A fast, fully validated GC‐MS method using a simplified pretreatment for the quantification of short and branched chain fatty acids in human stool

Abstract

The study of short (SCFAs) and branched chain fatty acids (BCFAs) in human stool related to gastrointestinal diseases, gut microbiota, metabolism, and diet has dramatically increased. As a result, a fast, reliable method with minimal pretreatment is needed for quantification of these metabolites (acetic, propionic, isobutyric, butyric, isovaleric, valeric, and caproic acid) in stool. Therefore, a GC-MS method meeting this criterion was developed. A bias sampling study showed no statistical difference (p > 0.05) in analyte means when comparing 100 mg subsamples of homogenized to non-homogenized samples (n = 6, p values 0.153-0.910). Stool samples were homogenized, diluted with 80:20 water:methanol (v/v), and adjusted to a pH of 1.5-2.5. Samples were vortexed, centrifuged, and directly injected into the GC-MS using pulsed splitless injection offering twofold-to-threefold signal enhancement over a 10:1 split injection. DB-FATWAX Ultra Inert Polyethylene Glycol (PEG) Column showed no peak tailing, reduced responses, or retention time shifts after 1,476 stool injections, while other columns failed before 361 injections. Intra-and inter-day accuracy for stool supernatant samples ranged from À10.21% to 8.88% and À13.25% to 9.91%, while intra-and inter-day precision ranged from 0.21% to 1.21% and 0.89% to 2.84% coefficient of variation (CV), respectively. This method demonstrates excellent linearity (0.9999-1.0000) and low limits of quantification (1.50-8.01 μM). Stool samples proved stable stored at À20 C up to 28 days, and recoveries ranged from 85.04% to 106.59%. Matrix effects in stool are non-significant determined by comparing standard and stool supernatant calibration curve slopes (p > 0.05).

Simultaneous determination of 22 fatty acids in total parenteral nutrition (TPN) components by gas chromatography-mass spectrometry (GC-MS)

“…Optimization of the sodium methoxide FAME derivatization technique. See DOI: https://doi.org/10.1039/d3ay00407d ionization detector (GC-FID) methods [3][4][5][6][7][8][9] to gas chromatography-mass spectrometry (GC-MS) methods [10][11][12][13][14][15][16][17][18][19] due to the higher sensitivity of the MS detectors, 20,21 giving a better estimate of FAs present in the analyte of interest.…”

“…29,30 Due to the complex chemistry of FAs, there is a multitude of analytical techniques developed for FA quantication, including several GC-MS methods. These developed methods, however, are either (1) targeting only essential fatty acids; [31][32][33] (2) imposing limitations to some fatty acids of interest; 11,13,23,[34][35][36] (3) mostly focused on fatty acid extraction from human samples; 13,15,18,23,34,[36][37][38] (4) have complicated sample preparation technique, 4,32,39,40 (5) merely evaluating dysregulated fatty acids; 23,41,42 and (6) needing analytical method validation. 10,12,14,20,43 Thus, there is a need for straightforward GC-MS methods that allow the analysis of FAs as classied by their chain length and degree of unsaturation and are suitable for industrial routine applications, particularly in evaluating the quality of the TPN products.…”

“…Moreover, establishing an appropriate method for analysing total parenteral nutrition (TPN) products, administered mostly for critically ill patients is crucial due to the chemical complexity of its components, particularly in lipid content. 1,2 For instance, analytical techniques in analysing fatty acids (FAs) in food and pharmaceutical products has evolved through the recent years from using gas chromatography-flame ionization detector (GC-FID) methods 3–9 to gas chromatography-mass spectrometry (GC-MS) methods 10–19 due to the higher sensitivity of the MS detectors, 20,21 giving a better estimate of FAs present in the analyte of interest.…”