Metabolomic study of capsaicinoid compounds in urine samples by dispersive liquid-liquid microextraction and ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry

This paper presents a new analytical method based on dispersive liquid-liquid microextraction (DLLME) and ultra-high performance liquid chromatography with high resolution mass spectrometry (UHPLC-HRMS) used for the determination of capsaicin (CAP), dihydrocapsaicin (DCAP) and N-vanillylnonanamide (PCAP) in human urine. The presence of these compounds in urine can be due to the topical administration of capsaicin-based pharmaceuticals, among other sources. For the preconcentration of analytes, 600 mu L of methyl isobutyl ketone (extractant) and 1500 mu L of ethanol (dispersant) were added to 7.5 mL sample. The enriched organic phase was evaporated, reconstituted in 150 mu L of acetonitrile and injected into the chromatographic system applying an isocratic elution program. Detection was carried out using electrospray ionisation in positive mode and quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS). A matrix effect was observed with a similar behaviour for the different urines studied, so a matrix-matched calibration method was applied for quantification purposes. Method validation showed limits of detection (LODs) of 8.0, 1.5 and 0.9 mu g L-1 for PCAP, CAP and DCAP, respectively. Repeatability was evaluated using the relative standard deviation, with values between 5.3 and 7.4%. DLLME procedure provided enrichment factors between 60 and 64 for the analytes. The developed method was applied to the analysis of urine collected from people treated with CAP-based topical drugs and none of the targeted analytes were found in the analysed samples, at least above the corresponding LODs. In addition, no capsaicinoid metabolites were detected in a non-targeted analysis under the same experimental conditions.

Automated summary

This paper presents a new analytical method using DLLME and UHPLC-HRMS for the determination of capsaicin in human urine. The method showed low detection limits and good repeatability, and was successfully applied to the analysis of urine from patients treated with capsaicin-based drugs.