109 / 670
ABBOTT NUTRITION DIVISION
TITLE
PAGE NO.
Determination of Biotin in Infant, Pediatric and Adult
Nutritionals by HPLC and Fluorescence Detection
9 of 11
Example: Level 7 (WS7) = 5 mL x 0.9962
P
g/mL
y
50 mL x 1000ng/
P
g = 99.62 ng/mL
d)
Working Standards (WS) Concentration Level 1-6
= (volume of WS7 used in mL) x (concentration of WS7 in ng/mL)
y
(WS volume in mL)
Example:
Level 1 (WS1) = 0.5 mL x 99.62 ng/mL
y
10 mL = 4.981 ng/mL
Level 2 (WS2) = 1 mL x 99.62 ng/mL
y
10 mL = 9.962 ng/mL
Level 3 (WS3) = 2 mL x 99.62 ng/mL
y
10 mL = 19.92 ng/mL
Level 4 (WS4) = 4 mL x 99.62 ng/mL
y
10 mL = 39.85 ng/mL
Level 5 (WS5) = 6 mL x 99.62 ng/mL
y
10 mL = 59.76 ng/mL
Level 6 (WS6) = 8 mL x 99.62 ng/mL
y
10 mL = 79.70 ng/mL
Level
Standard Concentration
(ng/mL)
HPLC peak area
1
4.981
0.2019
2
9.962
0.6600
3
19.92
2.2932
4
39.85
6.1743
5
59.76
9.2305
6
79.70
11.353
7
99.62
12.863
Polynomial regression: y = ax
3
+ bx
2
+ cx + d
y = peak area, x = concentration
a
-0.0000094
b
0.0007713
c
0.1482
d
-0.7451
R
2
=0.9994
2.
Calculation of biotin concentration in samples
a)
The biotin concentration in the injected product sample, C
i
, is calculated from its biotin peak area
and the standard curve generated from the standards.
b)
The original product concentration, C
p
, ( in
P
g/100g) is then calculated based on the dilution scheme
employed for the sample
C
p
= C
i
x 50
y
ss x D
1
x D
2
where C
p:
Original product concentration (
P
g/100g)
C
i
: Injected sample’s biotin concentration, from standard curve (ng/mL)
50: Dilution of original product prior to filtration in mL
Bio-03 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE