Detection method for dissolution rate of nicotine in nicotine bag and application

Abstract

The invention relates to a method for detecting the nicotine dissolution rate of a nicotine bag and application. The method comprises the following steps: immersing the nicotine bag in a solvent to obtain a nicotine bag content extracting solution, sampling the nicotine bag content extracting solution at intervals, centrifuging, and taking supernate to obtain a sample to be detected; pre-coating the nicotine antigen to the bottom of the pore plate by using a coating agent; adding the to-be-detected sample into a pore plate, adding at least two nicotine antibody enzyme markers at the same time, incubating, and washing after the incubation is finished; adding at least two color developing agents into the pore plate, uniformly mixing, and incubating and developing in a dark place; after the chromogenic reaction is finished, adding a stop solution into the pore plate, uniformly mixing, and measuring an absorbance value; and calculating the concentration of nicotine in the to-be-detected sample through the standard curve.

Description

Technical Field

[0001] The present application relates to the field of biochemical engineering technology, and in particular to a method for detecting the dissolution rate of nicotine in a nicotine bag and its application. Background Art

[0002] Nicotine pouches, a new type of smokeless tobacco product, have become widely popular in the international market. On March 15, 2022, the new U.S. Tobacco Control Act came into effect. This law revised the definition of tobacco products and redefined it as "any product manufactured or derived from tobacco, or containing nicotine from any source, for human consumption," further highlighting the unique and important role of nicotine in smokeless tobacco products. Furthermore, in 2015, Shihadeh et al. proposed the concept of nicotine flux—the rate of nicotine release—as a measure of nicotine release speed, with its fluctuations being used to characterize nicotine delivery stability. Furthermore, in May 2016, the U.S. FDA's Center for Tobacco Products proposed adding regulatory requirements for nicotine dissolution in snus, requiring new snus products to undergo an equivalence evaluation based on this metric before they can be marketed. These materials all indicate that nicotine is a core indicator in the development and sensory evaluation of nicotine pouches.

[0003] As an emerging tobacco product, nicotine detection methods for its core substance, nicotine, are still under development. Currently available methods for measuring nicotine flux in smokeless tobacco products only provide a general indication of nicotine content and fail to meet the diverse regulatory requirements brought about by the new era of diversified product formats. The most classic method is liquid chromatography, which offers advantages in precise quantification, complex matrix handling, and method standardization. This method can measure nicotine content to ±0.01 mg / g, meeting the stringent FDA / PMTA requirements for dosage consistency and effectively separating interfering substances (menthol, sweeteners, and over 30 other additives) in nicotine pouches. HPLC testing is mandatory in the internal quality control SOPs of tobacco giants such as British American Tobacco. However, sampling is difficult, making real-time detection impossible. Furthermore, the equipment investment and testing costs are high, the operation is complex, and the testing cycle is long. Therefore, the development of a rapid, effective, and accurate method for measuring nicotine flux in nicotine pouches is crucial. Summary of the Invention

[0004] Enzyme-linked immunosorbent assay (ELISA) is a qualitative and quantitative detection method that uses the specificity of antigen-antibody binding to conduct immune reactions. It is a classic experimental method in immunology.

[0005] In a first aspect, the present application provides a method for detecting the dissolution rate of nicotine in a nicotine pouch, comprising the following steps:

[0006] Step A: Immersing the nicotine bag in a solvent to obtain an extract of the contents of the nicotine bag, sampling the extract of the contents of the nicotine bag, centrifuging it, and collecting the supernatant to obtain a sample to be tested;

[0007] Step B: Pre-coat the nicotine antigen onto the bottom of the well plate using a coating agent;

[0008] Step C: Add the sample to be tested to the well plate, and at least two nicotine antibody enzyme markers, incubate, and wash after the incubation;

[0009] Step D: Add at least two color developing reagents to the well plate, mix well, and incubate in the dark to develop color;

[0010] Step E: After the color development reaction is completed, add the stop solution to the well plate, mix well, and measure the absorbance value;

[0011] Step F: Calculate the nicotine concentration in the sample to be tested using the standard curve.

