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ABBOTT NUTRITION DIVISION
TITLE
PAGE NO.
Determination of Biotin in Infant, Pediatric and Adult
Nutritionals by HPLC and Fluorescence Detection
5 of 11
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
Bio-03 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE