AOAC SPIFAN Review Team (August 2016)

LC-MS/MS Analysis of Nicotinic acid

and Nicotinamide

Chemistry Method

Version 2

Page 9/15

Prepared by

S. Bhandari

Approved by

Issue Date

Implementation Date

________________________________________________________________________________

Uncontrolled When Printed

e) Mobile Phase B

—

100% Acetonitrile

4.7

Procedure:

4.7.1 Sample Preparation.

Samples should be completely homogenous and well blended . NIST

1849a can be prepared as a slurry before the analysis 25 g and 200 mL of water and

mixed to uniform mixture.

4.7.2 Sample Extraction

1. Weigh approximately 0.2g

–

2.3 g in 50mL Centrifuge tube

2. Add 10mL Extraction Buffer (12mM Ammonium Formate pH 5.0)

3. Sonicate 30min (quick vortex every 10 min)

4. Add 0.1mL Protease Solution (50mg/mL)

5. Incubate in 60

C shaking water bath 30min

6. Boil 10min ; cool to R.T

7. Add 1mL Takadiastase Solution (1% in Extraction Buffer)

8. Incubate 3Hrs @ 45

C shaking water

9. Boil 5min; Cool to R.T

10. Bring to Final Volume 40mL with Extraction Buffer

11. Centrifuge 5min 8500rpm

12. PVDF filter into 16x25 disposable glass culture tube

13. Pipette 1mL into HPLC vial and add 30u: of IS Mix

14. Inject 2uL on LCMS/MS

4.7.3 Method Blank

: Process 0.5 mL of water just like sample as described

in the section 4.7.2.

4.7.4 Standard as a Sample

: Process 0.8 mL of mixed intermediate

standard just like a sample as described in the section 4.7.2.

4.7.5 Analysis of the Calibration, Blank, standard as a sample and sample

solutions:

Analyze by HPLC MS/MS.

4.8 LC-MS/MS Analysis

4.8.1. LC Analysis:

METHOD

FOR ERP USE ONLY

DO NOT DISTRIBUTE