Development and validation of QuEChERS-HPLC method for simultaneous analysis of 5-hydroxymethylfurfural, furfural, and p-cresol in brewery spent grain's hydrolysate.

Developing efficient methods to analyze inhibitors resulting from the breakdown of the lignocellulosic matrix in brewery spent grain is crucial for selecting optimal pretreatment strategies and monitoring these compounds during biodigestion processes. However, to date, these determinations have been based on separate methodologies that have not been subjected to the analytical validation stage. This study optimized QuEChERS extraction methods combined with High-Performance Liquid Chromatography (HPLC) analysis to determine 5-hydroxymethylfurfural (5-HMF), furfural, and p-cresol, simultaneously, in the semisolid and liquid fraction of the BSG hydrolysate. The analytical methods were validated by AOAC and INMETRO guidelines. All analytes showed good linearity, with correlation coefficients (R) greater than 0.97, and low limits of quantification. Recoveries at low, medium, and high levels were 94-116 %, repeatability was 0.6-6.8 %, and intermediate precision was 0.5-7.6 %. The method proved efficient when applied to a set of seven samples. It will contribute to monitoring inhibitory compounds present in biodigestion systems even at low concentrations, which is currently a challenge.