On the use of the Horwitz Ratio (HorRat) as an acceptance criterion for dietary fiber collaborative studies.

The quest for optimum methodology for any analyte often includes a study of the method in multiple independent laboratories. Evaluation of the results of these studies is typically subjective; however, various attempts have been made to increase the objectivity of the evaluation. Among the objective propositions is the use of the Horwitz Ratio (HorRat) as applied to collaborative study statistics. A historical review of fiber method validation studies performed since 1940 shows that the Horwitz curve does not effectively predict the results of dietary fiber collaborative studies, either retrospectively or prospectively. Consequently, use of the HorRat as a criterion for accepting or rejecting dietary fiber methods is contraindicated. An alternative, objective statistical approach is proposed that may also apply to collaborative studies of other empirical analytical methods in general.

Determination of insoluble, soluble, and total dietary fiber (CODEX definition) by enzymatic-gravimetric method and liquid chromatography: collaborative study.

A method for the determination of insoluble (IDF), soluble (SDF), and total dietary fiber (TDF), as defined by the CODEX Alimentarius, was validated in foods. Based upon the principles of AOAC Official Methods 985.29, 991.43, 2001.03, and 2002.02, the method quantitates water-insoluble and water-soluble dietary fiber. This method extends the capabilities of the previously adopted AOAC Official Method 2009.01, Total Dietary Fiber in Foods, Enzymatic-Gravimetric-Liquid Chromatographic Method, applicable to plant material, foods, and food ingredients consistent with CODEX Definition 2009, including naturally occurring, isolated, modified, and synthetic polymers meeting that definition. The method was evaluated through an AOAC/AACC collaborative study. Twenty-two laboratories participated, with 19 laboratories returning valid assay data for 16 test portions (eight blind duplicates) consisting of samples with a range of traditional dietary fiber, resistant starch, and nondigestible oligosaccharides. The dietary fiber content of the eight test pairs ranged from 10.45 to 29.90%. Digestion of samples under the conditions of AOAC 2002.02 followed by the isolation, fractionation, and gravimetric procedures of AOAC 985.29 (and its extensions 991.42 and 993.19) and 991.43 results in quantitation of IDF and soluble dietary fiber that precipitates (SDFP). The filtrate from the quantitation of water-alcohol-insoluble dietary fiber is concentrated, deionized, concentrated again, and analyzed by LC to determine the SDF that remains soluble (SDFS), i.e., all dietary fiber polymers of degree of polymerization = 3 and higher, consisting primarily, but not exclusively, of oligosaccharides. SDF is calculated as the sum of SDFP and SDFS. TDF is calculated as the sum of IDF and SDF. The within-laboratory variability, repeatability SD (Sr), for IDF ranged from 0.13 to 0.71, and the between-laboratory variability, reproducibility SD (SR), for IDF ranged from 0.42 to 2.24. The within-laboratory variability Sr for SDF ranged from 0.28 to 1.03, and the between-laboratory variability SR for SDF ranged from 0.85 to 1.66. The within-laboratory variability Sr for TDF ranged from 0.47 to 1.41, and the between-laboratory variability SR for TDF ranged from 0.95 to 3.14. This is comparable to other official and approved dietary fiber methods, and the method is recommended for adoption as Official First Action.

Determination of vitamin A (retinol) in infant formula and adult nutritionals by liquid chromatography: First Action 2011.15.

During the "Standards Development and International Harmonization: AOAC INTERNATIONAL Mid-Year Meeting," held on June 29, 2011, an Expert Review Panel (ERP) reviewed the method for the "Determination of Vitamins A (Retinol) and E (alpha-Tocopherol) in Foods by Liquid Chromatography: Collaborative Study," published by Jonathan W. DeVries and Karlene R. Silvera in J. AOAC Int. in 2002. After evaluation of the original validation data, an ERP agreed in June 2011 that the method meets standard method performance requirements (SMPRs) for vitamin A, as articulated by the Stakeholder Panel on Infant Formula and Adult Nutritionals. The ERP granted the method First Action status, applicable to determining vitamin A in ready-to-eat infant and adult nutritional formula. In an effort to achieve Final Action status, it was recommended that additional information be generated for different types of infant and adult nutritional formula matrixes at varied concentration levels as indicated in the vitamin A (retinol) SMPR. Existing AOAC LC methods are suited for specific vitamin A analytical applications. The original method differs from existing methods in that it can be used to assay samples in all nine sectors of the food matrix. One sector of the food matrix was powdered infant formula and gave support for the First Action approval for vitamin A in infant and adult nutritional formula. In this method, standards and test samples are saponified in basic ethanol-water solution, neutralized, and diluted, converting fats to fatty acids and retinol esters to retinol. Retinol is quantitated by an LC method, using UV detection at 313 or 328 nm for retinol. Vitamin concentration is calculated by comparison of the peak heights or peak areas of retinol in test samples with those of standards.

