2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? In Jan., 2006, this project was initiated and replaced 1935-42000-044-00D, Advanced Techniques for the Analysis of Chemical Residues in Foods. This new 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. This pertains to the ARS National Program 108 Food Safety 2006-2010 Action Plan Problem Statements 1.2.1 (Detection and Validation – “Detection and quantitation of … toxins, and chemical contaminants are the central challenge to any food safety [and food security] system.”) and 1.2.9 (Food Security – “At present the Program can and will only focus on detection and prevention, with detection technologies and their validation in food systems being the principal directive.”). Moreover, the research addresses Agency Performance Measure 3.1.2: Develop and transfer to Federal agencies and the private sector, methods and systems that rapidly and accurately detect, identify, and differentiate critical and economically important foodborne toxins and chemical residues. The health of consumers is adversely affected by the presence of harmful chemicals in food, and pesticide, veterinary drug, and other chemical residues in food are a serious concern of consumers in the U.S. and worldwide. Nearly all nations have set maximum residue limits for chemical residues in food which are enforced by regulatory agencies using methods of analysis. The project meets the needs of the USDA Food Safety Inspection Service (FSIS), Food and Drug Administration, and other organizations that monitor chemical residues in food, which also includes industry, consumer groups, and academic scientists. New analytical methods are needed to expand the range of veterinary drug, pesticide, and other chemical analytes of toxicological concern that can be detected in animal and plant derived food products in a more efficient process. The overall goal of this project is to develop better approaches using advanced technologies and techniques for the rapid and reliable analysis of chemical residues and biosecurity agents in foods. The investigators are attempting to meet this overall goal with 5 objectives: . 1)develop laboratory-based multiclass, multiresidue analytical methods for veterinary drugs in animal-derived foods, feeds, and marker matrices (e.g. tissues, blood, urine);. 2)develop field-based instruments and methods for analytical screening of chemicals of food safety/security concern (or nutritional interest) in foods;. 3)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 identifications 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. analysis of dioxins). The implementation of rapid and reliable approaches will serve to increase the percentage of the food supply monitored and thereby: improve food safety and security; increase productivity and/or lower costs; provide more statistically valid and accurate results for risk assessment and other purposes; overcome trade barriers associated with the analysis of chemical residues; provide more information to understand the effects and mechanisms of antimicrobial resistance and endocrine disruption; allow for better verification of organic food labeling; improve possible industrial HACCP programs; and reduce the potential for food that has been deliberately or accidentally adulterated by toxic chemicals to reach the consumer. 2.List by year the currently approved milestones (indicators of research progress) I – Develop laboratory-based multiclass, multiresidue analytical methods for veterinary drugs in animal-derived foods, feeds, and marker matrices (e.g. tissues, blood) A. Determine LC-MS/MS conditions and limits of detection for as many drug analytes as feasible - to be completed FY07 B. Determine stability of the drug analytes at different conditions - to be completed FY07 II - Develop field-based instruments and methods for analytical screening of chemicals of food safety/security concern (or nutritional interest) in foods. A. Fluorescence screening method development and instrumentation field testing - to be completed FY07 I – Develop laboratory-based multiclass, multiresidue analytical methods for veterinary drugs in animal-derived foods, feeds, and marker matrices (e.g. tissues, blood) C. Determine the intrinsic fluorescence of the drugs to help devise simple screening techniques and LC/fluorescence detection methods - to be completed FY08 II - Develop field-based instruments and methods for analytical screening of chemicals of food safety/security concern (or nutritional interest) in foods. B. Sorbent extraction and solid-matrix luminescence - to be completed FY08 III - Develop field-portable and laboratory-based analytical methods for the screening and reliable identification of biotoxins and phytochemicals in foods. A. Development of biosensor methods and low-tech immunoassays for the analysis of