Method for determining amino acid, protein and lignin in sample

Abstract

The invention belongs to the field of detection and analysis, and relates to a method for determining amino acid, protein and lignin in a sample, which comprises the following steps: extracting the sample by using an acetic acid solution containing ethyl acetate, and carrying out solid-liquid separation to obtain a first liquid phase and a first solid phase; measuring a first liquid phase by liquid chromatography-tandem mass spectrometry, and calculating the content of amino acid; extracting the first solid phase by using an ammonia-containing dimethyl sulfoxide solution to obtain extract liquor and residues; measuring the content of protein in the extract liquor by a coomassie brilliant blue method; carrying out acidolysis on the residues by using a trifluoroacetic acid solution to enable the concentration of trifluoroacetic acid in an acidolysis product to reach 8-12% by volume, carrying out acidolysis at the microwave power of 250-350W and the temperature of 115-130 DEG C, and carrying out solid-liquid separation to obtain a second liquid phase and a second solid phase; measuring the absorbance of the second liquid phase by using an ultraviolet and visible spectrophotometer to obtain the content of the acid-soluble lignin; performing second solid-phase roasting, and obtaining the content of the acid-insoluble lignin according to the mass change before and after roasting. The method disclosed by the invention can be used for rapidly and accurately determining the contents of amino acid, protein and lignin.

Description

Technical Field

[0001] This invention belongs to the field of detection and analysis, specifically relating to a method for determining amino acids, proteins and lignin in a sample. Background Technology

[0002] Tobacco is a perennial herbaceous plant, and lignin, amino acids, and proteins are important components of tobacco. The lignin content directly affects the quality of raw tobacco leaves, cigarette safety, and sensory quality. Amino acids and proteins increase the bitterness of the smoke and give it a burnt feather smell during smoking, which has an adverse effect on the smoking quality of tobacco. Therefore, the accurate determination and in-depth research of the lignin, amino acid, and protein content in tobacco are of great significance for improving the quality of cigarette products.

[0003] The proteins in tobacco are biological macromolecules composed of one or more polypeptide chains. Each polypeptide chain contains anywhere from twenty to several hundred amino acid residues (-R). The various amino acid sequences and the resulting three-dimensional structures contribute to the diversity of protein structures. Lignin is formed by the enzymatic dehydrogenation polymerization of three types of monomers: guaiayl (G-group), syringyl (S-group), and p-hydroxyphenyl (H-group). Furthermore, lignin contains numerous functional groups such as carboxyl, hydroxyl, methoxy, and aromatic groups. These different functional groups form various types of random chemical bonds through direct or indirect coupling and addition.

[0004] Proteins and lignins both belong to the macromolecular class and coexist within tobacco plants, further increasing the difficulty of separating and accurately measuring them. Therefore, there is an urgent need for a method that can accurately and rapidly determine the amino acid, protein, and lignin content in tobacco. Summary of the Invention

[0005] The purpose of this invention is to provide a method for determining the content of amino acids, proteins and lignin in a sample. This method can rapidly and accurately determine the content of amino acids, proteins, acid-soluble lignin, acid-insoluble lignin and total lignin in tobacco raw materials and / or tobacco products, with high sensitivity and good repeatability.

[0006] To achieve the above objectives, the present invention provides a method for determining amino acids, proteins, and lignin in a sample, comprising the following steps:

[0007] The sample was extracted with an aqueous solution of acetic acid containing 6%-8% ethyl acetate (e.g., 7% wt%), followed by solid-liquid separation to obtain a first liquid phase and a first solid phase; wherein the sample was tobacco raw material and / or tobacco product;

[0008] The first liquid phase was detected by liquid chromatography-tandem mass spectrometry, and the amino acid content of the sample was calculated based on the spectrum.

[0009] The first solid phase was extracted with an aqueous solution of dimethyl sulfoxide containing 0.1%-0.5% ammonia (e.g., 0.1%, 0.2%, 0.3%, 0.4%) to obtain an extract and a residue.

[0010] The protein content in the extract was determined using the Coomassie Brilliant Blue method, and the protein content of the sample was obtained based on the determination results.

[0011] The residue is subjected to a first acid hydrolysis at 25 ℃-45 ℃ (preferably 25 ℃-35 ℃, for example 30 ℃) using an aqueous solution of trifluoroacetic acid with a concentration of 60 volume%-70 volume% (for example 65 volume%) to obtain a first acid hydrolysis product.

