13 C CP / MAS NMR sample tube and its application, and method for determining pectin content

Abstract

The present invention belongs to the field of detection and analysis, and relates to a method for 13 The present invention also relates to a method for determining the pectin content in tobacco or tobacco products and the use of the sample tube. The method of the present invention can accurately determine the pectin content in tobacco or tobacco products with high precision and good repeatability.

Description

Technical Field

[0001] The present invention belongs to the field of detection and analysis, and relates to a method for 13 The invention relates to a sample tube for C CP / MAS NMR detection, and also relates to the application of the sample tube and a method for determining the pectin content in tobacco or tobacco products. Background Art

[0002] Pectin is a group of polygalacturonic acids found in tobacco at a concentration of 5% to 13%. While pectin helps maintain the moisture content of tobacco, incomplete combustion of pectin can lead to elevated levels of harmful substances such as methane, methanol, and carbonyl compounds in the smoke, potentially harming smokers' health. Furthermore, high-pectin tobacco leaves produce up to 1.2% to 1.5% acetic acid during fermentation, which can cause irritation such as coughing, acridity, and burning when smoked, reducing the quality of tobacco products. Therefore, determining the pectin content in tobacco and its products is of great significance.

[0003] Currently, the main methods for determining pectin content in tobacco include chemical methods, carbazole colorimetry, enzymatic hydrolysis-flow analysis, and enzymatic hydrolysis-ion chromatography. Chemical methods are cumbersome and require significant manpower and material resources, making them difficult to meet the tobacco industry's practical needs for rapid, batch determination. Carbazole colorimetry offers high accuracy, but the sample pretreatment steps are complex and the selectivity is poor. Enzymatic hydrolysis-flow analysis uses harsh enzymatic conditions, and the large amount of water-soluble sugars and pigments in tobacco can easily interfere, affecting measurement accuracy. Enzymatic hydrolysis-ion chromatography offers high selectivity and sensitivity, but the instrumentation is expensive and its applicability is limited.

[0004] Solid-state nuclear magnetic resonance spectroscopy is a rapid detection method that is widely used in the determination of material structure. Among them, carbon 13 cross-polarization / magic angle spinning solid-state nuclear magnetic resonance spectroscopy ( 13 C CP / MAS NMR) technology can effectively improve the sensitivity and resolution of nuclear magnetic spectra.

[0005] Currently, there is an urgent need for a method with high accuracy, high precision and good repeatability for determining the pectin content in tobacco or its products. Summary of the Invention

[0006] One of the purposes of the present invention is to provide a 13 A sample tube for C CP / MAS NMR detection maintains the relative positions of the outer and inner tubes in a fixed position during the detection process, thereby obtaining stable and accurate spectral peak data. On this basis, another object of the present invention is to provide a method for determining the pectin content in tobacco or tobacco products, which method has high accuracy, high precision and good repeatability.

[0007] To achieve the above-mentioned purpose, the first aspect of the present invention relates to a method for 13Sample tube for C CP / MAS NMR detection,

[0008] The device comprises an outer sleeve with an open top and an inner sleeve with an open top, wherein the inner sleeve is located inside the outer sleeve;

[0009] The lower portion of the inner wall of the outer sleeve protrudes inward to form a convex portion, and the outer wall of the inner sleeve extends from the lower portion to the bottom to form a groove, and the size of the convex portion matches the size of the groove;

[0010] The material of the inner sleeve is selected from at least one of glycine, tetrakis(trimethylsiloxy)silane, methyl hydroxybutyrate, adamantane and silica gel (such as silica gel). The inner sleeve is an internal standard, and the interior of the inner sleeve is used to hold the sample.

[0011] In some embodiments of the first aspect of the present invention, radial dimensions of the inner sleeve match those of the outer sleeve.

[0012] In some embodiments of the first aspect of the present invention, the protrusion is interference fit with the groove.

[0013] In some embodiments of the first aspect of the present invention, the outer diameter of the inner sleeve is 0.1 to 0.2 mm smaller than the inner diameter of the outer sleeve.

[0014] In some embodiments of the first aspect of the present invention, the mass of the protrusion is exactly equal to the mass reduced by forming the groove.

[0015] In some embodiments of the first aspect of the present invention, the outer sleeve fits the bottom of the inner sleeve.

[0016] In some embodiments of the first aspect of the present invention, the outer sleeve has a height of 19 to 21 mm (eg, 20 mm) and a wall thickness of 0.5 to 1 mm (eg, 0.5 mm, 1 mm).

