Validation and application of an ultrahigh-performance liquid chromatographic-Orbitrap mass spectrometric method for...

A simple and selective ultrahigh-performance liquid chromatographic-Orbitrap mass spectrometric (UHPLC-HR-MS) method was developed and validated for the simultaneous detection and quantification of short-chain fatty acids (SCFAs) such as acetic, propionic, butyric, isobutyric, valeric, isovaleric, 2-methyl-butyric (IS) and lactic, pyruvic and succinic acid in human faecal samples. A simple extraction procedure with 0.001% formic acid in water was performed on 40 samples. The extracts were centrifuged and analyzed by UHPLC-HR-MS on a sub-2μm column using gradient elution; meanwhile, the same samples were analyzed by GC-FID and HPLC-UV as reference methods The UHPLC-HR-MS method showed a recovery of 83-105%, a repeatability of 2.2-8.3% and an intermediate precision of 2.9-9.4%. The LOD and LLOQ were in the range of 0.04-0.23 and 0.2-0.5μg/ml, respectively. Regarding the SCFAs, statistical analysis showed a good correlation between the data obtained by UHPLC-HR-MS and those provided by GC-FID (p>0.05). On the contrary, the LC-UV data were not in agreement with those obtained by UHPLC-HR-MS determination (p<0.05). To the best of our knowledge, this is the first method available for the simultaneous extraction and quantification of SCFAs, lactic, pyruvic and succinic in faecal samples by UHPLC-HR-MS.