John Gilbert

Validation of analytical methods for determining mycotoxins in foodstuffs

The European Union (EU) has established demanding regulatory limits for controlling aflatoxins B1, B2, G1 and G2, in cereals, nuts, nut products and dried fruit, aflatoxin M1 in milk, and ochratoxin A in cereals. These limits are likely to be extended in the future to additional commodities and other mycotoxins. For enforcement purposes and in particular for resolving any disputes between parties, it is essential that validated methods are available, with performance characteristics that meet certain minimum criteria. As such methods were not available and had not previously been validated either for matrices of interest in Europe or at the low European limits compared to the USA, the EU funded a method-validation project to fulfil this requirement. Immunoaffinity column clean-up methods with HPLC determination were established for aflatoxins B1, B2, G1 and G2 in peanut butter, pistachios, fig paste and paprika, aflatoxin B1 in baby food, aflatoxin M1 in liquid milk, and ochratoxin A in roasted coffee and baby food. For patulin in apple juice and apple puree, solvent extraction and solid-phase clean-up HPLC methods were developed. To undertake collaborative studies, particular care was taken in preparation of naturally-contaminated test materials containing the toxins at levels close to regulatory limits and in demonstrating the homogeneity of batches of material. To ensure that participants in the validation exercise could follow the procedures to be tested, videos of the methods were prepared showing, in particular, any critical steps. Prior to undertaking the method validation, participants were invited to collaborative study workshops to ensure that they fully understood the methods and their role in the study. This care in planning and executing the collaborative studies led to impressive performance characteristics and adoption of six procedures by AOAC International as First Action Methods and seven methods by CEN as European standards. The valuable lessons learned in undertaking these validation exercises are now being put to further use in studies aimed at validating methods for mycotoxins in foodstuffs, which are appropriate for developing countries based on TLC as the end determination but use more modern sample clean-up techniques.

Bioactive compounds in foods

Introduction. Professor John Gilbert , Central Science Laboratory, Sand Hutton, York, UK and. Dr Hamide Z. Senyuva , Ankara Test and Analysis Laboratory, Scientific and Technical Research Council of Turkey, Ankara, Turkey. Part 1 - Natural toxicants . 1. Pyrrolizidine alkaloids . Colin Crews , Central Science Laboratory, Sand Hutton, York, UK and. Professor Dr Rudi Krska , Christian Doppler Laboratory for Mycotoxin Research, Center for Analytical Chemistry, Department for Agrobiotechnology (IFA-Tulln), University of Natural Resources and Applied Life Sciences, Tulln, Austria. 2. Glucosinolates . Dr Ruud Verkerk and Dr Matthijs Dekker, Department of Food Technology and Nutrition Science, Agricultural University Wageningen, The Netherlands. 3. Phycotoxins in seafoods . Dr John Leftley Scottish Association for Marine Science, Oban, Argyll, Scotland, UK and Integrin Advanced Biosystems Ltd., Marine Resource Centre, Barcaldine, Argyll, Scotland, UK and. Dr Fiona Hannah , University of London, University Marine Biological Station, Millport, Isle of Cumbrae, Scotland, UK. 4. Mushroom toxins. Professor Jana Hajslova , Institute of Chemical Technology, Prague, Czech Republic. 5. Mycotoxins . Mr Keith Scudamore , KAS Mycotoxins, Taplow, Berkshire, UK. 6. Phytoestrogens . Dr Don Clarke , Central Science Laboratory, Sand Hutton, York, UK. 7. ss-Carboline alkaloids . Dr T. Herraiz , Spanish Council for Scientific Research, Madrid, Spain. 8. Naturally-occuring Nitrates and Nitrites in foods. Dr M. Reinik , Estonian Health Protection Inspectorate, Tartu Laboratory, Tartu, Estonia. Dr T Tamme , Estonian University of Life Sciences, Department of Food Science and Hygiene, Tartu, Estonia. Dr M. Roasto , Estonian University of Life Sciences, Dept. of Food Science and Hygiene, Tartu, Estonia. Part II Man-made components . 9. Acrylamide in heated foods . Dr Hamide Z. Senyuva , Ankara Test and Analysis Laboratory, Scientific and Technical Research Council of Turkey, Ankara, Turkey. Dr Vural Gokmen , Food Engineering Department, Hacettepe University, Ankara, Turkey. 10. Furan in processed foods . Dr Imre Blank , Science Department Head, Nestle Product Technology Center, Orbe, Switzerland. 11. Chloropropanols and chloroesters. Dr Colin Hamlet , RHM Technology Ltd, The Lord Rank Centre, High Wycombe, Bucks, UK. 12. Hetrocyclic amines . Dr M. Knizel , University of California, Lawrence Livermore National Laboratory, Biology & Biotechnology Research Program, Livermore, CA, USA. 13. Polycyclic Aromatic Hydrocarbons . Dr M. Rose , Central Science Laboratory, Sand Hutton, York, UK. Dr Laura Cano-Lerida , Johnson Matthey Catalysis, Belasis Avenue, Chilton, Billingham, UK. Dr P. Walton , Department of Chemistry, University of York, Heslington, York, UK.

