UPLC method for simultaneous determination of free and protonated nicotine in oral products

Abstract

The present application belongs to the technical field of physicochemical detection of smokeless tobacco products, and relates to a UPLC detection method for simultaneously determining free-state and proton-state nicotine in a mouth-containing product. The mouth-containing bag sample is pretreated by an in-vitro oral cavity simulation release method to simulate the human oral cavity environment and realize nicotine dissolution. A 20 mmol / L acetic acid-ammonium acetate buffer solution is selected to prepare a mobile phase, and the pH value of the system is adjusted to 5.2±0.1. The buffer system is used to stabilize the dissociation equilibrium of nicotine, and effectively inhibit the form transformation of free-state and proton-state nicotine during the detection process. Single injection analysis can be realized, and baseline separation and accurate quantification of the two forms of nicotine can be simultaneously completed. The method is simple to operate and has high detection efficiency, and does not require complex pretreatment. The method can truly reflect the original form distribution and in-vitro release rule of nicotine in the mouth-containing product, and is suitable for daily quality detection, product research and development, and supervision and inspection work of smokeless nicotine products such as mouth-containing bags and mouth-containing tablets.

Description

Technical Field

[0001] This invention belongs to the field of physicochemical detection technology of smokeless tobacco products, specifically relating to a UPLC detection method for the simultaneous determination of free and protonated nicotine in oral products. Background Technology

[0002] Nicotine is the core functional component and safety control indicator of smokeless tobacco products such as lozenges, tablets, and cigarette packs. It exists stably in the saliva environment of the human oral cavity in a protonated state (ionic nicotine). + Nicotine exists in two coexisting forms: total nicotine (total nicotine) and protonated nicotine (molecule-state nicotine). Protonated nicotine, with its excellent lipid solubility, can rapidly penetrate the oral mucosa and be absorbed by the body, directly determining the product's onset rate, oral irritation, and addiction risk. Protonated nicotine, on the other hand, is highly water-soluble, has poor mucosal penetration, and its absorption and utilization rate by the body is extremely low; it exists only as a reserve form of nicotine. Therefore, simply measuring the total nicotine content cannot accurately characterize the actual release characteristics and bioavailability of the nicotine in the oral cavity; it is essential to independently and accurately quantify the two different forms of nicotine.

[0003] Current mainstream nicotine speciation detection methods have significant fundamental flaws and operational drawbacks: traditional detection methods mostly rely on indirect quantitative methods such as liquid-liquid extraction, stepwise pH adjustment, and sample testing. These methods convert protonated nicotine into a free state through strong alkali adjustment, followed by extraction with organic solvents to achieve quantification. This not only involves cumbersome procedures, high consumption of organic solvents, and long testing cycles, but also completely disrupts the original acid-base balance and natural nicotine speciation in the oral cavity during the extraction and pH adjustment processes. Consequently, the test results cannot accurately reflect the actual release state in the oral cavity. Meanwhile, conventional liquid chromatography detection often uses strongly acidic, neutral, or alkaline mobile phases. Strongly acidic and alkaline mobile phases cause nicotine to exist in a single proton or free state. The difference between a neutral mobile phase (pH around 7) and nicotine pKa (around 8) is less than 1.5, which can induce rapid dynamic interconversion between free and protonated nicotine, resulting in only a single chromatographic peak of total nicotine and failing to achieve dual-form detection. The few detection schemes that use mass spectrometry are expensive and inefficient, making it difficult to meet the needs of routine detection of batch samples.

[0004] More importantly, existing technologies cannot achieve breakthroughs in preserving the in-situ morphology of the oral environment, direct injection without extraction, and baseline separation of dual morphologies in a single injection. They generally either disrupt the original morphological balance of the sample or fail to achieve chromatographic separation of dual morphologies, resulting in distorted morphological detection data. This makes it difficult to support the optimization of taste, safety risk assessment, quality grading, and formula iteration and upgrading of oral pouch products.

