Validation of a fast-HPLC method for the separation of iridoid, glycosides to distinguish between the Harpagophytum species

A fast high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of the iridoid glycosides harpagoside (HS) and 8-p-coumaroyl-harpagide (8pCHG) in extracts and preparations of Harpagophytum procumbens and H. zeyheri. The ratio between 8pCHG and the sum of HS and 8pCHG can be used to distinguish between both species. Quantitation was accomplished with the internal standard (IS) method. The separation was performed on a monolithic silica column (Chromolith Performance RP-18e), under gradient conditions using a mobile phase of water (pH 2.0 adjusted with phosphoric acid) and acetonitrile. The elution of the analytes was monitored at 278 mn and conducted at a column temperature of 30 degrees C. Because of the high porosity of the monolithic column the mobile phase was able to be pumped at a flow rate of 5.0 mL/min. The retention time of 8pCHG, HS, and the IS was 1.9 min, 2.1 min, and 3.0 min, respectively, and the total run time of the assay was 5 min. The method was validated by specificity, linearity, accuracy, and precision. For the determination of method robustness a number of chromatographic parameters were varied.