A benchmark UPLC-based method for concurrent analysis of seventeen Stevia rebaudiana metabolites (steviol, isosteviol, and fifteen steviol glycosides) in a single analytical run and its implementation in diverse products.

Abstract

Stevia rebaudiana is a popular plant-derived noncaloric sweetener with numerous health benefits. The steviol glycosides (SGs) are regarded as primary sweetening components of this plant, having diverse flavour profiles and individual significance. Numerous ongoing research studies focusing on enhancing specific SGs' yields, improving sweetness, and reducing bitter aftertaste require a simple and efficient method involving SG precursors and as many of its glycosides in one go. The simultaneous analysis of SG precursors (steviol and isosteviol) and its glycosides are vital to ensure the quality, safety, and regulatory compliance of stevia-based products. This highlights the need for the development of an easy, reliable, and efficient method involving as many SGs and its precursors in one go. With this aim, a UPLC-PDA based method was developed involving fifteen key SGs along with their precursors steviol and isosteviol, which is the first report having both precursors and fifteen SGs in a single analytical run. The developed method was validated by incorporating numerous parameters such as linearity, accuracy, precision, sensitivity, specificity, peak resolution, peak asymmetry, theoretical plate numbers, peak height/area ratio, peak kurtosis, and peak skew. The developed method represents excellent linearity having regression coefficients (r2) value ranging from 0.998 to 0.999. The detection and quantification limits were 0.18-3.60 μg/mL and 0.60-12.00 μg/mL, respectively, indicating sensitivity for low-concentration analysis. Recovery values ranged from 94.45 % to 102.10 %, confirming the accuracy of method. The intra-day and inter-day precision, evaluated through RSD values for both peak area and retention time, also remained within acceptable limits. Overall method was found in accordance with ICH guidelines, having symmetric, well-resolved peaks with improved sensitivity and theoretical peak count numbers. The developed method was