WO2002012872A1 - Quality control and standardisation of tobacco by means of nmr and pattern recognition - Google Patents
Quality control and standardisation of tobacco by means of nmr and pattern recognition Download PDFInfo
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- WO2002012872A1 WO2002012872A1 PCT/GB2001/003540 GB0103540W WO0212872A1 WO 2002012872 A1 WO2002012872 A1 WO 2002012872A1 GB 0103540 W GB0103540 W GB 0103540W WO 0212872 A1 WO0212872 A1 WO 0212872A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
- G01R33/00—Arrangements or instruments for measuring magnetic variables
- G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
- G01R33/44—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
- G01R33/46—NMR spectroscopy
- G01R33/4625—Processing of acquired signals, e.g. elimination of phase errors, baseline fitting, chemometric analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
- G01N24/08—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using nuclear magnetic resonance
- G01N24/085—Analysis of materials for the purpose of controlling industrial production systems
Definitions
- the present invention relates to the use of NMR spectroscopy in combination with computer-based statistical procedures in the standardisation and quality control of tobacco.
- tobacco by either inhalation or ingestion, is a recreational activity enjoyed by a significant sector of most societies around the world.
- tobacco in leaf form is smoked, for instance in cigarettes, cigars or pipes, but it may also be chewed.
- the leaves can be pulverised for talcing in powder form as snuff.
- Tobacco is grown as a cash crop in many parts of the world including the Americas, southern Africa, Russia and India.
- the tobacco leaves are harvested and cured and are then typically bulked, graded and baled before being left to mature for one or two years prior to commercial processing.
- "Tobacco” refers to plants of the genus Nicotiana.
- N. tabacum is now the principal species used for commercial tobacco although N.rustica was the species originally introduced into Europe in the 17 th century.
- N.rustica contains a high level of nicotine and is now only cultivated on a commercial scale in Russia and northern India.
- Species of Nicotiana are in any event highly polymorphic and a given species is best regarded as an assembly of cultivars.
- Cultivars are divided into classes according to the method used for their curing which, as described below, may be (i) flue curing, (ii) fire-curing, (iii) air-curing, or (iv) sun-curing.
- the principal fire-cured type cultivar is Pry or, which originated from the flue-cured Orinico. Another fire-cured cultivar is Heavy Western. In the air-cured category Burley is the principal type cultivar. Others include Southern Maryland and Maryland Mammoth. Flue-curing is typically carried out in brick barns in which heat from wood, oil, gas or other sources is supplied in enclosed flues. It is essential for the temperature and the humidity in the barn to be controlled. Fire-curing is usually carried out in log or grass barns. The tobacco leaf is hung for 4 to 7 days to turn yellow and small open fires are then made in pits in the floor to provide smoke and to produce the creosotic and distinctive aroma.
- Air-curing is a natural process, being carried out under normal atmospheric conditions in a wood or grass barn.
- the tobacco leaf should yellow before it dries out after which the rate of drying is gradually increased by increasing the ventilation in the barn.
- sun-curing is a process of exposing tobacco leaves to the sun. It is useful to air-cure leaves for 2 to 3 days and ferment them it in heaps for 24 to 36 hours before exposing them to the sun. Sun-curing is used in the production of Turkish tobacco.
- the tobacco leaves are taken from the barn and bulked. This is typically carried out on platforms with weights placed on the bulks.
- the bulks should be large enough to allow some fermentation of the tobacco. Bulking needs to take place for at least a month before the tobacco leaves are graded (according to size, colour and texture) for marketing.
- the grade of the tobacco leaf depends partly on the position of the leaf on the plant.
- the bottom leaves are called lugs, the lower middle leaves cutters, and the upper, middle and top leaves are called leaf tobacco.
- the identity of the tobacco cultivar, the grade of the leaf and the method of curing all contribute to the characteristic properties of a given tobacco. Those properties will determine the appropriate end use for the tobacco.
