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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 #2
Title:
Determination of Biotin by HPLC coupled with EASI-EXTRACT Biotin
Immunoaffinity column cleanup extraction
Author:
George Joseph, Ranjani Devi, Raj Naganaboyina, AsureQuality
Summary of Method:
1.
Biotin extraction with sodium phosphate buffer, pH 7, and autoclaving at 121°C for 25 minutes.
2.
Sample cleanup and concentration with immunoaffinity cleanup columns
3.
Gradient HPLC analysis with Kinetex phenyl hexyl solid-core column and UV detection at 200 nm.
Method Scope/Applicability:
Method applicable for use with infant, adult, and pediatric formulas.
General comments about the method:
Because of the use of immunoaffinity columns the method is specific for biotin.
Method Clarity:
Overall method document was clear and easy to follow, but may need a little more detail and some
clarification. It would be helpful to explain how to back flush the column three times when eluting the
sample and system suitability parameters would be helpful.
Pros/Strengths:
•
1.
Method should specific for biotin because of the use of a monoclonal antibody.
2.
At least two immunoaffinity column suppliers were identified – R-Biopharm Biotin Easy Extract and
BioTeZ Immunoaffinity columns Berlin-Buch Germany- technology not proprietary.
Cons/Weaknesses
•
Although samples are autoclaved during the sample preparation procedure, they are autoclaved in a
pH 7 phosphate saline buffer which would not be expected to release bound biotin. For total biotin
analyses samples are typically autoclaved in sulfuric acid or hydrolyzed enzymatically.
Supporting Data
•
General Comment:
Bio-02 Review Forms