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Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analys
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The General MS parameters:
The MS parameters applicable for detection of all the analytes (all transitions) for Sciex 3200 in our
study are provided in the flowing section. The parameters worked out for Sciex 5500 and 6500 LC-
MS/MS are provided in the method.
Curtain gas (nitrogen):
30 psi;
Collision Assisted Dissociation: Medium
Ion Spray voltage:
5500 V;
Temperature:
400 °C;
Ion source gas1(zero air):
60 psi;
Ion source gas2(zero air):
50 psi;
Optimization of HPLC Conditions:
Selection of the Column which provides the 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 Shimadzu LC 20 HPLC 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
റ
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 Phases Evaluated:
Evaluation of the best Mobile Phase Buffer:
Different buffers were evaluated for best analytical response of different analytes in the scope in the
method.
AOAC method uses formic Acid in the mobile phase. 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 2). The following Table 8 below shows comparison of responses obtained for different
analytes with Formic and Acetic.
2011.06/Fol-22 (February 2016)
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