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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 #1
Title:
Determination of Biotin in Infant, Pediatric and Adult Nutritionals by HPLC and Fluorescence
Detection
Author:
Abbott Nutrition, Columbus, OH. USA
Summary of Method:
This reverse phase HPLC method with post column protein conjugation and fluorescence detection
allows for the quantitative determination of biotin in infant, pediatric, and adult nutritionals.
Sample of appropriate size is mixed with 6% meta-phosphoric acid to precipitate out the protein to
produce a filtrate. The filtrate is injected onto a C18 HPLC column where biotin and riboflavin are separated
with a mobile phase (20% methanol in 0.02 M phosphate buffer at pH7.0). The 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.
Method Scope/Applicability:
Applicability of this method includes determination of Biotin in Infant, Pediatric and Adult Nutritionals.
General comments about the method:
The method is suitable to determine biotin in a short time with the used of LC-FLD methodology.
The basis of this method is the strong affinity between biotin and streptavidin.
Method Clarity:
The description of the method is clear and easy to follow.
Pros/Strengths:
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.
Cons/Weaknesses
The method requests appropriate sample size for analysis. If the concentration of the prepared
sample is higher than 60 ng/ml, a further dilution with mobile phase is required.
It is necessary to equilibrate the system before standard and sample injections with several
standards injections. The fluorescence intensity of the post column reaction end product did not
show a linear relationship with biotin concentration
Supporting Data
General Comment:
Bio-03 Review Forms