Review Team (February 2016)

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

ABBOTT NUTRITION DIVISION TITLE

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

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Weigh 0.05000 (±.0050) g USP Biotin reference standard. Quantitatively transfer the standard to a 500 mL volumetric flask using 50% ethanol. Mix well. Transfer to a 500 mL amber bottle. Store refrigerated. Expiration: 3 months. b) Biotin Intermediate Solution, 1000 ng/mL Using a volumetric pipette, transfer 1 mL of the biotin stock solution into a 100 mL volumetric flask. Bring to volume with lab water. Mix well. Expiration: make fresh on day of use. c) Biotin Working Standards: 5, 10, 20, 40, 60, 80 and 100 ng/mL For working standard 100 ng/mL, use a volumetric pipette to transfer 5 mL of the biotin intermediate solution into a 50 mL volumetric flask. Bring to volume with biotin mobile phase and mix well. For working standard 5, 10, 20, 40, 60 and 80 ng/mL, use volumetric pipettes to transfer 0.5, 1, 2, 4, 6 and 8 mL of the 100 ng/mL working standard into 10 mL volumetric flasks, respectively. Bring the volumetric flasks to volume with biotin mobile phase and mix well. Expiration: make fresh on day of use. E. PROCEDURE It is recommended that all samples be prepared under UV shielded fluorescent lights (UV cutoff at 385 nm). 1. Sample Reconstitution Accurately weigh up to 4 grams of powder or up to 20 grams of liquid products in a 50 mL volumetric flask. The final concentration of biotin in the 50 mL solution should be in the range of 8 – 60 ng/mL. Add 20 mL of water to reconstitute powder samples or add appropriate amount of water to bring up to a total volume of 24 mL for liquid products. Swirl to mix well. 2. Sample Preparation a) Add 10 mL of 50% methanol into each sample. Swirl to mix well. b) Add 7.5 mL of 6% meta-phosphoric acid into each sample. Immediately swirl to mix well. c) Bring the volumetric flask to volume with lab water. Invert the flask to mix well. d) Transfer approximately 2 mL of each sample into a microcentrifuge tube. Centrifuge at 10,000 x g for 10 min. e) Filter the supernatant of each sample through syringe filter into an HPLC autosampler vial.

F. INSTRUMENT SET-UP 1. Instrumental Operating Conditions

a) Fluorescence detector wavelength parameter: Ex 495 nm, Em 518 nm. b) Run time: 15 min. c) Injection volume: 20 P L d) Column temperature: 20 °C