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

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

Max. 4.7e4 cps.

Intensity, cps

6.14

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

Figure 5: Folate Chromatograms with sodium ascorbate.

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 13 C 5 -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 ( 13 C 5 -FA and 13 C 5 -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 13 C 5 -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.

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