Sample preparation tobacco extracts

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. 

Supercritical fluid extraction (SEE) using supercritical carbon dioxide (SC-CO2) has been successfully used for isolation of volatile nitrosamines from different matrices such as tobacco and food products. This technique presents several advantages with respect to other extraction methods (e.g., mineral oil distillation or low-temperature vacuum distillation) currently used. Thus, SEE minimizes sample handling, provides fairly clean extracts, expedites sample preparation, and reduces the use of environmentally toxic solvents. Good results have also been obtained with the use of SPE in the analysis of food matrices combining extraction with Extrelut sorbent and purification with Florisil. This method is applicable for the analysis of a range of the most widely encountered volatile N-nitrosamines, including the poorly volatile NDBA, NDBzA, and N-nitroso-N-methylaniline in various food products. Active carbon is suitable for this preconcentration step due its low cost, versatility, and easy application. 

Use of the cELISA to determine methoprene content of tobacco residues prepared as described above, showed that the extracts contained materials which interfered with the assay (Figure 8). Extracts of tobacco not only contained methoprene, but also plant substances that interfered with the assay. Thin-layer chromatography (TLC) was then used to clean up crude extracts from tobacco samples to reduce and/or eliminate interference. 

Gas chromatographic analysis of tobacco alkaloids does not require derivation. The general procedures for GC analyses are as follows (1) use the smallest sample applicable for analysis (2) utilize preparations that clean up the sample without loss of alkaloids (3) pre-extract the sample prior to alkaloid extraction with hexane (removes pigments and lipids) (4) extract alkaloids from tobacco with an aqueous acid solution, filter, partition the aqueous acid extract with organic solvent (hexane, methylene chloride, chloroform, etc.) (5) increase pH of the aqueous extract to pH 10 (5N NaOH) and partition the basic solution with chloroform (6) dry the chloroform solution over Na2S04, and (7) concentrate the sample or analyze as is. 

Acetone, butanone, cyclobutanone and similar ketones have been used to prepare Schiff bases from long-chain amines, aromatic amines, diamines, anriino-phenols and catecholamines in biological media [29-34], and from hydrazines in air [35], prior to separation by GC. When electron capture detection was used, penta-fluorobenzaldehyde was the chosen reagent. This procedure was applied to the analysis of traces of amines in water samples [36] and biological extracts [37,38], as well as hydrazine in tobacco smoke, technical maleic hydrazide and pyrolysis products [39]. 

The practical potential of nonionic MLC was demonstrated by the use of micellar solutions of Brij 35 in the analysis of tobacco [18], Samples of smoking tobacco were extracted with an ueous solution of 30% Brij 35, and an aliquot of the extract was chromatographically separated without further preparation, with a 6% Brij 35 mobile phase. Comparison with an aromatic aldehyde standard mixture enabled verification of vanillin and ethylvanillin as two of the extract components. Brij 35 was chosen for this study over other nonionic surfactants (such as Tritons , Spans , Igepals or Tweens ) on the basis of its commercial availability, high purity, low cost, low toxicity, high cloud temperature, and low background absorbance, compared to the other types of surfactants mentioned. Brij 35 does not possess a strong chromophore and its absorption is minimal. 

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