Determination of vitamin B12 in fortified bovine milk-based infant formula powder, fortified soya-based infant formula powder, vitamin premix, and dietary supplements by surface plasmon resonance: collaborative study

A collaborative study was conducted on an inhibition-based protein-binding assay using the Biacore Q biosensor instrument and the Biacore Qflex Kit Vitamin B12 PI. The samples studied included infant formula, cereals, premixes, vitamin tablets, dietary supplements, and baby food. The collaborative study, which involved 11 laboratories, demonstrated that the assay showed an RSDr of 1.59-27.8 and HorRat values for reproducibility of 0.34-1.89 in samples with levels ranging from ppm to ppb. The assay studied is a label-free protein binding-based assay that uses the principle of surface plasmon resonance (SPR) to measure the interaction between vitamin B12 and a specific binding protein. A Biacore Q biosensor uses this principle to detect binding directly at the surface of a sensor chip with a hydrophilic gold-dextran surface. The instrument passes a mixture of prepared sample extract and binding protein solution across a covalently immobilized vitamin B12 chip surface, and the response is given as free-binding protein as the mixture binds to the immobilized surface. This technique uses the specificity and robustness of the protein-ligand interaction to allow minimal sample preparation and a wide range of matrixes to be analyzed rapidly. The reagents and accessories needed to perform this assay are provided as the ready-to-use format "Qflex Kit Vitamin B12 PI." The method is intended for routine use in the quantitative determination of vitamin B12 (as cyanocobalamin) in a wide range of food products, dietary vitamin supplements, and multivitamin premixes.     

Determination of vitamin B12 in infant formula and adult nutritionals by HPLC: First Action 2011.10

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 "Determination of Vitamin B12 in Infant Formula and Adult Nutritionals by HPLC." Under the new pathway to Official Methods, the ERP adopted the method as Official First Action. The method is applicable to the determination of vitamin B12 in infant formula and adult nutritionals. Data showed an average overall intermediate precision of 6.64% RSD, an estimated quantitation limit of 0.8 microg/kg, and a detection limit of 0.2 microg/kg in prepared samples. The standard range of the method is 2 to 200 microg/L, which corresponds to an analytical range of 0.8 to 500 microg/kg.     

Determination of vitamin A in infant formula and adult nutritionals by UPLC-UV: First Action 2011.07

Vitamin A, a fat-soluble vitamin, is essential for health and plays an important part in vision, bone growth, reproduction, regulating the immune system, cell function, and skin health. Due to the advances in technology and the expansion of its uses, LC technologies are being studied for effectiveness in detecting and quantifying vitamin A in an effort to help determine the amount of vitamin A in various types of samples. For this reason, an Expert Review Panel agreed on June 29, 2011, at the "Standards Development and International Harmonization: AOAC INTERNATIONAL Mid-Year Meeting," to approve "Determination of Vitamin A in Infant Formula and Adult Nutritionals by UPLC-UV" as AOAC Official Method 2011.07. To move from First to Final Action status, it was recommended that additional information be generated for all types of infant formulas and adult nutritional formula matrixes at varied concentration levels, as indicated in the standard method performance requirements. International units or retinol equivalents typically represent the concentration of vitamin A in food and supplements. However, for the purpose of this method, the concentration represented is presented in microg/100 g.     

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.     

Determination of vitamin B12 in infant formula and adult nutritionals by liquid chromatography/UV detection with immunoaffinity extraction: First Action 2011.08

At the "Standards Development and International Harmonization: AOAC INTERNATIONAL Mid-Year Meeting," on June 29, 2011, an Expert Review Panel agreed that the method "Determination of Vitamin B12 in Infant Formulas and Adult Nutritionals by Liquid Chromatography/UV Detection with Immunoaffinity Extraction" be adopted AOAC Official First Action status. The method is applicable for the determination of vitamin B12, which includes added cyanocobalamin and natural forms, making it applicable to both fortified and nonfortified products. Vitamin B12 is extracted from the sample in sodium acetate buffer in the presence of sodium cyanide (100 degrees C, 30 min). After purification and concentration with an immunoaffinity column, vitamin B12 is determined by LC with UV detection (361 nm). A single-laboratory validation study was conducted on a range of products, including milk- and soy-based infant formulas, cereals, cocoa beverages, health care products, and polyvitamin premixes. The method demonstrated linear response over a large range of concentrations, recovery rates of 100.8 +/- 7.5% (average +/- SD), repeatability RSD (RSDr) of 2.1%, and intermediate reproducibility (RSD(iR)) of 4.3%. LOD and LOQ values were 0.10 and 0.30 microg/100 g, respectively, and correlation with the reference microbiological assay was good (R2 = 0.9442). The results of the study were published in J. AOAC Int. 91, 786-793 (2008). The performance characteristics of the method met the standard method performance requirements set forth by the Stakeholder Panel on Infant Formula and Adult Nutritionals; thus, the method was determined to be appropriate for First Action status.     

