Progress 01/20/01 to 01/18/06
Outputs
Progress 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? This project terminated in Jan. 2006 and was replaced by CRIS 1935-42000- 055-00D Novel Technologies and Techniques for the Detection of Residues, Toxins, and Other Chemicals in Foods. This project addressed the problem related to the lack of rapid, automated, cost-effective, waste- minimizing, safe, and high-quality analytical methods to detect multiple chemical residues in foods. This pertains to Food Safety (National Program 108), Priority Objective 2.1.2.2 under the Toxic Chemicals section in the National Program 108 Action Plan. 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 in foods of both animal and plant origin. We met this overall goal with three broad objectives: I) develop and evaluate rapid and sensitive lab-based approaches to dependably detect multiple veterinary drug classes in animal-derived
foods predominantly using atmospheric pressure ionization/tandem mass spectrometry; II) develop and evaluate rapid lab-based quantitative and confirmatory analytical approaches for multiple pesticide classes in a variety of foods predominantly using gas chromatography/ tandem mass spectrometry; and III) develop and evaluate portable instrument-based screening techniques for chemical contaminants in foods at tolerance levels for use at the sample point of origin. The implementation of improved analytical approaches will (a) increase productivity and/or lower costs of analysis, (b) provide more statistically valid and accurate results for risk assessment and other purposes, (c) overcome trade barriers associated with the analysis of chemical residues, (d) provide more information to understand the effects and mechanisms of antimicrobial resistance and endocrine disruption, (e) allow for better verification of organic food labeling, (f) improve possible industrial food safety procedures,
and (g) 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) Year 1 (FY 2001) Objective 1, Milestone 1.1 The simultaneous determinative and confirmatory analysis of fluoroquinolones in egg and/or liver matrices using liquid chromatography(LC) / atmospheric pressure ionization (API) / tandem mass spectrometry (MS-MS) and fluorescence detection Objective 2, Milestone 2.1 Development of a method using gas chromatography (GC) / pulsed flame photometric detection (PFPD) + micro electron capture detection (mECD) for the analysis of organophosphorus and organochlorine pesticides in fatty matrices. Year 2 (FY 2002) Objective 1, Milestone 1.2a Rapid confirmatory screening of phenicols and beta-agonists, respectively, using LC/API/MS-MS and assessment of quantitation and confirmation capabilities of the approaches in real samples
Objective 2, Milestone 2.2a Development of MS-MS spectral libraries with chlorpyrifos as the example analyte Objective 1&2, Milestone 1.2b&2.2b Extraction and clean-up with different solvents, pH, and SPE sorbents of a variety of different food commodities. Year 3 (FY 2003) Objective 1, Milestone 1.3a Use of LC/APCI/MS-MS for the analysis of 2 classes of veterinary drugs Objective 1, Milestone 1.3b LC/API/MS-MS method development for the rapid analysis of aminoglycoside antibiotics in animal tissues Objective 2, Milestone 2.3a Development of a portable multiresidue screening analysis of OP pesticides at violative concentrations in nonfatty foods Objective 2, Milestone 2.3b Use of rapid and automated direct sample introduction (DSI) / low pressure-GC/MS, ideally for the reliable analysis of >100 pesticides in <15 min; NIST will likely to have completed the MS-MS spectral library for pesticides and developed a software program by this time; Also, Sandia may have prototypes of
micro- technological devices for us to evaluate Objective 3, Milestone 3.3 Biosensor detection of Staphylococcal enterotoxins. Year 4 (FY 2004) Objective 1, Milestone 1.4a Use of LC/API/MS-MS for the analysis of 3 classes of veterinary drugs Objective 1, Milestone 1.4b LC/API/MS-MS for the rapid analysis of peptide antibiotics in animal tissues. Objective 1&2, Milestone 1.4c and 2.4a Rapid, simple, representative, and reproducible sample preparation approaches for the application of micro-technological devices to chemical residue analysis in foods Objective 1, Milestone 1.4d Use of rapid and automated DSI/LP-GC/MS for GC-amenable veterinary drugs Objective 3, Milestone 3.4 Rapid and sensitive detection of aminoglycosides and biosensor detection of Staphylococcal enterotoxins. Year 5 (FY 2005) Objective 1, Milestone 1.5a Use of LC/API/MS-MS for the analysis of >3 classes of veterinary drugs Objective 1, Milestone 1.5b LC/API/MS-MS for the rapid analysis of 2 classes of
veterinary drugs in animal tissues Objective 1&2, Milestone 1.5c and 2.5a Use of a micro-technological device for chemical residue analysis in foods Objective 2, Milestone 2.5b Use of rapid API/MS-MS in the analysis of multiple thermally labile pesticides Objective 3, Milestone 3.5 Biosensor and multiresidue detection of aminoglycosides and multi-toxin detection of Staphylococcal enterotoxins. 4a List the single most significant research accomplishment during FY 2006. This project was terminated on 1-18-2006 and the replacement project was initiated on 1-19-2006. See report for 1935-42000-055-00D. 4b List other significant research accomplishment(s), if any. This project was terminated on 1-18-2006 and the replacement project was initiated on 1-19-2006. See report for 1935-42000-055-00D. 4d Progress report. For the reports on subordinate projects, see the separate reports for 1935-42000-055-01S and 1935-42000-055-02R. 5. Describe the major accomplishments to date and their
predicted or actual impact. Rapid method for detection of pesticides. In 2002, we developed the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method for multiple pesticide residues in foods. Using the method, a single analyst can extract hundreds of pesticides from 8 samples in about 30 min using $1 in materials/sample. This has become a standard method of pesticide analysis for dozens of labs around the world and reduces time, cost, waste, space needs for lab operations by about a factor of four. (NP108 Action Plans 2.1 and 2.1.2.2) Intralaboratory validation of the QuEChERS method. In 2003, the usefulness of the QuEChERS method was demonstrated in LC/MS-MS analysis, and the method was validated in a single laboratory for more than 200 pesticides in fruits and vegetables by ERRC scientists in collaboration with scientists in the Dutch Food Inspection Service, Amsterdam, The Netherlands. This accomplishment was important in showing that the method worked for so many
pesticides in different matrices. Validation experiments entailed fortification of fruit and vegetable samples with the pesticides in replicate analyses at 3 different levels. Also, many proficiency test samples were analyzed by the method, and side-by-side analyses were conducted to compare the QuEChERS results with those from a traditional method. The experiments pinpointed actions that needed to be taken to improve results for a few problematic base-sensitive pesticides, which was done in a follow-up study, and the successful validation of more than 200 pesticides in a thorough protocol convinced other laboratories to participate in further collaborative evaluation. (NP108 Action Plans 2.1 and 2.1.2.2) Adaptation of the QuEChERS method to recover problematic pesticides. Pesticide residue analysis of foods is a main function of many analytical laboratories around the world. The current methods used in analysis are time-consuming, laborious, and expensive. A quick, easy, cheap,
effective, rugged, and safe (QuEChERS) method for analysis of pesticide residues in food was recently introduced to provide a much more efficient way to better meet laboratory needs. However, a few problematic pesticides remained in the method, and this study demonstrates how the use of buffering in the procedure solves the problems associated with sample preparation of these pesticides. Excellent results were obtained for notoriously difficult-to-detect pesticides. This buffered approach was used in an interlaboratory validation study, and the QuEChERS method will likely replace the inefficient monitoring methods currently in use to save time and money. (NP108 Action Plans 2.1 and 2.1.2.2) Interlaboratory validation of the QuEChERS method. The quick, easy, cheap, effective, rugged, and safe (QuEChERS) method for pesticide residue analysis was further optimized and a finalized protocol was prepared for an interlaboratory collaborative study involving 13 laboratories in 7
countries. This accomplishment is important because hundreds of monitoring and contract laboratories around the world need rapid, inexpensive, and effective analytical methods for routine use, but the methods must be collaboratively validated for official purposes. The 13 collaborating laboratories analyzed 21 blind samples provided by the ARS lab, and results show that the method will become an approved method available for official use in regulatory applications. The impact of this accomplishment will be that the highly efficient and beneficial method will be used worldwide to provide reliable results more quickly and cheaply then before to ease trade and improve food safety. (NP108 Action Plans 2.1 and 2.1.2.2) Optimized sample extraction and clean-up for QuEChERS analysis of fatty foods. The QuEChERS method for pesticide analysis was favorably compared with a matrix solid-phase dispersion (MSPD) method for the analysis of fatty foods. Pesticide residue analysis of foods is a
main function of many analytical laboratories around the world, and the current methods used in analysis are time-consuming, laborious, and expensive, especially in the case of fatty foods due to the need to remove co-extracted lipids before the detection step. This accomplishment evaluated the two rapid methods for the analysis of 32 diverse pesticides fortified in milk, egg, avocado and other fatty foods. Excellent results were obtained for certain problematic residues by the QuEChERS method that were not recovered by the MSPD method. It is likely that the QuEChERS method will become commonly used in many pesticide monitoring applications for foods of less than 20% fat. (NP108 Action Plans 2.1 and 2.1.2.2) Multi-residue chromatographic/fluorescence detection and MS confirmation of fluoroquinones. The use of fluoroquinolone antibiotics, particularly in poultry, is being scrutinized by the Food and Drug Administration due to antimicrobial resistance concerns, thus analytical
methods for fluoroquinolones have become more important in the regulatory arena. A rapid extraction procedure and liquid chromatographic (LC) analysis with both fluorescence (FL) detection and tandem mass spectrometry (MS/MS) to analyze and confirm the presence of 8 fluoroquinolones in egg products (both white and/or yolk). A comparison study has been conducted in chicken muscle using this method and the currently used FSIS screening method, and this LC-FL-MS-MS method compared very favorably. The LC-FL- MS-MS method has also been compared with a bioassay (collaboration with Dan Donoghue) for analysis of chicken egg and muscle samples and it is now being extended to beef liver and shrimp. The method has been extensively validated internally and the method as applied to chicken samples was transferred to the FSIS Midwestern Laboratory. (NP108 Action Plans 2.1 and 2.1.2.2) Rapid multi-residue and confirmatory analysis of beta-lactam antibiotics. The Food Safety Inspection Service
(FSIS) and the Food and Drug Administration (FDA) have a problem with the differentiation between the beta-lactam antibiotics, ceftiofur and penicillin, in their current monitoring method. A rapid and simple, quantitative and confirmatory method of analysis for 10 beta-lactam antibiotics in beef kidney using liquid chromatography/tandem mass spectrometry (LC/MS-MS) was developed and compared with the FSIS 7-plate microbial assay for 45 incurred beef kidneys. This approach may be used to verify responses from the FSIS 7- plate assay, or to replace it altogether after it has been expanded to include other antibiotic drugs. (NP108 Action Plans 2.1 and 2.1.2.2). Screening Assay for detection of ceftiofur metabolites in incurred kidney samples. A selective hydrolysis method followed by a microbial inhibition assay can presumptively identify the presence of ceftiofur metabolites at parts per million levels. The method detects all beta- lactam compounds and can differentiate ceftiofur
metabolites from antimicrobial compounds classified as unidentified microbial inhibitors. This screening approach can be used to validate the presence of unknowns in the FSIS 7-plates assay or suspect ceftiofur metabolite prior to using instrumentation analysis. This test is rapid, simple and inexpensive where the sample preparation, hydrolysis and testing can be carried out in less than 4 hours. (NP108 Action Plans 2.1 and 2.1.2.2) Rapid screening method for enrofloxacin and tetracyclines. Simple rapid screening methods are desirable to allow a large number of samples to be analyzed for the presence of veterinary drugs. Such screens can greatly reduce the number of samples that would need to be analyzed by more extensive, quantitative and confirmatory methods, thus saving considerable time and expense. Last year, a rapid screening assay was developed which allows for detection of both enrofloxacin and tetracyclines in the same chicken muscle sample extract, at their respective
tolerance levels. This assay enables rapid screening for two classes of antibiotics, without the need for separate extraction methods, saving time and expense. (NP108 Action Plans 2.1 and 2.1.2.2) Designed, built, and tested prototype portable TRL fluorometer. A new methodology was developed that hyphenated sorbent extraction and solid matrix TRL. Its application was demonstrated using tetracycline (TC) in milk as a model analyte. A small format C18 sorbent strip was cut to extract tetracycline from milk in a 10-min immersion. Cleanup was a simple 3-min water immersion. After the C18 strip was spotted with a reagent solution and desiccated, TRL was measured directly on its surface. This rapid and simple method screened TC in milk at the 300 ppb tolerance level. No SPE cartridge or organic solvent was needed so it was of low-cost and friendly to both the operator and the environment. (NP108 Action Plans 2.1 and 2.1.2.2] Designed, built and tested prototype evanescent field
sensor. Tapered waveguides were developed for TRL sensing in evanescent field: (1) proof of the evanescent field sensing concept using tapered quartz-rod waveguides; (2) fabrication of tapered waveguides by acetylene torch drawing; (3) a waveguide accessory was designed and assembled for a commercial fluorescence spectrometer; (4) the TRL performance was compared on different taper geometries using TC as a model analyte; (5) the profile of the best performing exponential-linear tapered waveguide was further optimized by fluorescence imaging, a technique also developed in this lab that visually revealed the location and the intensity of light leaking from the core due to violation of total internal reflection. For tetracycline a 20 ppb limit of detection (LOD) was achieved. This innovative method was approved by the ARS Office of Technology Transfer (OTT) review panel for patent application (0032.05). (NP108 Action Plans 2.1 and 2.1.2.2) Non-instrumental and biosensor methods for
detection of SEA an SEB in foods. Developed a non-instrumental latex agglutination assay that can detect toxin contaminants from Staphylococcus aureus (SEA and SEB) in 1 g of food samples. Improved sensitivity of 1 2.5 ng/g in eggs, milk and ham samples was attained using a commercial biosensor instrument. FSIS expressed needs for these methods as alternatives to commercial assays. (NP108 Action Plans 2.1.2). Facile sample preparation using dispersive SPE. Developed the dispersive solid-phase extraction (dispersive-SPE) concept in which the cleanup sorbent is used to retain matrix co-extractives rather than the analytes, and the procedure is conducted in a small tube rather than a column. This fast and cheap technique is used in the QuEChERS approach, and is expanding to non-pesticide residue applications. (NP108 Action Plans 2.1 and 2.1.2.2) Development of the analyte protectants concept. In the U.S., the Food and Drug Administration (FDA) and Environmental Protection Agency