[0012] Furthermore, in step A:

[0013] The temperature of the solvent is 35.0-36.0°C, 36.0-37.0°C or 37.0-38.0°C;

[0014] The volume of solvent used in each nicotine bag is 5-15mL / bag, 15-50mL / bag, 50-100mL / bag or 100-200mL / bag;

[0015] Step A includes sampling the nicotine bag content extract multiple times at intervals of 1 minute to 2 minutes, 2 minutes to 5 minutes, or 5 minutes to 10 minutes;

[0016] The solvent is artificial saliva, which is prepared as follows: sodium carboxymethyl cellulose is added to 700 ml of distilled water and soaked overnight. After swelling, sorbitol, sodium chloride, magnesium chloride, potassium chloride, and paraben are added. After stirring to dissolve, calcium phosphate and sodium phosphate solutions are added. Distilled water is added to a sufficient volume. After stirring, the solution is adjusted to pH 7 with 0.01% hydrochloric acid or 0.05% sodium hydroxide. The solution is bottled and sterilized with circulating steam at 100 degrees for 30 minutes.

[0017] Furthermore, step B is specifically as follows: using hydroxypropyl cellulose to coat the nicotine antigen on the bottom of a 96-well plate, wherein the concentration ratio of hydroxypropyl cellulose to nicotine antigen is 1:1, and the concentration range of the two is controlled within 100 ng / ml-20 ug / ml.

[0018] Furthermore, step C is specifically as follows: adding the sample to be tested to the well plate, adding nicotine antibody alkaline phosphatase marker and nicotine antibody β-galactosidase marker at the same time, covering with a sealing film and incubating at 37°C for 30 minutes, and after the incubation is completed, repeating the washing 3 times.

[0019] Furthermore, step D is specifically as follows: adding nitrophenyl phosphate and o-nitrophenyl-β-D-galactoside to the well plate, mixing, covering with a sealing film, and incubating at 37° C. in the dark for 15 minutes for color development.

[0020] Furthermore, step E is specifically as follows: after the color development reaction is completed, concentrated sulfuric acid is added to the well plate, mixed, and the absorbance value is measured at a wavelength of 405 nm, 420 nm or 450 nm using a spectrophotometer within 5 minutes.

[0021] Furthermore, the standard curve was obtained by:

[0022] a) preparing a series of nicotine standard solutions;

[0023] b) pre-coating the nicotine antigen onto the bottom of the well plate using a coating agent;

[0024] c) adding a nicotine standard solution and at least two nicotine antibody enzyme markers to the well plate, incubating, and washing after the incubation;

[0025] d) adding at least two color developing reagents to the well plate, mixing, and incubating in the dark to develop color;

[0026] e) After the color development reaction is completed, add the stop solution to the well plate, mix well, and measure the absorbance value;

[0027] f) Establish a three-variable linear regression curve using the absorbance value as the abscissa and the nicotine concentration as the ordinate.

[0028] In a second aspect, the present application provides a nicotine detection kit for implementing the method according to the first aspect, which comprises at least: a solvent, a coating agent, a well plate, at least two nicotine antibody enzyme labels, at least two color developers and a stop solution.

[0029] In a third aspect, the present application provides a nicotine pouch screening method, which comprises screening a nicotine pouch from a group of candidate nicotine pouches using the method according to the first aspect.

[0030] Furthermore, the screening parameters include pH value, moisture or non-woven bag type.

[0031] This application has the following beneficial effects:

[0032] (1) The innovative use of an alkaline phosphatase and β-galactosidase dual enzyme system combined with a nitrophenyl phosphate and o-nitrophenyl-β-D-galactoside dual substrate system can enhance the colorimetric signal or expand the linear detection range through synergistic effects. Traditional ELISAs typically use a single enzyme and a single substrate. The dual substrate design can improve the detection sensitivity of low-concentration nicotine.

[0033] (2) By using a specific wavelength and curve fitting algorithm, a three-variable linear equation (multivariate regression) is used instead of a conventional standard curve to improve fitting accuracy, especially for complex matrices (nicotine bags containing multiple additives).

