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7 PHILIP MORRIS U. S. A. I N T E R' - O F' F I C E C O R' R E S P O N D E' N C E Richmond, Virginia To: Dr. A. Warfield Date: 7 Ftbruary 191819. From:.W. P. Hempfl!ing Swbject:' .1"Urnextracted Nicotine"'as: a possible precursor of pyrosynthetic. NNK. Isolation from tobacco and characterization of components that release nicotine upon treatment with hot alkali supercritical fluid extraction with1C07) following various treatments. Treatment Nicotine (ppm). 1. Extraction with methanol- 10 $ NH 4 ,OH, 3'0 °' C, 2'4 :hr 6,5'4' 2. Extraction with water, 90° C', 24 hr 57'2' 3. Extraction with 1 N NaOH, 90° C', 24 hr 14119 4. "Pyroliysis" in air, 10'50. Evidence exists that nicotine (or a direct precursor thereof) not otherwise detected is present in tobacco in a form ("Nic-X") convertible to nicotine upon treatment with hot alkali (1). Table 1 shows the amounts of nicotine detected (2) in a, tobacco blend (previously subjected to Table 1. Nicotine releasedifrom,SCFE-blend due to various.treatments Subsequent treatment of'hot water solubles from Treatment 2, above, with 1 N Na0'H (910° C, 241 hr) increased the amount of nicotine present by some 2'7'S ppm referred to the tobacco mass. Treatment with hot alkali of the marc remaining after Treatment 2I disclosed that about 492, ppm nicotine remained with the hot-water-extracted pulp. These findings suggest that at least part of the nicotine extracted from the tobacco by hot alkali is not present i~n, tobacco: as either free nicotine or a~s a salt dissociated in water. That "Nic-X" comprises two portions: one that is solub:le in hot water, and one that remain~s bound'to the insoluble matrix. I contend'.that we can take advantage of the water solubility of the first portion by isolating it using suitable fractionation procedures, and then, studying its chemical characteristics. In this manner we ought to be able to: identify the mode of bonding of nicotine to "X" and identify the nature of "X" as well. It may be that water-soluble Nic-X iss similar to water-insoluble Nic-X, but that the latter is further linked to water-insoluble components of the tobacco matrix. I suggest that the following,steps be taken to begin this study. The goal is the preparation of purified water-soluble Nic-X in sufficient

quantity for chemical characterization and for the determination of its possible role as.a precursor for NNK in smoke. li. Determine if'water-soluble Nic-X is soluble in dichloromethane. If not, extract free nicotine from CEL, andluse that extracted liquor as a source of Nic-X for fractionation. The assay for Nic-X is the difference of nicotine extractable into dichloromethane before an!d after treatment withihot alkali. Fractionations can be performed even in the presence of nicotine, but the accuracy of estimation of Nic-X will likely be diminished. 2. Separate Nic-X from neutral and anionic compounds by passing through a, sulfonic acid cation-exchange resin at pH 5. Preliminary results indicate that both nicotine and Nic-X will be bound to the resin, and can be eluted with dilute alkali in, the cold. Nic-X appears to be stable in 1 N HzSrJ, for at least 24' hr at 90° C. 3. . After neutralization and concentration (Roto-Vap), subject the preparation to gel permeation chromatography using BioGel P-4. Preliminary results sh~ow, that Nic-X is somewhat larger than nicotine. The relative enrichment with, Nic-X can be quantified by measuring the Nic-X and dry weight contents of appropriate fractions during elution. At this point highly enriched fractions can be subjected to mass spectrometric analysis,, the results of which will hopefully lead to systematic chemical characterization of Nic-X'and of "X". Such an examination ought also to include possible release of nicotine from Nic-X catalyzed by the enzyme "NADase".. 4. Upon accumulation of sufficient material, apply Nic-X to LTF or Oriental base: web and determine if NNK formation during smoking is increased in proportion to the load of Nic-X. 5. The amounts of water-soluble and water-insoluble Nic-X should be measured in tobacco preparations that differ significantly in pyrosynthetically formed NNK. I will be pleasedito participate in thiis study. 1. Hempfling;, W. Memorandum in preparation. 2. Hempflingy W. Extraction of nicotine from ART tobaccos. 17 June 1988. (Copy appended.) Copies to: C. Ellis R. Kinser -2-