Rapid and sensitive LC-MS/MS method for simultaneous quantification of capsaicin and dihydrocapsaicin in microdialysis samples following dermal application

Abstract

A bioanalytical LC-MS/MS method was developed and validated for the simultaneous quantification of capsaicin (CAPS) and dihydrocapsaicin (D-CAPS) in dermal microdialysis samples from rats. Capsaicinoids were separated by using a C18 column, with a mobile phase of water and acetonitrile, both with 0.1% of formic acid, eluted as a gradient. Compounds were detected by using an electrospray ionization source operating in the positive mode (ESI+) to monitor the m/z transitions of 306.1 > 137.0 for CAPS and 308.1 > 137.0 for D-CAPS. The method showed linearity in the concentration range of 0.5-100 ng/ml for CAPS and 0.25-100 ng/ml for D-CAPS, with coefficients of determination of ≥ 0.99. The inter- and intra-day precision, accuracy, and compound stability in different conditions were in accordance with the limits established by the US Food and Drug Administration guidelines. The recovery of the drugs by microdialysis were dependent on the flow rate, but independent of drug concentration. For CAPS, calibration of the in vitro microdialysis probes by dialysis and retrodialysis resulted in statistically similar drug recovery of 68.5% ± 5.9% and 77.8% ± 6.6%, respectively, at a flow rate of 0.5 μl/min. For D-CAPS, the recovery by dialysis was lower than by retrodialysis, at 51.4% ± 6.6% and 92.6% ± 2.4%, respectively. This difference was attributed to the binding of D-CAPS to the plastic tubing, which was experimentally evaluated and mathematically modeled. In vivo recoveries were 75.7% ± 6.3% for CAPS and 81.9% ± 1.5% for D-CAPS at the same flow rate. The analytical method showed high specificity, accuracy, and sensitivity, and suitability for dermatopharmacokinetic studies. These results will allow the determination of the actual free concentration of these drugs in dermatopharmacokinetic experiments, as shown in a pilot experiment with a commercial cream containing capsaicinoids.

Keywords: Bioanalytical validation; Capsaicin; Dermal microdialysis; Dihydrocapsaicin; LC–MS/MS.