toxins - to be completed FY08 V - 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) - to be completed FY08 I – Develop laboratory-based multiclass, multiresidue analytical methods for veterinary drugs in animal-derived foods, feeds, and marker matrices (e.g. tissues, blood) D. Develop sample preparation methods - to be completed FY09 II - Develop field-based instruments and methods for analytical screening of chemicals of food safety/security concern (or nutritional interest) in foods. C. Evanescent-field sensing with tapered and planar waveguides - to be completed FY09 I – Develop laboratory-based multiclass, multiresidue analytical methods for veterinary drugs in animal-derived foods, feeds, and marker matrices (e.g. tissues, blood) E. Validate methods and perform technology transfer - to be completed by end of FY10 II - Develop field-based instruments and methods for analytical screening of chemicals of food safety/security concern (or nutritional interest) in foods. D. Development of a detector based on whisper gallery mode resonance technology - to be completed FY10 III - Develop field-portable and laboratory-based analytical methods for the screening and reliable identification of biotoxins and phytochemicals in foods. B. Development of rapid methods for phytochemicals - to be completed FY10 IV - Devise a scientifically defensible and practical system to evaluate qualitative identifications of chemical residues in complex matrices - to be completed FY10 4a.List the single most significant research accomplishment during FY 2006. Simultaneous multiresidue determination of fluoroquinolones and tetracyclines in chicken muscle and in catfish muscle. Chemical methods which allow for simultaneous analysis of more than one class of residue, without the need for mass spectrometry, have generally not been available. In this work, a method for simultaneous analysis of five fluoroquinolones and three commonly used tetracyclines was developed. The method uses liquid chromatography-fluorescence and allows for good recoveries and satisfactory limits of detection. The work with chicken muscle was a collaborative effort with Dan Donoghue at the University of Arkansas, while the work with catfish was a collaborative effort with Ahmed Darwish and Donald Freeman at the ARS Aquaculture Research Center in Stuttgart, Arkansas. This approach to residue analysis in which more than one class can be simultaneously determined has great promise for increasing the efficiency of analysis by those monitoring the safety of the food supply. NP108 Drug Residues and Environmental Contaminants Action Plans 2.1 and 2.1.2.2). 4b.List other significant research accomplishment(s), if any. A rapid and sensitive method for detection of Staphylococcus aureus enterotoxin A. Food security has become more important during this time of heightened threats to the U.S., and rapid detection methods are needed for biotoxins, such as microbial enterotoxins. A rapid and sensitive method for detection of Staphylococcus aureus enterotoxin A (SEA) was developed using a competitive immunoassay and analyzing with a BIAcore biosensor assay, which resulted in detection of SEA from 1 – 40 ng/mL in spiked liquid egg. The sample preparation homogenizaton and centrifugation required less than 0.5 hours and the BIAcore analysis was completed in 15 min per sample or 4 samples per hour. The biosensor analysis is fully automated and it is anticipated that this method will be utilized for multi-toxin detection of other toxins in various food matrices. NP108 Drug Residues and Environmental Contaminants Action Plans 2.1 and 2.1.2.2) Fluorescent micro-particle immuno-assay (FLMIA). Food security has become more important during this time of heightened threats to the U.S., and rapid detection methods are needed for biotoxins, such as microbial enterotoxins. A rapid and inexpensive FLMIA method was developed for trace analysis (below 1 ng/ml) of Staphylococcus aureus enterotoxin B (SEB) in drinking water (0.125 ng/mL) and whole milk (0.25 – 0.5 ng/mL). The milk samples spiked with SEB were analyzed directly without sample preparation. The analysis can be completed in less than 2.5 h for 20 samples. This research also describes the preparation of the tracer and the covalent linking of the antibody to the latex. The cost of reagents (tracer and anti-SEB-latex) is less than $1 per test. This assay can be used for high throughput analysis and may be useful to screen foods suspected of SEB contamination for improved food safety and security. NP108 Drug Residues and Environmental Contaminants Action Plans 2.1 and 2.1.2.2. Solid-matrix time-resolved luminescence (TRL) of oxytetracycline (OTC) screening in catfish muscle. Most foods of animal origin are in solid tissue matrices with high protein and fat contents, which entails laborious multi-step methods