(EPA) policies do not permit the use of matrix-matched calibration standards to improve accuracy in the quantitation of pesticide residues for regulatory enforcement actions. To overcome this regulatory limitation, and to provide better gas chromatographic (GC) peak shapes and lower detection limits for problematic pesticides, a new concept that we call analyte protection was developed and investigated. The concept involves the addition of chemicals, such as sugar derivatives, to both calibration standards and extracts, and the chemicals serve to mask the active sites in the GC system that cause the inaccurate quantitation of certain pesticides. This simple approach may become standard practice for GC analysis of pesticides because it was shown to provide accurate results and low detection limits while still meeting EPA and FDA policies for enforcement actions. (NP108 Action Plans 2.1 and 2.1.2.2) Determined solvent suitability and pesticide stability for GC analysis. The
suitability of 6 different organic solvents (acetonitrile, acetone, ethyl acetate, toluene, isooctane, and hexane) for injection in GC and pesticide stability was thoroughly assessed. Selection of the ideal solvent is a key factor in the testing of pesticide residues in foods using GC, yet it has not been comprehensively discussed before. The ideal solvent should: (i) provide sufficient solubility and stability of the pesticides of interest; (ii) be used in the extraction and/or clean- up step to avoid an additional solvent exchange step; (iii) permit optimal GC analysis in terms of sensitivity, reproducibility, and speed; and (iv) be non-hazardous and inexpensive. Acetonitrile was found to be the most suitable solvent for extraction of a wide polarity range of pesticide residues from produce, and adding a small amount of acetic acid to it slows degradation of problematic pesticides for better analysis. We provided practical recommendations on selection of the most suitable solvent
for pesticide residue analysis and proposed solutions to several real-world problems associated with the use of particular solvents. (NP108 Action Plans 2.1 and 2.1.2.2) Rapid multiresidue analysis of pesticides using LP-GC/MS. In order to substantially speed up the analysis of pesticide residues in food, a new method of analysis using low-pressure gas chromatography/mass spectrometry (LP-GC/MS) was optimized and evaluated. An analytical method using LP-GC/MS for 20 challenging pesticides in carrots was demonstrated, which decreased the time of chromatographic analysis from approximately 20 min to 6 min. Limits of detection were also decreased by enabling a higher injection volume and increasing the ruggedness of the chromatographic approach. The outcome of this research is that LP- GC/MS can significantly increase the speed of multi-residue pesticide analysis and provide other benefits of larger sample capacity, lower detection limits, and reduced thermal degradation of pesticide
analytes. (NP108 Action Plans 2.1 and 2.1.2.2) Rapid methods for multi-residue detection and confirmatory analysis of thyreotats and beta-agonists in bovine tissues. Currently, regulatory and contract laboratories use 2 different time-consuming and intensive analytical methods for the determination of thyreostats and beta-agonists, which are banned growth promoters. A novel multiresidue analytical screening method for the isolation and detection of 14 thyreostats and beta-agonists using liquid chromatography-fluorescence-tandem mass spectrometry in meat and liver tissues was developed. The presence of the compounds can be confirmed and quantified (if necessary) at levels less than 100 ng/g within 1 hour. This approach is simpler, faster and more effective than the current methods used by U.S., Canadian, and the European Union regulatory agencies and the protocol was provided to the regulatory agencies. The method was transferred to FSIS Eastern and Western Labs and is being
adapted for their potential usage. (NP108 Action Plans 2.1 and 2.1.2.2) Rapid and sensitive method to detect spectinomycin aminoglycoside. Rapid screening methods are needed for detection of spectinomycin residues in animal tissues at the regulatory action levels. Reagents for a fluorescent immunoassay were synthesized, and the fluorescent latex immunoassay method was optimized for detection of spectinomycin at 5 100 ng/g in bovine kidney extract. This method can be used to identify compounds classified as an unidentified microbial inhibitor (UMI) in the 7-plate microbial assay used by the Food Safety Inspection Service (FSIS). The use of this method by FSIS could reduce the number of unidentified microbial inhibitors (UMI) compounds and enable better quantitation if these compounds appear above the action levels. (NP108 Action Plans 2.1 and 2.1.2.2) A fluorescent latex particle immunoassay (FLPIA) was developed with detection of staphylococcal enterotoxin below 1 ng/mL. This
assay has high throughput and 50 sample analyses can be completed in 3 hours. A rapid and cheap non-instrumental latex agglutination assay can be used to detect staphylococcal enterotoxins at 2.5 ng/mL in water samples. However, this approach presented difficulties in determining positive from negative findings, and is being modified. (NP108 Action Plans 2.1 and 2.1.2.2) Investigation of automated direct sample introduction with analyte protectants. In the gas chromatographic analysis of pesticide residues in food, maintenance of the instrument is a major limitation in routine practice. In this study, we investigated a novel approach called automated direct sample introduction (DSI) that can eliminate the need for frequent instrument maintenance while also improving detectablity for the pesticides in complex food matrices. The results indicated that DSI worked well for this purpose, especially in combination with a unique approach known as analyte protectants developed in our
laboratory to deactivate exposed glass surfaces in the system. Those laboratories that implement this approach will benefit by the improved analytical performance and ease of use to conduct trace residue analysis of pesticides in food. (NP108 Action Plans 2.1 and 2.1.2.2) Development and evaluation of the solvent in silicone tube extraction (SiSTEx) approach. Effective and efficient analytical methods are needed to detect pesticide residues in fruits and vegetables. Rapid methods usually do not detect very low amounts of the pesticides because it takes time to concentrate the residues in a final extract. Furthermore, other chemicals in the food sample are also concentrated to the same extent as the pesticides in traditional methods, which interfere in the detection. This research study evaluated a new way to concentrate the pesticides, but block the interfering chemicals. The new approach is called solvent in silicone tube extraction (SiSTEx), in which an organic solvent is
placed in a thin silicone tube surrounded by the watery sample. Many of the pesticides transfer into and through the tubing into the solvent, and most of the other chemicals stay in the watery sample. The pesticides are more highly concentrated in the solvent and very low levels can be detected from the sample. This new approach is simple, cheap, and effective, and can be used to lower detection limits of pesticide residues in food samples. (NP108 Action Plans 2.1 and 2.1.2.2) Further evaluation of low-pressure gas chromatography-mass spectrometry. Current methods of analysis for pesticide residues and other chemical contaminants in food are time-consuming. Low-pressure gas chromatography/mass spectrometry (LP-GC/MS) is a unique approach to speed the analysis of many types of chemical residues, including pesticides. A comparison was made between a wider analytical column of thicker film versus a narrower column of thinner film in LP-GC/MS. The narrower column configuration gave
a slightly faster separation of the pesticides, but the wider analytical column with a thicker film gave and greater sensitivity and ruggedness. The analysis of 57 pesticides was optimized and demonstrated using LP-GC/MS with separation time less than 6 min, which is 5-7 times faster than traditional methods of analysis. This form of fast-GC/MS approach shows promise to become a widely used tool to help increase sample throughput in the analysis of pesticide residues. (NP108 Action Plans 2.1 and 2.1.2.2) Multiresidue quantitation and confirmation of fluoroquinolones in shrimp. The liquid chromatography-fluorescence-mass spectrometryn method developed for use in chicken muscle, liver and eggs was applied successfully, after minor modification, to shrimp samples fortified with 8 fluoroquinolones, as well as samples incurred with enrofloxacin. This project was a collaborative effort with Luz Vazquez-Moreno at CIAD in Sonora, Mexico. The method would be suitable for CIADs efforts in
monitoring shrimp samples for fluoroquinolones residues. (NP108 Action Plans 2.1 and 2.1.2.2) Distribution of enrofloxacin residues in incurred chicken breast vs thigh muscle. When a tolerance level is established for veterinary drug residues in animal tissue, such as muscle, the type of muscle is typically not specified (e.g., breast vs. thigh). There is potential for drug residues to preferentially distribute within a selected type of muscle tissues. In this collaborative study with Dan Donoghue at the University of Arkansas, the liquid chromatography-fluorescence-mass spectrometryn method was used to supplement bioassay data in an effort to adddress this question. In this study, higher enrofloxacin concentrations were found in breast muscle samples, as compared to thigh muscle samples, after dosing of chickens with the drug. These types of studies can thus provide helpful guidance to those involved in setting tolerance levels for drugs in animal tissues. (NP108 Action Plans
2.1 and 2.1.2.2) 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? The results from research studies have been transferred to interested parties by means of collaborations, peer-reviewed publications, written reports, internet accounts, presentations at scientific meetings, and communications with scientists and administrators from regulatory agencies, industry, and other organizations. Research results of studies and recommendations designed to meet residue detection needs in the Food Safety Inspection Service (FSIS) for meat, poultry, and eggs have been provided to their laboratories. Three analytical methods using LC-MS/MS developed in ARS were transferred to FSIS (the analysis of beta-lactams, fluoroquinolones, and thyreostats in animal tissues),
and the methods were implemented after the FSIS internal validation and approval process. FSIS is also going to evaluate the ARS method for analysis of pesticide residues in egg and meat. State programs, U.S. federal agencies, foreign regulatory programs, industry and contract laboratories are particularly interested in the approaches developed for pesticide residue analysis. In Nov of 2004, the ERRC scientists co-organized and hosted a week-long EPA/USDA Pesticide Analytical Workshop for 17 chemists from 15 official state labs in the U. S. Several lectures and hands-on training were provided, and several of the labs have adopted the approaches developed from the ARS research. Laboratories in the USDA Agricultural Marketing Service and Food and Drug Administration are also using extraction and analytical techniques developed in ARS. The QuEChERS method was validated in an interlaboratory study involving 13 labs in 7 countries, and the protocol was disseminated to scores of
interested scientists around the world upon their request. Also, a noncommerical antibody for hygromycin developed at ERRC was provided to a scientist at Columbia University. The latex and biosensor immunoassay methods for detection of enterotoxins (SEA and SEB) in eggs are of high interest to FSIS. These methods will be transferred after validation for routine analysis. The technique that hyphenates sorbent extraction and solid matrix TRL has been filed with the ARS Technical Transfer Office for patent application (0062.04). The ARS Press Release on New Detection Methods Improve Food Safety attracted the attention of scientists at the Center for Advanced Sensor Technology, University of Maryland Baltimore County (UMBC). Our technical know-how on biosensor development for detection of staphylococcal enterotoxins is being applied to micro-sensor technology developed at UMBC through research collaboration. We are transferring immunochemical reagents synthesized at ERRC for their
application. 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). This project was terminated on 1-18-2006 and the replacement project was initiated on 1-19-2006. See report for 1935-42000-055-00D.
Impacts
(N/A)
Publications
- Lehotay, S.J., De Kok, A., Hiemstra, M., Van Bodegraven, P. 2005. Validation of a fast and easy method for the determination of 229 pesticide residues in fruits and vegetables using gas and liquid chromatography and mass spectrometric detection. Journal of Association of Official Analytical Chemists International. 2005. v. 88. p. 595-614.
- Lehotay, S.J., Mastovska, K., Lightfield, A.R. 2005. Use of buffering to improve results of problematic pesticides in a fast and easy method for residue analysis of fruits and vegetables. Journal of Association of Official Analytical Chemists International. 88.pp. 615-629.
- Lehotay, S.J., Mastovska, K., Jong, Y. 2005. Evaluation of two fast and easy methods for pesticide residue analysis in fatty food matrices. Journal of Association of Official Analytical Chemists International. 88. pp. 630-638.
- Cajka, T., Mastovska, K., Lehotay, S.J., Hajslova, J. 2005. Use of automated direct sample introduction with analyte protectants in the gc-ms analysis of pesticide resides. Journal of Separation Science. 28. pp. 1048- 1060.
- Medina, M.B. 2005. Development of a competitive immunoassay for detection of staphylococcal enterotoxin b ((seb) in milk. Journal of Rapid Methods and Automation in Microbiology. 13. pp. 37-55.
- Schneider, M.J. 2004. A rapid flourescence screening assay for enrofloxacin and tetracyclines in chicken muscle. Journal of Agricultural and Food Chemistry. 52. pp. 7809-7813.
- Schneider, M.J., Vazquez Moreno, L., Barraza Guardado, R., Ortega-Nieblas, M. 2005. Multiresidue determination of fluoroquinolone antibiotics in shrimp by liquid chromatography-fluorescence-mass spectrometry. Journal of the Association of Official Analytical Chemists. 88. pp. 1160-1166.
- Chen, G., Schneider, M.J., Darwish, A.M., Lehotay, S.J., Freeman, D.W. 2004. Europium-sensitized luminescence determination of oxytetracycline in catfish muscle. Talanta. 64. pp. 252-257.
- Mastovska, K., Hajslova, J., Lehotay, S.J. 2004. Ruggedness and other performance characteristics of low-pressure gas chromatography-mass spectrometry for the fast analysis of multiple pesticide residues in food crops. Journal of Chromatography A. 1054. 2004. p. 335-349.
- Fagerquist, C.K., Lightfield, A.R., Lehotay, S.J. 2005. Confirmatory and quantitative analysis of b-lactam antibiotics in bovine kidney tissue by dispersive solid-phase extraction and liquid chromatography-tandem mass spectrometry. Analytical Chemistry 5(77).1473-1482.
- Liu, L.S., Chen, G., Fishman, M.L. A single sorbent for tetracycline enrichment and following solid-matrix time-resolved luminescence. 2005. Analytica Chimica Acta:528: p.261-268.
- Liu, L.S., Chen, G., Fishman, M.L., Hicks, K.B. Pectin gel vehicles for citronellal delivery. Controlled Release Journal.2005. Drug Delivery. 12:p.149-157.
- Lehotay, S.J., Mastovska, K. Determination of pesticide residues. Methods of Analysis of Food Components and Additives. Taylor & Francis, Boca Raton, FL. 2005. pp. 329-359.