[0012] The concentration of trifluoroacetic acid in the first acid hydrolysis product is made to reach 8-12 vol% (e.g., 10 vol%). A second acid hydrolysis is carried out at a microwave power of 250-350W (e.g., 300W) and 115-130℃ (preferably 120-125℃, e.g., 121℃) to separate the solid and liquid phases, thereby obtaining a second liquid phase and a second solid phase.

[0013] The absorbance of the second liquid phase or its dilution at a wavelength of 200-210 nm (e.g., 205 nm) was detected using a UV-Vis spectrophotometer, and the acid-soluble lignin content in the sample was calculated based on the absorbance value.

[0014] The second solid is dried and then calcined at 620℃-650℃ (e.g., 630℃, 640℃) for 1-2 hours (preferably 1.2-1.5 hours, e.g., 1.2 hours, 1.4 hours). The acid-insoluble lignin content in the sample is calculated based on the mass change before and after calcination.

[0015] In any embodiment, the method further includes: calculating the total lignin content of the sample based on the acid-soluble lignin content and the acid-insoluble lignin content in the sample.

[0016] In any embodiment, the extraction temperature is 25°C-40°C, for example 30°C or 35°C.

[0017] In any implementation, the extraction time is 5 minutes to 25 minutes, for example, 10 minutes, 15 minutes, or 20 minutes.

[0018] In any embodiment, the extraction temperature is 40°C-60°C, for example 45°C, 50°C, or 55°C.

[0019] In any embodiment, the extraction time is 30 minutes to 100 minutes, for example, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, or 90 minutes.

[0020] In any embodiment, the first acid hydrolysis time is 1-4 hours (preferably 2-3 hours, for example 2 hours, 3.5 hours, 3.8 hours).

[0021] In any embodiment, the second acid hydrolysis time is 40-100 minutes (preferably 50-60 minutes, such as 60 minutes, 70 minutes, 80 minutes, or 90 minutes).

[0022] In any embodiment, the chromatographic column is a combination of an Acclaim Explosive E2 pre-column (Dionex, 10.0 mm × 4.3 mm, 120 Å) and an Acclaim Explosive E2 column (Dionex, 250.0 mm × 4.6 mm, 120 Å).

[0023] In any embodiment, mobile phase A is acetonitrile and mobile phase B is water.

[0024] In any implementation, the elution procedure for the mobile phase is shown in the table below:

[0025] In any embodiment, the flow rate of the mobile phase is 0.6 mL / min.

[0026] In any embodiment, the column temperature is 40°C.

[0027] In any embodiment, the injection volume for liquid chromatography is 5 μL.

[0028] In any implementation, the mass spectrometer is a quadrupole mass spectrometer.

[0029] In any implementation, the ion source for the mass spectrometer is an electrospray ionization source.

[0030] In any implementation, the mass spectrometry scanning mode is a positive ion scan.

[0031] In any implementation, the detection method of mass spectrometry is multiple reaction monitoring.

[0032] In any embodiment, the electrospray voltage of the mass spectrometer is 5000 V.

[0033] In any implementation, the ion source temperature for the mass spectrometer is 350°C.

[0034] In any embodiment, the pressure of the auxiliary gas Gas1 for mass spectrometry is 60 psi, and the pressure of the auxiliary gas Gas2 is 50 psi.

[0035] In any implementation, the declustering voltage (DP) of the mass spectrometer is 40 V.

[0036] In any implementation, the quantitative ion pairs Q1 / Q3 and collision energies for various amino acids are shown in the table below:

[0037] In any embodiment, before extraction, the sample is dried, crushed, sieved, and the pigments are removed from the resulting sieved material.

[0038] In any embodiment, drying is performed at 35°C-50°C, for example, 40°C.

[0039] In any implementation, the sample is passed through a 40-60 mesh sieve.

[0040] In any implementation, the amino acid content in the sample is calculated using external standard analysis based on the spectrum.

[0041] In any implementation, the acid-soluble lignin content in the sample is calculated using external standard analysis based on the absorbance value.

[0042] In any implementation, the protein content in the sample is obtained through the following steps:

[0043] The extract was mixed with Coomassie Brilliant Blue G-250 solution to obtain a mixture;

[0044] The absorbance of the mixture or its dilution at a wavelength of 630-650 nm (e.g., 640 nm) was detected using a UV-Vis spectrophotometer. The protein content in the mixture or its dilution was calculated using external standard analysis based on the absorbance value, and then the protein content in the sample was calculated.

[0045] In any embodiment, the volume ratio of Coomassie Brilliant Blue G-250 solution to extract is 5:1 to 7:1 (e.g., 6:1).

