Determination of stigmasterol, β-sitosterol and stigmastanol in oral dosage forms using high performance liquid chromatography with evaporative light scattering detection

A validated and repeatable high performance liquid chromatography (HPLC) method with online evaporative light scattering (ELSD) was developed for the analysis of two sterols, stigmasterol, β-sitosterol and a stanol, stigmastanol, found to be common in many herbal formulations and health care supplements. The method is based on the separation of the three marker compounds on a C8 column (Phenomenex Luna, 5 μm, 150 mm × 4.6 mm i.d.) using methanol:water (95:5 v/v) as the mobile phase, and a flow rate of 1 ml/min to separate all the marker compounds within 12 min. Cholesterol (50 μg/ml) was used as internal standard and methanol as the extraction solvent. The ELSD response parameters were optimised and the limits of detection (LOD) and quantification (LOQ) were calculated to be 2 and 5 μg/ml, respectively, which is more sensitive than obtained by photo diode array detection (5 and 7 μg/ml). Using ELSD, the percentage relative standard deviation (%R.S.D.) of intra-day and inter-day (3 days) precision for each marker was better than 3%, the accuracy data were within 97–103% and the recovery data were found to be within 95–107% for the five commercially available products examined. This method was used to assay commercially available products formulated as oral dosage forms purported to contain African Potato and associated sterols and stanol and proved to be suitable for the routine analysis and quality control of such products.