Elsevier

Journal of Chromatography A

Volume 1453, 1 July 2016, Pages 10-18
Journal of Chromatography A

An in situ derivatization – dispersive liquid–liquid microextraction combined with gas-chromatography – mass spectrometry for determining biogenic amines in home-made fermented alcoholic drinks

https://doi.org/10.1016/j.chroma.2016.05.052Get rights and content

Highlights

  • DLLME-GC–MS method was developed for separation of biogenic amines in home-made wine.

  • Derivatization and extraction processes are performed in the same time.

  • Two procedures of DLLME were proposed and examined.

  • Chemometric analysis was performed to choose better procedure and conditions of derivatization reaction.

  • The developed method was successfully applied to the analysis of home-made wine samples not regulated by low.

Abstract

A novel dispersive liquid–liquid microextraction (DLLME) gas chromatography mass-spectrometry (GC–MS) method was developed for the determination of 13 biogenic amines in home-made wine samples. The method allows to simultaneous extraction and derivatization of the amines providing a simple and fast mode of extract enrichment. During the study, two different procedures were examined. Statistical analysis was performed to choose better procedure, as well as the conditions of derivatization reaction. At least, a mixture of methanol (dispersive solvent; 215 μL), chloroform (extractive solvent; 400 μL), and isobutyl choloroformate (derivatizing reagent; 90 μL) was used as extractive/derivatizing reagent, added to 5 mL of sample. The addition of mixture of pyridine and HCl was necessary to eliminate the by-products. The proposed method showed good linearity (correlation coefficients >0.9961), good recoveries (from 77 to 105%), and good intra-day precision (below 13%) and inter-day precision (below 10%). Moreover, detection limits were never over 4.1 μg/L. The developed method was successfully applied to the analysis of 17 home-made wine samples not regulated by law. All of the biogenic amines analyzed were found in most of the wines.

Introduction

Biogenic amines (BAs) refer to all amines arising from the metabolism of living cells, whether they be animal, vegetable or microbial. BAs can be also found in a broad range of food products, mainly in protein-rich foods (plant and animal origin), however, they are also found in fermented products (foods produced or preserved by the action of microorganisms) such as wine, beer, kefir, etc. [1].

Although, BAs consumed at low concentrations are essential for many physiological functions, when consumed in large quantities, their pharmacological activity may become toxic [1], [2]. For this reason, the consumption of foods containing high levels of BAs may cause some adverse effects in sensitive individuals, such as nausea, headache, cardiac palpitations, hypo- or hypertension, renal intoxication and respiratory disorders [2], [3].

Biogenic amines are naturally present in wine. Due to the fact that ethanol and acetaldehyde can increase the risk effects on human health directly or indirectly inhibiting enzymes responsible for detoxification, the presence and the level of BAs in wine have received much attention [4], [5]. According to Ganic et al. [6], the toxic dose of BA in alcoholic beverages varies between 8 and 20 mg/L for histamine, between 25 and 40 mg/L for tyramine, and only 3 mg/L for phenethylamine. Biogenic amines in wine are of great interest not only for their potential risk to human health. Another reason of the interest for the BAs in wine is fact that they could have a role as chemical indicators of unwanted microbial contamination or deficient processing conditions [7]. The relevance of sensory and toxicological implications of BAs has contributed to the proliferation of analytical methods to quantify these compounds in wines. Various research papers concerning the determination of biogenic amines in wine have been published recently [8], [9], [10].

Although official regulation of the European Union (EU) about the maximum content of ABs in wine not exist, monitoring and analysis in order to determine the content of ABs is required by many importers of wine in the EU [11]. Unfortunately, this kind of regulations are not introduced in all countries. Moreover, production on a large-scale of wine or fermented alcoholic drinks made from different kind of fruits (also called home-made wine) is also carried out in home, and next, this kind of wine is admitted to trading as a regional product. Despite the fact that there are law regulations governing this type of production (for example in Poland, an Act made on production and bottling of wine products, marketing of these products and market organization for wine was introduced on 22 January, 2004 [12]), they often do not contain information on possible concentration limits of BAs. Therefore, in order to protect human health and life, it is important to control not only the wine industry, but also home-made wines, both produced as regional products and those produced for own use. For that reasons, the development of new analytical methods, which in a quick, inexpensive and environmentally safe allowed to qualitative and quantitative determination of each of biogenic amines in wine is essential.

Different analytical techniques have thus been used to determine BAs in wine samples, particularly these include, liquid chromatography (LC) [8], [13], [14], capillary electrophoresis (CE) [10], [15] and an enzymatic method [16]. Although, gas chromatography is not that common to BAs determination, several methods with application of this method have also been developed [17], [18].