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Progress 10/01/04 to 09/30/05
Outputs
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? What does it matter? 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 in foods. This pertains to Food Safety (National Program 108), Priority Objective 2.1.2. 2 under the Toxic Chemicals section in the National Program 108 Action Plan. 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 in foods of both animal and plant origin. We are meeting this overall goal with three broad objectives: I) develop and evaluate rapid and sensitive lab-based approaches to dependably detect multiple veterinary drug classes in animal-derived foods predominantly using atmospheric pressure ionization/tandem mass spectrometry; II) develop and evaluate rapid lab-based quantitative and confirmatory analytical approaches for multiple pesticide
classes in a variety of foods predominantly using gas chromatography/ tandem mass spectrometry; and III) develop and evaluate portable instrument-based screening techniques for chemical contaminants in foods at tolerance levels for use at the sample point of origin. Eventually, we hope to devise the most effective and efficient overall analytical scheme to monitor for chemical residues in foods. The implementation of improved analytical approaches will (a) increase productivity and/or lower costs of analysis, (b) provide more statistically valid and accurate results for risk assessment and other purposes, (c) overcome trade barriers associated with the analysis of chemical residues, (d) provide more information to understand the effects and mechanisms of antimicrobial resistance and endocrine disruption, (e) allow for better verification of organic food labeling, (f) improve possible industrial food safety procedures, and (g) reduce the potential for food that has been deliberately
or accidentally adulterated by toxic chemicals to reach the consumer. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2001) Objective 1 Milestone 1.1 -- The simultaneous determinative and confirmatory analysis of fluoroquinolones in egg and/or liver matrices using liquid chromatography(LC) / atmospheric pressure ionization (API) / tandem mass spectrometry (MS-MS) and fluorescence detection. Objective 2 Milestone 2.1 -- Development of a method using gas chromatography (GC) / pulsed flame photometric detection (PFPD) + micro electron capture detection (mECD) for the analysis of organophosphorus and organochlorine pesticides in fatty matrices. Year 2 (FY 2002) Objective 1 Milestone 1.2A -- Rapid confirmatory screening of phenicols and beta- agonists, respectively, using LC/API/MS-MS and assessment of quantitation and confirmation capabilities of the approaches in real samples Milestone 1.2B -- Extraction and clean-up with different solvents, pH and SPE
sorbents of a variety of different food commodities. Objective 2 Milestone 2.2A -- Development of MS-MS spectral libraries with chlorpyrifos as the example analyte. Milestone 2.2B -- Extraction and clean-up with different solvents, pH, and SPE sorbents of a variety of different food commodities. Year 3 (FY 2003) Objective 1 Milestone 1.3A -- Use of LC/APCI/MS-MS for the analysis of 2 classes of veterinary drugs Milestone 1.3B -- LC/API/MS-MS method development for the rapid analysis of aminoglycoside antibiotics in animal tissues Objective 2 Milestone 2.3A -- Development of a portable multiresidue screening analysis of OP pesticides at violative concentrations in nonfatty foods Milestone 2.3B -- Use of rapid and automated direct sample introduction (DSI) / low pressure-GC/MS, ideally for the reliable analysis of >100 pesticides in <15 min; NIST will likely to have completed the MS-MS spectral library for pesticides and developed a software program by this time; Also, Sandia may have
prototypes of micro-technological devices for us to evaluate. Objective 3 Milestone 3.3 -- Biosensor detection of Staphylococcal enterotoxins. Year 4 (FY 2004) Objective 1 Milestone 1.4A -- Use of LC/API/MS-MS for the analysis of 3 classes of veterinary drugs. Milestone 1.4B -- LC/API/MS-MS for the rapid analysis of peptide antibiotics in animal tissues. Milestone 1.4C -- Rapid, simple, representative, and reproducible sample preparation approaches for the application of micro-technological devices to chemical residue analysis in foods. Milestone 1.4D -- Use of rapid and automated DSI/LP-GC/MS for GC- amendable veterinary drugs. Objective 2 Mileston 2.4A -- Rapid, simple, representative, and reproducible sample preparation approaches for the application of micro-technological devices to chemical residue analysis in foods. Objective 3 Milestone 3.4 -- Rapid and sensitive detection of aminoglycosides and biosensor detection of Staphylococcal enterotoxins. Year 5 (FY 2005) Objective 1
Milestone 1.5A -- Use of LC/API/MS-MS for the analysis of >3 classes of veterinary drugs. Milestone 1.5B -- LC/API/MS-MS for the rapid analysis of 2 classes of veterinary drugs in animal tissues. Milestone 1.5C -- Use of a micro-technological device for chemical residue analysis in foods. Objective 2 Milestone 2.5A -- Use of a micro-technological device for chemical residue analysis in foods. Milestone 2.5B -- Use of rapid API/MS-MS in the analysis of multiple thermally labile pesticides. Objective 3 Milestone 3.5 -- Biosensor and multiresidue detection of aminoglycosides and multi-toxin detection of Staphylococcal enterotoxins. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Milestone 1.5A -- Use of LC/API/MS-MS for the analysis of less than 3 classes of veterinary drugs. Milestone Substantially Met 2. Milestone 1.5B -- LC/API/MS-MS for the rapid analysis
of 2 classes of veterinary drugs in animal tissues. Milestone Fully Met 3. Milestone 1.5C -- Use of a micro-technological device for chemical residue analysis in foods. Milestone Fully Met 4. Milestone 2.5A -- Use of a micro-technological device for chemical residue analysis in foods. Milestone Fully Met 5. Milestone 2.5B -- Use of rapid API/MS-MS in the analysis of multiple thermally labile pesticides. Milestone Fully Met 6. Milestone 3.5 - Biosensor and multiresidue detection of aminoglycosides and multi-toxin detection of Staphylococcal enterotoxins. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? A new 5-year project plan was proposed in accordance with the OSQR process in ARS. The proposal is being reviewed at this time, and no substantial revisions are expected. In the proposal, the milestones listed for the
next 3 years are listed as follows. Objective 1: Develop laboratory-based multiclass, multiresidue analytical methods for veterinary drugs in animal-derived foods, feeds, and marker matrices (e.g. tissues, blood, urine) Objective 2: Develop field-based instruments and methods for analytical screening of chemicals of food safety/security concern (or nutritional interest) in foods Objective 3: Develop field-portable and laboratory-based analytical methods for the screening and reliable identification of biotoxins and phytochemicals in foods Objective 4: Devise a scientifically defensible and practical system to evaluate qualitative identifications of chemical residues in complex matrices Objective 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). The anticipated impacts/outcomes/results of the research will be a more efficient and effective
monitoring system for chemical residues and toxins in food, which will increase food safety and security. 4a What was the single most significant accomplishment this past year? Multiresidue quantitation and confirmation of fluoroquinolones in shrimp. The liquid chromatography-fluorescence-mass spectrometryn method developed for use in chicken muscle, liver and eggs was applied successfully, after minor modification, to shrimp samples fortified with 8 fluoroquinolones, as well as samples incurred with enrofloxacin. This project was a collaborative effort with Luz Vazquez-Moreno at CIAD in Sonora, Mexico. The method would be suitable for CIADs efforts in monitoring shrimp samples for fluoroquinolones residues. 4b List other significant accomplishments, if any. A fluorescent latex particle immunoassay (FLPIA) was developed with detection of staphylococcal enterotoxin below 1 ng/mL. This assay has high throughput and 50 sample analyses can be completed in 3 hours. A rapid and cheap
non-instrumental latex agglutination assay can be used to detect staphylococcal enterotoxins at 2.5 ng/mL in water samples. Investigation of automated direct sample introduction with analyte protectants. In the gas chromatographic analysis of pesticide residues in food, maintenance of the instrument is a major limitation in routine practice. In this study, we investigated a novel approach called automated direct sample introduction (DSI) that can eliminate the need for frequent instrument maintenance while also improving detectablity for the pesticides in complex food matrices. The results indicated that DSI worked well for this purpose, especially in combination with a unique approach known as analyte protectants developed in our laboratory to deactivate exposed glass surfaces in the system. Those laboratories that implement this approach will benefit by the improved analytical performance and ease of use to conduct trace residue analysis of pesticides in food. Development and
evaluation of the solvent in silicone tube extraction (SiSTEx) approach. Effective and efficient analytical methods are needed to detect pesticide residues in fruits and vegetables. Rapid methods usually do not detect very low amounts of the pesticides because it takes time to concentrate the residues in a final extract. Furthermore, other chemicals in the food sample are also concentrated to the same extent as the pesticides in traditional methods, which interfere in the detection. This research study evaluated a new way to concentrate the pesticides, but block the interfering chemicals. The new approach is called solvent in silicone tube extraction (SiSTEx), in which an organic solvent is placed in a thin silicone tube surrounded by the watery sample. Many of the pesticides transfer into and through the tubing into the solvent, and most of the other chemicals stay in the watery sample. The pesticides are more highly concentrated in the solvent and very low levels can be
detected from the sample. This new approach is simple, cheap, and effective, and can be used to lower detection limits of pesticide residues in food samples. Further evaluation of low-pressure gas chromatography-mass spectrometry. Current methods of analysis for pesticide residues and other chemical contaminants in food are time-consuming. Low-pressure gas chromatography/mass spectrometry (LP-GC/MS) is a unique approach to speed the analysis of many types of chemical residues, including pesticides. A comparison was made between a wider analytical column of thicker film versus a narrower column of thinner film in LP-GC/MS. The narrower column configuration gave a slightly faster separation of the pesticides, but the wider analytical column with a thicker film gave and greater sensitivity and ruggedness. The analysis of 57 pesticides was optimized and demonstrated using LP-GC/MS with separation time less than 6 min, which is 5-7 times faster than traditional methods of analysis.
This form of fast-GC/MS approach shows promise to become a widely used tool to help increase sample throughput in the analysis of pesticide residues. Multiresidue quantitation and confirmation of fluoroquinolones in shrimp. The liquid chromatography-fluorescence-mass spectrometryn method developed for use in chicken muscle, liver and eggs was applied successfully, after minor modification, to shrimp samples fortified with 8 fluoroquinolones, as well as samples incurred with enrofloxacin. This project was a collaborative effort with Luz Vazquez-Moreno at CIAD in Sonora, Mexico. The method would be suitable for CIADs efforts in monitoring shrimp samples for fluoroquinolones residues. Distribution of enrofloxacin residues in incurred chicken breast vs thigh muscle. When a tolerance level is established for veterinary drug residues in animal tissue, such as muscle, the type of muscle is typically not specified (e.g., breast vs. thigh). There is potential for drug residues to
preferentially distribute within a selected type of muscle tissues. In this collaborative study with Dan Donoghue at the University of Arkansas, the liquid chromatography-fluorescence-mass spectrometryn method was used to supplement bioassay data in an effort to adddress this question. In this study, higher enrofloxacin concentrations were found in breast muscle samples, as compared to thigh muscle samples, after dosing of chickens with the drug. These types of studies can thus provide helpful guidance to those involved in setting tolerance levels for drugs in animal tissues. 4d Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and the Dept. of Biosciences and Biotechnology at Drexel University, entitled Development of New Methods to Detect and Control Acrylamide Formation in Deep-Fat Fried Foods. Additional details of research can be found in the report for the parent CRIS 1935-42000-044-00D, Advanced Techniques
for the Analysis of Chemical Residues in Foods The objective of this cooperative research project is 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 in which different frying techniques will be assessed in order to minimize the generation of acrylamide in the food. Acrylamide has become an important food safety concern, especially for regulators and the food industry, since the report of its presence in several types of processed foods in 2002, and 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 are being pursued by Dr. Phil Handel at Drexel University and ARS is analyzing the generated samples. Furthermore, Dr. Xuetong Fan of ARS is studying 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. This report serves to document research conducted under U.S.-Israel Binational Agricultural Research and Development (BARD) grant US-35000-03 shared equally between ARS and the the School of Chemistry at Tel Aviv University in Tel Aviv, Israel, entitled Fast, Practical and Effective Approach for the Analysis of Hazardous Chemicals in the Food Supply. Additional details of research can be found in the report for the parent CRIS 1935-42000-044-00D, Advanced Techniques for the Analysis of
Chemical Residues in Foods Dr. Lehotay collaborates with Prof. Aviv Amirav of Tel Aviv University on the 3-year grant. 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 will be 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. 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 to conduct the
research objectives described in the project. Initial experiments have been conducted pertaining to sample preparation in veterinary drug analysis, which show the QuEChERS approach for pesticides to be feasible after modifications depending on the matrix and analytes. 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. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Rapid method for detection of pesticides. In 2002, we developed the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method for multiple pesticide residues in foods. Using the method, a single analyst can extract hundreds of pesticides from 8 samples in about 30 min using $1 in materials/sample. (Milestones 2.2 and
2.3), NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2, Performance Measures in the ARS Strategic Plan, 3.1.2). Intralaboratory validation of the QuEChERS method. In 2003, the usefulness of the QuEChERS method was demonstrated in LC/MS-MS analysis, and the method was validated in a single laboratory for more than 200 pesticides in fruits and vegetables by Lehotay in collaboration with de Kok, Hiemstra, and van Bodegraven in the Dutch Food Inspection Service, Amsterdam, The Netherlands. This accomplishment was important in showing that the method worked for so many pesticides in different matrices. Validation experiments entailed fortification of fruit and vegetable samples with the pesticides in replicate analyses at 3 different levels. Also, many proficiency test samples were analyzed by the method, and side- by-side analyses were conducted to compare the QuEChERS results with those from a traditional method. The experiments pinpointed actions that
needed to be taken to improve results for a few problematic base- sensitive pesticides, which was done in a follow-up study, and the successful validation of more than 200 pesticides in a thorough protocol convinced other laboratories to participate in the collaborative study discussed in 4A. (Milestones 2.2, 2.3, and 2.5, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2, Performance Measures in the ARS Strategic Plan, 3.1.2). Adaptation of the QuEChERS method to recover problematic pesticides. Pesticide residue analysis of foods is a main function of many analytical laboratories around the world. The current methods used in analysis are time-consuming, laborious, and expensive. A quick, easy, cheap, effective, rugged, and safe (QuEChERS) method for analysis of pesticide residues in food was recently introduced to provide a much more efficient way to better meet laboratory needs. However, a few problematic pesticides remained in the method, and this
study demonstrates how the use of buffering in the procedure solves the problems associated with sample preparation of these pesticides. Excellent results were obtained for notoriously difficult-to-detect pesticides. This buffered approach will be used in an interlaboratory validation study in the future, and the QuEChERS method will likely replace the inefficient monitoring methods currently in use. (Milestones 2.2, 2.3, and 2.5, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2, Performance Measures in the ARS Strategic Plan, 3.1.2). Interlaboratory validation of the QuEChERS method. The quick, easy, cheap, effective, rugged, and safe (QuEChERS) method for pesticide residue analysis was further optimized and a finalized protocol was prepared for an interlaboratory collaborative study involving 13 laboratories in 7 countries. This accomplishment is important because hundreds of monitoring and contract laboratories around the world need rapid,
inexpensive, and effective analytical methods for routine use, but the methods must be collaboratively validated for official purposes. The 13 collaborating laboratories analyzed 21 blind samples provided by the ARS lab, and results show that the method will become an approved method available for official use in regulatory applications. The impact of this accomplishment will be that the highly efficient and beneficial method will be used worldwide to provide reliable results more quickly and cheaply then before to ease trade and improve food safety. (Milestones 2.2, 2.3 and 2.5, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2, Performance Measures in the ARS Strategic Plan, 3.1.2). Optimized sample extraction and clean-up for QuEChERS analysis of fatty foods. The QuEChERS method for pesticide analysis was favorably compared with a matrix solid-phase dispersion (MSPD) method for the analysis of fatty foods. Pesticide residue analysis of foods is a