[0005] To address the industry pain points of existing technologies, such as inability to simulate the real oral release environment, morphological detection distortion, complex operation, and low detection efficiency, there is an urgent need to develop a highly efficient detection method that can accurately reproduce the real morphology of oral nicotine, requires no complex pretreatment, and can simultaneously quantify dual-morphological nicotine in a single injection. Summary of the Invention

[0006] The purpose of this invention is to overcome the technical defects of existing oral products (nicotine lozenges, tablets, etc.) in nicotine speciation detection, which rely on acid-base adjustment and solvent extraction, disrupt the original oral morphological balance, cannot achieve in-situ quantification, and are cumbersome and inefficient. This invention provides a UPLC (ultra-high performance liquid chromatography) detection method for the simultaneous determination of free and protonated nicotine in oral products. Based on in vitro oral simulated release, this method eliminates the need for solvent extraction and pH adjustment, and allows for the simultaneous quantification of free and protonated nicotine released in the oral cavity from oral products with a single injection.

[0007] The technical solution adopted by this invention to solve its technical problem is: a UPLC detection method for simultaneous determination of free and protonated nicotine in oral products. This method constructs a highly adaptable in vitro oral cavity simulated release system, utilizes a pH 5.2 buffer system to fix the existence of free and protonated nicotine forms, avoiding interconversion. The sample solution requires no extraction, pH adjustment, or sample derivatization; a single injection can achieve baseline separation and simultaneous accurate quantification of the two forms of nicotine actually released in the oral cavity, accurately reproducing the distribution pattern of nicotine release forms in the human oral cavity. The method includes the following steps:

[0008] S1. In vitro oral cavity simulation release: Using standard artificial saliva as the release medium, the temperature and slow-release environment of the human oral cavity are simulated. The oral product sample is released at a constant temperature and constant state to obtain the nicotine release liquid that completely restores the real state of the oral cavity, and retains the original ratio of free nicotine to protonated nicotine.

[0009] S2. Sample pretreatment: After the released original solution is allowed to stand, the supernatant is taken and filtered through a microporous membrane to obtain the test solution whose original form is completely preserved.

[0010] S3. Establishment of standard curves: Prepare free nicotine standard solutions and protonated nicotine standard solutions respectively, and establish corresponding quantitative standard curves;

[0011] S4, UPLC chromatographic separation: The test solution is injected once, and a weakly acidic buffer mobile phase system is used to maintain the original state equilibrium of the sample. The difference in chromatographic kinetics between the two forms of nicotine is utilized to achieve baseline in-situ separation of protonated nicotine and free nicotine.

[0012] S5. Simultaneous Quantitative Calculation of Two Forms: The protonated nicotine chromatographic peak and the free nicotine chromatographic peak obtained by separation are integrated separately. Based on the integration results of the two peaks in the chromatogram and the corresponding standard curve, the content of protonated nicotine and free nicotine in the sample are calculated; the total nicotine content and the proportion of free nicotine are calculated.

[0013] S6. Methodology Validation: Through recovery rate and precision validation, confirm that the detection results are accurate and effective, and output the final quantitative data.

[0014] Further, the specific process of step S1, in vitro oral cavity simulation release, is as follows: A standard artificial saliva at pH 6.8 is selected as the release medium, and an aqueous solution is prepared from the following components at the specified mass concentrations: anhydrous magnesium chloride 100 mg / L, calcium chloride dihydrate 220 mg / L, disodium hydrogen phosphate heptahydrate 1350 mg / L, potassium dihydrogen phosphate 680 mg / L, potassium chloride 750 mg / L, urea 600 mg / L, and sodium chloride 600 mg / L. The solution is kept at a constant temperature of 37°C to accurately simulate the temperature environment of the human oral cavity. 10 mL of artificial saliva is precisely measured and placed in a sealed centrifuge tube, then added to an intact oral bag sample. The sample is kept at a constant temperature and allowed to stand for 2–8 minutes to simulate the state of nicotine in the mouth, ensuring a natural and stable release of nicotine and forming a stable dual-form equilibrium system, thus maximally replicating the real release scenario in the human oral cavity.

[0015] Furthermore, the sample pretreatment in step S2 specifically involves: letting the release stock solution stand for 2 minutes, taking the upper clear release solution, filtering it through a 0.22μm organic microporous membrane, and using the resulting filtrate directly as the test solution; without adjusting the pH, adding acid or alkali reagents, extracting with organic solvents, performing sample derivatization, or diluting the sample throughout the entire process, thus completely preserving the original dual-form coexistence system of nicotine formed in the oral cavity simulation environment.

