Review Team (February 2016)

Bio-02 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

APPENDIX 1 DETERMINATION OF BIOTIN BY LIQUID CHROMATOGRAPHY COUPLED WITH IMMUNOAFFINITY COLUMN CLEAN-UP EXTRACTION George Joseph, Ranjani Devi and Raj Naganaboyina AsureQuality Ltd, PO Box 41, Shortland Street, Auckland 1140, New Zealand Elaine C. Marley and David Leeman R-Biopharm Rhône Ltd, West of Scotland Science Park, Glasgow, Scotland G20 0XA Corresponding author’s email: george.joseph@asurequality.com 1. INTRODUCTION 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. The method also has an assured limit of quantification of 0.1μg/100g (1ppb) based on a simple mathematical relationship between lowest standard and dilution. The method involves immunoaffinity column (R-Biopharm Rhone, EASI- EXTRACT biotin column or equivalent) clean-up and extraction followed by liquid chromatography with UV detection at 200nm. 2. PRINCIPLE / METHODOLOGY The sample is dispersed in phosphate buffered saline and autoclaved at 121±2 ͼ C for 25 minutes. The sample is cooled to room temperature and then diluted to 100mL in a volumetric flask. The extract is centrifuged and filtered using a Whatman glass microfiber filter paper. Clear filtrate is collected for clean-up and extraction. Biotin immunoaffinity column is mounted onto a 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 phosphate buffered saline 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. 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 re-constituted in 1mL of water. The biotin in the reconstituted sample is quantified by HPLC using a UV detector set at 200nm.

3. CHEMICALS 1. Laboratory Reagent Grade Water

2. Sodium Dihydrogen Phosphate Dihydrate 3. Disodium Hydrogen Phosphate Dihydrate 4. Sodium Hydroxide

5. Methanol, HPLC grade 6. Acetonitrile, HPLC grade 7. Ortho-phosphoric acid, 85% 8. Phosphate buffered saline (PBS) pH 7.4 (Life Technologies 100100.31 or equivalent) 9. Biotin, purity ≥ 99% (Sigma B4501 or equivalent)

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