Simultaneous ion removal and quantitation of low-molecular-weight dietary fiber from high-molecular-weight dietary fiber filtrates using liquid chromatography.

Dietary fiber and its quantitation in foods have been of significant interest in the nutrition community for over 50 years. A number of AOAC Official Methods of Analysis have been adopted for the analysis of dietary fiber and some of its fractions and components commensurate with the evolving discoveries of dietary fiber nutrition research. Quantitation of low-molecular-weight soluble dietary fiber (LMWSDF) has been difficult due to high solubility in a precipitating solvent mixture of four parts alcohol and one part water. AOAC Method 2001.03 effectively quantitates LWMSDF subsequent to gravimetric removal of high-molecular-weight dietary fiber using LC. However, deionization and concentration of the enzymatic digestate, necessary to assure accurate LC quantitation, requires substantial time and manual labor. A modification to the method and resulting method performance is presented that describes a means of simultaneously deionizing the digestate and quantitating the LMWSDF in a single LC injection, eliminating a number of time-consuming manual preparation steps.

Determination of total dietary fiber (CODEX definition) by enzymatic-gravimetric method and liquid chromatography: collaborative study.

A method for the determination of total dietary fiber (TDF), as defined by the CODEX Alimentarius, was validated in foods. Based upon the principles of AOAC Official Methods 985.29, 991.43, 2001.03, and 2002.02, the method quantitates high- and low-molecular-weight dietary fiber (HMWDF and LMWDF, respectively). In 2007, McCleary described a method of extended enzymatic digestion at 37 degrees C to simulate human intestinal digestion followed by gravimetric isolation and quantitation of HMWDF and the use of LC to quantitate low-molecular-weight soluble dietary fiber (LMWSDF). The method thus quantitates the complete range of dietary fiber components from resistant starch (by utilizing the digestion conditions of AOAC Method 2002.02) to digestion resistant oligosaccharides (by incorporating the deionization and LC procedures of AOAC Method 2001.03). The method was evaluated through an AOAC collaborative study. Eighteen laboratories participated with 16 laboratories returning valid assay data for 16 test portions (eight blind duplicates) consisting of samples with a range of traditional dietary fiber, resistant starch, and nondigestible oligosaccharides. The dietary fiber content of the eight test pairs ranged from 11.57 to 47.83%. Digestion of samples under the conditions of AOAC Method 2002.02 followed by the isolation and gravimetric procedures of AOAC Methods 985.29 and 991.43 results in quantitation of HMWDF. The filtrate from the quantitation of HMWDF is concentrated, deionized, concentrated again, and analyzed by LC to determine the LMWSDF, i.e., all nondigestible oligosaccharides of degree of polymerization > or =3. TDF is calculated as the sum of HMWDF and LMWSDF. Repeatability standard deviations (Sr) ranged from 0.41 to 1.43, and reproducibility standard deviations (S(R)) ranged from 1.18 to 5.44. These results are comparable to other official dietary fiber methods, and the method is recommended for adoption as Official First Action.

Total Protein Methods and Their Potential Utility to Reduce the Risk of Food Protein Adulteration.

