Green and novel ultrasonic extraction with UHPLC-MSMS analysis of natural sweetener (Glycyrrhizic acid) from Glycyrrhiza glabra; a multifactorial mechanistic evaluation based on statistical analysis

Abstract

A novel, green and eco-friendly, cost-effective, fast, and reliable high energy ultrasonication (US) extraction with UHPLC-MSMS (Ultrahigh performance liquid chromatography with mass spectrometry) quantification of Glycyrrhizic acid (GZA) is reported herein for the first time. The study provides useful insights regarding the effect of US-factors with statistical analysis and mechanisms, involved in GZA-extraction and analysis. An US-extraction method (US-MD) was developed using three levels of US factors: solvents (AC (acetone), EtOH (ethanol), H₂O (water)), time (1, 2, 3 min), amplitudes (30, 40, 50%), pulse (10/0.5, 20/0.5, 30/0.5 sec), particle sizes (0.5, 1, 1.4 mm), and temperatures (20, 30, 40 °C). The US-MD was further validated with high accuracy 98.96 ± 6.82 and r² = 0.995 whereas, an in-house analytical method (UHPLC-MSMS) was developed and validated to quantify the GZAamount. UHPLCMSMS-MD resulted in a retention time of 0.31 min with MSMS (821.400 > 351.200) in a 1 min run time whereas, UHPLCMSMS-MV showed high accuracy and precision with r² = 0.998 for GZA. Statistical analysis of K-mean clustering finalized US-set-of-factors showing optimum extract yield (mg/1mg) of 0.48 with sum (2.41 ± 014) and mean (0.27) along with a high GZA-amount (μg/mg) of 8.23 with sum (43.31 ± 2.07) and mean (4.81) for H₂O in 3 min at 40 °C using particle size (1.4 mm), amplitude (50%), and pulse (30/0.5). Large scale application of US-UHPLCMSMS confirmed the evaluation power of the method showing the order for GZA amount; Egypt > Pakistan > Syria > India > Palestine > America > Georgia > Morocco. A significant effect for US factors Vs extract yield and GZA amount was observed however, solvent*GZA-amount and extract yield*particle size were more significantly correlated compared to time*temperature*amplitude*pulse analyzed via PCA, GLM-UniANOVA, K-mean, and Pearson's correlation (P ≤ 0.05). A combined mechanism of shear stress, macroturbulence due to acoustic cavitation and implosions, sonochemical, and sonocapillary effect were noted for the US technique producing higher extract yield and GZA amount from licorice.

Keywords: Cavitation; Glycyrrhizic acid; PCA; Sonochemical; UHPLC-MSMS; Ultrasonication.