title: A reversed-phase HPLC-UV method developed and validated for simultaneous quantification of six alkaloids from Nicotiana spp.
abstract: 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 13min with mobile phase pH 10.5 and a small gradient of 9-13% acetonitrile, and detected using UV at 260nm. 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 1.6μg/mL for nicotine and below 1μg/mL for all other alkaloids. The limit of quantification was 2.8 and 4.8μg/mL for nornicotine and nicotine respectively, and below 2μg/mL for all other alkaloids. The method was successfully applied for simultaneous analysis of alkaloids in leaves of Nicotiana benthamiana.
authors.affiliation: School of Pharmacy, The University of Queensland, Brisbane, Qld 4072, Australia.
authors.forename: Nahid
authors.initials: N
authors.lastname: Moghbel
authors.affiliation: School of Pharmacy, The University of Queensland, Brisbane, Qld 4072, Australia.
authors.forename: BoMi
authors.initials: B
authors.lastname: Ryu
authors.affiliation: School of Pharmacy, The University of Queensland, Brisbane, Qld 4072, Australia. Electronic address: k.steadman@uq.edu.au.
authors.forename: Kathryn J
authors.initials: KJ
authors.lastname: Steadman
mesh_terms: D000470:Alkaloids / Q000032:analysis* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase; D057230:Limit of Detection; D016014:Linear Models; D010936:Plant Extracts / Q000737:chemistry; D018515:Plant Leaves / Q000737:chemistry; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet; D014026:Nicotiana / Q000737:chemistry*
chemical_list: D000470:Alkaloids; D010936:Plant Extracts
keywords: Alkaline mobile phase; Nicotiana benthamiana; Quantitative method; Reversed-phase HPLC; Tobacco alkaloids