A Luna HILIC column (2.0 ×
50 mm, 200 Å, Phenomenex, Torrance, CA) was used to separate
the LMWHs. Mobile phase A was 5 mM ammonium acetate prepared with
HPLC grade water. Mobile B was 5 mM ammonium acetate prepared in 98%
HPLC grade acetonitrile with 2% of HPLC grade water. The gradient
was used from 5% A to 70% A in 7 min then reset to 5% A at a flow
rate of 250 μL/min. The LC column was directly connected online
to the standard ESI source of LTQ-Orbitrap XL FT MS (Thermo Fisher
Scientific, San-Jose, CA). The source parameters for FTMS detection
were optimized using Arixtra (a synthetic ultra LMWH from Sanofi-Aventis,
Paris, France) to minimize the insource fragmentation and sulfate
loss and maximize the signal/noise in the negative-ion mode. The optimized
parameters, used to prevent in-source fragmentation, included a spray
voltage of 4.2 kV, a capillary voltage of −40 V, a tube lens
voltage of −50 V, a capillary temperature of 275 °C, a
sheath flow rate of 30 L/min, and an auxiliary gas flow rate of 6
L/min. External calibration of mass spectra routinely produced a mass
accuracy of better than 3 ppm. All FT mass spectra were acquired at
a resolution 60 000 with 200–2000 Da mass range.
50 mm, 200 Å, Phenomenex, Torrance, CA) was used to separate
the LMWHs. Mobile phase A was 5 mM ammonium acetate prepared with
HPLC grade water. Mobile B was 5 mM ammonium acetate prepared in 98%
HPLC grade acetonitrile with 2% of HPLC grade water. The gradient
was used from 5% A to 70% A in 7 min then reset to 5% A at a flow
rate of 250 μL/min. The LC column was directly connected online
to the standard ESI source of LTQ-Orbitrap XL FT MS (Thermo Fisher
Scientific, San-Jose, CA). The source parameters for FTMS detection
were optimized using Arixtra (a synthetic ultra LMWH from Sanofi-Aventis,
Paris, France) to minimize the insource fragmentation and sulfate
loss and maximize the signal/noise in the negative-ion mode. The optimized
parameters, used to prevent in-source fragmentation, included a spray
voltage of 4.2 kV, a capillary voltage of −40 V, a tube lens
voltage of −50 V, a capillary temperature of 275 °C, a
sheath flow rate of 30 L/min, and an auxiliary gas flow rate of 6
L/min. External calibration of mass spectra routinely produced a mass
accuracy of better than 3 ppm. All FT mass spectra were acquired at
a resolution 60 000 with 200–2000 Da mass range.