ABOUT OUR TESTING

Who is Testing Our Pet Food?

Midwest Laboratories: Midwest Laboratories in Omaha, NE is an independent laboratory highly skilled at testing for contaminants. Starting as a testing facility for agricultural analysis of soil, plant tissue and feeds, Midwest has expanded into pesticide analysis, water analysis and microbiological analysis for food, pet food, fertilizer, nematodes, biosolids, petroleum, and hazardous waste. Their chemists, biologists and environmental scientists are able to address new analytical challenges with the latest instruments and methods of testing.

NB Laboratories: Natural Balance has hired Research Biochemist, Dr. Ali Haghighi (left) to create the protocols at NB Laboratories for Melamine and Cyanuric Acid Testing, as well as oversee all lab testing procedures.

Dr. Haghighi received his Ph.D. in Biochemistry at Cleveland State
University. He is also a professor of sciences in Surgical Technology, and teaches Medical Assistant Certification for the State of California. He also has certification as a criminalist from the California Justice Department. Dr. Haghighi has developed an ELISA-based diagnostic assay for Polygenetics, Co., using Cavilink polymers.

NB Laboratories has also hired Quality Control Chemist, Roummel (Rome) Ruvolo (right). Mr. Ruvolo has earned his Bachelor of Science in Pharmacy and has been working in the biotechnology field for over two years. His chief research is based on Bio-inspired Oxygen and Light-driven Homogenous Catalysts. It is his responsibility to implement the testing procedures developed by Dr. Haghighi.

Our standard testing protocol includes testing for Aflatoxin, DON (Vomitoxin), Ochratoxin, Zearalenone (ZEA), and Fumonisin. As of September, 2008, we have added screening for E. coli and Salmonella. Stringent research, development and quality control practices have led to proven reliability and consistency of our tests. The accuracy and reproducibility of these testing protocols have inspired wide acceptance and use throughout the food industry. These tests have also earned official approvals and third party validations, including:

• AOAC International
• AOAC Research Institute
• IUPAC
• USDA/GIPSA (FGIS)
• USDA/FSIS

We have also added testing for Melamine and Cyanuric acid to our protocol. For months these tests were being done through an independent laboratory, and since October, 2007, we have started testing for Melamine and Cyanuric Acid in our own Laboratory, using a Mass Spectrometer, which is used at many major Universities.

What kinds of Test Methods are being used? (Click HERE to learn about the Toxins we test for.)

Testing for Aflatoxin/DON (Vomitoxin):

ELISA Test
NB Laboratories and Midwest Labs, our independent laboratory, use ELISA tests for Aflatoxin, DON (Vomitoxin), Ochratoxin, Zearalone (ZEA), and Fumonisin. ELISA tests are quantitative tests that compare up to 19 samples at a time against test controls. Through the use of a microwell reader, the tests provide accurate sample results in parts per million (for Aflotoxin) or parts per billion (for Vomitoxin). Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. The Enzyme ImmunoAssay (EIA) is a synonym for the ELISA.

Neogen GeneQuence® Test
NB Laboratories uses Neogen GeneQuence® tests for E. coli and Salmonella.

Testing for E. coli: GeneQuence E. coli O157:H7 is a double antibody (sandwich) enzyme-linked immunosorbent assay (ELISA) utilizing specific anti- E. coli O157:H7 antibodies coated to microwells. The controls and samples are added to the antibody wells. After incubation, the wells are washed with a wash buffer and conjugate is added. After a second incubation and wash, substrate is added, which develops a blue color. A final incubation is followed by the addition of the stop solution, which turns the blue color to yellow. Results are read using a microwell plate or strip reader by comparing the optical densities of the samples to the optical densities of the negative and positive controls.

Testing for Salmonella: The DNA hybridization test employs Salmonella-specific DNA probes which are directly labeled with horseradish peroxidase. A colorimetric endpoint is then used for the detection of Salmonella spp. in food samples following broth culture enrichment. A sample is considered negative for the presence of Salmonella if the absorbance value obtained is less than 0.10. A sample is considered presumptively positive for the presence of Salmonella if the absorbance value obtained is greater than or equal to 0.10.

Testing for Melamine/Cyanuric Acid
Traditional crude protein and non-protein nitrogen tests are not melamine specific. The possibility of new kinds of protein tests which will be able to separate natural protein from added nitrogen is still being explored, but they have yet to be implemented in standard protein testing. Our testing for melamine is to make sure that the level, if any, does not exceed 2.5 ppm in accordance with FDA and European Union guidelines. Midwest Labs, our independent laboratory, uses two different methods to detect Melamine and Cyanuric Acid. One is Liquid Chromatography Mass Spectrometry (LC/MS) and the second is Gas Chromatograph fitted with a Nitrogen-Phosphorus Detector (GC/NPD). NB Laboratories uses Spectrophotometric testing. Details on each method are below.

Liquid Chromatography (LC):
Chromatography studies the separation of molecules based on differences in their structure and/or composition. The most common technique is Liquid Chromatography, which is used to separate the target molecule from undesired contaminants, as well as to analyze the final product for the requisite purity established with governmental regulatory groups (such as the FDA).

Mass Spectrometry (MS):
Mass spectrometry is a powerful analytical technique that is used to extract the structure and chemical properties of molecules to identify known and unknown compunds. Compounds can be identified at very low concentrations in chemically complex mixtures. LC/MS is a powerful combination for tests requiring the most thorough chemical analysis.

Gas Chromatograph fitted with a Nitrogen-Phosphorus Detector (GC/NPD):
This instrument and method is very accurate, with the ability to detect melamine at very low levels (10 part per million). The instrument is set up with dual NP detectors and the GC is fitted with two different GC columns. Both columns must detect melamine and deviate less than 5% from each other for a sample to be confirmed as containing melamine.

Spectrophotometric Testing:
Spectrophotometry involves the use of a spectrophotometer, which can measure intensity as a function of the color, or more specifically, the wavelength of light in a compound. This method of chemical analysis based on the absorption by matter of electromagnetic radiation of a specified wavelength or frequency.The radiation interacts with specific features of the molecular species being determined, such as the vibrational or rotational motions of the chemical bonds. Using UV-microscopy, absorbance spectra of melamine can be detected.