[0017] In some embodiments of the first aspect of the present invention, the inner sleeve has a height of 17 to 19 mm (eg, 18 mm) and a wall thickness of 0.5 to 1 mm (eg, 0.5 mm, 1 mm).

[0018] In some embodiments of the first aspect of the present invention, the upper edge of the protrusion is 2.7 to 3.3 mm, for example, 3 mm, away from the bottom of the outer sleeve.

[0019] In some embodiments of the first aspect of the present invention, the height of the raised portion is 1.8 to 2.2 mm (for example, 2 mm), the width is 0.8 to 1.2 mm (for example, 1 mm), and the thickness is 0.4 to 0.6 mm (for example, 0.5 mm); wherein the thickness of the raised portion refers to the radial dimension of the raised portion along the outer sleeve.

[0020] In some embodiments of the first aspect of the present invention, the upper edge of the groove is 4.8 to 5.2 mm away from the bottom of the inner sleeve, for example, 5 mm.

[0021] In some embodiments of the first aspect of the present invention, the groove has a height of 4.8 to 5.2 mm (eg, 5 mm), a width of 1.2 to 1.8 mm (eg, 1.5 mm), and a depth of 0.5 to 0.7 mm (eg, 0.6 mm).

[0022] In some embodiments of the first aspect of the present invention, the outer diameter of the outer sleeve is 6.8-7.3 mm, for example, 7 mm.

[0023] In some embodiments of the first aspect of the present invention, the inner diameter of the inner cannula is 5.3-5.8 mm, for example, 5.5 mm.

[0024] In some embodiments of the first aspect of the present invention, the outer sleeve is made of at least one material selected from polytetrafluoroethylene, glass, quartz, zirconium dioxide, and aluminum oxide, such as polytetrafluoroethylene.

[0025] A second aspect of the present invention relates to a method for determining the pectin content in tobacco or tobacco products, comprising the following steps:

[0026] (1) Washing tobacco or tobacco products with water to separate the solid phase;

[0027] (2) extracting the solid phase with water, adding an acid solution to adjust the pH value to 1.5 to 2.5 (e.g., the pH value is adjusted to 2.0), and centrifuging to separate the supernatant;

[0028] (3) adjusting the pH value of the supernatant to 3-4 (e.g., pH value to 3.5), precipitating with alcohol, separating the sediment, drying, crushing, and collecting the powder;

[0029] (4) Powder was placed in the sample tube described in the first aspect of the present invention, sodium chloride powder was added to fill the sample tube and mixed, and the mixture was used. 13 C CP / MAS NMR detection, to obtain the spectrum; wherein the detection conditions are: 13 C 100.63MHz and 1 The test frequency is H400.15MHz, using a 7mm H / X probe, a rotor speed of 3-5KHz (e.g., 4KHz), a contact time of 1-3ms (e.g., 2ms), a sampling time of 20-30ms (e.g., 25ms, 28ms), a sampling interval of 1-3s (e.g., 2s), a scan number of 2048, a field strength of 9.4T, a magic angle of 53°-56° (e.g., 53.5°, 54°, 54.7°, 55°, 55.5°), 13 C and 1The 90° pulse widths of H are 3 to 5 μs (e.g., 4 μs) and 3.4 to 5.5 μs (e.g., 4 μs, 4.4 μs, 5 μs);

[0030] (5) Deconvolute the peak of δ171ppm to δ174ppm (preferably δ171ppm to δ173ppm or 171ppm to δ172.6ppm, for example, δ171.381ppm, δ172.406ppm) in the spectrum to obtain three quantitative peaks of δ170ppm to δ171ppm (for example, δ171ppm), δ172ppm to δ174ppm (for example, δ174ppm), and δ175ppm to δ176ppm (for example, δ176ppm); deconvolute the peak of the internal standard in the spectrum to obtain the quantitative peak of the internal standard; calculate the pectin content in the tobacco or tobacco product based on the ratio of the sum of the three quantitative peak areas to the quantitative peak area of the internal standard.

[0031] In some embodiments of the second aspect of the present invention, in step (5), the calculation is performed by the internal standard method.

