First comprehensive validation of an HPLC-DAD method for dihydrocapsaicin quantification in Capsicum chinense germplasm accessions

Capsaicinoids are alkaloid compounds found exclusively in peppers of the genus Capsicum . Among them, capsaicin (CAP) and dihydrocapsaicin (DHC) are primarily responsible for the pungency of the fruit, which is the characteristic burning sensation. The species Capsicum chinense produces fruits with the highest pungency levels. The aim of this study was to validate, according to the SANTE/11,312/2021 and ICH Q2(R1) guidelines, an analytical method for quantifying DHC in 53 C. chinense accessions from the Capsicum Active Germplasm Bank at the Montes Claros State University (UNIMONTES), Minas Gerais, Brazil, to identify high-DHC accessions (high-pungency candidates) for breeding prioritization. Validation included the parameters of selectivity, limit of detection (LOD), limit of quantification (LOQ), linearity, matrix effect, precision, and accuracy. Analyses were performed by high-performance liquid chromatography with diode-array detection (HPLC-DAD), a technique whose analytical performance had not yet been fully validated for DHC quantification in C. chinense . The validated method showed satisfactory accuracy, with recovery rates ranging from 87.85% to 96.78%, and good precision, with a relative standard deviation (RSD) of 2.33%. To the best of our knowledge, this is the first comprehensive validation of an HPLC-DAD method for DHC determination in C. chinense accessions from a germplasm bank. The C. chinense accessions were quantified and grouped into 28 clusters according to their mean DHC concentrations, demonstrating high variability of pungency within the germplasm, with six accessions identified as presenting the highest DHC contents. Accessions with high DHC contents represent promising candidates for use in breeding programs aimed at developing cultivars with greater pungency. Validated HPLC-DAD method for dihydrocapsaicin (DHC) quantification in Capsicum chinense ; large-scale analysis reveals high pungency diversity (28 clusters) and identifies six elite high-DHC accessions for breeding applications.