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Silliker
Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis
20
Table 13: 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
%RSD
0.7%
8.6%
6.5%
4.7%
11.6%
1.1%
ACN/MeOH
118.9%
78.3%
75.3%
100.1%
67.7%
85.5%
HPLC Conditions: Flow: 0.5 mL/min; Column Temperature: 40
°
C
All the HPLC columns listed above (pages 15-16) were evaluated using different and relevant mobile
phase combinations. Waters ACQUITY UPLC HSS T3 Column (100Å, 1.8 µm, 2.1 mm X 50 mm)
with 1% acetic acid and methanol as mobile phase with a varied flow rate provided the best sensitivity
and separation. The gradient is shown below:
The Pumping gradient of different solvents and flow at different time
Time (min)
%B
Flow
(mL/min)
0
0
0.5
0.5
0
0.5
5.00
40
0.5
5.05
100
0.5
6.50
100
0.5
6.55
0
0.5
9.00
0
0.5
Investigation of different Injection Solvents:
Various solvent mixture were evaluated for their effect on chromatography and resolution of the
analytes. The most important solvent mixture tried are mixtures A and G and are listed in the following
section. The solvent A is used for elution of the analytes during solid-phase extraction. We tried a step
of the evaporation of the extract concentrated and purified by SPE to improve sensitivity of the method.
The extract residue following the evaporation is reconstituted in the following solvent mixture G which
is some what similar to the HPLC mobile phase used in the analysis.
A: 90% MeOH + 10% Formic acid + 1% ascorbic acid
2011.06 (Fol-22) w/SLV
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