[0016] Furthermore, the principle of establishing and quantifying the standard curve in step S3: This invention adopts the core strategy of preparing dual standard curves using differentiated phase-fixing solvents to eliminate the mixing and cross-interference of standard forms from the source, and achieve independent, accurate and absolute quantification of the two forms of nicotine. Specifically, free nicotine standard solutions and protonated nicotine standard solutions with mass concentrations ranging from 0.1 to 100 μg / mL were prepared. Both types of standards were prepared using dedicated phase-fixing solvents to achieve absolute locking of a single form: ① The free nicotine standard solution was prepared using a sodium hydroxide aqueous solution with a pH ≥ 9.5. The high pH water environment can completely suppress the protonation equilibrium of nicotine, stably locking 100% pure molecular free state without the generation of protonated side peaks. Moreover, this solvent is a pure aqueous phase, which is highly matched with the polarity and elution intensity of the chromatographic mobile phase, completely eliminating solvent effects such as peak precession, peak broadening, and peak shape distortion caused by organic solvents; ② The protonated nicotine standard solution was prepared using a 0.1 wt% acetic acidic aqueous solution. The sufficient proton environment can lock nicotine 100% in the single protonated ionic form without interference from free state side peaks, and is specifically used for the quantitative standardization of characteristic peaks of protonated nicotine. A linear standard curve was fitted with the peak area of ​​the characteristic single peak as the ordinate and the concentration of the standard as the abscissa, and the correlation coefficient R² ≥ 0.999 was obtained.

[0017] Furthermore, the ultra-high performance liquid chromatography (UHPLC) detection conditions in step S4 UPLC chromatographic separation are as follows: a BEHC18 column with dimensions of 2.1 mm × 100 mm and a particle size of 1.7 μm is used; the column temperature is 33–37 °C, the flow rate is 0.28–0.32 mL / min, the detection wavelength is 260 nm, and the injection volume is 2 μL; the buffer mobile phase system is acetonitrile-20 mM acetate-ammonium acetate buffer solution with a volume ratio of 35:65, the pH of the buffer solution is precisely adjusted to 5.2 ± 0.1, and the isocratic elution time is 10 min.

[0018] The pH 5.2 ± 0.1 weakly acidic buffer mobile phase system is the core form-locking system of this invention: the critical dissociation constant pKa of nicotine is 8.02, and the pH 5.2 system has a pKa difference of 2.82, far from the rapid form-change sensitive range of nicotine pKa ± 1.5. More importantly, this invention uses a 20mM acetate-ammonium acetate buffer system, which can stably clamp the hydrogen ion activity of the system, significantly reduce the dynamic exchange rate of nicotine protonation and deprotonation, prolong the form equilibrium half-life, and make the form-change reaction extremely slow. Combined with the short residence time of the sample in the chromatographic column of only 2-6 min, the form conversion reaction can be completely ignored, and the components only undergo physical differential migration. At the same time, there are significant differences in chromatographic retention between the two forms of nicotine. Protonated nicotine has high polarity, weak chromatographic retention, and an earlier peak time, while free nicotine has low polarity, strong chromatographic retention, and a later peak time. The spatial isolation effect in the column completely freezes the proportion of the original oral nicotine forms, eliminating the problem of form-change distortion during detection.

[0019] Qualitative rules for chromatograms: The symmetrical independent chromatographic peak with the earlier retention time is the characteristic peak of protonated nicotine, and the symmetrical independent chromatographic peak with the later retention time is the characteristic peak of free nicotine. The resolution between the two peaks is R≥1.5, achieving complete baseline separation without tailing, impurities, or cross-interference.

[0020] Furthermore, the dual-form simultaneous quantitative calculation method in step S5: This invention establishes a dedicated, interference-free quantitative system of "pure form standard calibration - in-situ dual-peak detection of samples," ensuring the standard curve is free from form-related interference and that the sample retains the true morphological balance of the oral cavity. Quantification strictly adheres to the principle of one-to-one dedicated calibration of pure form: a pure free nicotine standard curve prepared with an alkaline aqueous solution of pH ≥ 9.5 is used to quantify the characteristic peak of free nicotine in the sample chromatogram; a pure protonated nicotine standard curve prepared with a 0.1 wt% dilute acetic acid aqueous solution is used to quantify the characteristic peak of protonated nicotine in the sample chromatogram. The actual content of protonated and free nicotine in the sample is accurately calculated, where the total nicotine content is the sum of the protonated and free nicotine contents, and the free nicotine percentage is the percentage of the free nicotine content to the total nicotine content. This method solves the industry technical problems of form-related interference, quantitative distortion, and cross-interference caused by traditional single-solvent standard preparation.

