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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-03 #2
Title:
Determination of Biotin in Infant, Pediatric and Adult Nutritionals by HPLC and Fluorescence Detection
Author:
Abbott
Summary of Method:
Sampleis mixed with meta-phosphoric acid to precipitate out the protein with filtrate is injected onto a C18
HPLC column where biotin and riboflavin are separated. After elution biotin after eluting from the column
binds with the streptavidin fluorescein to become a fluorescent conjugate. The conjugate is then
detected by fluorescence at an excitation wavelength of 495 nm and an emission wavelength of 518 nm.
A column switch is used in the method to shorten the run time from 30 min to 15 min, by eluting out
riboflavin at a higher flow rate.
Method Scope/Applicability:
Infant Formula and Adult/Pediatric Formulas
General comments about the method:
Well establish technique based on the high affinity of streptavidin for biotin
Method Clarity:
Clear and well written method description.
Method Safety Concerns:
Pros/Strengths:
Cons/Weaknesses
Non-linear response of formation of biotin-streptavidin complex.
Column switching not used in all labs.
Supporting Data
Values obtained for NIST 1849a are acceptable against certified values.
SPIFAN kit was used in the validation of the method.
Performance Characteristics:
Analytical Range:
1.66-142 mcg/100g reconstituted final product
Bio-03 Review Forms