Molecularly imprinted polymers selective for nitrosamines and methods of using the same

Abstract

A class of molecularly imprinted polymers that specifically recognizes and binds to nitrosamines members of which class are useful, for example, in analysis and separation of nitrosamines from biological fluids. Such molecularly imprinted polymers are also useful in methods of treating and manufacturing tobacco products and materials.

Claims

1. A molecularly imprinted polymer selective for at least one tobacco-specific nitrosamine, wherein said molecularly imprinted polymer is obtained using a functional or structural analogue of a tobacco-specific nitrosamine as a template, and includes an imprint of the functional or structural analogue of a tobacco-specific nitrosamine.

2. The molecularly imprinted polymer according to claim 1, wherein said at least one tobacco-specific nitrosamine is derived from nicotine, nornicotine, anabasine or anatabine.

3. The molecularly imprinted polymer according to claim 1, selective for the nicotine-derived nitrosamine NNAL.

4. The molecularly imprinted polymer according to claim 1, wherein the polymer has been prepared using an isosteric analogue of a tobacco-specific nitrosamine as a template.

5. The molecularly imprinted polymer according to claim 1, wherein the polymer has been prepared using 4-(methylpropenyl-amino)-1-pyridin-3-yl-butan-1-ol as a template.

6. The molecularly imprinted polymer according to claim 1, wherein the polymer has been prepared using pyridine carbinol as a template.

7. The molecularly imprinted polymer according to claim 1, wherein the polymer has been prepared using a compound that includes a substructure of the tobacco specific nitrosamine.

8. A kit, comprising: the molecularly imprinted polymer according to claim 1; and instructions for using the molecularly imprinted polymer to perform at least one of detecting, quantifying, and separating nitrosamines in a sample.

9. A smoking article, comprising: a smoking material; and the molecularly imprinted polymer according to claim 1, selective for at least one tobacco-specific nitrosamine found in the thermall decomposition products of the smoking material.

10. The smoking article according to claim 9, wherein the molecularly imprinted polymer is selective for at least one volatile nitrosamine found in the vapor phase of the thermal decomposition products of the smoking material.

11. A smoke filter, comprising: filter material; and the molecularly imprinted polymer according to claim 1.

12. The smoke filter according to claim 11, wherein the molecularly imprinted polymer is selective for at least one volatile nitrosamine found in the vapor phase of the thermal decomposition products of tobacco or a tobacco substitute.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows an outline of the procedure for synthesis of an imprinted polymer;

(2) FIG. 2 shows the nitrosamine functional group and examples of nicotine related-nitrosamine targets;

(3) FIG. 3 shows isosteric analougues of nitrosamines;

(4) FIG. 4A shows examples of amide and sulfonamide based target analogs;

(5) FIG. 4B shows an enamine target analogue (MPAPB) used as a template to prepare a MIP for extraction of NNAL;

(6) FIG. 4C shows pyridine carbinol used as a template prepare a MIP for extraction of NNAL;

(7) FIGS. 5A and 5B show recovery rates of NNAL using an NNAL-selective MIP;

(8) FIG. 6 shows chromatograms obtained after analysis of 1 mL human urine spiked with 0.25 μg NNAL (represented by solid line) and 1 mL blank human urine (represented by bold line);

(9) FIG. 7 show an overlay of chromatograms obtained after sample analysis in the presence of nicotine where the solid line represents NNAL and nicotine-spiked sample, dashed line represents eluent collected from loading 1 mL of the NNAL and nicotine-spiked sample, and the long dashed line represents a wash of (NH.sub.4)H.sub.2PO.sub.4, pH 4.5;

(10) FIG. 8 is a side elevation, partly longitudinal cross-section and partially broken away view of a smoking article with a smoke filter according to the invention;

(11) FIG. 9 is a similar view to FIG. 8 of a smoking article with an alternative smoke filter according to the invention;

(12) FIG. 10 shows the chemical structure and boiling point for three products described in Example 6; and

(13) FIG. 11 shows the chemical structure and boiling point for select volatile nitrosamines.