main function of many analytical laboratories around the world, and the current methods used in analysis are time-consuming, laborious, and expensive, especially in the case of fatty foods due to the need to remove co-extracted lipids before the detection step. This accomplishment evaluated the two rapid methods for the analysis of 32 diverse pesticides fortified in milk, egg, avocado and other fatty foods. Excellent results were obtained for certain problematic residues by the QuEChERS method that were not recovered by the MSPD method. It is likely that the QuEChERS method will become commonly used in many pesticide monitoring applications for foods of less than 20% fat. (Milestones 2.1, 2.3, and 2.5, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2, Performance Measures in the ARS Strategic Plan, 3.1.2). Multi-residue chromatographic/fluorescence detection and MS confirmation of fluoroquinones. The use of fluoroquinolone antibiotics, particularly
in poultry, is being scrutinized by the Food and Drug Administration due to antimicrobial resistance concerns, thus analytical methods for fluoroquinolones have become more important in the regulatory arena. A rapid extraction procedure and liquid chromatographic (LC) analysis with both fluorescence (FL) detection and tandem mass spectrometry (MS/MS) to analyze and confirm the presence of 8 fluoroquinolones in egg products (both white and/or yolk). A comparison study has been conducted in chicken muscle using this method and the currently used FSIS screening method, and this LC-FL-MS-MS method compared very favorably. The LC-FL- MS-MS method has also been compared with a bioassay (collaboration with Dan Donoghue) for analysis of chicken egg and muscle samples and it is now being extended to beef liver and shrimp. The method has been extensively validated internally and the method as applied to chicken samples was transferred to the FSIS Midwestern Laboratory. (Milestone 1. 1),
NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2, Performance Measures in the ARS Strategic Plan, 3.1.2). Rapid multi-residue and confirmatory analysis of beta-lactams. The Food Safety Inspection Service (FSIS) and the Food and Drug Administration (FDA) have a problem with the differentiation between the beta-lactam antibiotics, ceftiofur and penicillin, in their current monitoring method. A rapid and simple, quantitative and confirmatory method of analysis for 10 beta-lactam antibiotics in beef kidney using liquid chromatography/tandem mass spectrometry (LC/MS-MS) was developed and successfully compared with the FSIS 7-plate microbial assay for 45 incurred beef kidneys. This approach may be used to verify responses from the FSIS 7-plate assay, or to replace it altogether after it has been expanded to include other antibiotic drugs. (Milestone 1.3, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2, Performance Measures in
the ARS Strategic Plan, 3.1.2). Screening Assay for detection of ceftiofur metabolites in incurred kidney samples. A selective hydrolysis method followed by a microbial inhibition assay can presumptively identify the presence of ceftiofur metabolites at parts per million levels. The method detects all ?-lactam compounds and can differentiate ceftiofur metabolites from antimicrobial compounds classified as unidentified microbial inhibitors. This screening approach can be used to validate the presence of UMI or suspect ceftiofur metabolite prior to using instrumentation analysis. This test is rapid, simple and inexpensive where the sample preparation, hydrolysis and testing can be carried out in less than 4 hours. (Milestone 3.4, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2. 1.2.2, Performance Measures in the ARS Strategic Plan, 3.1.2). Rapid screening method for enrofloxacin and tetracyclines. Simple rapid screening methods are desirable to allow a
large number of samples to be analyzed for the presence of veterinary drugs. Such screens can greatly reduce the number of samples that would need to be analyzed by more extensive, quantitative and confirmatory methods, thus saving considerable time and expense. Last year, a rapid screening assay was developed which allows for detection of both enrofloxacin and tetracyclines in the same chicken muscle sample extract, at their respective tolerance levels. This assay enables rapid screening for two classes of antibiotics, without the need for separate extraction methods, saving time and expense. (Milestone 1.4, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2, Performance Measures in the ARS Strategic Plan, 3.1.2). Designed, built, and tested prototype portable TRL fluorometer. A new methodology was developed that hyphenated sorbent extraction and solid matrix TRL. Its application was demonstrated using tetracycline (TC) in milk as a model analyte. A
small format C18 sorbent strip was cut to extract tetracycline from milk in a 10-min immersion. Cleanup was a simple 3-min water immersion. After the C18 strip was spotted with a reagent solution and desiccated, TRL was measured directly on its surface. This rapid and simple method screened TC in milk at the 300 ppb tolerance level. No SPE cartridge or organic solvent was needed so it was of low-cost and friendly to both the operator and the environment. (Milestones 1.2, 1.5, and 2.5, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2, Performance Measures in the ARS Strategic Plan, 3.1.2). Designed, built and tested prototype evanescent field sensor. Tapered waveguides were developed for TRL sensing in evanescent field: (1) proof of the evanescent field sensing concept using tapered quartz-rod waveguides; (2) fabrication of tapered waveguides by acetylene torch drawing; (3) a waveguide accessory was designed and assembled for a commercial fluorescence
spectrometer; (4) the TRL performance was compared on different taper geometries using TC as a model analyte; (5) the profile of the best performing exponential-linear tapered waveguide was further optimized by fluorescence imaging, a technique also developed in this lab that visually revealed the location and the intensity of light leaking from the core due to violation of total internal reflection. For tetracycline a 20 ppb limit of detection (LOD) was achieved. This innovative method was approved by the ARS Office of Technology Transfer (OTT) review panel for patent application (0032.05). (Milestone 1.5 and modified 2.5, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2, Performance Measures in the ARS Strategic Plan, 3. 1.2). Non-instrumental and biosensor methods for detection of SEA an SEB in foods. Developed a non-instrumental latex agglutination assay that can detect toxin contaminants from Staphylococcus aureus (SEA and SEB) in 1 g of food
samples. Improved sensitivity of 1 2.5 ng/g in eggs, milk and ham samples was attained using a commercial biosensor instrument. FSIS expressed needs for these methods as alternatives to commercial assays. (Milestones 3.4 and 3.5, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1.2, Performance Measures in the ARS Strategic Plan, 3.1.2). Facile sample preparation using dispersive SPE. Developed the dispersive solid-phase extraction (dispersive-SPE) concept in which the cleanup sorbent is used to retain matrix co-extractives rather than the analytes, and the procedure is conducted in a small tube rather than a column. This fast and cheap technique is used in the QuEChERS approach, and is expanding to non-pesticide residue applications. (Milestones 1.2 and 2.2, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2, Performance Measures in the ARS Strategic Plan, 3.1.2). Development of the analyte protectants concept. In the U.S., the Food
and Drug Administration (FDA) and Environmental Protection Agency (EPA) policies do not permit the use of matrix-matched calibration standards to improve accuracy in the quantitation of pesticide residues for regulatory enforcement actions. To overcome this regulatory limitation, and to provide better gas chromatographic (GC) peak shapes and lower detection limits for problematic pesticides, a new concept that we call analyte protection was developed and investigated. The concept involves the addition of chemicals, such as sugar derivatives, to both calibration standards and extracts, and the chemicals serve to mask the active sites in the GC system that cause the inaccurate quantitation of certain pesticides. This simple approach may become standard practice for GC analysis of pesticides because it was shown to provide accurate results and low detection limits while still meeting EPA and FDA policies for enforcement actions. (Milestones 2.1 and 1.4, NP108 Drug Residue sand
Environmental Contaminants Action Plans 2.1 and 2.1.2.2, Performance Measures in the ARS Strategic Plan, 3.1.2). Determined solvent suitability and pesticide stability for GC analysis. The suitability of 6 different organic solvents (acetonitrile, acetone, ethyl acetate, toluene, isooctane, and hexane) for injection in GC and pesticide stability was thoroughly assessed. Selection of the ideal solvent is a key factor in the testing of pesticide residues in foods using GC, yet it has not been comprehensively discussed before. The ideal solvent should: (i) provide sufficient solubility and stability of the pesticides of interest; (ii) be used in the extraction and/or clean- up step to avoid an additional solvent exchange step; (iii) permit optimal GC analysis in terms of sensitivity, reproducibility, and speed; and (iv) be non-hazardous and inexpensive. Acetonitrile was found to be the most suitable solvent for extraction of a wide polarity range of pesticide residues from produce,
and adding a small amount of acetic acid to it slows degradation of problematic pesticides for better analysis. We provided practical recommendations on selection of the most suitable solvent for pesticide residue analysis and proposed solutions to several real-world problems associated with the use of particular solvents. (Milestones 2.2 and 1.4, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2, Performance Measures in the ARS Strategic Plan, 3.1.2). Rapid multiresidue analysis of pesticides using LP-GC/MS. In order to substantially speed up the analysis of pesticide residues in food, a new method of analysis using low-pressure gas chromatography/mass spectrometry (LP-GC/MS) was optimized and evaluated. An analytical method using LP-GC/MS for 20 challenging pesticides in carrots was demonstrated, which decreased the time of chromatographic analysis from approximately 20 min to 6 min. Limits of detection were also decreased by enabling a higher
injection volume and increasing the ruggedness of the chromatographic approach. The outcome of this research is that LP- GC/MS can significantly increase the speed of multi-residue pesticide analysis and provide other benefits of larger sample capacity, lower detection limits, and reduced thermal degradation of pesticide analytes. (Milestones 2.3, and 2.4, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2, Performance Measures in the ARS Strategic Plan, 3.1.2). Rapid methods for multi-residue detection and confirmatory analysis of thyreotats and beta-agonists in bovine tissues. Currently, regulatory and contract laboratories use 2 different time-consuming and intensive analytical methods for the determination of thyreostats and beta-agonists, which are banned growth promoters. A novel multiresidue analytical screening method for the isolation and detection of 14 thyreostats and beta-agonists using liquid chromatography-fluorescence-tandem mass
spectrometry in meat and liver tissues was developed. The presence of the compounds can be confirmed and quantified (if necessary) at levels less than 100 ng/g within 1 hour. This approach is simpler, faster and more effective than the current methods used by U.S., Canadian, and the European Union regulatory agencies and the protocol was provided to the regulatory agencies. The method was transferred to FSIS Eastern and Western Labs and is being adapted for their potential usage. (Milestone 1.2, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2, Performance Measures in the ARS Strategic Plan, 3.1.2). Rapid and sensitive method to detect spectinomycin aminoglycoside. Rapid screening methods are needed for detection of spectinomycin residues in animal tissues at the regulatory action levels. Reagents for a fluorescent immunoassay were synthesized, and the fluorescent latex immunoassay method was optimized for detection of spectinomycin at 5 100 ng/g in
bovine kidney extract. This method can be used to identify compounds classified as an unidentified microbial inhibitor (UMI) in the 7-plate microbial assay used by the Food Safety Inspection Service (FSIS). The use of this method by FSIS could reduce the number of unidentified microbial inhibitors (UMI) compounds and enable better quantitation if these compounds appear above the action levels. (Milestone 3.4, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2, Performance Measures in the ARS Strategic Plan, 3.1.2). 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? The results from research studies have been transferred to interested parties by means of collaborations, peer-reviewed publications, written reports, internet
accounts, presentations at scientific meetings, and communications with scientists and administrators from regulatory agencies, industry, and other organizations. Research results of studies and recommendations designed to meet residue detection needs in the Food Safety Inspection Service (FSIS) for meat, poultry, and eggs have been provided to their laboratories. Three analytical methods using LC-MS/MS developed in ARS were transferred to FSIS (the analysis of beta-lactams, fluoroquinolones, and thyreostats in animal tissues), and the methods were implemented after the FSIS internal validation and approval process. FSIS is also going to evaluate the ARS method for analysis of pesticide residues in egg and meat. State programs, U.S. federal agencies, foreign regulatory programs, industry and contract laboratories are particularly interested in the approaches developed for pesticide residue analysis. In Nov of 2004, the ERRC scientists co-organized and hosted a week-long EPA/USDA
Pesticide Analytical Workshop for 17 chemists from 15 official state labs in the U. S. Several lectures and hands-on training were provided, and several of the labs have adopted the approaches developed from the ARS research. Laboratories in the USDA Agricultural Marketing Service and Food and Drug Administration are also using extraction and analytical techniques developed in ARS. The QuEChERS method was validated in an interlaboratory study involving 13 labs in 7 countries, and the protocol was disseminated to scores of interested scientists around the world upon their request. Also, a noncommerical antibody for hygromycin developed at ERRC was provided to a scientist at Columbia University. The latex and biosensor immunoassay methods for detection of enterotoxins (SEA and SEB) in eggs are of high interest to FSIS. These methods will be transferred after validation for routine analysis. The technique that hyphenates sorbent extraction and solid matrix TRL has been filed with
the ARS Technical Transfer Office for patent application (0062.04). The ARS Press Release on New Detection Methods Improve Food Safety attracted the attention of scientists at the Center for Advanced Sensor Technology, University of Maryland Baltimore County (UMBC). Our technical know-how on biosensor development for detection of staphylococcal enterotoxins is being applied to micro-sensor technology developed at UMBC through research collaboration. We are transferring immunochemical reagents synthesized at ERRC for their application. 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). New Detection Methods Improve Food Safety Agricultural Research, Vol. 53, No. 1, January, 2005, pp. 15-17. STORY LEAD: New Detection Methods Improve Food Safety. ARS News Service. January 6, 2005. Schneider, M.J. 2005. Review of Practical High-Performance Liquid
Chromatography, 4th Ed., Inside Lab. Management, Jul/Aug, p. 14. Lehotay, S.J. 2005. Review of Modern Practice of Gas Chromatography, 4th Ed. Inside Lab. Management, Jan/Feb, pp. 17-18. Matovska, K, 2005. Review of Chromatography: Concepts and Contrasts, 2nd Ed. Inside Lab. Management, May/Jun, p. 9. Chen, G. 2005. Versatile Portable Fluorometer for Time-Resolved Luminescence Analysis. Rev. Sci. Instrum. 76, 063107. Lehotay, S.J. 2005. Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) Approach for Determining Pesticide Residues. in Methods in Biotechnology, Vol. 19, Pesticide Protocols, Martinez Vidal, J.L. and Garrido Frenich, A. (Eds.), Humana Press, Totowa, NJ, pp. 239-261.
Impacts
(N/A)
Publications
- Schneider, M.J. 2004. A rapid flourescence screening assay for enrofloxacin and tetracyclines in chicken muscle. Journal of Agricultural and Food Chemistry. 52. pp. 7809-7813.
- Schneider, M.J., Vazquez Moreno, L., Barraza Guardado, R., Ortega-Nieblas, M. 2005. Multiresidue determination of fluoroquinolone antibiotics in shrimp by liquid chromatography-fluorescence-mass spectrometry. Journal of the Association of Official Analytical Chemists. 88. pp. 1160-1166.
- Chen, G., Schneider, M.J., Darwish, A.M., Lehotay, S.J., Freeman, D.W. 2004. Europium-sensitized luminescence determination of oxytetracycline in catfish muscle. Talanta. 64. pp. 252-257.
- Mastovska, K., Hajslova, J., Lehotay, S.J. 2004. Ruggedness and other performance characteristics of low-pressure gas chromatography-mass spectrometry for the fast analysis of multiple pesticide residues in food crops. Journal of Chromatography A. 1054. 2004. p. 335-349.
- Fagerquist, C.K., Lightfield, A.R., Lehotay, S.J. 2005. Confirmatory and quantitative analysis of b-lactam antibiotics in bovine kidney tissue by dispersive solid-phase extraction and liquid chromatography-tandem mass spectrometry. Analytical Chemistry 5(77).1473-1482.
- Liu, L.S., Chen, G., Fishman, M.L. A single sorbent for tetracycline enrichment and following solid-matrix time-resolved luminescence. 2005. Analytica Chimica Acta:528: p.261-268.
- Liu, L.S., Chen, G., Fishman, M.L., Hicks, K.B. Pectin gel vehicles for citronellal delivery. Controlled Release Journal.2005. Drug Delivery. 12:p.149-157.
- Lehotay, S.J., Mastovska, K. Determination of pesticide residues. Methods of Analysis of Food Components and Additives. Taylor & Francis, Boca Raton, FL. 2005. pp. 329-359.
- Lehotay, S.J., De Kok, A., Hiemstra, M., Van Bodegraven, P. 2005. Validation of a fast and easy method for the determination of 229 pesticide residues in fruits and vegetables using gas and liquid chromatography and mass spectrometric detection. Journal of Association of Official Analytical Chemists International. 2005. v. 88. p. 595-614.
- Lehotay, S.J., Mastovska, K., Lightfield, A.R. 2005. Use of buffering to improve results of problematic pesticides in a fast and easy method for residue analysis of fruits and vegetables. Journal of Association of Official Analytical Chemists International. 88.pp. 615-629.
- Lehotay, S.J., Mastovska, K., Jong, Y. 2005. Evaluation of two fast and easy methods for pesticide residue analysis in fatty food matrices. Journal of Association of Official Analytical Chemists International. 88. pp. 630-638.
- Cajka, T., Mastovska, K., Lehotay, S.J., Hajslova, J. 2005. Use of automated direct sample introduction with analyte protectants in the gc-ms analysis of pesticide resides. Journal of Separation Science. 28. pp. 1048- 1060.
- Medina, M.B. 2005. Development of a competitive immunoassay for detection of staphylococcal enterotoxin b ((seb) in milk. Journal of Rapid Methods and Automation in Microbiology. 13. pp. 37-55.
