AOAC SPIFAN ERP Meeting Book-March 16, 2016

2011.06 (Fol-22) w/SLV FOR ERP USE ONLY DO NOT DISTRIBUTE

Silliker Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis

a.) Pipette and appropriate amount of each folate stock solution (EXCPET THE INTERNAL STANDARD STOCK SOLUTION AND PTEROLYTRI-y-GLUTAMIC ACID) into a 10mL low-actinic volumetric flask. b.) Three separate solutions (one mixture and two individual solutions) are prepared as detailed in the following table. c.) Add approximately 5mL of Folate Intermediate Standard Solution Solvent and sonicate in water bath set at 40 o C for about 10min to thoroughly mix. d.) Bring to volume with Folate Intermediate Standard Solvent and store approximately -20 o C. Stable for up to 30days.

Table 3: Preparation intermediate standard solutions

Mixture Intermed or Individual std. Soln

Stock std. Concn (mcg/mL)

Stock std. Volume (uL)

Intermed std Soln #

Folate compd #

Final Volume* (mL)

Concen (mcg/mL)

Stock Standard

Shircks (6S)-5-Formyl-5,6,7,8- tetrahydrofolic acid

1

607

356

21.6092

2

10-Formylfolic acid

597

335

19.9995

Mixture Intermed Std. Soln

4

10-methylfolic acid

529

378

19.9962

I

10

Shircks (6R,S)-5,10-methenyl- 5,6,7,8-tetrahydrofolic acid

5

587

368

21.6016

Shircks (6S)-5,6,7,8- tetrahydrofolic acid

7

542

369

19.9998

Indiv. Intermed. Std soln Indiv. Intermed. Std soln Conjugase check soln

Shircks (6R,S)-5-methyl-5,6,7,8- tetrahydrofolic acid

II

3

584

371

10

21.6664

III

8

Shircks Folic acid

555

360

10

19.98

Conjugase check soln

6

pteroyltri-y-L-glutamic acid

911

222

10

20.2242

*Make up final volume to 10 mL with Folate Intermediate std. solvent.

7.) Internal Standard Intermediate Standard Solution (20ug/mL):

8