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Determination of catecholamine concentration in blood of patients on cardiac ventricular assist support using Liquid Chromatography & Tandem Mass Spectrometry

Abstract: "Elevation in catecholamine blood concentrations has long been reported in patients with heart failure. Norepinephrine is believed to be of cardiac origin and its elevation to reflect impaired presynaptic uptake in excessively stimulated hearts. In patients with left ventricular assist support, normalisation of norepinephrine concentration could indicate regeneration of failing heart and suggest possibility to remove ventricular assist device. However, determination of catecholamines is challenging due to the low circulatory concentrations. The goal of the project was to develop a sensitive liquid chromatography mass spectrometry (LC-MS/MS) procedure, with limited sample preparation, for the determination of catecholamines using a tandem quadrupole mass analyser system linked to UHPLC system. Plasma and serum samples were collected from 8 control healthy subjects, from 6 patients in mild heart failure (Class I-II) and from 10 patients in severe heart failure (Class IV), early post implantation of ventricular assist device. Catecholamines were adsorbed on Alumina and extracted with acetic acid. Separation was performed using an Agilent 1200SL UHPLC system with Zorbax Eclipse Plus C18 column, 1.8µ, 2.1x100mm. Mass spectrometry analysis was performed using an Agilent 6410B triple quadrupole mass analyser operated in multiple reaction monitoring mode (MRM). Electropsray ionisation was used throughout. Norepinephrine was detected and quantitated from m/z transitions 170>152 and 170>107, DOPA from 154>91 and adrenaline from transition 184>77 and 166>77. Dihydrobenzylamine that was used as internal standard was detected from transition 140>123. The method was highly reproducible with within assay coefficients of variation of ranging from 4 to 9%. Detection limit was sufficient for determination of all compounds in clinical samples with signal-to-noise ratio for norepinephrine >100. Recoveries were >85% and response was linear up to micromolar concentrations. Plasma and serum concentrations followed similar pattern, although serum concentrations tended to be about 10% higher. In healthy controls concentration of norepinephrine was 2.1±0.4, epinephrine 1.2±0.3 and DOPA 0.46±0.03 nM (±SD, n=8). Concentration of norepinephrine was 2.0±0.6 (n=6) in patients with mild heart failure – similar to healthy controls. Norepinephrine was markedly elevated in patients with ventricular assist device for end-stage heart failure 3.1±0.8 (n=10). Determination of norepinephrine and other catacholamines with the method described was highly accurate, reproducible and robust. Results obtained could greatly aid assessment of cardiac dysfunction and in particular could help to make clinical decision. Clinically relevant LCMSMS assay to monitor low level circulatory catecholamines in blood with minimal sample preparation to determine cardiac dysfunction"

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