[0034] (3) It was proposed that the nicotine dissolution curve of nicotine bags can be used as a screening and evaluation indicator for nicotine bags. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solution of this application, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of this patent and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 Schematic diagram of 96-well enzyme-labeled plate sample loading during detection;

[0037] Figure 2 This is the nicotine dissolution curve of Philip Morris International's nicotine pouch ZYN (6mg / pouch);

[0038] Figure 3 The standard curve obtained by detecting different concentrations of nicotine standards using a wavelength of 420 nm (the horizontal axis is nicotine concentration (ng / ml), and the vertical axis is absorbance);

[0039] Figure 4 Specific values ​​for plotting a standard curve for different concentrations of nicotine standards using a wavelength of 420 nm, and the specific regression equation for the standard curve;

[0040] Figure 5 This is the nicotine dissolution curve of the Hubei China Tobacco R&D sample (coffee, mint, 5mg nicotine / bag) (the vertical axis is concentration, the horizontal axis is time);

[0041] Figure 6 To use high performance liquid chromatography to detect the nicotine dissolution curve of Hubei China Tobacco's research and development sample (mint, 5mg / bag). DETAILED DESCRIPTION

[0042] " Scope " disclosed herein is limited in the form of lower limit and upper limit, and given range is limited by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of special range. The scope limited in this way can be to include end value or not include end value, and can be arbitrarily combined, that is, any lower limit can form a scope with any upper limit combination. For example, if the scope of 60-120 and 80-110 is listed for a particular parameter, it is understood that the scope of 60-110 and 80-120 is also expected. In addition, if the minimum range value 1 and 2 are listed, and if the maximum range value 3,4 and 5 are listed, then the following range can all be expected: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In this application, unless otherwise specified, the numerical range " ab " represents the abbreviation of any real number combination between a and b, wherein a and b are all real numbers. For example, a numerical range of "0-5" indicates that all real numbers between "0-5" are listed herein, and "0-5" is simply an abbreviation for these numerical combinations. Furthermore, when a parameter is expressed as an integer ≥ 2, this is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0043] In this document, unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined with each other to form a new technical solution.

[0044] In this document, unless otherwise specified, all technical features and preferred features mentioned herein can be combined with each other to form a new technical solution.

[0045] In this document, unless otherwise specified, all steps mentioned herein may be performed sequentially or randomly, but are preferably performed sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order, for example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0046] In this document, unless otherwise specified, the terms "include" and "comprising" may be open-ended or closed-ended. For example, "include" and "comprising" may mean that other components not listed may also be included or that only the listed components are included.

[0047] In the description of this article, it should be noted that, unless otherwise specified, “above” and “below” are inclusive of the number themselves, and “several” in “one or several” means two or more.

[0048] In the description herein, unless otherwise indicated, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B." More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0049] Unless otherwise specified herein, percentages (%) refer to percentages by mass relative to the composition.

[0050] Herein, unless otherwise stated, the sum of the contents of the various components in the composition is 100%.

[0051] In this context, unless otherwise stated, "a combination thereof" means a multi-component mixture of the elements mentioned, for example a mixture of two, three, four and up to the maximum possible multi-component mixture.

[0052] If not specifically stated, the terms "a" and "an" used in this specification mean "at least one".

[0053] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature specified as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "plurality" means at least two, for example, two, three, etc., unless otherwise specifically defined.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0055] The detailed features and advantages of the present application are described in detail below in the specific implementation methods, and the content is sufficient to enable any technical personnel in this field to understand the technical content of the present application and implement it accordingly. Based on the description, claims and drawings disclosed in this specification, technical personnel in this field can easily understand the relevant purposes and advantages of the present application.

[0056] Nicotine pouch contents extraction

[0057] As used herein, "nicotine pouch" generally refers to a small fiber bag that absorbs nicotine through the lining of the mouth, the main component of which is nicotine powder or nicotine salt. They are usually placed between the gums and the upper lip, and nicotine is absorbed by the body through the oral mucosa.

[0058] As used herein, "nicotine" (also known as nicotine) has a chemical formula of C 10 H 14 N2, belongs to the pyridine alkaloids. In this article, the term "nicotine" and "nicotine" can be used interchangeably.

[0059] In some aspects, the method of the present invention comprises immersing a nicotine bag in a solvent to obtain an extract of the contents of the nicotine bag, sampling the extract of the contents of the nicotine bag at intervals, and collecting the supernatant after centrifugation to obtain a sample to be tested.

[0060] The temperature of the solvent may be 35.0-36.0° C., 36.0-37.0° C., or 37.0-38.0° C. Preferably, the temperature of the solvent is 37.0° C.

