hilic
63
hilic
High-performance liquid chromatography–tandem mass spectrometry
(HPLC-MS/MS) has become a powerful tool when detection sensitivity
is an issue. HILIC offers unique advantages for MS detection of very po-
lar compounds when compared to reversed phase mode. The higher or-
ganic content of the eluent in HILIC mode supports efficient evaporation
of the solvent thus enhancing sensitivity and altering ion suppression.
HILIC separations are performed with gradients starting with high per-
centage of organic solvent and ending with a high portion of aqueous
solvent - opposite to typical reversed phase gradients. The elution order
of compounds is usually inversed as well. As a result polar compounds
are very well separated according to increased polarity in HILIC mode.
At the same time the portion of organic solvent in the mobile phase is
relatively high.
Figure 6
shows the analysis of basic drug substances using a TSKgel
Amide-80 3 µm column compared to a reversed phase TSKgel ODS-100V
3 µm column. Ranitidine, a histamine H2 receptor antagonist, Ondanse-
tron, an antiemetic serotonin receptor antagonist, and Labetalol, an
alpha-1 and beta adrenergic blocker were selected to demonstrate the
differences in selectivity and MS-signal response when applying diffe-
rent chromatographic modes.
Ranitidine has the highest number of polar groups among these molecu-
les and as a result shows the highest retention in HILIC and the lowest
retention in RPC mode. Signal intensity is almost doubled for Ranitidine
in HILIC mode. For Labetalol a tenfold increase in signal height can be
achieved by using HILIC instead of RPC.
figure 6
LC-MS/MS Analysis of basic drugs in HILIC and RPC mode
Column: TSKgel Amide-80 3 µm (2.0 mm ID x 15 cm L)
Eluent : A: 10 mM Ammoniumformiate (pH 3.75); B: ACN
Gradient: 0 min (B 90%) -> 10 min (B 40%) ->13 min (B 40%)
Flow rate: 0.2 mL/min; Inj. volume : 5 µL (50 µg/L)
Detection : QTrap® LC-MS/MS (Applied Biosystems), ESI+
Column: TSKgel ODS-100V 3 µm (2.0 mm ID x 15 cm L)
Eluent: A: 10 mM Ammoniumformiate (pH 3.75); B: ACN
Gradient: 0 min (B 0%) -> 10 min (B 80%) ->13 min (B 80%)
Flow rate: 0.2 mL/min; Inj. volume: 5 µL (50 µg/L)
Detection: QTrap® LC-MS/MS (Applied Biosystems), ESI+
time (min)
0
2
4
6
8
0.0
1.0e5
intensity, cps
1.5e5
10
4.19
time (min)
0
2
4
6
8
0.0
1.0e5
intensity, cps
10
5.47
0
2
4
6
8
0.0
1.0e5
intensity, cps
1.6e5
10
4.67
time (min)
0
2
4
6
8
0.0
1.0e4
intensity, cps
1.5e4
10
7.23
time (min)
time (min)
0
2
4
6
8
0.0
5.0e4
intensity, cps
7.3e4
10
7.10
1.00e5
time (min)
0
2
4
6
8
0.0
5.00e4
intensity, cps
10
5.25