[0046] In any embodiment, the volume of the diluent is 1.3 to 1.7 times the volume of the mixture, for example, 1.4 times, 1.5 times, or 1.6 times.

[0047] In any embodiment, the mixture or its dilution is allowed to stand for 1-10 minutes, for example 5 minutes, before testing.

[0048] In any embodiment, during the extraction, the ratio of the aqueous acetic acid solution to the sample is 70:1 mL / g - 120:1 mL / g, for example 80:1 mL / g, 90:1 mL / g, 100:1 mL / g, or 110:1 mL / g.

[0049] In any embodiment, the concentration of acetic acid in the aqueous acetic acid solution used for extraction is 0.1-0.5% by mass, for example, 0.2% by mass, 0.3% by mass, 0.34% by mass, or 0.4% by mass.

[0050] In any embodiment, before detection by liquid chromatography-tandem mass spectrometry, the first liquid phase is filtered through a 0.2-0.3 μm filter membrane, for example, a 0.22 μm filter membrane.

[0051] In any embodiment, the ratio of dimethyl sulfoxide aqueous solution to sample used in the extraction is 140:1 mL / g to 180:1 mL / g, for example, 150:1 mL / g, 160:1 mL / g, or 170:1 mL / g.

[0052] In any embodiment, the concentration of the dimethyl sulfoxide aqueous solution used for extraction is 15%-30% by mass, for example 20% or 25% by mass.

[0053] In any embodiment, the residue is washed with ethanol and dried before the first acid hydrolysis.

[0054] In any embodiment, the ratio of the trifluoroacetic acid aqueous solution to the sample used in the first acid hydrolysis is 5:1 mL / g to 12:1 mL / g, for example, 6:1 mL / g, 7:1 mL / g, 8:1 mL / g, 9:1 mL / g, 10:1 mL / g, 11:1 mL / g.

[0055] In any embodiment, the trifluoroacetic acid concentration in the first acidolysis product is diluted to 8-12 vol%, for example, 9-10-11 vol%.

[0056] In any embodiment, the drying temperature of the second solid phase is 110°C-130°C (preferably 115°C-120°C, for example 118°C).

[0057] The present invention has achieved at least one of the following beneficial effects:

[0058] 1. The method of the present invention can quickly and accurately determine the content of amino acids, proteins, acid-soluble lignin, acid-insoluble lignin and total lignin in a sample.

[0059] 2. The method of the present invention solves the problems of difficulty in separating protein and lignin in tobacco raw materials or tobacco products and inaccurate determination. It has the characteristics of high sensitivity, good repeatability and simple operation, and is suitable for quality control and component analysis of various tobacco raw materials or tobacco products. Attached Figure Description

[0060] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0061] Figure 1 This is a flowchart of the method for determining the amino acid content, protein content, acid-soluble lignin content, acid-insoluble lignin content, and lignin content in tobacco stem samples according to Example 1 of the present invention.

[0062] Figure 2 This is the standard curve for determining protein content in Example 1 of the present invention. Detailed Implementation

[0063] The embodiments of the present invention will now be clearly and completely described in conjunction with examples. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0064] The materials or reagents used in the following examples:

[0065] Coomassie Brilliant Blue G-250 solution: Weigh 0.01 g of Coomassie Brilliant Blue G-250, dissolve it in 5 mL of 95% ethanol aqueous solution, add 10 mL of 85% phosphate aqueous solution, and finally dilute to 100 mL with 20% dimethyl sulfoxide aqueous solution. Store in a brown bottle for later use.

[0066] Example 1

[0067] Methods for determining the amino acid content, protein content, acid-soluble lignin content, acid-insoluble lignin content, and lignin content in a sample, such as... Figure 1 As shown, it includes:

[0068] (1) Sample pretreatment

[0069] Tobacco stem sample 1 was dried at 40℃ until it could be crushed, ground, and passed through a 40-60 mesh sieve. The sieved material was collected as the sample to be tested. 0.2500 g (accurate to 0.0001 g) of the sample to be tested was accurately weighed and placed in a sintered funnel. After decolorization with isooctane, 25 mL of an aqueous solution of acetic acid containing 7% ethyl acetate (acetic acid concentration in the solution was 0.34% by mass, pH=2 of the system) was added. The solution was ultrasonically treated at 30℃ for 15 min, filtered through a sintered funnel, and the extract and filter residue were obtained. Then, 40 mL of an aqueous solution of dimethyl sulfoxide containing 0.1% ammonia (dimethyl sulfoxide concentration in the solution was 20% by mass, pH=11 of the system) was added to the filter residue, and the solution was ultrasonically extracted at 50℃ for 60 min to separate the dimethyl sulfoxide extract and filter residue.

