Simultaneous determination of sesquiterpenes and pyrrolizidine alkaloids from the rhizomes of Petasites hybridus (L.) G.M. et Sch. and dietary supplements using UPLC-UV and HPLC-TOF-MS methods

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Abstract

UPLC-UV and HPLC-TOF-MS methods have been developed for the analysis of major sesquiterpenes and pyrrolizidine alkaloids from rhizomes of Petasites hybridus (L.) G.M. et Sch. (Family, Asteracea) and dietary supplements claiming to contain P. hybridus. The best results were obtained with Acquity UPLC™ HSS T3 (100 mm × 2.1 mm, I.D., 1.8 μm) column system using a gradient elution with a mobile phase consisting of ammonium formate (50 mM) and acetonitrile (0.05% formic acid) at a constant flow rate of 0.25 mL/min via UPLC-UV. The newly developed method was validated according to the ICH guidelines with respect to specificity, linearity, accuracy and precision. The limits of detection were found to be 5 μg/mL and 0.1 μg/mL for pyrrolizidine alkaloids and sesquiterpenes, respectively by UPLC-UV and 0.001 and 0.01 μg/mL, respectively using HPLC-TOF-MS. The methods were successfully used to analyze different P. hybridus market products, as well as to distinguish between two other Petasites species. The total content of petasins was found to be in the range of 0.02–11.6 mg/dosage form for 15 dietary supplements and no petasins were detected in an additional six dietary supplements. Additionally, pyrrolizidine alkaloids, which are considered to be toxic for the liver, were detected in seven dietary supplements. The amount of petasin in seven dietary supplements was found to be within limits of label claim and no pyrrolizidine alkaloids were detected. HPLC–mass spectrometry coupled with electrospray ionization (ESI) interface method is described for the identification and confirmation of sesquiterpenes and pyrrolizidine alkaloids from plant extracts and dietary supplements that claim to contain P. hybridus as well as different species of Petasites.

Introduction

Common Butterbur (Petasites hybridus (L.) G. Gaertn., B. Mey. & Scherb.) is a herbaceous perennial plant in the family Asteraceae, native to Europe and northern Asia [1]. P. hybridus exists in two chemo-varieties: those containing petasins and those with furano-petasins [2]. These two chemotypes of P. hybridus cannot be distinguished using classical morphological techniques. The pharmacological active substances of P. hybridus are believed to be sesquiterpene esters of the petasin and the furanopetasin chemotype [2], [3]. Butterbur has been reported to be effective in reducing the occurrence or severity of migraine headaches [4], [5]. Additionally, a peer-reviewed journal published a Swiss study showing butterbur extract as an effective treatment for hay fever without the sedative effect of the antihistamine cetirizine [6]. The extracts of rhizomes and leaves have been traditionally used as spasmolytic agents, mainly against gastro-intestinal tract cramps, anti-inflammatory activities and hay fever [7], [8]. Butterbur naturally contains components called pyrrolizidine alkaloids. Pyrrolizidine alkaloids (PAs) and their N-oxides are well established as hepatotoxins in animals and humans [9]. The concentrations are often highest in the rhizomes and stalks, and lowest in the leaves, and vary depending on where the plants are grown. Furthermore, the rhizomes of P. hybridus been found to contain trace amounts of toxic PAs mainly senecionine and intergerrimine [10] which have demonstrated a carcinogenic and mutagenic potential [11]. Therefore, plants intended to be used for pharmaceutical purposes should be low in PAs. After a comprehensive risk assessment of toxic pyrrolizidine alkaloids, the German Federal Health Bureau established regulations, on the basis of their potential to be genotoxic and carcinogenic, that restricts oral exposure to pyrrolizidine alkaloids or their N-oxides in herbal preparations to 1.0 μg/day (intake restricted to 6 weeks only) or 0.1 μg/day (no restrictions of intake) with the exclusion of pregnant and lactating women for which zero exposure is recommended [12], [13].

