Filling the AOAC triangle with food-matrix standard reference materials

Well-characterized reference materials are needed by laboratories in the food testing and nutrition communities to facilitate compliance with nutritional labeling laws, to provide traceability for food exports needed for acceptance in many foreign markets, and to improve the accuracy of nutrition information that is provided to assist consumers in making sound dietary choices. As a result of the enactment of the Nutrition Labeling and Education Act of 1990 and the Infant Formula Act of 1980, the National Institute of Standards and Technology (NIST) has developed a suite of food-matrix Standard Reference Materials (SRMs) characterized for nutrient concentrations. These include SRM 1544 Fatty Acids and Cholesterol in a Frozen Diet Composite, SRM 1546 Meat Homogenate, SRM 1548a Typical Diet, SRM 1566b Oyster Tissue, SRM 1846 Infant Formula, SRM 1946 Lake Superior Fish Tissue, SRM 2383 Baby Food Composite, SRM 2384 Baking Chocolate, SRM 2385 Spinach, and SRM 2387 Peanut Butter. Many of these materials were developed at the request of the food industry to populate a nine-sectored fat-protein-carbohydrate triangle developed by AOAC International. With the completion of SRM 2387, SRMs representing each sector of the triangle are now available. These food-matrix reference materials are intended primarily for validation of analytical methods for the measurement of proximates, fatty acids, vitamins, minerals, and so on in foods of similar composition. They may also be used as "primary control materials" in the value-assignment of in-house, secondary, control materials to confirm accuracy as well as to establish traceability to NIST.     

Alternative AOAC sporicidal test carrier for evaluating peracetic acid-based sterilants (modification of AOAC official method 966.04)

Dacron suture loops were demonstrated to be inert, consistent carriers in the presence of peracetic acid-based sterilants, whereas black silk sutures had a variable preparation process and interacted with peracetic acid. In addition, Dacron suture loops provided comparable spore loading to black silk suture loops and an HCI resistance of > or = 2 min. These results indicate that black silk suture loops are not appropriate carriers for assessing peracetic acid-based sterilants, and Dacron loops are an acceptable alternative. This finding is consistent with the Office of Science and Technology Laboratory (Center for Devices and Radiological Health) study which determined that "polyester suture material is a viable alternative to silk for the AOAC sporicidal test for liquid disinfectants."     

An update on AOAC INTERNATIONAL new program activities

Significant progress has been made with the introduction of new AOAC INTERNATIONAL programs since plans were announced at BERM-5 in Aachen, Germany. The AOAC^® Technical Division on Reference Materials has been formed, the AOAC^® Peer-Verified Methods Program has been established and ready to accept methods for study, and the AOAC^® Test Kit Performance Testing Program has become operational.     

Matching reference materials with AOAC International methods of analysis

Proper implementation and use of validated analytical methodology with use of appropriate reference materials (RM) is a preferred means of helping to ensure equivalent analytical method performance in diverse laboratories. Choice of an appropriate RM that not only matches the analyte and matrix of the required determination, but also has been demonstrated to be within the applicability of a specific analytical method, are key factors. In response to numerous requests since its founding in 1993, the Technical Division on Reference Materials (TDRM), AOAC International is implementing a program for recognizing the matching of specific reference materials to specific AOAC methods of analysis. This recognition is accomplished by means of a thorough peer-reviewed selection system, under the auspices of the AOAC official methods board and the executive committee of the TDRM. Potential RM/method matching (RM/MM) proposals will be submitted to an RM/MM committee. After technical review of the suitability of the proposed RM by the RM/MM committee, acceptable matches are recommended for review by the current AOAC process responsible for review and recognition of new methods and modifications to existing AOAC methods of analysis. Several trial matches have been used to develop and test this system. The end product of this effort will ultimately be made available as either a stand-alone document, a section of the AOAC Official Methods of Analysis, or a site within the AOAC web site listing recognized matches.     

Culture age and drying time as variables of the AOAC Sporicidal Test

In the United States, the AOAC Sporicidal Activity of Disinfectants Method 966.04 is the standard for identifying a liquid chemical germicide as a sterilant. Furthermore, the highest level of a disinfectant must also be a sterilant as defined by Method 966.04, when used in its sterilant mode for a longer exposure time. The AOAC Sporicidal Test is also used as a part of the standard test methods to define a sterilant for Australia and the European Union. Many laboratories have identified variables of this test that can affect the sterilization exposure time for sterilants, or even the ability to classify a chemical as a sterilant. Method 966.04 requires spore-labeled porcelain penicylinders (cylinders) and silk suture loops, collectively referred to as carriers, to be dried for 24 h, but allows these carriers to be used for at least 7 days, in effect allowing a drying time of 24 h to at least 7 days. We tested the resistance of cylinders that had been labeled with Bacillus subtilis spores cultured for 72, 96, and 120 h, and dried for 24, 48, and 72 h against a 60 min exposure to 2.0% alkaline glutaraldehyde, and 2, 5, 10, 15, and 20 min exposures to 2.5N HCl. All the culture incubation and drying times met the standard of resistance to 2.5N HCI for at least 2.0 min at 20 degrees C, and all carriers contained at least 10(5) colony-forming units (CFU) of B. subtilis per carrier. However, for 3 repeated tests, regardless of incubation time, an average of 96% of the carriers were sterilized by the 2.0% glutaraldehyde after drying for 24 h, and an average of 61 % were sterilized after drying for 48 or 72 h. We propose that the variable of drying time be eliminated from Method 966.04.     

