Critical ReviewAnalysis of 2-alkylcyclobutanones for detection of food irradiation: Current status, needs and prospects
Highlights
► Review describes formation and analysis of 2-alkylcyclobutanones in irradiated food. ► Techniques for extraction of lipids and 2-alkylcyclobutanones from foods are compared. ► Detection techniques and method performance are compared. ► Particular attention is given to the widely adopted European Standard EN1785. ► The need to improve analytical methods is explained and approaches proposed.
Introduction
Irradiation of food is useful for the sterilisation of products that are prone to microbial attack, especially for foods that are sold without heat treatment, such as raw poultry, meat, and seafood. It is also useful in slowing ripening of fruit and for inhibiting sprouting of plant products such as potatoes. However, there is a need to detect the use of food irradiation to ensure that food is labelled correctly and to allow informed consumer choice. It is a legal requirement in many countries that foods containing an irradiated ingredient be labelled as such. The use of irradiation as a preservation procedure requires the existence of tests capable of distinguishing between irradiated foodstuff and non-irradiated foods in order to regulate international trade and to meet labelling requirements. The legal aspects of food irradiation were outlined in 1999 (Masotti and Zonta, 1999).
Several methods are available to detect food irradiation. For fatty foods, the major methods are based on the chemical determination of compounds formed from the irradiation of lipid components. The preferred method for such foods is the European Committee for Standardization (CEN) method EN1785 (CEN, 2003). This method is based on measurement of 2-alkylcyclobutanones (2-ACBs), that are produced by the irradiation of fatty acids and glycerides. Since this detection test was first developed in the 1990s there have been advances in analytical techniques, and new methods of isolating and detecting 2-ACBs are available. Until recently there have been no reports of the presence of 2-ACBs in food that has not been irradiated, but a single contrary finding has been published recently (Variyar et al., 2008) which will be described in detail later in this review.
Improvements to method EN1785 are required to verify whether 2-ACBs can be detected in non-irradiated food or in food processed by means other than irradiation, i.e. to eliminate any false positive results. Improvements could also usefully widen the scope of the method and permit detection of the use of irradiated ingredients in composite foods. Conversely, lowering of the limit of detection would increase the need to confirm the absence of 2-ACBs in foods that have not been irradiated and possibly the need to derive a threshold value to distinguish irradiated from non-irradiated foods with confidence.
This paper reviews the current literature in respect of the various analytical methods available to detect the irradiation of food that are based on 2-ACB determination. It describes the limitations of and modifications to the methods, and offers some alternative approaches that are within the capabilities of most regulatory laboratories.
Several approaches have been made to detecting irradiation of foods. The methods include: photostimulated luminescence and thermoluminescence, Electron Spin Resonance Spectroscopy, tests of microbiological quality, detection of damaged DNA by the comet assay, and detection of 2-ACBs by chemical analysis. We have identified about 20 published reviews of analytical methods for the detection of irradiation, varying in their depth and scope. However, only four make any significant mention of 2-ACBs (Stevenson, 1994, Delincee, 1998, Fielding, 2007, Arvanitoyannis et al., 2009a, Arvanitoyannis et al., 2009b, Arvanitoyannis, 2010), and these are described later in the relevant sections of this review.
Section snippets
Formation of 2-ACBs
Food irradiation is carried out by the use of accelerated electron beams, X-rays, or gamma radiation (60Co or 137Cs). The irradiation dose varies with application. Ionising radiation induces the formation of a variety of hydrocarbons and 2-ACBs from lipids (Kim et al., 2004a). Their formation was first reported by Le Tellier and Nawar (1972) who irradiated triacylglycerols (triglycerides) at a high dose (60 kGy). The formation was dose related, with both hydrocarbons and 2-ACBs increasing with
Occurrence of 2-ACBs
A list of the major 2-ACBs found in irradiated foods is given in Table 1. A number of acronyms have been used in various publications that describe 2-ACBs, and unfortunately as the number of known 2-ACBs and the number of publications increase the variety of acronyms used may lead to confusion. In earlier studies and in the bulk of reports published to date, the major 2-ACBs that are 2-dodecylcyclobutanone (C12 chain) and 2-tetradecylcyclobutanone (C14 chain) were referred to as 2-DCB and 2-TCB
Analysis of 2-ACBs
The detection of 2-ACBs was based on research that showed that lipid degradation products (2-ACBs and hydrocarbons) could be used to detect irradiated foods that contain fat, including meat, fish, shrimp, cheese, and liquid egg products (Morehouse and Ku, 1990).
