Method Development and Validation of Anabasine and Nornicotine in Human Plasma by LC-MS/MS

Aim: For the Method development and validation of Anabasine and Nornicotine in human plasma LC-MS/MS technique is used. Methodology: Operated in positive electron spray ionization which removes the hardness in ions. Metoprolol is used as an internal standard. Mass analyzers are used to determine the mass to charge ratio. Liquid chromatography separates mixture components in the basis of differences in affinity for stationary and mobile phase. It removes undesired impurities. It increases reproducibility, sensitivity, robustness, detection of low-level proteins.  C18 Column (Phenomenex Luna C18, 5 μm, 50*4.6mm ID) is used to for high resolution and peak area. Calibration curve is constructed with the help of linear regression. During the entire process no instability is found. Results: m/z values of Anabasine are 162.84(Q1), 134.68(Q3) and for Nornicotine are 148.90(Q1), 79.82(Q3). Back calculated curve is calculated at different concentrations from 1-1000ng/ml and their accuracy range from 97-105% for Anabasine and 93-108% for Nornicotine. In Anabasine and Nornicotine the precision and accuracy are calculated by Intra-batch and inter-batch. In the process of multi-step preparation, consider n=6 for intra-batch and n=18 for inter-batch the values obtained are between the nominal concentrations i.e., 90-110%. For each analyte the coefficients of variation are below nominal criteria <15%.  For Anabasine and Nornicotine the average recovery rate is 98.3% and 94.3%.