Green RP-HPLC method for estimation of trigonelline: AQbD based development, validation, and application to nanoformulations

Trigonelline is an alkaloidal plant derived bioactive compound reported for its various pharmacological activities. Recent advances in the utilization of this compound in drug development have highlighted the importance of establishing a reproducible and efficient analytical method for estimating trigonelline (TRG) in both its pure form and formulated nano systems. Current research aims to develop and validate a green RP-HPLC method for estimating TRG by integrating Analytical Quality by Design (AQbD) with the principles of Green Analytical Chemistry. Optimization of the chromatographic conditions was done by employing a rotatable central composite design with amount of mobile phase and its flow rate selected as critical variables and tailing factor (T_f), retention time (R_t) and theoretical plates as the responses. Optimal separation was achieved using ethanol and water (40:60, v/v) on a Phenomenex C18 (250 × 4.6 mm, 5 μm) column at 264 nm, yielding a sharp, symmetric peak at 5.60 min at a flow rate of 1.5 mL/min. Developed method exhibited excellent linearity over the range of 5-15 µg/mL (r² = 0.9986) with %RSD less than 2% and LOD & LOQ were found to be 0.628 µg/mL and 1.90 µg/mL, respectively. Forced degradation studies showed 12% degradation in acidic media and 9% in alkaline media after exposure of 8 h indicating moderate susceptibility to hydrolysis. Further, the validation was performed for the developed method according to ICH Q2(R1) guidelines. A comprehensive greenness assessment was performed using AES, GAPI, AGREE, AMGS, and AGSA tools, confirming that the newly developed method demonstrates superior greenness and its suitability for sustainable routine analysis compared to existing methods.