Determination of vitamin B12 in infant formula and adult nutritionals using HPLC after purification on an immunoaffinity column: first action 2011.09

During the "Standards Development and International Harmonization: AOAC INTERNATIONAL Mid-Year Meeting," on June 29, 2011, the method "Determination of vitamin B12 in infant formula and adult nutritionals using HPLC after purification on an immunoaffinity column" was recommended by an Expert Review Panel and adopted as AOAC Official First Action status. The method is applicable for the determination of vitamin B12 in milk-based infant formula. Vitamin B12 is extracted from the sample in sodium acetate buffer in the presence of potassium cyanide. After purification and concentration with an immunoaffinity column (IAC), vitamin B12 is determined by LC with UV detection (361 nm). Data supplied by CLF demonstrated linear response over a wide range of concentrations (1.4-39 microg/100 mL). The analytical range is 0.2-10 microg/100 g, depending on the capacity of the IACs (0.01-0.5 microg), the input weight, and dilutions. Recovery rates were assessed using National Institute of Standards and Technology SRM 1849, and determined to be 95.1%, with SD of 0.34 and CV of 9.0. Measurement uncertainty (UE) was 0.8 microg/100 g, which was calculated from the validation data. It is an expanded measurement uncertainty and was obtained through multiplication with a coverage factor k. LOQ values were reported as 0.10 microg/100 g. The performance characteristics of the method met the standard method performance requirements set forth by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals; thus, the method was determined to be appropriate for First Action status.     

Determination of vitamins D2 and D3 in infant formula and adult nutritionals by ultra-pressure liquid chromatography with tandem mass spectrometry detection (UPLC-MS/MS): First Action 2011.12

The method for the "Determination of Vitamins D2 and D3 in Infant Formula and Adult Nutritionals by Ultra-Pressure Liquid Chromatography with Tandem Mass Spectrometry Detection (UPLC-MS/MS)" was adopted as AOAC Official First Action during the "Standards Development and International Harmonization: AOAC INTERNATIONAL Mid-Year Meeting" held June 29, 2011. During the meeting, an Expert Review Panel (ERP) evaluated the available validation information against standard method performance requirements (SMPRs) articulated by stakeholders. The method, approved by the ERP, is applicable for the determination of vitamin D (total vitamins D2 and D3). A range of products had been tested during a single-laboratory validation study. The products included butter, National Institute of Standards and Technology SRM 1849, eggs, cheese, yogurt, ready-to-eat cereal, bread, mushrooms, and tuna. The testing of the method established linearity in the range of 0.005-50 microg/mL. The recovery range was 93.4-100.9% for vitamin D2 and 102.4-106.2% for vitamin D3. The LOD and LOQ for vitamin D2 were reported as 0.20 and 0.61 microgl100 g, respectively; for vitamin D3, the reported values were 0.47 and 1.44 microg/100 g, respectively. The method met the SMPRs set by the Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN). It was, therefore, decided that the method was appropriate for Official First Action Method status.     

Simultaneous determination of vitamins D2 and D3 by LC-MS/MS in infant formula and adult nutritionals: First Action 2011.13

During the "Standards Development and International Harmonization: AOAC INTERNATIONAL Mid-Year Meeting" held on June 29, 2011, an Expert Review Panel (ERP) on behalf of AOAC INTERNATIONAL adopted the method "Simultaneous Determination of Vitamins D2 and D3 by LC-MS/MS in Infant Formula and Adult Nutritionals" as an AOAC Official First Action method. Vitamins D2 and D3 are extracted from the sample using pentane-ether; the extract is collected and dried under nitrogen. Vitamin D is separated from interfering compounds using UPLC, and quantitated using tandem mass spectrometry (MS/MS). Preliminary data showed the intermediate precision ranged from 3.34-8.05% and an accuracy range of 98.5-111% over the samples tested for vitamin D3. For vitamin D2, the intermediate precision ranged from 2.37-5.45% and accuracy ranged from 96.4-104% over the four matrixes evaluated. The analytical range for the method is bounded by the concentrations of the working standards, 21-270 ng/mL, and is equivalent to 0.168-2.16 mcg/100 g in ready-to-feed product. The practical method quantitation limit is 0.168 mcg/100 g product with method detection limit of 60 ng/100 g product. The ERP reviewed the data and determined that the performance characteristics of the method met the standard method performance requirements, and therefore the method was granted First Action status.     