for sample preparation (homogenization, centrifugation, and solid phase extraction or liquid-liquid extraction). To simplify this process, a methodology was developed that combines sorbent extraction and solid-matrix time-resolved luminescence (TRL), illustrated by oxytetracycline (OTC) screening in catfish muscle. Extraction and enrichment are accomplished by immersing small sorbent strips into tissue homogenates for 20 minutes, followed by 3-minute water rinse cleanup and 2-minute dipping in a reagent solution. TRL is measured directly on the sorbent surface after desiccation, with a 0.026 µg/g limit of detection for OTC in muscle, which is far below the 2 µg/g U.S. regulatory tolerance level. By eliminating acids and organic solvents, this method has the potential to improve throughput and reduce assay costs. NP108 Drug Residues and Environmental Contaminants Action Plans 2.1 and 2.1.2.2. A portable specific tetracycline analyzer based on europium-sensitized luminescence. To improve the cost-effectiveness and practicality of a prototype portable analyzer built previously in our lab, a 385 nm light emitting diode (LED) was used in pulsed mode replacing the xenon flashlamp as the excitation source. Its narrow emission band and clean post-pulse extinction reduce background and hence improve sensitivity. The instrument operation and data processing are controlled by a laptop computer running a custom LabVIEW program. The time-resolved luminescence (TRL) signal is detected by a photomultiplier tube. Individual LED pulses are monitored using a photodiode to correct pulse-to-pulse energy fluctuation. The detector is gated to minimize its response to the LED pulse. Data acquisition is performed at 4-µs time resolution and 12-bit amplitude resolution. The analyzer has two interchangeable configurations: TRL in liquid phase using a sample cuvette, or solid-matrix TRL on a sorbent surface after extraction and cleanup. The analyzer weighs 15 lbs and is readily deployable to the field for usage by regulators or producers. NP108 Drug Residues and Environmental Contaminants Action Plans 2.1 and 2.1.2.2. Oxytetracycline (OTC) detection of in surface water. The occurrence and distribution of antibiotics and their impact on the environment create concerns, in particular, on microbial resistance. OTC is the only tetracycline drug approved by the FDA to treat certain fish and lobster diseases in aquaculture. More than 99% of it, however, enters aquacultural water and sediment due to poor GI absorption and leaching from fish feed. It would be useful if the producer or a researcher could quickly monitor the OTC in the water during and after dosage. OTC is extracted and cleaned up using Oasis HLB solid phase extraction (SPE) cartridges, and detected by europium-sensitized luminescence. The integrated time-resolved luminescence (TRL) intensity reveals linear dependence on OTC concentrations in the 0-3000 ng/g range (r2 = 0.9988) without SPE and 0-100 ng/g range (r2 = 0.9997) with SPE. The limit-of-detection is 0.045 ng/mL with a typical less than or equal to 5% relative standard deviation. The instrument and method are highly deployable allowing sensitive and specific in-situ detection of OTC residues in the field. Screening and quantitative methods in food matrices are currently underway to extend the approach to additional applications. NP108 Drug Residues and Environmental Contaminants Action Plans 2.1 and 2.1.2.2. 4c.List significant activities that support special target populations. None. 4d.Progress report. 1935-42000-055-01S: This report serves to document research conducted under a specific cooperative agreement between ARS and the Dept. of Biosciences and Biotechnology at Drexel University. The objective of this cooperative research project was to develop a new analytical method for acrylamide in foods that is faster, easier, and cheaper than the current methods, and to use this method for monitoring acrylamide in deep-fat fried foods and to attempt to minimize the generation of acrylamide in the food. Acrylamide has become another food safety concern, especially for the food industry, since the report of its presence in several types of processed foods in 2002. Effective methods are needed for its efficient analysis by many laboratories worldwide. In 2004, ARS developed a new rapid method using the QuEChERS concept, and optimized and evaluated the method in proficiency test samples in which results compared favorably versus other methods in use. The new, highly streamlined method uses only a single vessel in an easy liquid-liquid partitioning extraction and dispersive solid-phase cleanup approach for a variety of food types followed by LC/MS-MS analysis. Acrylamide mitigation strategies were pursued by Dr. Phil Handel at Drexel University and ARS analyzed the generated samples. Furthermore, Drs. Xuetong Fan and Katerina Mastovska of ARS studied the effect of irradiation as another mitigation possibility. If the presence of acrylamide in processed foods can be avoided, this would solve a food safety dilemma and controversy surrounding the issue. Research was completed this year and two manuscripts on the topics were submitted to the Journal of Agricultural and Food Chemistry in April and May of 2006: . 