Selected product ion monitoring for quantification of 5-hydroxymethylfurfural in food products by capillary zone electrophoresis-tandem ion trap mass spectrometry
Introduction
During heat treatment of foods, complex chemical reactions take place, contributing to desired color, taste and aroma of heated foodstuff. The advanced stage of the Maillard reaction (MR) is characterized by the accumulation of undesirable compounds, such as furfurals, that are useful tools used to evaluate the severity of heat treatment applied and the effect of storage (Rada-Mendoza et al., 2004, Ramírez-Jiménez et al., 2001). In details, two pathways leading to furfural production start from sugar decomposition: the caramelisation, where the reducing carbohydrates suffer 1–2 enolisation, dehydration and cyclisation reactions, and the MR, where the Amadori product is submitted to enolisation and dehydration of the sugar moiety, releasing the intact amino acid (Cardenas Ruiz et al., 2004, Kroh, 1994, Ramírez-Jiménez et al., 2000).
5-hydroxymethylfurfural (HMF) is a common product of these two reactions. In addition to temperature, the amount of HMF in foods is dependent on the type of sugar, pH, water activity and cations concentration (Capuano & Fogliano, 2011).
HMF represents a recognized parameter for evaluating food freshness and quality, especially for honey and apple juice (Directive 2001/110/EC, 2001, Khalil et al., 2010). Several food matrices can form furfurals, and the amount of HMF is directly related to the food processing and storage. A common source of HMF is represented by ingredients used in the formulation such as caramel solutions or honey. In 2011, EFSA revised a scientific opinion about caramel colors (E 150 a, b, c, d), defining that some of its constituents, including 5-HMF and furans, may be present in food products at levels that may be of concern. Therefore it considers that the specifications should include maximum levels for these constituents (EFSA, 2011).
Although the concentrations in some food items are extremely high, bread and coffee are the most important contributors to dietary intake (Murkovic & Pichler, 2006). The estimated exposure is several orders of magnitude higher than the daily intake for other heat-induced food toxicants such as acrylamide and furan (Morehouse et al., 2008, Svensson et al., 2003). Based on data reported in literature it is not clear whether human exposure to HMF represents a potential health risk (Capuano & Fogliano, 2011): the major concern is related to its enzymatic conversion to sulphoxymethylfurfural (SMF) which has been reported to be mutagenic (Lee et al., 1995, Surh et al., 1994), as also reported by EFSA in a report concerning its genotoxic potential (EFSA, 2005). Moreover, SMF in the blood of mice after HMF intravenous administration has been recently detected (Monien, Frank, Seidel, & Glatt, 2009), and associated risk for humans may be higher since human enzymes are more active than in rodents (Capuano & Fogliano, 2011). Although the correlation of adverse health effects and exposure to HMF is not conclusive (Abraham et al., 2011, Janzowski et al., 2000), analytical measurements of HMF in foods seem opportune for an objective risk assessment, as well as for quality evaluation of food. Therefore, new methods with a simplified protocol and lower cost are still in demand to ensure the high-throughput screening and the high efficiency. The common analytical technique employed is HPLC-UV (Aquino et al., 2006, Gaspar and Lucena, 2009, Pereira et al., 2011, Spano et al., 2009, Zappalá et al., 2005), reference method of the Association of the Official Analytical Chemists (AOAC, 1996). During the last years several analytical methods have been developed. Liquid chromatography with pulsed amperometric detection (Xu, Templeton, & Reed, 2003), refractive index detection (Xu et al., 2003), or coupled to mass spectrometry (Gökmen and Senyuva, 2006, Teixidó et al., 2008) have been used. Recently, gas chromatography coupled to mass spectrometry (Teixidó, Santos, Puignou, & Galceran, 2006), and electrochemical biosensors (Lomillo, Campo, & Pascual, 2006) have been proposed for HMF analysis in honey, baby-foods, jam, orange juice and bakery products. However, LC methods proposed often require long analysis time and consume considerable amounts of solvents, while a derivatization procedure is mandatory in GC analysis to increase volatility and overcome adsorption to the column.
In a recent paper, a detailed study of the Direct Analysis in Real Time mass spectra of carbohydrates and HMF using a single quadrupole mass spectrometer has been reported. However, the accurate DART-MS quantitation of HMF in carbohydrates-rich matrices was possible only with a high resolution-mass spectrometer and/or tandem MS (Chernetsova & Morlock, 2012).
Capillary electrophoresis (CE) with UV detection has been selected as an alternative technique to LC for the quantification of HMF in breakfast cereals, toast, honey, orange and apple juice, jam, coffee, chocolate, by employing the micellar electrokinetic capillary chromatography (MECK) mode (Chen and Yan, 2009, Corradini and Corradini, 1992, Corradini and Corradini, 1994, Morales and Jimenez-Perez, 2001, Rizelio et al., 2012, Teixidó et al., 2011, Wong et al., 2012). To the best authors' knowledge, no applications were carried out by capillary-electrophoresis tandem mass spectrometry (CE-MS2) for quantitation of HMF in food. This technique represents an interesting challenge as allows the combination of low costs in terms of solvent, short time of analysis and good results regarding selectivity and sensibility, as shown by recent papers (Bignardi et al., 2012, Bonvin et al., 2012, Huhn et al., 2010).
In this work, a new analytical method employing CE-MS2 for determination of HMF in different foodstuffs was developed and validated. Selected MS/MS ion monitoring (SMIM) was chosen as acquisition mode, programming the detector to perform continuous MS/MS scans on a selected precursor. This acquisition mode has been shown to be convenient when dealing with complex matrices, because it provides the MS/MS spectrum of the analyte to confirm its nature (Jorge et al., 2007).
Analytical conditions and sample preparation (without any pretreatment as SPE purification, often employed) were optimized and applied to different kind of food products. The results were also compared to those obtained with a HPLC-UV method.
Section snippets
Chemicals
All chemicals were of analytical reagent grade. Formic acid, trichloroacetic acid, ammonium hydroxide (25%, w/w), hydrochloric acid (37%, w/w), acetic acid (100%) and sodium hydroxide (50%, w/w) were obtained from J. T. Baker (Deventer, The Netherlands). Methanol (Chromasolv) was purchased from Sigma Aldrich (Milan, Italy). HPLC water was obtained with a MilliQ element A10 System (S. Francisco, CA, USA). 2-Furylmethylketone (FMK) used as internal standard (IS), and 5-hydroxymethylfurfural (99%
Sample preparation
In the first part of the work, the most opportune sample extraction method was adopted on the basis of the best HMF recovery rate. Different combinations of extractant/clarifying agent proposed in previous papers (Cardenas Ruiz et al., 2004, Ramírez-Jiménez et al., 2000) have been studied by comparing them in terms of repeatability of analysis, recovery rate and better protein precipitation (Ameur, Trystram, & Birlouez-Aragon, 2006). In our work we selected the method proposed by Chen and Yan
Conclusion
Capillary electrophoresis is well known to present several positive features such as high efficiency, simple instrumentation, rapid method development, relatively short analysis time, and low solvent/sample consumption. In this context, the combination of capillary electrophoresis with mass spectrometry provides an attractive perspective and presents some major benefits. Among them, MS enhances the selectivity of CE and enables the determination of co-migrating compounds with different m/z
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