[0032] In some embodiments of the second aspect of the present invention, in step (5), the standard working curve used in the internal standard method is established by the following steps:

[0033] 1) Put different masses of polygalacturonic acid standards into a plurality of sample tubes according to the first aspect of the present invention, fill each sample tube with sodium chloride powder and mix well, and then 13 CCP / MAS NMR detection to obtain a spectrum; wherein the detection conditions are the same as step (4);

[0034] 2) Deconvolute the spectral peak of δ171ppm~δ174ppm (preferably δ171ppm~δ173ppm or 171ppm~δ172.6ppm, for example, δ171.381ppm, δ172.406ppm) in the spectrum to obtain three standard quantitative peaks of δ170ppm~δ171ppm (for example, δ171ppm), δ172ppm~δ174ppm (for example, δ174ppm), and δ175ppm~δ176ppm (for example, δ176ppm); deconvolute the internal standard peak in the spectrum to obtain the internal standard quantitative peak; perform regression analysis with the mass of the polygalacturonic acid standard as the independent variable and the ratio of the sum of the quantitative peak areas of the three standards to the quantitative peak area of the internal standard as the dependent variable to obtain a standard working curve.

[0035] In some embodiments of the second aspect of the present invention, in step (5) and / or step 2), the δ171ppm~δ174ppm peak in the spectrum is deconvoluted using the III-dimensional curve fitting of PeakFit software combined with the Lorentz function, and the iteration factor is 7.

[0036] In some embodiments of the second aspect of the present invention, in step (5) and / or step 2), the internal standard peak in the spectrum is deconvoluted using I-dimensional fitting of PeakFit software with an iteration factor of 7.

[0037] In some embodiments of the second aspect of the present invention, the method further comprises step (6): dividing the three quantitative peak areas of δ170ppm~δ171ppm (e.g., δ171ppm), δ172ppm~δ174ppm (e.g., δ174ppm), and δ175ppm~δ176ppm (e.g., δ176ppm) in step (5) by the sum of the three quantitative peak areas to obtain the ratios of protonated galacturonic acid units, methylated galacturonic acid units, and ionized galacturonic acid units in pectin, respectively.

[0038] In some embodiments of the second aspect of the present invention, the method further comprises step (7): multiplying the ratios of the protonated galacturonic acid units, the methylated galacturonic acid units and the ionized galacturonic acid units in the pectin by the pectin content in the tobacco or tobacco product, respectively, to obtain the protonated pectin content, the methylated pectin content and the ionized pectin content in the tobacco or tobacco product, respectively.

[0039] In some embodiments of the second aspect of the present invention, in step (4) and / or step 1), the detection conditions further include one or more of the following:

[0040] (A) Detected using an AVANCE III 400 MHz superconducting Fourier digital nuclear magnetic resonance spectrometer;

[0041] (B) Pulse sequence cptoss;

[0042] (C) spectral width 300ppm;

[0043] (D) Magic angle spin speed 4 kHz.

[0044] In some embodiments of the second aspect of the present invention, in step (4), before loading, the powder is passed through a 30-50 mesh sieve, such as a 40 mesh sieve.

[0045] In some embodiments of the second aspect of the present invention, in step (4), the amount of powder loaded into the sample tube is 100-200 mg, for example, 170 mg.

[0046] In some embodiments of the second aspect of the present invention, in step (5) and / or step 2), the spectrum is baseline corrected and smoothed before deconvolution.

[0047] In some embodiments of the second aspect of the present invention, in step (1), the immersion is performed two to four times, for example, three times or four times.

[0048] In some embodiments of the second aspect of the present invention, in step (1), the first immersion under ultrasonic conditions lasts for 20 to 40 minutes, for example, 30 minutes.

[0049] In some embodiments of the second aspect of the present invention, in step (1), the amount of water used per gram of tobacco or tobacco product is 5 to 20 mL, for example, 10 mL or 15 mL.

[0050] In some embodiments of the second aspect of the present invention, in step (2), the concentration of the acid solution is 0.5 to 2M, for example, 1M.

[0051] In some embodiments of the second aspect of the present invention, in step (2), the extraction is carried out at 70°C to 95°C (e.g., 80°C, 85°C, 90°C) for 1 to 5 hours (e.g., 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours).

[0052] In some embodiments of the second aspect of the present invention, in step (3), an alkaline solution is used to adjust the pH value; preferably, the concentration of the alkaline solution is 0.5 to 2M, for example, 1M.

[0053] In some embodiments of the second aspect of the present invention, in step (3), before drying, the precipitate is washed sequentially with anhydrous ethanol, acetone, and anhydrous ethanol.

[0054] In some embodiments of the second aspect of the present invention, in step (3), drying is performed at 35°C to 45°C (eg, 40°C) under vacuum conditions.

[0055] In some embodiments of the second aspect of the present invention, in step (3), ethanol is used for alcohol precipitation, and preferably the volume ratio of ethanol to supernatant is 1:1.

[0056] In some embodiments of the second aspect of the present invention, the pectin content in the tobacco is determined, preferably the pectin content in the tobacco dust is determined.

