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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