docTargeted screening of botanicals in herbal dietary supplements by
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Targeted screening of botanicals in herbal dietary supplements by
Targeted screening of botanicals in herbal dietary supplements by LC MS/MS LC-MS/MS Caroline Mathon, PhD student School S h l off Ph Pharmaceutical ti l S Sciences, i U University i it off G Geneva, S Switzerland it l d Official Food Control Authority of Geneva, Switzerland SCAHT, Swiss Centre of Applied Human Toxicology [email protected] Presentation outline Aim of this work Definition of a food supplement Some problems encountered with HFS Methodology Analytical approach Results and perspectives Aim of this work Consumption of food supplements is increasing every year No rigorous testing process To check the composition of food supplements to protect consumers Public health : Does this food supplement contain any undesirable plant ? Fraud : Is the composition consistent with the labelling and current legislation on herbal food supplements ? Presentation outline Aim of this work Definition of a food supplement Some problems encountered with HFS Methodology Analytical approach Results and perspectives Definition of a food supplement Europe : European p Food Safety y Authority y ((EFSA)) "Food Food supplements are concentrated sources of nutrients or other substances with a nutritional or physiological effect, whose purpose is to supplement the normal diet." [1] [1] Directive 2002/46/EC Definition of a food supplement Switzerland : Equivalent definition[1] to EFSA List of plants[2] edited by swissmedic and FOPH Ginger Gi k Gingko 72 plants [1] RS 817.022.104, article 22 [2] Guidance Composition of food supplements Switzerland : Mandatory : vitamins and/or minerals Vitamin C, C Mg, Mg Fe… Fe S Some plants l t are authorized th i d Forbidden : plants with therapeutic effects Presentation outline Aim of this work Definition of a food supplement Some problems encountered with HFS Methodology Analytical approach Results and perspectives Some problems encountered with HFS When labelling is not in accordance with the regulation Typical illegal health claims HDS contain unauthorized ingredients When labelling does not correspond to the composition … Absence of a p plant omission, mistake, … or fraud Presence of a plant not listed adulteration or contamination Addition of synthetic chemicals adulteration Objective j To develop a generic method able to detect several plants (e.g. list Swissmedic) by means of adequate biomarkers. Currently numerous methods exist: But usually y they y are specific p for a p particular species p or dedicated to a class of compounds. Presentation outline Aim of this work Definition of a food supplement Some problems encountered with HFS Methodology Analytical approach Results and perspectives Methodology gy Ginkgo biloba L. Specific Biomarker(s) Plant Concentration Ginkgolide g A 0.2-0.02% in Ginkgo Commercially available 3 MS/MS spectra Intensity, cps 3.0e8 RT 7.17 2.0e8 0.0 Analysable by LC-MS/MS LOD 33 ng/ml 1.0e8 1.0 3.0 5.0 7.0 9.0 Ginkgolide g A 11.0 13.0 Time, min 106 / 10 mg Presentation outline Aim of this work Definition of a food supplement Some problems encountered with HFS Methodology Analytical approach Results and perspectives Analytical approach Analysis in 2 steps : Qualitative identification of biomarkers Id tifi ti off biomarkers Identification bi k Quantitative Dosage of specific biomarkers Only one crude extract HPLC-MS/MS : electrospray ionisation, ionisation Qtrap Analytical y approach pp Sample preparation 200 mg of food supplements MeOH-H2O (1-1) 10' sonication at RT Dilution with buffer solution Filtration Ready for injection by LC-MS/MS Qualitative analysis MRM (Multiple Reaction Monitoring) : one