Quantitative determination of short chain organic acids by RP-HPLC/ESI(-)-MS using post column phase modification
2 Ecological Microbiology, BayCEER
P 2.3 in Research in its Prime: First Results of Ongoing Research
Microbial fermentation products include short chain organic acids in environmental samples. Analyses of carbon flow via microbial fermentation utilizing 13C-labelled tracers depend on a sensitive detection of short chain organic acids and their isotopic composition. Thus, a high performance liquid chromatography/mass spectrometry (HPLC-MS) method with selected ion monitoring was developed and optimized to analyze several short chain organic acids (acetic, propionic, butyric, iso-butyric, 2-methylbutyric, 3-methylbutyric, valeric, lactic, succinic, pyruvic, oxalic, and acrylic acids) in environmental samples. Chromatographic separation and peak shape optimization require protonation of short chain organic acids by a mobile phase approximating pH 3, whereas a sensitive detection in atmospheric pressure electrospray (APS-ES) negative mode requires charged carboxylate anions occurring at higher pH. Such a dilemma was resolved by a post column phase modification of the eluent pH (i.e., the continuous addition of base in post column flow increasing the pH of the mobile phase from 3 to 9-10), maximizing the formation of carboxylate anions and thus the detection sensitivity under APS-ES negative ionization mode. Three methods for selected organic acids differing in detection sensitivity, analysis time, and chromatographic separation were developed. Baseline separation of nine organic acids was achieved at a flow rate of 0.2 mL min-1. Limits of detection (LOD) and quantification (LOQ) ranged from 0.01 to 1 µM and 0.05 to 5 µM, respectively. LODs and LOQs were highest for succinic acid. Calibration curves were linear over three orders of magnitude of organic acid concentrations. A 4-fold increase in detection sensitivity for acetic acid was achieved at a flow rate of 0.1 mL/min. Recoveries of representative organic acids in peat extracts were reduced on average by approximately 20 % due to matrix effects. Representative 13C-labelled acids were likewise quantified. This method is accurate, sensitive, simple, and can be used to analyze diverse short chain organic acids including their isotopic composition in various environmental samples.