A rapid identification of anthocyanins in various plants from Burkina Faso by HPTLC/MS

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1 A rapid identification of anthocyanins in various plants from Burkina Faso by HPTLC/MS Presented by Rosella SPINA July 2-4, 2014 Lyon

2 Background- Anthocyanidins chemistry A B

3 Background- Anthocyanins > 300 structurally different anthocyanins Sugars Aglycon Acyl moiety 2-Phenylbenzopyrylium cation

4 Interest and biological properties Are used as a food-colorant E163 Have antioxidant properties Potential therapeutic role to: - Cardiovascular diseases - Cancer - Inhibition of several virus - Improvement of visual acuity Ref. Kong et al. Phytochemistry 64 (2003)

5 Burkina Faso s plants studied HPTLC separation conditions for qualitative and quantitative analysis of anthocyanins Tapinanthus dodoneifolius Loranthaceae Flowers Burkina Faso Eichhornia crassipes Pontederiaceae Flowers TLC/MS INTERFACE CAMAG SCANER TLC 4 CAMAG

6 Screening stationary and mobil phase Crude Extract LINOMAT 5 HPTLC plate silica gel 60 F 254 HPTLC plates RP-18 WF 254 s Different solvents systems: -) n-butanol-acetic acid-water v/v -) Ethyl acetate-isopropanol-water v/v -) Ethyl acetate-toluene-water-formic acid v/v -) Ethyl acetate-butanone-water-formic acid v/v -) Ethyl acetate-acetic acid-formic acid, water v/v Different solvents systems: -) Methanol-water- formic acid v/v -) Methanol-water-TFA v/v -) Methanol-water-TFA v/v -) Ethanol-water-TFA 20/80/2 v/v -) Acetonitrile-water-TFA v/v

7 Optimized conditions of separation HPTLC plates RP-18 WF 254 s LINOMAT 5 Acetonitrile-water-TFA (v/v) Eichhornia crassipes Tapinanthus dodoneifolius

8 Results by HPTLC/MS from crude extract of Tapinanthus dodoneifolius [M] + [M-H+Na] + Tapinanthus dodoneifolius Loranthaceae Flowers MicroTofQ Bruker Spot 1 [M] + [M+Glu] TLC/MS interface [M-H+Na] + Spot 2

9 Results by HPTLC/MS from crude extract of Eichhornia crassipes [M] + MS/MS Spot 11 Spot 10 No detectable - - Delphinidin-3,5-diglucoside / Spot 9 Delphinidin diglucoside + malic acid Spot 8 Delphinidin diglucoside +succinic acid / / / Spot 7 Delphinidin derivatives Spot 6 Delphinidin diglucoside +succinic acid / / Spot 5 Delphinin- derivatives Spot 4 Delphinidin-3-glucoside Spot 3 Pelargonidin-3-glucoside Spot 2 Pelargonidin 3- (6 coumaroyl) glucoside Spot 2 Delphinidin Spot 1 Cyanidin Eichhornia crassipes Pontederiaceae Flowers Results from HRMS

10 Example of HPTLC/MS from Eichhornia crassipes Spot 6 Eichhornia crassipes Pontederiaceae Flowers [M] + MicroTofQ Bruker 6 MS and MS/MS spectra TLC/MS interface Delphinidin Glucose Succinic acid

11 Quantitation of anthocyanins in the samples Sample Compound nm ng/spot r sdv % Tapinanthus dodoneifolius Eichhornia crassipes Cyanidin Cyanidin Tapinanthus dodoneifolius Cyanidin-3- glucoside Eichhornia crassipes Pelargonidin Eichhornia crassipes Pelargonidin-3- glucoside Eichhornia crassipes Delphinidin SCANNER TCL 4 CAMAG Eichhornia crassipes Eichhornia crassipes Delphinidin-3- glucoside Delphinidin-3,5- diglucoside Correlation coefficient > 0.99 Regression via height: polynomial; Volume applied of samples 10 µl; Concentration of solutions of crude extract: 5 mg/0,5 ml in MeOH

12 Quantitation of anthocyanins in the samples Substance: 530 nm Regression via height: Polynomial Y = * X e-005 * X2 r = sdv = 2.06 Cyanidin HPTLC 3D-densitogram of absorbance measurement at 530nm for all tracks Polynomial calibration of cyanidin via peak height by absorption at 530 nm

13 Conclusion Methanolic extract Methanolic extract Linomat 5 CAMAG TLC-MS interface CAMAG RP-18 WF 254 s Scanner TLC 4 CAMAG MicroTofQ BRUKER Quantification of 7 anthocyanins Determination of 14 anthocyanins

14 Paris Nancy Pr. Dominique LAURAIN-MATTAR Service Commun de Spectrométrie de Masse: François DUPIRE Dr. Fabien LACHAUD THANKS Université Ouagadougou Burkina faso Dr. Adama HÉMA Dr. Eloi PALÉ Pr. Mouhoussine NACRO Pierre BERNARD-SAVARY Jérémy MERCIER René de VAUMAS

15 THANKS FOR YOUR ATTENTION