transition i i iis targeted d ffor each h bi biomarker k IDA (Information Dependent Acquisition) Threshold : 1000 cps MS/MS spectrum CE : 20V MS/MS spectrum CE : 35V MS/MS spectrum CE : 50V C Comparison i with i h an iin-house h lib library off mass spectra Comparison with an in-house library of MS/MS spectra Analysis A l i : Gingkolide A CE 20V CE 35V Library : Gingkolide A Matching : 90% 78% Quantitative analysis MRM (Multiple Reaction Monitoring) : Identification points : retention time (max ± 0.2 min) two transitions are targeted for each compound Intensity cps Intensity, the h ratio i between b the h 2 transitions ii 3.0e8 RT 7.17 2.0e8 409 → 345 10 8 1.0e8 409 → 115 Calibration curve (weighted 1/x) llowestt concentration t ti is i the th LOQ LOQ between 1 ng/ml to 500 ng/ml 0.0 1.0 3.0 5.0 7.0 9.0 11.0 13.0 Time, min Presentation outline Aim of this work Definition of a food supplement Some problems encountered with HFS Methodology Analytical approach Results and perspectives Chromatograms with all biomarkers DOS GUG SMI VIN VIT COL Time, min GAR FIP 3.0e4 13.0 THC ALA GKA COU 11.0 DAI Q QIN QID Q COC GEN GUA SOL SIL FOR POD W PAR KAW CLO OLE CHE E ATO TAX Intensity, cps 9.0 STY SPT CAF EMT DIM 7.0 SPO SEN S RET 5.0 AMY CYT HTP ALL THE 3.0 Chromatogram in positive ESI 65 BM at 100 ng/ml MEZ LOB B 0.0 1.0 MEZ SCH BEB SNT T CHE C PAM LOB 1.0e5 RUC VIC YOH Y SKK CIM EPH 2.0e5 BOL ATR HEL 3.0e5 MET LAS 4.0e5 CAP HAR R Intensity cps Intensity, EMO O Chromatogram in negative ESI 26 BM at 50 ng/ml 5.0 9.0 RHE ASI HPF QRC C 7.0 SNA 3.0 VEB 1.0 ANI CYN 0.0 ALI 1.0e4 ECH SCD HOM AGN LEO 2.0e4 11.0 13.0 Column : Synergi polar RP 50 x 2 mm, 2.5 mm particle size 15.0 Time, min State of the current method lasiocarpine amygdalin vincamine agnuside schizandrin procyanidin sinensetin 5 HTP stevioside garcinol leiocarposide sesamin chelidonine parthenolide kawaine atropine heliotrine harmine 89 biomarkers areruscin targeted and registered chelerythrine boldine theanine-Ll in the MS/MS library y digitoxin coumarin Hyperforin kaempferol quercitrin ruscogenin curcumin taxol 3,5bilobalide dimethoxyphenolsmilagenin capsaicin echinacoside vinpocetine strychine Rhein cynarin Ginkgolide A senecionine vitexicarpin colchicine lobeline(-) senkirkin daidzein coumestrol THC allantoine cytisine cimifugin i if i 72 plants Allow to screen Gugglesterone Aloin A Ginsenoside 31 "Swissmedic" plants caffeine silybin asiaticoside sennoside alliin y g nordihydroguaiaretic Toxic plants l p yohimbin diosgenin h d hydroxyvalerenic i di i palmatine acid quinidineAuthorized plants with some limitations aconitine quinine alantolactone formononetin scopolamine mezerein sparteine emodin emetine i ld h d cinnamaldehyde podophyllotoxin melatonine genistein ephedrine acteoside convallatoxin helenalin retrorsine berberine Confusion or contamination This method allowed to identify a toxic contamination A child was hospitalized in Switzerland Between Chinese and Japanese p star anise Illicium verum Hook. f. and Illicium anisatum L. Toxic level of the neurotoxin anisatine in the Japanese star anise Structure of anisatine Perspectives ! Lack of specificity of 10 biomarkers Improve the specificity of this generic method increase the number of BM per plant search for alternative confirmatoryy methods Test sample p p preparation p to obtain a better sensitivity y for the BM Implement new plants of interest in the method Acknowledgements Official Food Control Authority of Geneva, Switzerland SCAHT Swiss Centre of Applied Human Toxicology SCAHT, Swiss Centre of Applied Human Toxicology Mrs Duret, Dr Kohler, Dr Bieri, Dr Bugey and Dr Edder Pharmacognosy, University of Geneva, Switzerland Dr Christen Thank you for your attention! Caroline Mathon, PhD student [email protected]