The major analytical methods are based on extraction of the lipid fraction of food using a solvent such as hexane, fractionation of the lipid phase on a column of deactivated Florisil or silica, separation of the 2-ACBs by gas
Gas chromatography columns
The EN1785 method suggests use of a non-polar (100% dimethyl polysiloxane) column of short length (e.g. 12 m). In practice, longer (25–30 m) columns of slightly higher polarity (e.g. 5% phenyl) have more often been used. A longer and more polar (OV-20-MS) column was used by Horvatovich et al. (2005) with both the EN1785 method and with SFE to detect monounsaturated 2-ACBs. The column separated 2-ACBs from aldehydes that coeluted when columns recommended in EN1785 were used.
Electron impact mass spectra of 2-ACBs
The mass spectra of
Method performance comparisons
A brief summary of the performance of some of the analytical methods is provided in Table 3. However, reliable comparison of method performance is made difficult by the lack of data provided by many authors. Statistical studies of method performance are so uncommon that detailed comparison is impossible. The performance of the methods depends mainly on the limit of detection and the recovery of 2-ACBs. As most methods assume that 2-ACBs are absent from non-irradiated foods, there has been
Effects of storage and cooking
Storage at 10 °C did not have a significant effect on the 2-TCB content of irradiated mangoes; the concentration remained constant over 14 days by which time the mangoes had started to deteriorate (Stewart et al., 2000). Similarly, 30 mango samples were correctly identified as non-irradiated or irradiated (0.1–1 kGy) detecting 2-TCB, even after storage of half the samples for 14 days at 10 °C. For papaya, 2-DCB could be used to detect doses as low as 0.1 kGy but only soon after irradiation whereas
Effect of food processing on 2-ACB formation
The possibility should be considered that food processing technologies other than irradiation can form ACBs.
In model system studies aqueous suspensions of the triacylglycerols glyceryl tricaproate (tricaprin), glyceryl trilaurate (trilaurin), glyceryl trimyristate (trimyristin), glyceryl tripalmitate (tripalmitin) and glyceryl tristearate (tristearin) subjected to the various physical treatments listed below did not form 2-ACBs (Ndiaye et al., 1999b).
Microwave heating for 20 min, 750 W output,
2-ACBS in non-irradiated foods
Until recently, the analysis of non-irradiated foods, for example in all the controlled trials of foods likely to be treated with irradiation, has provided no evidence of 2-ACB without irradiation. Recently, however, evidence has been presented for the natural existence of 2-DCB, 2-tetradecenylcyclobutanone, and 2-tetradecylcyclobutanone in cashew nuts, and of 2-decylcyclobutanone and 2-DCB in nutmeg (Variyar et al., 2008). The 2-ACBs could not be detected using a Soxhlet extraction of 30 g nuts
Discussion
Many alternative procedures and variations to EN1785 have been proposed or adopted. However, few of these alternative procedures have been validated by interlaboratory trials.
Ndiaye et al. (1999a) have stated that in order to determine 2-ACBs in foods with low (<1%) fat content irradiated at low doses (0.5 kGy). Where a food sample contains 1% fat a 20 g sample would be required to provide the necessary 0.2 g fat. The 2-ACBs are extracted from the fat into 0.2 ml solvent. The detection limit of
Recommendations
In practical terms, improvement of the limit of detection of the 2-ACB analytical method in ways available to a large number of laboratories can best be achieved by:
- 1.
Increasing (if necessary) the sample size to provide about 2 g of fat extract.
- 2.
Permitting a variety of extraction procedures. Replacement of the Soxhlet method is unnecessary but the use of direct extraction with acetonitrile is worthy of further investigation.
- 3.
Optimising the separation of 2-ACBs from the extracted fat. This should
Knowledge gaps
Knowledge is lacking regarding the true quantitative performance of the methods currently used for extracting 2-alkylcyclobutanones (2-ACBs) for detecting irradiation in food, namely in terms of the optimum procedures for separating 2-ACBs from fat, the limits of detection of irradiated ingredients in foods, the relative yield of 2-ACBs from fatty acids at different positions of triacylglycerols, the stability and fate of 2-ACBs on storage and food processing, and most importantly the possible
Acknowledgement
Financial support for the preparation of the review was provided by the UK Food Standards Agency.
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