AOAC SPIFAN Review Team Meeting Book-July 22, 2015

Silliker

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

Total Process Effect:

It was found that all six folate compounds behaved differently while going through the whole sample

processing procedure for extraction purposes including enzymatic reaction and SPE purification.

Labeled folic acid was used as internal std. not only for folic acid but also for 10-CH3-FA and 10-

CHO-FA and

-5-CH3-THF was used as internal std. not only for 5-CH3-THF but also for 5-

CHO-THF and THF estimation by the method. The measured concentrations of Folic acid and 5-CH3-

THF based on internal standard calculation against solvent standard curve matching closely to the

target values as there were isotopic labeled internal standards for both of these measured folate

compounds (

-FA and

-5-CH3-THF respectively) to compensate for the process and stability

loss and the impact of matrix on ionization during MS analysis. However, the compensations for the

other four analytes (labeled folic acid used internal std. for 10-CH3-FA and 10-CHO-FA whereas 5-

CHO-THF and THF used

-5-CH3-THF as internal std.) did not function as good as Folic acid and

5-CH3-THF especially for THF, which is shown in

Table 3

in triplicates. The reason for this

observation may be that all folate compounds differ significantly in their stability during the sample

processing required for the analysis. Therefore, it is necessary that the calibration standard(s) used for

the calculation is(are) subjected to the same treatment as the sample so that impact of the sample

processing treatment on stability of individual folate compound can be compensated in the estimation

by the method.

Intensity,

cps

Max. 4.7e4

cps.

4.0

4.2

4.4

4.6

4.8

5.0

5.2

5.4

5.6

5.8

6.0

6.2

6.4

6.6

6.8

7.0

7.2

7.4

7.6

7.8

8.0

Time, min

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

6.14

Figure 5: Folate Chromatograms with sodium ascorbate.

2011.06 (Fol-22) w/SLV

FOR ERP USE ONLY

DO NOT DISTRIBUTE