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 ractopamine in swine, bovine, and turkey tissues by HPLC with fluorescence detection: first action 2011.22

A candidate LC method proposed by the Expert Review Panel (ERP) for ractopamine was evaluated in a single-laboratory validation (SLV) study. The matrixes examined included bovine liver, kidney, muscle, and fat; swine liver, kidney, muscle, and fat; and turkey liver and muscle. Solution standards were shown to provide a linear response with an unweighted regression. The method demonstrated acceptable precision (HorRat, values 0.25 to 1.38) and recovery (75.4 to 88.8%) in all fortified matrixes. Method precision was verified with incurred residue tissues (bovine liver, kidney, and muscle; swine liver, kidney, and muscle; and turkey liver and muscle), which yielded RSDr values below 16% for all tissues and below 7% for most tissues. Estimated LOQ values ranged from 1.8 to 20.7 ng/g and support the utility of the method in the range of the maximum residue limits or tolerances for the various tissues. The data satisfy the requirements of the AOAC Stakeholder Panel on Veterinary Drug Residue for SLV studies, and the method was adopted Official Methods ofAnalysis First Action 2011.22 by the AOAC ERP on Veterinary Drug Residues.     

Extension of AOAC official method 999.14 (choline in infant formula and milk) to the determination of choline in dietary supplements

AOAC Official Method 999.14 is applicable for the determination of choline in milk and infant formulas. To date, its use has not been extended beyond these matrixes. We modified Official Method 999.14 and applied it to the determination of choline in a range of choline-containing dietary supplements. Dietary supplement tablets, capsules, wafers, softgels, liquid products, and drink powders were included. We found that the standard curve could be extended to cover a wider range of choline concentrations and defined a procedure for the use of Norit for samples in which the vitamin C content was high enough to interfere with the analysis. Recoveries of choline added to infant formula powders and to representative dietary supplement tablets, capsules, powdered drink mix, and wafer products were 85-114%. The use of Norit during the procedure did not affect the recovery of choline added to infant formula powders or to dietary supplements. An alkaline digestion was included for use with a product containing lecithin as the sole source of choline. Ten of 11 dietary supplement products analyzed by the modified method contained amounts of choline at or above declarations found on the product labels. The remaining product contained about 40% of the label-declared amount of choline.     

Determination of vitamin A (retinol) in infant and medical nutritional formulas with AOAC method 992.06 using a modified extraction procedure: single-laboratory validation

The applicability of AOAC Official Method 992.06, vitamin A (retinol) in milk-based infant formula can be extended to specialty infant formulas, and medical and adult nutritional products with a few minor modifications to the sample preparation procedure. Currently, AOAC Official Method 992.06 is only applicable to milk-based infant formulas containing >500 IU vitamin A per reconstituted quart. When this method is used as written to test specialty infant formulas, vitamin A recoveries are low compared to results generated with alternate validated vitamin A methods. AOAC Method 992.06 vitamin A recoveries can be improved significantly in specialty infant formulas if the amount of potassium hydroxide used during the saponification step is doubled. With this one minor modification to the sample preparation procedure, AOAC Method 992.06 demonstrates acceptable precision and accuracy for the quantitation of vitamin A (retinol) in specialty infant formulas, milk- and soy-based infant formulas, and adult and medical nutritionals. Because increasing the amount of potassium hydroxide can cause emulsions to form, 2-4 mL aliquots of reagent alcohol may need to be added to some samples to separate the organic and aqueous layers during the extraction step. A single-laboratory validation of these modifications was completed. During validation, 15 different product matrixes were analyzed. The intermediate precision averaged 2.70% RSD, and spike recovery data averaged 96.3%.     

Method for the rapid determination of protein in meats using the CEM Sprint protein analyzer: collaborative study

A collaborative study was conducted to determine the protein content of raw and processed meat products by a protein-tagging and colorimetric technique. Meat products were prepared following AOAC Official Method 983.18 and analyzed using CEM Corporation's Sprint Rapid Protein Analyzer. Sprint provides protein results by combining an accurately weighed test portion with a known amount of dye-binding agent. The dye-binding agent binds with the lysine, histidine, and arginine, as well as the n-terminus of the proteins commonly found in raw meat and processed meat products. Results are displayed and reported by the Sprint as a percentage (g/100 g) of protein. Ten blind duplicate study samples were sent to 10 collaborating laboratories in the United States. The within-laboratory (repeatability) relative standard deviation (RSD(r)) ranged from 0.91 to 3.04%, and between-laboratories (reproducibility) relative standard deviation (RSDR) ranged from 1.50 to 3.41% for protein. The method is recommended for Official First Action.     

