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Silliker
Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis
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Ion source gas1(zero air):
60 psi;
Ion source gas2(zero air):
50 psi;
Peak Area Ratio
The peak area ratio of the two MRM transitions of each analyte was determined by the average value of
those at difference concentration levels
( Table ). All of the following data, including peak area and
calculated concentration, are based on the quantification (more intense MRM transitions).
Table 11: Peak Area Ratios of the six analytes in solvent
.
Optimization of HPLC Conditions:
Selection of the Column provides best performance:
Attempts were made to get the best resolution possible of the analytes in scope. A series of HPLC
column were selected for evaluation purposes based on the AOAC method and the available literature.
We used HPLC (Shimadzu LC 20) for this study. The Medallion used Waters UPLC in developing the
AOAC method. The column used by the AOAC method
is Waters ACQUITY UPLC HSS T3 Column,
100Å, 1.8 µm, 2.1 mm X 150 mm. The following columns were evaluated by us for the best possible
resolution of the analytes in scope.
Poly LC PolyHYDROXYETHYLA
™
200x 2.1 mm, 5 um
Agilent Zorbax Eclipse xdb-c8, 4.6x150 mm, 3.5 um
Phenomenex Kinetex C18, 2.1x50 mm, 1.7 um
Phenomenex Kinetex HILIC, 2.1x50 mm, 1.7 um
The Nest Group Zic HILIC 2.1x100 mm, 3.5 um
Waters ACQUITY UPLC BEH C18, 2.1 mm X 50 mm, 1.7 µm
Waters ACQUITY UPLC HSS T3 Column, 100Å, 1.8 µm, 2.1 mm X 50 mm
Thermo Hypersil GOLD aQ, 2.1 mm X 50 mm, 1.9 µm
2. Mobile Phase Tested:
Evaluation of the best Mobile Phase Buffer:
Different buffers were evaluated for best analytical response of different analytes in the scope in the
Conc (ng/mL)
FA
5-CH3-THF 5-CHO-THF
THF
10-CH3-FA 10-CHO-FA
1
2.7853
3.0872
4.1603
1.7700
1.8942
1.9322
2
3.0791
3.4956
5.3346
1.4988
1.8534
2.0930
5
1.9507
3.4167
4.4296
2.5811
1.9350
1.7459
20
2.0907
3.7024
4.4652
2.3251
2.1522
1.6303
50
2.1094
3.9070
4.0784
2.4488
1.8941
1.7760
200
2.1657
4.0045
4.1413
2.2653
2.0275
1.7895
500
2.2014
3.9217
4.4295
2.1387
2.0199
1.7018
Average
2.3403
3.6479
4.4341
2.1468
1.9680
1.8098
2011.06 (Fol-22) w/SLV
FOR ERP USE ONLY
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