(14) In the drawings, similar features are gives like reference numerals.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(15) Molecular imprinting typically consists of the following steps: (1) a template compound, which may be the targeted molecule or a structural analogue thereof, is allowed to interact with a selected functional monomer, or monomers, in solution to form a template-monomer complex; (2) the template-monomer complex is co-polymerized with a cross-linking monomer resulting in a polymeric matrix incorporating the template compound; (3) the template compound is extracted from the polymer matrix to form a MIP that can be used for selective binding of the targeted molecule. Prior to step (3), where the MIP is prepared as a solid polymer (or monolith) it is typically crushed and sieved to obtain a desired size fraction of particulate material. When prepared by either suspension or emulsion polymerization methods, such crushing and sieving is unnecessary since the particle size can be controlled within the desired limits during the polymerization process. Particulate material prepared by any of the aforementioned methods can be packed into a chromatographic or solid phase extraction column and used for chromatographic separation of the template from other components of a mixture, including molecules with similar structures or functionalities.

(16) The reactive sites on the molecularly imprinted polymer exposed by removal of the template composed will be in a stereo-chemical configuration appropriate for reaction with fresh molecules of the targeted molecule. As a result, the polymer can be used for selective binding of the targeted molecule.

(17) Currently the most widely applied technique to generate molecularly imprinted binding sites is via the ‘non-covalent’ roots. This makes use of non-covalent self-assembly of the template compound and functional monomers to form the template-monomer complex, followed by free radical polymerization in the preserve of a cross-linking monomer and finally template compound extraction. Covalent imprinting, in which the template molecule and a suitable monomer or monomers are covalently bound together prior to polymerization can also be carried out according to known methods. The birthing properties of the MIPs formed by either of the above methods can be examined by reminding of the template molecule.

(18) The polymerization is performed in the presence of a pore-forming solvent called a porogen. In order to stabilize the electrostatic interactions between the functional momomers and the template compound the porogen is often chosen from among aprotic solvents of low to moderate polarity. Ideally, template compounds exhibit moderate to high solubility in the polymerization media and these, or their structural analogues, can therefore be used directly using this standard procedure.

(19) While it is possible to use the targeted molecule itself as the template, a structural analog of the target molecule is commonly preferred because: (a) the targeted molecule may be unstable under the polymerization conditions or may inhibit the polymerization; (b) the targeted molecule may not be available in sufficient quantities due to complexity of its synthesis or cost, or both; (c) the template may be insoluble or poorly soluble in the pre-polymerization mixture; (d) the MIP may remain contaminated by low levels of the targeted molecule retained in poorly accessible regions of the polymer matrix, which may bleed from the MIP during use; and/or (e) the target analyte(s) may present a significant health risk and should not be used as a template(s).

(20) In the case of nitroso-compounds, particularly the compounds known as TSNAs described below, it is often more convenient to use functional analogues thereof as template compounds. For example, glucose derivatives of TSNAs may be particularly useful as template compounds, see FIG. 2.

(21) Where the MIP is derived using a functional analog of the targeted compound, the functional analogue should be isosteric and preferably also isoelectronic with the targeted compound, or it may contain a substructure of the targeted compound where strong interactions may be likely.

(22) Nitroso-containing compounds, particularly nitrosamines, which have the general formula O═N—N(R.sub.1)(R.sub.2) are among the numerous ingredients of tobacco and tobacco smoke that have been suggested as having a harmful effect on consumers.

(23) One particular class of nitroso compounds to which the present invention is applicable is the group of nitrosamines that occur naturally in tobacco, known as tobacco-specific nitrosamines (TSNAs), which are derived from the alkaloids that occur naturally in tobacco namely nicotine, nornicotine, anabasine and anatabine. TSNAs include:

(24) ##STR00001##

(25) In addition, a group of compounds known as volatile nitrosoamines is found in the vapor phase of tobacco smoke. This group includes the following compounds:

(26) ##STR00002##

(27) Other nitroso-containing compounds have also been identified in chemical studies of tobacco or tobacco smoke, for example:

(28) ##STR00003##

(29) Possible isosteric analogs for the targeting of nitrosamines are seen in FIG. 3. The molecules shown are all derivatives of the parent amine and can be synthesized in a single step from the secondary amine and corresponding aldehyde or acid chloride. Molecular models of the enamine (FIG. 4A) have shown a good steric complementarily with one of the nitrosamines of interest, NNAL.

