Abstract
•Validation of GC–MS and LC-HRMS methods to determine in vivo metabolites/catabolites.•GC–MS is not suitable for phenolic sulfate and glucuronide metabolites analysis.•HPLC-HRMS analysis provides a more detailed profile on phase II metabolites.•SDB-L and HLB sample purification is not suitable for sulfate phenolic derivatives.•Inappropriate analytical protocol can adversely affect bioavailability studies.
The present study compared and validated two analytical methods, HPLC-HRMS, and GC–MS using MSTFA as derivatization agent, for the analysis of microbiota-derived phenolic acids and aromatic compounds accumulating in urine, collected over a 24 h period after the consumption of 500 mL of orange juice. In addition, purification procedures using SDB-L and HLB solid phase cartridges were compared when HPLC-HRMS technique was used. Both HPLC-HRMS and GC–MS methodologies were successfully validated in terms of specificity, sensitivity, limit of detection and quantification, recovery and matrix effects. HPLC-HRMS, unlike GC–MS, does not require sample derivatization prior to analysis. GC–MS was not suitable for the analysis of phenolic sulfate and glucuronide metabolites because of their lack of volatility. These phase II metabolites could, however, be analysed by HPLC-HRMS which, as a consequence, provided more detailed and complete information on the phenolic compounds derived from microbiota-mediated degradation of orange juice (poly)phenols. Furthermore, the use of SDB-L and HLB cartridges for sample purification prior to HPLC-HRMS analysis is suitable for free phenolics and glucuronide metabolites but not sulfate derivatives. These findings highlight that the use of an inappropriate analytical protocol can adversely affect studies on the bioavailability of dietary (poly)phenols in which microbiota-derived phenolic catabolites play an important role.