Eight naturally occurring capsaicinoids have been determined in Capsicum by use of high-purity standards, with norcapsaicin as an internal standard. The solid standards were rigorously checked for purity. The sensitivity of electrospray ionization (ESI), atmospheric-pressure chemical ionization (APCI), and coordination ion-spray (CIS; with silver) toward the capsaicinoids were measured and compared. The highest sensitivity was found for positive-ion ESI. Method validation of the liquid chromatography-ESI-mass spectrometry (LC-ESI-MS) determination is reported, including tests for repeatability (4%), detection limit (5 pg injected), linear range (20-6 ng injected), quantitation (excellent linearity; < 2% relative standard deviation), and recovery (99-103%). The major and minor capsaicinoids in a commercial plant extract and in chili pepper fruits were quantified.