HPLC separation of blood 11C-nicotine from its radioactive metabolites

1618 Objectives PET imaging with 11C-nicotine (11C-NIC)-loaded cigarettes is a valuable tool to understand the role of fast brain nicotine accumulation in tobacco dependence. To accurately assess 11C-NIC kinetics, it is necessary to quantify the amount of parent compound in plasma apart from its two major metabolites, radioactive cotinine (COT) and 3’-hydroxycotinine (3HC). Previously such separation was achieved by extraction of radioactive substances from plasma using acetonitrile (ACN) and by reversed-phase HPLC at pH 6. To achieve higher yield of extraction and better separation of 11C-NIC, we sought to implement plasma deproteinization with perchloric acid and performing the HPLC separation using alkaline pH. Methods After denaturing proteins with 5% perchloric acid, 1.5mL of supernatant was adjusted to pH 10 by adding 0.25mL 28% NH4OH and 0.25mL solvent A (18% ACN + 82% pH 10 buffer (volume ratio: 493: 7: 2.42 for water, triethylamine, and acetic acid)). HPLC separation was performed on PRP-1 semi-preparative column using gradient elution (4 mL/min): 100% solvent A 4 min; 100% A ~ 100% B (30% ACN + buffer) 1 min linear; 100% B 6 min. Test-retest reliability was evaluated in venous blood samples from 20 subjects (13 males, 7 females; mean age 41yrs, 20 cigarettes/day) collected during PET studies (2 sessions/day with a 2 h interval), at 15 min after inhaling one puff from 11C-NIC-labelled cigarettes (ca. 75 MBq). Results Extracted radioactivity averaged 90 ± 7% (mean ± SD) of that in the plasma. Peak retention times for 3HC, COT, and NIC were 3.1, 4.4, and 10.4 min, respectively. Mean percent radioactivity attributed to 11C-NIC were 45 ± 7% of total radioactivity. Mean deviation of non-metabolized fraction of 11C-NIC from the average of repeated blood sample measurements was 6 ± 4%. Conclusions These results demonstrate that the introduced method provides efficient extraction, good peak separation and has high test-retest reliability. Research Support NIH grant RC2DA028948