Determination of calcium, copper, iron, magnesium, manganese, potassium, phosphorus, sodium, and zinc in fortified food products by microwave digestion and inductively coupled plasma-optical emission spectrometry: single-laboratory validation and ring trial

A single-laboratory validation (SLV) and a ring trial (RT) were undertaken to determine nine nutritional elements in food products by inductively coupled plasma-optical emission spectrometry in order to modernize AOAC Official Method 984.27. The improvements involved extension of the scope to all food matrixes (including infant formula), optimized microwave digestion, selected analytical lines, internal standardization, and ion buffering. Simultaneous determination of nine elements (calcium, copper, iron, potassium, magnesium, manganese, sodium, phosphorus, and zinc) was made in food products. Sample digestion was performed through wet digestion of food samples by microwave technology with either closed- or open-vessel systems. Validation was performed to characterize the method for selectivity, sensitivity, linearity, accuracy, precision, recovery, ruggedness, and uncertainty. The robustness and efficiency of this method was proven through a successful RT using experienced independent food industry laboratories. Performance characteristics are reported for 13 certified and in-house reference materials, populating the AOAC triangle food sectors, which fulfilled AOAC criteria and recommendations for accuracy (trueness, recovery, and z-scores) and precision (repeatability and reproducibility RSD, and HorRat values) regarding SLVs and RTs. This multielemental method is cost-efficient, time-saving, accurate, and fit-for-purpose according to ISO 17025 Norm and AOAC acceptability criteria, and is proposed as an extended updated version of AOAC Official Method 984.27 for fortified food products, including infant formula.     

Analysis of 5'-mononucleotides in infant formula and adult/pediatric nutritional formula by liquid chromatography: First Action 2011.20

A method for the routine determination of 5'-mononucleotides (uridine 5'-monophosphate, inosine 5'-monophosphate, adenosine 5'-monophosphate, guanosine 5'-monophosphate, and cytidine 5'-monophosphate) in infant formula and adult nutritionals is described. After sample dissolution and addition of internal standard, potential interferences were removed by anion-exchange SPE followed by HPLC-UV analysis. Single-laboratory validation performance parameters include recovery (92-101%) and repeatability (1.0-2.3% RSD). The method was approved for Official First Action status by an AOAC expert review panel.     

Extension of AOAC Official Method 996.01 to the analysis of Standard Reference Material (SRM) 1846 and infant formulas.

There is currently no official method for the analysis of fatty acids (including trans fatty acids) in infant formulas. AOAC Official Method 996.01 for Fat Analysis in Cereal Products was extended to the analysis of milk-based infant formula Standard Reference Material (SRM)1846 to determine its applicability for use with infant formulas. Following the analysis of SRM 1846, 2 infant formulas, one milk-based liquid and one soy-based powdered infant formula, were analyzed for total fatty acid composition. Fatty acid methyl esters were prepared and analyzed by gas chromatography. The results of the analysis of SRM 1846 show that the mean analyzed values were highly reproducible as indicated by low coefficients of variation (CV). The CVs were <5% for the major fatty acids. Mean analyzed values for individual fatty acids in SRM 1846 were within +/- 1 standard deviation of the certificate values. The analyzed value for total fat as triglycerides (26.27 +/- 0.25%) agreed well with the certificate value (27.1 +/- 0.59%). Analyses of infant formulas showed that the concentrations of linoleic acid and fat meet the requirements for such formulas.     

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.     

Evaluation of VIDAS Immuno-Concentration Salmonella (ICS)/VIDAS Salmonella (SLM) immunoassay method for detection of Salmonella in selected foods (Method Modification 2001.09): collaborative study

A new method for detection of Salmonella in foods in a minimum of 24 h was adopted as an AOAC Official First Action Method for selected foods (2001.09) using both the VIDAS Immuno-Concentration Salmonella (ICS) and VIDAS Salmonella (SLM) methods.     

Determination of methylcellulose and hydroxypropyl methylcellulose food gums in food and food products: collaborative study

A collaborative study was performed to determine the reproducibility of a method for the determination of methylcellulose (MC) and hydroxypropyl methylcellulose (HPMC) in food. These widely used food gums possess unusual solubility characteristics and cannot accurately be determined by existing dietary fiber methods. The new method uses the enzyme-digestion procedure of AOAC Official Method 991.43. Digestate solutions must be refrigerated to fully hydrate MC or HPMC. The chilled solutions are filtered and analyzed by size-exclusion liquid chromatography. Collaborating laboratories received 28 samples containing MC or HPMC in the range of 0-100%. The sample set included blind duplicates of 5 food matrixes (bread, milk, fish, potato, and powdered juice drink). Cochran and Grubbs tests were used to eliminate outliers. For food samples containing MC, values for within-laboratory precision, repeatability relative standard deviation (RSDr), ranged from 4.2 to 16%, and values for among-laboratories precision, reproducibility relative standard deviation (RSDR), ranged from 11 to 20%. For HPMC samples, RSDr values ranged from 6.4 to 27%, and RSDR values ranged from 17 to 39%. Recoveries of MC and HPMC from the food matrixes ranged from 78 to 101%. These results show acceptable precision and reproducibility for the determination of MC and HPMC, for which no Official AOAC Methods exist. It is recommended that this method be adopted as AOAC Official First Action.     

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.