[0070] (2) Determination of amino acid content

[0071] (2.1) Plot the standard curve:

[0072] Standards of proline (Pro), alanine (Ala), isoleucine (Ile), leucine (Leu), arginine (Arg), cystine (Cys), histidine (His), methionine (Met), phenylalanine (Phe), serine (Ser), threonine (Thr), tyrosine (Tyr), valine (Val), glycine (Gly), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), and asparagine (Asn) (purity >99%, Beijing Bailingwei Technology Co., Ltd.) were prepared into a 1000 mg / L stock solution with ultrapure water. Standard solutions with concentration gradients of 0.05, 1, 2, 4, 6, 8, and 10 μg / L were obtained by serial dilution with ultrapure water. The peak areas of the quantitative ions in each standard solution were determined by liquid chromatography-tandem quadrupole mass spectrometry [operating conditions see (2.2)]. The linear equations for each amino acid were plotted with the concentration of the standard solution as the x-axis and the peak area of ​​the quantitative ion pair as the y-axis, as shown in Table 1.

[0073] Table 1. Linear relationships and detection limits of various amino acids

[0074] (2.2) Determination of amino acid content:

[0075] The extract was filtered through a 0.22 μm filter membrane and then injected into a high-performance liquid chromatography-tandem quadrupole mass spectrometry system for detection.

[0076] Operating conditions for high performance liquid chromatography (HPLC): The chromatographic columns were Acclaim Explosive E2 pre-columns (Dionex, 10.0 mm × 4.3 mm, 120 Å) and Acclaim Explosive E2 columns (Dionex, 250.0 mm × 4.6 mm, 120 Å), connected in series; the flow rate was 0.6 mL / min; the column temperature was 40℃; the injection volume was 5 μL; mobile phase A was acetonitrile, and mobile phase B was water; the gradient elution program for the mobile phases is shown in Table 2.

[0077] Table 2 Gradient elution program for high performance liquid chromatography

[0078] Mass spectrometry operating conditions: ion source was electrospray ionization (ESI); scanning mode was positive ion scanning; detection mode was multiple reaction monitoring (MRM); electrospray voltage was 5000 V; ion source temperature was 350 °C; auxiliary gas Gas1 pressure was 60 psi; auxiliary gas Gas2 pressure was 50 psi; declustering voltage (DP) was 40 V; the structural formulas, quantitative ion pairs Q1 / Q3, possible daughter ion structures, and collision energies (CE) of various amino acids are shown in Table 3.

[0079] Table 3 Mass Spectrometry Analysis and Parameters of Various Amino Acids

[0080] The peak area of ​​each amino acid quantitative ion pair in the filtered extract was determined. Based on the linear equation in Table 1, the concentration of each amino acid in the filtered extract was calculated in μg / L. Each sample was measured in triplicate, and the average value was taken.

[0081] The content of each amino acid in the tobacco stem sample was calculated using the following formula:

[0082] W = 100% × (C × V) / m0

[0083] Where: C is the concentration of amino acids in the filtered extract (μg / L); V is the volume of the filtered extract (L); M0 is the mass of the tobacco stem sample (μg).

[0084] The amino acid content in tobacco stem sample 1 was calculated to be 1.84 by mass.

[0085] (3) Determination of protein content:

[0086] (3.1) Plotting the standard curve

[0087] Bovine serum protein standard (purity >99%, Beijing Bailingwei Technology Co., Ltd.) was used to prepare a stock solution with a concentration of 100 μg / mL in ultrapure water. 0.2, 0.4, 0.8, 1.2, and 1.6 mL of the stock solution were taken, and 6 mL of Coomassie Brilliant Blue G-250 solution was added to each. The solution was then diluted to 10 mL with ultrapure water and mixed thoroughly. After standing for 5 min, the absorbance of each standard solution at 640 nm was measured using a UV-Vis spectrophotometer. A standard curve was plotted with the concentration of the standard solution on the x-axis and the absorbance on the y-axis, yielding the curve y = 6.035x + 0.13. Figure 2 As shown.

[0088] (3.2) Determination of protein content

[0089] Accurately transfer 1.0 mL of dimethyl sulfoxide extract, add 6 mL of Coomassie Brilliant Blue G-250 solution and dilute to 10 mL with ultrapure water, let stand for 5 min, and measure the absorbance at 640 nm using a UV-Vis spectrophotometer.