[0057] In some embodiments of the second aspect of the present invention, the water is selected from the group consisting of purified water, distilled water and deionized water.

[0058] In some embodiments of the second aspect of the present invention, the acid solution is selected from the group consisting of sulfuric acid solution, hydrochloric acid solution and acetic acid solution.

[0059] In some embodiments of the second aspect of the present invention, the acid solution is an aqueous solution.

[0060] In some embodiments of the second aspect of the present invention, the alkaline solution is selected from the group consisting of sodium hydroxide solution, sodium carbonate solution, sodium bicarbonate solution, potassium hydroxide solution, potassium carbonate solution and potassium bicarbonate solution.

[0061] In some embodiments of the second aspect of the present invention, the alkaline solution is an aqueous solution.

[0062] The third aspect of the present invention relates to the sample tube according to the first aspect of the present invention. 13 Application of C CP / MAS NMR in the determination of pectin content in tobacco or tobacco products.

[0063] For purposes of this invention, "tobacco" refers to a plant of the genus Nicotiana in the Solanaceae family, an annual or limited perennial herb covered entirely with glandular hairs; its roots are robust. The stem is 0.7-2 meters tall, slightly lignified at the base. The leaves are oblong-lanceolate, lanceolate, oblong, or ovate, gradually tapering at the apex and tapering to an auricular, semi-amplexicaul shape. The inflorescence is terminal, conical, and multiflorous; the pedicel is 5-20 mm long. The capsule is ovate or oblong, approximately as long as the persistent calyx. The seeds are round or broadly oblong, approximately 0.5 mm in diameter, and brown. Flowers and fruits bloom in summer and autumn. Native to South America, it is widely cultivated in provinces throughout northern and southern China.

[0064] In the present invention, "tobacco products" refer to addictive consumer products made from tobacco as raw material, which are divided into combustible tobacco products and non-combustible tobacco products.

[0065] The beneficial effects achieved by the present invention are:

[0066] 1. The present invention is used for 13 The sample tube for C CP / MAS NMR detection can keep the outer and inner tubes in relatively fixed positions during detection to obtain stable and accurate spectral peak data.

[0067] 2. The method of the present invention can accurately determine the pectin content in tobacco or tobacco products with high precision and good repeatability.

[0068] 3. The method of the present invention can accurately determine the content of pectin in different forms in tobacco or tobacco products. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0070] Figure 1 This is an assembly diagram of the sample tube in Example 1;

[0071] Figure 2 is a three-dimensional diagram of the outer sleeve in Example 1;

[0072] Figure 3 is a cross-sectional view of the outer sleeve in Example 1;

[0073] Figure 4 is a three-dimensional diagram of the inner sleeve in Example 1;

[0074] Figure 5is a cross-sectional view of the inner sleeve in Example 1;

[0075] Figure 6 This is the NMR spectrum of the standard product processed by MestReNova6.1.1 software in Example 2;

[0076] Figure 7 For Example 2 Figure 6 The C-6 peak in the spectrum was deconvoluted using PeakFit v4.12 software;

[0077] Figure 8 For Example 2 Figure 6 The internal standard peak in the spectrum was deconvoluted using PeakFit v4.12 software;

[0078] Figure 9 It is the standard working curve in Example 2;

[0079] Figure 10 This is the NMR spectrum of the sample processed by MestReNova6.1.1 software in Example 2;

[0080] Among them: 1 represents the outer sleeve, 2 represents the inner sleeve, 3 represents the protrusion, and 4 represents the groove. DETAILED DESCRIPTION

[0081] The embodiments of the present invention will be clearly and completely described below with reference to the examples. Obviously, the examples described are only some of the embodiments of the present invention, rather than all of them. The following description of at least one exemplary embodiment is actually only illustrative and is in no way intended to limit the present invention and its application or use. All other embodiments obtained by ordinary technicians in this field based on the examples in the present invention without making any creative efforts are within the scope of protection of the present invention.

