Background: A sensitive, rapid and selective UHPLCââ?¬â??MS/MS method has been developed and validated for the\nquantification of Nicotine (NT) and Cotinine (CN) using Continine-d3 as internal standard (IS) as per FDA guidelines.\nSample preparation involved simple protein precipitation of 20 Ã?¼L mouse plasma or brain homogenate using acetonitrile\nat 1:8 ratio. Mass Spectrometer was operated in positive polarity under the multiple reaction-monitoring mode\nusing electro spray ionization technique and the transitions of m/z 163.2 ââ? â?? 132.1, 177.2 ââ? â?? 98.0 and 180.2 ââ? â?? 101.2\nwere 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,\nrespectively. This was achieved with a gradient mobile phase consisting of 5 mM ammonium bicarbonate, acetonitrile\nand 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\nlower limit of quantitation 3.0 ng/mL in mouse plasma and brain for both the analytes.\nResults: A linear response function was established for the range of concentrations 3ââ?¬â??200 (r > 0.995) for NT and\n3ââ?¬â??600 ng/mL (r > 0.995) for CN. The intra- and inter-day precision values met the acceptance criteria. NT and CN are\nstable in the battery of stability studies viz., stock solution, bench-top and auto-sampler.\nConclusion: This method was successfully utilized to validate a newly developed preclinical smoking model in mice.
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