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LC-MS/MS Analysis of Nicotinic acid

and Nicotinamide

Chemistry Method

Version 2

Page 8/15

Prepared by

S. Bhandari

Approved by

Issue Date

Implementation Date

________________________________________________________________________________

Draft Date: 3/2/16 Copyright © Merieux NutriSciences, 2016

Uncontrolled When Printed

Std Level

Final Concen.

(ng/mL, ppb)

Vol. of Std (mL)

Internal Std Mix

Solution (IS-

Mix) (mL)

Final Concen.

(ng/mL, ppb)

6

175

1.00

0.030

175

5

100

1.00

0.030

100

4

45

1.00

0.030

45

3

17.5

1.00

0.030

17.5

2

5.0

1.00

0.030

5.0

1

0.9

1.00

0.030

0.9

The net concentration of different internal standards in the calibration standards is

provided in the following Table.

The net concentration of different nicotinic acid and nicotinamide internal standards in

the calibration standards are as the following.

Name

ppb

(ng/mL)

Added to

each

Calibration

std. (mL)

Internal std.

final conc. in

each calibration

std. (ng/mL)

Nicotinic Acid

13

C

3

1000.0

0.03

30.0

Nicotinamide

13

C

3

4000.0

120.0

4.6

Reagent Solutions and mixtures

a)

Extraction Buffer 12.0 mM Ammon

ium Formate; pH 5.0

.

In a 1L Solvent reservoir

bottle add 0.771g Ammonium formate and 1L Milli-Q water. Stir until dissolved completely. Adjust

pH with formic acid to pH 5.0. (

1-2 drops

)

b)

Pro

Protease Solution

50mg/mL. Weigh 100 mg of protease in a test tube and add 2 mL of

water. Gently mix to dissolve/suspend and make sure no foaming. 0.1mL/sample needed. Make

fresh for each batch.

c)

T

akadiastase

weigh 3g of taka-diastase and add 30ml 10mM ammonium formate pH 5.0

extraction buffer (1mL/sample needed) Make fresh each batch.

d) Mobile Phase A 12.0 mM Ammonium Formate; pH 3.0

.

I

n a 1L Solvent reservoir bottle add

0.771g Ammonium formate and 1L Milli-Q water. Stir until dissolved completely. Adjust pH with

formic acid to pH 3.0

METHOD

FOR ERP USE ONLY

DO NOT DISTRIBUTE