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

13 C 5

-FA

447.2 465.2

295.2 313.2

20 35

4.5

39

31 29

6 6

13 C 5

-5-CH3- THF

5

23.9

DP: Declustering potential; EP Entrance potential; CEP collision cell entrance potential; CE Collision cell; CXP collision cell exit potential.

The General MS parameters for API 5500 (Qtrap):

Curtain gas (nitrogen): 25 psi; Collision Assisted Dissociation: Medium Ion Spray voltage: 5500 V; Temperature: 700 °C; Ion source gas1 (zero air): 60 psi; Ion source gas2 (zero air): 50 psi;

Calculations :

Calculations and reporting

Ratio of the folate vitamer with respective internal standard is determined by HPLC-MS analysis. 5-CH3-THF, 5-CHO-THF and THF use labeled 5-CH3-THF as internal std. Folic acid and 10-CH3-Folic acid 10-CHO-Folic acid use labeled Folic acid as internal std. Calibration curve is drawn based on concentration of calibration standard of specific folate compound plotted against ratio of peak area (for the analyte) to the relevant internal standard. The calibrations curve are prepared for all the targeted folate compounds. The ratio peak areas of the folate compounds and respective internal standard is calculated in the standard sample which was processed like sample. Concentration of each of the folate compound as ng/mL in the standard processed like sample is calculated based on calibration curve. This is usually determined by the MS software (multquant). The ratio of peak areas of the detected folate compounds and respective internal standard is calculated in the blank which was processed like sample in the batch. Concentration of each of the detected folate compound as ng/mL is calculated in blank based on calibration curve. . Concentration of each of the folate compound as ng/mL in the standard processed like sample is adjusted by substracting the amount of respective folate compound detected in blank. Recovery factors (Rfs) for all the targeted folate compounds are determined based on adjusted concentration of respective analyte determined in standard processed like sample using the following relationship.

Rf = Adjusted concentration (ng/mL) of folate compound estimated in std. /amount of the specific folate compound expected to be present in the std.

The ratio peak areas of the folate compounds and respective internal standard is calculated in a sample. Concentration of each of the folate compound as ng/mL

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