2009 Annual Report 1a.Objectives (from AD-416) The objectives include:. 1)To develop laboratory-based multiclass, multiresidue analytical methods for veterinary drugs in animal-derived foods, feeds, and marker matrices (e.g., tissues, blood, urine);. 2)to develop field-based instruments and methods for analytical screening of chemicals of food safety/security concern (or nutritional interest) in foods;. 3)to develop field-portable and laboratory-based analytical methods for the screening and reliable identification of biotoxins and phytochemicals in foods;. 4)devise a scientifically defensible and practical system to evaluate qualitative identification of chemical residues in complex matrices; and. 5)employ and adapt progressive analytical concepts previously developed for pesticides (QuEChERS and related approaches) to meet other food safety and security applications (e.g. dioxins). 1b.Approach (from AD-416) The project involves the application and evaluation of rapid and sensitive lab-based approaches to dependably detect mutliple veterinary drug classes in animal-derived foods predominantly using atmospheric pressure ionization/tandem mass spectrometry. Similarly, for multiple chemical residues and toxins in meats, poultry, eggs, fruits, vegetables, and grains, the approach involves the development and evaluation of rapid lab-based quantitative and confirmatory analytical approaches predominatly using as chromatography/tandem mass spectrometry. Furthermore, field-based screening techniques for chemical contaminants in foods at tolerance levels will be developed. The results obtained by these novel methods will be compared to those obtained by traditional techniques currently in use. 3.Progress Report This project addresses the problem related to the lack of rapid, automated, cost-effective, waste-minimizing, safe, and high-quality analytical methods to detect multiple chemical residues and other toxic compounds in foods. The project is devised to meet the needs of the USDA Food Safety Inspection Service (FSIS), Food and Drug Administration (FDA), and other organizations that monitor chemical residues in food, which also includes industry, consumer groups, and academic scientists. We have made very good progress for each milestone. In the case of Milestone 1, we have mostly completed the method development phase of the project to effectively and efficiently monitor >75 drug residues in animal-derived foods, and now we are in the process of method validation and technology transfer of the approach with FSIS. We are devising a turn-key protocol with training for FSIS to implement the method in their monitoring laboratory as a replacement of the 7-plate bioassay that they currently use. In the case of Milestone 2, a field-portable residue screening instrument was built and tested in the field for analysis of tetracyclines in aquacultural food products. A new system has been built for fluoroquinolones and malachite green for screening analysis of catfish in particular. For Milestone 3, different rapid and simple methods for Staphylococcal Enterotoxins A (SEA) and B (SEB) were completed, and the project is transitioning as planned into new methods for the analysis of phytochemicals and phenolics in foods. A rapid and inexpensive test has been devised and tested, and it is ready for technology transfer activities. Milestone 4 was substantially completed with respect to presenting a proposed approach to identify the presence or absence of chemical residues monitored by mass spectrometry. Members of the analytical and regulatory community may choose to adopt this approach if they find it advantageous as we do. Milestone 5 was also completed in that a rapid, qualitative and quantitative screening method for trace determination of dioxins in fatty foods (cod liver oil) was developed and single-laboratory validated. It does not meet the needs for ultratrace level risk assessment purposes, but it can be used for regulatory and process control purposes. 