- the main commercial products in which tobacco is used are cigarettes, cigars and pipe tobacco.
- Cigarettes are produced mainly from flue-cured tobacco, optionally with a proportion of light air-cured or Vietnamese tobacco added. During manufacture various additives may be used as flavouring and/or conditioning agents. Typically these are formed into a "sauce” which is applied to the tobacco leaves either by immersion or by spraying.
- the types of additive used include glycerine, licorice, sugar, molasses, menthol and tonka bean (Dipterix odorata).
- Cigars are typically made from air-cured tobacco leaf and special cultivars are used.
- Pipe tobacco is usually made from blended flue and air-cured leaf.
- Snuff is also typically prepared from dark air and/or fire-cured tobacco.
- Manufacturers of tobacco products set great store by achieving a consistent taste and quality for the goods which they market under particular brand names. Indeed, most tobacco users favour one or two commercial brands and will choose those to the exclusion of most others on the grounds that the strength, flavour and other characteristics of the product are best suited to their own individual tastes. For this reason any given brand of cigarette, cigar or pipe tobacco tends to have a loyal following of consumers.
- counterfeit branded goods present a particular threat to the authentic manufacturers. Whilst undercutting price, counterfeit manufacturers also undermine the reliability and consistency of the genuine branded tobacco products because the quality and properties of the cigarettes, cigars and pipe tobacco which they sell in lookalike packaging are significantly different from those of the genuine article.
- a related problem is that of adulteration, whereby the tobacco of the required grade and quality for a given branded product is supplemented by unauthorised manufacturers with cheaper tobacco of lower quality. As discussed above, this practice serves to undermine the reliability and consistency of the branded product as perceived by the consumer as well as undercutting the authentic manufacturer.
- tobacco can be analysed chemically for three of its constituents. These are nicotine, chloride (ions) and total sugars.
- the three analyses are performed by HPLC and GLC.
- an additional organoleptic evaluation is frequently carried out, the purpose of which is to distinguish between different batches of a given blend of tobacco or between different blends.
- this relies on an individual's qualitative discernment of subtle differences in aroma and is thus purely subjective.
- Neither the chemical analysis nor the organoleptic evaluation provides a reliable and consistent means of distinguishing between types and blends of tobacco and tobacco products. There is therefore no technique available at present which permits authentic tobacco products to be reliably and consistently differentiated from ostensibly identical products sold by counterfeit producers under the same brand name. There is no convenient method of verifying the authenticity of a branded tobacco product or of confirming that a suspected counterfeit tobacco product is indeed a fake.
- tobacco plant material encompasses tobacco plants as such, processed tobacco leaves and finished tobacco products such as cigarettes, cigars and pipe tobacco.
- the process of the present invention provides a means of defining a standard for a given tobacco plant material on the basis of a known sample of the material which possesses the particular property desired for the standard.
- a specification for the standard is established by submitting the known sample to a combination of NMR spectroscopy and a computer-based pattern recognition technique and defining the results thus obtained as the standard specification.
- Subsequent "candidate" samples of the said tobacco plant material can then be tested for compliance with the standard. They can be accepted or rejected depending on whether they give analytical results which fall within or outside either part or all of the established specification,
- the present invention accordingly provides a process (A) for establishing a standard specification for a tobacco plant material, the process comprising: (i) preparing a test solution or test extract of a sample of the medicinal tobacco plant material which is known to possess the or each property desired for the standard; (ii) submitting the said solution or extract to analysis by a combination of NMR spectroscopy and a computer-based pattern recognition technique; (iii) obtaining results from the analysis of step (ii); and
- step (iv) defining a standard specification for the said plant material on the basis of the results obtained in step (iii).
- the standard specification resulting from step (iv) is thus based on the results of NMR spectroscopy and computer-based pattern recognition.
- the invention further provides a process (B) for providing a sample of a tobacco plant material, which sample complies with a previously defined standard specification for that plant material, the process comprising:
- step (i') preparing a test solution or test extract of a candidate sample of the tobacco plant material; (if) submitting the said solution or extract to analysis by a combination of NMR spectroscopy and a computer-based pattern recognition technique; (iii') obtaining results from the analysis of step (ii'); and (iv') selecting the candidate sample if the results obtained in step (iii') comply with the standard specification for the said material established in step (iv) of the process above.
- the invention provides a process (C) for identifying and rejecting a sample of a tobacco material which fails to comply with a previously defined standard specification for that material, the process comprising.
- Processes (B) and (C) are conveniently carried out on a high-throughput batch scale. Candidate samples are taken from batches of the same tobacco plant material and submitted to steps (i') to (iv'). Process (C) is especially useful for detecting a sample of a tobacco product which is suspected of being a counterfeit or of being adulterated with non-authentic tobacco.
- the product may be, for instance, a branded cigarette, cigar or pipe tobacco.
- the "property desired for the standard " in the context of above process (A) of the present invention may be any property or quality possessed by, or attributed to, a tobacco plant material. Examples of this include a recognised commercial quality, for instance that of a branded tobacco product; a particular tobacco cultivar or variety; an authenticated origin (in terms of either tobacco growing location or commercial batch); and a particular pathological state.
- the pathological state in question may be a given level of maturity, dictated for instance by the time of harvesting, the position of the leaf on the tobacco plant or an established resistance to a parasite, herbicide, insecticide or other agent with potential for damage to the tobacco plant in question.
- the sample of tobacco plant material which is "known to possess the or each property desired for the standard" is a sample of authenticated and audited tobacco plant or finished tobacco product of which the provenance is known.
- it may be a sample of a specified tobacco cultivar, a sample of tobacco of a specific leaf grade, a sample of tobacco grown in a particular geographic location or a sample of tobacco which has been cured by a particular technique. It may alternatively be a sample of an authentic branded finished tobacco product.
- a standard specification is established by submitting that sample to NMR spectroscopy/pattern recognition as described above. Subsequent samples of the same tobacco plant material, the origin or quality of which is not known or is in doubt, can then be tested for compliance with the standard specification thus established for the authenticated and audited material.
- the process of the invention represents a significant departure from the conventional technique of analysing tobacco for specific isolated components such as nicotine or sugar.
- the present process yields data which take account of more than one compound, or class of compound, present in the tobacco plant material.
- the solvent used for the test solution or test extract is typically one which can dissolve more than one compound or class of compound present in the tobacco plant material. Preferably it is one which can solubilise members of the maximum number of different classes of compound present in the tobacco plant material .
- the classes of compound mentioned in this context include phytochemicals and other categories of compound and may be, for instance, classes based on structure or on function.
- the test solution or test extract therefore contains more than one compound present in the tobacco plant material.
- the test solution or test extract contains members of more than one class of compound present in the tobacco plant material, for instance members of more than one structural class or functional class.
- the test solution or test extract will preferably contain representatives of the maximum number of classes of compound present in the tobacco plant material which respond to the NMR technique being used. It is thus possible for the NMR data obtained in the process of the invention to reflect the maximum number, preferably the totality, of compounds or classes of compound present in the tobacco plant material which are responsive to the NMR technique being used.
- test extract is a total extract.
- a preferred solvent for the test solution or test extract is methanol.
- Nuclear magnetic resonance spectroscopy is known by itself as an analytical tool in the investigation of plant materials.
- One example of its application is in the verification of the authenticity of drinks derived from fruit.
- hydrogen-2 NMR spectroscopy has been employed with the technique of site- specific natural isotope fractionation (SNIF-NMR) as a means of establishing the authenticity of fruit juices.
- SNIF-NMR site- specific natural isotope fractionation
- the technique relies on the fact that, when a fruit juice or fruit concentrate is fermented, the proportion of the resulting ethanol molecules which are mono- deuterated on the methyl site decreases with the addition of beet sugar. Thus any fruit juice sample to which beet sugar has been added will have a significantly lower (D/H) isotope ratio than a corresponding authentic sample.
