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FOR EXPERT REVIEW PANEL USE ONLY
NOT FOR DISTRIBUTION
1
Expert Review Panel for Infant Formula and Adult Nutrition
Evaluation of Method Bio-02
Title:
DETERMINATION OF BIOTIN BY LIQUID CHROMATOGRAPHY COUPLED WITH
IMMUNOAFFINITY COLUMN CLEAN-UP EXTRACTION
AOAC FIRST ACTION OFFICIAL METHOD 2016.02 - Multi laboratory Test
Author:
George Joseph - AsureQuality New Zealand
Summary of Method:
The sample is dispersed in phosphate buffered saline and autoclaved at 121 ±2⁰C for 25 minutes. 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. 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. Biotin from
the column is eluted with methanol. 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 1mL of water.
The biotin in the reconstituted sample is quantified by HPLC using a UV detector set at 200nm.
Method Scope/Applicability:
The study was carried out using all SPIFAN matrices.
Fourteen different product types were provided to each laboratory which includes two practice samples
and 12 samples for multi laboratory testing over two separate days. Two split samples of each matrix were
included in the package as blind coded duplicates to be analyzed on the same day.
General comments about the method:
The objective of the MLT study is to establish the precision and accuracy of the method in various
laboratories to demonstrate the suitability of the method.
The study was divided in two parts:
Part 1:
The participating laboratories were requested to analyze two practice samples in duplicate using the
method provided and report the results to the study director. It was communicated to the laboratories that
any deviation must be reported. Electronic templates were provided to the participants for data reporting.
Part 2: