The chromatographic apparatus consisted of an Agilent 1260 HPLC system
equipped with a binary pump (Agilent model G1312C), VWD UV detector (G1314F) set
at 341 nm, standard autosampler with thermostat (G1329B), online vacuum degasser
(G4225A), and column head (G1316A). Chromatographic separation was carried out
using a Waters XBridge™ BEH 130 C18 (3.5 μm, 2.1×150 mm)
column coupled with a Waters XBridge™ C-18 (3.5 μm,
2.1×10 mm) guard column maintained at 25°C. The analysis was
performed at a column temperature of 25°C using a mobile phase of
acetonitrile (ACN) and distilled water (65:35, v/v), an isocratic mode, pumped
at a flow rate of 0.18 ml/min.
2, 3-Diphenylquinoxaline, which is structurally unrelated but has a
reasonable absorbance at detection wavelength, was used as an internal standard
for the determination of SHetA2 in plasma. The sample (70 μl) was
injected into the HPLC system through the autosampler, which was maintained at
4°C through the thermostat and eluted for a run time of 40 min. All
biological samples were filtered using 0.2-μm polypropylene Captiva ND
plates, (Part # A5969002, Agilent Technologies), 1-ml Captiva 96-deep
well collection plates (Part # A696001000, Agilent Technologies), and a
CaptiVac vacuum collar (Part # A796, Agilent Technologies).
equipped with a binary pump (Agilent model G1312C), VWD UV detector (G1314F) set
at 341 nm, standard autosampler with thermostat (G1329B), online vacuum degasser
(G4225A), and column head (G1316A). Chromatographic separation was carried out
using a Waters XBridge™ BEH 130 C18 (3.5 μm, 2.1×150 mm)
column coupled with a Waters XBridge™ C-18 (3.5 μm,
2.1×10 mm) guard column maintained at 25°C. The analysis was
performed at a column temperature of 25°C using a mobile phase of
acetonitrile (ACN) and distilled water (65:35, v/v), an isocratic mode, pumped
at a flow rate of 0.18 ml/min.
2, 3-Diphenylquinoxaline, which is structurally unrelated but has a
reasonable absorbance at detection wavelength, was used as an internal standard
for the determination of SHetA2 in plasma. The sample (70 μl) was
injected into the HPLC system through the autosampler, which was maintained at
4°C through the thermostat and eluted for a run time of 40 min. All
biological samples were filtered using 0.2-μm polypropylene Captiva ND
plates, (Part # A5969002, Agilent Technologies), 1-ml Captiva 96-deep
well collection plates (Part # A696001000, Agilent Technologies), and a
CaptiVac vacuum collar (Part # A796, Agilent Technologies).