- This technique has also been applied to the detection of wine chaptalisation using hydrogen-2 NMR spectroscopy, as reported for example in J.Chim. Phys.-Chim Biol. 1983, vol 80, pp 293 - 297 by Martin et al.
- PCA principal component analysis
- PCs are ordered according to their ability to explain the variance contained in the original data.
- a projection of the samples into a space spanned by the first PCs provides an insight into the similarity or dissimilarity of the samples with respect to their biochemical composition.
- Unknown or test samples can also be projected onto this space and can thus often visually be compared with the reference samples (Vogels et al, J. Agric. Food Chem. 44, 175 - 180, 1996).
- the combination of hydrogen- 1 NMR spectroscopy with pattern recognition techniques has been applied as a screening tool in determining the authenticity of orange juice (Vogels et al, 1996 loc. cit.). The adulteration of suspect samples could be detected by this means.
- the identity of the responsible contaminants was then determined by correlation of the PCA results with particular resonances present in the original NMR spectrum.
- the NMR spectroscopic and pattern recognition procedure employed in the process of the present invention requires neither an investigation into the biochemistry of the tobacco plant material being analysed nor a subsequent correlation of the pattern recognition results with particular NMR spectroscopic resonances attributed to specific component(s) of the tobacco plant material. Instead it relies upon the information presented by the inherent pattern of clusters derived from NMR data, those data in turn reflecting the totality of the compounds in the tobacco plant material which respond to the NMR spectroscopic technique being used.
- NMR spectroscopy/pattern recognition employed in the process of the invention typically comprises:
- step (a) submitting the test solution or test extract to NMR spectroscopy and recording one or more NMR spectra; and (b) submitting the data obtained from the or each NMR spectrum to a multivariate analysis to generate one or more points on a score plot.
- a sphere of acceptability is typically defined around the point or points on the score plot generated in step (ii) above when the NMR spectroscopy/pattem recognition analysis is being used to establish a standard specification for a tobacco plant material. That sphere then constitutes part of the specification.
- Candidate samples of the same material are subsequently accepted or rejected depending whether, when submitted to the NMR spectroscopy/pattern recognition analysis defined above, they give points in step (ii) which fall within or outside the sphere.
- the present invention provides a process for providing a sample of a tobacco plant material which complies with a previously established standard specification for that material, the process comprising:
- the standard specification in this aspect of the invention may be provided by a process which comprises: (i " ' ) preparing a test solution or test extract of a sample of the said tobacco plant material which is known to possess the or each property desired for the standard; (ii” ') submitting the test solution or test extract to NMR spectroscopy and recording one or more spectra; (iii'") submitting the data obtained from the or each said NMR spectrum to a computer-based multi-variate analysis to generate one or more points on a score plot; and (iv'") defining a sphere of acceptability around the points generated in step (iii'") as the, or as part of the, standard specification for the said tobacco plant * material.
- the total data is the product of the scores multiplied by the loadings.
- the loadings plot can be used to define the contribution of each of the variables (spectral descriptors).
- a "score plot” is a graphic representation in which samples are projected into the space spanned by two or more principal component axes.
- Principal component analysis (PCA) is a particular method used to analyse data included in a multivariate analysis. In PCA the position of the samples can be plotted in a score plot in two dimensions where similar samples will tend to form clusters while dissimilar samples will tend to spread over large distances (Kowalski & Bender, 1972, loc. cit. and Trevisan, 1997, loc. cit.).
- the context in which the points are generated on the score plot in the process of the present invention must be the same when establishing the standard specification as when analysing candidate samples for compliance with the standard.
- the components of the methodology used to establish the positioning of the point or points on the score plot for the known sample used to define the standard must be present when the NMR spectroscopic data from the candidate test samples are processed.
