Individual Tobacco Alkaloids

Pyridine alkaloids in tobacco are usually determined by gas chromatography. The parent alkaloids - nicotine, nornicotine, anatabine, anabasine, and myosmine - may be determined in a single extract. A capillary GC procedure we use (Andersen et al. 1990) is based on that of Severson et al. (1981) but with modification. 

Quantitative results obtained during storage, aging, or fermentation are expressed as pg/g dry wt and because of dry weight changes may be normalized on the basis of calcium concentration at the start of storage. 

Nitrosated, acylated, and oxidized alkaloids are extracted and separated from the bulk of the parent alkaloids by liquid-liquid partitioning at pH 5 

Quantifications are by internal standardization with azobenzene after calibration of retentions (see Table 2) and nitrogen-phosphorus detector (NPD) response factors for authentic compounds. Response factors are determined by these steps  

Tndex for the DB-5 capillary column used with conditions described in Section 3.2. 

---

A series of reviews describing nicotine metabolism has recently appeared [2], Specific topics covered include the biosynthesis and metabolism of nicotine and related alkaloids [3], an overview of mammalian nicotine metabolism [4], the role of cytochrome P450 in nicotine metabolism [5], nicotine metabolism beyond cotinine [6], N-oxidation, A -methylation, and N-conjugation reactions of nicotine [7], extrahepatic metabolism of nicotine and related compounds [8], metabolism of the minor tobacco alkaloids [9], analysis and levels of nicotine and metabolites in body fluids [10], kinetics of nicotine and its metabolites in animals [11], pharmacokinetics of (S)-nicotine and metabolites in humans [12], and sources of inter-individual variation in nicotine pharmacokinetics [13]. Another recent review described variables which affect nicotine metabolism [14]. Several compilations of studies or reviews on the tobacco-specific A-nitrosamines are available [15-18]... 

Thin-layer chromatography is frequently used to separate pyridine alkaloids from tobacco extracts. This method is frequently used by tobacco breeders to screen plant populations and individual tobacco plants to insure seed purity. Thin-layer is considerably faster than paper chromatography and can also be used as a preliminary cleanup for more sensitive methods for analytical analysis such as capillary gas chromatography (GC). Semi-... 

In a study for precursor determination, we stem-fed individual Burley leaves with nicotine-2 - C or nornicotine-2 - C (29). Subsequently, the leaves were air cured, dried and analyzed for NNN and NNN- C. Recovery of the p-activity in the form of NNN- C amounted to 0.009% and 0.007%, respectively of the stem-fed label. This demonstrates that both alkaloids give rise to NNN. More importantly, it points to the fact that the tertiary amine, nicotine, which constitutes 0.5-2.6% of commercial tobaccos (26,27), is the major precursor for the carcinogenic tobacco-specific NNN, while the secondary amine, nornicotine is of lesser importance because it amounts to only 0.005-0.06% in tobacco (Figure 8). 

Quin, L.D. and N.A. Pappas Quantitative determination of individual alkaloids in tobacco by gas chromatography 1. Agr. Food Chem. 10 (1962) 79-82. 

The flue-cured leaf is the only portion of the plant used in manufacturing tobacco products. Distribution of alkaloids in flue-cured tobacco leaves varies by stalk position and within individual leaves regardless of stalk position. Alkaloids readily form organic salts with dicarboxylic acids and amino acids alkaloids also form complexes with proteins, polyphenols, carbonyls, and iron to produce color in tobacco leaves (Dawson 1945). Alkaloid complexes in the cured leaf are water-soluble however, aqueous... 

Analysis is most efficient with a Griffith Still which consists of a series of six stills connected together with a master electrical control. An experienced operator can run 40 samples/h (Griffith and Jeffery 1948 Griffith 1957). The Griffith method is an AO AC (Association of Official Analytical Chemists)-approved method for determination of total alkaloids in tobacco (Williams 1984). Steam distillation is not the method of choice for determining secondary alkaloids which include nornicotine, anabasine, anatabine, and myosmine. In cases where individual alkaloids in a sample must be analyzed, steam distillation is not dependable. However, in cases where total alkaloids determined as nicotine will suffice, steam distillation is adequate and the use of more sensitive equipment is not necessary. 

Tobacco breeders have found it necessary to use wild species of Nicotiana in breeding programs to introduce disease and insect resistance into new flue-cured varieties. Such practices often result in tobaccos that produce undesirably high levels of nornicotine, anabasine, and anatabine. Colorimetric analyses used to determine nornicotine do not give accurate results if anabasine and anatabine are produced in high amounts, since the methods used for determining nornicotine do not account for anabasine, anatabine, and myosmine quantitatively. Chromatographic procedures were developed that allowed tobacco breeders to separate complex mixtures of alkaloids into individual alkaloids. 

Substances toxic to all individuals are called toxic substances or toxins. Once a toxic substance has contacted the body it may have either acute (immediate) or chronic (long term) effects. Most of food-born toxins are substances with low acute toxicity (such as the pungent alkaloid piperine in black pepper), although some may present chronic effects, such as hepatotoxic pyrrohzidine alkaloids in plants (such as comfrey and coltsfoot species) or cause pathological changes to the respiratory system (e.g., tobacco smoke). 

---