[0021] Furthermore, the nicotine spiked recovery rate of this method was 95.0%–105.0%, and the relative standard deviation of the detection results was ≤2.0%.

[0022] The present invention has the following beneficial effects:

[0023] 1. In-situ reproduction of the true oral cavity morphology, accurate and reliable test data: This invention uses artificial saliva at pH 6.8 and a constant temperature system of 37℃ to simulate the real release environment of the human oral cavity, completely replicating the human oral scenario, accurately preserving the natural coexistence balance of proton and free states of nicotine in the oral cavity, and completely solving the core pain points of traditional extraction methods and acid-base adjustment methods that destroy the original form and distort test results. The test data can truly reflect the oral release characteristics and human bioavailability of oral products.

[0024] 2. Extraction-free single-pass dual-form detection, significantly improving detection efficiency: Completely eliminating the cumbersome operations of traditional liquid-liquid extraction, step-by-step pH adjustment, and sample separation detection, samples only need simple filtration before direct injection. A single 10-minute isocratic elution can simultaneously output three core detection data: free state, proton state, and total nicotine. The sample pretreatment efficiency is greatly improved, making it suitable for high-throughput and accurate detection scenarios of large batches of samples.

[0025] 3. The invention employs a pH 5.2 buffer system to stabilize nicotine forms, demonstrating significant innovation: The preferred pH 5.2 ± 0.1 acetate-ammonium acetate weak buffer mobile phase overcomes the industry's bias of relying solely on thermodynamic pH differences to determine form stability. By utilizing buffer salts to suppress hydrogen ion activity and reduce the rate of form interconversion, coupled with a short chromatographic residence time window, the invention achieves stable proportions of original oral nicotine forms. This solves the problems of rapid form interconversion, overlapping peaks, and distorted proportions in conventional systems. The technical principle is novel and highly original.

[0026] 4. Differentiated Phase-Fixing Dual-Standard Curve System, Completely Eliminating Quantitative Cross-Interference: This invention addresses the differences in the stable environments of the two nicotine forms by using dedicated phase-fixing solvents to prepare standard curves for pure free and pure protonated nicotine states. This eliminates the problem of mixed standard forms from the source, significantly improving quantitative specificity and accuracy. No expensive isotope internal standard is required, there is no interference from exogenous reagents, and the original oral release form balance of the sample is fully preserved. The methodology exhibits excellent performance, with a spike recovery rate of 95.0%–105.0% and a relative standard deviation ≤5.0%, demonstrating good recovery.

[0027] 5. Low cost, versatility, and wide applicability: Detection can be completed using conventional UPLC-PDA detection equipment, eliminating the need for high-end mass spectrometry instruments, complex derivatization reagents, and large amounts of organic solvent extraction. It is environmentally friendly and cost-effective. It is widely applicable to the detection of nicotine speciation, formulation optimization, and quality control in various base-based oral products, such as nicotine tartrate and nicotine lactate.

[0028] This invention constructs a highly adaptable in vitro oral cavity simulated release system. Utilizing a pH 5.2 buffer system, it fixes the existence of nicotine in both free and protonated states, avoiding interconversion between the two states. The sample solution requires no extraction, pH adjustment, or sample derivatization. A single injection can achieve baseline separation and simultaneous precise quantification of the two forms of nicotine released in the oral cavity, accurately restoring the distribution pattern of nicotine release forms in the human oral cavity. Attached Figure Description

[0029] Figure 1 This is the UPLC spectrum of the test solution in Example 1 of the present invention. Detailed Implementation

[0030] The following are specific embodiments of the present invention, which further describe the technical solution of the present invention. However, the scope of protection of the present invention is not limited to these embodiments. All changes or equivalent substitutions that do not depart from the concept of the present invention are included within the scope of protection of the present invention.

[0031] Example 1: Detection of dual-form nicotine release from a nicotine lozenge in the oral cavity

[0032] S1. In vitro oral cavity simulated release: Prepare a standard artificial saliva solution with pH 6.8, composed of the following components at the specified mass concentrations: anhydrous magnesium chloride 100 mg / L, calcium chloride dihydrate 220 mg / L, disodium hydrogen phosphate heptahydrate 1350 mg / L, potassium dihydrogen phosphate 680 mg / L, potassium chloride 750 mg / L, urea 600 mg / L, and sodium chloride 600 mg / L. Take 10 mL of the pH 6.8 standard artificial saliva solution and place it in a sealed centrifuge tube. Preheat the tube in a water bath at a constant temperature of 37°C. Place a complete commercially available nicotine lozenge sample inside and allow it to stand at a constant temperature for 2 minutes to simulate the process of oral administration of nicotine, thus obtaining the nicotine release concentrate.