- the data derived from the NMR spectrum of the sample used as the standard are subjected to appropriate manipulation by multivariate statistical methods, for example principal component analysis or canonical variation, together with those of the standard.
- the sphere of acceptability is defined by limits in the score plot which have been established on the basis of the position in the score plot of points derived from one or more extracts of the known sample.
- the multivariate analysis is performed using an unsupervised methodology.
- the NMR spectroscopic technique used in the invention may involve carrying out hydrogen- 1 NMR spectroscopy at high fields in combination with multivariate analysis.
- the NMR spectra are typically measured at 400 to 700 MHz.
- the data derived from them are then analysed by multivariate analysis software, for instance the commercially available "Pirouette" package. Examples of the high resolution hydrogen- 1 NMR spectroscopic and pattern recognition analysis are discussed by M. L. Anthony et al in Biomarkers 1996, 1, 35- 43 and Molecular Pharmacology 46, 199- 211, 1994, and by J.O.T. Gibb et al in Comp. Biochem. Physiol. vol. 118B No. 3, pp 587- 598, 1997.
- 1 -dimensional NMR spectroscopy using other NMR-active nuclei such as carbon- 13 or hydrogen-2 may be used in the present invention. It is also possible to use a range of 2-dimensional pulse sequence spectroscopic investigations with hydrogen- 1 or other NMR-active nuclei such as those mentioned above. The same principles apply in each case, though, and the results are analysed by appropriate multivariate analysis.
- the NMR spectra may be normalised or non-normalised before the computer- based pattern recognition is carried out. Normalisation has the effect of removing peak intensity, which is a purely quantitative parameter of the spectra, as a discriminating factor. Normalisation is therefore typically carried out when the main objective of the procedure is to highlight qualitative differences between spectra obtained from different samples. However, in some cases peak intensity may be required as a discriminating factor when absolute quantitative values, for instance potency, are required. In such situations the spectra are non-normalised. .
- NMR spectroscopy/pattern recognition is that it is not limited by a selective delivery or detection system. Spectra can be recorded without prior purification of the test solution or test extract, thus allowing all components of the tobacco material sample which possess a proton to contribute to the overall NMR spectrum. Analysis of the spectrum by the multivariate analytical techniques discussed above reveals potential valuable discriminating features of the spectra which can be used with a high degree of precision for the description of the complex mixtures of components contained in tobacco plant materials.
- the process of the invention therefore includes the additional initial step of purifying the test solution or test extract of the candidate sample of tobacco plant material prior to submitting it to NMR spectroscopy.
- Tea provides a convenient illustration of this principle. Signals due to caffeine dominate the hydrogen- 1 NMR spectrum of tea and so when NMR spectroscopic results of tea are processed by multivariate analysis the points on the score plot for samples of the same tea do not form clusters. If caffeine is removed from tea prior to carrying out NMR spectroscopy, for instance by reverse phase chromatography, proper clustering of the samples occurs and the similarities between like samples on the score plot become clear. This is illustrated in Example 2 and accompanying Figures 4 A and 4B. Figure 4A is a score plot for untreated tea samples where clustering is indistinct.
- Figure 4B is a score plot for pre-treated tea where, in contrast, there is clear clustering which allows positive discrimination.
- the NMR spectroscopy/patte recognition analysis is highly sensitive and has the capacity to differentiate samples of tobacco plant material which appear to be identical when analysed by other methodologies. This principle has again been illustrated using tea.
- Comparative Example 1 describes the analysis by high performance liquid chromatography (HPLC) of extracts of two different types of tea. The resulting chromatograms are shown in accompanying Figures 2 and 3.
- HPLC high performance liquid chromatography
- NMR spectroscopy and pattern recognition technique of the present invention can be used in the standardization and differentiation of blends of tobacco plant material.
- the technique may thus be particularly useful in distinguishing between batches of of a given tobacco blend, and between different blends of tobacco.