4.Accomplishments 1. Developed methods for multiresidue analysis of aminoglycoside antibiotics. Aminoglycosides are widely applied antimicrobial drugs in current veterinary practice. In the U.S., the USDA Food Safety and Inspection Service (FSIS) uses a semi-quantitative microbial assay to determine violative levels of aminoglycosides (and some other antibiotics) in animal tissues. For positive findings, this assay is followed by a confirmatory LC-MS/MS method based on a laborious sample preparation using cartridge-based solid-phase extraction (SPE). The aim of this project was to develop a streamlined approach for multiresidue LC-MS/MS analysis of aminoglycosides that would provide fast screening and eliminate the microbial assay and the SPE step. The streamlined sample preparation method involves simple extraction followed by DPX for cleanup, which is faster, reduces material cost and solvent use, and enables easy automation. After elution from the DPX tip, 12 target aminoglycosides are analyzed using a 3 min UPLC-MS/MS method, which should allow much greater sample throughput than the current approach used by FSIS. 2. A new instrument approach provides a multiclass, multiresidue lab-based screening method for veterinary drug residues. For lab-based testing of veterinary drug residues in the US food supply, the USDA Food Safety and Inspection Service (FSIS) is predominantly using microbial inhibition assays, immunoassays, and thin-layer chromatography. Liquid chromatography-mass spectrometry (LC-MS) is employed mainly for confirmation of positive responses and in some specific methods to analyze a small number of selected veterinary drugs. The goal of this work is to develop a fast screening method for a large number of veterinary drugs using ultra-performance LC (UPLC)-tandem MS (MS/MS) to replace multiple non-specific tests performed in the FSIS laboratories. UPLC-MS/MS conditions were optimized for nearly 200 veterinary drugs from different classes (antibiotics, anthelmintics, coccidiostats, beta-agonists, corticosteroids, NSAIDs, tranquilizers, thyreostats, etc.). Our current streamlined sample preparation method for beta-lactam antibiotics was tested for a group of more than 100 veterinary drugs in bovine kidney. Evaluation and optimization of the sample preparation method for the larger group of veterinary drugs is being done in various target matrices using fast UPLC-MS/MS analysis. Method validation and technology transfer is being done to provide a more efficient and effective drug residue monitoring system than currently used by FSIS. 3. Developed an improved pesticide residue analysis. Additional investigations were conducted of the “quick, easy, cheap, effective, rugged, and safe” (QuEChERS) approach for the extraction of hundreds of pesticides from many kinds of foods. The approach typically provides 4-fold reductions in sample preparation time, labor, waste, and material expenses over previous methods. Major analytical supply companies such as Restek, Sigma-Aldrich, UCT, ThermoFisher, Agilent, Waters, and Varian sell QuEChERS sample preparation products that generate income on the order of hundreds of thousands of dollars for the vendors. This year, our comparison studies of different versions of QuEChERS showed that AOAC Official Method 2007.01 provided the best results for the most pesticides in different fruits and vegetables. Regulatory laboratories in the USDA, FDA, EPA, states in the U.S., and countries on 6 continents are routinely using official versions or adaptations of QuEChERS approaches (as are many private contract and university labs). 4. Developed a rapid and easy detection method for phenolic antioxidants. Antioxidants are generally recognized to be important for improving human health and increasing longevity. Fast and simple detection methods are needed for the detection of phenolic antioxidants in foods and beverages. We developed a novel method for direct measurement of polyphenol anti-oxidants in foods and beverages through a chemical interaction. The method involves mixing a unique dye with the samples containing phenolic compounds followed by addition of a buffer, and the resulting color change is measured. Analysis of 20 samples can be completed in less than 3 hours (automation can increase throughput), and the cost of the reagent is less than one cent per analysis. Results from analysis of many beverages, fruits, grains, and other foods of interest by this new method were compared against the 100 year-old standard method. Our method shows higher polyphenol content. Further investigations may demonstrate that the new method may be used in place of the current standard method. The analytical results are beneficial to regulatory agencies, producers, and consumers interested in knowing relative polyphenolic content of foods. The novel method may also provide economic benefit to consumers if utilized in food labeling. 