Kjeldahl and combustion (Dumas) methods are widely accepted for total protein determination but lack analytical selectivity for protein because they measure protein on the basis of sample nitrogen content. Adulteration incidents exploiting this analytical vulnerability (for example, melamine) demonstrate that these methods are no longer sufficient to protect the public health. This article explores the challenges and opportunities to move beyond total nitrogen based methods for total protein measurement. First, it explores the early history of protein measurement science, complexities of current global protein measurement activities, and ideal analytical performance characteristics for new methods. Second, it comprehensively reviews the pros and cons of current and emerging approaches for protein measurement, including their selectivity for protein, ability to detect adulteration, and practicality for routine use throughout the supply chain. It concludes that some existing highly selective methods for food protein measurement have potential for routine quality control. It also concludes that their successful implementation will require matrix-specific validation and the use of supporting reference materials. These methods may be suitable only for food ingredients that have a low degree of compositional variability and are not complex finished food products.

Cleaning and other control and validation strategies to prevent allergen cross-contact in food-processing operations.

Food allergies affect an estimated 10 to 12 million people in the United States. Some of these individuals can develop life-threatening allergic reactions when exposed to allergenic proteins. At present, the only successful method to manage food allergies is to avoid foods containing allergens. Consumers with food allergies rely on food labels to disclose the presence of allergenic ingredients. However, undeclared allergens can be inadvertently introduced into a food via cross-contact during manufacturing. Although allergen removal through cleaning of shared equipment or processing lines has been identified as one of the critical points for effective allergen control, there is little published information on the effectiveness of cleaning procedures for removing allergenic materials from processing equipment. There also is no consensus on how to validate or verify the efficacy of cleaning procedures. The objectives of this review were (i) to study the incidence and cause of allergen cross-contact, (ii) to assess the science upon which the cleaning of food contact surfaces is based, (iii) to identify best practices for cleaning allergenic foods from food contact surfaces in wet and dry manufacturing environments, and (iv) to present best practices for validating and verifying the efficacy of allergen cleaning protocols.

Microbiological assay-trienzyme procedure for total folates in cereals and cereal foods: collaborative study.

In 1996, U.S. Food and Drug Administration regulations mandated the fortification of enriched cereal-grain products with folic acid, thereby emphasizing the need for validated methods for total folates in foods, particularly cereal products. The AOAC Official Methods (944.12, 960.46) currently used for the analysis of folate in foods for compliance purposes are microbiological methods. When the fortification regulations were finalized, no Official AOAC or Approved AACC methods for folate in cereal-grain products were in place. The AOAC Official Method (992.05) for folic acid in infant formula does not incorporate important improvements in the extraction procedure and was not considered suitable for the analysis of folates in foods in general. A microbiological assay protocol using a trienzyme extraction procedure was prepared and submitted for comments to 40 laboratories with recognized experience in folate analysis. On the basis of comments, the method was revised to have the conjugase (gamma-glutamyl-carboxy-peptidase) treatment follow a protease treatment, to include the use of cryoprotected inoculum, and to include the spectroscopic standardization of the standard and optional use of microtiter plates. Thirteen laboratories participated in a collaborative study of 10 required and 10 optional cereal-grain products, including flour, bread, cookies, baking mixes, and ready-to-eat breakfast cereals. The majority of the participating laboratories performed the assay by the standard test tube method; others used the microtiter plate modification for endpoint quantitation with equal success. For the required products, the relative standard deviation between laboratories (RSD(R)) ranged from 7.4 to 21.6% for 8 fortified (or enriched) products compared with expected (Horwitz equation-based) values of 11-20%. RSD(R) values were higher (22.7-52.9%) for 2 unfortified cereal-grain products. For the optional products, the RSD(R) ranged from 1.8 to 11.2% for 8 fortified products. RSD(R) values were higher (27.9-28.7%) for 2 unfortified cereal-grain products. Based on the results of the collaborative study, the microbiological assay with trienzyme extraction is recommended for adoption as Official First Action.

Determination of beta-carotene in supplements and raw materials by reversed-phase high pressure liquid chromatography: collaborative study.

