6. AOACSPIFANMethods-2018Awards

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1110 J oseph et al .: J ournal of aoaC I nternatIonal V ol . 99, n o . 4, 2016 OFFICIAL METHODS

Determination of Biotin by Liquid Chromatography Coupled with Immunoaffinity Column Cleanup Extraction: Single- Laboratory Validation, First Action 2016.02 G eorGe J oseph and r anJani D evi AsureQuality Ltd, PO Box 41, Shortland St, Auckland 1140, New Zealand e laine C. M arley and D aviD l eeMan R-Biopharm Rhône Ltd, West of Scotland Science Park, Glasgow, Scotland G20 0XA

S takeholder P anel on I nfant f ormula and a dult n utrItIonalS E xpErt r EviEw p anEl for Spifan n utriEnt M EthodS Darryl Sullivan (Chair) , Covance Laboratories

Introduction

The AsureQuality Auckland Laboratory has initiated a method to facilitate a specific, precise, accurate, and robust procedure for the analysis of biotin from infant formula and adult/pediatric nutritional formulas (1-8). The method also has an assured limit of quantification of 0.1 μg/100 g (1 part per billion; ppb) based on a simple mathematical relationship between lowest standard and dilution. The method involves an immunoaffinity column (R-Biopharm Rhone, EASI-EXTRACT biotin column or equivalent) cleanup and extraction followed by LC–UV set at 200 nm.

John Austad , Covance Laboratories Sneh Bhandari , Silliker Laboratories Esther Campos-Gimenéz , Nestlé

Adrienne McMahon , Nestlé Scott Christiansen , Perrigo

Hans Cruijsen , FrieslandCampina Wil van Loon , FrieslandCampina Jon DeVries , General Mills/Medallion Laboratories Brendon Gill , Fonterra Don Gilliland , Abbott Nutrition Karen Schimpf , Abbott Nutrition Min Huang , Frontage Laboratories Estela Kneeteman , Instituto Nacional de Tecnología Industrial Maria Ofitserova , Pickering Laboratory Shay Phillips , Mead Johnson Guenther Raffler , Central Laboratories Friedrichsdorf–Eurofins Kate Rimmer , National Institute of Standards and Technology (NIST) Melissa Phillips , NIST David Woollard , Hill Laboratory Jinchuan Yang , Waters Corp.

AOAC Official Method 2016.02 Biotin Liquid Chromatography Coupled with Immunoaffinity Column Cleanup Extraction First Action 2016

A. Principle/Methodology

The sample is dispersed in phosphate-buffered saline (PBS) and autoclaved at 121 ± 2°C for 25 min. The sample is cooled to room temperature and then diluted to 100 mL in a volumetric flask. The extract is centrifuged and filtered using Whatman glass microfiber filter paper (GE Healthcare Life Sciences, Buckinghamshire, UK). Clear filtrate is collected for cleanup and extraction. The biotin immunoaffinity column is mounted onto an SPE manifold. A disposable syringe barrel is connected to the immunoaffinity column as a reservoir. The buffer in the affinity column is drained and the sample filtrate is loaded through the reservoir and allowed to flow through by gravity. The column is washed with PBS followed by water. Air is passed through the column to remove residual liquid. Biotin from the column is eluted with methanol and collected in a Reacti-Vial (Cat. No. 13223, Thermo Scientific). The eluent is evaporated to dryness using a heating block set at 85 ± 5°C under a gentle stream of nitrogen, and the sample is reconstituted in 1 mL water. The biotin in the reconstituted sample is quantified by HPLC–UV set at 200 nm.

B. Chemicals

Submitted for publication May 9, 2016. Adopted as a First Action Official Method SM by the Expert Review Panel on Biotin. Corresponding author’s e-mail: george.joseph@asurequality.com Approved March 17, 2016. DOI: 10.5740/jaoacint.16-0155

(a) Laboratory reagent-grade water. (b) Sodium dihydrogen phosphate dihydrate. (c) Disodium hydrogen phosphate dihydrate. (d) Sodium hydroxide. (e) Methanol. —HPLC grade.