[0090] The protein concentration in the solution after the dimethyl sulfoxide extract was diluted to a fixed volume was calculated by substituting the measured absorbance into the standard curve. The protein content of the tobacco stem sample was then calculated using the following formula.

[0091]

[0092] in: This indicates the protein content (mass %) in the tobacco stem sample. This indicates the protein concentration (g / mL) in the solution after the dimethyl sulfoxide extract has been diluted to a fixed volume. The volume (mL) of the solution after dimethyl sulfoxide extract has been diluted to the desired volume; n represents the ratio of the total volume of the dimethyl sulfoxide extract to the volume transferred. This indicates the dried mass (g) of the tobacco stem sample.

[0093] The protein content in tobacco stem sample 1 was 4.21%.

[0094] (4) Determination of lignin content

[0095] (4.1) Two-step acid hydrolysis of filter residue:

[0096] Rinse the filter residue obtained in step (1) with anhydrous ethanol until the filtrate is colorless, and air dry in a fume hood; transfer the air-dried filter residue to a polytetrafluoroethylene digestion vessel, add 2 mL of 65% trifluoroacetic acid aqueous solution, gently shake until the sample is completely immersed in the acid solution, and acidify for 2 hours under a 30°C water bath; at room temperature, add ultrapure water to dilute the trifluoroacetic acid concentration in the digestion vessel to 10% by volume, place the digestion vessel in a microwave digestion apparatus, acidify for 1 hour at a microwave power of 300W and 121°C, cool to room temperature, and vacuum filter the acidification product using dried and weighed ashless filter paper, and collect the filtrate and filter residue;

[0097] (4.2) Determination of acid-soluble lignin content

[0098] (4.2.1) Plotting the standard curve:

[0099] Alkali-reduced lignin standards (purity >99%, TCI Corporation, Japan) were used as solvents. A series of standard solutions with concentrations of 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, and 12.0 mg / L were prepared using 10% (v / v) trifluoroacetic acid aqueous solution. The absorbance of each standard solution at a wavelength of 205 nm was measured using a UV-Vis spectrophotometer. Regression analysis was performed on the absorbance (Y) of the standard solution and its corresponding concentration (X) to obtain the standard curve, as shown in Table 4.

[0100] Table 4 Standard curve and detection limit of acid-soluble lignin

[0101] (4.2.2) Determination of acid-soluble lignin content

[0102] Add the filtrate collected in step (4.1) to ultrapure water and make up to 1L. Use a UV-Vis spectrophotometer to measure the absorbance of the solution after making up to 205 nm. Substitute the absorbance value into the standard curve to calculate the concentration of acid-soluble lignin in the solution after making up to 1L. Calculate the mass content of acid-soluble lignin in the tobacco stem sample according to the following formula.

[0103]

[0104] in, Indicates the mass content (%) of acid-soluble lignin in the tobacco stem sample. This indicates the protein concentration (g / mL) in the solution after volume adjustment. This indicates the volume (mL) of the solution after dilution. This indicates the dried mass (g) of the tobacco stem sample.

[0105] The mass content of acid-soluble lignin in tobacco stem sample 1 was 1.97%.

[0106] (4.3) Determination of acid-insoluble lignin content

[0107] The filter paper with filter residue obtained in step (4.1) (the mass of the dried and weighed ashless filter paper is recorded as m1) is placed in a ceramic crucible (the ceramic crucible needs to be calcined at 510 ℃ for 3 h to constant weight), dried in an oven at 118 ℃ to constant weight, naturally cooled to room temperature, and accurately weighed and recorded as m2. Then, it is transferred to a muffle furnace and calcined at 630 ℃ for 1.4 h, naturally cooled to room temperature, and accurately weighed and recorded as m3. The acid-insoluble lignin content in the tobacco stem sample is calculated according to the following formula.

[0108]

[0109] in, m0 is the mass content of acid-insoluble lignin in the tobacco stem sample (%); w is the mass of the tobacco stem sample (g); m1 is the mass of the dried ash-free filter paper (g); m2 is the total weight of the filter paper with filter residue after drying in the crucible (g); m3 is the weight of the crucible and ash after calcination (g).

[0110] The mass content of acid-insoluble lignin in tobacco stem sample 1 was 0.52%.

[0111] (4.4) The mass content of acid-soluble lignin and the mass content of acid-insoluble lignin were added together to obtain the lignin content of tobacco stem sample 1 as 2.49%.

