Solid-phase extraction of plasma vasopressin: evaluation, validation and application.
A new solid-phase extraction method using octyl-silica columns to extract vasopressin-like immunoreactivity from plasma has been developed. The extraction was followed by a radioimmunoassay on the vacuum-dried extracts, which were reconstituted in assay buffer. The total recovery of synthetic vasopressin was ca. 100%. Based on co-elution with synthetic vasopressin after separation by reversed-phase high-performance liquid chromatography of plasma extracts from normal Wistar and Brattleboro rats, and the cross-reactivity of the antiserum used in the radioimmunoassay system, the extracted material was found to be indistinguishable from authentic vasopressin. Unknown experimental samples were interpolated on a standard curve established in "zero" plasma (plasma derived from rats subjected to waterload) spiked with known amounts of synthetic vasopressin, and not on a standard curve established in assay buffer. The limit of detection was 1 fmol of vasopressin equivalent per millilitre. The intra- and inter-assay coefficients of variance were 10-16% and 16%, respectively. The procedure reliably showed that osmotic challenge and 24-h dehydration increased, whereas ethanol ingestion decreased vasopressin-like immunoreactivity plasma levels in the rat, compared with normally hydrated controls.
Van de Heijning
B J
BJ
Department of Pharmacology, Rudolf Magnus Institute, University of Utrecht, The Netherlands.
Koekkoek-van den Herik
I
I
Iványi
T
T
Van Wimersma Greidanus
T B
TB
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008297:Male; D011863:Radioimmunoassay; D051381:Rats; D011913:Rats, Inbred BB; D011919:Rats, Inbred Strains; D014667:Vasopressins / Q000097:blood*
D014667:Vasopressins