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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3. Column and System Maintenance The column, post column reactor and system may be cleaned using 50% methanol at a lower flow rate.

G. CALCULATIONS Quantification is obtained by using a seven-level external standard consisting of the following concentrations 5, 10, 20, 40, 60, 80, and 100 ng/mL. The calibration of the standards is determined by using a polynomial regression curve (cubic-fit) on Agilent OpenLAB. The curve generated is presented in the figure below:

1. Calculation Scheme for Standards a) Stock Standard (SS) Concentration SS = S w x P y 500 x D 1

where: SS: Stock Standard Concentration ( P g/mL) S w : Standard weight (g) P: Purity (g/g) 500: volume of the stock solution in mL D 1 : Unit conversion factor, D 1

= 1,000,000 P g/g

Example: SS = 0.4986 g x 0.999 y 500 mL x 1,000,000 P g/g SS = 99.62 P g/mL b) Intermediate Standard (IS) Concentration = (volume of SS used ) x (SS concentration) y dilution volume Example: 1 mL x 99.62 P g/mL y 100 mL = 0.9962 P g/mL c) Working Standards Concentration Level 7 (WS7) = (volume of IS used in mL) x (concentration of IS in P g/mL) y (WS volume in mL) x 1000 ng/ P g