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Progress 10/01/03 to 09/30/04
Outputs
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? What does it matter? 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 in foods. This pertains to Food Safety (National Program 108), Priority Objective 2.1.2. 2 under the Toxic Chemicals section in the National Program 108 Action Plan. 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. These needs are being met by the use of modern methods and approaches which are being evaluated to screen, quantify, and/or confirm chemical contaminants. These advanced technologies include hyphenated gas or liquid
chromatography/mass spectrometry (or tandem mass spectrometry), fluorescence, solid phase extraction, surface plasmon resonance biosensor, field-based instruments, immunoassays and other techniques. 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) and other organizations that monitor chemical residues in food, which also includes industry, consumer groups, and academic scientists. Current methods of detection are often too time- consuming, expensive, and labor-intensive. Minimization of the time, effort, and cost, and improvement in the quality of the results of these analyses, would
provide a significant benefit to governments, industry, academic scientists, and consumers. The implementation of improved analytical approaches will (a) increase productivity and/or lower costs of analysis, (b) provide more statistically valid and accurate results for risk assessment and other purposes, (c) overcome trade barriers associated with the analysis of chemical residues, (d) provide more information to understand the effects and mechanisms of antimicrobial resistance and endocrine disruption, (e) allow for better verification of organic food labeling, (f) improve possible industrial food safety procedures, and (g) reduce the potential for food that has been deliberately or accidentally adulterated by toxic chemicals to reach the consumer. 2. List the milestones (indicators of progress) from your Project Plan. The milestones originally listed in the approved project plan did not have any numbers. Also, projected contributions from SYs hired after the plan was in place were
used as milestones in the approved project plan. For reporting purposes, the milestones are numbered for easy reference. Milestone 1. 2001: 2.1.1. Publication by Schneider et al. concerning the simultaneous determinative and confirmatory analysis of fluoroquinolones in egg and/or liver matrices using liquid chromatography(LC) / atmospheric pressure ionization (API) / tandem mass spectrometry (MS-MS) and fluorescence detection. 2. 1.2. Publication by Lehotay et al. on the use of gas chromatography (GC) / pulsed flame photometric detection (PFPD) + micro electron capture detection (mECD) for the analysis of organophosphorus and organochlorine pesticides in fatty matrices. Milestone 2. 2002: 2.2.1. Publications by Vacant 3/2000 et al. and 2.2. 2. Schneider et al. on the rapid confirmatory screening of phenicols and beta-agonists, respectively, using LC/API/MS-MS and assessment of quantitation and confirmation capabilities of the approaches in real samples. 2. 2.3. Publication by
Lehotay, Klassen, et al. on the development of MS-MS spectral libraries with chlorpyrifos as the example analyte. 2.2.4. Publication by Lehotay et al. and Schenck and Lehotay on the extraction and clean-up with different solvents, pH, and SPE sorbents of a variety of different food commodities. Milestone 3. 2003: 2.3.1. Publication by Vacant 3/2000 et al. on the use of LC/APCI/MS-MS for the analysis of 2 classes of veterinary drugs. 2. 3.2 Publication by Schneider et al. on LC/API/MS-MS for the rapid analysis of aminoglycoside antibiotics in animal tissues. 2.3.3. Publication by Vacant 11/2000 et al. on the portable multiresidue screening analysis of OP pesticides at violative concentrations in nonfatty foods. 2.3.4. Publication by Lehotay et al. on the use of rapid and automated direct sample introduction (DSI) / low pressure-GC/MS, ideally for the reliable analysis of >100 pesticides in <15 min. NIST will likely to have completed the MS-MS spectral library for pesticides and
developed a software program by this time. Also, Sandia may have prototypes of micro-technological devices for us to evaluate. Modified 2. 3.5. Publication by Medina in biosensor detection of Staphylococcal enterotoxins Milestone 4. 2004: 2.4.1. Publications by Vacant 3/2000 et al. on the use of LC/API/MS-MS for the analysis of 3 classes of veterinary drugs, and (2.4.2 )by Schneider et al. on LC/API/MS-MS for the rapid analysis of peptide antibiotics in animal tissues. 2.4.3. Publication by Vacant 11/2000 et al. on rapid, simple, representative, and reproducible sample preparation approaches needed for the application of micro-technological devices to chemical residue analysis in foods. 2.4.4 . Publication by Lehotay et al. on the use of rapid and automated DSI/LP-GC/MS for GC- amenable veterinary drugs. Modified 2.4.5. Publication by Medina in rapid and sensitive detection of aminoglycosides and biosensor detection of Staphylococcal enterotoxins. Milestone 5. 2005: 2.5.1.
Publications by Vacant 3/2000 et al. on the use of LC/API/MS-MS for the analysis of >3 classes of veterinary drugs, and (2.5.2) by Schneider et al. on LC/API/MS-MS for the rapid analysis of 2 classes of veterinary drugs in animal tissues. 2.5.3. Publication by 2.5.4. Vacant 11/2000 et al. on the use of a micro-technological device for chemical residue analysis in foods. 2. 5.5. Publication by Lehotay et al. on the use of rapid API/MS-MS in the analysis of multiple thermally labile pesticides. Modified 2.5.6. Publications by Medina in biosensor and multiresidue detection of aminoglycosides and multi-toxin detection of Staphylococcal enterotoxins. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. Milestone 4. 2004: 2.4.1. Publications by Vacant 3/2000 et al. on the use of
LC/API/MS-MS for the analysis of 3 classes of veterinary drugs, and (2.4.2) by Schneider et al. on LC/API/MS-MS for the rapid analysis of peptide antibiotics in animal tissues. 2.4.3. Publication by Vacant 11/2000 et al. on rapid, simple, representative, and reproducible sample preparation approaches needed for the application of micro-technological devices to chemical residue analysis in foods. 2.4.4. Publication by Lehotay et al. on the use of rapid and automated DSI/LP-GC/MS for GC- amenable veterinary drugs. 2.4.5. Publication by Medina in rapid and sensitive detection of aminoglycosides and biosensor detection of Staphylococcal enterotoxins. Over the course of this CRIS project, we have substantially met 6 of the milestones originally listed in the approved 5-year project plan proposed in May of 2000. In 2004, 3 of the 4 envisioned milestones ( 2.4.2, 2.4.3 and 2.4.4)are being pursued as described in 2000, and substantial progress has been made. The Vacant 3/2000 position
listed in the project plan was filled by Dr. C. Keith Fagerquist in 2001, and in 2003, Dr. Fagerquist transferred to another ARS location. Despite this, Lehotay, Schneider, et al. have completed the optimization of LC/API/MS-MS conditions for more than 100 veterinary drugs from more than 5 different classes. Substantial progress has been made in all of the major objectives listed in the CRIS project, including new priorities for investigation that could not have been envisioned in 2000. Dr. Medina was reassigned to this CRIS in FY 2002. The eventualities of unforeseen research results and changing personnel and resources were described as a strong possibilities in the Project Plan, thus the appropriate contingencies were pursued. Milestones 2.3.2 and 2.4.2 were modified. Dr. Guoying Chen filled the position described as Vacant 11/2000 in the Project Plan. In 2004, Chen et al. designed, prototyped, and tested a portable fluorometer capable of time-resolved luminescence (TRL)
measurement in the field. Major progress made in 2004 include: (a) the optics and electronic hardware were finalized (second version); (b) the custom software was developed in LabVIEW (third version); (c) the packaging was completed in a hard-plastic suitcase; (d) the gating technique was implemented to overcome saturation of photomultiplier tube by intense xenon flashes; and (e) pulse-to-pulse fluctuations of the lamp were monitored and overcome. This marked for the first time the field deployment of TRL technology and instrument to test and analyze drug residues in foods. Another milestone in instrument development has been achieved that uses a tapered waveguide for evanescent-field sensing with the potential to simplify sample preparation in analysis of animal muscle. Rapid, simple, representative, and reproducible sample preparation approaches are being developed for the application of micro-technological devices to chemical residue analysis in foods (Milestone 2.4.3). An
automated DSI device was not available in the commercial market until 2003, and the system was purchased for our investigations in the summer of 2004. Arrangements have been made for a Visiting Scientist to work with Dr. Lehotay to pursue the 2004 milestone later in the year. The milestone on the LC/API/MS-MS publication for the rapid analysis of peptide antibiotics in animal tissues was not pursued because our communications with stakeholders indicated that rapid chemical screening assays have greater potential for significant cost and time savings in the residue monitoring process. Thus, Schneider et al. opted to pursue this promising avenue of research rather than the analysis of peptide antibiotics in order to provide potentially higher impact results for our stakeholders, such as FSIS and the food processing industry. A rapid chemical screening assay for enrofloxacin in chicken tissue was developed and published in 2003, which was the first in a series of methods illustrating
the potential for success. (Milestone 2.4.4) In addition to the original milestones listed in 2000, Medina et al. recently completed development and dissemination of results on methods for detection of Staphylococcus aureus enterotoxin B (SEB) in milk. Methods for detection of Staphylococcus aureus enterotoxin A (SEA) in eggs using biosensor and latex agglutination assays will be completed and published in the near future, and screening assays for detection of beta- lactams using enzyme hydrolysis to confirm presence of the drugs in meat tissues will also be completed. Dr. Medina also completed and disseminated results on a rapid screening assay for spectinomycin aminoglycoside (Modified Milestone 2.3.5 and 2.4.5) B. List the milestones that you expect to address over the next 3 years. What do you expect to accomplish, year by year, over the next 3 years under each milestone? 2005: Development of LC/API/MS-MS methods for the rapid analysis of multiple classes of veterinary drugs in
animal tissues. This milestone represents a reasonable goal for 2005, as a step in the multiclass multiresidue approach to residue analysis to which we are committed. Analysis of more than one residue, and ultimately, more than one class of residues simultaneously has the potential for major cost and time savings in residue analysis, increasing international trade of U.S. food products, and providing improved food safety for the consumer. Chen et al. will field-test the portable fluorometer for the analysis of veterinary drugs in animal tissues and optimize the tapered-waveguide accessory for commercial fluorescence spectrophotometers. Publications will be written on the design of the portable fluorometer and the scheme of evanescent field sensing based on tapered waveguide devices. The impact of this research will lead to possible commercialization of the devices and their use in the field to aid regulatory agencies and industry. Medina will determine the feasibility of using
lysozyme or lactalbumin for multi-residue rapid assay of the aminoglycoside antibiotics, spectinomycin, gentamicin, neomycin, streptomycin, and complete and publish results on development of latex fluorescent assays for sensitive detection of SEA and/or SEB. Lehotay et al. will have completed investigations and interlaboratory validation of the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method for multiclass, multiresidue applications of pesticide residue analysis in foods. The approach will be available for official usage by monitoring laboratories around the world 2006 - 2007: A new 5-year CRIS project plan will be proposed in 2005 for approval. Discussions are underway with stakeholders to determine their potential future needs in chemical contaminant analysis, as well as in analysis of select agents. Outcomes from these discussions will help to shape plans for the next Project Plan covering these years. Expected milestones involving the continuation of the aims
given in the current project plan are given as follows. Chen et al. will apply the tapered waveguide to analysis of animal tissues for veterinary drugs residues; integrate the tapered waveguide into the portable, time-resolved fluorometer; and publish results on the tapered waveguide application of enrofloxacin and oxytetracycline analysis. Medina will complete development and optimization of a rapid assay method for multi-toxin detection of staphylococcal enterotoxins in food samples and publish results. It is anticipated that our multiclass multiresidue analytical approach will be pursued further during this time in an effort to maximize the number of veterinary drugs which can be analyzed with the minimum number of separate methods. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004: Rapid and non-instrumental method for detection of pesticides. The quick, easy, cheap, effective, rugged, and safe (QuEChERS)
method for pesticide residue analysis was further optimized and a finalized protocol was prepared for an interlaboratory collaborative study involving 15 laboratories in 7 countries. This accomplishment is important because hundreds of monitoring and contract laboratories around the world need rapid, inexpensive, and effective analytical methods for routine use, but the methods must be collaboratively validated for official purposes. The highly streamlined QuEChERS method was modified by using buffering to achieve high recoveries and greater stability of a few problematic base- sensitive pesticides. The 15 collaborating laboratories analyzed 21 blind samples provided by the ARS lab, and initial results show promise that the method will become an approved method available for official use in regulatory applications. The impact of this accomplishment will be that the highly efficient and beneficial method will be used worldwide to provide reliable results more quickly and cheaply
then before to ease trade and improve food safety. .(Milestones 2.3.4 and 2.4.4, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2) B. Other significant accomplishment(s), if any. Validation of QuEChERS method. The usefulness of the QuEChERS method was demonstrated in LC/MS-MS analysis, and the method was validated in a single laboratory for more than 200 pesticides in fruits and vegetables by Lehotay in collaboration with de Kok, Hiemstra, and van Bodegraven in the Dutch Food Inspection Service, Amsterdam, The Netherlands. This accomplishment was important in showing that the method worked for so many pesticides in different matrices. Validation experiments entailed fortification of fruit and vegetable samples with the pesticides in replicate analyses at 3 different levels. Also, many proficiency test samples were analyzed by the method, and side-by-side analyses were conducted to compare the QuEChERS results with those from a traditional method.
The experiments pinpointed actions that needed to be taken to improve results for a few problematic base-sensitive pesticides, which was done in a follow-up study, and the successful validation of more than 200 pesticides in a thorough protocol convinced other laboratories to participate in the collaborative study discussed in 4A. .(Milestones 2.3. 4 and 2.4.4, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2) Rapid screening method for enrofloxacin and tetracyclines. Simple rapid screening methods are desirable to allow a large number of samples to be analyzed for the presence of veterinary drugs. Such screens can greatly reduce the number of samples that would need to be analyzed by more extensive, quantitative and confirmatory methods, thus saving considerable time and expense. This year, a rapid screening assay was developed which allows for detection of both enrofloxacin and tetracyclines in the same chicken muscle sample extract, at their
respective tolerance levels. This assay enables rapid screening for two classes of antibiotics, without the need for separate extraction methods, saving time and expense. [Milestone 2.4.2 (modified), NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2] Design, build and test prototype portable TRL fluorometer. A new methodology was developed that hyphenated sorbent extraction and solid matrix TRL. Its application was demonstrated using tetracycline (TC) in milk as a model analyte. A small format C18 sorbent strip was cut to extract tetracycline from milk in a 10-min immersion. Cleanup was a simple 3-min water immersion, the C18 strip was then desiccated and TRL was measured directly on its surface. This rapid and simple method screened TC in milk at the 300 ppb tolerance level. No SPE cartridge or organic solvent was needed so it was of low-cost and friendly to both the operator and the environment. This method was filed for patent application
(0062.04) to the ARS Technical Transfer Office. [Milestone 2. 4.3 and modified 2.4.2, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2] Design, build and test prototype evanescent field sensor. Tapered waveguides were developed for TRL sensing in evanescent field: (1) proof of the evanescent field sensing concept using tapered quartz-rod waveguides; (2) fabrication of tapered waveguides by acetylene torch drawing; (3) a waveguide accessory was designed and assembled for a commercial fluorescence spectrometer; (4) the TRL performance was compared on different taper geometries using TC as a model analyte; (5) the profile of the best performing exponential-linear tapered waveguide was further optimized by fluorescence imaging, a technique also developed in this lab that visually revealed the location and the intensity of light leaking from the core due to violation of total internal reflection. For tetracycline a 20 ppb limit of detection (LOD) was
achieved. This innovative method has patent potential and opportunities for commercialization. .(Milestone 2.4.3 and modified 2.4.2, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2) Develop non-instrumental and biosensor methods for detection of SEA an SEB in foods. Developed a non-instrumental latex agglutination assay that can detect toxin contaminants from Staphylococcus aureus (SEA and SEB) in 1 g of food samples. Improved sensitivity of 1-2.5 ng/g in eggs, milk and ham samples was attained using a commercial biosensor instrument. FSIS expressed needs for these methods as alternatives to commercial assays. (modified Milestones 2.3.5 and 2.4.5, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1.2). Optimize sample extraction and clean-up for QuEChERS analysis of fatty foods. The QuEChERS method for pesticide analysis was favorably compared with a matrix solid-phase dispersion (MSPD) method for the analysis of fatty foods.