[0082] Example 1 Sample tube for detecting pectin using carbon-13 cross-polarization magic angle spinning nuclear magnetic resonance

[0083] like Figure 1-5 As shown, the sample tube includes an outer sleeve with an open top and an inner sleeve with an open top, wherein the inner sleeve is located inside the outer sleeve; the lower portion of the inner wall of the outer sleeve protrudes inward to form a raised portion, and the outer wall of the inner sleeve extends from the lower portion to the bottom to form a groove, and the size of the raised portion and the groove match and have an interference fit; the material of the inner sleeve is silicone, the inner sleeve is an internal standard, and the interior of the inner sleeve is used to hold the sample;

[0084] The outer sleeve fits in contact with the bottom of the inner sleeve; the radial dimensions of the inner sleeve match those of the outer sleeve, and the outer diameter of the inner sleeve is 0.1 to 0.2 mm smaller than the inner diameter of the outer sleeve; the outer sleeve has an outer diameter of 7 mm, a height of 20 mm, and a wall thickness of 1 mm; the inner sleeve has an inner diameter of 5.5 mm, a height of 18 mm, and a wall thickness of 1 mm;

[0085] The mass of the raised portion is exactly equal to the mass reduced by forming the groove; the height of the raised portion is 2 mm, the width is 1 mm, and the thickness is 0.5 mm; the upper edge of the raised portion is 3 mm away from the bottom of the outer sleeve; the height of the groove is 5 mm, the width is 1.5 mm, and the depth is 0.6 mm; the upper edge of the groove is 5 mm away from the bottom of the inner sleeve; the material of the outer sleeve is polytetrafluoroethylene.

[0086] Example 2 Determination of pectin content in tobacco

[0087] (1) Establishment of standard working curve:

[0088] Weigh 98.7 mg, 121.2 mg, 141.8 mg, 160.5 mg, and 184.1 mg of polygalacturonic acid standard and add them to multiple sample tubes (see Example 1 for structure). Fill all the sample tubes with NaCl powder and mix them thoroughly. Then, perform carbon 13 cross-polarization magic angle spinning nuclear magnetic resonance (CMS) on the tubes. 13 C CP / MAS NMR) detection, the obtained NMR spectrum was baseline corrected and smoothed by MestReNova6.1.1 software as shown below Figure 6 As shown;

[0089] 13 C CP / MAS NMR detection conditions: AVANCEⅢ 400MHz superconducting Fourier digital nuclear magnetic resonance spectrometer (purchased from Bruker, Switzerland) was used. 13 C 100.63MHz and 1 The H frequency was 400.15 MHz, using a 7 mm H / X probe, a rotor speed of 4 kHz, a cross-polarization time (contact time) of 2 ms, a sampling time of 25 ms, a sampling interval of 2 s, a number of scans of 2048, a field strength of 9.4 T, a pulse sequence of cptoss, a spectral width of 300 ppm, a magic angle of 54.7°, and a magic angle spin speed of 4 kHz. 13 C and 1 The 90° pulse widths of H are 4 μs and 4.4 μs respectively;

[0090] right Figure 6The C-6 peak (δ172.406 ppm) in the spectrum was subjected to III-dimensional curve fitting combined with Lorentz function deconvolution using PeakFit v4.12 software, with an iteration factor of 7, to obtain three standard quantitative peaks at δ171 ppm, δ174 ppm, and δ176 ppm, as shown in Figure 2. Figure 7 shown; Figure 6 The internal standard peak of the spectrum was deconvoluted by 1-dimensional fitting using PeakFit v4.12 software with an iteration factor of 7. The quantitative peak of the internal standard obtained was as follows: Figure 8 shown.

[0091] The ratio of the sum of the quantitative peak areas of the three standards to the quantitative peak area of the internal standard is plotted as the ordinate, and the absolute mass of each standard is plotted as the abscissa to obtain the standard working curve, as shown in the following example: Figure 9 shown.

[0092] (2) Preparation of test samples:

[0093] Weigh 10 g of cigarette dust sample into a 250 mL beaker, add 100 mL of distilled water, soak under ultrasonic conditions for 30 minutes, filter to remove the supernatant, and then wash the filter residue with distilled water 2 to 3 times to remove soluble sugars and pigments in the cigarette dust; add 100 mL of distilled water to the washed filter residue and stir and extract at 85°C for 1.5 hours, then add 2 ml of 1 M dilute sulfuric acid solution to adjust the pH value to 2.0, centrifuge, and retain the supernatant; add 1 M NaOH solution to the supernatant to adjust the pH value to 3.5, then add ethanol at a volume ratio of 1:1 to precipitate, centrifuge, and wash the precipitate with anhydrous ethanol, acetone, and anhydrous ethanol in sequence, finally dry in a vacuum drying oven at 40°C to constant weight and grind into powder, pass through a 40-mesh sieve to obtain the sample to be tested, and store at -18°C for later use.

[0094] (3) Detection:

[0095] 170 mg of the sample to be tested was placed in the sample tube of Example 1, and NaCl powder was added and mixed until the sample tube was filled. 13 C CP / MAS NMR detection, the detection conditions are the same as those in item (1), the obtained NMR spectrum is baseline corrected and smoothed by MestReNova6.1.1 software as shown below Figure 10 shown.

