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Silliker
Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analys
8
%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: 0.2-0.3 mL/min; Column Temperature: 40
r
C
All the HPLC columns listed above 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.
Mobile Phase: A: 1% acetic acid and B: MS grade methanol. The mobile phase components are
varied in different ratios with a varied flow rate as detailed in the following section.
Time (min)
%B
Flow
(mL/min)
0.10
0.1
0.2
1.00
0.1
0.2
8.00
100
8.01
0.2
8.02
0.3
10.00
100
10.10
0.1
11.00
0.3
11.01
0.2
12.00
0.1
15.00
(Stop)
A = 1%Acetic Acid in Water
B = 100% Methanol
Column Temperature: 40
r
C
Autosampler Temperature: 6
r
C
01112
Injection Volume = 2 uL
These HPLC conditions were revised to provide a similar or better chromatographic separation of the
targeted folate compounds and response using Dionex Ultimate 3000 LC or Agilent 1290 Infinity LC
with the detector Sciex 5500 or 6500 LC-MS/MS systems. The revised conditions are the following
and also listed in the method
.
LC Column: Waters ACQUITY UPLC HSS T3 Column (100Å, 1.8 μm, 2.1 mm X 50 mm).
Mobile Phase: A: 1% acetic acid and B: MS grade methanol as mobile phase components in different
ratios at different time of the analysis. The Pumping gradient of two solvents at different time is as the
following.
2011.06/Fol-22 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE