A reversed-phase HPLC-UV method developed and validated for simultaneous quantification of six alkaloids from Nicotiana spp.

A reversed-phase HPLC-UV method was developed, optimized, and validated for the separation and quantitation of six target alkaloids from leaves of Nicotiana species (nicotine, nornicotine, anatabine, anabasine, myosmine, and cotinine). A bidentate reversed-phase C18 column was used as stationary phase and an alkaline ammonium formate buffer and acetonitrile as mobile phase. The alkaloids were well separated in a short run time of 13 min with mobile phase pH 10.5 and a small gradient of 9-13% acetonitrile, and detected using UV at 260 nm. Peak parameters were acceptable for all six closely related alkaloids. The proposed method has enough linearity with correlation coefficient >0.999 within the investigated range for all tested alkaloids. Satisfactory precision was achieved for both intra- and inter-day assay, with RSD less than 2% for all alkaloid standards. Reproducibility was also within the acceptable range of RSD <2%. Limit of detection was 1.6 μg/mL for nicotine and below 1 μg/mL for all other alkaloids. The limit of quantification was 2.8 and 4.8 μg/mL for nornicotine and nicotine respectively, and below 2 μg/mL for all other alkaloids. The method was successfully applied for simultaneous analysis of alkaloids in leaves of Nicotiana benthamiana.