Impact of HPLC Parameters on Chiral Separation of Nornicotine Enantiomers

Given the well-documented pharmacological differences between nicotine and nornicotine enantiomers, understanding their distribution is essential. This knowledge also helps authenticate the enantiomers’ source (natural or synthetic). To improve the quantitative determination methods for these distributions, the influence of additives, modifiers, and sample matrices on the chiral HPLC separation of the nornicotine enantiomers using UV detection is discussed. Changes on the order of 5–10% in selected alcohols as modifiers and selected amines (0.1% changes) as additives were found to have significant effect on the resolution and retention times of nornicotine enantiomers, while sample matrices demonstrated an impact on nornicotine enantiomer resolution (R). Systematic variation in the concentration of ethanol and isopropanol, as modifiers, along with variations in the concentration of diethylamine, triethylamine, and isopropylamine, as additives, revealed that the resolution (R) of the nornicotine enantiomers could be adjusted to values much greater than 2, using mobile phase flow rates of 0.8 and 1 mL/min. The retention times of the nornicotine enantiomer pairs could be varied between ~8 and 20 min, through modification of the mobile phase with the additives and modifiers. As expected, faster mobile phase flow rates of 1 mL/min moderately reduced retention times when compared with influences on retention time caused by changes in the amounts of modifier and additive, with an accompanying slight decrease in the R values. The %RSD values for both nornicotine resolution and retention times consistently remained below 3%. The detection limits for the nornicotine enantiomers were approximately 1 ng on column for each enantiomer. With the judiciously selected optimization of the levels of the hexane mobile phase additive, diethylamine, and modifier, ethanol, coupled with results from previously published results on nicotine and nornicotine alkaloid enantiomer separations, a simultaneous separation of both nornicotine and nicotine enantiomers having R values greater than 2 and an overall retention time of less than 15 min was attained. Noticeable influences on nornicotine enantiomer resolution and co-elution of tobacco extract components with nornicotine enantiomers as a function of mobile phase composition were illustrated and discussed. Previous published results in combination with this current body of work culminate in a well-rounded understanding of the influences of modifier and additive structure and concentration on the resolution as well as a simultaneous separation of nicotine and nornicotine enantiomers employing high performance liquid chromatography.