Tobacco extracts analysis

Tobacco extracts, analysis of 957 Tobacco smoke, phenohc compounds in 932 of-Tocopherol 844-850, 861, 872, 883-888, 892-895, nil, 1335 oxidation of 1080... 

This method requires about 40 g of tobacco which are extracted with ethyl acetate in the presence of ascorbic acid. A trace amount of C-NDELA is added as an internal standard for quantitative analytical work. The filtered extract is concentrated and NDELA is enriched by column chromatography of the concentrate on silica gel. The residues of fractions with p-activity are pooled and redissolved in acetonitrile. Initially, we attempted to separate NDELA on a 3% OV-225 Chromosorb W HP column at 210 C using a GC-TEA system with direct interface similar to the technique developed by Edwards a. for the analysis of NDELA in urine (18). We found this method satisfactory for reference compounds however, it was not useful for an optimal separation of NDELA from the crude concentrate of the tobacco extract (Figure 4). Therefore, we silylated the crude concentrate with BSTFA and an aliquot was analyzed by GC-TEA with direct interface. The chromatographic conditions were 6 ft glass column filled with 3% OV-... 

Figure 9 shows the curve for tobacco residues purified by TLC before analysis by the assay. It is clear that the interference seen previously was practically eliminated. However, such extensive sample preparation makes use of this assay in its present form cumbersome, at best. We are presently investigating an alternative form of immunoassay, the enzyme immunoassay (EIA) ( , 21) In this assay methoprene is conjugated directly to an enzyme and the anti-methoprene antibody is bound to the solid support. Free methoprene and methoprene-enzyme conjugate are in solution and compete for immobilized antibody binding sites. Unbound methoprene is washed from the assay prior to addition of substrate. Preliminary results under these conditions indicate that tobacco extracts of acetonitrile/water (9 1) do not require further purification steps prior to application to the EIA. 

Fig. 8-77. Analysis of inorganic and organic anions in a tobacco extract. — Separator column IonPac AS4A eluent 0.0017 mol/L NaHC03 + 0.0018 mol/L Na2C03 flow rate 2 mL/min detection suppressed conductivity injection 50 pL of a 1 25 diluted 1% extract sample components organic acids (1), chloride (2), nitrate (3), orthophosphate (4), and sulfate (5).

Hsieh, S.T. Status of phosphate compounds in tobacco leaves associated with phosphorus deficiency Chung Kuo Nung Yeh Hua Hsueh Hui Chih (1970) 105-109. Hsu, S.C., R.L. Pollack, and R.D. Going Gas-liquid chromatographic analysis of sugars present in tobacco extracts Am. Assoc. Dental Res. 45 (1981) 787-788. Hsu, W.P. and G.W. Miller Coproporphyrinogenase in tobacco Nicotiana tabacum L.) Biochem. J. 117 (1970) 215-220. 

FIGURE 7.2 Flow cytometric analysis of cell cycle distribution and apoptosis in human oral keratinocytes in response to increasing concentrations of smokeless tobacco extract. Primary keratinocytes were grown to approximately 70% confluence and then treated with various concentrations of STE for 25h. Following incubation, the cells were removed from the culture surfaces by trypsinization, and DNA content and apoptosis were determined using the method of Telford et al. Raw data were modeled using ModFit cell cycle... 

Fig. 1. Diagram of autoanalyzer manifold for analysis of nicotine in tobacco extracts...

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-... 

This is demonstrated here with the analysis of an aqueous tobacco extract. When analyzing this extract under isocratic conditions with a carbonate/bicarbonate eluant mixture, the chromatogram illustrated in Fig. 9-172 is obtained. The group of peaks close to the system void points to the presence of short-chain organic acids. In addition to chloride, nitrate and orthophosphate can be identified. The peak appearing at = 5.4 min, which normally should be assigned to sulfate, is unusually broad. This imphes that at least one more component (possibly a short-chain dicarboxyhc acid) elutes at that time. 

The first step in the analysis is extraction of the tobacco with buffer solution (pH 4.5) containing 20 mM ascorbic acid. The nitrosamines are then concentrated by partition with dichloromethane, and a chromatographic clean-up on alumina. In the final step, the concentrate is analyzed by GC-TEA and confirmation of the nitrosamines is obtained by GC-MS (O. If isolated amounts of the nitrosamines are below levels needed for GC-MS confirmation, we employ confirmatory techniques proposed by Krull et a. ( 5). 

