Heparin Digests Analysis by LC-MS

The liquid chromatography/mass spectrometry (LC/MS) method used to analyze heparin digests was as described previously (8 (link)), and used an Acquity UPLC BEH C18 column, 150 × 2.1 mm, 1.7 μm (Waters SAS, En Yvelines Cedex, France). Mobile phase A was water, and mobile phase B water:acetonitrile (30:70). The ion pairing reagent, heptyl amine (HPTA; 7.5 mM), and a buffering agent, hexafluroisopropanol (50 mM), were added to both A and B. A linear gradient (t_0min B% 1; t_70min B% 70) was applied for elution at a flow rate of 0.22 mL/min. Column temperature was set at 30°C and diode array detection used. Double UV detection was performed at 265 nm and 232 nm. Electrospray ionization mass spectra were obtained using a Waters Xevo Q-Tof mass spectrometer. The electrospray interface was set in negative ion mode with a capillary potential of 2000 V and a sampling cone potential of 20 V. Source and desolvation temperatures were 120 and 300°C, respectively. Nitrogen was used as the desolvation (750 L/min) and cone (25 L/min) gas. The mass range was 50–2500 Da (scan rate = 0.8 s). Acquisition was performed in MSE mode with low energy at 7 V and a high energy ramp from 30 to 50 V.