Abstract：
Feces is a highly complex matrix containing thousands of metabolites. It also contains live bacteria and enzymes, and does not have a static chemistry. Consequently, proper control of pre-analytical parameters is critical to minimize unwanted variations in the samples. However, no consensus currently exists on how fecal samples should be stored/processed prior to analysis.
The effects of sample handling conditions on fecal metabolite profiles and abundances were examined using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOFMS).
Solid-phase microextraction (SPME) and derivatization via trimethylsilylation (TMS) were employed as complementary techniques to evaluate fresh, frozen, and lyophilized fecal samples with expanded coverage of the fecal metabolome. The total number of detected peaks and the signal intensities were compared among the different handling conditions.
Our analysis revealed that the metabolic profiles of fecal samples depend greatly on sample handling and processing conditions, which had a more pronounced effect on results obtained by SPME than by TMS derivatization. Overall, lyophilization resulted in a greater amount of total and class-specific metabolites, which may be attributed to cell lysis and/or membrane disintegration.
A comprehensive comparison of the sample handling conditions provides a deeper understanding of the physicochemical changes that occur within the samples during freezing and lyophilization. Based on our results, snap-freezing at -80 °C would be preferred over lyophilization for handling samples in the field of fecal metabolomics as this imparts the least change from the fresh condition.
The effects of sample handling conditions on fecal metabolite profiles and abundances were examined using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOFMS).
Solid-phase microextraction (SPME) and derivatization via trimethylsilylation (TMS) were employed as complementary techniques to evaluate fresh, frozen, and lyophilized fecal samples with expanded coverage of the fecal metabolome. The total number of detected peaks and the signal intensities were compared among the different handling conditions.
Our analysis revealed that the metabolic profiles of fecal samples depend greatly on sample handling and processing conditions, which had a more pronounced effect on results obtained by SPME than by TMS derivatization. Overall, lyophilization resulted in a greater amount of total and class-specific metabolites, which may be attributed to cell lysis and/or membrane disintegration.
A comprehensive comparison of the sample handling conditions provides a deeper understanding of the physicochemical changes that occur within the samples during freezing and lyophilization. Based on our results, snap-freezing at -80 °C would be preferred over lyophilization for handling samples in the field of fecal metabolomics as this imparts the least change from the fresh condition.
摘要：
粪便是含有数千种代谢物的高度复杂的基质。它还含有活细菌和酶，并且没有静态化学。因此,对分析前参数的适当控制对于最小化样品中不需要的变化是关键的。然而,目前在分析前应如何储存/处理粪便样本方面尚无共识.
使用全面的二维气相色谱结合飞行时间质谱（GC×GC-TOFMS）检查了样品处理条件对粪便代谢物谱和丰度的影响。
固相微萃取（SPME）和通过三甲基甲硅烷基化（TMS）的衍生化被用作评估新鲜，冷冻,和冻干的粪便样本，扩大了粪便代谢组的覆盖范围。在不同处理条件之间比较检测到的峰的总数和信号强度。
我们的分析表明，粪便样品的代谢谱在很大程度上取决于样品处理和加工条件，与TMS衍生化相比，这对SPME获得的结果具有更明显的影响。总的来说,冻干导致更大量的总代谢物和类别特异性代谢物，这可能归因于细胞裂解和/或膜崩解。
样品处理条件的综合比较提供了对冷冻和冻干期间样品内发生的物理化学变化的更深入理解。根据我们的结果,在-80°C下快速冷冻对于处理粪便代谢组学领域中的样品而言比冻干更优选,因为这对新鲜条件的变化最小。
使用全面的二维气相色谱结合飞行时间质谱（GC×GC-TOFMS）检查了样品处理条件对粪便代谢物谱和丰度的影响。
固相微萃取（SPME）和通过三甲基甲硅烷基化（TMS）的衍生化被用作评估新鲜，冷冻,和冻干的粪便样本，扩大了粪便代谢组的覆盖范围。在不同处理条件之间比较检测到的峰的总数和信号强度。
我们的分析表明，粪便样品的代谢谱在很大程度上取决于样品处理和加工条件，与TMS衍生化相比，这对SPME获得的结果具有更明显的影响。总的来说,冻干导致更大量的总代谢物和类别特异性代谢物，这可能归因于细胞裂解和/或膜崩解。
样品处理条件的综合比较提供了对冷冻和冻干期间样品内发生的物理化学变化的更深入理解。根据我们的结果,在-80°C下快速冷冻对于处理粪便代谢组学领域中的样品而言比冻干更优选,因为这对新鲜条件的变化最小。