[0096] right Figure 10 The C-6 peak (δ171.381ppm) of the spectrum was fitted with a III-dimensional curve using PeakFit v4.12 software and combined with Lorentz function deconvolution, with an iteration factor of 7, to obtain three quantitative peaks of the sample at δ171ppm, δ174ppm, and δ176ppm. Figure 10The internal standard peak of the spectrum was deconvoluted using PeakFit v4.12 software with an iteration factor of 7 to obtain the internal standard quantitative peak. The absolute mass of pectin in the test samples was calculated by substituting the ratio of the sum of the quantitative peak areas of the three test samples to the quantitative peak area of the internal standard into the standard working curve, and then the pectin mass content in the tobacco dust samples was calculated.

[0097] The pectin content in tobacco samples 1-7 was determined according to the above method. The results are shown in Table 1.

[0098] Table 1 Pectin content test results of each tobacco sample

[0099] Cigarette dust sample number Pectin mass content (%) 1 7.16 2 7.24 3 7.49 4 7.66 5 9.14 6 7.18 7 7.05

[0100] Example 3 Determination of the content of three forms of pectin in tobacco

[0101] The quantitative peaks at δ171ppm, δ174ppm and δ176ppm represent protonation (-COOH), methylation (-COOCH3) and ionization (-COO - ) galacturonic acid units. The quantitative peak areas of δ171ppm, δ174ppm, and δ176ppm obtained in Example 2 (3) were divided by the sum of the three quantitative peak areas to calculate the protonated (-COOH), methylated (-COOCH3), ionized (-COOCH3) and ionized (-COOH) in pectin. - ) and then calculated the mass content of the three forms of pectin in the sample. The results are shown in Table 2.

[0102] Table 2 Detection results of three pectin forms in each tobacco sample

[0103]

[0104] Example 4 Spiked Recovery Test

[0105] To evaluate the accuracy of the assay method in Example 2, three 10 g samples of cigarette dust with a known pectin content of 43.81 mg / g were prepared. A polygalacturonic acid standard was added to each of the three samples at respective amounts of 50.6 mg / g, 80.6 mg / g, and 120.4 mg / g to produce spiked samples. The pectin content in the cigarette dust sample and each spiked sample was determined according to the method in Example 2. The spiked sample recovery and RSD of the spiked sample were calculated for five replicates. The results are shown in Table 3.

[0106] Table 3 Results of spike recovery test

[0107]

[0108] As shown in Table 3, the spiked recovery of the method of the present invention is 94.33% to 102.77%, the average recovery is 98.11%, and the RSD (n=5) is 1.57% to 4.93%. This shows that the method of the present invention has good precision and high accuracy, and is a method for accurately quantitatively analyzing the pectin content in tobacco.

[0109] Example 5

[0110] Preparation of six types of tobacco powder: Six types of flue-cured tobacco produced in Chizhou, Anhui, Kunming, Yunnan, Qujing, Yunnan, Yanshan, Yunnan, Gui'an, Guizhou, and Zimbabwe were dried, ground, and passed through a 40-mesh sieve to obtain six types of tobacco powder;

[0111] The pectin content of the six kinds of tobacco dust was detected by the method of Example 2, external standard method and spectrophotometry. The results are shown in Table 4.

[0112] The spectrophotometric method is carried out in accordance with the industry standard NY / T 2016-2011 “Determination of pectin content in fruits and their products—Spectrophotometric method”.

[0113] The external standard method uses a conventional sample tube for testing, that is, the inner sleeve is not included and the outer tube without a raised portion is used as the sample tube. The specific experimental method is as follows:

[0114] 1) Establishment of standard working curve: Weigh 98.7 mg, 121.2 mg, 141.8 mg, 160.5 mg, and 184.1 mg of polygalacturonic acid standard and place them in several conventional sample tubes respectively. Add NaCl powder to all the sample tubes and mix them until the sample tubes are filled. Then, perform the following steps: 13 CCP / MAS NMR detection, the detection conditions are the same as those in Example 2, item (1);

[0115] The NMR spectrum was baseline corrected and smoothed using MestReNova 6.1.1 software. PeakFit v4.12 software was used to perform III-dimensional curve fitting of the C-6 peak (δ172.406 ppm) in the processed spectrum, combined with Lorentz deconvolution, with an iteration factor of 7. This yielded three standard quantitative peaks at δ171 ppm, δ174 ppm, and δ176 ppm. The standard working curve was plotted using the sum of the three standard quantitative peak areas as the ordinate and the absolute mass of the standards as the abscissa.

