Determination of nicotine and its metabolites in rat brain using microdialysis coupled with UHPLC-Q-Exactive high resolution mass spectroscopy
In order to accurately determine nicotine (Nic) and its metabolites in rat brain, a method of microdialysis coupled with ultra high performance iquid chromatography-high resolution quadrupole-Orbitrap (UHPLC-Q-Exactive) mass spectrometry was established. Microdialysis probes were inserted into the brain striatum of Wistar rats, and dialysates were collected every 15 min after nicotine (1.5 mg/kg, i.p.) administration. Target analytes were separated on a hydrophilic interaction liquid chromatography column (HILIC 3.0 ×150 mm, 2.7 µm) and detected by Q-Exactive under Full MS / Targeted-MS2 mode. The results showed that Nic access to brain tissues could generate eleven metabolites such as cotinine (Cot), nicotine-N’-oxide (NNO), nornicotine (NorNic), cotinine-N-oxide (CNO), norcotinine (NorCot), trans-3’-hydroxycotinine (OH-Cot), nicotine-N-glucuronide (Nic- Gluc), cotinine-N-glucuronide (Cot-Gluc), trans-3’-hydroxycotinine-O-glucuronide (OH- Cot-Gluc), 4-oxo-4-(3-pyridyl)-butanoic acid (OxPyBut) and 4-hydroxy-4-(3-pyridyl)- butanoic acid (HyPyBut). The limits of detection of Nic and its metabolites ranged from 0.012 to 0.081 ng/mL and the intra-day precision ranged from 1.19 % to 9.20% with the accuracy of -8.77% to 8.61%. The brain concentration-time profiles of nicotine metabolites and pharmacokinetic results indicate that cotinine is generated as the main metabolite and the second most abundant metabolites were OH-Cot and Nic-Gluc; meanwhile, the other eight metabolites were present in minor amounts in rat brain. The method is suitable for the simultaneous analysis of Nic and its metabolites in rat brain for pharmacokinetic application.