Using High Performance Liquid Chromatography for the Universal Analysis of Phytochemicals

stevia leaves

Phytochemicals are used by plants to organically combat potentially lethal bacteria, viruses, and fungi. When harvested for human consumption, many of these biological compounds have been reported to play a protective role against both chronic and acute human ailments. Many phytochemicals behave as antioxidants, and some are believed to be anticarcinogenic. With the continually increasing popularity of botanicals and natural products, a simple, universal approach for the analysis of products containing botanical components is indicated.

Unfortunately, many botanical compounds of interest lack chromophores or resist ionization, rendering conventional analytical methods, such as mass spectrometry, UV absorbance or evaporative light scattering detection, less than ideal. For this reason, high-performance liquid chromatography (HPLC) with charged aerosol detection (CAD) may be a preferable vehicle for analysis.

Using this approach, Acworth et al.¹ established and evaluated specific methods for testing the following phytochemicals derived from botanical sources: triterpene glycosides from black cohosh, ginkgolides and bilobalides from ginkgo biloba, ginsenosides from ginseng, silibinins in milk thistle, ursane and oleanane triterpenes from gotu kola, and diterpene glycosides from stevia. While clinical and laboratory results vary and no supplement should be taken without the advice of a medical provider, some individuals use these compounds to treat menopause (black cohosh), increase memory and concentration (ginkgo biloba), reduce stress and fatigue (ginseng), decrease and repair liver damage (milk thistle), stimulate blood flow and the healing of wounds and skin conditions (gotu kola), and act as a low-calorie sweetener (stevia).¹

HPLC with CAD allows researchers to nebulize analyte and produce droplets that are dried and charged prior to measurement by an on-board electrometer. This process is both sensitive and reproducible since it functions at low levels of detection and achieves <2% reproducibility. For analytes without chromophores, this method outperforms traditional approaches, including both evaporative light scattering detection and UV absorbance. Specifically, the compounds that are generally analyzed using HPLC with evaporative light scattering (black cohosh and gotu kola) achieved results that demonstrated greater sensitivity and linearity and alleviated baseline issues. The analytes that usually undergo HPLC with UV detection (ginseng and stevia) also showed higher sensitivity and resolution while minimizing reaction times.

Acworth et al. report that HPLC with CAD demonstrates a dynamic range of approximately four orders of magnitude as well as a consistent response pattern among compounds with generally less than 10% variability between analytes. Other benefits of CAD over other methods include the ability to analyze both non-volatile and semi-volatile compounds, minimal impact by chemical structure on analyte response, and opportunity to estimate analytes even in the absence of external standards.

In terms of product and food safety and testing, the clinical impact of a universal approach to analyzing natural compounds may be as far-reaching as the botanicals themselves. This sensitive, reproducible method could allow interested parties to ensure that botanical products meet not only consumer safety standards but also surpass the threshold of potency for each phytochemical.

References

Acworth, Ian et al. ‘Novel, Universal Approach for the Measurement of Natural Products in a Variety of Botanicals and Supplements.’ Thermo Fisher Scientific, Application Note PN70021_E 03/12S, pp. 1-6.

To read the full application note, please follow the link: http://www.dionex.com/en-us/webdocs/113457-Pittcon12_1780-2_INAcworth_PN70021_NatProd.pdf. If you’d like to know more, let us know, and feel free share this post with your colleagues.