Estimation of steviol glycosides in food matrices by high performance liquid chromatography

Steviol glycosides (SGs) are non-caloric, natural sweetener obtained from plant Stevia rebaudiana and are used as sugar substitute in foods. The level of SGs in foods should not exceed maximum permissible limit defined by regulatory agencies. Thus analytical methods are required for assay of stevioside (Stev) and rebaudioside A (Reb A), which are two major constituents of SGs, in foods. A method for extraction of Stev and Reb A from dairy viz., flavoured milk, flavoured yoghurt and non-dairy foods viz., carbonated water, jam, chewing gum and estimation of these by HPLC has been described. Extraction of SGs from dairy samples was achieved by treating samples with 20% acetonitrile in presence of Carrez solutions while these can be simply extracted with water from non-dairy samples. Separation and estimation of these two glycosides was achieved on C18 column (length: 4.6 × 250 mm, particle size: 5 μm) using isocratic mobile phase prepared by mixing of acetonitrile and 10 mM sodium phosphate buffer (pH 2.6) in ratio of 32:68 (v/v). Recovery of two SGs was quantitative. Separation and estimation of SGs by HPLC was robust. Limit of detection and limit of quantitation for Reb A in different food was in range from 1.057-1.834 to 3.525-6.114 mg kg^-1 while that of Stev was from 1.679-2.912 to 5.596-9.707 mg kg^-1, respectively. Neotame, an artificial sweetener can be used as internal standard for separation of SGs.