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

摘要

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?→?132.1, 177.2?→?98.0 and 180.2?→?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.