Systematic Optimization of Acid Hydrolysis Parameters for Diosgenin Extraction Coupled with UHPLC-DAD Analysis

Diosgenin (DG) is a steroidal sapogenin of considerable industrial and pharmaceutical importance, commonly present in fenugreek seeds in glycosidically bound forms that require hydrolysis for efficient quantification. Despite numerous extraction approaches reported in the literature, systematic optimization of acid hydrolysis parameters under green and analytically reproducible conditions remain limited. In this study, a stepwise optimization strategy was developed to maximize diosgenin yield from fenugreek seeds using sustainable solvents and controlled hydrolytic conditions, coupled with a rapid UHPLC-DAD analytical method. A reflux condenser was used for extraction of DG whereas UHPLC-DAD was used for the identification and quantification of DG in the fenugreek samples. Numerous factors were optimized to maximize the DG yield, including the use of green solvents [ethanol (EtOH), water (H₂O)], different acids [hydrochloric acid (HCl), nitric acid (HNO₃), sulphuric acid (H₂SO₄)], acid strengths [1N (normality), 2N, 3N], extraction times [0.5 h (hour), 1 h], extraction temperatures [80 °C, 100 °C, 120 °C], and sample concentrations [1 mg/mL, 2 mg/mL, 4 mg/mL]. Model-1 exhibited more yield for DG in EtOH whereas H₂SO₄ resulted more DG amount in the extracts as compared to the acids of HNO₃ and HCl. The strength of 1N for H₂SO₄ at a temperature of 120 °C for 0.5 h were the optimized factors resulting in the highest extract yield of 36% with a maximized DG yield of 3.346%, as witnessed in this study. The UHPLC-DAD resulted in a short and effective analytical method with a retention time of 2.58 min. The validation of the method produced an LOD and LOQ of 30.43 ppm and 92.22 ppm with r² value of 0.998 in the linearity range of 5–500 ppm. The samples from Indian (F1), Egypt (F5), and Iran (F10) origins were noticed with more extract and DG yield. The study identified optimal conditions within the tested experimental range for maximizing the DG yield in fenugreek seed samples with quality evaluation of fifteen different origin fenugreek seed samples in terms of DG amount. The developed method may serve as a practical analytical approach for DG quantification under similar laboratory conditions to be employed for the quality variation and standardization of DG-containing food, herbs, dietary supplements, cosmeceuticals, and pharmaceutical products.