5. To meet regulatory needs, determined distribution of penicillin G residues in culled dairy cow muscle. The U.S. Food and Drug Administration sets tolerance levels for veterinary drug residues in muscle, but does not specify which type of muscle should be monitored. In this work, we compared the levels of penicillin G residues in varied muscle types from dosed, culled dairy cows. Levels of penicillin G in the corresponding muscle juice samples were also determined, in order to establish the acceptability of using the readily accessible juice as opposed to the more difficult to extract muscle samples. Results show that penicillin G levels can vary substantially depending on the muscle group and animal. This work will assist federal and other regulatory agencies in making decisions regarding sampling for monitoring purposes. 6. Extended an advantageous pesticide residue analysis method to additional commodities. A research and technology transfer project was established with General Mills to extend and validate a state-of-the-art approach to detect chemical residues in cereals, grains, and similar commodities to high fatty matrices, such as doughs, flaxseeds, and peanuts. The QuEChERS sample preparation method was successfully adapted for the commodities of interest. The company chemists have validated and implemented the approach for ca. 200 pesticides in the new matrices. This project has substantially improved the pesticide residue monitoring program within the company while saving time, labor, and other expenses in the process. 7. Developed a sorbent to reduce chlorophyll from vegetable extracts. Chlorophyll from green vegetables can pose problems in the gas chromatographic analysis of pesticide residues. A research and technology transfer project was established with UCT to prepare a new sorbent material to selectively remove chlorophyll during pesticide residue analysis. We developed a polymeric material that removes 90% of the chlorophyll from QuEChERS extracts, but which still yields 100% recoveries of hundreds of pesticides. UCT has devised a large-scale production protocol for the material and is marketing it as ChloroFiltrTM along with their current scope of QuEChERS products. This will reduce the maintenance needed on instruments for routine pesticide residue monitoring. 6.Technology Transfer Number of the New/Active MTAs (providing only) 1 Number of Invention Disclosures Submitted 1 Review Publications Martos, P.A., Lehotay, S.J., Shurmer, B. 2008. Ultra-Trace Analysis of Nine Macrolides, including Tulathromycin A (Draxxin), in Edible Animal Tissues with Mini-Column Liquid Chromatography Tandem Mass Spectrometry. Journal of Agriculture and Food Chemistry. 56(19):8844-8850. Schneider, M.J., Mastovska, K., Lehotay, S.J., Lightfield, A.R., Kinsella, B., Shultz, C.2009. Comparison of screening methods for antibiotics in beef kidney juice and serum. Analytica Chimica Acta. 637:290-297. Kinsella, B., Lehotay, S.J., Mastovska, K., Lightfield, A.R., Furey, A., Danaher, M. 2009. New Method for the Analysis of Flukicide and Other Anthelmintic Residues in Bovine Milk and Liver using Liquid Chromatography - Tandem Mass Spectrometry. Anal. Chim. Acta. (637):196-207. Hoh, E., Lehotay, S.J., Mastovska, K., Ngo, H., Vetter, W., Pangallo, K., Reddy, C. 2009. Simultaneous quantitation of multiple classes of organohalogen compounds in fish oils with direct sample introduction comprehensive two-dimensional gas chromatography and time-of-flight mass spectrometry. Journal of Agricultural and Food Chemistry. 57(7):2653-2660. Lehotay, S.J., Mastovska, K., Amirav, A., Fialkov, A.B., Alon, T., Martos, P.A., Kok, A., Fernandez-Alba, A.R. 2009. Identification and confirmation of chemical residues by chromatography-mass spectrometry and other techniques. Trends in Analytical Chemistry. 27(11):1070-1090. Mastovska, K., Lightfield, A.R. 2008. Streamlining methodology for the multiresidue analysis of beta-lactam antibiotics in bovine kidney using liquid chromatography-tandem mass spectrometry. Journal of Chromatography A. 1202:118-123. Hoh, E., Lehotay, S.J., Mastovska, K., Huwe, J.K. 2008. Evaluation of automated direct sample introduction with comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry for the screening analysis of dioxins of fish oil. Journal of Chromatography A. 1201(1):69-77. Cajka, T., Hajslova, J., Mastovska, K. 2008. Mass spectrometry and hyphenated instruments in food analysis. In: Otles, S., editor. CRC Press. p. 119-144. Hoh, E., Lehotay, S.J., Mastovska, K., Ngo, H., Vetter, W., Pangallo, K.C., Reddy, C.M. 2009 Capabilities of Direct Sample Introduction - Comprehensive Two-Dimensional Gas Chromatgraphy-Time-of-Flight Mass Spectrometry to Analyze Organic Chemicals of Interest in Fish Oils. Environmental Science and Technology. 43:3240-3247.