Journal of Pharmaceutical and Biomedical Analysis 88 (2014) 7–15 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba Combined HPLC-DAD–MS, HPLC–MS n and NMR spectroscopy for quality control of plant extracts: The case of a commercial blend sold as dietary supplement A. Karioti a,∗ , E. Giocaliere b , C. Guccione a , G. Pieraccini b , E. Gallo c , A. Vannacci c , A.R. Bilia a a Department of Chemistry, Building of Pharmaceutical Sciences, University of Florence, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Firenze, Italy b Mass Spectrometry Center (CISM), University of Florence, Viale Pieraccini 6, 50139 Florence, Italy c University of Florence, Department of Preclinical and Clinical Pharmacology, Centre for Molecular Medicine (CIMMBA), Viale Pieraccini 6, 50139 Florence, Italy article info Article history: Received 27 March 2013 Received in revised form 26 July 2013 Accepted 29 July 2013 Available online 7 August 2013 Keywords: Quality control Dietary supplements NMR HPLC-UV–MS HPLC–MS n Phytovigilance 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 con- stituents 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 antihyperten- sive 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. © 2013 Elsevier B.V. All rights reserved. 1. 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 mar- ket contributes to marketing of herbal products of questionable quality. Adulteration of unprocessed bulk herbs and herbal medic- inal products with undeclared synthetic drugs is a serious problem [3–5] 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 anti- hypertensive in the Mediterranean medicine. The attention for this herbal drug preparation was drawn after several cases of collapses ∗ Corresponding author. Tel.: +39 0554573709. E-mail addresses: anastasiakarioti@yahoo.it, akarioti@gmail.com (A. Karioti). 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 Superi- ore di Sanità (Rome, Italy) since April 2002 [6]. “Olivis” preparation reports in the label to be a blend of four herbal drugs, namely Cratae- gus 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 addi- tion 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. 2. Materials and methods 2.1. Chemicals Dimethylsulfoxide-d6 (99.9% D), methanol-d4 (99.8% D) and D 2 O (99.9% D) were purchased from Sigma–Aldrich. LC–MS grade 0731-7085/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2013.07.040