Development of a solid-phase extraction technique for α-human atrial natriuretic peptide in human plasma

Abstract

A reliable extraction method was developed for α-human atrial natriuretic peptide (α-hANP) using Bond Elut C 8 columns in tandem. This involved activation of the columns using methanol followed by a water wash to remove the excess methanol. Plasma (1 ml) was then added and a vacuum applied until all was drawn through. Excess protein and other endogenous compounds were removed by washing the columns with water and elution of the α-hANP was achieved with 0.75 ml acetonitrile—water—trifluoroacetic acid (80:19.8:0.2, v/v/v). Samples were evaporated under nitrogen and reconstituted in radioimmunoassay buffer ready for analysis. The recovery of α-hANP from plasma using this method was found to be 90% ± 0.6% [mean ± standard error of the mean (S.E.M.); coefficient of variation (C.V.) = 1.5%] which will allow more precise measurement of the peptide than is presently available. With this high precision of analysis available, having a limit of detection of 0.4 fmol/ml and a range of 0 to 32 fmol/ml, a low-dose infusion of α-hANP was conducted and the changes in plasma concentration were followed.