AOAC Final Action Methods in 2017

1112  C ampos G iménez & M artin : J ournal of AOAC I nternational V ol . 101, N o . 4, 2018

Infant Formula and Adult Nutritionals

Vitamin B 12 (Cyanocobalamin) in Infant Formula Adult/Pediatric Nutritional Formula by Liquid Chromatography with Ultraviolet Detection: Collaborative Study, Final Action 2014.02 E ster C ampos G iménez and F rédéric M artin Nestlé Research Center, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland Collaborators: F. Arella; J. Austad; S. Bandhari; S.H. Chooi; S. Christiansen; A. Chua; J.L. Deborde; M. Dole; J. Frueh; T. Gallegos; B. Gill; H. Indyk; G. Jaudzems; W.E. Jobgen; M. Jones; G. Joseph; E. Kneteeman; D. Labitan; G. Larkin; L. LeCours; H.X. Lim; K. Liu; I. Malaviole; E. Marley; A. McMahon; S. Meng-Jensen; S. Nielsen; R. Nijman; R.N. Nipales; T. Noorloos; C. Otto; A. Ruosh; B.L. Tan; G. Weerasekera; P. White; L.K. Yap

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V itaminB 12 is the termgenerallyused todesignate a family of cobalamins showing the same biological activity. Among them, hydroxycobalamin, adenosylcobalamin, and methylcobalamin are the major naturally occurring forms. Because of its greater stability, cyanocobalamin is the form used in food fortification. Daily vitamin B 12 requirements vary with age and gender, from 0.4 μg in infants (0–6 months) to 2.4 μg in adults, and those requirements increase during pregnancy and lactation up to 2.8 μg (1). Codex has established a lower limit of 0.1 μg/100 kcal and a guidance upper level of 1.5 μg/100 kcal in infant formula and foods for specialmedical purposes (2). SeveralAOAC OfficialMethods SM exist for the analysis of vitamin B 12 in foods and, in particular, in infant formula (3).All of them require heat treatment in the presence of cyanide to convert all native forms into cyanocobalamin, which is then detected and quantified in the food extract by using microbiological assay (4), surface plasmon resonance (5), or liquid chromatography (6–9). In the current International Organization for Standardization method (10), vitamin B 12 is extracted at pH 4.5 in the presence of cyanide at 105°C. Extracts are then purified and concentrated by C 8 or C 18 solid-phase extraction. The determination of cyanocobalamin is made by two-dimensional liquid chromatography with visible detection at 550 nm. In 2008, we published a method applicable to the quantification of vitamin B 12 in fortified food products by using an immunoaffinity clean up prior to reversed-phase liquid chromatography followed by ultraviolet detection. Among the matrices validated were infant formula and nutritional products (11). The method was proposed to the Stakeholder Panel for Infant Formula and Adult Nutritionals (SPIFAN) and was approved as a First Action Official Method 2011.08 method in 2011 (12). An improvement of the method in terms of LOQ and faster chromatography was proposed in 2014 and single validation was published (13). The method was granted AOAC First Action 2014.02 . In an effort to collect reproducibility data, a collaborative study of the method was organized; this paper presents the results of such study.

To determine the repeatability and reproducibility figures of the AOAC First Action Official Method  SM 2014.02 ( Vitamin B 12 in Infant Formula and Adult/ Pediatric Formula by Liquid Chromatography with UV Detection ), a collaborative study was organized. Twenty-one laboratories located in 13 different countries agreed to participate. The study was divided into two parts. During the first part, the laboratories analyzed two samples in duplicate by using the method described in the protocol. The laboratories that provided results within the expected range were qualified for part two, during which they analyzed 10 samples in blind duplicates. Eighteen laboratories managed to provide results on time for reporting. The results were compared with the Standard Method Performance Requirement (SMPR ® 2011.005) established for vitamin B 12 . The precision results met the requirements stated in the SMPR except for one sample. Repeatability and reproducibility relative standard deviation ranged from 1.1 to 6.5% and from 6.0 to 23.8%, respectively, with only one matrix showing reproducibility values higher than the required 11%. Horwitz ratio values were all well below 2 (0.17–0.78). The AOAC Expert Review Panel (Stakeholder Panel for Infant Formula and Adult Nutritional Expert Review Panel) determined that the data presented met the SMPR and, hence, recommended the method to be granted Final Action status in September 2016. Received June 12, 2017. Accepted by SG September 13, 2017. The method was approved by the AOAC Official Methods Board as Final Action. See “Standards News,” (2017) Inside Laboratory Management, July/August issue. The AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) invites method users to provide feedback on the Final Action methods. Feedback from method users will help verify that the methods are fit for purpose and is critical to gaining global recognition and acceptance of the methods. Comments can be sent directly to the corresponding author. Corresponding author’s e-mail: esther.campos-gimenez@rdls. nestle.com

Samples

The study took place using SPIFAN matrices representing most of the products in the scope of the project (Infant Formula andAdult Nutritionals made from any combination of milk, soy,

DOI: https://doi.org/10.5740/jaoacint.17-0452