[0061] The volume of the solvent used in each nicotine bag can be 5-15 mL / bag, 15-50 mL / bag, 50-100 mL / bag or 100-200 mL / bag. Preferably, the volume of the solvent used in each nicotine bag is 50 mL / bag.

[0062] In some respects, During the extraction process of the nicotine bag contents, multiple samples can be taken. The interval time can be 1 minute to 2 minutes, 2 minutes to 5 minutes or 5 minutes to 10 minutes. Thus, a nicotine dissolution curve can be obtained, that is, the change of nicotine concentration dissolved by the nicotine bag in the solvent over time.

[0063] The solvent is artificial saliva, which may contain water and one or more of the following components: sodium carboxymethyl cellulose, sorbitol, potassium chloride, sodium chloride, magnesium chloride, calcium phosphate, sodium phosphate, paraben, distilled water, a pH adjuster, α-amylase, lysozyme, lipase, and peroxidase. The artificial saliva is prepared as follows: sodium carboxymethyl cellulose is added to 700 ml of distilled water and soaked overnight. After swelling, sorbitol, sodium chloride, magnesium chloride, potassium chloride, and paraben are added. After stirring to dissolve, calcium phosphate and sodium phosphate solutions are added. Distilled water is added to a sufficient volume. After stirring, the pH is adjusted to 7 with 0.01% hydrochloric acid or 0.05% sodium hydroxide, bottled, and sterilized with circulating steam at 100 degrees for 30 minutes.

[0064] Competitive enzyme-linked immunosorbent assay

[0065] As used herein, "enzyme-linked immunosorbent assay" (ELISA) is the most widely used enzyme immunoassay technique. Its basic method involves adsorbing a known antigen or antibody onto the surface of a solid support, allowing the enzyme-labeled antigen-antibody reaction to proceed on the solid surface, and then washing away free components in the liquid phase.

[0066] As used herein, "pre-coating" means that the nicotine-conjugated antigen is fixed to a solid support (such as a microplate) so that it can react with other molecules and will not be removed by detergents during washing.

[0067] As used herein, a "nicotine-conjugated antigen" (also referred to herein as a nicotine antigen) is an antigen that is bound to a nicotine molecule. Simply put, this conjugated antigen contains nicotine (nicotine) and is combined with other molecules (usually proteins) to form a molecule that can be recognized by the immune system.

[0068] Regarding "nicotine antibody enzyme markers": Nicotine antibodies are antibodies that can recognize and bind to nicotine. Antibodies are part of the immune system, specifically designed to identify foreign substances (such as bacteria, viruses, or toxins). Enzyme markers involve binding enzyme molecules to antibodies, enabling the antibodies to react with substrates during detection, producing a measurable signal (such as a color change). The signal intensity of this enzyme reaction can be used to infer the nicotine concentration in the sample. Advantageously, the present invention utilizes at least two nicotine antibody enzyme markers, such as a nicotine antibody alkaline phosphatase marker and a nicotine antibody β-galactosidase marker.

[0069] As used herein, "substrate" (also known as developer) refers to a chemical substance that can react with an enzyme in this process. When the enzyme-labeled antibody reacts with the substrate, a certain color change occurs, and this color change can be measured by an instrument. The product after the reaction of the enzyme marker (a combination of an antibody and an enzyme) with the substrate is usually colored and can be quantified by absorbance measurement. The absorbance value can be measured at a wavelength of 405nm, 420nm or 450nm, preferably at a wavelength of 420nm. In this article, "developer" and "substrate" can be used interchangeably. Advantageously, the present invention uses at least two developers, such as nitrophenyl phosphate and o-nitrophenyl-β-D-galactoside.

[0070] About “competitive integration”:

[0071] (1) “Three roles” in the experimental system:

[0072] Pre-coated antigen: Nicotine-coupled antigen (complex of nicotine molecule and carrier protein) fixed to the bottom of a 96-well plate.

[0073] Free nicotine: The free nicotine released from the sample to be tested (target analyte).

[0074] Enzyme-labeled antibodies: Nicotine-specific antibodies labeled with an enzyme (such as alkaline phosphatase).

