Combined HPLC-DAD–MS, HPLC–MSn and NMR spectroscopy for quality control of plant extracts: The case of a commercial blend sold as dietary supplement

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Highlights

  • HPLC-DAD–MS, HPLC–MSn and NMR analyses revealed the fraudulous content of a dietary supplement.

  • Rauwolfia sp. alkaloids (ajmaline, reserpine) were found.

  • Reserpine was in the therapeutic range and responsible for the collapses of the patients.

Abstract

The efficiency of 1D and 2D NMR spectroscopy along with HPLC-DAD–MS analyses in characterising the content of a dietary supplement is demonstrated. Experiments directly performed on a lyophilised sample of a commercial product gave details on the quality control of the product. The lack of the marker constituents of some of the declared plant species (Crataegus oxyacantha, Olea europea, Capsella bursa-pastoris and Fumaria officinalis) and the presence of banned adulterants, responsible for the strong antihypertensive effect of the supplement were established. The analyses proved the presence of indole alkaloids belonging to the group of Rauwolfia sp., such as ajmaline, reserpine and yohimbine. Quantitative HPLC analysis showed that the content of reserpine in the product was in the therapeutic range and therefore responsible for the collapses of the patients.

Introduction

In Europe, the dietary supplement segment accounts for 15–20% and the rest is represented by registered drugs, by contrast, dietary supplements hold the largest share of the whole Italian botanical market [1]. Dietary supplements have simplified regulations for both production and marketing authorisation in comparison with herbal medicinal products, with a positive list (botanical resources admitted) and a negative list (botanicals banned from ingredients) notified by the Health Ministry [2]. The lack of strict regulations and effective controls of dietary supplements available on the market contributes to marketing of herbal products of questionable quality. Adulteration of unprocessed bulk herbs and herbal medicinal products with undeclared synthetic drugs is a serious problem [3], [4], [5] which puts in risk consumers’ health. The commercial blend “Olivis” is sold as a dietary supplement and the name is associated to Olea europea and olive suggesting the product as a hypotensive remedy because olive leaf decoction is traditionally used as an antihypertensive in the Mediterranean medicine. The attention for this herbal drug preparation was drawn after several cases of collapses after ingestion of this preparation by patients under treatment with hypotensive drugs, which were communicated to “the alternative medicine products signalling system” working at Istituto Superiore di Sanità (Rome, Italy) since April 2002 [6]. “Olivis” preparation reports in the label to be a blend of four herbal drugs, namely Crataegus oxyacantha L., Olea europea L., Capsella bursa-pastoris L. and Fumaria officinalis L. and from a preliminary analysis by LC–MS we have reported a short letter concerning the presence of reserpine added to the blend [6]. Aims of the present study were to evaluate the presence of the declared plants in the preparation and in addition to characterise unexpected natural or synthetic constituents which have contributed or were responsible to the adverse effects of this dietary supplement. Powerful analytical techniques such as HPLC-DAD–MS, NMR and HPLC–MS/MS were employed for the screening of this complex mixture and revealed the presence of fraudulent plant extract responsible for the undesirable effects. To the best of our knowledge this is the first case of an adulteration of a herbal remedy with an extract of a banned plant.

Section snippets

Chemicals

Dimethylsulfoxide-d6 (99.9% D), methanol-d4 (99.8% D) and D2O (99.9% D) were purchased from Sigma–Aldrich. LC–MS grade water, methanol and acetonitrile were supplied by Mallinckrodt Baker (Deventer, The Netherlands). The HPLC column used for HPLC-UV–MS analysis was a Luna C18, 150 × 3 mm 5 μm (Phenomenex, Torrance, CA, USA). HPLC–MS/MS analysis was performed using a Gemini C18 column, 100 × 2 mm 5 μm (Phenomenex).

All solvents used for the isolation procedure were of HPLC grade; chloroform (CHCl3),

Results and discussion

Purposes of the study were to evaluate the presence of the declared plants in the preparation and in addition to characterise unexpected natural or synthetic constituents which have contributed or were responsible to the adverse effects of this dietary supplement. Different extracts of the herbal drugs declared in the label were firstly analysed by HPLC-DAD–MS and NMR to identify the constituents and a common chromatographic method was developed in order to obtain a complete fingerprint of

Conclusions

Based on the aforementioned data the presence of the four herbal drugs in the product mixture is dubious. Among the herbal drugs reported as the ingredients of Olivis, only O. europea could be related to hypotensive effects of the product. However, both NMR and HPLC-DAD–MS analyses evidenced the lack of olive leaf extracts and in addition, their concentration of the other herbal drugs in the final product were so low that it could not justify the collapses. The cause of the side effects instead

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