In order to ascertain that the NDELA formation does not occur as a result of trapping of the smoke or during the analysis, we added diethanolamine to tobacco prior to extraction with ethyl acetate in the presence of ascorbic acid. The control value for NDELA was 121 ppb and the experiment with 5.5 iqg diethanolamine addition yielded 113 ppb NDELA. For control of the smoke analysis we added 5.5 mg of DELA in the solvent trap and smoked cigarettes known to be free of DELA. Analysis of the trapped material showed no significant quantities of NDELA, so that artifactual formation of this nitrosamine during smoke collection and analysis can be ruled out. 

Nonvolatile Nitrosamines In Tobacco. A method which we developed several years ago for the analysis of tobacco-specific nitrosamines (TSNA 31) involves extraction of tobacco with buffered ascorbic acid TpH 4.5) followed by partition with ethyl acetate, chromatographic clean-up on silica gel, and analysis by HPLC-TEA (Figure 9). Results obtained with this method for a large spectrum of tobacco products (Table IV), strongly support the concept that the levels of nitrate and alkaloids, and especially the methods for curing and fermentation, determine the yields of TSNA in tobacco products. Recent and as yet preliminary data from snuff analyses indicate that aerobic bacteria play a role in the formation of TSNA during air curing and fermentation. 

Shen, J. and Shao, X., Comparison of accelerated solvent extraction, Soxhlet extraction, and ultrasonic-assisted extraction for analysis of terpenoids and sterols in tobacco. Ana/. Bioanal. Chem., 383, 1003, 2005. 

Fig. 11.3 Purification ofSOl-lOOxELP-proteins from transgenic tobacco plants by inverse transition cycling and analysis by SDS-PAGE. 1 15 pg of total soluble leaf protein extracted in raw extract buffer 2 cleared supernatant of original 15 pg total soluble leaf protein after heat treatment (60 min, 95 °C) 3 cleared supernatant of original 300 pg leaf protein after heat treatment 4 cleared supernatant of original 300 pg leaf protein after heat treatment (60 min, 60 °C) with 2 M NaCI 5 redissolved spider silk-elastin protein pellet from original 300 pg of total soluble leaf protein after heat treatment (60 min, 60 °C) with 2 M NaCI.

A new approach to the analysis of the carcinogenic TSNA in moist snuff tobacco is based on SFE with methanol-modified carbon dioxide. Extracted TSNA are trapped across a glass cartridge filled with Tenax GR, from which they are subsequently released by thermal desorption and analyzed by capillary GC-TEA LOD was <2 ng/g. The technique is fast, reproducible, highly selective and sensitive617. SFE with carbon dioxide was also used in the analysis of TSNA in smokeless tobacco. It revealed the presence of higher levels of 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (300) than had been determined earlier by conventional methods618. 

P. Faxar says that the salt urao crystallizes on the bottom of the lake in the valley La Lagunilla in Venezuela, and that it is collected by the natives every two years, and used in the manufacture of mo—a dried extract of tobacco used by the natives as a stimulant. J. B. J. D. Boussingault says this urao crystallizes in prismatic needles arranged in radiating masses it is a little softer than calcite the taste is alkaline and it does not effloresce. His analysis is C02, 39 00 per cent. Na20, 4122 H20, 18 80 and impurities, 0 98 per cent. He thus reports on the peculiar way in which the natives obt ain the salt ... 

Bush determined the four most important tobacco alkaloids (nicotine, nornicotine, anabasine and anatabine) using a 10 % DC 550 packed column on Chromosorb 60-80, and using isoquinoline as an internal standard. The alkaloids were extracted with benzene-chloroform (9 1) after treatment of the tobacco sample (1 g) with bariumhydroxide and water. The organic phase was concentrated and used for the gas chromatographic determination. Because of the great differences in the amounts of the minor alkaloids and nicotine in most tobacco samples, two extractions, each with an appropriate amount of internal standard, were required for a complete assay. The precision of the quantitative analysis on tobacco samples of different alkaloids is given in Table 5.6. 

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