[0075] (2) Competitive binding process

[0076] Limited antibody binding sites: The binding sites of enzyme-labeled antibodies are "limited" and can only choose to bind to one of the following two antigens:

[0077] oPre-coated antigen (fixed on the bottom of the well plate)

[0078] o Free nicotine (dissolved in solution)

[0079] Dynamic competitive relationship:

[0080] oIf the free nicotine concentration is low, the enzyme-labeled antibody will preferentially bind to the pre-coated antigen to form a "pre-coated antigen-enzyme-labeled antibody" complex.

[0081] If the free nicotine concentration is high, the free nicotine will bind to the enzyme-labeled antibody in large quantities, preventing the antibody from binding to the pre-coated antigen, resulting in a decrease in the antibody bound to the bottom of the well plate.

[0082] About the “chromogenic process”: Correlation between chromogenic signals and competition outcomes

[0083] (1) Signal generation mechanism

[0084] Enzymatic reaction triggers color development:

[0085] oOnly the enzyme-labeled antibody bound to the bottom of the well plate (i.e., the "pre-coated antigen-enzyme-labeled antibody" complex) can react with the subsequently added substrate to catalyze the generation of a colored product (such as yellow).

[0086] Free enzyme-labeled antibody (antibody not bound to the plate) is removed during the wash steps and does not participate in color development.

[0087] (2) Negative correlation of signal intensity

[0088] The more free nicotine there is, the less enzyme-labeled antibody will bind to the plate, the less enzyme will participate in color development, and the lighter the color will be (the lower the absorbance value).

[0089] The less free nicotine, the more enzyme-labeled antibodies bind to the plate, and the stronger the color signal (higher absorbance value).

[0090] In some embodiments, a dual enzyme system of alkaline phosphatase and β-galactosidase plus a dual substrate system of nitrophenyl phosphate and o-nitrophenyl-β-D-galactoside is used.

[0091] Reaction Principle: The reaction mechanism and process of alkaline phosphatase (ALP) and nitrophenyl phosphate (e.g., p-nitrophenyl phosphate, pNPP) are as follows: Nitrophenyl phosphate (e.g., pNPP) is a common artificial substrate for ALP and is hydrolyzed by ALP under alkaline conditions (pH 9-10). ALP catalyzes the hydrolysis of pNPP, releasing p-nitrophenol (p-Nitrophenol, pNP) and inorganic phosphate (Pi).

[0092] p-Nitrophenyl Phosphate(pNPP)+ALP→p-Nitrophenol(pNP)+Phosphate(Pi)

[0093] p-Nitrophenol (pNP) turns yellow under alkaline conditions and its absorbance can be detected.

[0094] Reaction principle: The reaction principle of β-galactosidase (β-Galactosidase, β-Gal) and ortho-Nitrophenyl-β-D-galactopyranoside (ortho-Nitrophenyl-β-D-galactopyranoside, ONPG) is: ONPG is a colorless synthetic substrate that can be hydrolyzed by β-galactosidase to release yellow o-nitrophenol (o-Nitrophenol, ONP) and galactose.

[0095] ONPG+β-Gal→o-Nitrophenol(ONP)+Galactose

[0096] ONP turns yellow under alkaline conditions (pH>7) and its absorbance can be detected.

[0097] Standard curve drawing

[0098] The standard curve can be obtained by:

[0099] a) preparing a series of nicotine standard solutions;

[0100] b) pre-coating the nicotine antigen onto the bottom of the well plate using a coating agent;

[0101] c) adding the nicotine standard solution and at least two nicotine antibody enzyme markers to the well plate, incubating, and washing after the incubation;

[0102] d) adding at least two color developing reagents to the well plate, mixing, and incubating in the dark to develop color;

[0103] e) After the color development reaction is completed, add the stop solution to the well plate, mix well, and measure the absorbance value;

[0104] f) Establish a three-variable linear regression curve using the absorbance value as the abscissa and the nicotine concentration as the ordinate.

[0105] The absorbance value can be measured at a wavelength of 405 nm, 420 nm or 450 nm, preferably at a wavelength of 420 nm.

[0106] Nicotine standards are solutions of known nicotine concentrations used to establish a standard curve. By comparing the standard curve, the nicotine concentration in the sample can be accurately determined.

