Abstract
Abstract
A robust, fast and sensitive capillary column switching reversed-phase (RP) liquid chromatography (LC) method with back flushing of the pre-column and ultraviolet (UV) detection has been developed to investigate the stability of angiotensin II and bradykinin stored in different matrices and by different temperatures. The loading mobile phase (MP) containing acetonitrile (AcN) – H2O – formic acid (FA) (2 : 97.95 : 0.05, v/v/v) was delivered isocratically at a flow rate of 0.200 mL/min. Manual injections of 200 μL were made with an external loop, and the samples were loaded on a HotSep Tracy column (Kromasil C18; 5 x 1 mm (inner diameter (ID)), 5 μm). After a loading time of 3.5 min the analytes were back flushed from the pre-column and transferred to the separation column, a HotSep Kromasil C18 column (50 x 0.3 mm (ID), 3.5 μm), using an eluent consisting of AcN – H2O (gradient) with 20 mM NH4+HCOO- and 0.05 % FA added, delivered at a flow rate of 0.005 mL/min. In-line UV detection was performed at 210 nm. The limits of detection (LOD) were ~10 ng/mL for bradykinin and below 1 ng/mL for angiotensin II. The robustness of the system is discussed.
An isocratic and a gradient eluting nano LC-UV system and a column switching gradient nano LC-UV method, all with a Kromasil C18 nanocolumn (150 x 0.1 mm (ID), 3.5 μm), were developed for bradykinin determination.
The signal responses for bradykinin dissolved in H2O and 20 % AcN with FA added in different amounts were compared using direct infusion electrospray time-of-flight mass spectrometry (direct infusion-ESI-TOF-MS) at different capillary voltages.
The ratio of theoretical dilution of two different capillary columns was calculated and compared to the experimental dilution ratio obtained with those columns using capillary RPLC coupled to an electrospray time-of flight mass spectrometer (ESI-TOF-MS).