Identification and Characterisation of UV Inactive Acid Degradation Impurities of Rebaudioside A Using LC-HRMS/MS,...

Rebaudioside A is a predominant steviol glycoside, extracted from Stevia rebaudiana Bertoni leaves and is used as an alternative to sucrose in food and beverages. It undergoes degradation in various conditions, resulting in the formation of degradation products. The identification and characterisation of these degradation impurities are essential for understanding compound stability and product safety, especially the UV-inactive degradation impurities, which are not detected in UV-HPLC by previously reported methods.

A robust HPLC method was developed and validated using an XBridge BEH amide column with 10 mM ammonium acetate and acetonitrile (20:80% v/v) as the mobile phase, at 1.0 mL/min flow,10 μL injection, and 40 °C column temperature. Degradation products (DPs) formed under acid and base degradation were identified using LC-HRMS/MS. In silico ADME and toxicity profiling were performed using ProTox 3.0 and Pre ADMET. The environmental impact of the method was assessed using the AGREE tool.

The method was linear over 0.10-5.0 mg/mL with LOD and LOQ of 0.025 mg/mL and 0.10 mg/mL, respectively. Five DPs were identified in acid degradation and four DPs were identified under base degradation. The DP-3 was identified as (3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl acetate, which was not reported previously. The toxicity analysis of the degradation impurities showed that the acid DPs were mostly class 4, moderately toxic, except DP-3, which was class 6 and non-toxic, and the entire base DPs were non-toxic. The AGREE tool gave the method a greenness score of 0.75, indicating strong environmental compatibility.

The study provides a valuable understanding of the degradation behaviour of rebaudioside A in various conditions. All the degradation products were identified, including DP-3, which was identified as(3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl acetate. Additionally, in silico toxicity and ADMET studies provided a safety profile of degradation impurities. The AGREE score of the validated method indicates that the developed method is suitable as an environmentally friendly analytical approach.