[0107] To establish an accurate standard curve, the nicotine standard needs to be diluted in a gradient, i.e., nicotine solutions of varying concentrations are prepared. Typically, this is done starting with a high concentration and then gradually diluting the solution to obtain a series of samples with known concentrations.

[0108] By measuring the absorbance of standards at varying concentrations, a curve is plotted showing the relationship between absorbance and concentration. Curve fitting uses mathematical models (such as linear regression) to find the optimal match between absorbance and concentration, ensuring that the nicotine concentration in the sample can be accurately calculated based on the absorbance value during actual testing.

[0109] Example

[0110] Standard curve drawing

[0111] 1. Nicotine antigen pre-coating treatment

[0112] Nicotine-coupled antigen was pre-coated on the bottom of a 96-well plate using hydroxypropyl cellulose, where the ratio of hydroxypropyl cellulose to nicotine antigen concentration was 1:1, and the concentration range of both was controlled between 100 ng / ml and 20 ug / ml.

[0113] 2. Add 50 μL of nicotine standards of varying concentrations (0, 0.05, 0.1, 0.2, 0.4, and 0.8 ng / mL) to each well. Also add nicotine antibody alkaline phosphatase and nicotine antibody β-galactosidase [0.2 mg / mL, 50 μL per well]. Cover with a sealing film and incubate at 37°C for 30 minutes. After incubation, repeat the wash three times.

[0114] 3. Color development: Add 50 μL each of nitrophenyl phosphate and o-nitrophenyl-β-D-galactoside to each well, mix gently, cover with sealing film and incubate at 37°C in the dark for 15 minutes.

[0115] 4. Stop the reaction: After the color development reaction is completed, add 50 μL of concentrated sulfuric acid (stop solution) to each well, mix gently, and use a UV micro-spectrophotometer Nanodrop 2000 to select three wavelengths of 405 nm, 420 nm, and 450 nm, respectively, to read the absorbance value and record it.

[0116] 5. Regression curve fitting: With the absorbance value as the horizontal axis and the nicotine concentration as the vertical axis, the most suitable and stable three-variable linear equation regression curve is established.

[0117] Among the three wavelengths, the reading at 420 nm is more suitable for standard curve preparation and has the best regression effect (including r 2 >0.999), and the regression effects of other wavelengths are poor. Figure 3 The standard curve obtained by detecting different concentrations of nicotine standards at a wavelength of 420 nm is shown, with the horizontal axis representing nicotine concentration (ng / ml) and the vertical axis representing absorbance. Figure 4 The specific values ​​of the standard curve drawn by detecting different concentrations of nicotine standards at a wavelength of 420nm are shown, as well as the specific regression equation of the standard curve.

[0118] Table 1. Absorbance readings at 420 nm

[0119] 1 2 3 4 A 0.038 0.037 0.037 0.042 B 0.344 0.309 0.038 0.039 C 0.551 0.572 0.045 0.045 D 0.932 1.094 0.04 0.041 E 1.747 1.967 0.044 0.044 F 2.659 2.767 0.041 0.043 G 0.038 0.036 0.05 0.054 H 0.039 0.04 0.049 0.045

[0120] Table 2. Absorbance readings at 405 nm (regression fitting not possible)

[0121] 1 2 3 4 A 0.035 0.034 0.034 0.031 B 0.026 0.032 0.031 0.031 C 0.034 0.033 0.027 0.034 D 0.032 0.031 0.028 0.032 E 0.032 0.034 0.036 0.036 F 0.032 0.034 0.034 0.035 G 0.039 0.04 0.035 0.035 H 0.039 0.04 0.038 0.04

[0122] Table 2. Absorbance readings at 450 nm (regression fitting not possible)

[0123] 1 2 3 4 A 0.039 0.041 0.043 0.041 B 0.038 0.036 0.041 0.043 C 0.043 0.045 0.05 0.042 D 0.036 0.041 0.042 0.043 E 0.041 0.052 0.047 0.042 F 0.04 0.042 0.044 0.041 G 0067 0055 0054 0043 H 0.048 0.045 0.047 0.041

[0124] Nicotine bag sample testing

[0125] (1) Preparation of candidate nicotine bags

[0126] The preparation method of the nicotine bag comprises the following steps:

[0127] Step 1): a nicotine source, a filler, and an adjuvant were stirred at 45° C. for 3 h at a stirring speed of 300 rpm until uniform, to obtain a mixed solution;

[0128] Step 2): drying the mixed solution in step 1) at 60° C., sterilizing it under ultraviolet conditions for 7 hours, and then placing it at room temperature of 25° C. for 25 hours to obtain a dry mixed material;

[0129] Step 3): Weigh 0.8 g of the dry mixture from step 2), place it in a long non-woven bag, and heat-seal it to obtain a nicotine bag.