Pesticide residue analysis of foods is a main function of many analytical laboratories around the world, and the current methods used in analysis are time-consuming, laborious, and expensive, especially in the case of fatty foods due to the need to remove co-extracted lipids before the detection step. This accomplishment evaluated the two rapid methods for the analysis of 32 diverse pesticides fortified in milk, egg, avocado and other fatty foods. Excellent results were obtained for certain problematic residues by the QuEChERS method that were not recovered by the MSPD method. It is likely that the QuEChERS method will become commonly used in many pesticide monitoring applications for foods of less than 20% fat. The suitability of 6 different organic solvents (acetonitrile, acetone, ethyl acetate, toluene, isooctane, and hexane) for injection in GC and pesticide stability was thoroughly assessed. Selection of the ideal solvent is a key factor in the testing of pesticide residues
in foods using GC, yet it has not been comprehensively discussed before. The ideal solvent should: (i) provide sufficient solubility and stability of the pesticides of interest; (ii) be used in the extraction and/or clean- up step to avoid an additional solvent exchange step; (iii) permit optimal GC analysis in terms of sensitivity, reproducibility, and speed; and (iv) be non-hazardous and inexpensive. Acetonitrile was found to be the most suitable solvent for extraction of a wide polarity range of pesticide residues from produce, and adding a small amount of acetic acid to it slows degradation of problematic pesticides for better analysis. We provided practical recommendations on selection of the most suitable solvent for pesticide residue analysis and proposed solutions to several real-world problems associated with the use of particular solvents. . (Milestones 2.3.4 and 2.4.4, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2) C. Significant
activities that support special target populations. None. D. Progress Report: 1935-42000-044-02S This report serves to document research conducted under a specific cooperative agreement between ARS and the Dept. of Biosciences and Biotechnology at Drexel University, entitled "Development of New Methods to Detect and Control Acrylamide Formation in Deep-Fat Fried Foods". The objective of this cooperative research project is 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 in which different frying techniques will be assessed in order to minimize the generation of acrylamide in the food. Acrylamide has become an important food safety concern, especially for regulators and the food industry, since the report of its presence in several types of processed foods in 2002, and 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 are being pursued by Dr. Phil Handel at Drexel University and ARS is analyzing the generated samples. Furthermore, Dr. Xuetong Fan of ARS is studying 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. 1935-42000-044-03R This report serves to document research conducted under U.S.-Israel Binational Agricultural Research and Development (BARD) grant US-35000-03 shared
equally between ARS and the School of Chemistry at Tel Aviv University in Tel Aviv, Israel, entitled "Fast, Practical and Effective Approach for the Analysis of Hazardous Chemicals in the Food Supply". Dr. Lehotay collaborates with Prof. Aviv Amirav of Tel Aviv University in the 3-year grant. 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 University, 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 will be 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. In
2004, an automated DSI device was purchased for evaluation to meet ARS objectives A and B. Arrangements have been made for Visiting Scientists to conduct the research objectives described in the project. Initial experiments have been conducted pertaining to sample preparation in veterinary drug analysis, which show the QuEChERS approach for pesticides to be feasible after modifications depending on the matrix and analytes. 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. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Rapid and non-instrumental method for detection of pesticides. Developed the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method for multiple pesticide
residues in foods. Using the method, a single analyst can extract hundreds of pesticides from 8 samples in about 30 min using $1 in materials/sample. This method is being validated and transferred in interlaboratory trials. .(Milestones 2.3.4 and 2.4.4), NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2) Rapid multi-residue and confirmatory analysis of beta-lactams. The Food Safety Inspection Service (FSIS) and the Food and Drug Administration (FDA) have a problem with the differentiation between the beta-lactam antibiotics, ceftiofur and penicillin, in their current monitoring method. A rapid and simple, quantitative and confirmatory method of analysis for 10 beta-lactam antibiotics in beef kidney using liquid chromatography/tandem mass spectrometry (LC/MS-MS) was developed and compared with the FSIS 7-plate microbial assay for 30 incurred beef kidneys. The results agreed in 28 instances, with the 2 contradictory samples being mis-identified by the
FSIS microbial assay. This approach may be used to verify responses from the FSIS 7-plate assay, or to replace it altogether after it has been expanded to include other antibiotic drugs. .(Milestone 2.3.1. NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2) Quantitative and confirmatory analysis of pesticides. In the U.S., the Food and Drug Administration (FDA) and Environmental Protection Agency (EPA) policies do not permit the use of matrix-matched calibration standards to improve accuracy in the quantitation of pesticide residues for regulatory enforcement actions. To overcome this regulatory limitation, and to provide better gas chromatographic (GC) peak shapes and lower detection limits for problematic pesticides, a new concept that we call 'analyte protection' was developed and investigated. The concept involves the addition of chemicals, such as sugar derivatives, to both calibration standards and extracts, and the chemicals serve to mask the
active sites in the GC system that cause the inaccurate quantitation of certain pesticides. This simple approach may become standard practice for GC analysis of pesticides because it was shown to provide accurate results and low detection limits while still meeting EPA and FDA policies for enforcement actions (Milestones 2.1.2 ,2.2.3 and 2.2.4, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2). Rapid multiresidue analysis of pesticides using LP-GC/MS. In order to substantially speed up the analysis of pesticide residues in food, a new method of analysis using low-pressure gas chromatography/mass spectrometry (LP-GC/MS) was optimized and evaluated. An analytical method using LP-GC/MS for 20 challenging pesticides in carrots was demonstrated, which decreased the time of chromatographic analysis from approximately 20 min to 6 min. Limits of detection were also decreased by enabling a higher injection volume and increasing the ruggedness of the
chromatographic approach. The outcome of this research is that LP- GC/MS can significantly increase the speed of multi-residue pesticide analysis and provide other benefits of larger sample capacity, lower detection limits, and reduced thermal degradation of pesticide analytes (Milestones 2.3.4, and 2.4.4, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2). Facile sample preparation using dispersive SPE. Developed the dispersive solid-phase extraction (dispersive-SPE) concept in which the cleanup sorbent is used to retain matrix-co-extractives rather than the analytes, and the procedure is conducted in a small tube rather than a column. This fast and cheap technique is used in the QuEChERS approach, and is expanding to non-pesticide residue applications (Milestones 2.4.4), NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2) . Multi-residue chromatographic/fluorescence detection and MS confirmation of fluoroquinones. The use
of fluoroquinolone antibiotics, particularly in poultry, is being scrutinized by the Food and Drug Administration due to antimicrobial resistance concerns, thus analytical methods for fluoroquinolones have become more important in the regulatory arena. A rapid extraction procedure and liquid chromatographic (LC) analysis with both fluorescence (FL) detection and tandem mass spectrometry (MS/MS) to analyze and confirm the presence of 8 fluoroquinolones in egg products (both white and/or yolk). A comparison study has been conducted in chicken muscle using this method and the currently used FSIS screening method, and this LC-FL-MS-MS method compared very favorably. The LC-FL- MS-MS method has also been compared with a bioassay (collaboration with Dan Donoghue) for analysis of chicken egg and muscle samples and it is now being extended to beef liver and shrimp. The method has been extensively validated internally and the method as applied to chicken samples was transferred to the FSIS
Midwestern Laboratory (Milestones 2.1. 1, and modified 2.2.2 & 2,23.2, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2). Design and build portable TRL fluorometer and test prototype for rapid screening of fluoroquinones and tetracycline in foods. Developed lanthanide-sensitized time-resolved luminescence (TRL) methods for the screening of trace amounts of fluoroquinolones or tetracyclines in chicken, turkey, and fish tissues. The detectable concentrations are well below U.S. tolerance levels for the approved drugs, but the low detection limits are needed for monitoring in the E.U. This detection approach is being incorporated into a portable TRL detection instrument for field applications (Milestones 2.3.3, 2.3.4, 2.4.3 and 2.4.4 ), NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2) . Design, build and test prototype of evanescent field sensor. Developed methods using steady state fluorescence and time-resolved
luminescence for screening and quantitative analysis of fluoroquinolone and tetracycline in chicken, catfish and milk evanescent field sensing using tapered quartz-rod waveguides. Established a method that hyphenates sorbent extraction and solid matrix time-resolved luminescence; and completion of a portable, time-resolved fluorometer (Milestones 2.3.3, and 2.4.3, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2). Rapid methods for multi-residue detection and confirmatory analysis of thyreotats and beta-agonists in bovine tissues. Currently, regulatory and contract laboratories use 2 different time-consuming and intensive analytical methods for the determination of thyreostats and beta-agonists, which are banned growth promoters. A novel multiresidue analytical screening method for the isolation and detection of 14 thyreostats and beta-agonists using liquid chromatography-fluorescence-tandem mass spectrometry in meat and liver tissues was developed.
The presence of the compounds can be confirmed and quantified (if necessary) at levels less than 100 ng/g within 1 hour. This approach is simpler, faster and more effective than the current methods used by U.S., Canadian, and the European Union regulatory agencies and the protocol was provided to the regulatory agencies. The method was transferred to FSIS Eastern and Western Labs and is being adapted for their potential usage (Milestones 2. 2.1 and 2.3.1, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2). Develop non-instrumental and biosensor methods for detection of SEA an SEB in foods. Staphylococcus aureus enteric toxins cause a major type of food-borne gastroenteritis, and the food industry needs rapid methods for detection of the most common Staphylococcus aureus A and B (SEA and SEB) toxins, but commercially available methods are expensive for routine use. We developed and optimized a latex particle agglutination assay for detection of SEA and
SEB. The assay is simple and non-instrumental, and the detectability is as little as 10 ng/g. Improved detectability was attained using a commercial instrumental-based biosensor immuno-assay with detection of less than 1 to 2.5 ng/g of toxins in eggs, milk and ham. This gives potential users of the detection methods, depending on their needs and priorities, a choice between the inexpensive screening method at higher levels or expensive instrument-based method at lower levels (modified Milestones 2.3.5 and 2.4.5, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1.2). Rapid and sensitive method to detect spectinomycin aminoglycoside. Rapid screening methods are needed for detection of spectinomycin residues in animal tissues at the regulatory action levels. Reagents for a fluorescent immunoassay were synthesized, and the fluorescent latex immunoassay method was optimized for detection of spectinomycin at 5-100 ng/g in bovine kidney extract. This method can be used
to identify compounds classified as an unidentified microbial inhibitor (UMI) in the 7-plate microbial assay used by the Food Safety Inspection Service (FSIS). The use of this method by FSIS could reduce the number of 'unidentified microbial inhibitors' (UMI) compounds and enable better quantitation if these compounds appear above the action levels (Milestones 2.3.5, NP108 Drug Residue sand Environmental Contaminants Action Plans 2.1 and 2.1.2.2) . 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? The results from research studies have been transferred to interested parties by means of collaborations, peer-reviewed publications, written reports, internet accounts, presentations at scientific meetings, and communications with scientists and
administrators from regulatory agencies, industry, and other organizations. Research results of studies and recommendations designed to meet residue detection needs in the Food Safety Inspection Service (FSIS) for meat, poultry, and eggs have been provided to their laboratories. The technology is available currently, but FSIS must decide whether to implement the methodology which involves the purchase of instrumentation, training of personnel, and method validation studies. FSIS laboratories have limited resources and personnel and many other types of analyses to conduct, but it is possible that the FSIS will implement the more efficient methods in the future to extend the analytical range of their current methods and increase laboratory productivity. State programs, U.S. federal agencies, foreign regulatory programs, industry and contract laboratories are particularly interested in the approaches developed for pesticide residue analysis. Specifically, written protocols in FSIS
format were provided to FSIS in 2002 and 2003 for the LC/MS-MS methods developed in ARS-ERRC (analysis of beta-lactams, fluoroquinolones, beta-agonists and thyreostats in animal tissues). The QuEChERS method was disseminated to scores of interested scientists around the world upon request, and specific protocols were provided to key laboratories for evaluation. Also, a noncommerical antibody for hygromycin developed at ERRC was provided to a scientist at Columbia University. The latex and biosensor immunoassay methods for detection of enterotoxins (SEA and SEB) in eggs are of high interest to FSIS. These methods will be transferred after validation for routine analysis. The technique that hyphenates sorbent extraction and solid matrix TRL has been filed with the ARS Technical Transfer Office for patent application (0062.04).
Impacts
(N/A)
Publications
- Medina, M.B. 2004. Binding interactions of extracelluar membrane and muscle proteins with immobilized salmonella typhimurium using an spr biosensor. International Journal of Food Microbiology. 93. p. 63-72.
- Schneider, M.J., Donoghue, D.J. 2004. Comparison of a bioassay and a liquid chromatography - fluorescence-mass spectrometry(n) method for the detection of incurred enrofloxacin residues in chicken tissues. Poultry Science. p. 830-834. 2004.
- MEDINA, M.B. DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B (SEB) WITH SURFACE PLASMON RESONANCE BIOSENSOR. JOURNAL OF RAPID METHODS AND AUTOMATION IN MICROBIOLOGY. p. 225-243. 2003.
- Medina, M.B. 2004. Development of a fluorescent latex immunoassay for detection of spectinomycin antiobiotic. Journal of Agricultural and Food Chemistry. p. 3231-3236.
- Oaks, A., Blackwood, C.B., Buyer, J.S. 2004. Molecular analysis of basidiomycete communities in soils from different land uses [abstract]. American Society of Microbiology Meeting Abstracts. Abstract N-004.
- Medina, M.B. 2004. Development of competitive immunoassay for detection of staphylococcal enterotoxin b(seb) in milk. International Food Technology Meeting Abstracts. Paper number 491-4.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? 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 in foods. New analytical methods are needed to expand the range of veterinary drug and pesticide analytes that can be detected in animal and plant derived food products in a more efficient process. These needs are being met by the use of modern methods and approaches which are being evaluated to screen, quantify, and/or confirm chemical residues. These advanced technologies include hyphenated gas or liquid chromatography/mass spectrometry (or tandem mass spectrometry), fluorescence, solid phase extraction, surface plasmon resonance biosensor, field-based instruments, immunoassays and other techniques. 2. How serious is the problem? Why does it matter? The health of consumers is adversely affected by
the presence of harmful chemicals in food, and pesticide and veterinary drug 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) and other organizations that monitor chemical residues in food, which also includes industry, consumer groups, and academic scientists. Current methods of detection are often too time-consuming, expensive, and labor-intensive. Minimization of the time, effort, cost, and improvement in the quality of the results of these analyses, would provide a significant benefit to governments, industry, academic scientists, and consumers. The implementation of improved analytical approaches will (a) increase productivity and/or lower costs of analysis, (b) provide more statistically valid and accurate results for
risk assessment and other purposes, (c) overcome trade barriers associated with the analysis of chemical residues, (d) provide more information to understand the effects and mechanisms of antimicrobial resistance and endocrine disruption, (e) allow for better verification of organic food labeling, (f) improve possible industrial HACCP programs, and (g) reduce the potential for food that has been deliberately or accidentally adulterated by toxic chemicals to reach the consumer. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? The national program component to which this project has been assigned is Food Safety (#108). By providing better methods for risk assessment, increasing laboratory productivity, and potentially reducing the amount of chemical residues in food, the research findings and approaches help improve and maintain a safe food supply. 4. What were the most significant accomplishments this past year? A.
Single Most Significant Accomplishment during FY 2003: The Food Safety Inspection Service (FSIS) and the Food and Drug Administration (FDA) have a problem with the differentiation between the beta-lactam antibiotics, ceftiofur and penicillin, in their current monitoring method. A rapid and simple, quantitative and confirmatory method of analysis for 10 beta-lactam antibiotics in beef kidney using liquid chromatography/tandem mass spectrometry (LC/MS-MS) was developed and compared with the FSIS 7-plate microbial assay for 30 incurred beef kidneys. The results agreed in 28 instances, with the 2 contradictory samples being mis-identified by the FSIS microbial assay. This approach may be used to verify responses from the FSIS 7-plate assay, or to replace it altogether after it has been expanded to include other antibiotic drugs. B. Other Significant Accomplishment(s), if any: Although chemists have suspected that desfuroylceftiofur (DFC: a metabolite of ceftiofur) covalently attaches
to peptides and proteins through disulfide bonds, this was not demonstrated with certainty previously. This bonding mechanism was shown to occur in actuality by using electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD FT-ICR MS) in a collaborative study with Florida State University scientists. Furthermore, a technique for the detection/identification of DFC covalent adducts attached to peptides was devised using collision-activated dissociation tandem ion trap mass spectrometry, in which a unique gas phase dissociation intra-molecular disulfide bond rearrangement was observed and mechanistically described. This may lead to a method to analyze for the covalent adducts in the sample without the need to perform extensive sample preparation, and thus speed and improve the analysis. To meet regulatory needs, different types of commercial screening kits need to be adapted and evaluated for the detection of drug residues in meat tissue.
