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United States Department of Agriculture

Agricultural Research Service


Location: Residue Chemistry and Predictive Microbiology Research

2013 Annual Report

1a. Objectives (from AD-416):
1. Develop methods to increase sample throughput in order to improve monitoring of chemical contaminants in foods. Specifically: investigate faster screening and/or analytical approaches, and faster data processing without loss of reliability. 1A. Develop and evaluate new, useful analytical multiresidue methods that are faster than existing methods without sacrificing quality of results. 1B. Investigate practical means of processing analytical results in multiresidue analysis that lead to reliable, objective decisions with minimal human review. 1C. Develop and evaluate improved analytical screening methods in the analysis of chemicals of interest in foods. 1D. Study and implement robotic sample preparation systems to increase speed and reduce manual labor for routine analysis of chemical residues in foods. 2. Conduct research to evaluate the validity of new approaches for regulatory monitoring of veterinary drug residues in foods and feeds. Specifically: an assessment of sampling procedures and addressing matrix effects in quantification. 2A. Lead and conduct an AOAC International Collaborative Study to update and harmonize the QuEChERS method for analysis of chemical residues in a wide variety of foods. 2B. Investigate sampling and sample processing of appropriate matrices, taking matrix effects into account, for veterinary drug residue analysis to yield meaningful results in a fast and practical procedure. 2C. Lead and conduct an interlaboratory validation study to quantitatively and qualitatively assess our multiclass, multiresidue method for veterinary drugs in food tissue matrices. 3. Develop biosensor methods with multi-analyte capability for biological toxins of concern. 4. Modify/refine novel method for routine measurement of total phenolics, phytoestrogens and/or estrogenic endocrine disruptors. 4A. Expand application of our total phenolics method to other food and dietary products; and identify the reaction products of the key reagent with select phenolic compounds. 4B. Modify the method to screen for estrogenic compounds, including bisphenol A. 5. Develop novel analytical methods for inorganic and organometallic heavy metals (for example forms of mercury (Hg) and arsenic (As)) in foods and supplements. 5A. Develop modern analytical methods for mercury speciation and quantification in foods and supplements. 5B. Develop modern analytical methods for arsenic speciation and quantification in foods and supplements.

1b. Approach (from AD-416):
1a) Evaluate novel mass spectrometric methods such as ambient mass spectrometry and supersonic molecular beam mass spectrometry combined with fast gas chromatography; 1b) use robotic systems to develop fast, automated sample cleanup approaches for routine monitoring purposes; 1c) devise data handling software that is faster and better at compiling the needed information for validating multi-class, multiresidue methods for analysis; 2a) multi-laboratory AOAC International collaborative studies will be pursued to achieve the gold standard quality in method validation; 2b) both gas chromatography and liquid chromatography (LC) combined with tandem mass spectrometry (MS/MS) will be used to assess and address matrix effects in chemical residue analysis of foods; 3) surface plasmon resonance biosensing instrumentation and techniques will be used to develop advanced new methods for multiple toxins of concern; 4) both LC-MS/MS and novel, quick chemical test methods will be used to measure total phenolics, phytoestrogens and endocrine disruptors; and 5) LC combined with atomic fluorescence spectroscopy will be used to rapidly and accurately determine different organometallic species of Hg and As in foods and supplements.

3. Progress Report:
The objective of this project is to develop, validate, and implement effective and useful, high-throughput analytical methods in monitoring labs (in the U.S. and elsewhere) for chemical residues, biotoxins, endocrine disruptors, organometallics, and other chemicals of concern in agricultural food products to better ensure food safety, security, and/or nutritional aspects. This project addresses the problem related to the lack of rapid, automated, cost-effective, waste-minimizing, safe, and high-quality analytical methods to detect multiple chemical residues and other toxic compounds in foods. The project is devised to meet the needs of the USDA Food Safety Inspection Service (FSIS), Food and Drug Administration (FDA), and other organizations that monitor chemical residues in food, which also includes industry, consumer groups, and academic scientists. Development of a faster and less expensive method for the analysis of inorganic arsenic in rice. Progress was made on all other Objectives and Milestones in the Project Plan except Objective 3 due to the retirement of the responsible scientist. Highlights of this additional progress are described below: Screening using dispersive liquid-liquid microextraction followed by terbium-sensitive luminescence detection of two fluoroquinolones (enrofloxacin and its metabolite, ciprofloxacin) in swine liver was accomplished at the 500 ng/g tolerance level set by the FDA. Screening of sarafloxacin, oxolinic acid, and flumequine residues is being developed in finfish at European Union maximum residue limits of 30, 100, and 600 ng/g, respectively, using a portable fluorometer and solid-phase sorbent strips. To meet a special request from FSIS, a new method for nitrosamines in bacon is being developed and used for toxicological risk assessment. The quick, easy, cheap, effective, rugged, and safe (QuEChERS) method has been shown to work well, and analyses on different instruments are being compared to achieve the lowest detection limits possible. A new state-of-the-art triple quadrupole - linear ion trap mass spectrometer has been obtained through a material transfer agreement with an instrument manufacturer. The new instrument will be used to adapt the multiclass, multiresidue method transferred to FSIS which will provide lower detection limits, reduced matrix effects, and more ruggedness in routine analysis. The instrument will also be used in flow-injection analysis mode to speed analyses even further. A photochemical reactor using Teflon coil was built to perform speciation of mercury and methylmercury in seafood followed by cold-vapor generation quantification using atomic fluorescence spectroscopy. A sample mixture is prepared form a hydrophobic complex which is swept by argon and enriched on the inner surface of the coil reactor. A lamp is turned on to photoreduce the complex to mercury vapor for detection.

