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