Determination of Capsaicinoids in Habanero Peppers by Chemometric Analysis of UV Spectral Data CHRISTOPHER B. DAVIS,CAROLYN E. MARKEY,MARIANNA A. BUSCH, AND KENNETH W. BUSCH* Center for Analytical Spectroscopy, Department of Chemistry & Biochemistry, Baylor University, One Bear Place #97348, Waco, Texas 76798 A novel spectrophotometric method for the determination of capsaicinoids in habanero pepper extracts is described that does not require prior analyte separation. The method uses partial-least-squares (PLS-1) multivariate regression modeling techniques in conjunction with ordinary UV absorption spectral data obtained on alcoholic extracts of habanero peppers (Capsicum chinese). The PLS-1 regression models were developed by correlating the known total concentration of the two major capsaicinoids (capsaicin and dihydrocapsaicin) in the extracts as determined by high-performance liquid chromatography with the spectral data. The regression models were subsequently validated with laboratory-prepared test sets. The validation studies revealed that the root-mean-square error of prediction varied from 4 to 8 ppm, based on the results obtained from models prepared from nine test sets. Once a regression model has been developed and validated, analyses of the extracts can be accomplished rapidly by ordinary spectrophotometric procedures without any prior separation steps. KEYWORDS: Capsaicin; multivariate regression modeling; UV-vis spectrophotometry; pungency INTRODUCTION Capsaicin {N-[(4-hydroxy-3-methoxy-phenyl)methyl]-8-methyl- non-6-enamide} and dihydrocapsaicin {N-[(4-hydroxy-3-meth- oxy-phenyl)methyl]-8-methyl-nonanamide} are two members of a family of naturally occurring capsaicinoids (Figure 1), which make up the pungent components of Capsicum fruits. Capsaicinoids are all N-vanillylamides of fatty acids. When evaluating the pungency of a pepper, capsaicin and dihydro- capsaicin constitute approximately 80-90% of the total cap- saicinoids present (1, 2) and account for the majority of the “hotness”. The concentrations of capsaicin and dihydrocapsaicin in a chile pepper vary, depending on the species of the pepper, its growing conditions, and the time when the pepper was harvested (3, 4). The capsaicinoids are not evenly distributed throughout the pepper, and substantial amounts of the capsai- cinoids exist in the pericarp and placenta of the individual fruits (5). Capsicum fruits have long been utilized in food preparation (6), medicinal applications (7, 8), and personal protection aerosols (3). Chile peppers and spices, such as paprika, with different capsaicin contents are used routinely in salsas and other food products (5). Pungent sauces in Mexican and Asian cuisine often contain pungent peppers such as the habanero (9). Capsaicin has been used in the treatment of rheumatoid arthritis, osteoarthritis, and other peripheral neuropathic disorders as a therapeutic pain reliever (10). Capsaicin is also used to gauge a patient’s cough threshold after ingestion of an antitussive agent (7). Recent studies have used capsaicin in other applications, such as neurobiological research (8), weight management (11, 12), local/topical analgesia (13), and antimicrobial defense (14, 15). Personal self-defense aerosols rely on the lachrymator-like effects caused when capsaicin is introduced to the mucous membranes (3, 16). In the past, organoleptic tests, such as the Scoville heat test, introduced in 1912 by Wilbur Scoville, were used to determine pepper pungency (17). These methods have been replaced with modern instrumental analyses (1, 18). Analytical methods employed for the determination of capsaicinoids have included colorimetry (19, 20), gas chromatography (GC) (21-24), liquid chromatography (LC) (9, 16, 25-35), GC/LC-MS (3, 36), NMR-flow probe analysis (37), spectrophotometry (38-45), amperometric titration (46), micellar electrokinetic capillary chromatography (47), and sensory methods such as an electronic nose (48). Of these methods, the most widely used is high-performance liquid chromatography (HPLC), which offers sufficient accuracy and precision. The standard analytical method, as described by the American Spice Trade Association, employs HPLC for testing the pungency of capsicums and their oleoresins (49). In this method, a 4.6 mm × 250 mm C-18 column (10 μm packing) is used with a mobile phase consisting of a mixture of acetonitrile, dioxane, water, methanol, and perchloric acid depending on the sample concentration. For samples greater than 700 ppm, absorption UV detection at 280 nm is used. For samples less than 700 ppm, fluorescence detection is employed * To whom correspondence should be addressed. Tel: +12547103311. Fax: +12547104272. E-mail: kenneth_busch@baylor.edu. J. Agric. Food Chem. 2007, 55, 5925-5933 5925 10.1021/jf070413k CCC: $37.00 © 2007 American Chemical Society Published on Web 07/04/2007