4. Accomplishments
1. Screening method for fluoroquinolone residues in swine. Fluoroquinolone antibiotics are used to treat humans and they are often the last defense against antibiotic-resistant microorganisms. Thus, the use of the drugs in veterinary medicine is of particular concern in regulatory monitoring programs worldwide. Current screening tests using microbial inhibition for antibiotics do not respond well to fluoroquinolones, and a new screening method is needed for these drugs. ARS researchers at Wyndmoor, Pennsylvania developed a novel application of dispersive liquid-liquid microextraction followed by terbium-sensitized luminescence for screening the approved fluoroquinolones, enrofloxacin (ENRO) and its metabolite ciprofloxacin (CIPRO), in swine liver (the regulatory target tissue). The approach met the US-tolerance detection level of 500 ng/g and was demonstrated in analyses of spiked samples. This method may be used by monitoring labs in the US and worldwide to screen for these important drugs to better ensure use of proper veterinary practices and reduce the chances of transfer of antibiotic microbial resistance.

2. Speciation of inorganic arsenic in rice. Arsenic is one of the most toxic elements, and it is a particular concern in rice. Inorganic forms of arsenic are the most toxic, and analytical methods must distinguish the different forms in the samples. The current method used for speciation of arsenic is time-consuming and expensive due to the use of chromatography and inductively-coupled plasma – mass spectrometry for analysis. ARS researchers at Wyndmoor, Pennsylvania developed a faster and less costly method to determine inorganic forms of arsenic in rice using solid-phase extraction in batches and atomic fluorescence spectroscopy, which is very selective and sensitive. Excellent results were achieved with the method for standard reference materials of rice flour, demonstrating equal performance to established methods. This provides higher sample throughput and lower costs, and others are already starting to use the method for monitoring of rice in their labs.

3. Development of a new fast and simple method for the analysis of polyphenolic compounds. Polyphenolic compounds are naturally occurring chemicals with a variety of biological activities, including anti-oxidant properties, and numerous potential health benefits. However, some of these compounds demonstrate estrogenic activity by mimicking the actions of naturally occurring estrogens. Because of both benefits and threats to human health associated with the consumption of polyphenolic compounds, reliable data on their content in food is critical, however few data are available. A new simple and fast method for high throughput analysis of a wide range of polyphenolic compounds was developed. The method is based on simple “dilute and shoot” approach and analysis with liquid chromatography tandem triple quadrupole mass-spectrometry (LC-MS/MS). The developed method for simultaneous determination of multiple polyphenols is fast, simple, and cost-effective. The method was successfully applied to the analysis of juice, tea, and coffee samples. The generated information on occurrence of these chemicals in foods is critical for estimating their content in food, effects on human health and population intake.

4. New high throughput method for the multi-residue analysis of pesticides in fish. Simple and sensitive analytical methods are needed to monitor pesticide residues and ensure that the food is safe for consumption. To achieve low cost, highly selective and sensitive monitoring of a wide range of pesticides with high sample throughput, ARS researchers at Wyndmoor, Pennsylvania developed and evaluated a laboratory-based multi-residue analytical method for simultaneous determination of over 140 pesticide residues in fish. The sample preparation is based on the QuECHERS (quick, easy, cheap, effective, rugged, safe) method using extract clean-up with a novel zirconium-based sorbent. Sample preparation for a batch of 10 homogenized samples took about an hour per analyst, and low-pressure gas chromatography – tandem mass spectrometry gave fast analysis within 9 minutes. The developed method was successfully applied for analysis of fish samples from the market. This technology has been demonstrated for use by regulatory and industrial testing laboratories to help assure the safety of the food supply.

