recommended doses according to the products label. Intakes varied from 0.57 to 15.8 mg/day for magnoflorine and from 5.97 to 302.4 mg/day for total saponins. LC–mass spectrometry coupled with electrospray ionization (ESI) method is described for the identification and confirmation of nine compounds in plant samples and dietary products. A HPTLC method was also developed for the fast chemical fingerprint analysis of C. thalictroides samples.

Introduction

Caulophyllum thalictroides (L.) Michx. (Berberidaceae), known as blue cohosh, is an indigenous perennial herb commonly found in eastern North America where the roots are used in diuretic, uterine antispasmodic and laxative preparations [1]. Traditionally the roots and rhizomes of C. thalictroides are used for the treatment of menstrual difficulties and inducing uterine contractions [2]. Earlier studies have revealed blue cohosh rhizomes and roots contain two types of saponins, caulosaponins and caulophyllosaponins [3], [4], [5], [6]. The caulosaponins (caulosides A, C, D, G) contain caulosapogenin (later corrected to hederagenin [3]) as aglycon [4], [5] whereas caulophyllosaponins (caulosides B, H, leonticin D) show caulophyllosapogenin as aglycon [4], [5], [6]. The saponins in blue cohosh are considered to be responsible for the uterine stimulant effects together with teratogenic alkaloids [2]. Between 1882 and 1905, blue cohosh was listed in the United States Pharmacopoeia as a labor inducer [6] and sold as an herbal supplement that can aid in childbirth. There is considerable concern about the safety of blue cohosh with reports of new born babies having heart attacks or strokes after the mother consumed blue cohosh to induce labor [6], [7], [8], [9]. Blue cohosh is avoided during the first trimester (three months) of pregnancy [6]. In addition to the quinolizidines, the aporphine alkaloid magnoflorine is found in substantial quantities [10], [11] which have been implicated as teratogens. Based on available scientific evidence, there is no proven safe or effective dose for blue cohosh [6].

Few published analytical methods have been reported for the analysis of alkaloids alone or for alkaloids and saponins using GC, HPLC and densitometry for blue cohosh [1], [2], [12], [13]. Levels of the major quinolizidine alkaloids in herbal preparations have been determined by gas chromatography, HPLC and densitometry [1], [12]. Betz et al. [12], developed a GC methodology for the quantitative determination of alkaloids from the roots of C. thalictroides L. Woldemariam et al. [1], developed a TLC densitometric method and an HPLC method for the quantification of alkaloids from extracts of C. thalictroides L. roots. The studies by Woldemariam et al. [1], and Betz et al. [12], focused on isolation and determination of alkaloids including N-methylcystisine, baptifoline, anagyrine and magnoflorine. The saponins present in C. thalictroides L. have received less attention than the alkaloids. Ganzera et al. [2], developed an HPLC method for the quantitative analysis of primary alkaloids and saponins from C. thalictroides L. roots. Subramaniam et al. [13], also developed a HPLC method for the separation and quantification of three alkaloids and three saponins from extracts of blue cohosh roots and dietary supplements. The newly developed UPLC method for quantitative determination of one major alkaloid [magnoflorine (1)] and eight triterpene saponins [cauloside H (2), leonticin D (3), cauloside G (4), cauloside D (5), cauloside B (6), cauloside C (7), cauloside A (8) and saponin PE (9)] (Fig. 1) from the roots of C. thalictroides is found to be capable of giving shorter retention times while maintaining good resolution and sensitivity. Detection of the saponins was achieved with the use of an ELS detector. The compounds were numbered by the order of elution using LC–UV–ELSD method. A comparison of chromatographic performance of HPLC and UPLC was performed. A simple and fast HPTLC method was also developed for the chemical fingerprint analysis of alkaloid (magnoflorine) and saponins. The highly sensitive UPLC–MS method was used to identify and confirm the compounds in blue cohosh root samples and dietary supplements that claim to contain C. thalictroides. These methods are useful in establishing the quality and safety of herbal products claiming to contain C. thalictroides.

Section snippets

UPLC–UV–ELSD

All analyses were performed on a Waters Acquity UPLC™ system (Waters Corp., Milford, MA, USA) including binary solvent manager, sampler manager, column compartment, PDA (Waters Acquity model code UPD), ELS detector (Waters Acquity model code UPE), and MS detector (Waters Acquity model code SQD), all connected to Waters Empower 2 data station. An Acquity UPLC™ BEH Shield RP18 column (50 mm × 2.1 mm I.D., 1.7 μm) also from Waters was used. The column and sample temperature were maintained at 35 °C and

Chromatographic conditions optimization

Optimal chromatographic conditions were obtained after running different mobile phases with a reversed phase C18 column. The different columns tried for UPLC were Acquity UPLC BEH C18 (100 mm × 2.1 mm I.D., 1.7 μm), Acquity UPLC BEH C18 (50 mm × 2.1 mm I.D., 1.7 μm) and Acquity UPLC BEH Shield RP18. The best results were observed with BEH shield RP18 column (50 mm × 2.1 mm I.D., 1.7 μm) using ammonium acetate (pH 7.2) and acetonitrile as the mobile phase. Acetonitrile was preferred over methanol as the mobile

Conclusion

The newly developed UPLC method for the chemical analysis of nine compounds from blue cohosh was found to be capable of providing short retention times while maintaining good resolution as compared to conventional HPLC. The new UPLC technique allowed for the reduction in the mobile phase flow rate and an increase in acquisition rate with the benefit of decreased injection volumes to achieve good peak shapes. The method is suitable for rapid analysis of magnoflorine and saponins and for chemical

Acknowledgements

This research is supported in part by “Science Based Authentication of Dietary Supplements” and “Botanical Dietary Supplement Research” funded by the Food and Drug Administration grant numbers 5U01FD002071-10 and 1U01FD003871-02, and the United States Department of Agriculture, Agricultural Research Service, Specific Cooperative Agreement No. 58-6408-2-0009 and the authors would like to thank Annette Ford for samples preparation.

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