Chile habanero

J. Agric. Food Chem. 2006, 54, 9303−9311

9303

Determination of Capsaicin and Dihydrocapsaicin in Capsicum Fruits by Liquid Chromatography−Electrospray/Time-of-Flight Mass Spectrometry
ANA GARCES-CLAVER,† MARIA S. ARNEDO-ANDRES,† JAVIER ABADIA,§ Ä Ä Ä Ä † AND ANA ALVAREZ-FERNANDEZ*,§ RAMIRO GIL-ORTEGA, Ä Ä
Technology for Plant Production Department, Centro de Investigacion y TecnologıaAgroalimentaria ´ ´ (CITA), Apartado 727, E-50080 Zaragoza, Spain; and Plant Nutrition Department, Estacion ´ Experimental de Aula Dei, Consejo Superior de Investigaciones Cientıficas (CSIC), Apartado 202, ´ E-50080 Zaragoza, Spain

A simple, highly selective, sensitive, and reproducible liquid chromatography-electrospray ionization/ time-of-flight mass spectrometry method has been developed forthe direct and simultaneous determination of capsaicin and dihydrocapsaicin in Capsicum fruit extracts. Capsaicin and dihydrocapsaicin are the two major members of the so-called capsaicinoid family, which includes other minor analogues, and usually account for at least 90% of the pungency trait in Capsicum fruits. Chromatographic separation of capsaicin and dihydrocapsaicin was achieved with areversed-phase chromatography column, using a gradient of methanol and water. Quantification was done using as an internal standard (4,5-dimethoxybenzyl)-4-methyloctamide, a synthetic capsaicin analogue not found in nature. Analytes were base-peak resolved in less than 16 min, and limits of detection were 20 pmol for capsaicin and 4 pmol for dihydrocapsaicin. The intraday repeatability values werelower than 0.5 and 12% for retention time and peak area, respectively, whereas the interday repeatability values were lower than 0.6 and 14% for retention time and peak area, respectively. Analyte recoveries found were 86 and 93% for capsaicin and dihydrocapsaicin, respectively. The method developed has been applied to the identification and quantification of capsaicin and dihydrocapsaicin infruit extracts from different Capsicum genotypes, and concentrations found ranged from 2 to 6639 mg kg-1.
KEYWORDS: Capsaicin; Capsicum; dihydrocapsaicin; liquid chromatography; mass spectrometry; pepper; time-of-flight

INTRODUCTION

Fruits of pepper (Capsicum) plants are among the most consumed spices throughout the world. These fruits contain capsaicinoids, a family of compounds that givethem the characteristic pungent taste. The two major capsaicinoids, capsaicin and dihydrocapsaicin, are responsible for up to 90% of the total pungency of pepper fruits (1, 2). Besides capsaicin and dihydrocapsaicin, at least nine minor capsaicinoids (Figure 1) have been shown to occur in peppers (3-7). Capsaicinoid compounds have been widely studied and are currently used in the food industry, formedical purposes, as pharmaceuticals, and in defensive sprays (8-15). Capsaicinoids are alkaloids produced by a condensation reaction between an aromatic moiety and a C9-C11 branchedchain fatty acid. The aromatic moiety is vanillylamine, a
* Corresponding author (e-mail ana.alvarez@eead.csic.es; telephone +34976716052; fax +34976716145). † Centro de Investigacion y Tecnologıa Agroalimentaria(CITA). ´ ´ § Consejo Superior de Investigaciones Cientıficas (CSIC). ´

phenylalanine derivative. Branched-chain fatty acids are biosynthesized from valine and leucine, and in the case of capsaicin and dihydrocapsaicin those acids differ in the degree of unsaturation of the 9-carbon fatty acid side chain (1, 16). Suzuki and Iwai (17) reviewed aspects related to the chemistry, analysis,biochemistry, and pharmacology of the pungency principle of Capsicum spp. Further work on the chemistry and analysis methods for total and individual capsaicinoids has been reviewed by Govindarajan (18), Anu et al. (19), and Pruthi (20). Capsaicinoids are synthesized exclusively in the epidermal cells of the placenta of Capsicum fruits and are accumulated in blisters along the epidermis (21-23). Their...