Critical Review
Analysis of 2-alkylcyclobutanones for detection of food irradiation: Current status, needs and prospects

https://doi.org/10.1016/j.jfca.2011.11.006Get rights and content

Abstract

It is believed that 2-alkylcyclobutanones (2-ACBs) are formed in food only by irradiation, and they are therefore considered as useful markers for detecting the irradiation of food. This review compares the numerous techniques available for the extraction of the 2-ACB-containing lipid fraction of foods and the isolation of 2-ACBs from the fat matrix, along with the detection techniques used. Particular attention is given to the widely adopted European Standard EN1785 procedure, and the performance of variations and similar methods. The paper highlights the need to improve analytical methods in order to detect irradiated ingredients in composite foods, and to resolve the emerging possibility that some non-irradiated foods may contain 2-ACBs. Approaches are proposed that might best enhance the sensitivity of the EN1785 method.

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.

References (81)

  • M. Miesch et al.

    Efficient reaction pathway for the synthesis of saturated and monounsaturated 2-alkylcyclobutanones

    Radiation Physics and Chemistry

    (2002)
  • M. Miesch et al.

    2-Alkylcyclobutanones as markers for irradiated foodstuffs. I. Synthesis of saturated and unsaturated standards

    Radiation Physics and Chemistry

    (1999)
  • K.M. Morehouse et al.

    A gas-chromatographic method for the identification of gamma-irradiated frog legs

    Radiation Physics and Chemistry

    (1990)
  • B. Ndiaye et al.

    2-Alkylcyclobutanones as markers for irradiated foodstuffs – III. Improvement of the field of application on the EN1785 method by using silver ion chromatography

    Journal of Chromatography A

    (1999)
  • B. Ndiaye et al.

    2-Alkylcyclobutanones as markers for irradiated foodstuffs II. The CEN (European Committee for Standardization) method

    Radiation Physics and Chemistry

    (1999)
  • T. Pena et al.

    Optimization of a microwave-assisted extraction method for the analysis of polycyclic aromatic hydrocarbons from fish samples

    Journal of Chromatography A

    (2006)
  • F. Priego-Capote et al.

    Focused microwave-assisted Soxhlet extraction: a convincing alternative for total fat isolation from bakery products

    Talanta

    (2005)
  • F. Priego-Capote et al.

    Fast method for the determination of total fat and trans fatty-acids content in bakery products based on microwave-assisted Soxhlet extraction and medium infrared spectroscopy detection

    Analytica Chimica Acta

    (2004)
  • R. Rahman et al.

    A rapid method (SFE-TLC) for the identification of irradiated chicken

    Food Research International

    (1996)
  • I.H. Tewfik et al.

    A rapid supercritical fluid extraction method for the detection of 2-alkylcyclobutanones in gamma-irradiated beef and chicken

    Food Science and Technology-Lebensmittel-Wissenschaft & Technologie

    (1998)
  • M. Virot et al.

    New microwave-integrated Soxhlet extraction an advantageous tool for the extraction of lipids from food products

    Journal of Chromatography A

    (2007)
  • Anon, 2003. EN 1785. Foodstuffs – detection of irradiated food containing fat – gas chromatographic/mass spectrometric...
  • I.S. Arvanitoyannis

    Detection of irradiated foods

  • I.S. Arvanitoyannis et al.

    Irradiation applications in vegetables and fruits: a review

    Critical Reviews in Food Science and Nutrition

    (2009)
  • L.S. Arvanitoyannis et al.

    Impact of irradiation on fish and seafood shelf life: a comprehensive review of applications and irradiation detection

    Critical Reviews in Food Science and Nutrition

    (2009)
  • D.R. Boyd et al.

    Synthesis, characterization, and potential use of 2-dodecylcyclobutanone as a marker for irradiated chicken

    Journal of Agricultural and Food Chemistry

    (1991)
  • CEN

    European Standard (EN 1785). The Detection of Irradiated Foods Containing Fat – Gas Chromatography/Mass Spectrometric Analysis of 2-alkylcyclobutanone

    (2003)
  • S. Chen et al.

    Identification of irradiated prawn (Penaeus monodon) using thermoluminescence and 2-alkylcyclobutanone analyses

    Journal of Agricultural and Food Chemistry

    (2011)
  • A.V.J. Crone et al.

    Effect of storage and cooking on the dose–response of 2-dodecylcyclobutanone, a potential marker for irradiated chicken

    Journal of the Science of Food and Agriculture

    (1992)
  • A.V.J. Crone et al.