- the process of the invention thus allows tobacco blends to be differentiated and standardized.
- the principle of applying the invention to blends of tobacco plant material is illustrated in Example 5, which describes how samples of a Traditional Chinese Medicine remedy, which is a mixture of plant materials, may be differentiated in accordance with the invention.
- the tobacco plant material typically consists of, or is derived from, a whole tobacco plant, a part of a tobacco plant, a tobacco plant extract, a tobacco plant fraction or a finished commercial tobacco product such as cigarettes, cigars, pipe tobacco or snuff.
- Figure 1 is a principal component analysis (PCA) score plot of factor 3 (y axis) against factor 2 (x axis) for six samples of Panax ginseng obtained from different suppliers as described in Reference Example 1 .
- PCA principal component analysis
- Figure 2 shows two HPLC chromatograms for Kemmun tea, using a system optimised for the separation of catechins as described in Comparative Example 1, in which A represents an untreated tea sample and B represents a tea sample which has been previously treated by passage through a solid phase extraction column.
- Figure 3 shows two HPLC chromatograms for Lapsang Souchong tea, using a system optimised for the separation of catechins as described in Comparative Example 1, in which A represents an untreated tea sample and B represents a tea sample which has been previously treated by passage through a solid phase extraction column.
- Figure.5 is a PCA score plot of factor 3 (y axis) against factor 1 (x axis) for the five different commercial samples of Tanacetum parthenium (feverfew) capsules obtained in Example 3.
- Figure 6 is a PCA score plot of factor 2 (y axis) against factor 1 (x axis) for the seven samples of Tanacetum parthenium harvested at different intervals after planting out, as described in Example 4.
- Figure 7 is a PCA score plot of factor 2 (y axis) against factor 1 (x axis) for the Traditional Chinese Medicine remedy analysed in Example 5.
- White Ginseng is derived from the root of Panax ginseng CA. Meyer. The root is put in boiling water briefly and then soaked in the sugar juice. It is subsequently exposed and dried in the sun. White Ginseng is also known as Sugar Ginseng.
- the dried plant root material was ground to a fine powder using an "Illico" blender for five minutes. 1 g of the powder was mixed with 20mL of cold water and stirred continuously for 2 hours on a shaker at 150 rpm at room temperature (22 °C). The extract was filtered through Whatman No. 1 filter paper, the filtrate collected and freeze-dried overnight. lOmg of the sample was dissolved in 1 mL of deuterated water for NMR spectroscopic analysis. 800 ⁇ L were used for NMR spectroscopic analysis and samples were referenced internally to TSP at 0.00 ppm. Protocol for NMR spectroscopy
- Spectra were a result of 64 scans of 20 ppm sweep width and were collected into 49152 data points. Acquisition time per scan was 2.0 seconds. Prior to transformation, a line broadening of 0.3 Hz was applied and the spectra were Fourier transformed. Referencing was to TSP at 0.00 ppm. NMR spectra were analysed using AMIX software to reduce the spectra into "histograms" containing buckets of
- Untreated samples Dried commercial tea sample (50 g) was ground to a fine powder for approximately 1 minute using a Moulinex "Illico' blender, to produce a homogeneous sample. 5g of the powder was removed and placed in a 100 mL glass conical flask at room temperature (22°C). 50 mL of boiling distilled water was poured over the tea sample which was stirred with a plastic rod and left at room temperature for 30 minutes. The extract was filtered through a Whatman No. 1 filter paper, the filtrate collected and freeze dried overnight. 10 mg of the sample were dissolved in 1 mL of distilled water.
- HPLC equipment Hewlett Packard series II 1090 Liquid Chromatograph
- HPLC column C18 RP Hypersil 5 ⁇ , 150 x 4.6 mm
- HPLC mobile phase Methanol: Water: Orthophosphoric acid 20:79.9:0.1 v/v, flow rate 0.9 ml/minute, absorption wavelength 21 Onm.
