Detection of CITES-listed <i>hoodia</i> species and extracts thereof in herbal supplements using LC-HRMS/MS: validation study and qPCR method comparison

This study describes the validation of a approach to detect in herbal supplements regulated Hoodia species or their extracts, targeting the oxypregnane steroidal glycoside P57AS3, using liquid chromatography coupled to tandem mass spectrometry with a high-resolution acute mass detection (LC-HRMS/MS). The method was validated by assessing the selectivity and specificity by analysing other related Apocynaceae species, particularly Stapeliinae subtribe members, and various plants commonly found in dietary supplements promoting weight loss. The limit of detection (LOD), equal to 8 µg P57AS3 per gram powder, matrix effects, and the ratio of false positives (0%) were determined. Next, the analytical sensitivity of the LC-HRMS/MS methodology was compared to a recently developed quantitative PCR (qPCR) method by comparing the results obtained from 20 commercial herbal supplements, often originating from rogue internet pharmacies, that either claimed or were reported to contain Hoodia sp. material or extracts thereof. The study results highlight the complementary nature of LC-HRMS/MS and qPCR. While qPCR provides high molecular sensitivity for powdered material, LC-HRMS/MS delivers chemical confirmation and detection of metabolites when DNA is absent or degraded. The integration of both techniques creates a comprehensive detection framework, enhancing accuracy, reliability, and confidence in the identification of regulated plant material across different and complex herbal matrices and mixtures thereof.