[0112] Example 2

[0113] The amino acid content, protein content, acid-soluble lignin content, acid-insoluble lignin content, and lignin content in tobacco stem samples 2 to 4 were determined using the method in Example 1. The results are shown in Table 5.

[0114] Table 5. Amino acid content, protein content, acid-soluble lignin content, acid-insoluble lignin content, and lignin content in tobacco stem samples 2 to 4

[0115] Example 3: Validation of the accuracy, sensitivity, and repeatability of the method

[0116] Two or more tobacco stem samples were taken, and standards for amino acids, proteins and lignin (including acid-soluble and acid-insoluble lignin) were added to them respectively. The amount added is shown in Table 6. Then, the content of amino acids, proteins and lignin in the spiked samples was tested according to the method in Example 1. Each test was performed in parallel three times to obtain the measured values ​​after spiking. Then, the average spiked recovery rate and RSD were calculated. The results are shown in Table 6.

[0117] Table 6. Average spiked recoveries and RSD results of the method in Example 1

[0118] The results showed that the average spiked recovery rate for determining the amino acid content of tobacco samples by the method of the present invention was 99.65% with an RSD of 0.79%; the average spiked recovery rate for determining the protein content of tobacco samples was 99.2% with an RSD of 1.39%; and the average spiked recovery rate for determining the lignin content of tobacco samples was 98.5% with an RSD of 1.95%. This indicates that the method of the present invention has high accuracy, good repeatability, and good reliability.

[0119] Comparative Example 1: Investigating the effect of acetic acid aqueous solution containing ethyl acetate on the sample detection effect.

[0120] Based on Example 1, the "acetic acid aqueous solution containing 7% ethyl acetate (acetic acid concentration in the solution is 0.34% by mass, pH=2 of the system)" was replaced with ultrapure water, and the rest of the operation was the same as in Example 1.

[0121] Two or more tobacco stem samples were taken, and amino acid and protein standards were added to them respectively. The amount added was the same as in Table 6. Then, the amino acid and protein content of the spiked samples were tested according to the above method. Each test was performed in parallel three times to obtain the measured value after spiking. Then, the average spiked recovery rate and RSD were calculated. The results are shown in Table 7.

[0122] Table 7. Average spiked recoveries and RSDs of the method in Comparative Example 1

[0123] As shown in the table above, the amino acid content of tobacco samples determined by the method in Comparative Example 1 was low, with an average recovery rate of only 82.10%, significantly lower than the 99.65% of our method, and a relative standard deviation greater than 5%. This indicates that the acetic acid aqueous solution containing ethyl acetate has a better extraction effect on amino acids in tobacco. Furthermore, the protein recovery rate was also slightly lower. During the initial amino acid extraction process, acetic acid can provide hydrogen ions, making the solution slightly alkaline (pH=2), which has a certain destructive effect on the tobacco cell wall and to some extent helps in the later protein extraction. Therefore, the accuracy and repeatability of the method in Comparative Example 1 are significantly lower than those of the method of this invention, and the reliability of the method in Comparative Example 1 is significantly reduced.

[0124] Comparative Example 2: Investigating the effect of ammonia in dimethyl sulfoxide aqueous solution on the detection effect of samples.

[0125] Based on Example 1, the "dimethyl sulfoxide aqueous solution containing 0.1% ammonia (dimethyl sulfoxide concentration in the solution is 20% by mass, pH=11)" was replaced with "dimethyl sulfoxide aqueous solution (dimethyl sulfoxide concentration in the solution is 20% by mass, pH=7)", and the remaining operations were the same as in Example 1. Since this substitution occurred after amino acid extraction, it did not affect the detection of amino acids. In this comparative example, only the effect on the detection of protein and lignin was examined.

[0126] Two or more tobacco stem samples were taken, and protein and lignin (including acid-soluble and acid-insoluble lignin) standards were added to them respectively. The amount added was the same as in Table 6. Then, the protein and lignin content of the spiked samples were tested according to the above method. Each test was performed in parallel three times to obtain the measured value after spiking. Then, the average spiked recovery rate and RSD were calculated. The results are shown in Table 8.