[0033] S2. Simplified sample pretreatment: Take the sample with the bag at the outlet, let the released original liquid stand for 2 minutes, take the clear liquid from the top, filter it through a 0.22μm organic filter membrane, and use the filtrate directly as the test solution. There is no pH adjustment, extraction, dilution or internal standard addition operation throughout the process.

[0034] S3. Preparation of Standard Curves: A series of standard solutions with different concentration gradients were prepared using specialized solvents for phase determination: Free nicotine standards were prepared using sodium hydroxide aqueous solution with pH ≥ 9.5 to avoid buffer salt interference, ensuring pure free nicotine, no protonation conversion, and that the pure aqueous system had no solvent effect and regular peak shape; Protonated nicotine standards were prepared using 0.1 wt% acetic acid aqueous solution to ensure pure protonated nicotine; Free nicotine standard gradient solutions and protonated nicotine standard gradient solutions with mass concentrations of 0.1–100 μg / mL were prepared, and the correlation coefficients R² of the curves were 0.9998 and 0.9996, respectively.

[0035] S4, UPLC injection and detection: Chromatographic conditions: ACQUITY BEH C18 column (2.1 mm × 100 mm, 1.7 μm), column temperature 35 ℃, mobile phase acetonitrile: 20 mM acetate-ammonium acetate buffer (pH 5.2) = 35:65, flow rate 0.3 mL / min, detection wavelength 260 nm, injection volume 2 μL, isocratic elution for 10 min.

[0036] S5, Dual-morphology simultaneous quantification: The chromatogram shows two symmetrical chromatographic peaks with complete baseline separation, such as... Figure 1 As shown, the peaks with earlier retention times are characteristic of protonated nicotine, while those with later retention times are characteristic of free nicotine. Substituting the peak areas of the two peaks into the corresponding standard curves and integrating, the protonated nicotine content was calculated to be 9.26 μg / mL, the free nicotine content to be 0.58 μg / mL, the total nicotine content to be 9.84 μg / mL, and the proportion of free nicotine in the oral environment to be 5.89%.

[0037] S6. Method validation: Nicotine reference standard was added to the blank solution to prepare three spiked test solutions of different concentrations. The contents of free and protonated nicotine were calculated, and the total nicotine content was further calculated and compared with the theoretical amount. The spiked recovery rate was found to be between 97.8% and 101.2%, and the relative standard deviation was no greater than 1.9%. The results are shown in Table 1, indicating that the repeatability and recovery rate of different concentrations are good.

[0038] Table 1. Recovery results of spiked test solutions at different concentrations

[0039]

[0040] Example 2: Detection of Nicotine Forms Released from Oral Pouches at Different Release Times

[0041] This embodiment was used to verify the versatility and stability of the proposed method. Two different sustained-release time gradients (5 min and 8 min) were set using the same nicotine lozenge product. All other experimental operations and parameters were identical to those in Example 1. The test results showed that as the simulated oral holding time increased, the release of both free and protonated nicotine increased synchronously, but the proportion of each form remained relatively stable, consistent with the actual nicotine release pattern in the human oral cavity. This demonstrates the excellent stability and versatility of the proposed method, making it suitable for testing scenarios with different sustained-release durations.

[0042] Table 2 Results of nicotine form release from oral pouches at different release times

[0043]

[0044] Comparative Example 1: A comparative experiment on the effect of different mobile phase pH on the separation of nicotine dual forms

[0045] This comparative example is used to verify that the proprietary mobile phase system of pH 5.2 ± 0.1 is the only effective condition for achieving baseline separation and form freezing of nicotine in both forms. It demonstrates that deviations from this pH range cannot effectively distinguish between free and protonated nicotine, highlighting the innovation and necessity of the mobile phase pH parameter in this invention. Except for the mobile phase pH, all other sample pretreatment, standard curves, UPLC chromatographic conditions, and elution parameters in this comparative example are completely consistent with Example 1. Only the pH of the acetate-ammonium acetate buffer was adjusted, using either acetic acid or ammonia to adjust the mobile phase pH. Three control systems were set up: strongly acidic (pH 3.5), neutral (pH 7.0), and alkaline (pH 10) to compare the nicotine form separation effect under different pH conditions.

