6. AOACSPIFANMethods-2018Awards

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1656 Butler-Thompson et al.: J ournal of AOAC I nternational V ol. 98, N o. 6, 2015 AOAC 2011.10 uses a pH 4.5 sodium acetate buffer and potassium cyanide at 105°C to extract and convert all forms of vitamin B 12 present to cyanocobalamin, C 8 or C 18 SPE cartridges

sample analyses, standard curve information, calculations, and completed reporting of analysis forms with dilution and sample weights. Participants were also asked to report any deviations to the method and any relevant comments based on their experiences with the method. All data were statistically analyzed using AOAC INTERNATIONAL guidelines to determine overall mean, repeatability SD (s r ), RSD r , reproducibility SD (s R ), RSD R , and Horwitz ratio (HorRat; 9). Cochran ( P = 0.025, one-tail) and Grubbs (single and double, P = 0.025, two-tail) tests were used to determine statistical outliers. Vitamin B 12 SPIFAN SMPRs for repeatability were ≤15% RSD at vitamin B 12 concentrations of 0.01 µg/100 g RTF liquid and ≤7% RSD at vitamin B 12 concentrations of 0.2–5.0 µg/100 g RTF liquid. Requirements for reproducibility were ≤11% RSD in products with vitamin B 12 concentrations ranging from 0.3 to 5.0 µg/100 g RTF liquid. A few minor modifications were made to AOAC Official First Action method 2011.10 before it was sent to the study participants in the collaborative study protocol. These changes included increasing the concentration of the sodium acetate buffer from 0.1 to 0.25 M, adding more RP column options, providing guidance for the preparation of samples containing free amino acids or no intact protein, and providing guidance for choosing appropriate size SPE cartridges. After completion of the study, the modifications noted above were incorporated in the Final Action method along with a few additional modifications based on study results and feedback from study participants and the ERP. In addition to the modifications listed above, procedures for safely handling potassium cyanide, qualifying SPE cartridges, and establishing appropriate elution gradients to adequately resolve vitamin B 12 on the RP column were added to the Final Action method. The option for using SPE cartridges smaller than 900 mg and a guidance for choosing appropriate size SPE cartridges were removed from the method since many laboratories may not have enough information about the samples that they are testing to use smaller size SPE cartridges. Method

to purify and concentrate cyanocobalamin, a combination of size-exclusion and RPLC to isolate cyanocobalamin, and visible absorbance at 550 nm to detect and quantitate cyanocobalamin. This method is applicable to the determination of vitamin B 12 , including cyanocobalamin, aquocobalamin, hydroxycobalamin, methylcobalamin, and adenosylcobalamin, in all forms of infant, adult, and pediatric formula (powders, RTF liquids, and liquid concentrates.) Initially 17 laboratories expressed interest in participating in the AOAC 2011.10 vitamin B 12 collaborative study, but only 11 laboratories were able to complete the study. The 11 participating laboratories were located in eight different countries. The remaining six laboratories were not able to participate because of time and resource constraints and issues with the importation of samples into their countries. Two of the participating laboratories only received partial sample shipments. Before actual multilaboratory collaborative study samples were analyzed, each participating laboratory was asked to analyze two practice samples in duplicate to identify and resolve any testing issues that the laboratories may have had executing the method. The practice samples included National Institute of Standards and Technology (NIST; Gaithersburg, MD) standard reference material (SRM) 1849a and a high protein adult nutritional RTF product. After approval of the practice sample results by the study directors, laboratories began testing the study samples. Blind duplicates of the 12 SPIFAN matrixes were shipped to each participating laboratory. The matrixes included SRM 1849a, an adult nutritional milk protein-based powder, an infant formula partially hydrolyzed milk-based powder, an infant formula partially hydrolyzed soy-based powder, an adult nutritional low-fat powder, a child formula powder, an infant elemental powder, an infant formula milk-based powder, an infant formula soy-based powder, an infant formula milk-based RTF liquid, an adult high-fat nutritional RTF liquid, and an adult high protein nutritional RTF liquid. Participants were asked to reconstitute all powders prior to analysis. SRM 1849a was reconstituted by dissolving the entire contents of the sachet (10 g) in 90 mL water. All other powders were reconstituted by dissolving 25 g powder in 200 mL laboratory water. Participants were asked to split the 24 test samples into two groups of 12 according to the data reporting sheets included in the protocol and to test each group on a separate day. Although the original AOAC 2011.10 First Action method allowed for the use of different size SPE cartridges, depending on the sample type being analyzed, collaborative study participants were required to test all samples with cartridges containing at least 900 mg resin since the study samples were blinded. Most laboratories used the 900 mg octylsilyl (C 8 ) or C 18 cartridges referenced in the method, but one participating laboratory used 1 g C 18 cartridges. Upon completion of the sample analyses, participating laboratories were asked to send all of their data to the study directors. This included all standard and sample chromatograms for the instrument check, practice sample analyses, test Multilaboratory Collaborative Study

AOAC Official Method 2011.10

in Infant and Pediatric Formulas and Adult Nutritionals HPLC

Vitamin B 12

First Action 2011 Final Action 2014

ISO-AOAC Method

(Applicable to the determination of vitamin B 12 pediatric formulas and adult nutritionals.) Caution : Refer to Material Safety Data Sheets of chemicals prior to use. Use the suggested personal protective equipment and follow good laboratory practices. Note : Potassium cyanide is highly toxic. When handling this chemical, wear gloves and appropriate personal protective equipment. Weigh chemical and dispense solutions in a fume hood. Perform test in a well-ventilated area. Treat sample waste with sodium hypochlorite and dispose of waste according to in infant and