A study on the equivalence of olive oil and the EU official substitute test media for migration testing at high temperatures

As a consequence of an increasing awareness and findings about the technical and analytical difficulties related to the employment of triglycerides as fatty food simulants in migration testing, EU Directive 82/711/ EEC on the basic rules of migration has been amended for the second time and adapted with the recent Commission Directive 97/48/EC. The major merit of this important amendment is to authorize alternative tests such as extraction tests and to allow substitute tests for the fat test with concrete indication of corresponding test conditions to be applied. As a novelty in food law compliance testing it authorizes a modified polyphenylene oxide (MPPO), well-known as Tenax®, a thermostable and highly adsorptive porous polymer, as a 'solid' matrix to substitute fat simulant D at temperatures equal to or higher than 100°C. In the study presented here, comparative migration testing overall and specific was carried out between fat simulants olive oil and ¹⁴C-labelled HB 307 and the substitute test media iso-octane, 95% ethanol and MPPO using polypropylene materials as examples at test temperatures of 100°C to 120°C and prescribed corresponding substitute test conditions. As a consequence, a number of conclusions were drawn which are relevant for overall and/or specific migration testing under the investigated test conditions. One of the major findings was that MPPO was generally more equivalent to the fat test than the other two substitute test media. The presence of volatile hydrocarbons in the test samples was found to play a crucial role with respect to the equivalence of the iso-octane-based substitute test. At high volatile concentrations in the test sample the iso-octane test underestimated the olive oil benchmark whereas low volatile concentrations led to satisfying equivalence. It is therefore recommended that the procedure is modified and GC-FID screening of the isooctane extract is carried out. As a logical consequence, the possibility of quantitative determination of the overall extract by GC-FID is recommendable and should be investigated in more depth. Concerning the overall migration test procedure in olive oil, the vacuum pre-conditioning at 60°C should be given re-consideration to avoid the possibility of loss of volatile migrants whose determination should be within the scope of the overall migration test method itself. Finally, the specific migration related equivalence between olive oil, MPPO and iso-octane needs further investigations to clarify the net effect of strongly increased diffusion rates at the regular high temperature versus the substitute test solvent accelerated extraction effect at 60°C. Stability aspects of migrants at the different test temperatures deserve further consideration.     

Evaluation and validation of two fluorometric HPLC methods for the determination of aflatoxin B1 in olive oil

Two methods for the determination of aflatoxin B₁(AFB₁) in olive oil were tested and compared. In method A the oil sample was mixed with methanol + water (60 + 40), extracted with hexane and then with chloroform. Chloroform was evaporated and the residue was dissolved with dichloromethane which was then transferred for clean-up onto a silica 'Sep-Pak' cartridge. The cartridge was pre-washed with hexane, ethyl ether and dichloromethane. AFB₁ was eluted with chloroform + acetone (9 + 1) and evaporated to dryness. In method B, the oil sample was mixed with methanol + water (80 + 20), shaken and centrifuged. The supernatant was diluted 1:10 with water and 10ml of the diluted mixture transferred to an 'Aflaprep' immunoaffinity column for the clean-up step. AFB₁ was eluted with acetonitrile and evaporated to dryness. AFB₁ from both methods was derivatized to its hemiacetal (AFB_2a ) and then quantitated by HPLC using a C₁₈ (60 A 4.6 x 250 mm) column with fluorescence detection. Both methods are simple, reliable and efficient, but method A showed a lower detection limit (2.8 ng/kg) than method B (56 ng/kg). With a 95% confidence level there was no significant difference in recovery between the two methods, which was 87.2% for method A and 84.8% for method B. In addition, application of a two-tailed F-test to the variances within spiked samples at concentrations 1, 2, 5 and 10 mu g/kg separately showed that there was no significant difference in the precisions of the two methods. Fifty samples of olive oil of Greek origin produced between 1995 and 1998 were examined with both methods for the presence of AFB₁. When analysing the samples with method B, the presence of AFB₁ was not detected. The use of method A revealed the presence of AFB₁ in 72% of the samples. The range of contamination was generally found to be very low (2.8-15.7 ng/kg), however one sample was contaminated with 46.3 ng/kg.     

Looking through the qualities of a fluorimetric assay for the total phenol content estimation in virgin olive oil,olive fruit or leaf polar extract

As an alternate to the Folin-Ciocalteu assay (F-C) we propose a fluorimetric estimation of the total phenol content in virgin olive oil (VOO), olive fruit and leaf polar extracts. Phenol content was determined at excitation/emission wavelengths set at 280/320 nm. Standard operational procedures (slit widths, temperature, pH) and method validation were carried out according to Eurachem guidelines. The qualities of the proposed assay are better than those of the F-C one, as the procedure is more sensitive (LOD and LOQ values 10-fold lower), three times faster, needs no reagents and most importantly, is not destructive for the sample that can be further used in HPLC or other assays. Data for VOO extracts correlated well with the colorimetric ones (r = 0.69, n = 65). HPLC coupled with diode array and fluorescence detectors supported the above findings. Good correlations were also found between the respective data for olive fruit and leaf extracts (r = 0.96, n = 18).     

An internet compendium of analytical methods and spectroscopic information for monomers and additives used in food packaging plastics

An internet website (http://cpf.jrc.it/smt/) has been produced as a means of dissemination of methods of analysis and supporting spectroscopic information on monomers and additives used for food contact materials (principally packaging). The site which is aimed primarily at assisting food control laboratories in the European Union contains analytical information on monomers, starting substances and additives used in the manufacture of plastics materials. A searchable index is provided giving PM and CAS numbers for each of 255 substances. For each substance a data sheet gives regulatory information, chemical structures, physico-chemical information and background information on the use of the substance in particular plastics, and the food packaging applications. For monomers and starting substances (155 compounds) the infra-red and mass spectra are provided, and for additives (100 compounds); additionally proton NMR are available for about 50% of the entries. Where analytical methods have been developed for determining these substances as residual amounts in plastics or as trace amounts in food simulants these methods are also on the website. All information is provided in portable document file (PDF) format which means that high quality copies can be readily printed, using freely available Adobe Acrobat Reader software. The website will in future be maintained and up-dated by the European Commission's Joint Research Centre (JRC) as new substances are authorized for use by the European Commission (DG-ENTR formerly DGIII). Where analytical laboratories (food control or other) require reference substances these can be obtained free-ofcharge from a reference collection housed at the JRC and maintained in conjunction with this website compendium.