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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Weigh 27.800 (±2.780) g of sodium phosphate monobasic into a 1 liter volumetric flask. Add about 800 mL of laboratory water, mix until all solids dissolve. Dilute to volume with laboratory water. Mix well. Expiration: 1 month at room temperature. b) 0.2 M Sodium Phosphate Dibasic (SPD) Weigh 28.390 (±2.840) g of sodium phosphate dibasic into a 1 liter volumetric flask. Add about 800 mL of laboratory water, mix until all the solids dissolve. Dilute to volume with laboratory water. Expiration: 1 month at room temperature. c) 0.02 M Phosphate Buffer - pH 7.0 Place a 400 mL beaker on a stir plate. Using graduated cylinders, transfer 78 mL of 0.2 M sodium phosphate monobasic (SPM) and 168 mL of 0.2 M Sodium phosphate dibasic (SPD) into the beaker. Adjust pH of the solution in the beaker to 7.00 (±0.05) using SPM and SPD. This solution is 0.2 M phosphate buffer (pH 7) and is made fresh for mobile phase preparation. Using a graduated cylinder, transfer 200 mL of the 0.2 M phosphate buffer (pH 7) into a 2 liter volumetric flask. Dilute to volume with laboratory water and mix well. This is 0.02 M phosphate buffer (pH 7). Expiration: 1 week at room temperature. d) Biotin Mobile Phase Using a graduated cylinder, transfer 400 mL of methanol into a 2 liter volumetric flask. Bring to volume with the 0.02 M phosphate buffer (pH 7). Mix well, filter, and degas in sonicator for 5 minutes. Transfer to a suitable mobile phase container. Expiration: 1 week at room temperature. e) 6% Meta Phosphoric Acid Weigh 60 (±6) g of meta-phosphoric acid into a 1 liter beaker. Add about 800 mL of laboratory water and mix until all solids dissolve. Quantitatively transfer the solution to a 1 liter volumetric flask, dilute to volume with laboratory water and mix well. Store refrigerated. Expiration: 1 week. f) 50% Ethanol Using a graduated cylinder, transfer 250 mL of ethanol to a 500 mL volumetric flask. Bring this to volume with laboratory water. Expiration: 1 month at room temperature. g) 50% Methanol Using a graduated cylinder, transfer 500 mL of methanol into a 1 liter volumetric flask. Bring this to volume with laboratory water. Expiration: 1 month at room temperature. h) Post Column Reagent Using a volumetric pipette, transfer 0.8 mL of streptavidin fluorescein into a 1 liter volumetric flask and dilute to volume with biotin mobile phase. Expiration: 1 week at room temperature.

3. Standards a) Biotin Stock Standard Solution, 100 P g/mL