[0127] Table 8. Average spiked recoveries and RSDs of the method in Comparative Example 2

[0128] As shown in the table above, the detection values ​​of both protein and lignin decreased in Comparative Example 2, and the average spiked recovery rate also decreased. During protein extraction, ammonia provides an alkaline environment, aiding in the lysis of cell wall pectin and facilitating protein dissolution. Furthermore, ammonia readily forms ammonium ions in aqueous solution, acting as a salt-dissolving agent and inhibiting protein adsorption and precipitation. When the protein extraction rate is insufficient, especially when the extraction of protein from the cell wall is inadequate, lignin cross-linked with cell wall proteins is also insufficiently extracted. Therefore, the accuracy and repeatability of Comparative Example 2 are significantly lower than those of the method of this invention, and the reliability of Comparative Example 2 is significantly reduced.

[0129] Comparative Example 3: Investigating the effect of trifluoroacetic acid on lignin detection.

[0130] Based on Example 1, the trifluoroacetic acid aqueous solution was replaced with sulfuric acid aqueous solution of equal concentration, while the rest of the operation remained unchanged. Since the acidolysis step occurs after the extraction and detection of amino acids and proteins, the substitution of the type of acid does not affect the detection effect of amino acids and proteins. This comparative example only examines the detection effect on lignin.

[0131] Two or more tobacco stem samples were taken and lignin (including acid-soluble and acid-insoluble lignin) standards were added to them respectively. The amount added was the same as in Table 6. Then, the lignin content of the spiked samples was tested according to the above method. Each test was performed in parallel three times to obtain the measured value after spiking. Then, the average spiked recovery rate and RSD were calculated. The results are shown in Table 9.

[0132] Table 9. Average spiked recoveries and RSDs of the method in Comparative Example 3

[0133] As shown in the table above, replacing the trifluoroacetic acid solution with sulfuric acid solution may degrade lignin, resulting in lower detection values. The spiked recovery rate was only 80.43%, with a high RSD value. Therefore, the accuracy and repeatability of the method in Comparative Example 3 are significantly lower than those of the method of this invention, and the reliability of the method in Comparative Example 3 is significantly reduced.

[0134] The effect of measuring wavelength in step (4.2.2) of Comparative Example 4

[0135] Based on the method in Example 1, in steps (4.2.1) and (4.2.2), the absorbance at a wavelength of 320 nm was measured using a UV-Vis spectrophotometer, and the remaining operations were the same as in Example 1. This wavelength is for the detection of acid-soluble lignin. At this wavelength, a new standard curve was plotted, and the correlation coefficient and detection limit of the standard curve were calculated. The results are listed in Table 10.

[0136] Table 10 Standard curves and limits of detection for acid-soluble lignin obtained by the method in Comparative Example 4

[0137] As shown in the table above, the sensitivity (detection limit) of the method in Comparative Example 4 deteriorated to more than twice that of the method of the present invention. This indicates that the detection sensitivity of the method in Comparative Example 4 is significantly lower than that of the method of the present invention.

[0138] Furthermore, the detection effects of Example 1 and Comparative Example 4 on acid-soluble lignin in tobacco stem sample 2 were compared. The results were measured in parallel three times and are listed in Table 11.

[0139] Table 11 Detection results of acid-soluble lignin using different detection methods (mass %)

[0140] As can be seen from the table above, the standard deviation of the results measured in Comparative Example 4 is relatively large, and the test results are less stable, which is inferior to the method in Example 1 of this invention.

[0141] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A method for determining amino acids, proteins, and lignin in a sample, comprising the following steps: The sample was extracted using an aqueous solution of acetic acid containing 6%-8% ethyl acetate, followed by solid-liquid separation to obtain a first liquid phase and a first solid phase; wherein, The samples are tobacco raw materials and / or tobacco products; The first liquid phase was detected by liquid chromatography-tandem mass spectrometry, and the amino acid content of the sample was calculated based on the spectrum. The first solid phase was extracted with an aqueous solution of dimethyl sulfoxide containing 0.1%-0.5% ammonia to obtain an extract and a residue. The protein content in the extract was determined using the Coomassie Brilliant Blue method, and the protein content of the sample was obtained based on the determination results. The residue was subjected to a first acid hydrolysis at 25 ℃-45 ℃ using an aqueous solution of trifluoroacetic acid with a concentration of 60 vol%-70 vol% to obtain the first acid hydrolysis product. The concentration of trifluoroacetic acid in the first acid hydrolysis product is made to reach 8%-12% by volume. A second acid hydrolysis is carried out at a microwave power of 250-350W and 115℃-130℃, and solid-liquid separation is performed to obtain a second liquid phase and a second solid phase. The absorbance of the second liquid phase or its dilution at a wavelength of 200-210 nm was detected using a UV-Vis spectrophotometer, and the acid-soluble lignin content in the sample was calculated based on the absorbance value. The second solid was dried and then calcined at 620℃-650℃ for 1-2 hours. The acid-insoluble lignin content in the sample was calculated based on the mass change before and after calcination.