[0130] In the following examples, the specific raw material names and purchasing manufacturers are shown in Table 4:

[0131] Table 4 Raw material names and purchasing manufacturers

[0132]

[0133]

[0134] Five groups of nicotine pouches with different pH values ​​were prepared according to the nicotine pouch recipes in Table 5.

[0135] Table 5 Nicotine bag formulas with different pH values

[0136]

[0137] Nicotine pouches were prepared from five groups of nicotine pouches with different moisture contents according to the nicotine pouch recipes in Table 6.

[0138] Table 6 Nicotine bag formulas with different moisture content

[0139]

[0140]

[0141] 5 sets of nicotine bags with different types of non-woven fabrics

[0142] Nicotine pouches were prepared according to the nicotine pouch recipe in Table 6.

[0143] Table 6 Non-woven fabric nicotine bag formulas of different

[0144]

[0145] (2) Extract the contents of the nicotine pouch using artificial saliva as a solvent.

[0146] Artificial saliva preparation: Sodium carboxymethyl cellulose is added to 700 ml of distilled water and soaked overnight. After swelling, sorbitol (1.5 g / L), sodium chloride (0.4 g / L), magnesium chloride (0.4 g / L), potassium chloride (0.4 g / L), and paraben (0.02%) are added. After stirring to dissolve, calcium phosphate and sodium phosphate solutions are added. Distilled water is added to the sufficient volume. After stirring, the pH is adjusted to 7 with 0.01% hydrochloric acid or 0.05% sodium hydroxide. Bottle the solution and sterilize it with circulating steam at 100°C for 30 minutes.

[0147] Content extraction: Place each nicotine bag in artificial saliva: Take a complete nicotine bag sample and place it in 50 mL of constant-temperature artificial saliva at 37°C. Use a 1 mL pipette to take 0.5 mL of the solution into an EP tube every two minutes. After sampling, centrifuge at 3000 rpm for 1 minute and take the supernatant as the nicotine bag content extraction sample.

[0148] (3) Enzyme-linked immunosorbent assay

[0149] See " Standard curve drawing Steps in

[0150] result:

[0151] Nicotine can be protonated in different pH environments, forming monoprotonated or diprotonated forms. The pH of the sample has a significant impact on the onset of nicotine release and taste. By screening different combinations and ratios of acid-base modifiers, we studied the effect of pH on nicotine release and found that controlling the sample pH to 7.5-9 resulted in a rapid onset of physiological satisfaction and a better taste.

[0152] Experiments determined that the ideal moisture content for the formulation and process is between 8% and 40%, and samples with varying moisture contents were compared. The higher the moisture content, the faster the nicotine release in the first minute, the higher the concentration of released nicotine, and the faster the onset of physiological satisfaction. Furthermore, the preparation of low-moisture-content nicotine pouch samples with optimal efficacy requires significant technical expertise.

[0153] Screening of the three available nicotine salts revealed that nicotine tartrate had the best dissolution onset and the highest sustained concentration, while nicotine resinate performed poorly in nicotine pouch products.

[0154] Comparison with liquid chromatography detection

[0155] The above method was used to test the nicotine dissolution of our company's experimental samples, including mint and coffee flavors. The results are as follows Figure 5 As shown in FIG, it can be found that the nicotine dissolution curve is more accurate than that of liquid chromatography detection (such as Figure 6 shown) has a better effect.

[0156] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this patent pertains. The terms used herein in the specification of this patent are for the purpose of describing specific embodiments only and are not intended to limit this patent. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0157] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, this patent will not further describe various possible combinations.

[0158] In addition, the various implementation methods of this patent can be arbitrarily combined, as long as they do not violate the concept of this patent, they should also be regarded as the content disclosed by this patent.

