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

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

Mobile Phase

5-CH3-

THF

5-CHO-

THF

10-CH3-

10-CHO-

10 mM

NH4Frmt

pH=2.8

2.22E+05

6.00E+05

6.54E+05

2.42E+05

3.96E+05

6.15E+05

2.14E+05

6.07E+05

6.35E+05

2.61E+05

4.01E+05

6.00E+05

2.18E+05

6.01E+05

6.32E+05

2.55E+05

3.62E+05

5.83E+05

Average

2.18E+05

6.03E+05

6.40E+05

2.53E+05

3.86E+05

5.99E+05

%RSD

1.8%

0.6%

1.9%

3.8%

5.5%

2.7%

1% Acetic

Acid

3.79E+05

1.67E+06

1.39E+06

5.87E+05

5.84E+05

1.38E+06

3.67E+05

1.67E+06

1.35E+06

5.89E+05

6.29E+05

1.36E+06

4.15E+05

1.69E+06

1.31E+06

6.24E+05

5.83E+05

1.35E+06

Average

3.87E+05

1.68E+06

1.35E+06

6.00E+05

5.99E+05

1.36E+06

%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.

2011.06 (Fol-22) w/SLV

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