Development of a solid-phase extraction technique for alpha-human atrial natriuretic peptide in human plasma.
A reliable extraction method was developed for alpha-human atrial natriuretic peptide (alpha-hANP) using Bond Elut C8 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 alpha-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 alpha-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 alpha-hANP was conducted and the changes in plasma concentration were followed.
O'Flynn
M A
MA
Department of Clinical Pharmacology, Royal Postgraduate Medical School, Hammersmith Hospital, London, U.K.
Causon
R C
RC
Brown
J
J
Kageyama
S
S
D009320:Atrial Natriuretic Factor / Q000097:blood*; D006801:Humans; D007202:Indicators and Reagents; D011863:Radioimmunoassay
D007202:Indicators and Reagents; D009320:Atrial Natriuretic Factor