(30) During the design of a suitable template compound for the target analyte NNAL, a particularly interesting template was identified, corresponding to the pyridine carbinol substructure but surprisingly lacking the nitrosamine moiety (FIG. 4B). If sufficient binding affinity and selectivity can be obtained for sub-structural templates, this is the preferred approach. In fact, the binding affinity, selectivity and recoveries obtained with this pyridine carbinol MIP are superior to the MIPs obtained, with the more complex enamine template. Thus, the invention provides a surprisingly effective MIP which comprises a simple template lacking certain key features of the target but providing for effective binding with those target nitrosamines which contain the pyridine-methanol moiety.

(31) Using the functional monomer methacrylic acid (MAA), either of two crosslinkers, ethylene glycol dimethacrylate (EDMA) or trimethylopropane trimethacrylate (TRIM) and either of the two NNAL analogs, 4-(Methylpropenyl-amino)-1-pyridin-3-yl-butan-1-ol (4MPAPB, FIG. 4A) and pyridine carbinol (FIG. 4B) as templates, two different polymers are obtained both exhibiting strong affinity and selectivity for NNAL in organic and aqueous solvent environments.

(32) This invention includes an extraction method for quantitative recovery of the nicotine analog NNAL that entails the steps of preparation of an NNAL-selective MIP in a chromatographic material format, column conditioning, application of a urine sample, removal of interfering compounds and finally selective elution of the NNAL analyte.

(33) By way of explanation and not of limitation, the invention will be further described in more detail with reference to a number of examples. The invention refers to template molecules, polymer materials designed to bind nitrosamines deriving from nicotine and present in organic or aqueous systems, and finally use of said materials in, for example, analytical or preparative separations, in chromatography, for analytical sample pre-treatment and in chemical sensors.

(34) Unless otherwise described, materials are commercially available or can be prepared by conventional techniques. See, for example, B. Sellergren (Ed.) Molecularly-imprinted Polymers: Man made mimics of antibodies and their application in analytical chemistry, part of the series Techniques and Instrumentation in Analytical Chemistry, Elsevier Science, Amsterdam, Netherlands, 2001.

Example 1: Synthesis of Enamine Template (MPAPB)

(35) Anhydrous toluene (freshly dried over sodium) 2 ml was added to a vial containing 4-methylamine-1-(3-pyridyl)-1-butanol (100 mg). 500 mg freshly dried Molecular sieve was added to it. The mixture was stirred for 1 hour under N.sub.2. To the mixture 100 μl propionaldehyde was added. The mixture was stirred at 55° C. for 4 hours. The reaction was monitored by HPLC after 1.5 hours. The color of the product was orange-yellow in toluene. The crude product in toluene was directly used for the synthesis of the MIP alter filtration without publication. Template MPAPB yield was around 90%.

Example 2: Synthesis of MIP Using Pyridine Carbinol as Template

(36) To pyridine methanol (97 μl) 3.74 ml of purified TRIM (purified over basic alumina), functional monomer MAA (1020 μl), porogenic solvent toluene (7.1 ml) and finally initiator ABDV (63 mg) were added and stirred until a clear solution was obtained. The solution was transferred to a glass vial, purged with nitrogen for 5 minutes and flame sealed. Heal induced polymerization was carried out at 45° C. for 24 hours. The polymer mixture was then cured at 70° C. for a further 24 hours.

(37) Processing of the crude MIP material was as follows: the MIP was coarsely crashed and transferred to a Soxhlet thimble. It was excessively washed first with methanol for 12 hours and then with acetic acid for 12 hours in order to remove any remaining template and other non-reacted monomers. After these first extraction steps, the polymer was vacuum dried and then ground and sieved to a line powder within a size range of 20 to 90 μm. As a final extraction step, the finely ground MIP was subjected to a 40 minutes microwave assisted solvent extraction using formic acid as the extraction solution. After drying, the MIP was ready for use.

Example 3: Use of MIPs as Selective Sorbents in SPE

(38) In one embodiment of the invention, the MIP can be packed into solid phase extraction columns for the selective extraction of NNAL from a biological matrix. First, a polypropylene frit was placed in an appropriate SPE column (typically 10 ml capacity for analytical uses), 25 mg of the MIP was then added on top to form a MIP bed and the second frit was firmly pressed onto the surface of the MIP bed. Conditioning of the column was carried out in the following order: 1 ml DCM, 1 ml MeOH and finally 1 ml distilled water were added to the MIPSPE.

(39) The sample, e.g. human urine (5 mL) containing low amounts of the analyte was allowed to pass through the conditioned MIPSPE column. The column was then subjected to vacuum in order to remove the water until the material was dry. Then, polar interfering substances that may have non-specifically associated with the MIP were eluted by a wash with 1 ml distilled water. Again, a drying step using several minutes of vacuum was performed in order to enable the so-called phase-switch (change of the environment from aqueous to organic). At this point, non-polar interfering substances were removed by washes with each 1 ml toluene, toluene:DCM (9:1) and toluene:DCM (4:1). The final selective elution of NNAL was carried out in 3 times elution steps, each of 1 ml DCM.

(40) After solvent evaporation, the samples were reconstituted in the mobile phase and analyzed on an HPLC system: e.g. Merck-Hitachi (L-7000 system) using a beta-basic C18 column, 5 μm, 150×2.1 mm+pre-column 10×2.1 mm. Flow was at 0.25 mL/min, injection volume 100 μL, temperature 30° C. and detection at UV 262 nm. The mobile phase consists of 50 mM NH4PO4 pH 3, 5 mM octanesulfonic acid 20% methanol.

(41) Under these conditions, NNAL was obtained as a clearly distinguishable double peak eluting at about 8-10 minutes (see FIG. 6, where a 1 ml sample of human urine spiked with 0.25 μg NNAL is compared with NNAL-free urine). The double peak is characteristic for NNAL as it corresponds to its two rotamers. From the structure of NNAL, it can be demonstrated that the side chain on the pyridine ring can have different conformational states. The preferred conformations are called rotamers and for NNAL there are two major conformations. The retention of these two rotamers on an HPLC column will differ. As shown in FIG. 6, the NNAL peak is cleanly separated from interfering substances. It can therefore be easily and accurately quantified. Recovery rates for NNAL (defined as Amount recovered/Amount loaded×100) are typically up to 90%, depending on the initial levels of NNAL in the biological sample. Recovery rates of close to 100% have been seen with samples containing 50 pg/ml and 500 pg/ml of NNAL (FIGS. 5A and 5B).

Example 4: Use of MIPs as Selective Sorbents in SPE in the Presence of Nicotine

(42) Another application of the invention is the use of the MIP as a selective sorbent for NNAL where there are high levels of nicotine present. This illustrates the wide scope of applications of the MIP material and how the selective nature of the MIP can be finetuned for particular samples.

(43) SPE columns were prepared as described in Example 3. Conditioning of the SPE column was carried out in the following order: 1 ml DCM followed by 1 ml MeOH followed by 1 ml 50 mM (NH.sub.4)H.sub.4PO.sub.4, pH 4.5. The sample, in this example 5 mL human urine-containing low amounts of the analyte was allowed to pass through the conditioned MIPSPE column. The column was then subjected to a mild vacuum (e.g., 10-80 kPa) to remove water until the material was dry. Polar interfering substances that may have nonspecifically associated with the MIP were eluted by a wash with 1 ml 50 mM (NH.sub.4)H.sub.2PO.sub.4, pH 4.5. Another drying step of several minutes of mild vacuum was performed. Further, washes with 1 ml each toluene, toluene:DCM (9:1) and toluene:DCM (4:1) were performed in that order. The final selective elution of NNAL was carried out in 3 elution steps each of 1 ml DCM.

(44) After solvent evaporation the samples were reconstituted in the mobile phase and analyzed on an HPLC system similar to that described in Example 3. An example chromatogram is shown in FIG. 7, which illustrates how the NNAL is selectively retained on the MIP while the nicotine is removed in the buffer wash.

Example 5: Smoking Articles Incorporating MIPs

(45) Referring to the drawings, FIGS. 8 and 9 illustrate smoking articles in the form of cigarettes having a rod 1 of tobacco encased in a wrapper 2 attached to a smoke filter 3 by means of a tipping paper 4. For clarity, the tipping paper 4 is shown spaced from the wrapper 2, but in fact they will lie in close contact.

(46) In FIG. 8, the smoke filter 3 comprises three cylindrical filter elements 3a, 3b, 3c. The first filter element 3a at the mouth end of the filter is 7 mm in length, composed of cellulose acetate tow impregnated with 7% by weight of triacetin plasticizer having a 25 mm water gauge pressure drop over its length. The second filter element 3b, positioned centrally is a cavity 5 mm in length containing 150 mg of activated carbon granules. The third filter element 3c adjacent the rod 1 is 15 mm in length, has a 90 mm water gauge pressure drop over its length, and comprises 80 mg cellulose acetate tow. The tow is impregnated with 4% by weight of triacetin and has 80 mg of MIP specific for volatile nitroamines, produced as described in Example 6 below, distributed evenly throughout its volume in a “Dalmatian” style.

(47) The cigarette shown in FIG. 9 is similar to that of FIG. 8 except that the smoke filter 3 has four coaxial, cylindrical filter elements 3a, 3b, 3c and 3d. The first filter element 3a at the month end of the cigarette is 5 mm in length, and composed of cellulose acetate tow impregnated with 7% by weight of triacetin plasticizer. The second filter element 3b, positioned adjacent the first filter element 3a is a cavity 5 mm in length containing 200 mg of molecularly-imprinted polymer specific for volatile nitrosamines, produced as described in Example 6 below. The third filter element 3c adjacent the second filter element 3b is 10 mm in length and comprises cellulose acetate tow impregnated with 7% by weight of triacetin. The fourth filter element 3d lies between the third filter element 3c, is 7 mm in length and comprises 80 mg of granular activated carbon. A ring of ventilation holes 5 is formed in the tipping paper 4 in a radial plane A-A which deliver air into the third filter element 3c about 3 mm downstream of the junction with the fourth filter 5 element 3d when smoke is inhaled through the cigarette.

(48) The following Examples further illustrative this aspect of the invention.

(49) ##STR00004##

(50) Two equivalents of an appropriate secondary amine, e.g. dimethylamine, diethyl amine, pyrrolidine, piperidine or morpholine, are dissolved in anhydrous ether and freshly dried molecular sieves (50 g/mole amine) are added. The mixture is then cooled to −5° C. and stirred. One equivalent of propionaldehyde is then added drop-wise to the cooled mixture, maintaining the temperature at 0±5° C. The mixture is allowed to stand in a cold bath overnight and is then filtered. The product is obtained in approximately 50% yield by distillation of the filtrate under reduced pressure, depending on the boiling point of the product. By way of example, structures and boiling points are shown in FIG. 10. (See, Brannock, et. al., J. Org. Chem., 1964, 29, 801-812.)

(51) By using a strong acid functional monomer, the enamine is protonated, thus creating the necessary non-covalent interaction during the imprinting step. The positive charge resides on the carbon atom attached to the nitrogen, a structure stabilized due to derealization to give an iminium ion. This positions the acidic functional monomers correctly for later recognition of volatile nitrosamines. As there is no opportunity to delocalise the positive charge, protonation of the enamine nitrogen is disfavored. (See, Cook, et al., J. Org. Chem., 1995, 60, 3169-3171.)

(52) It may be preferred to use a more strongly-acidic functional monomer than MAA. Further 5 embodiments incorporate 4-vinylbenzoic acid or 4-vinyl benzene sulphonic acid as functional monomers.

Example 7: Synthesis of a MIP Using an Enamine as Template

(53) A pre-polymerization solution is prepared by dissolving the desired enamine (1 mmol), an acidic functional monomer (4 mmol), a cross-linking monomer (20 mmol) and a free-radical initiator (1% w/w total monomers) in a appropriate porogenic solvent. The functional monomer is either MAA or trifluoromethacrylic acid (TFMAA), the cross-linker is either EDMA or TRIM, the free-radical initiator is ABDV and the porogenic solvent is one of chloroform, toluene, acetonitrile or acetonitrile/toluene (1/1 v/v). The solution is transferred to a polymerization vessel, cooled to 0° C. and then purged with N.sub.2 for 5 minutes, after which the vessel is flame sealed. Polymerization is initiated at 45° C. and allowed to continue at this temperature for 24 hours. The polymer is then cured at 70° C. for a further 24 hours.

(54) The crude MIP material is then processed. The MIP is coarsely crushed and transferred to a Soxhlet thimble. It is then extensively extracted (i) with methanol for 12 hours and (ii) with acetic acid for 12 hours, in order to remove the template molecule and any unreacted monomers. After these first extraction steps, the polymer is vacuum dried, ground, and sieved to give particles of the desired size range, e.g. 25-36 μm. The finely-ground MIP is then subjected to a final extraction step, involving 40 minutes microwave assisted extraction using formic acid as the extraction solvent. The MIP is then dried in vacuo for 24 hours.

