A convenient UHPLC-MS/MS method for routine monitoring of plasma and brain levels of nicotine and cotinine as a tool to validate newly developed preclinical smoking model in mouse.

BACKGROUND: A sensitive, rapid and selective UHPLC-MS/MS method has been developed and validated for the quantification of Nicotine (NT) and Cotinine (CN) using Continine-d 3 as internal standard (IS) as per FDA guidelines. Sample preparation involved simple protein precipitation of 20 µL mouse plasma or brain homogenate using acetonitrile at 1:8 ratio. Mass Spectrometer was operated in positive polarity under the multiple reaction-monitoring mode using electro spray ionization technique and the transitions of m/z 163.2 â†’ 132.1, 177.2 â†’ 98.0 and 180.2 â†’ 101.2 were used to measure the NT, CN and IS, respectively. The elution of NT, CN and IS are at 1.89, 1.77 and 1.76 min, respectively. This was achieved with a gradient mobile phase consisting of 5 mM ammonium bicarbonate, acetonitrile and methanol (3:1, v/v) at a flow rate of 0.3 mL/min on a Kinetex EVO C18 column. The method was validated with a lower limit of quantitation 3.0 ng/mL in mouse plasma and brain for both the analytes. RESULTS: A linear response function was established for the range of concentrations 3-200 (r > 0.995) for NT and 3-600 ng/mL (r > 0.995) for CN. The intra- and inter-day precision values met the acceptance criteria. NT and CN are stable in the battery of stability studies viz., stock solution, bench-top and auto-sampler. CONCLUSION: This method was successfully utilized to validate a newly developed preclinical smoking model in mice.

Nicotine exposure can be detected in cerebrospinal fluid of active and passive smokers.

A simple, rapid and sensitive liquid chromatography/mass spectrometry (LC/MS) method has been utilized for the quantitative determination of nicotine and its major metabolite cotinine (COT) in human cerebrospinal fluid (CSF) of active and passive smokers. CSF samples from 18 smokers, 15 non-smokers, 15 children, 15 infants, and 9 neonatal were analyzed for nicotine (NIC) and cotinine content. Cotinine levels in the CSF of smokers ranged from 27.3 to 457.1 ng/ml, whereas nicotine levels were considerably lower (6.0-215.1 ng/ml). Cotinine could be detected in 4 of the 15 CSF samples from non-smokers (3.5-30.4 ng/ml), and a few other passive smokers, including neonates from smoking mothers (15.6-81.1 ng/ml). The concentrations of cotinine in CSF samples suggests that nicotine easily passes into the CSF, which makes it an excellent CSF marker for tobacco-smoke exposure.