The frozen plasma samples were thawed on ice and centrifuged at 4°C for 10 min at 3000 × g. The supernatants were collected, diluted (1:5) with 0.1 M formic acid and 0.018 M pyridine (buffer I) and separated on minicolumns (1 ml) packed with SP-Sephadex C-25 gel according to a previously outlined procedure [66]. The columns were washed with 10 ml buffer I and then, after application of the sample, washed with additional 5 ml of 0.1 M formic acid/0.1 M pyridine, pH 4.4 (buffer II). The peptide-containing fractions were then eluted with 4 ml of 1.6 M formic acid/1.6 M pyridine, pH 4.4 (buffer V). All buffers contained 0.01% mercaptoethanol. The eluted samples were then evaporated in a Speed Vac centrifuge (Savant, Hicksville, NY, USA).
