Abstract
This work presents the optimization of a miniaturized liquid-liquid extraction (mLLE) followed by reversed-phase liquid chromatography-electrospray tandem mass spectrometry (RP-HPLC-MS/MS) for the determination of ethyl carbamate (EC) in fortified wine, without using derivatizing agents. The mLLE was optimized by an experimental design. Thus, 15 mL of wine and 8 mL of ethyl acetate were used for the extraction. After concentration, each extract was injected into the HPLC-MS/MS equipment and the characteristic secondary ion transition of EC (m/z = 90.10 → 62.05) was used for the quantification purposes. The proposed method presented a good linearity (R 2 = 0.9999) and a high sensitivity with low limits of detection (LOD) and quantification (LOQ), 0.17 and 0.52 μg L−1, respectively. The precision (repeatability and reproducibility) never exceeded 8% of variation, and the recoveries varied between 93 and 114%. The applicability of the method was checked through the analysis of 24 fortified wines, with EC values ranging between 23 ± 1 and 194 ± 5 μg L−1. All chromatograms revealed good peak resolutions. This new method is efficient for the simple, fast, and reliable determination of EC in fortified wines, providing great sensitivity without using derivatizing agents or large volumes of organic solvents.
Highlights
Ethyl carbamate (EC), known as urethane (PubChem CID 5641), is the ethyl ester of carbamic acid that is formed in fermented foods and beverages, namely fortified wines, by the reaction of nitrogen compounds with ethanol (JECFA 2005)
The aim of this study was the development of a simple, efficient, and sensitive method to quantify EC in fortified wines, without using derivatizing agents, minimizing the reagent consumption, and reducing the use of toxic organic solvents, through the optimization of a miniaturized liquid-liquid extraction (mLLE) procedure combined with reversed-phase liquid chromatographyelectrospray tandem mass spectrometry (RP-HPLC-MS/MS)
The influence of salts and buffers on the extraction yield was studied. Since they did not increase the extraction yield of EC, these were removed from the extraction procedure and the extraction was transformed into a mLLE, with the advantage of being cheaper and user-friendly
Summary
Ethyl carbamate (EC), known as urethane (PubChem CID 5641), is the ethyl ester of carbamic acid that is formed in fermented foods and beverages, namely fortified wines, by the reaction of nitrogen compounds with ethanol (JECFA 2005). It is recognized as a toxic compound and was reclassified in 2007 by the International Agency of Research on Cancer (IARC) as probably carcinogenic to humans (group 2A) (Baan et al 2007). The most relevant pathways for the formation of EC in alcoholic beverages have already been identified. The chemical family of EC’s main precursors are cyanide and carbamoyl compounds, namely urea, carbamoyl phosphate, citrulline, and hydrogen cyanide (Jiao et al 2014). EC is mainly formed during and after fermentation, by the reaction of carbamoyl compounds with ethanol.
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