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Simultaneous Determination of Thiamine HCl, Riboflavin, Niacin (Nicotinic Acid and Nicotinamide),

Pantothenic acid, Vitamin B6 (Pyridoxine, Pyridoxal and Pyridoxamine) and Biotin in Infant

Formula and Adult Nutritionals

CONFIDENTIAL METHOD FOR ERP ASSESSMENT ONLY

PAGE 10 OF 13

IV. CALCULATION and REPORT

The LCMSMS software will automatically calculate the concentration of the sample in ng/mL for Thiamin

HCl, Riboflavin, Nicotinic Acid, Nicotinamide, Pantothenic acid, Pyridoxine, Pyridoxal, Pyridoxamine and

Biotin, provided the concentration of the working standards in ng/mL, sample weight / powder equivalent

(g) or sample volume (mL), dilution factor and conversion to 100g or 100 mL are entered correctly. Report

in 2 decimal places or as customer requirement.

100

1000

xDFx

W

CxV

))ml or(g 100 / mcg ( WSV .

Conc

Where:

C

=

Concentration of water-soluble vitamin from working curve (ng/mL)

V

=

Sample volume (ml)

= 100 mL

DF

=

Dilution Factor

= 1/0.5

W

=

Sample weight (2.78 g for powder or 15 g Liquid, or 1 g for SRM1849a) or sample

volume (15 mL)

1000

=

conversion factor from

ng to mcg

100

=

conversion factor from

g to 100 g or from mL to 100 mL

V. DISCUSSION

a. Niacin (mcg/100g(or mL)) = Nicotinamide(mcg/100g(or mL))+Nicotinic acid (ng/100g (or

mL))

b. Vitamin B6 (mcg/100g(or mL)) = Pyridoxine (mcg/100g(or ml)) + Pyridoxal (mcg/100g(or

ml)) + Pyridoxamine (mcg/100g(or mL))

c. Vitamin B6 (as Pyridoxine) (mcg/100g(or mL)) = Pyridoxine (mcg/100g(or ml)) + Pyridoxal

(mcg/100g(or ml))x(169.18/167.162) + Pyridoxamine (mcg/100g(or mL))x(169.18/168.19)

Where

Molecular weight of Pyridoxine = 169.18 g/mol

Molecular weight of Pyridoxal = 167.162 g/mol

Molecular weight of Pyridoxamine = 168.19 g/mol

d. If Thiamine (C

12

H

17

N

4

OS) is reported, Thiamine Hydrochloride (C

12

H

18

C

l2

N

4

OS) is

converted by multiplying with 0.787.

Thiamine (mcg/100g (or mL) = Thiamine HCl (mcg/100g (or mL)) x 0.787

Note : 0.787 = MW of Thiamine (265.4 g/mol)/MW of Thiamine HCl (337.3 g/ mol)

e. Comments:

Comment No.

1

The gradient given can be altered as required to maximize the analytical separation and

avoid interferences. This should only be done by an authorized analyst.

BVit-01

FOR ERP USE ONLY

DO NOT DISTRIBUTE