New method for the analysis of heterocyclic amines in meat extracts using pressurised liquid extraction and liquid chromatography–tandem mass spectrometry
Introduction
In recent years, many studies have focused on the role of certain heterocyclic amines (HAs) as mutagenic/carcinogenic compounds found in protein rich foods, such as meat and fish, when processed at home or in a restaurant [1], [2]. These compounds are of major concern in the aetiology of human cancers such as colon, prostate and breast cancers [3], [4]. To measure the effects of these mutagens/carcinogens in human consumption, it is essential to isolate, purify and identify these compounds. With information on the amount of mutagen in protein rich foods, risks to human health can be assessed. Currently, more than 20 HAs have been identified and isolated as potent mutagens from various food samples [5].
The analysis of HAs is quite challenging for food researchers because they are present at very low concentrations in heat-treated foods. An intensive clean-up procedure is required to adequately determine the concentration of these compounds. Treatment can involve a variety of purification techniques: liquid–liquid extraction [6], solid phase extraction (SPE) [7], [8], [9], [10], followed by various separation and detection techniques, such as, liquid chromatography with both UV (LC-UV) [11] and fluorescence detection (LC-Flu) [12], gas chromatography–mass spectrometry (GC–MS) [13], liquid chromatography–mass spectrometry (LC–MS) [14], [15], capillary electrophoresis [16] and ELISA immunoassay [17].
In recent years purification methods developed by Gross [9] and modified by Toribio et al. [10] have been widely used as reference methods. However, such procedures require large amounts of organic solvents and involve laborious purification steps. In an attempt to overcome these limitations, an alternative extraction technique, pressurised liquid extraction (PLE) was developed. In general, this technique is just as effective as conventional extraction techniques but with the advantage of an important reduction in time and solvent requirement. Nowadays, this technique is considered a simple and fast sample preparation technique [18], [19], [20] that combines elevated temperatures (50–200 °C) and high pressures (1.0 × 104–1.4 × 104 kPa) with conventional liquid solvents allowing both improved mass transfer and increased extraction efficiency [21], [22]. This technique has been used for the extraction of organic compounds from food, environmental samples and pharmaceutical products [23], [24], [25], [26], [27], [28], [29]. Hence, this method is more appropriate for routine analysis of HAs in meat samples.
The aim of this study was to develop a rapid method based on PLE to extract HAs from meat samples. By using a mixed organic solvent as the extractant, at high pressure and temperature, the sample can be injected directly, without the need for any further clean-up. To demonstrate the suitability of the new method, it was used to analyse a laboratory reference material. Following this, several cooked meat samples, including roast beef loin, grilled beef fillet and fried beef chop, were analysed to further demonstrate the applicability of the method.
Section snippets
Chemicals and materials
Gradient-grade methanol, acetonitrile and HPLC-grade dichloromethane, ethyl acetate and acetone, were purchased from Merck (Darmstadt, Germany). Water was purified in a Milli-Q Simplicity 185 system (Millipore, Bedford, MA, USA). Analytical grade ammonia solution (25%, w/w) was purchased from Fluka (Buchs, Switzerland). Hydrochloric acid (HCl) (37%, w/w) and sodium hydroxide (NaOH) were supplied by Panreac (Barcelona, Spain). Diatomaceous earth and anhydrous sodium sulphate were provided by
Optimisation of PLE method
As there are no previous studies into the extraction of HAs from meat samples using PLE and liquid chromatography–tandem mass spectrometry (LC–MS/MS) preliminary studies were carried out on standard solutions of HAs. Solvent type, temperature and extraction time are the most critical factors affecting extraction efficiency [33]. To find optimum conditions for HA extraction from meat samples dichloromethane, and mixtures of dichloromethane with ethyl acetate and acetone were tested. Once the
Conclusions
LC–MS/MS has proved to be a robust method for the analysis of HAs in food samples. In addition, this selected extraction method offers advantages in eliminating the extensive clean-up process of extracts prior to analysis, reducing the sample manipulation and total extraction time (by nearly four-fold compared to the conventional SPE technique). This increases the possibilities of process automation. The optimised PLE method produced high extraction efficiency for HAs with only a minor presence
Acknowledgements
This study was carried out with financial support from the Ministerio de Ciencia y Tecnologia, Spain, research project AGL2003-03100. The authors would like to thank the Serveis Científico Técnics of the University of Barcelona for their technical support. M.R.K. is also very grateful to the Spanish Ministerio de Ciencia y Tecnologia for his Ph.D. grant.
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