LC-MS and LC-MS/MS as a tool in standardizing extracts of medicinal

plants

N. Harding, G. Fouche, V.J. Maharaj, N. Moodley, D. Naidoo,

P.A.Steenkamp, and S. van Rooyen.

CSIR, Biosciences, P.O. Box 395, Pretoria, 0001, South Africa

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Drug Discovery

Competency Area

Bioprospecting Discovery Chemistry Specialty groups

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Information Identification

of active

components

Extract(s)

Biological

screening

Activity

NCE/mixture

Formulation

Preclinical

Workup

*

*

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What do we mean by standardization?

• Standardize: to cause to conform to a standard.

• Why? - Establish a ‘baseline’ chemical profile.

- Establish variations between cultivation/harvesting sites.

- Stability studies.

- Final product specification – QC.

• What? - All compounds in the crude extract?

- Herbal mixture: how many components?

- Final formulation, actives, chemical ‘markers’

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Consideration I

• Concentration of the sample. - Too high – overload column, contaminate system, distorted

spectra. - Too little – don’t see all the components. - Impossible to estimate the concentration of individual

compounds in an unknown sample.

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UV chromatogram

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A ‘simple’ extract …..

• Diethyl ether extract of a plant.

• Final formulation, diethyl ether not pharmaceutically acceptable, solubility problems, ether presents health and safety issues for technology/knowledge transfer.

• Ethanol used to extract plant material, pharmaceutically acceptable, safer.

• Chromatogram of ethanol extract is similar, ‘active’ present in approximately the same concentration.

• The challenge …….

- The ethanol extract has no activity!

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Overlay of UV chromatograms

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Overlay of ESI + chromatograms

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Overlay of ESI + and ESI-

chromatograms

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Overlay of ESI- chromatograms

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Consideration II

• Separation is important! - Co elution – combined spectra.

- Completive ionization.

- Choice of column chemistries

- Mobile phase considerations.

• LC-MS: - Sensitivity is due to selectivity and efficient ionization. - How do you set up for true unknowns?

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Consideration III

• Data processing and interpretation.

• It is easy to produce data, but how do you turn that data into useful information?

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Stacked plot of UV, ESI+ and ESI- Chromatograms of an extract.

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Stacked plot of 191 (neg) and 162 (pos) chromatograms

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Peak at 17.8

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• By using product ion scan of 515m/z and information published by Michael Clifford in the Journal of Agricultural and Food Chemistry we were able to identify the isomers of Dicaffeoylquinic acids present in the sample.

(Clifford, M. N. Discriminating between the six Isomers of Dicaffeoylquinic Acid by LC-MS. J. Agric. Food Chem. 2005, 53,

3821-3832)

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Stacked plot of 191 (neg) and 162 (pos) chromatograms

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Product ions of 515m/z

Thank you