Utilizing a method previously developed in our laboratory to screen for beta-lactams and unidentified microbial inhibiting (UMI) compounds, lactamase enzymes from new sources were optimized for their selective hydrolysis of beta-lactams in bovine tissues. Three commercial beta-lactam assays originally developed for milk analysis were adapted for tissue analysis. The results from this study will help kit manufacturers improve their products and regulatory agencies to choose kits that best suit their needs. Easy, fast, and inexpensive screening methods are desirable to permit a large number of samples to be analyzed for the presence of antibiotic drug residues. A simple extraction followed by fluorometry was investigated as a rapid screening method for the presence of the fluoroquinolone antibiotic, enrofloxacin, in chicken muscle. The method works well to determine whether enrofloxacin is present at the tolerance level (300 ng/g) or above. This approach has also been applied to
tetracyclines in chicken muscle at the 2000 ng/g tolerance level, taking advantage of the fluorescence of tetracyclines in the presence of base. This approach may prove very useful to FSIS and other organizations for screening of fluorescent drugs in a variety of animal-derived foods. Simple, rapid, and cheap screening methods are needed to detect chemical residues in a variety of foods. A rapid time-resolved fluorimetric method was developed to monitor for enrofloxacin in chicken muscle. By implementing a simple extraction and eliminating cleanup, sample throughput was considerably improved over current methods. A sensitive europium-sensitized luminescence screening method was also developed for tetracyclines in chicken muscle. This method improved throughput and achieved a 200 ng/g screening sensitivity, and similar performance was achieved in aquaculture-farmed fish. This approach may prove very useful to FSIS and other organizations for screening of fluorescent antibiotics in
a variety of matrices. Regulatory monitoring programs in the U.S. and worldwide, as well as contract labs and industry, need improved methods to extract, cleanup, and detect pesticide residues in raw agricultural commodities. A quick, easy, cheap, effective, rugged, and safe method, that we call QuEChERS (pronounced "catchers"), was developed in 2002, and its utility and effectiveness was further demonstrated in 2003. The method was shown to give >90% recoveries for more than 200 pesticides so far. This method has already generated great interest among the pesticide residue analytical community worldwide, and several laboratories in the regulatory arena and industry are evaluating the method to save time and money in their pesticide monitoring programs. C. Significant Accomplishments/Activities that Support Special Target Populations None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. In the U.S., the Food and Drug
Administration (FDA) and Environmental Protection Agency (EPA) policies do not permit the use of matrix-matched calibration standards to improve accuracy in the quantitation of pesticide residues for regulatory enforcement actions. To overcome this regulatory limitation, and to provide better gas chromatographic (GC) peak shapes and lower detection limits for problematic pesticides, a new concept that we call "analyte protection" was developed and investigated. The concept involves the addition of chemicals, such as sugar derivatives, to both calibration standards and extracts, and the chemicals serve to mask the active sites in the GC system that cause the inaccurate quantitation of certain pesticides. This simple approach may become standard practice for GC analysis of pesticides because it was shown to provide accurate results and low detection limits while still meeting EPA and FDA policies for enforcement actions. The use of fluoroquinolone antibiotics, particularly in poultry,
is being scrutinized by the Food and Drug Administration due to antimicrobial resistance concerns, thus analytical methods for fluoroquinolones have become more important in the regulatory arena. A rapid extraction procedure and liquid chromatographic (LC) analysis with both fluorescence detection and tandem mass spectrometry (MS/MS) to analyze and confirm the presence of 8 fluoroquinolones in egg products (both white and/or yolk). This hyphenated approach takes advantage of the strengths of each detection method, and the issue of unambiguous confirmation using this multi-faceted approach has been investigated. The Food Safety Inspection Service's Midwestern Laboroatory has requested and received the method and is planning to test it for their purposes. In order to substantially speed up the analysis of pesticide residues in food, a new method of analysis using low-pressure gas chromatography/mass spectrometry (LP-GC/MS) was optimized and evaluated. An analytical method using
LP-GC/MS for 20 challenging pesticides in carrots was demonstrated, which decreased the time of chromatographic analysis from approximately 20 min to 6 min. Limits of detection were also decreased by enabling a higher injection volume and increasing the ruggedness of the chromatographic approach. The outcome of this research is that LP- GC/MS can significantly increase the speed of multiresidue pesticide analysis and provide other benefits of larger sample capacity, lower detection limits, and reduced thermal degradation of pesticide analytes. Currently, regulatory and contract laboratories use 2 different time- consuming and intensive analytical methods for the determination of thyreostats and beta-agonists, which are banned growth promotants. A novel multiresidue analytical screening method for the isolation and detection of 14 thyreostats and beta-agonists using liquid chromatography- fluorescence-tandem mass spectrometry in meat and liver tissues was developed. The presence of
the compounds can be confirmed and quantified (if necessary) at levels less than 100 ng/g within 1 hour. This approach is simpler, faster and more effective than the current methods used by U. S., Canadian, and the European Union regulatory agencies and the protocol was provided to the regulatory agencies. Staphylococcus aureus enteric toxins cause a major type of food-borne gastroenteritis, and the food industry needs rapid methods for detection of the most common Staphylococcus aureus A and B (SEA and SEB) toxins, but commercially available methods are expensive for routine use. We developed and optimized a Latex particle agglutination assay for detection of SEA and SEB. The assay is simple and non-instrumental, and the detectability is as little as 100 ng/g, and for comparison, a commercial instrumental-based biosensor immuno-assay was optimized and resulted in a detection of less than 5 ng/g of toxins. This gives potential users of the detection methods, depending on their
needs and priorities, a choice between the inexpensive screening method at higher levels or expensive instrument-based method at lower levels. Rapid screening methods are needed for detection of spectinomycin residues in animal tissues at the regulatory action levels. Reagents for a fluorescent immunoassay were synthesized, and the fluorescent latex immunoassay method was optimized for detection of spectinomycin at 5 '100 ng/g in bovine kidney extract. This method can be used to identify compounds classified as an unidentified microbial inhibitor (UMI) in the 7-plate microbial assay used by the Food Safety Inspection Service (FSIS). The use of this method by FSIS could reduce the number of UMI compounds and enable better quantitation if these compounds appear above the action levels. 6. What do you expect to accomplish, year by year, over the next 3 years? We plan to make the following accomplishments in the next two years (when the current project plan expires in 2005): Year 1a)
use fluorescence to screen for multiple fluorescent drugs in the field after a simple extraction procedure in meat and other pertinent animal-derived foods; Year 1b) comparison of commercial assays to detect antibiotics in animal tissues (kidney); Year 1c) interlaboratory evalutaion of the QuEChERS method for pesticide residue analysis of fruits and vegetables; Years 1- 2a) expand the scope of analysis of the QuEChERS approach using chromatography/mass spectrometry for detection to also be useful for veterinary drug residues as well as pesticides; Years 1-2b) develop bioassays for veterinary drugs and biotoxins in food using surface plasmon resonance spectroscopy and mass spectrometry instruments; Years 1- 2c) build a working prototype of a time-resolved luminescence device that can be used in the field to detect chemical residues in food. Our overarching goal is to develop multiresidue methods for as many compounds as possible in an efficient and effective overall chemical
monitoring program. The use of mass spectrometry should allow for a single multiresidue method for multiple compounds and may also provide quantitation and confirmation of the results simultaneously. Fluorescence detection provides additional information for those analytes which fluoresce. Current common methods often detect single or only a few analytes and require separate injections for confirmation of the identity of the chemicals. We expect to accomplish the development of a multiclass, multiresidue overall approach for veterinary drugs in animal tissues that is suitable for transfer to other laboratories within 3 years. In the case of pesticides, research will focus on rapid and simple, laboratory-based methods, such as direct sample introduction, for trace-level pesticide residues in a variety of foods. 7. 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? The results from research studies have been transferred to interested parties by means of collaborations, peer-reviewed publications, written reports, internet accounts, presentations at scientific meetings, and communications with scientists and administrators from regulatory agencies, industry, and other organizations. Research results of studies and recommendations designed to meet residue detection needs in the Food Safety Inspection Service (FSIS) for meat, poultry, and eggs have been provided to their laboratories. The technology is available currently, but FSIS must decide whether to implement the methodology which involves the purchase of instrumentation, training of personnel, and method validation studies. FSIS laboratories have limited resources and personnel and many other types of analyses to conduct, but it is possible that the FSIS will implement the
more efficient methods in the future to extend the analytical range of their current methods and increase laboratory productivity. State programs, U.S. federal agencies, foreign regulatory programs, industry and contract laboratories are particularly interested in the approaches developed for pesticide residue analysis. Specifically, written protocols in FSIS format were provided to FSIS in 2002 and 2003 for the LC/MS-MS methods developed in ARS-ERRC (analysis of beta-lactams, fluoroquinolones, beta-agonists and thyreostats in animal tissues). The QuEChERS method was disseminated to scores of interested scientists around the world upon request, and specific protocols were provided to key laboratories for evaluation. Also, a noncommerical antibody for hygromycin developed at ERRC was provided to a scientist at Columbia University. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does
not replace your peer-reviewed publications listed below). "QuEChERS Method Catches Pesticide Residues" Agricultural Research Magazine, July, 2003, p. 9.
Impacts
(N/A)
Publications
- Schneider, M.J., Donoghue, D.J. Multiresidue Analysis of Fluoroquinolone Antibiotics in Chicken Tissue using Liquid Chromatography-Fluorescence- Multiple Mass Spectrometry. Journal of Chromatography B. 2002. v. 780. p. 83-92.
- Schneider, M.J., Donoghue, D.J. Multiresidue Determination of Fluoroquinolone Antibiotics in Eggs using Liquid Chromatography- Fluorescence-Mass Spectrometry(n). Analytica Chimica Acta. 2003. v. 483. p. 39-49.
- Chen, G., Schneider, M.J. A Rapid Spectrofluorometric Screening Method for Enrofloxacin in Chicken Muscle. Journal of Agricultural and Food Chemistry. 2003. v. 51. p. 3249-3253.
- Schenck, F.J., Callery, P., Gannett, P.M., Daft, J.R., Lehotay, S.J. Comparison of Magnesium Sulfate with Sodium Sulfate for the Removal of Water from Pesticide Residue Extracts of Food. Journal of AOAC International. 2002. v. 85. p. 1177-1180.
- Lehotay, S.J. Determination of Pesticide Residues in Nonfatty Foods by Supercritical Fluid Extraction and Gas Chromatography/Mass Spectrometry: Collaborative Study. Journal of AOAC International. 2002. v. 85. p. 1148- 1166.
- Lehotay, S.J., Hajslova, J. Application of Gas Chromatography in Food Analysis. Trends in Analytical Chemistry. 2002. v. 21. p. 686-697.
- Krynitsky, A.J., Lehotay, S.J. Overview of Analytical Technologies that are Available to Regulatory Laboratories for the Determination of Pesticide Residues. Lee, P., editor. John Wiley Sons, Chichester, UK. Residue Analytical Methods Handbook for Agrochemicals. 2002. p. 753-786.
- Kochman, M., Gordin, A., Goldshlag, P., Lehotay, S.J., Amirav, A. Fast, High Sensitivity, Multi-Pesticide Analysis of Complex Mixtures with the Supersonic GC-MS. Journal of Chromatography A. 2002. v. 974. p. 185-212.
- Schenck, F.J., Lehotay, S.J., Vega, V. Comparison of Solid-Phase Extraction Sorbents for Cleanup in Pesticide Residue Analysis of Fresh Fruits and Vegetables. Journal of Separation Science. 2002. v. 25. p. 883- 890.
- Anastassiades, M., Lehotay, S.J., Stajnbaher, D., Schenck, F.J. Fast and Easy Multiresidue Method for the Determination of Pesticide Residues in Produce by Acetonitrile Extraction/Partitioning and Dispersive Solid-Phase Extraction. Journal of AOAC International. 2003. v. 86. p. 412-431.
- Anastassiades, M., Lehotay, S.J., Stajnbaher, D. Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) Approach for the Determination of Pesticide Residues. 18th Annual Waste Testing Quality Symposium Proceedings. 2002. p. 231-241.
- Bethem, R., Boison, J., Gale, J., Heller, D., Lehotay, S., Loo, J., Musser, S., Price, P., Stein, S. Establishing the Fitness for Purpose of Mass Spectrometric Methods. Journal of the American Society for Mass Spectrometry. 2003. v. 14, p. 528-541.
- Mastovska, K., Lehotay, S.J. Practical Approaches to Fast Gas Chromatography-Mass Spectometry. Journal of Chromatography A. 2003. v. 1000. p. 153-180.
- Fagerquist, C.K., Lightfield, A.R. Confirmatory Analysis of beta-Lactam Antibiotics in Kidney Tissue by Liquid Chromatography/Electrospray Ionization Selective Reaction Monitoring Ion Trap Tandem Mass Spectrometry. Rapid Communications in Mass Spectrometry. 2003. v. 17. p. 660-671.
- Fagerquist, C.K., Hudgins, R.R., Emmett, M.R., Hakansson, K., Marshall, A. G. An Antibiotic Linked to Peptides and Proteins is Released by Electron Capture Dissociation Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Journal of the American Society for Mass Spectrometry. 2003. v. 14. p. 302-310.
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Progress 10/01/01 to 09/30/02
Outputs
1. What major problem or issue is being resolved and how are you resolving it? 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 in foods. New analytical methods are needed to expand the range of veterinary drug and pesticide analytes that can be detected in animal and plant derived food products in a more efficient process. These needs are being met by the use of modern methods and approaches which are being evaluated to screen, quantify, and/or confirm chemical residues. These advanced technologies include hyphenated gas or liquid chromatography/mass spectrometry (or tandem mass spectrometry), solid phase extraction, surface plasmon resonance biosensor, field-based instruments, immuno-assays and other techniques. 2. How serious is the problem? Why does it matter? The health of consumers is adversely affected by the presence
of harmful chemicals in food, and pesticide and veterinary drug 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 detection. The project meets the needs of the USDA Food Safety Inspection Service (FSIS)and other organizations that monitor chemical residues in food, which also includes industry, consumer groups, and academic scientists. Current methods of detection are often too time-consuming, expensive, and labor-intensive. Minimization of the time, effort, and cost, and improvement in the quality of the results of these analyses, would provide a significant benefit to governments, industry, academic scientists, and consumers. The implementation of improved analytical approaches will (a) increase productivity and/or lower costs of analysis, (b) provide more statistically valid and accurate results for risk
assessment and other purposes, (c) overcome trade barriers associated with the analysis of chemical residues, (d) provide more information to understand the effects and mechanisms of antimicrobial resistance and endocrine disruption, (e) allow for better verification of organic food labeling, (f) improve possible industrial HACCP programs, and (g) reduce the potential for food that has been deliberately or accidentally adulterated by toxic chemicals to reach the consumer. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? The national program component to which this project has been assigned is food safety (#108). By providing better methods for risk assessment, increasing laboratory productivity, and potentially reducing the amount of chemical residues in food, the research findings and approaches help improve and maintain a safe food supply. 4. What was your most significant accomplishment this past year? A. Single
Most Significant Accomplishment during FY 2002: Regulatory monitoring programs in the U.S. and worldwide, as well as contract labs and industry, need improved methods to extract, cleanup, and detect pesticide residues in raw agricultural commodities. A quick, easy, cheap, effective, rugged, and safe method, that we call QuEChERS (pronounced "catchers"), was developed by an ARS scientist at the Eastern Regional Research Center and foreign visiting scientist. The method provides high quality results for a wide range of pesticides, and using the method, a single chemist can prepare 6-12 extracts in 20-30 minutes using <$1 of materials per sample (and only a single reusable piece of glassware). This method has already generated great interest among the pesticide residue analytical community worldwide, and several laboratories in the regulatory arena and industry are planning to evaluate the method to save time and money in their pesticide monitoring programs. B. Other Significant
Accomplishment(s): Rapid screening methods are needed for the detection of spectinomycin residues in animal tissues at regulatory action levels. Researchers at the Eastern Regional Research Center have optimized a fluorescent latex immuno-assay method for detection of spectinomycin at 5-100 ng/g in bovine kidney extract. This method can be used to identify compounds classified as unidentified microbial inhibitors (UMIs) by the Food Safety Inspection Service (FSIS). The use of this method by FSIS would reduce the number of UMI compounds and would enable FSIS to quantitate these compounds if these compounds exist below or above action levels. In the U.S., the Food and Drug Administration (FDA) and Environmental Protection Agency (EPA) policies do not permit the use of matrix-matched calibration standards to improve accuracy in the quantitation of pesticide residues for regulatory enforcement actions. To overcome this regulatory limitation, and to provide better gas chromatographic
(GC) peak shapes and lower detection limits for problematic pesticides, a new concept that we call "analyte protection" was developed and investigated by visiting scientists and an ARS scientist at the Eastern Regional Research Center. The concept involves the addition of chemicals, such as sugars, to both calibration standards and extracts, and the chemicals serve to mask the active sites in the GC system that cause the inaccurate quantitation of certain pesticides. This simple approach is likely to become standard practice for GC analysis of pesticides because it was shown to provide accurate results and low detection limits while still meeting EPA and FDA policies for enforcement actions. The use of fluoroquinolone antibiotics, particularly in poultry, is being scrutinized by the Food and Drug Administration due to antimicrobial resistance concerns, thus analytical methods for fluoroquinolones have become more important in the regulatory arena. A researcher at the Eastern
Regional Research Center investigated a rapid extraction procedure and liquid chromatographic (LC) analysis with both fluorescence detection and tandem mass spectrometry (MS/MS) to analyze and confirm the presence of 8 fluoroquinolones in egg products (both white and/or yolk). This hyphenated approach takes advantage of the strengths of each detection method, and the issue of unambiguous confirmation using this multi-faceted approach has been investigated. The Food Safety Inspection Service's Midwestern Laboratory has requested and received the method and is planning to test it for their purposes. The Food Safety Inspection Service (FSIS) and the Food and Drug Administration have a problem with the differentiation between the beta- lactam antibiotics, ceftiofur and penicillin, in their current detection methods. A scientist at the Eastern Regional Research Center evaluated a new approach for beef kidney analysis using LC/MS-MS primarily for confirmation, but also for quantitation.
