Rapid Isolation and Identification of Betaine From Massa Medicata Fermentata and Its Quantification by High Performance Liquid Chromatography Coupled With Solid‐Phase Extraction

ABSTRACT Liu Shenqu (Massa Medicata Fermentata [MMF]) is a natural fermented product of flour (and/or wheat bran) and five medicinal herbs, Prunus armeniaca , Vigna umbellata , Artemisia annua , Polygonum hydropiper , and Xanthium strumarium . Unprocessed, stir‐fried, and charred MMF has been used therapeutically in China for over a thousand years to treat conditions such as dyspepsia, abdominal distension, and diarrhea. Variations in quantities, fermentation conditions, and processing procedures across different production regions can influence the quality of MMF products, ultimately affecting their efficacy. Therefore, it is essential to establish robust qualitative and quantitative methods for the quality control of MMF. In this study, betaine, a bioactive compound, was identified via TLC, isolated using preparative TLC and analyzed using NMR spectroscopy in MMF. Ultrasonic extraction of betaine from MMF samples was optimized using orthogonal experimental design. Following aluminum oxide (OH − type) solid‐phase extraction, a simple, rapid, and accurate HPLC method was developed for the quantification of betaine, utilizing a HILIC column. Analysis of 58 commercially available MMF batches revealed that betaine concentrations in 49 batches (84.5%) ranged from 0.75 to 4.0 mg/g, whereas only 9 samples contained concentrations below 0.5 mg/g. Notably, betaine levels were significantly lower in charred products compared with unprocessed ones ( p < 0.001), suggesting that betaine may serve as a potential biomarker for the quality control of MMF.