HPLC separation of the enantiomers of nicotine and nicotine-like compounds

A sensitive and reproducible HPLC method utilizing a commercially available chiral α₁-acid glycoprotein (AGP) phase has been developed to separate and quantify the enantiomers of nicotine. The method is suitable for routine use as indicated by column life. The quantification of (R/S:0.05/99.95)-nicotine or (R/S:99/1)-nicotine was possible. In addition, the separation or at least partial separation of the enantiomers of nornicotine and nornicotine-derived compounds was achieved. © 1993 Wiley-Liss, Inc.     

Alkaloid production in cultured roots of three species of Duboisia

Cultured roots were obtained from calluses of Duboisia leichhardtii, D. myoporoides and D. hopwoodii. Cultured roots of all these species produced both tropane and pyridine-type alkaloids. The selected cultured roots of D. leichhardtii showed high contents of tropane alkaloids (hyoscyamine 0.53%, scopolamine 1.16%, on a dry weight basis).     

The alkaloid contents of sixty Nicotiana species

The alkaloid content (nicotine, nornicotine, anabasine, and anatabine) in leaves and roots of 60 Nicotiana species were analyzed by GC. All species contained alkaloids, the amounts varying with the species. There was no clearcut correlation between alkaloid amounts and subgeneric or sectional classification. The alkaloid content in the floral parts and immature and mature fruits of Nicotiana tabacum were also analyzed.     

Spermidine: an indirect precursor of the pyrrolidine rings of nicotine and nornicotine in Nicotiana glutinosa

Spermidine, which was labeled asymmetrically in its four-carbon moiety ([6-¹⁴C]-1,5,10-triazadecane), was administered to Nicotiana glutinosa plants. After 7 days the plants were harvested, yielding radioactive nicotine (0.43 % incorporation) and nornicotine (0.07 % inc.). A systematic degradation of the alkaloids indicated that they were labelled equally at C-2′ and C-5′ of their pyrrolidine rings. These results are consistent with the hypothesis that spermidine is degraded to putrescine prior to its incorporation into the pyrrolidine rings of nicotine and nornicotine.     

Modified alkaloid pattern in developing tobacco callus

Developing Nicotiana tabacum L. cv. Wisconsin-38 callus grown on modified Murashige-Skoog (MS) medium with Kao organic acids (pyruvic, citric, malic and fumaric acids) contains abnormally high levels of nornicotine and total alkaloids when compared with the leaves of the donor plant. Nornicotine/nicotine ratios observed during callus development suggest that nicotine is converted into nornicotine in the callus, with subsequent movement of alkaloids into roots formed on the callus and into the agar medium. Addition of Kao organic acids to the medium increases alkaloid levels, but cannot account for the abnormal increase in nicotine demethylation. This study thus reports two new findings: (a) that the total alkaloid content of tobacco callus can be greatly enhanced to 3.75% on a dry weight basis by exogenous organic acids, and (b) that endogenous nornicotine can accumulate in tobacco tissue cultures.     

烟草去甲基尼古丁产生的机理

烟叶的采收后处理和加工过程中,大量的尼古丁经去甲基化作用生成了去甲基尼古丁,后者是烟草特有的亚硝胺类（tobacco-specific nitrosamines,TSNAs）致癌物——亚硝基去甲基尼古丁（nitrosonomicoline,NNN）的前体,与人类健康息息相关。由于尼古丁去甲基化反应在基础理论研究和商业上的重要价值,长期以来关于这个反应的机制研究一直是学术界的热点。本文讨论了尼古丁去甲基化反应研究的历史概况、反应机制假说的演变及影响该反应的因素,期望通过对烟草尼古丁去甲基化反应研究的总结,为烟草品质提高和低毒害烟草制品的研究与开发提供一定的参考。     

Separation of nicotine and nornicotine enantiomers via normal phase HPLC on derivatized cellulose chiral stationary phases

This paper describes the enantiorecognition of (±)nicotine and (±)nornicotine by high-performance liquid chromatography using two derivatized cellulose chiral stationary phases (CSPs) operated in the normal phase mode. It was found that different substituents linked to the cellulose backbone significantly influence the chiral selectivity of the derivatized CSP. The results showed that, in general, the tris(4-methylbenzoyl) cellulose CSP (Chiralcel OJ) surpasses tris(3,5-dimethylphenyl carbamoyl) cellulose CSP (Chiralcel OD). On the former column, the resolution (±)nicotine and (±)nornicotine enantiomers depended largely on mobile phase compositions. For the separation of the nicotine enantiomers, the addition of trifluoroacetic acid to a 95:5 hexane/alcohol mobile phase greatly improved the enantioresolution, probably due to enhanced hydrogen bonding interactions between the protonated analytes and the CSP. For (±)nornicotine separation, a reduction in the concentration of alcohol in the mobile phase was more effective than the addition of trifluoroacetic acid. Possible solute-mobile phase-stationary phase interactions are discussed to explain how different additives in the mobile phase and different substituents on the cellulose glucose units of the CSPs affect the separation of both pairs of enantiomers. Chirality 10:364–369, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Separation and measurement of plant alkaloid enantiomers by RP-HPLC analysis of their Fmoc-Alanine analogs