Twelve laboratories representing 4 countries participated in an interlaboratory study conducted to determine all-trans-veta-carotene and total beta-carotene in dietary supplements and raw materials. Thirteen samples were sent as blind duplicates to the collaborators. Results obtained from 11 laboratories are reported. For products composed as softgels and tablets that were analyzed for total beta-carotene, the reproducibility relative standard deviation (RSDR) ranged from 3.35 to 23.09% and the HorRat values ranged from 1.06 to 3.72. For these products analyzed for trans beta-carotene, the reproducibility relative standard deviation (RSDR) ranged from 4.28 to 22.76% and the HorRat values ranged from 0.92 to 3.37. The RSDr and HorRat values in the analysis of a beadlet raw material were substantial and it is believed that the variability within the material itself introduced significant variation in subsampling. The method uses high pressure liquid chromatography (LC) in the reversed-phase mode with visible light absorbance for detection and quantitation. If high levels of alpha-carotenes are present, a second LC system is used for additional separation and quantitation of the carotene species. It is recommended that the method be adopted as an AOAC Official Method.

Historical perspective as a guide for identifying and developing applicable methods for dietary fiber.

A review is presented describing the nature and evolving definition of dietary fiber. The historical development of the current definition is discussed as are the efforts to develop analytical methods to support food labeling regulations. Also considered are the characterization and quantitation of resistance starch, a dietary starch that does not digest in the small intestine, behaves like dietary fiber and therefore may have potential as a health-related ingredient in foods. The current status of AOAC methodology is discussed along with the possibility of updating the definition of dietary fiber. The potential impacts of changing the dietary fiber definition on analytical issues and on food composition databases are also considered.

Dietary fiber: the influence of definition on analysis and regulation.

Since 1953 when the term "dietary fiber" was coined, there has been concern about accurately defining this macronutrient component of the human diet. Proper and adequate analytical methodology and food labeling regulations are dependent upon an accurate definition. Health impact studies also depend upon an accurate and meaningful definition along with relevant methodology to provide data of adequate quality for epidemiological and clinical studies. The scientific communities associated with dietary fiber within AOAC INTERNATIONAL have been the leaders in bringing consensus to the dietary fiber definition and method validation for over a quarter of a century. The consensus definition and subsequent methodology have served as the base for regulations worldwide with regard to dietary fiber labeling and health claims. Recently, there has been renewed interest in reviewing the dietary fiber definition and updating it if the review indicates such a need. The American Association of Cereal Chemists completed an effort that provides a continuum for the historical scientific and regulatory efforts while allowing for inclusion of future discoveries into a framework based upon the knowledge gained in the past. Such a definition will provide for transparent and workable regulations with regard to dietary fiber, and will allow the dietary fiber scientific community of AOAC to validate relevant methods. The Food Nutrition Board of the Institute of Medicine of the National Academies has published a set of definitions disconnected from the historical scientific base that do not provide a relevant basis for either adequate methodology, health studies or workable regulations.

On defining dietary fibre.

Establishing a definition for dietary fibre has historically been a balance between nutrition knowledge and analytical method capabilities. While the most widely accepted physiologically-based definitions have generally been accurate in defining the dietary fibre in foods, scientists and regulators have tended, in practice, to rely on analytical procedures as the definitional basis in fact. As a result, incongruities between theory and practice have resulted in confusion regarding the components that make up dietary fibre. In November 1998 the president of the American Association of Cereal Chemists (AACC) appointed an expert scientific review committee and charged it with the task of reviewing and, if necessary, updating the definition of dietary fibre. The committee was further charged with assessing the state of analytical methodology and making recommendations relevant to the updated definition. After due deliberation, an updated definition of dietary fibre was delivered to the AACC Board of Directors for consideration and adoption (Anon, 2000; Jones 2000b). The updated definition includes the same food components as the historical working definition used for approximately 30 years (a very important point, considering that the majority of the research of the past 30 years delineating the positive health effects of dietary fibre is based on that working definition). However, the updated definition more clearly delineates the make-up of dietary fibre and its physiological functionality. As a result, relatively few changes will be necessary in analytical methodology. Current methodologies, in particular AACC-approved method of analysis 32-05 (Grami, 2000), Association of Official Analytical Chemists' official method of analysis 985.29 (Horwitz, 2000a) or AACC 32-07 (Grami, 2000) Association of Official Analytical Chemists 991.43 (Horwitz, 2000a) will continue to be sufficient and used for most foods. A small number of additional methods will be necessary to quantify the dietary fibre levels in foods containing fibres such as fructans (polymers and oligomers of fructose, inulin), modified dextrins, and/or synthetic dietary fibre analogues.