Determination of Capsaicin, Nonivamide, and Dihydrocapsaicin in Blood and Tissue by Liquid Chromatography-Tandem Mass Spectrometry

A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the analysis of capsaicin, nonivamide, and dihydrocapsaicin in blood and tissue has been developed. The method utilized a one-step liquid-liquid extraction that yielded an approximate 90% recovery of capsaicinoids from blood. Chomatographic separation of the capsaicinoids was achieved using a reversed-phase high-performance liquid chromatography column and a stepwise gradient of methanol and distilled water containing 0.1% (v/v) formic acid. Identification and quantitation of the capsaicinoids was achieved using electrospray ionization-tandem mass spectrometry monitoring the precursor-to-product-ion transitions for the internal standard octanoyl vanillamide (m/z 280 --> 137), capsaicin (m/z 306 --> 137), dihydrocapsaicin (m/z 308 -->137), and nonivamide (m/z 294 --> 137). Calibration curves, 1.0 to 250 ng/mL, were constructed by plotting concentration versus peak-area ratio (analyte/internal standard) and fitting the data with a weighted quadratic equation. The accuracy of the assay ranged from 90% to 107% for all analytes. The intra-assay precision (%RSD) for capsaicin was 4% at 2.5 ng/mL, 3% at 10 ng/mL, and 7% at 100 ng/mL. The interassay precision (% RSD) for capsaicin was 6% at 2.5 ng/mL, 6% at 10 ng/mL, and 7% at 100 ng/mL. Similar values for inter- and intra-assay precision were obtained for nonivamide and dihydrocapsaicin. This method was used to assay for capsaicinoids in blood and tissue samples collected from rats exposed to capsaicinoids via nose-only inhalation. The concentration of capsaicin in these samples ranged from < 1.0 to 90.4 ng/mL in the blood, < 5.0 to 167 pg/mg in the lung, and < 2.0 to 3.4 pg/mg in the liver.