2. The method according to claim 1, further comprising: The total lignin content of the sample is calculated based on the acid-soluble lignin content and the acid-insoluble lignin content.

3. The method according to claim 1 or 2, wherein, The extraction temperature is 25℃-40℃; and / or, The extraction time is 5-25 minutes.

4. The method according to any one of claims 1 to 3, wherein, The extraction temperature is 40℃-60℃; and / or, The extraction time is 30-100 minutes.

5. The method according to any one of claims 1 to 4, wherein, The first acid hydrolysis lasts for 1-4 hours; and / or, The second acid hydrolysis takes 40-100 minutes.

6. The method according to any one of claims 1 to 5, wherein, The operating conditions for the liquid chromatography-tandem mass spectrometry include one or more of the following: The chromatographic column consisted of a series of Acclaim Explosive E2 pre-columns and Acclaim Explosive E2 columns; Mobile phase A is acetonitrile, and mobile phase B is water; The elution program for the mobile phase is shown in the table below: The flow rate of the mobile phase was 0.6 mL / min; The column temperature is 40℃; The injection volume for liquid chromatography is 5 μL; The mass spectrometer is a quadrupole mass spectrometer; The ion source for mass spectrometry is an electrospray ionization source; The mass spectrometer uses a positive ion scan mode. The detection method of mass spectrometry is multiple reaction monitoring; The electrospray voltage for mass spectrometry is 5000 V; The ion source temperature for mass spectrometry is 350℃; The pressure of the auxiliary gas Gas1 for mass spectrometry is 60 psi, and the pressure of the auxiliary gas Gas2 is 50 psi. The declustering voltage for mass spectrometry is 40 V; The quantitative ion pairs Q1 / Q3 and collision energies of various amino acids are shown in the table below:

7. The method according to any one of claims 1 to 6, wherein, Before extraction, the sample is dried, crushed, and sieved to remove pigments from the sieved material. Optionally, drying can be carried out at 35℃-50℃; Optionally, pass through a 40-60 mesh sieve.

8. The method according to any one of claims 1 to 7, wherein, The amino acid content of the sample was calculated using external standard analysis based on the spectral data; and / or, The acid-soluble lignin content in the sample was calculated using external standard analysis based on the absorbance value.

9. The method according to any one of claims 1 to 8, wherein, The protein content in the sample is obtained through the following steps: The extract was mixed with Coomassie Brilliant Blue G-250 solution to obtain a mixture; The absorbance of the mixture or its dilution at wavelengths of 630-650 nm was detected using a UV-Vis spectrophotometer. The protein content in the mixture or its dilution was calculated using external standard analysis based on the absorbance values, and then the protein content in the sample was calculated.

10. The method according to claim 9, characterized in that... One or more of the following: (1) The volume ratio of Coomassie Brilliant Blue G-250 solution to extract is 5:1-7:1; (2) The volume of the diluent is 1.3 to 1.7 times the volume of the mixed liquid; (3) Before testing, let the mixture or its dilution stand for 1-10 minutes.

11. The method according to any one of claims 1 to 10, characterized in that... One or more of the following: 1) During the extraction, the ratio of the acetic acid aqueous solution to the sample is 70:1 mL / g - 120:1 mL / g, for example, 100:1 mL / g; 2) The concentration of acetic acid in the aqueous acetic acid solution used for extraction is 0.1-0.5% by mass, for example, 0.34% by mass; 3) Before detection by liquid chromatography-tandem mass spectrometry, the first liquid phase is filtered through a 0.2-0.3 μm filter membrane, for example, a 0.22 μm filter membrane; 4) The ratio of dimethyl sulfoxide aqueous solution to sample used in the extraction is 140:1 mL / g - 180:1 mL / g, for example 160 mL / g; 5) The concentration of the dimethyl sulfoxide aqueous solution used for extraction is 15%-30% by mass, for example, 20% by mass; 6) Before the first acid hydrolysis, the residue was washed with ethanol and dried; 7) The ratio of trifluoroacetic acid aqueous solution to sample used in the first acid hydrolysis is 5:1 mL / g - 12:1 mL / g, for example 8 mL / g; 8) Dilute the concentration of trifluoroacetic acid in the first acid hydrolysis product to 8%-12% (v / v). 9) The drying temperature of the second solid is 110℃-130℃, for example 118℃.

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