A fast,fully validated GC-MS method using a simplified pretreatment for the quantification of short and branched chain fatty acids in human stool |
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Authors: | Justin Gray Baochuan Guo Rebecca Bearden Julie Manka |
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Institution: | 1. Department of Chemistry, Cleveland State University, Cleveland, Ohio, USA;2. Department of Chemistry, John Carroll University, University Heights, Ohio, USA |
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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 ?10.21% to 8.88% and ?13.25% 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 ?20°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). |
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Keywords: | branched chain fatty acids direct injection gas chromatography–mass spectrometry short chain fatty acids stool |
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