Confirmation of hydrazone formation in HYNIC-peptide conjugate preparation, and its hydrolysis during labeling with 99mTc

Because of its monodenticity, 6-hydrazinopyridine-3-carboxylic acid (HYNIC) is of interest as a bifunctional chelator for labeling peptide with Tc-99m. Here, we confirm the formation of hydrazone in HYNIC-conjugated peptide. The preparative HPLC was used to purify the HYNIC conjugated somatostatin-based peptide and the result showed two peaks, even after two consecutive purifications. Analysis of these peaks by mass spectrometry indicated the presence of hydrazone, produced during preparation conjugate. Further, we have shown that presence of hydrazone really does not matter because under Tc-99m-labeling conditions, hydrazone is hydrolyzed back to HYNIC that then chelates Tc-99m. A HYNIC-peptide conjugate freeze-dried kit was also prepared in a mildly acidic or neutral condition with a final pH of 6-7. The kit was then labeled by Tc-99m and incubated in 100 degrees C for 10 min, and a labeling yield of >95% was obtained. (C) 2007 Elsevier Ltd. All rights reserved.