Work

Development of an LC-MS Method for the Quantitative Determination of Six Organic Acids Produced by Bacterial Fermentation In Vitro and In Vivo

Year: 2025

Type: article

Abstract: Due to the strong polarity and volatility of organic acids, there is often a lack of effective quantitative methods for organic acids in vivo or in vitro. This study introduced a robust and scientifically validated LC-MS methodology for quantitatively analyzing six organic acids, encompassing five short-chain fatty acids and lactate, observed in in vitro fermentation and human fecal samples. This method was able to achieve precise quantification through the monitoring of mass-to-charge (m/z) ratios of the deprotonated negative ions. After optimization, a 2:1 (v/v) dichloromethane/acetonitrile mixture was utilized to extract the crude acid mix from fermentation or fecal samples. Subsequently, the organic acids were isolated using 3 mL of 3.5 M ammonium hydroxide solution from mixtures. The calibration curves for six organic acids demonstrated linearity with R2 > 0.991 across the concentration ranges of 0.01–5.0 or 0.01–20 mM. The coefficient of variation and accuracy were 2–13% and 95–128%, respectively, which exhibited good precision and sensitivity. Accurate quantification of organic acids would aid in metabolic analysis in bacterial culture supernatants and human fecal matter.

Source: Processes

DOI: 10.3390/pr13030697