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

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.