Review Team (February 2016)

Bio-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

ABBOTT NUTRITION DIVISION TITLE

PAGE NO.

Determination of Biotin in Infant, Pediatric and Adult Nutritionals by HPLC and Fluorescence Detection

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A. SAFETY ALL REAGENTS AND EQUIPMENT SHOULD BE HANDLED IN A MANNER CONSISTENT WITH SAFE LABORATORY PRACTICE. REAGENTS AND SAMPLES SHOULD BE TREATED WITH THE RESPECT AND CARE THAT ANY CHEMICAL REQUIRES. REAGENTS WHICH ARE ALLOWED TO COME IN CONTACT WITH THE SKIN, EYES, OR CLOTHING SHOULD BE FLUSHED IMMEDIATELY WITH LARGE VOLUMES OF WATER. IMMEDIATE MEDICAL ATTENTION SHOULD BE OBTAINED IF ANY REAGENT IS INGESTED OR ALLOWED TO COME IN CONTACT WITH THE EYES. THE REAGENTS USED IN THIS METHOD (META-PHOSPHORIC ACID, METHANOL) POSE HEALTH AND/OR FIRE HAZARDS. THOROUGHLY FLUSH ANY AREA COMING IN CONTACT WITH ANY REAGENTS USED IN THIS METHOD WITH WATER. SEEK QUALIFIED MEDICAL ATTENTION IMMEDIATELY SHOULD ANY REAGENT COME IN CONTACT WITH THE EYES OR SHOULD ANY REAGENT BE INGESTED. B. THEORY 1. Biotin is one of the most active biological substances known. It functions in the vitally important metabolic process of glucose and fat synthesis as a coenzyme. It acts as a CO 2 carrier in carboxylation, transcarboxylation and decarboxylation reactions. Biotin is also known as yeast growth factor, vitamin H, protective X factor, egg white injury factor, Bios II B, and coenzyme R. 2. Biotin is a monocarboxylic acid containing a cyclic urea structure with the sulfur atom in a thioether linkage. The chemical formula for biotin is C 10 H 16 O 3 N 2 S, its molecular weight is 244.31, and its structural formula is:

3. Biotin molecule has three asymmetric centers. However, of the eight possible stereo isomers, only the (+) isomer (d-biotin) has biological activity. Biotin is a white crystalline substance that, in dry form, is fairly stable to air, heat and light. In solution, however, it is sensitive to degradation under strong acidic or basic conditions. Biotin can withstand autoclaving in concentrated sulfuric acid (4M, 120 o C, 2 hours), condition commonly used to extract biotin from biological samples. Avidin, present in egg white, binds biotin and renders it unavailable to host, as does streptavidin produced by Streptomyces . 4. The basis of this method is the strong affinity between biotin and streptavidin. 5. This method is applicable to infant, pediatric and adult nutritional products. Sample of appropriate size, is mixed with 6% meta-phosphoric acid to precipitate out the proteins to produce a filtrate, which is subjected to subsequent HPLC analysis.