The Measurement of Nicotine in Human Plasma by High-Performance Liquid Chromatography-Electrospray-Tandem Mass Spectrometry

Abstract

The authors describe a reverse-phase high-performance liquid chromatography-electrospray-tandem mass spectrometry method for the measurement of nicotine in human plasma. Samples (500 microL) with added deuterium-labeled d3-nicotine as an internal standard (IS) were treated with a 2-step process of ether extraction (6 mL) followed by back-extraction into 0.1% formic acid (50 microL). Chromatography was performed on a phenyl Novapak column with a mobile phase consisting of 50% 10 mM ammonium formate (pH 3.3) and acetonitrile (50:50, vol/vol). A flow rate of 0.2 mL/min resulted in a total analysis time of 5 minutes per sample. Mass spectrometric detection was by selected reactant monitoring (nicotine m/z 163.2 --> 130.2; IS m/z 166.2 --> 87.2). The assay was linear from 0.5 to 100 microg/L (r > 0.993, n = 9). The accuracy and imprecision of the method for quality control samples were 87.5% to 113% and <10.2%, respectively. Interday accuracy and imprecision at the limit of quantification (0.5 microg/L) was 113% and 7.2% (n = 4). The process efficiency for nicotine in plasma was >75%. The method described has good process efficiency, stabilized nicotine, avoided concentration steps, and most importantly minimized potential contamination. Further, we have established that water-based standards and controls are interchangeable with plasma-based samples. This method was used successfully to measure the pharmacokinetic profiles of subjects involved in the development of an aerosol inhalation drug delivery system.