Introduction . Ammodendrine ( 1 ), anabasine ( 2 ) and coniine ( 3 ) can cause congenital malformations in livestock. They appear naturally in both enantiomeric forms, and can cause variable physiological responses. A method to measure the enantiomeric ratio of these natural toxins is needed. Objective . To develop a simple and economical method in order to determine the enantiomeric ratios of piperidine and pyrrolidine alkaloids in small samples of plant material. Methodology . Mixtures of isolated or purified plant alkaloids were converted to their Fmoc‐l ‐Ala‐alkaloid analogues forming diastereomeric mixtures, which were then analysed by high pressure liquid chromatography (HPLC) with mass spectrometry (MS) and ultraviolet (UV) detection to determine enantiomeric ratios. Results . The diastereomeric analogs for ammodendrine, anabasine and nornicotine could be separated and the enantiomeric ratios determined. The Fmoc‐l ‐Ala‐coniine analogue was not resolved under the HPLC conditions studied. The enantiomeric ratios of the selected plant alkaloids were measured and found to differ between both location within a species and location between species. Conclusion . A low‐cost HPLC method to analyse the enantiomeric ratio of plant alkaloids containing primary or secondary amine nitrogens via conversion to their respective diastereomeric analogues has been developed. Published in 2008 by John Wiley & Sons.     

Effect of auxin on alkaloids, K+ and free amino acid content in cultured tobacco callus

Callus cultures derived from the petiole of Nicotiana tabacum L. cv. Burley 21 were grown at 25°C in the dark on two different basal media containing: (1) 11.5 μ M α-naphthaleneacetic acid and 1 μ M kinetin, and (2) 1 μ M α-naphthaleneacetic acid and 1 μ M kinetin. The contents of alkaloids, K⁺ and free amino acids of callus tissues were determined. The tissues were also examined microscopically for organization when organogenesis was not apparent. The first medium limited nicotine synthesis and stimulated its N-demethylation to nornicotine. The second medium stimulated nicotine synthesis and limited tissue growth. Although significantly higher concentrations of K⁺ were observed in calli grown on the high-auxin medium, both cultures were K⁺ deficient. The fact that the low-auxin medium limited K⁺ uptake to a higher degree would account for the lower growth observed in calli cultured on this medium, and it is possible that the effect of auxin concentration on nicotine production may be mediated through its effects on K⁺ uptake by cells of the culture. The free amino acid concentration increased in the calli grown on the low-auxin medium. Glutamic acid and proline, known as initial precursors of nicotine, increased significantly. Histological examination showed that the occurrence of meristematic areas in calli without organogenesis promoted nicotine synthesis. The relation between the accumulation of nicotine and formation of roots or shoot-buds is discussed.     

Effect of polyamine biosynthetic inhibitors on alkaloids and organogenesis in tobacco callus cultures

We studied the effects of inhibitors of ornithine decarboxylase (ODC), arginine decarboxylase (ADC) and spermidine synthase (Spd synthase) on organogenesis and the titers of polyamines (PA) and alkaloids in tobacco calli. DL--difluoromethylarginine (DFMA) and D-arginine (D-Arg), both inhibitors of ADC activity, were more effective than DL--difluoromethylornithine (DFMO), an inhibitor of ODC, in reducing titers of PA and the putrescine (Put)-derived alkaloids (nornicotine and nicotine). Dicyclohexylammonium sulfate (DCHA), an inhibitor of Spd synthase, was also more efficient than DFMO in reducing PA and alkaloid levels. Root organogenesis is inversely related to the titers of Put and alkaloids. Thus, DFMA and D-Arg, which strongly inhibit Put and alkaloid biosynthesis, markedly promote root organogenesis, while control callus with high Put and alkaloid content showed poor root organization. These results suggest that morphological differentiation is not required for activation of secondary metabolic pathways and support the view that ADC has a major role in the generation of Put going to the pyrrolidine ring of tobacco alkaloids.