Determination of catecholamines in sheep plasma by high-performance liquid chromatography with electrochemical detection: comparison of deoxyepinephrine and 3,4-dihydroxybenzylamine as internal standard

Abstract

3,4-Dihydroxybenzylamine (DHBA) is commonly used as the internal standard in HPLC catecholamine assays. Sheep are frequently used in studies of cardiovascular physiology and in such studies measurement of catecholamines is important. The recovery of DHBA from sheep plasma is, however variable and poor. Therefore, we have developed a reliable and sensitive HPLC-ED method with alumina extraction for measurement of catecholamines in sheep plasma using deoxyepinephrine (epinine) as the internal standard. Separation was performed on a μBondapak C 18 column (300×3.9 mm, 10 μm) with a mobile phase containing 2% acetonitrile and 98% buffer (0.05% sodium acetate-0.02% EDTA-0.013% sodium heptanesulfonate), pH 3.25. The extraction of epinine from water, human plasma, dog plasma and sheep plasma did not differ ( p>0.05), but extraction of DHBA from sheep plasma was significantly impaired ( p<0.0001). The R 2 of regression curves ( n=5) of norepinephrine (NE) (25.02 pg/ml-1.00 ng/ml) and epinephrine (E) (25.82 pg/ml-1.03 ng/ml), using epinine as internal standard were greater than 0.99. The intra- and inter-day coefficients of variation were 2.11–11.15 and 0.88–12.60% for NE and 1.12–10.91 and 2.88–12.60% for E, respectively. The detection limits for NE and E are 12 pg/ml. The technique described has the advantage that it allows the simultaneous determination of both endogeneous and [ 3H]norepinephrine in sheep plasma using a sensitive and reproducible HPLC technique.