AOAC SPIFAN ERP Reviewer Forms (March 16, 2017)

FOR EXPERT REVIEW PANEL USE ONLY NOT FOR DISTRIBUTION

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. 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: General comments about the method:

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