Previously analytical methods including HPLC [14], [15], [16], [17], [18], and GC–MS [18], [19], [20] have been used for the analysis of P. hybridus. Wildi et al., reported the HPLC method [14] for the determination of mean content of petasins and pyrrolizidine alkaloids from various populations of rhizomes and leaves of various locations in Switzerland. Qualitative analysis, using HPLC, of butterbur leaves and rhizomes found in different sites in Switzerland indicated varying levels of petasin and PAs. A simple HPLC procedure [15] for the separation and isolation of six furanoeremophilanes from P. hybridus was reported by Siegenthaler et al. A normal phase LC method was presented by Debrunner et al., which allows separation of sesquiterpenes of P. hybridus [16], [17]. Essential oils of butterbur rhizomes obtained by hydrodistillation were determined by gas chromatography, mass spectrometry, nuclear magnetic resonance [18]. In another report natural variability in PA content in P. hybridus rhizomes and the distribution of the two chemotypes from populations was studied [19] and the main PA detected are senecionine and integerrimine. The alkaloid content of butterbur leaves were also determined by enzyme immunoassay [20]. For the analysis of PAs in natural samples GC–MS methods are often applied but these methods are not suitable for N-oxides which are unstable at GC method conditions. Over the years several other PA extraction methods [21], [22], [23], [24], [25] and techniques for analyzing PAs have been applied such as spectrophotometry [26], TLC, HPLC-UV, ion pair HPLC, GC–MS [27] and NMR [22], [28]. Recently liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been introduced for measuring PAs in plant material [24], [25], [29]. Unlike GC-related methods, LC–MS can detect both free amines and N-oxides without the time-consuming and tedious reduction step. These methods can all be used for the analysis of sesquiterpenes or for PAs from plant sample, but they often involve long run times. Most of the reported methods show the mean content for petasins however there is not a good analytical method described for the analysis of PAs from Petasites species. Some of the reported publications, report data, but lack information on the quality of their quantitative data; e.g., no linearity, no precision, and no information on the method used to obtain LOD's. Thus, it was necessary to develop and validate an assay with appropriate sensitivity, selectivity, accuracy, and precision. Additionally, any developed method might be utilized for pre-clinical studies that can generate potentially large number of samples which will require a rapid and reliable assay. An ideal method should have simple sample preparation, fast oncolumn separation, and sensitive and specific detection. UPLC-UV has become an analytical tool that meets most of the above needs. In order to reduce the run time and increase the chromatographic resolution the UPLC technology was investigated in combination with HPLC-TOF-MS to provide accurate MS analysis. To date there is not an analytical method reported which can detect PAs and sesquiterpenes from P. hybridus in a single procedure and also for the quality evaluation of dietary supplements. The outlined chemical fingerprinting method together with the target compounds will be able to provide further information about the quality of botanicals as well as herbal supplements using UPLC-UV.

A simple and rapid UPLC-UV method was developed to evaluate the quality of dietary supplements through a simultaneous determination of four active sesquiterpenoids (petasin, isopetasin, S-petasin and 8β-H-eremophilanolide) and five pyrrolizidine alkaloids (seneciphyllin N-oxide, integerrimine, senecionine, senecionine N-oxide and senkirkin) (Fig. 1). The wavelengths 220 and 230 nm were chosen for the determination of these constituents. A more selective and sensitive HPLC-TOF-MS procedure is also reported in this paper for further identification and confirmation purposes. To our knowledge, this is the first report to simultaneously determine the sesquiterpenes and pyrrolizidine alkaloids using UPLC-UV and HPLC-TOF-MS methods. These methods were also applied to the analysis of three Petasites species (P. hybridus, P. vulgaris and P. frigidus). The quantitation of petasins and PAs can be useful for both the assessment of the commercial value of the product, and to determine the daily intake of these compounds, in relation with the possible pharmacological actions that they may have. There is a relationship between active marker content and sample form/extraction procedure. The bulk plant material contains PAs, which are toxic and should be removed during extraction procedure to ensure safe products. Different manufacturers use different extraction procedures that influence the content of sesquiterpene lactones and PAs in the resulting extract. Further the formulation, e.g. capsules, tablets, softgels may influence the content of isopetasin as well. Variations can result directly from production (e.g. due to pressure, heat) or indirectly from a poor analytical recovery caused by insufficient extraction during sample preparation. Therefore, the determination of petasins and PAs content can represent a parameter for authenticity and quality control of the product. Information obtained from UPLC-UV and HPLC-TOF-mass spectra allowed the quantification and identification of sesquiterpenes and pyrrolizidine alkaloids of various samples.

Section snippets

Ultra performance-liquid chromatography-UV (UPLC-UV)

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), connected to Waters Empower 2 data station. An Acquity UPLC™ HSS T3 column (100 mm × 2.1 mm I.D., 1.8 μm) also from waters was used. The column and sample temperature were maintained at 40 °C and 15 °C, respectively. The column was equipped with a LC-18 guard column (Vanguard 2.1 mm × 5 mm, Waters Corp.,

UPLC-UV

In the UPLC-UV analysis, various mobile phase compositions and chromatographic conditions were tested to find the optimal chromatographic conditions. The different columns tried were Acquity UPLC™ Bridged Ethyl-siloxane/silica Hybrid (BEH) C18 (100 mm × 2.1 mm I.D., 1.7 μm), Acquity UPLC™ Bridged Ethyl-siloxane/silica Hybrid (BEH) C8 (straight chain alkyl column) (50 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 (embedded polar group column)

Conclusions

The newly developed UPLC-UV and HPLC-TOF-MS methods were found to be capable for the simultaneous determination of sesquiterpenes (S-petasin, 8β-H-eremophilanolide, petasin, isopetasin) and pyrrolizidine alkaloids (seneciphylline N-oxide, integerrimine, senecionine, senecionine N-oxide, senkirkin) from the roots of Petasites species and dietary supplements that claim to contain P. hybridus. Petasin, isopetasin and 8β-H-eremophilanolide was found to be a major compound for all samples analyzed.

Acknowledgements

This research is supported in part by the United States Department of Agriculture, Agricultural Research Service, Specific Cooperative Agreement No. 58-6408-2-0009, and the Global Research Network for Medicinal Plants (GRNMP), King Saud University. The authors would like to thank Annette Ford, R&D Biologist, University of Mississippi, University, MS, USA for extraction of samples.

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