Claims

1. A method for detecting nicotine dissolution in nicotine pouches, characterized in that: The following steps are involved: Step A: immersing the nicotine bag in a solvent to obtain an extract of the contents of the nicotine bag, sampling the extract of the contents of the nicotine bag, centrifuging it, and collecting the supernatant to obtain a sample to be tested; Step B: Pre-coat the nicotine antigen onto the bottom of the well plate using a coating agent; Step C: adding the sample to be tested to the well plate, and simultaneously adding at least two nicotine antibody enzyme markers, incubating, and washing after the incubation; Step D: Add at least two color developing reagents to the well plate, mix well, and incubate in the dark to develop color; Step E: After the color development reaction is completed, add the stop solution to the well plate, mix well, and measure the absorbance value; Step F: Calculate the nicotine concentration in the sample to be tested using the standard curve.

2. The method according to claim 1, characterized in that In the step A: The temperature of the solvent is 35.0-36.0°C, 36.0-37.0°C or 37.0-38.0°C; The volume of the solvent used in each nicotine bag is 5-15 mL / bag, 15-50 mL / bag, 50-100 mL / bag or 100-200 mL / bag; The step A comprises sampling the nicotine bag content extract multiple times at intervals, wherein the intervals are 1 minute to 2 minutes, 2 minutes to 5 minutes, or 5 minutes to 10 minutes; The solvent is artificial saliva, which is prepared as follows: sodium carboxymethyl cellulose is added to 700 ml of distilled water and soaked overnight. After swelling, sorbitol, sodium chloride, magnesium chloride, potassium chloride, and paraben are added. After stirring, calcium phosphate and sodium phosphate solutions are added. Distilled water is added to a sufficient amount. After stirring, the solution is adjusted to pH 7 with 0.01% hydrochloric acid or 0.05% sodium hydroxide. The solution is bottled and sterilized with circulating steam at 100 degrees for 30 minutes.

3. The method according to claim 1, characterized in that The step B specifically comprises: coating the nicotine antigen on the bottom of a 96-well plate using hydroxypropyl cellulose, wherein the concentration ratio of hydroxypropyl cellulose to nicotine antigen is 1:1, and the concentration range of the two is controlled within a range of 100 ng / ml-20 ug / ml.

4. The method according to claim 1, wherein The step C specifically comprises: adding the sample to be tested to the well plate, and simultaneously adding a nicotine antibody alkaline phosphatase marker and a nicotine antibody β-galactosidase marker, covering the plate with a sealing film and incubating at 37° C. for 30 minutes. After the incubation is completed, the plate is washed three times.

5. The method according to claim 1, wherein The step D is specifically as follows: adding nitrophenyl phosphate and o-nitrophenyl-β-D-galactoside to the well plate, mixing, covering with a sealing film, and incubating at 37° C. in the dark for 15 minutes to develop color.

6. The method according to claim 1, characterized in that The step E specifically includes: after the color development reaction is completed, adding concentrated sulfuric acid to the well plate, mixing, and measuring the absorbance value at a wavelength of 405 nm, 420 nm or 450 nm using a spectrophotometer within 5 minutes.

7. The method according to claim 1, characterized in that The standard curve is obtained by: a) preparing a series of nicotine standard solutions; b) pre-coating the nicotine antigen onto the bottom of the well plate using a coating agent; c) adding the nicotine standard solution and at least two nicotine antibody enzyme markers to the well plate, incubating, and washing after the incubation; d) adding at least two color developing reagents to the well plate, mixing, and incubating in the dark to develop color; e) After the color development reaction is completed, add the stop solution to the well plate, mix well, and measure the absorbance value; f) Establish a three-variable linear regression curve using the absorbance value as the abscissa and the nicotine concentration as the ordinate.

8. A nicotine detection kit for implementing the method according to any one of the preceding claims, comprising at least: Solvent, coating agent, well plate, at least two nicotine antibody enzyme labels, at least two color developing agents and stop solution.

9. A method for screening nicotine pouches, the method comprising screening a nicotine pouch from a group of candidate nicotine pouches using the method according to any one of claims 1 to 7.

10. The nicotine bag screening method according to claim 9, wherein the screening parameters include pH value, moisture content, or non-woven bag type.

Images