RECOVERY OF STANDARD I125 PEPTIDES AT THE PICOGRAM AND FEMTOGRAM LEVEL BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Average recoveries of standard [125I]angiotensin II are 92-103% when amounts of this peptide ranging from 10 pg to 50 fg are analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) employing a C8-bonded silica packing, whether an old (1 yr of prior use) or previously unused column is employed. In one case this recovery is 68% on a column giving a 10-fold higher retention of this peptide with the usual mobile phase of acetonitrile-aqueous 0.01 M sodium perchlorate (40:60); the same mobile phase except 0.1 M sodium perchlorate shortens the retention 10-fold on this column and gives an average recovery of 103%. Similar recoveries are obtained for [125I]bradykinin (105%) and [125I]LHRH (107%), tested at the 1-pg level. Although 2 commercial preparations of [125I]vasoactive intestinal peptide are recovered only to the extent of 75 and 85%, the HPLC chromatograms also display several peaks. Aside from the potential for complications in the analysis of real samples, this work establishes that RP-HPLC is a viable technique for sample clean-up/characterization in the ultratrace analysis of at least some peptides.