- Treated samples Dried sea sample (50g) was ground to a fine powder for approximately 1 minute using a Moulinex "Illico" blender, to produce a homogenous sample, 50 mL of boiling distilled water was poured over the tea sample which was stirred with a plastic rod and left at room temperature for 30 minutes. The extract was filtered through a Whatman No. 1 filter paper, the filtrate collected and freeze dried overnight. 10 mg of the sample was dissolved in 1 mL of distilled water which was run through a Reverse Phase (RP) Cl 8 Isolute R column under vacuum. 20 ⁇ L were used for the HPLC analysis, following the method described above for the untreated samples.
- RP Reverse Phase
- the teas used were Gunpowder, Darjeeling, Kemmun, Assam, Lapsang Souchong and Oolong. Untreated and treated samples of these teas were prepared as described in Comparative Example 1.
- the 10 mg tea samples were dissolved in 1 mL of deuterated water (D 2 O). 800 ⁇ L were used for NMR spectroscopic analysis and samples were referenced internally to TSP at O.OOppm. The procedure described in Reference Example 1 was carried out on each of the tea extracts.
- feverfew Teacetum parthenium
- the capsule contents from each sample were placed in a Moulinex "Illico” blender and the material ground to a fine powder for two minutes to produce a homogeneous sample.
- Water extracts were prepared for NMR spectroscopic analysis by dissolving 500 mg of the powder in 50 mL cold water and stirring the extract for four hours on a shaker at 150 rpm at room temperature (22°C). The extract was filtered through Whatman No. 1 filter paper, the filtrate collected and freeze dried overnight.
- FIG. 5 attached, in which each point corresponds to one capsule.
- the plot shows that samples 1, 4 and 5 gave good clustering that reflects consistency between individual capsules in those batches of products. Samples 2 and 3 gave poorer clustering, reflecting greater variability in those particular products from capsule to capsule.
- the plot also highlights distinct differences between different batches of one product from one manufacturer (samples 4 and 5) which are claimed to be identical.
- Tanacetum parthenium (Feverfew) was grown in the UK from warranted seed obtained from CN. Seeds, Ely, UK. Seeds were sown on 18 th April and raised under glass. Plantlets emerged on 25 th April and were planted out on 24 th May in plots laid out on a grid basis to provide a randomised sampling regime.
- Dried plant material was collected and immediately placed in a freezer at -20°C. After freezing, the material was placed in a freeze drier for 12 hours after which samples were checked to ensure they were dry. The material was placed in a Moulinex "Illico" blender, and the material ground to a fine powder for two minutes to produce a homogeneous sample.
- Water extracts were prepared for NMR spectroscopic analysis by dissolving 500 mg of plant material in 50 mL cold water and stirring the extract for four hours on a shaker at 150 rpm at room temperature
- Example 1 Use of NMR Spectroscopic and Multivariate Analysis to discriminate between sources and batches of tobacco
Abstract
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EP01954180A EP1307730A1 (en) | 2000-08-04 | 2001-08-06 | Quality control and standardisation of tobacco by means of nmr and pattern recognition |
AU2001276526A AU2001276526A1 (en) | 2000-08-04 | 2001-08-06 | Quality control and standardisation of tobacco by means of nmr and pattern recognition |
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GB0019264A GB0019264D0 (en) | 2000-08-04 | 2000-08-04 | Process for quality control and standardisation of tobacco |
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- 2001-08-06 TW TW90119144A patent/TW544286B/en active
- 2001-08-06 WO PCT/GB2001/003540 patent/WO2002012872A1/en not_active Application Discontinuation
- 2001-08-06 EP EP01954180A patent/EP1307730A1/en not_active Withdrawn
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Also Published As
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TW544286B (en) | 2003-08-01 |
EP1307730A1 (en) | 2003-05-07 |
AU2001276526A1 (en) | 2002-02-18 |
GB0019264D0 (en) | 2000-09-27 |
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