5. Effects of UV-B radiation levels on compositional profile of mushroom powder used as dietary supplements. Mushrooms exposed to UV-B radiation yield enhanced levels of vitamin D, and the powder is used as a dietary supplement to provide nutritional benefits. However, it is unknown if certain other chemicals (both beneficial and toxic) are also produced. Chemical composition changes of four types of mushroom powder dietary supplement exposed to different treatments of UV-B irradiation were analyzed by ARS researchers in Wyndmoor, Pennsylvania for the bioactive naturally occurring mushroom anti-oxidant, ergothioneine, other natural polyphenolic anti-oxidants: e.g. flavonoids, lignans, and others, and selected phytosterols. This study showed that UV-B radiation used to elevate vitamin D levels in mushrooms did not cause compositional changes of investigated bioactive compounds. These results provide strong evidence that irradiated mushrooms can be an excellent source of nutrients.

Review Publications
Geis-Asteggiante, L., Lehotay, S.J., Heinzen, H. 2012. Effects of temperature and purity of magnesium sulfate during extraction of pesticides residues using the QuEChERS method. Official Methods of Analysis of AOAC International. 95(5)1311-1318.

Kwon, H., Lehotay, S.J., Geis-Asteggiante, L. 2012. Variability of matrix effects in liquid and gas chromatography - mass spectrometry analysis of pesticide residues after QuEChERS sample preparation of different food crops. Journal of Chromatography A. 1270:235-245.

Lehotay, S.J., Lightfield, A.R., Geis-Asteggiante, L., Schneider, M.J., Dutko, T., Ng, C., Bluhm, L., Mastovska, K. 2012. Development and validation of a streamlined method designed to detech residues of 62 veterinary drugs in bovine kidney using ultrahigh performance liquid chromatography - tandem mass spectrometry. Drug Testing and Analysis Journal. 4:75-90.

Schneider, M.J., Lehotay, S.J., Lightfield, A.R. 2012. Evaluation of a multiclass, multiresidue liquid chromatography-tandem mass spectrometry method for analysis of 120 veterinary drugs in bovine kidney. Drug Testing and Analysis Journal. 4(1):91-102.

Sapozhnikova, Y.V., Lehotay, S.J. 2013. Multi-class, multi-residue analysis of pesticides, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, polybrominated diphenyl ethers and novel flame retardants....mass spectrometry. Analytica Chimica Acta. 758:80-92.

O'Mahony, J., Clarke, L., Whelan, M., O'Kennedy, R., Lehotay, S.J., Danaher, M. 2013. The use of ultra-high pressure liquid chromatography with tandem mass spectrometric detection of analysis of agrochemical residues and mycotoxines in food - challenges and applications. Journal of Chromatography A. 1292:83-95.

Schneider, M.J., Yun, L., Lehotay, S.J. 2013. Terbium-sensitized luminescence screening method for fluoroquinolones in beef serum. Food Additives & Contaminants. 30(4):666-669.

Hoh, E., Dodder, N.G., Lehotay, S.J., Pangallo, K.C., Reddy, C.M., Maruya, K.A. 2012. A non-targeted comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry method and software for inventorying persistent and bioaccumulative contaminants in marine environments. Journal of Environmental Science and Technology. 46(15):8001–8008.

Hausler, K., Godden, S.M., Schneider, M.J., Lightfield, A.R., Bulthaus, M., Haines, D. 2013. Hot study: Investigating the risk for violative meat residues in bob veal calves fed colostrum from cows treated at dry-off with cephapirin benzathine. Journal of Dairy Science. 96(4):2349-2355.

Geis-Asteggiante, L., Lehotay, S.J., Lightfield, A.R., Dutko, T., Ng, C., Bluhm, L. 2012. Ruggedness testing and validation of a practical analytical method for > 100 veterinary drug residues in bovine muscle by ultrahigh performance liquid chromatography – tandem mass spectrometry. Journal of Chromatography A. 1258:43-54.

Chen, G., Li, Q. 2013. Luminescence screening of enrofloxacin and ciprofloxacin residues in swine liver after dispersive liquid - liquid microextraction cleanup. Journal of Agricultural and Food Chemistry. 61:98-102.

Ozbay, G., Babu, B., Chen, G. 2012. Prevalence of veterinary drug residues and heavy metals in catfish nuggets. Journal of Food Processing and Technology. doi:10.4172/2157-7110.S11-005.

Last Modified: 07/27/2017
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