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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
2016.02 MLT
Text in
RED
: SKC review for First Action, 3/2016.
Text in
BLUE
: SKC review for Final Action, 3/2017.
Title:
DETERMINATION OF BIOTIN BY LIQUID CHROMATOGRAPHY COUPLED WITH
IMMUNOAFFINITY COLUMN CLEAN-UP EXTRACTION
Author:
George Joseph, Ranjani Devi and Raj Naganaboyina
AsureQuality Ltd, PO Box 41, Shortland Street, Auckland 1140, New Zealand
Summary of Method:
A portion of sample is dispersed in phosphate buffered saline(pH 7.4) and autoclaved at 121±2⁰C for 25
minutes, cooled to room temperature and diluted to 100mL. The extract is clarified (centrifuged, filtered)
and a portion of the resulting solution loaded to a Biotin immunoaffinity column mounted in an SPE
manifold. The SPE column is washed w PBS then biotin is eluted with methanol, which is evaporated, and
the residue re-constituted in water.
A portion of the resulting sample preparation is then injected onto a reversed-phase HPLC system using an
isocratic separation (0.1% phosphoric acid / acetonitrile) with a column wash performed after elution of
the peak of interest. Detection is by UV at 200nm. Quantification is by external standard against a multi-
point standard curve. The method does not specify whether peak height or peak area is used.
Method Scope/Applicability:
Updated 3/2017: The SMPR refers to the analysis of “total biotin”. The fortificant used in formulated
nutritional products is free biotin. Biotin in foods may be covalently bound to L-lysine, as biocytin, or to
proteins. The method used for the SLV addressed only free biotin.
The method used for the MLT was updated to include quantification of biocytin: a separate standard is
prepared and its response is monitored. The conditions of the separation are unchanged.
Biocytin elutes
early in the run and is positioned atop a baseline disturbance.
General comments about the method:
Method Clarity:
Sample Preparation, Table 1, specifies the mass of product to process based on the expected biotin content
in “ug/100g”; the values appear to be “as fed” or “as reconstituted” (rather than “as powder e.g.”); the
basis of the expression should be stated.
Updated 3/2017: The 3/2016 review of the Bio-02 method listed several points requiring clarification.
These have been largely addressed in the method which accompanied the MLT: