Stevia rebaudiana is a plant well-known as a source of dozens of high potency non-caloric natural sweeteners, many of them still uncharacterized. Herein, we describe the development of several routine HPLC (high performance liquid chromatography) analytical techniques to better separate steviol glycosides. Different stationary phases/columns were used: reverse phase C-18, amino, HILIC (hydrophilic interaction chromatography), Sepaxdiol and Synergi. The advantages and disadvantages of each method for resolving the very polar and less polar elution regions are demonstrated. Strategies for scale up of the purification process of steviol glycosides to make it more economic are also summarized, emphasizing the advantages of using high efficiency normal phase chromatography in relation with reverse phase. Additionally, we also developed an easy and useful way for predicting yields and composition of the pools resulting from the preparative chromatography. The development of these analytical and preparative liquid chromatography techniques allowed the isolation of several steviol glycosides in gram quantities at relatively low cost.

To assess topical delivery studies of glycoalkaloids, an analytical method by HPLC-UV was developed and validated for the determination of solasonine (SN) and solamargine (SM) in different skin layers, as well as in a topical formulation. The method was linear within the ranges 0.86 to 990.00 µg/mL for SN and 1.74 to 1000.00 µg/mL for SM (r = 0.9996). Moreover, the recoveries for both glycoalkaloids were higher than 88.94 and 93.23% from skin samples and topical formulation, respectively. The method developed is reliable and suitable for topical delivery skin studies and for determining the content of SN and SM in topical formulations.