[0116] 2) The pretreatment of the cigarette sample was the same as that of item (2) in Example 2; 170 mg of the treated sample was placed in a conventional sample tube, NaCl powder was added and mixed to fill the sample tube, and then 13C CP / MAS NMR detection, the detection conditions are the same as those in Example 2, item (1); the NMR spectrum is baseline corrected and smoothed by MestReNova6.1.1 software, and the C-6 peak (δ171.381 ppm) in the processed spectrum is subjected to III-dimensional curve fitting combined with Lorentz function deconvolution using PeakFit v4.12 software, with an iteration factor of 7, to obtain three quantitative peaks of the sample to be tested, namely δ171 ppm, δ174 ppm, and δ176 ppm; the sum of the quantitative peak areas of the three samples to be tested is substituted into the standard working curve to calculate the pectin mass in the sample to be tested, and then the pectin content in the tobacco powder sample is calculated.

[0117] Table 4 Detection results of three methods (n=3)

[0118]

[0119]

[0120] *Indicates the relative error compared with the spectrophotometric test results.

[0121] As shown in Table 4, the detection results of the method of the present invention are closer to the detection results of the spectrophotometric standard method than the detection results of the external standard method. In addition, the RSD (n=3) of the method of the present invention is 1.5% to 3.85%, and the RSD (n=3) of the external standard method is 1.74% to 4.06%. Therefore, the method of the present invention has higher accuracy, better repeatability and higher precision than the external standard method.

[0122] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for 13 The sample tube for C CP / MAS NMR detection is characterized by: The device comprises an outer sleeve with an open top and an inner sleeve with an open top, wherein the inner sleeve is located inside the outer sleeve; The lower portion of the inner wall of the outer sleeve protrudes inward to form a convex portion, and the outer wall of the inner sleeve extends from the lower portion to the bottom to form a groove, and the size of the convex portion matches the size of the groove; The material of the inner sleeve is selected from at least one of glycine, tetrakis(trimethylsiloxy)silane, methyl hydroxybutyrate, adamantane and silica gel. The inner sleeve is an internal standard, and the interior of the inner sleeve is used to hold the sample.

2. The sample tube according to claim 1, wherein The radial dimensions of the inner sleeve match those of the outer sleeve.

3. The sample tube according to claim 1, wherein The outer diameter of the inner sleeve is 0.1 to 0.2 mm smaller than the inner diameter of the outer sleeve.

4. The sample tube according to any one of claims 1 to 3, characterized in that One or more of the following: (a) The mass of the protrusion is exactly equal to the mass reduced by forming the groove; (b) the outer sleeve is in contact with the bottom of the inner sleeve; (c) the outer sleeve has a height of 19 to 21 mm and a wall thickness of 0.5 to 1 mm; (d) the inner sleeve has a height of 17 to 19 mm and a wall thickness of 0.5 to 1 mm; (e) the upper edge of the protrusion is 2.7 to 3.3 mm from the bottom of the outer sleeve; (f) the height of the protrusion is 1.8 to 2.2 mm, the width is 0.8 to 1.2 mm, and the thickness is 0.4 to 0.6 mm; (g) the upper edge of the groove is 4.8 to 5.2 mm from the bottom of the inner sleeve; (h) the groove has a height of 4.8 to 5.2 mm, a width of 1.2 to 1.8 mm, and a depth of 0.5 to 0.7 mm; (i) the outer diameter of the outer sleeve is 6.8 to 7.3 mm; (j) the inner diameter of the inner sleeve is 5.3 to 5.8 mm; (k) The outer sleeve is made of at least one material selected from polytetrafluoroethylene, glass, quartz, zirconium dioxide and alumina.

5. A method for determining the pectin content in tobacco or tobacco products, comprising the following steps: (1) Washing tobacco or tobacco products with water to separate the solid phase; (2) extracting the solid phase with water, adding an acid solution to adjust the pH value to 1.5-2.5, and centrifuging to separate the supernatant; (3) The pH value of the supernatant is adjusted to 3-4, and the precipitate is separated by alcohol precipitation, dried, crushed, and the powder is collected; (4) Powder is placed in the sample tube according to any one of claims 1 to 4, sodium chloride powder is added to fill the sample tube and mixed, and the mixture is used. 13 C CP / MAS NMR detection, the spectrum is obtained; wherein, The test conditions are: 13 C 100.63MHz and 1 The test was conducted at H400.15MHz frequency, using a 7mm H / X probe, a rotor speed of 3-5KHz, a contact time of 1-3ms, a sampling time of 20-30ms, a sampling interval of 1-3s, a scan number of 2048, a field strength of 9.4T, and a magic angle of 53°-56°. 13 C and 1 The 90° pulse widths of H are 3–5 μs and 3.4–5.5 μs respectively; (5) Deconvolute the δ171ppm~δ174ppm peak in the spectrum to obtain three quantitative peaks of δ170ppm~δ171ppm, δ172ppm~δ174ppm, and δ175ppm~δ176ppm; deconvolute the internal standard peak in the spectrum to obtain the internal standard quantitative peak; calculate the pectin content in the tobacco or tobacco product based on the ratio of the sum of the three quantitative peak areas to the internal standard quantitative peak area.