Rapid analysis of fungal cultures and dried figs for secondary metabolites by LC/TOF-MS.

A liquid chromatography-time-of-flight mass spectrometry (LC/TOF-MS) method has been developed for profiling fungal metabolites. The performance of the procedure in terms of mass accuracy, selectivity (specificity) and repeatability was established by spiking aflatoxins, ochratoxins, trichothecenes and other metabolites into blank growth media. After extracting, and carrying out LC/TOF-MS analysis, the standards were correctly identified by searching a specially constructed database of 465 secondary metabolites. To demonstrate the viability of this approach 11 toxigenic and four non-toxigenic fungi from reference collections were grown on various media, for 7-14 days. The method was also applied to two toxigenic fungi, A. flavus (200-138) and A. parasiticus (2999-465) grown on gamma radiation sterilised dried figs, for 7-14 days. The fungal hyphae plus a portion of growth media or portions of dried figs were solvent extracted and analysed by LC/TOF-MS using a rapid resolution microbore LC column. Data processing based on cluster analysis, showed that electrospray ionization (ESI)-TOF-MS could be used to unequivocally identify metabolites in crude extracts. Using the elemental metabolite database, it was demonstrated that from culture collection isolates, anticipated metabolites. The speed and simplicity of the method has meant that levels of these metabolites could be monitored daily in sterilised figs. Over a 14-day period, levels of aflatoxins and kojic acid maximised at 5-6 days, whilst levels of 5-methoxysterigmatocystin remained relatively constant. In addition to the known metabolites expected to be produced by these fungi, roquefortine A, fumagillin, fumigaclavine B, malformins (peptides), aspergillic acid, nigragillin, terrein, terrestric acid and penicillic acid were also identified.

Advances in Sampling and Analysis for Aflatoxins in Food and Animal Feed

Advances in analytical methods and novel detection systems are reviewed from publications from 1995 onwards. The review covers aflatoxins B1, B2, G1, G2, and their metabolites in food, animal feed and biological matrices such as blood and urine. Improved extraction techniques, new clean‐up methods and optimized methods for specific matrices are summarized. This review highlights methods such as thin layer chromatography (TLC) and high performance‐TLC (HPTLC), which are particularly suited to developing countries, and advances that have been made in TLC quantification through low cost detection and scanning systems. Novel developments in detection of aflatoxins are assessed such as the application of surface plasmon resonance biosensors, flow injection monitoring, fibre optic sensors, capillary electrokinetics, electrochemical transduction, and immunological‐based rapid test kits. Recent advances in confirmatory techniques such as liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (MS/MS) for aflatoxins are also covered. This review summarizes performance data from recent collaborative studies, and assesses the overall quality of analytical data for aflatoxins being produced worldwide, as evidenced by results from various proficiency testing schemes. Although there are only a few recent studies on sampling for aflatoxins, the recent progress in this area is also assessed.