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Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analys
7
Table 2 : Analytical Response of different analytes with different mobile phase buffers
Mobile Phase
FA
5-CH3-
THF
5-CHO-
THF
THF
10-CH3-
FA
10-CHO-
FA
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 9 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 (Table 3).
Table 3: Response (peak area) of targeted folate compounds at 50 ppb levels as impacted by change
in Organic Phase of the HPLC mobile phase during the analysis
Organic Phase
FA
5-CH3-
THF
5-CHO-
THF
THF
10-CH3-
FA
10-CHO-
FA
Methanol
2.26E+04 1.19E+05
7.31E+04 4.69E+04 5.01E+04
9.43E+04
2.34E+04 1.13E+05
7.02E+04 4.88E+04 5.07E+04
9.69E+04
2.34E+04 1.18E+05
7.25E+04 4.93E+04 5.08E+04
9.81E+04
Average
2.31E+04 1.17E+05
7.19E+04 4.83E+04 5.05E+04
9.64E+04
%RSD
2.0%
2.8%
2.1%
2.6%
0.7%
2.0%
Acetonitrile
2.73E+04 9.73E+04
5.82E+04 4.63E+04 3.88E+04
8.16E+04
2.77E+04 9.44E+04
5.25E+04 5.08E+04 3.19E+04
8.24E+04
2.75E+04 8.24E+04
5.17E+04 4.80E+04 3.19E+04
8.34E+04
Average
2.75E+04 9.14E+04
5.41E+04 4.84E+04 3.42E+04
8.25E+04
2011.06/Fol-22 (February 2016)
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