The method was demonstrated to work well for samples fortified with 11 beta-lactams, and it has also been shown to distinguish between ceftiofur and penicillin in a number of suspected samples provided by FSIS Midwestern Lab. This method is being provided to the FSIS Midwestern Laboratory and will likely be used in their programs. Staphylococcus aureus enteric toxins cause a major type of food-borne gastroenteritis, and the food industry needs rapid methods for detection of the most common Staphylococcus aureus A and B (SEA and SEB) toxins, but commercially available methods are expensive for routine use. A scientist at the Eastern Regional Research Center developed and optimized a Latex particle agglutination assay for detection of SEA and SEB. The assay is simple and non-instrumental, and the detectability is as little as 100 ng/g, and for comparison, a commercial instrumental-based biosensor immuno-assay was optimized and resulted in a detection of less than 5 ng/g of toxins.
This gives potential users of the detection methods, depending on their needs and priorities, a choice between the inexpensive screening method at higher levels or expensive instrument- based method at lower levels. C. Significant Activities that Support Special Target Populations: None. 5. Describe your major accomplishments over the life of the project, including their predicted or actual impact? Direct sample introduction for gas chromatography/tandem mass spectrometry (DSI/GC/MS-MS) is a novel approach for the analysis of multiple pesticides in a variety of food and other matrices; advantageous traits of the approach include: rapid, sensitive, quantitative, confirmatory, simple, inexpensive, safe, and rugged. Over the years, a scientist at the Eastern Regional Research Center has developed procedures using DSI/GC/MS-MS to analyze approximately 50 pesticides at levels as low as 1 ng/g in eggs, fruits, vegetables, spices, and other foods. This approach avoids the costly,
time-consuming and labor- intensive clean-up and solvent evaporation steps associated with traditional methods. This approach has been automated and commercialized and is starting to make an impact in the analysis of many types of pesticides and other semi-volatile chemicals in a variety of matrices, including food. In order to substantially speed up the analysis of pesticide residues in food, a new method of analysis using low-pressure gas chromatography/mass spectrometry (LP-GC/MS) was optimized and evaluated. A visiting scientist and an ARS scientist at the Eastern Regional Research Center developed an analytical method using LP-GC/MS for 20 challenging pesticides in carrots, which decreased the time of chromatographic analysis from approximately 20 min to 6 min. Limits of detection were also decreased by enabling a higher injection volume and increasing the ruggedness of the chromatographic approach. The outcome of this research is the demonstration that LP-GC/MS is able to
significantly increase the speed of multiresidue pesticide analysis and provide other benefits of larger sample capacity, lower detection limits, and reduced thermal degradation of pesticide analyses. Investigation of techniques for the determination of fluoroquinolone antibiotic residues in chicken tissue has remained a high priority due to the increased microbial resistance observed for these compounds and the resultant growing concern that has led the Food and Drug Administration to recently withdraw approval for a fluoroquinolone in poultry. A scientist at the Eastern Regional Research Center has investigated very promising, efficient techniques for the analysis of fluoroquinolones: automated microdialysis-liquid chromatography, and liquid chromatography- fluorescence-tandem mass spectrometry. Use of the automated techniques and an approach that allows simultaneous confirmation and quantitation provides a highly efficient method which should facilitate monitoring and analysis of
these compounds in poultry. Currently, regulatory and contract laboratories use 2 different time- consuming and intensive analytical methods for the determination of thyreostats and beta-agonists, which are banned growth promotants. A scientist at the Eastern Regional Research Center developed a novel multiresidue analytical screening method for the isolation and detection of 14 thermostats and beta-agonists using liquid chromatography- fluorescence-tandem mass spectrometry in meat and liver tissues. The presence of the compounds can be confirmed and quantified (if necessary) at levels less than 100 ng/g within 1 hour. This approach is simpler, faster and more effective than the current methods used by U.S., Canadian, and the European Union regulatory agencies and may supplant the current methods. 6. What do you expect to accomplish, year by year, over the next 3 years? We plan to make the following accomplishments in the next 3 years: 1) expand the scope of analytical methods
using gas chromatography/mass spectrometry for pesticide residues to also be useful for veterinary drug residues such as anthelmintics, analgesics, and tranquilizers; 2) expand the scope of single-class method for veterinary drugs in meat products to also be useful for multi-class multi residue analysis by liquid chromatography/tandem mass spectrometry; 3) develop instrument-based screening methods for chemical contaminants such as pesticides and veterinary drugs in food for use in the field; 4) develop an analytical method for arsenic containing drugs using gas chromatography with a pulsed-flame photometric detector; 5) evaluate fluorescence for the screening analysis of fluorescent drugs in the field after a simple extraction procedure; 6) develop bioassays for veterinary drugs and biotoxins in food using surface plasmon resonance spectroscopy and mass spectrometry instruments. Our overarching goal is to develop multiresidue methods for as many compounds as possible in an efficient
and effective overall chemical monitoring program. The use of mass spectrometry should allow for a single multiresidue method for multiple compounds and may also provide quantization and confirmation of the results simultaneously. Fluorescence detection provides additional information for those analyses which fluoresce. Current common methods often detect single or only a few analyses and require separate injections for confirmation of the identity of the chemicals. We expect to accomplish the development of a multicast, multiresidue overall approach for veterinary drugs in animal tissues that is suitable for transfer to other laboratories within 4 years. In the case of pesticides, research will focus on rapid and simple, laboratory-based methods, such as direct sample introduction, for trace-level pesticide residues in a variety of foods. The goal of this approach is almost met to analyze more than 100 pesticides in a 15 min procedure. The approach will be evaluated and
validated and should be ready for transfer to other laboratories within 2 years. Furthermore, investigations of novel, portable screening techniques are planned for the detection of multiple chemical contaminants at regulatory tolerance levels. These will utilize the fluorescent latex immunoassay method to detect spectinomycin in tissues (uncooked edible chicken tissues, bovine or swine kidney and muscle tissues). Collaborations will be pursued with regulatory agencies to help them identify unidentified microbial inhibitors (UMIs) and to validate the results of the screening methods. Also, a multiresidue biosensor screening method will be developed to detect and tentatively identify UMI compounds (spectinomycin, streptomycin, and dehydrostreptomycin). 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the
technology? The results from research studies have been transferred to interested parties by means of collaborations, peer-reviewed publications, written reports, internet accounts, presentations at scientific meetings, and communications with scientists and administrators from regulatory agencies, industry, and other organizations. Research results of studies and recommendations designed to meet residue detection needs in the Food Safety Inspection Service (FSIS) for meat, poultry, and eggs have been provided to their laboratories. The technology is available currently, but FSIS must decide whether to implement the methodology which involves the purchase of instrumentation, training of personnel, and method validation studies. FSIS laboratories have limited resources and personnel and many other types of analyses to conduct, but it is possible that the FSIS will implement the more efficient methods in the future to extend the analytical range of their current methods and increase
laboratory productivity. State programs, U.S. federal agencies, foreign regulatory programs, industry and contract laboratories are particularly interested in the approaches developed for pesticide residue analysis.
Impacts
(N/A)
Publications
- Schneider, M.J. Multiresidue analysis of fluoroquinolone antibiotics in chicken tissue using automated microdialysis-liquid chromatography. Journal of Chromatrographic Science. 2001. v. 39. p. 351-356.
- Lehotay, S.J., Lightfield, A.R. Harman-Fetcho, J.A., Donoghue, D.J. Analysis of pesticide residues in eggs by direct sample introduction/gas chromatography/tandem mass spectrometry. Journal of Agricultural Food Chemistry. 2001. v. 49. p. 4589-4596.
- Mastovska, K., Lehotay, S., Hajslova, J. Optimization and evaluation of low-pressure gas chromatography-mass spectrometry for the fast analysis of multiple pesticide residues in a food commodity. Journal of Chromatography. 2001. v. 926. p. 291-308.
- Klassen, J.K., Lehotay, S.J., Pu, Q-L., Conny, J.M. Development of searchable pesticide MS and MS-MS libraries. Proceedings of the 49th ASMS Conference. 2001. p. A010960.
- Schneider, M.J. Multireside LC-Fluorescence-MS-MS analysis of fluoroquinolone antibiotics in chicken tissue. Proceedings of the 49th ASMS Conference. 2001. p. A011213.
- Lehotay, S.J. Chemical Residues. National Conference on Animal Production Food Safety Proceedings. 2001. p. 108-115.
- Schenck, F.J., Lehotay, S.J., Vega, V. What is the optimal solid-phase extraction (SPE) cleanup for the GC analysis of pesticides in fresh fruits and vegetables? FDA Laboratory Information Bulletin. 2001. v. 17. #4262.
- Schenck, F.J., Callery, P., Gannett, P.M., Daft, J.R., Lehotay, S.J. How Dry is Dry? Removal of water from pesticide extracts of foods after salting out. FDA Laboratory Information Bulletin. 2001. v. 17(11). #4264.
- Schneider, M.J., Donoghue, D.J. Multiresidue determination of fluoroquinolone antibiotics in eggs using liquid chromatography- fluorescence-mass spectrometry. 4th International Symposium on Hormone and Veterinary Drug Residue Analysis Abstract Book. 2002. p. 178. Abstract No. T68.
- Lehotay, S.J., Lightfield, A.R., Anastassiades, M., Smith, D.J. Analysis of veterinary growth promoting drugs in animal tissues by LC/MS-MS and fluorescence. 223rd ACS National Meeting. 2002. Abstract No. AGRO-30.
- Zrostlikova, J., Lehotay, S.J., Hajslova, J. Simultaneous analysis of organophosphorus and organochlorine pesticides in animal fat by gas chromatography with pulsed flame photometric and micro-electron capture detectors. 4th European Pesticide Residue Workshop Book of Abstracts. 2002. p. 302. Abstract No. P-207.
- Lehotay, S.J., Lightfield, A.R., Anastassiades, M., Smith, D.J. Simultaneous analysis of beta-agonists and thyreostats in animal tissues by LC/MSn and In-Line fluorescence. 4th International Symposium on Hormone and Veterinary Drug Residue Analysis Abstract Book. 2002. p. 130. Abstract No. T20.
- Medina, M.B. SPR Biosensor applications in food analysis. Biacore Symposium 2002: Food Analysis Series. 2002. Abstract 1.
- Medina, M.B. Development of a fluorescent latex immunoassay for detection of spectinomycin antibiotic. Institute of Food Technologists Annual Meeting. 2002. Abstract No. 46I-11.
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Progress 10/01/00 to 09/30/01
Outputs
(N/A)
Impacts
(N/A)
Publications
- Fiddler, W., Pensabene, J.W., Shadwell, R.J., Lehotay, S.J. Potential artifact formation of dioxins in ball clay during supercritical fluid extraction. Journal of Chromatography. 2000. v. 902. p. 427-432.
- Pensabene, J.W., Lehotay, S.J., Fiddler, W. Method for the analysis of thyreostats in meat tissue using gas chromatography with nitrogen phosphorus detection and tandem mass spectrometric confirmation. Journal of Chromatographic Science. 2001. v. 39(5). p. 195-199.
- Mastovska, K., Lehotay, S.J., Hajslova, J. Optimization and evaluation of low-pressure GC/MS for the fast analysis of multiple pesticide residues in a food commodity. XIII California Pesticide Residue Workshop. 2001. Abstract p. GC-10.
- Lehotay, S.J., Gates, R.A. Evaluation of qualitative and quantitative accuracy in the GC/MS analysis of pesticide residues in fruit and vegetable extracts. XIII California Pesticide Residue Workshop. 2001. Abstract p. GC-11.
- Amirav, A., Kochman, M., Gordin, A., Lehotay, S.J. Fast, high sensitivity, multi-pesticide analysis of complex mixtures with supersonic GC/MS. XIII California Pesticide Residue Workshop. 2001. Abstract p. GC-12.
- Zhou, E., Ting, K.C., Lehotay, S.J., Results from a challenge to analyze pesticide residues in spices. XIII California Pesticide Residue Workshop. 2001. Abstract p. OT-04.
- Schenck, F.J., Vega, V., Lehotay, S.J. The effect of solid phase extraction cleanup on the GC analysis of pesticides in foods at low part per billion levels. XIII California Pesticide Residue Workshop. 2001. Abstract p. Food-05.
- Lehotay, S.J., Schenck, F.J. Encyclopedia of Separation Science. Wilson, I.D., Adlard, T.R., Poole, C.F., Cook, M., editors. Academic Press, London, UK. Multiresidue Methods: Extraction. 2000. v. III. p. 3409-3415.
- Lehotay, S.J. Encyclopedia of Analytical Chemistry: Instrumentation and Applications. Meyers, R.A., editor. John Wiley & Sons, Chichester, UK. Multi-Class, Multi-Residue Analysis of Pesticides and Strategies. 2000. v. 7. p. 6344-6384.
- Lehotay, S.J. Principles and Practices of Method Validation. Fajgelj, A., Ambrus, A., editors. Royal Society of Chemistry, Cambridge, UK. AOAC International Collaborative Study on the Determination of Pesticide Residues in Nonfatty Foods by Supercritical Fluid Extraction and Gas Chromatography/Mass Spectrometry. 2000. p. 89-99.
- Lehotay, S.J. Herramientas avanzadas para el analisis de los residuos de los plaguicidas (Advanced tools for pesticide residue analysis). Phytoma Espana. 2001. v. 129. p. 8-16.
- Reeves, V.B., Schneider, M.J., Clark, S.B., Podhorniak, L.V., Righter, H.F. Liquid chromatographic procedure for the determination of fluoroquinolone residues in bovine milk: interlaboratory study. Association of Official Analytical Chemists. 2000. Abstract p. 72.
- Lehotay, S.J. Fast multiresidue analysis of pesticides. Association of Official Analytical Chemists. 2000. Abstract p. 72.
- Lehotay, S.J. Advanced tools for pesticide residue analysis. 2nd MGPR International Symposium of Pesticides in Food and the Environment in Mediterranean Countries. 2001. Abstract p. 39.
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