Evaluation of the VIDAS Listeria (LIS) immunoassay for the detection of Listeria in foods using demi-Fraser and Fraser enrichment broths, as modification of AOAC Official Method 999.06 (AOAC Official Method 2004.06)

A multilaboratory study was conducted to compare the VIDAS LIS immunoassay with the standard cultural methods for the detection of Listeria in foods using an enrichment modification of AOAC Official Method 999.06. The modified enrichment protocol was implemented to harmonize the VIDAS LIS assay with the VIDAS LMO2 assay. Five food types--brie cheese, vanilla ice cream, frozen green beans, frozen raw tilapia fish, and cooked roast beef--at 3 inoculation levels, were analyzed by each method. A total of 15 laboratories representing government and industry participated. In this study, 1206 test portions were tested, of which 1170 were used in the statistical analysis. There were 433 positive by the VIDAS LIS assay and 396 positive by the standard culture methods. A Chi-square analysis of each of the 5 food types, at the 3 inoculation levels tested, was performed. The resulting average Chi square analysis, 0.42, indicated that, overall, there are no statistical differences between the VIDAS LIS assay and the standard methods at the 5% level of significance.     

Extension of the validation of AOAC Official Method 2005.06 for dc-GTX2,3: interlaboratory study

AOAC Official Method(SM) 2005.06 for the determination of saxitoxin (STX)-group toxins in shellfish by LC with fluorescence detection with precolumn oxidation was previously validated and adopted First Action following a collaborative study. However, the method was not validated for all key STX-group toxins, and procedures to quantify some of them were not provided. With more STX-group toxin standards commercially available and modifications to procedures, it was possible to overcome some of these difficulties. The European Union Reference Laboratory for Marine Biotoxins conducted an interlaboratory exercise to extend AOAC Official Method 2005.06 validation for dc-GTX2,3 and to compile precision data for several STX-group toxins. This paper reports the study design and the results obtained. The performance characteristics for dc-GTX2,3 (intralaboratory and interlaboratory precision, recovery, and theoretical quantification limit) were evaluated. The mean recoveries obtained for dc-GTX2,3 were, in general, low (53.1-58.6%). The RSD for reproducibility (RSD(r)%) for dc-GTX2,3 in all samples ranged from 28.2 to 45.7%, and HorRat values ranged from 1.5 to 2.8. The article also describes a hydrolysis protocol to convert GTX6 to NEO, which has been proven to be useful for the quantification of GTX6 while the GTX6 standard is not available. The performance of the participant laboratories in the application of this method was compared with that obtained from the original collaborative study of the method. Intralaboratory and interlaboratory precision data for several STX-group toxins, including dc-NEO and GTX6, are reported here. This study can be useful for those laboratories determining STX-group toxins to fully implement AOAC Official Method 2005.06 for official paralytic shellfish poisoning control. However the overall quantitative performance obtained with the method was poor for certain toxins.     

Recovery and sporicidal resistance of various B. subtilis spore preparations on porcelain penicylinders compared with results from AOAC test methods

Sporicidal test results obtained from carriers inoculated with 4 types of defined Bacillus subtilis spore preparations were compared with the standard AOAC sporicidal test using soil extract nutrient broth (SENB) B. subtilis 19659 spores. Recoveries of spores inoculated on penicylinders from B. subtilis clean spores (washed and suspended in water) and B. subtilis 19659 spores inoculated from culture filtrates according to the AOAC method were compared. Spores were exposed to 6 concentrations (0.5-3.0% w/v) of glutaraldehyde in phosphate buffer (pH 7.5) for 10 h. Concentrations were established by titrimetry and liquid chromatography. Recoveries of surviving spores were determined for 3 types of clean B. subtilis var. niger preparations, one clean B. subtilis 19659 preparation, and the SENB B. subtilis 19659 filtrates. Spore carriers, inoculated by the standard AOAC protocol, resulted in as much as a 2-log number difference in runs 1-12, but not more than 0.5 log number for each clean spore preparation. The SENB spores varied most in resistance to glutaraldehyde, with no growth in recovery media from 3 different batches of 1, 1.5, and 2% glutaraldehyde. Separate batches of SENB preparations of B. subtilis 19659 were resistant and destroyed by 1.0% glutaraldehyde, with 3.98 and 6.0 log numbers of spores on penicylinders, respectively. Clean spore preparations of B. subtilis 19659 on porcelain penicylinders were more resistant to glutaraldehyde than were SENB spores. Nutrient agar/Mg/Ca and nutrient agar/Mg spore preparations of B. subtilis var. niger showed the most uniform resistance to glutaraldehyde. Spores with calcium added showed increased resistance to glutaraldehyde. B. subtilis 19659 spores from the Columbia broth spore preparation were the most resistant and were recovered after exposure to 3.0% glutaraldehyde.