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

method. AOAC method uses formic Acid. We explored the use of formic acid and acetic acid at various concentrations and pH’s. The use of 1%Acetic Acid (approx. pH 2.8) gave the best response. Table 12 below shows comparison between Formic and Acetic.

Table 12 : Analytical Response of different analytes with different mobile phase buffers

5-CH3- THF

5-CHO- THF

10-CH3- FA 3.96E+05 4.01E+05 3.62E+05 3.86E+05 5.84E+05 6.29E+05 5.83E+05 5.99E+05 5.5%

10-CHO- FA 6.15E+05 6.00E+05 5.83E+05 5.99E+05 1.38E+06 1.36E+06 1.35E+06 1.36E+06 2.7%

FA

THF

Mobile Phase

2.22E+05 2.14E+05 2.18E+05 2.18E+05 3.79E+05 3.67E+05 4.15E+05 3.87E+05 1.8%

6.00E+05 6.07E+05 6.01E+05 6.03E+05 1.67E+06 1.67E+06 1.69E+06 1.68E+06 0.6%

6.54E+05 6.35E+05 6.32E+05 6.40E+05 1.39E+06 1.35E+06 1.31E+06 1.35E+06 1.9%

2.42E+05 2.61E+05 2.55E+05 2.53E+05 5.87E+05 5.89E+05 6.24E+05 6.00E+05 3.8%

10 mM NH4Frmt pH=2.8 Average 1% Acetic Acid Average %RSD %RSD

6.5%

0.7%

3.0%

3.5%

4.4%

1.1%

Evaluation of different solvents in the mobile phase to get higher analytical response of the analytes in the method: The analytical response of the targeted analytes was determined with different solvents as a part of the mobile phase. The following solvents were evaluated for the stated purpose. Methanol Acetonitrile AOAC method states the use of methanol as Mobile Phase B. Both solvents provide a good chromatography and a satisfactory resolution of the analytes. We determined the analytical response of the targeted analytes at 50 ppb level with either Methanol or Acetonitrile as a part of the mobile phase. Table 13 below shows comparison of peak areas when either one of these solvents were used as a part of the mobile phase. The analytes which are expected at lower concentrations in food samples , 5-methyl-THF, 5-formyl- THF and 10-methyly folic acid provided a higher analytical response with Methanol in the mobile phase in comparison when it was replaced by acetonitrile. Analytical response of other analytes was more or less same with both solvents.

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