The determination of amines in a complex matrix such as wine is very difficult because they are usually present at low concentrations (a few mg/L to about 90 mg/L). Moreover, they absorb at low wavelengths, a region where numerous interfering substances are present, such as polyphenols. It also must be mentioned that many ABs do not possess such properties that allow their determination using the appropriate techniques coupled with specific detectors (e.g. GC–MS). These problems have been solved by using a derivatization process, for example to displace the absorbance wavelength to higher values, thus improving the detection limit of the method. Derivatization process also allows to reduce the polarity and reactivity and increase the volatility of the amine, as is desirable for the analysis of a sample by GC. This helps to increase the sensitivity and selectivity, and thus to reduce the limit of quantification.

To obtain optimal analysis conditions, an appropriate sample preparation that could include an extraction cleanup step or concentration and derivatization procedures, are usually mandatory. To overcome drawbacks of liquid–liquid extraction technique, alternative methods have been applied, such as solid-phase extraction [19], solid-phase microextraction [20], hollow fibre liquid-phase microextraction [21] or dispersive liquid–liquid microextraction (DLLME) [22]. Taking into account advantages of this extraction technique, the most simple and rapid approach seems to be the DLLME procedure. The advantages of DLLME method are simplicity of operation, rapidity, low cost, high recovery, and enrichment factor. Moreover, due to the small aliquots of solvents used, this technique is numbered among environmental friendly methods of extraction, and has been successfully applied for extraction of analytes with high or moderate lipophilic properties [23], [24] including amines [25]. For high hydrophilic compounds, hardly extracted with the water-immiscible solvents used in DLLME, in situ derivatization is required to increase the yields [25], [26]. To the best of our knowledge the use of a DLLME procedure for the simultaneous extraction and derivatization of BAs in wine samples was not yet reported.

The main objective of this study was to develop and validate a reliable DLLME/GC–MS method for simultaneous determination of selected biogenic amines that are expected to be present in wine samples. It needs to be noted, that wine samples that have been analyzed are coming from home-made production that not regulated by law.

During the optimization of DLLME procedure, special attention was given on the careful evaluation of the nature and amount of extractive and dispersive solvents as well as the type and amount of derivatizing reagent (DR) and the reaction time. The developed method was used to assess the occurrence of selected biogenic amines in fermented alcoholic drinks so called home-made wine samples. To the best of our knowledge, there is no data concerning the biogenic amines analysis in wine samples by using DLLME method with simultaneous derivatization by using isobutyl chloroformate, therefore, this study enriches knowledge in the field of wine analysis. Moreover, there is no data on home-made wine analysis.

Experimental design (full factorial design on two levels of variation of the input factors) was used to help in the effort to find optimal experimental conditions.

Section snippets

Reagents and materials

The amine standards (butylamine, cadaverine, dietyloamine, dimetyloamine, etyloamine, histamine, metyloamine, propylamine, putrescine, spermine, tryptamine, tyramine, and 2-phenyletyloamine) and internal standard (hexylamine, IS1) were obtained, mostly as hydrochloride salts, from Sigma Aldrich (Steinheim, Germany). Stock standard solutions (1.0 mg/mL) of each free compound were prepared by weighing and dissolving in 0.1 M HCl (Procedure 1) or in deionized water (Procedure 2). The solutions were

Optimization of extraction and derivatization conditions

Due to the reason that nature of the extractive and dispersive solvents, solvents volume, type and volume of DR, and extraction and derivatization time can affect the DLLME efficiencies, these parameters were systematically studied in order to achieve a good sensitivity, selectivity and precision for all biogenic amines in study.

Conclusions

A simultaneous DLLME coupled with derivatization procedure followed by GC–MS analysis was developed and validated for a reliable determination of 13 biogenic amines in home-made wine samples. The developed methods enable extraction and derivatization processes being carried out simultaneously with acceptable enrichment factors. The main conclusions of the study can be summarized as follows. An efficient and high reproducible extraction was achieved by DLLME-derivatization procedure for all

Acknowledgments

The research is funded by the Polish Ministry of Science and Higher Education within the “Iuventus Plus” program in years 2015–2017, project no: IP2014 037573. The authors would like to thank Vineyard Anna de Croy from Słupsk, Poland.

References (26)

  • M. Rezaee et al.

    Determination of organic compounds in water using dispersive liquid–liquid microextraction

    J. Chromatogr. A

    (2006)
  • C. Almeida et al.

    A novel dispersive liquid–liquid microextraction (DLLME) gas chromatography mass spectrometry (GC–MS) method for the determination of eighteen biogenic amines in beer

    Food Control

    (2012)
  • A. Naila et al.

    Control of biogenic amines in food—existing and emerging approaches

    J. Food Sci.

    (2010)
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