    Synthesis, characterization and use of 2-tetradecylcyclobutanone together with other cyclobutanones as markers for irradiated liquid whole egg

    Journal of the Science of Food and Agriculture

    (1993)
  • M. Eissen et al.

    Electrophilic bromination of alkenes: environmental, health and safety aspects of new alternative methods

    Chemistry – A European Journal

    (2008)
  • C.T. Elliott et al.

    Detection of irradiated chicken meat by analysis of lipid extracts for 2-substituted cyclobutanones using an enzyme-linked-immunosorbent-assay

    Analyst

    (1995)
  • European Commission

    Regulation (EEC) No 2568/91 of 11 July 1991 on the characteristics of olive oil and olive–residue oil and on the relevant methods of analysis

    Official Journal

    (1991)
  • Fielding, L., 2007. The Safety of Irradiated Foods: A Literature Review. Technical Report. Food Standards Agency,...
  • P. Gadgil et al.

    2-Alkylcyclobutanones as irradiation dose indicators in irradiated ground beef patties

    Journal of Agricultural and Food Chemistry

    (2002)
  • P. Gadgil et al.

    Evaluation of 2-dodecylcyclobutanone as an irradiation dose indicator in fresh irradiated ground beef

    Journal of Agricultural and Food Chemistry

    (2005)
  • L.E. Garcia-Ayuso et al.

    Accelerated extraction of the fat content in cheese using a focused microwave-assisted Soxhlet device

    Journal of Agricultural and Food Chemistry

    (1999)
  • F.D. Gunstone et al.

    Occurrence and characteristics of oils and fats

  • L. Hamilton et al.

    Detection of 2-substituted cyclobutanones as irradiation products of lipid-containing foods: synthesis and applications of cis- and trans-2-(tetradec-5′-enyl)cyclobutanones and 11-(2′-oxocyclobutyl)-undecanoic acid

    Journal of the Chemical Society-Perkin Transactions

    (1996)
  • L. Hamilton et al.

    The use of 2-substituted cyclobutanone in the development of an enzyme-linked immunosorbant assay (ELISA) for the detection of irradiated foods

  • Cited by (35)

    • An investigation using a validated method based on HS-SPME-GC-MS detection for the determination of 2-dodecylcyclobutanone and 2-tetradecylcyclobutanone in X-ray irradiated dairy products

      2022, LWT
      Citation Excerpt :

      This approach contributes to overcome the strong reluctance of consumers and to obtain some acceptance of this treatment in the food field. Nowadays, in Europe, screening and confirmatory validated or standardized protocols are available to identify whether or not food products have been treated with ionising radiation (Crews, Driffield, & Thomas, 2012; Delincée, 2002; Mangiacotti, Marchesani, Floridi, Siragusa, & Chiaravalle, 2013; Zanardi, Caligiani, & Novelli, 2018). Some procedures are chemical methods, based on determination of specific radio-induced markers, as 2-Alkylcyclobutanones (2-ACBs) (Driffield et al., 2014).

    • Low-energy X-ray inactivation of Salmonella Enteritidis on shell eggs in mono-/co-culture biofilms with Pseudomonas fluorescens

      2021, Food Control
      Citation Excerpt :

      Despite of containing much lower lipid content (1.6%) than yolk (29.36 ± 3.29%), 2-DCB was detected in irradiated unpolished rice at the dose of only 100 Gy using electron beam (2.2 MeV) (Horvatovich, Miesch, Hasselmann, & Marchioni, 2002). The formation of 2-ACBs requires a high and fast energy input to the CO- group of fatty acid molecules and only ionizing irradiation is considered to meet this demand (Crews, Driffield, & Thomas, 2012). Conventional irradiation methods exclusively refer to 60Co gamma-ray (~2.5 MeV), electron beam (~10 MeV), and high-energy X-ray (~5 MeV), whose energy levels are approximately 10–100 times higher than that of the low-energy X-ray (~150 KeV) used in this study.

    • Inactivation of Salmonella Typhimurium, Escherichia coli O157:H7, Staphylococcus aureus, and Listeria monocytogenes in cardamom using 150 KeV low-energy X-ray

      2021, Innovative Food Science and Emerging Technologies
      Citation Excerpt :

      Ionizing irradiation can result in triglycerides breakage and generate 2-alkylcyclobutanones (i.e., 2-ACBs) in fat-containing food (Zanardi et al., 2007). To date, 2-ACBs are not detected in natural foodstuff or formed after any processing treatments other than irradiation (Crews, Driffield, & Thomas, 2012). Accordingly, these compounds are often used as marker molecules to identify ionizing irradiated foods.

    View all citing articles on Scopus
    View full text