Sensitive and quantitative determination of short-chain fatty acids in human serum using liquid chromatography mass spectrometry

Abstract

Short-chain fatty acids (SCFAs) are increasingly being monitored to elucidate the link between gut health and disease. These metabolites are routinely measured in faeces, but their determination in serum is more challenging due to their low concentrations. A method for the determination of eight SCFAs in serum is described here. High-resolution mass spectrometry and gas chromatography were used to identify the presence of isomeric interferences, which were then overcome through a combination of chromatographic separation and judicious choice of MS fragment ion. The SCFAs were derivatised to form 3-nitrophenylhydrazones before being separated on a reversed-phase column and then detected using liquid chromatography tandem mass spectrometry (LC-QQQ-MS). The LODs and LOQs of SCFAs using this method were in the range 1 to 7 ng mL−1 and 3 to 19 ng mL−1, respectively. The recovery of the SCFAs in serum ranged from 94 to 114% over the three concentration ranges tested.

Plasma Short-Chain Fatty Acids and Their Derivatives in Women with Gestational Diabetes Mellitus

“…The LC-MS/MS method, including sample preparation and analysis conditions, was applied according to the validated protocols of Jaochico et al and Shafaei et al [16,30]. The upper limit of quantification (ULOQ), linearity, LOD (S/N > 3) and LOQ (S/N > 10) for each analyte were determined, as shown in Table S2.…”

“…Many sample preparation procedures have been developed for SCFA analysis, which differ in the use of derivatization reagent, concentration of reagents, extraction time or extraction temperature [12,16,30,34,40]. These approaches were used to optimize the sample preparation for the best efficiency.…”

“…These approaches were used to optimize the sample preparation for the best efficiency. The final derivatization and the extraction were performed according to the validated protocol of Jaochico et al and Shafaei et al [16,30] with slight modifications. Plasma sample or calibration mixture (20 µL), in a plastic tube, was mixed with 20 µM IS (10 µL).…”

“…Determination of SCFA in biological materials allows a better understanding of their role, including outside of diabetes mellitus. Multiple analytical approaches for the detection of SCFA have been developed covering advanced chromatographic, mass spectrometric, electromigration and spectroscopic methods [16,[30][31][32][33][34][35][36][37][38][39]. The most common methods combine gas chromatography (GC), or liquid chromatography (LC) coupled with mass spectrometry (MS), most often with a derivatization step, which is time consuming but provides better chromatographic behavior.…”

“…In particular, GC-MS offers high sensitivity [31][32][33]. In recent years, LC-MS approaches that allow fast and efficient sample preparation and analysis were introduced [16,30,34]. Despite developments in SCFA analysis, the methods remain laborious and, therefore, SCFA determination is still not utilized in routine clinical practice.…”