6. The method according to claim 5, wherein: In step (5), calculation is performed by the internal standard method.

7. The method according to claim 6, wherein: The standard working curve adopted by the internal standard method is established by the following steps: 1) Put different masses of polygalacturonic acid standards into a plurality of sample tubes according to any one of claims 1 to 4, fill each sample tube with sodium chloride powder and mix well, and use 13 CCP / MAS NMR detection to obtain a spectrum; wherein the detection conditions are the same as step (4); 2) Deconvolute the δ171ppm~δ174ppm peak in the spectrum to obtain three standard quantitative peaks of δ170ppm~δ171ppm, δ172ppm~δ174ppm, and δ175ppm~δ176ppm; deconvolute the internal standard peak in the spectrum to obtain the internal standard quantitative peak; perform regression analysis with the mass of the polygalacturonic acid standard as the independent variable and the ratio of the sum of the quantitative peak areas of the three standards to the quantitative peak area of the internal standard as the dependent variable to obtain a standard working curve.

8. The method according to claim 5 or 7, wherein: In step (5) and / or step 2), the δ171ppm to δ174ppm peak in the spectrum is deconvoluted using the III-dimensional curve fitting of PeakFit software combined with the Lorentz function, with an iteration factor of 7.

9. The method according to claim 5 or 7, wherein: In step (5) and / or step 2), the internal standard peak in the spectrum is deconvoluted using I-dimensional fitting of PeakFit software with an iteration factor of 7.

10. The method according to any one of claims 5 to 7, further comprising step (6): dividing the three quantitative peak areas of δ170ppm-δ171ppm, δ172ppm-δ174ppm, and δ175ppm-δ176ppm in step (5) by the sum of the three quantitative peak areas to obtain the proportions of protonated galacturonic acid units, methylated galacturonic acid units, and ionized galacturonic acid units in pectin.

11. The method according to claim 10, further comprising step (7): multiplying the ratios of the protonated galacturonic acid units, the methylated galacturonic acid units and the ionized galacturonic acid units in the pectin by the pectin content in the tobacco or tobacco product, respectively, to obtain the protonated pectin content, the methylated pectin content and the ionized pectin content in the tobacco or tobacco product, respectively.

12. The method according to claim 5 or 7, wherein: In step (4) and / or step 1), the detection conditions may further include one or more of the following: (A) Detected using an AVANCE III 400 MHz superconducting Fourier digital nuclear magnetic resonance spectrometer; (B) Pulse sequence cptoss; (C) spectral width 300ppm; (D) Magic angle spin speed 4 kHz.

13. The method according to any one of claims 5 to 7, characterized in that One or more of the following: a. In step (4), before loading, sieve the powder through a 30-50 mesh sieve; b. In step (4), the amount of powder loaded into the sample tube is 100 to 200 mg; c. In step (1), immerse two to four times; d. In step (1), the amount of water used per gram of tobacco or tobacco product is 5 to 20 mL; e. In step (2), the concentration of the acid solution is 0.5 to 2 M; f. In step (2), extracting at 70 ℃ ~ 95 ℃ for 1 to 5 hours; g. In step (3), the pH value is adjusted using an alkaline solution; h. In step (3), before drying, the sediment was washed sequentially with anhydrous ethanol, acetone and anhydrous ethanol; i. In step (3), drying is carried out at 35°C to 45°C under vacuum conditions.

14. The method according to claim 13, wherein: In item c, in step (1), the first immersion is continued under ultrasonic conditions for 20 to 40 minutes.

15. The method according to claim 13, wherein In item g, in step (3), the concentration of the alkaline solution is 0.5 to 2M.

16. The method according to claim 5 or 7, wherein: In step (5) and / or step 2), the spectrum is baseline corrected and smoothed before deconvolution.

17. The sample tube according to any one of claims 1 to 4 13 Application of C CP / MAS NMR in the determination of pectin content in tobacco or tobacco products.

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