Multivariate optimization of human fecal fatty acids determination by HS-SPME-GC-MS

The gut microbiota plays a significant role in human health and compositional changes of the microbiota are associated with many diseases. Short chain fatty acids (SCFAs), which are abundant end products of microbial metabolism, are carboxylic acids with 1–6 carbons, and include acetic, butyric, propionic, pentanoic, isopentanoic and isobutyric acids. Most SCFAs are absorbed by the colon and transported to the liver through the portal vein. Subsequently, SCFAs are drained into the bloodstream and arrive at various organs where they influence host physiologies, such as affecting blood pressure, mitigating inflammation, and regulating cellular metabolism. The imbalance of SCFA levels has been related to various diseases, including inflammatory bowel diseases (IBDs), colorectal cancer (CRC), non-alcoholic fatty liver disease (NAFLD), type 2 diabetes (T2D), and hypertension. Owing to their biological prevalence and their close association with the pathophysiology of diseases, from the perspective of disease monitoring the quantitative determination of SFCAs in biological fluids has gained much attention in the last years. However, most of studies have focused solely on the determination of the SCFAs arising from the symbiotic gut microbiota metabolism. It has been recently hypothesized that medium-chain fatty acids (MCFAs) may be associated to gastrointestinal diseases, including colorectal cancer (CRC). The quantification of both SCFAs and MCFAs could be useful to reveal the interconnection between microbiota and metabolic alterations during disease. Fecal samples have gained a major role as the most accessible biological matrix, which can directly probe the connection between intestinal bacteria and the physiology of the holobiont. To this aim, a head space-solid phase microextraction-gas chromatography–mass spectrometry (HS-SPME-GC-MS) method for the simultaneous analysis of SCFAs and MCFAs in human feces was developed. A pool of faeces from healthy paediatric donors was use as test sample for the optimization of the procedure of extraction. A quantitative analytical procedure was set up using the stable isotope labelled internal standards normalization approach. Response Surface Methodology was undertaken to conduct a comprehensive investigation into the effect of the parameters influencing HS-SPME yield (extraction temperature and time, equilibration time). The coefficients of the quadratic model linking the factors to the responses were estimated by a Central Composite Design. The Method Operable Design Region (MODR) was identified by means of the calculated models and by Monte-Carlo simulations. The MODR represents a multivariate zone which ensures that the considered responses meet specific requirements with a selected probability of failure.