V.L. Beloborodov, N.G. Zakharova, A.M. Savvateev, V.K. Kolkhir, I.V. Voskoboynikova

Prostanorm is a domestically produced multi-component herbal drug, which is an extract of the mixture of the following herbal materials: St. John-s Wort (Нypericum perforatum L.) grass, Golden rod (Solidago Canadensis L.) grass, licorice (Glycyrrhiza glabra L.) roots and Echinacea (Echinacea purpurea (L.) Moench) roots and rhizome taken in equal quantities. We have previously identified marker substances of each of the drug herbal components, and set grounds for the possibility of their analysis for determination of the drug authenticity. Here we present the development of a combined method of assay of the above mentioned markers based on solid phase extraction (sample processing) and subsequent HPLC. Solid phase extraction using Discovery DSC-18 LT SPE cartridge was chosen for effective isolation of marker substances from Prostanorm extract. Extract aliquots were applied onto the cartridge, and strong polar substances (caphtaric acid, chlorogenic acid, caffeic acid, chicory acid, 2-hydroxycinnamic acid, rutin and hyperoside) were eluted by 0.2 % orthophosphoric acid (fraction 1), while less polar substances (quercetin, kempferol and glycirrhyzinic acid) were subsequently eluted by ethanol (fraction 2). Both fractions were subjected to two-stage gradient reverse-phase chromatography. Optimal chromatography conditions for separation of fraction components were determined. Method specificity, repeatability and reproducibility were evaluated. The following marker substance levels (in mg/ml) were found in Prostanorm extract (batch 130510) using the developed assay method: caphtaric acid 0.9, chlorogenic acid 1.2, caffeic acid 0.2, rutin 2.1, hyperoside 1.8, chicory acid 3.0, 2-hydroxycinnamic acid 0.5, quercetin 1.0, kempferol 0.06, glycirrhyzinic acid 15.7.