Analytical Methods Headspace gas chromatography–mass spectrometry determination of alkylpyrazines in cocoa liquor samples María del Rosario Brunetto a, * , Yelitza Delgado Cayama a , Lubin Gutiérrez a , Sabrina Clavijo Roa a , Yaritza Contreras Méndez a , Máximo Gallignani a , Alexis Zambrano b , Álvaro Gómez b , Gladys Ramos b a Laboratorio de Espectroscopia Molecular, Departamento de Química, Facultad de Ciencias, Universidad de Los Andes, Apartado Postal 3, Ipostel La Hechicera, Mérida 5101-A, Venezuela b Proyecto ICCO-CFC-INIA, Instituto Nacional de Investigaciones Agrícolas (INIA), Avenue Urdaneta Edificio Ministerio de Agricultura y Tierras, P.O. Box 425 Ipostel, Mérida 5101-A, Venezuela article info Article history: Received 27 November 2007 Received in revised form 26 May 2008 Accepted 27 May 2008 Keywords: Gas chromatography Headspace Cocoa Alkylpyrazines Mass spectrometer detection Maillard reaction products abstract This paper proposes a fast, accurate, and quantitative method for alkylpyrazines determination in cocoa liquors samples using gas chromatography with ion trap mass spectrometry detection (GC/IT-MS) and on-line headspace (HS) sample introduction. The optimization of the experimental conditions of the headspace gas chromatography–mass spectrometry (HS–GC–MS) system was carried out using the uni- variate method, aiming to find a compromise between time of the analysis, sensitivity and pyrazine res- olution in a Carbowax (AV-WAX) fused silica capillary column. The procedure was validated through accuracy and precision studies. The results showed a highly satisfactory accuracy rate for the method with a relative standard deviation (RSD) during 1 day and between days of <5.0% (n = 5). On the other hand, the recovery percentages (94–99%) were quantitative in all cases, with a RSD of <4.0% (n = 5). Finally, the use the on-line headspace extraction step simultaneously with the analytes separation and detection of the previous sample injection increased the analysis frequency to 3 samples/h. A total of 120 cocoa liquor samples were analyzed. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction During the last few years the conditions to produce cocoa with a strong aromatic potential have been tried to be optimized, and this is why some researchers point out that the optimization of the co- coa’s roasting conditions will allow the maximum use of the bean’s aromatic potential. It is well known that aroma precursors in cocoa beans developed in fermentation are converted to cocoa specific aroma during the roasting via the Maillardbrowning reaction (Barel, Leon, & Vincent, 1985). Barel, Gallois and Silwar (Barel et al., 1985; Gallois, 1984; Silwar, 1988), among others, have shown that the principal cocoa aroma compounds are formed after a thermal treatment by non-enzymatic browning reactions be- tween reducing sugars and amino acids. These reactions produce, particularly the pyrazines (Hashim & Chaveron, 1994; Serra Bonve- hi & Ventura Coll, 2002). There are many pyrazines in the cocoa ar- oma, whose concentrations may be variable depending on the time and temperature of the thermal treatment. In order to use these compounds as an indicator of the degree of cocoa roasting it is nec- essary to count with selective and sensible methods that allow evaluating the pyrazines in cocoa liquor samples. In that sense, most identification and determination methods of pyrazines in foods, for example, cocoa, have been carried out by gas or liquid chromatography (Holm, 1991). In most of them, a sample process- ing was carried out included a series of extraction steps, where va- por dragging or distillation systems followed by liquid–liquid extractions and more recently solid-phase micro extraction were generally used (Blanch, Tabera, Herraiz, & Reglero, 1993; Counet, Callemien, Ouwerx, & Collin, 2002; Pawliszyn, 1997; Schermann & Schieberle, 1997; Stevenson, Chen, & Mills, 1996). Supercritical fluid extraction (SFE) has proved to be suitable for the extraction of pyrazines and has been used as an experimental set-up for extracting cocoa beans from Ghana and Malaysian cocoa beans (Marsin Sanagi, Hung, & Yasir, 1997). However, in general, the whole sample processing may be tedious, it takes a lot of time and provides low recovery percentages from some of the analytes of interest. The HS manipulation procedure may be indicated for the isola- tion and pre-concentration of volatile analytes (Steinhart, Stephan, & Bucking, 2000) from different matrixes. Compared to solvent extraction, equilibrium HS–GC reduces significantly sample prepa- ration time for the analysis of volatile flavor components. This is why, in order to carry out the alkylpyrazine analysis, in this work a quantitative method using a GC-MS system introduc- ing an on-line sample processing using the HS technique was 0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2008.05.082 * Corresponding author. Tel.: +58 414 0787449; fax: +58 274 2714223. E-mail address: brunetto@ula.ve (María del Rosario Brunetto). Food Chemistry 112 (2009) 253–257 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem