Methods for reducing matrix-bound nicotine-derived nitrosamine ketone in tobacco plant material

Abstract

A method of reducing the amount of matrix-bound NNK in cured tobacco plant material is provided comprising reducing the amount of lignin in the cured tobacco plant material. A further method of reducing the formation of matrix-bound NNK during the curing of tobacco plant material is described comprising reducing the amount of lignin therein prior to curing.

Description

FIELD OF THE INVENTION

[0001] The present invention relates, in general, to methods for reducing the amount of nicotine-derived nitrosamine ketone or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in tobacco plant material.BACKGROUND OF THE INVENTION

[0002] During the manufacture and processing of tobacco products, by-products—such as tobacco stems, and leaf scraps—are produced. Tobacco stems and tobacco fines from manufacturing processes are unsuitable for use directly in the manufacturing of tobacco products. Since the stems and fines represent a substantial amount of raw material investment, processes have been developed to further convert these stems and fines into products—such as reconstituted tobacco materials (eg. reconstituted tobacco sheets)—which are then useable in relatively large amounts in a mixture with acceptable processed tobacco leaf. Reconstituted tobacco can be manufactured in a slurry or cast sheet process wherein pulp of mashed tobacco stems and other parts ...

Examples

example 1

Method for Analysis of Free and Matrix-Bound NNK in Tobacco

[0116]Aliquots of tobacco samples (for example, about 750 mg) are extracted with about 30 mL of Tris-HCl buffer (50 mM; pH 7.4) by shaking for about one hour at approximately room temperature. Internals standard (100 ng / mL NNK-d4) are added. Samples (0.4 mL) of the extracts are filtered using a 0.2 μM filter and the NNK content is analysed using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The sample concentrations calculated from these extract concentrations correspond to the “free NNK” concentrations in the sample. After treating the extraction mixtures (for example, by heating to about 130° C. for about 4 hours) and filtering aliquots of the extracts, NNK concentrations are again measured by UPLC-MS / MS. From these values, the “total NNK” concentration in the samples can be calculated. The “matrix-bound NNK” concentration is the difference between the “total NNK” and the “free NNK” concent...

example 2

UPLC Analysis

[0118]The column used is Waters Acquity BEH C18, 1.7 μm, 2.1×50 mm. The eluents used are: (A) ammonium bicarbonate (10 mM; adjusted to pH 9.8 with ammonia)+2% (v / v) acetonitrile; (B) acetonitrile. The gradient used is 0 min—5% B; 0.5 min—5% B; 3.3 min—18.3% B. The flow that is used is 0.5 mL / min. The column temperature that is used is 50° C.

example 3

MS / MS Methodology

[0119]This analysis is carried out on a Waters TQ spectrometer using the following MRM transitions: NNK: 208.2→122.2; dwell time 100 ms; NNK-d4: 212.2→126.2; dwell time 100 ms; Capillary voltage: 0.6 kV; Cone voltage: 25 V; Collision energy: 11 eV; Source temperature: 120° C.; Desolvation temperature: 400° C.; Desolvation gas flow: 800 L / h.

FIELD OF THE INVENTION

[0001] The present invention relates, in general, to methods for reducing the amount of nicotine-derived nitrosamine ketone or 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in tobacco plant material.BACKGROUND OF THE INVENTION

[0002] During the manufacture and processing of tobacco products, by-products—such as tobacco stems, and leaf scraps—are produced. Tobacco stems and tobacco fines from manufacturing processes are unsuitable for use directly in the manufacturing of tobacco products. Since the stems and fines represent a substantial amount of raw material investment, processes have been developed to further convert these stems and fines into products—such as reconstituted tobacco materials (eg. reconstituted tobacco sheets)—which are then useable in relatively large amounts in a mixture with acceptable processed tobacco leaf. Reconstituted tobacco can be manufactured in a slurry or cast sheet process wherein pulp of mashed tobacco stems and other parts ...

Examples

example 1

Method for Analysis of Free and Matrix-Bound NNK in Tobacco

[0116]Aliquots of tobacco samples (for example, about 750 mg) are extracted with about 30 mL of Tris-HCl buffer (50 mM; pH 7.4) by shaking for about one hour at approximately room temperature. Internals standard (100 ng / mL NNK-d4) are added. Samples (0.4 mL) of the extracts are filtered using a 0.2 μM filter and the NNK content is analysed using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). The sample concentrations calculated from these extract concentrations correspond to the “free NNK” concentrations in the sample. After treating the extraction mixtures (for example, by heating to about 130° C. for about 4 hours) and filtering aliquots of the extracts, NNK concentrations are again measured by UPLC-MS / MS. From these values, the “total NNK” concentration in the samples can be calculated. The “matrix-bound NNK” concentration is the difference between the “total NNK” and the “free NNK” concent...

example 2

UPLC Analysis

[0118]The column used is Waters Acquity BEH C18, 1.7 μm, 2.1×50 mm. The eluents used are: (A) ammonium bicarbonate (10 mM; adjusted to pH 9.8 with ammonia)+2% (v / v) acetonitrile; (B) acetonitrile. The gradient used is 0 min—5% B; 0.5 min—5% B; 3.3 min—18.3% B. The flow that is used is 0.5 mL / min. The column temperature that is used is 50° C.

example 3

MS / MS Methodology

[0119]This analysis is carried out on a Waters TQ spectrometer using the following MRM transitions: NNK: 208.2→122.2; dwell time 100 ms; NNK-d4: 212.2→126.2; dwell time 100 ms; Capillary voltage: 0.6 kV; Cone voltage: 25 V; Collision energy: 11 eV; Source temperature: 120° C.; Desolvation temperature: 400° C.; Desolvation gas flow: 800 L / h.

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