Review Team (February 2016).pdf

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AsureQuality Laboratory Services

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

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.

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)

Bio-02 (February 2016)

FOR ERP USE ONLY

DO NOT DISTRIBUTE