(55) Alternatively, the target TSNA may be used in place of the enamine. The boiling points of select volatile nitrosamines at normal atmospheric pressure are shown in FIG. 11.

Example 8: Use of the MIP Material of Example 2 and/or Example 7 in the Treatment of Tobacco Extracts

(56) The polymer produced in accordance with the method of Example 2 or Example 7 is incorporated into a solid phase extraction column, and the column is conditioned by passing through dichloromethane (DCM), methanol and finally distilled water.

(57) Shredded Burley tobacco leaf is extracted with water for 15 minutes at 60° C. The tobacco is separated from the solution by filtration and dried. The solution is passed through the column and allowed to adsorb TSNA from the extract. The column is then drained and the solution concentrated by film evaporation, the concentrate is then recombined with the extracted tobacco and dried in air.

(58) TSNA adsorbed by the polymer can be eluted from the column using DCM.

Example 9: Use of the MIP Material of Example 2 or Example 7 in the Treatment of Tobacco Extracts

(59) Flue-cured shredded tobacco leaf is extracted with water for 15 minutes at 60° C. The tobacco is separated from the solution by filtration and dried. The solution is mixed with the MIP of Example 2 or Example 7, during which period the polymer adsorbs the TSNAs selectively from the solution. The MIP is then mechanically separated from the extract by filtration or by centrifugation. The solution is concentrated by evaporation; the concentrate is then recombined with the extracted tobacco and dried in air.

(60) The MIP can be regenerated by elution with DCM, methanol and finally deionised water or pH 4 buffer, for reuse.

Example 10: Use of the MIP Material of Example 2 or Example 7 in the Treatment of Tobacco Extracts

(61) Using a continuous extraction process, US Blend-type shredded tobacco leaf is loaded into a first extraction chamber into which super-critical carbon dioxide is fed. After contacting the tobacco, the carbon dioxide is fed into a second extraction chamber containing a MIP produced as described in Example 2 or Example 7. Having contacted the polymer, the carbon dioxide is returned to the first extraction chamber and contacted again with the tobacco. The cyclic process is continued until the TSNA content of the tobacco has been reduced to a desired level, whereupon the carbon dioxide is vented from the system, and the tobacco removed from the first chamber. The MIP in the second chamber is then regenerated using DCM, methanol and acetic acid.

Example 11: Use of the Molecularly-Imprinted Polymer Material Developed for 4-methylnitrosoamino-1-(3-pyridyl)-1-butanol (NNAL), in the Treatment of an NNAL and Nicotine Containing Solution

(62) The polymer produced in accordance with the method of Example 2 was incorporated into a solid phase extraction column, and the column was conditioned by passing through phosphate buffer solution.

(63) Aqueous standard solutions of NNAL and nicotine were prepared in phosphate buffers over the pH range 3.0-7.5. The buffered standard solution was passed through the column, this fraction was collected and analyzed for NNAL and nicotine content. A buffered wash solution was passed through the column, this fraction was also collected and analyzed for NNAL and nicotine content.

(64) The solutions were analyzed by HPLC with UV detection. Optimum conditions for the MIP to retain NNAL and recover nicotine are observed at the pH range 4.0-4.5. At lower pH values the nicotine is protonated and has little interaction with the polymer, so is carried through with the aqueous buffer.

Example 12: Use of the MIP material developed for 4-methylnitrosoamino-1-(3-pyridyl)-1-butanol (NNAL), in the Treatment of a NNAL and TSNA Containing Solution

(65) The polymer produced in accordance with the method of Example 2 is incorporated into a solid phase extraction column, and the column was conditioned by passing through dichloromethane (DCM), methanol and finally distilled water.

(66) Aqueous standard solutions of NNAL and TSNAs (NAB, NAT, NNK and NNN) were acidified with glacial acetic acid to pH 3. The standard solution was passed through the column, followed by three glacial acetic acid solution washes. This fraction was analyzed for NNAL and TSNA content by GC-TEA. Three washes of dichloromethane were passed through the column, this fraction was also analyzed for NNAL and TSNA content.

(67) The MIP retained 91% of the NNAL, 65% of the NNK and an efficiency of about 20-30% for the other (less structurally similar) TSNAs.

(68) The foregoing disclosure has been set forth merely to illustrate the invention and is not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art, the invention should be construed to include everything within the scope of the appended claims and equivalents thereof.