1)Rapid Sample Preparation Method for LC-MS/MS or GC-MS Analysis of Acrylamide in Various Food Matrices, and. 2)Reduction of Furan and Acrylamide in Foods by Ionizing Radiation. The main conclusions of the research include: . 1)acrylamide can be detected at low or high levels in a variety of foods using a simple and rapid sample preparation method followed by analysis using LC-MS/MS and/or GC-MS/MS;. 2)this method was useful in testing mitigation strategies for acrylamide formation in deep-fat fried foods;. 3)irradiation at low doses can destroy virtually all of the acrylamide in water, but in real foods, irradiation even at higher doses had very limited effect on the acrylamide levels or its formation;. 3)different deep-fat frying experiments also did not provide a useful approach to mitigate the acrylamide formation in the food without adversely affecting food quality. 1935-42000-055-02R: This report serves to document research conducted under a U.S.-Israel Binational Agricultural Research and Development (BARD) grant shared equally between ARS and the the School of Chemistry at Tel Aviv University in Tel Aviv, Israel. Dr. Lehotay collaborates with Prof. Aviv Amirav of Tel Aviv University on the 3-year grant. The grant was extended 7 months until its termination on Jan. 31, 2007. The ARS objectives of the project are to: A) expand the QuEChERS approach to veterinary drugs and environmental pollutants; and B) integrate automated DSI into the QuEChERS approach for GC/MS analysis. For Tel Aviv Universtiy, the objectives include: A) develop fast methods for difficult analytes using Supersonic GC/MS; and B) develop a Supersonic LC/MS instrument for the analysis of polar and labile analytes. In the final year of the grant, the goals of both collaborators are to: A) combine the expanded, automated QuEChERS approach with automated DSI + fast Supersonic GC/MS and Supersonic LC/MS analysis; and B) evaluate the expanded, automated QuEChERS approach for real samples. Objectives A and B for Tel Aviv University have already been demonstrated for certain difficult pesticides and drugs. Good progress is also being made by Prof. Amirav toward making continual improvements in the prototype Supersonic GC/MS and LC/MS instruments used in his laboratory. In 2005, 2 automated DSI devices were purchased from competing vendors for evaluation to meet ARS objectives A and B. Arrangements have been made for a postdoc and visiting scientists to conduct the research objectives described in the project. The automated DSI conditions have been set for the detection of selected pesticides, environmental contaminants, and veterinary drugs in GC/MS. Sample preparation in veterinary drug analysis show the QuEChERS approach to be feasible for the multiclass, multiresidue analysis of antibiotics in beef kidney samples. Real samples of olives, olive oil, tea, and honey have been successfully conducted using the approaches. 5.Describe the major accomplishments to date and their predicted or actual impact. See the report for 1935-42000-044-00D. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? An ARS scientist visited the USDA AMS Science and Technology Division laboratory in Gastonia, NC in March 2006 to help implement analytical approaches originally developed in ARS. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). None. Review Publications Chen, G., Smith, E., Qin, F., Liu, L.S. 2006. Time-resolved luminesence screening of antibiotics in tissue matrices without centrifugation and filtration. Journal of Agricultural and Food Chemistry. 54:3225-3230. Medina, M.B. 2006. A biosensor method for detection of staphylococcal enterotoxin a (sea) in raw whole egg. Biotechnology Letters. 2006. v.14. pg.119-132. Mastovska, K., Lehotay, S.J., Anastassiades, M. 2005. Study of a combination of analyte protectants to overcome matrix effects in routine gc analysis of pesticide residues in food matrices. Analytical Chemistry. 77(24):129-8137 Medina, M.B. 2006. Development of a fluorescent latex microparticle immunoassay for detection of staphylococcal enterotoxin b (seb). Journal of Agricultural and Food Chemistry.54(14):4937-4942. Janska, M., Lehotay, S.J., Mastovska, K., Hajslova, J., Alon, T., Amirav, A. 2006. A simple and inexpensive "solvent in silicone tube extraction" approach and its evaluation in the gas chromatographic analysis of pesticdes in fruits and vegetables. Journal of Separation Science.(29)66-88.