Department of Chemistry, Cleveland State University, Cleveland, Ohio, USA.
Department of Chemistry, John Carroll University, University Heights, Ohio, USA.
J Mass Spectrom. 2022 Apr;57(4):e4817. doi: 10.1002/jms.4817.
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).
关于与胃肠道疾病、肠道微生物群、代谢和饮食相关的短链脂肪酸（SCFAs）和支链脂肪酸（BCFAs）在人类粪便中的研究已经大大增加。因此，需要一种快速、可靠且预处理最少的方法来定量分析粪便中的这些代谢物（乙酸、丙酸、异丁酸、丁酸、异戊酸、戊酸和己酸）。因此，开发了一种符合这一标准的 GC-MS 方法。偏采样研究表明，当比较均质化和非均质化样品的 100mg 亚样本时，分析物平均值没有统计学差异（p>0.05）（n=6，p 值为 0.153-0.910）。粪便样品经过均质化处理，用 80:20 的水:甲醇（v/v）稀释，并将 pH 值调节至 1.5-2.5。样品经涡旋、离心，然后直接通过脉冲不分流进样注入 GC-MS，与 10:1 分流进样相比，信号增强了两倍至三倍。DB-FATWAX Ultra Inert Polyethylene Glycol (PEG) 柱在进行了 1476 次粪便进样后没有出现峰拖尾、响应降低或保留时间偏移的情况，而其他柱子在进行了 361 次进样前就出现了问题。粪便上清液样品的日内和日间准确度范围为-10.21%至 8.88%和-13.25%至 9.91%，而日内和日间精密度范围分别为 0.21%至 1.21%和 0.89%至 2.84%（变异系数，CV）。该方法表现出极好的线性（0.9999-1.0000）和较低的定量下限（1.50-8.01μM）。粪便样品在-20°C 下储存 28 天内稳定，回收率范围为 85.04%至 106.59%。通过比较标准品和粪便上清液校准曲线斜率，确定粪便中的基质效应无显著性差异（p>0.05）。
