Abstract
IntroductionHeadspace gas chromatography–mass spectrometry (HS-GC–MS) is widely considered the gold standard of quantitative fecal VOC analysis. However, guidelines providing general recommendations for bioanalytical method application in research and clinical setting are lacking.ObjectivesTo propose an evidence-based research protocol for fecal VOC analysis by HS-GC–MS, based on extensive testing of instrumental and sampling conditions on detection and quantification limits, linearity, accuracy and repeatability of VOC outcome.MethodsThe influence of the following variables were assessed: addition of different salt solutions, injection temperature, injection speed, injection volume, septum use, use of calibration curves and fecal sample mass. Ultimately, the optimal sample preparation was assessed using fecal samples from healthy preterm infants. Fecal VOC analysis in this specific population has potential as diagnostic biomarkers, but available amount of feces is limited here, so optimization of VOC extraction is of importance.ResultsWe demonstrated that addition of lithium chloride enhanced the release of polar compounds (e.g. small alcohols) into the headspace. Second, a linear relationship between injection volume, speed and temperature, and fecal sample mass on the abundance of VOC was demonstrated. Furthermore, the use of a septum preserved 90% of the non-polar compounds. By application of optimal instrumental and sampling conditions, a maximum of 320 unique compounds consisting of 14 different chemical classes could be detected.ConclusionsThese findings may contribute to standardized analysis of fecal VOC by HS-GC–MS, facilitating future application of fecal VOC in clinical practice.
Highlights
Headspace gas chromatography–mass spectrometry (HS-Gas Chromatography-Mass Spectrometry (GC–MS)) is widely considered the gold standard of quantitative fecal volatile organic compounds (VOC) analysis
VOC analyses demonstrated potential as noninvasive early diagnostic biomarkers in diseases in which the intestinal microbiota is considered to play a role in the pathogenesis, such as inflammatory bowel disease (IBD), colorectal cancer, necrotizing enterocolitis (NEC) and late onset sepsis (LOS) (Berkhout et al 2017; Berkhout et al 2019; Bosch et al 2019; Bosch et al 2018; de Meij et al 2015; van Gaal et al 2017)
Applying headspace technology to the GC–MS is limited in its sensitivity, due to the concentration of the VOC in the headspace
Summary
Headspace gas chromatography–mass spectrometry (HS-GC–MS) is widely considered the gold standard of quantitative fecal VOC analysis. VOC analyses demonstrated potential as noninvasive early diagnostic biomarkers in diseases in which the intestinal microbiota is considered to play a role in the pathogenesis, such as inflammatory bowel disease (IBD), colorectal cancer, necrotizing enterocolitis (NEC) and late onset sepsis (LOS) (Berkhout et al 2017; Berkhout et al 2019; Bosch et al 2019; Bosch et al 2018; de Meij et al 2015; van Gaal et al 2017) In these diseases, feces is considered as the most suitable bodily excrement to be analyzed (El Manouni el Hassani et al 2018). Fecal VOC are considered to reflect the gut microbiota composition and function, (patho)physiological metabolic processes of the host and the interaction between host and microbiota (De Lacy Costello et al 2008)
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