[0046] 1. Separation results of a strongly acidic mobile phase system at pH 3.5: The mobile phase has an extremely high hydrogen ion concentration and a sufficient supply of protons. The original free nicotine in the sample undergoes rapid protonation during the chromatographic separation process and is completely converted into protonated nicotine. The chromatogram shows only a single characteristic peak of protonated nicotine, with no peak of free nicotine. It is impossible to distinguish between the two forms of nicotine. Only the total nicotine content can be detected, and quantitative determination of the form is not possible.

[0047] 2. Separation results in a pH 7.0 neutral mobile phase system: The pH difference with nicotine's pKa is only 1.02, falling within the pKa ± 1.5 range, a sensitive region for rapid speciation. Within the column, free and protonated nicotine undergo millisecond-level instantaneous dynamic exchange. During chromatographic migration, the components alternate between rapidly eluting protonated and slowly eluting free states. The chromatographic system cannot distinguish between the two forms, outputting only a single, broad, fused peak with severe tailing and asymmetry, making baseline separation and independent quantification of both speciations impossible.

[0048] 3. Separation results of pH10 alkaline mobile phase system: The pH value is higher than the pKa of nicotine, and the system is alkaline. The protonated nicotine in the sample is rapidly deprotonated and converted into free nicotine. The final chromatogram only shows a single characteristic peak of free nicotine, with no protonated nicotine chromatographic peak. The original protonated nicotine component information in the oral cavity is completely lost, and the speciation detection is completely distorted.

[0049] The experimental results show that the pH 5.2±0.1 weak buffer mobile phase system specified in this invention can effectively isolate nicotine from its rapid form-change range, transforming the protonation and deprotonation reactions into slow equilibrium reactions. Combined with the differential migration effect within the column, this maintains the original ratio of the two nicotine forms, achieving baseline separation, regular peak shapes, no form conversion, and no peak overlap between the two forms. Deviations from this pH range result in complete form conversion, double peak overlap, and peak distortion, making simultaneous and accurate detection of both forms impossible. This fully verifies the uniqueness, scientific validity, and inventiveness of the core parameters of this invention.

[0050] This invention is not limited to the above-described embodiments. Anyone should know that any structural changes made under the guidance of this invention, and any technical solutions that are the same as or similar to this invention, fall within the protection scope of this invention.

[0051] The technologies, shapes, and structures not described in detail in this invention are all known technologies.

Claims

1. A UPLC method for simultaneous determination of free and protonated nicotine in oral products, characterized in that, Includes the following steps: S1. In vitro oral cavity simulation release: Using standard artificial saliva as the release medium, the temperature and slow-release environment of the human oral cavity are simulated. The oral product sample is released at a constant temperature and constant state to obtain the nicotine release liquid that completely restores the real state of the oral cavity, and retains the original ratio of free nicotine to protonated nicotine. S2. Sample pretreatment: After the released original solution is allowed to stand, the supernatant is taken and filtered through a microporous membrane to obtain the test solution whose original form is completely preserved. S3. Establishment of standard curves: Prepare free nicotine standard solutions and protonated nicotine standard solutions respectively, and establish corresponding quantitative standard curves; S4, UPLC chromatographic separation: The test solution is injected once, and a weakly acidic buffer mobile phase system is used to maintain the original state equilibrium of the sample. The difference in chromatographic kinetics between the two forms of nicotine is utilized to achieve baseline in-situ separation of protonated nicotine and free nicotine. S5. Simultaneous quantification of dual forms: The protonated nicotine chromatographic peak and the free nicotine chromatographic peak obtained by separation are integrated respectively, and the contents of protonated nicotine and free nicotine in the sample are calculated by combining the corresponding standard curve; S6. Methodology Validation: Through recovery rate and precision validation, confirm that the detection results are accurate and effective, and output the final quantitative data.

2. The detection method according to claim 1, characterized in that, In step S1, artificial saliva with pH 6.8 is used as the release medium, and the water bath is kept at a constant temperature of 37°C. The oral bag sample is kept at a constant temperature for 2-8 minutes to simulate the temperature of the human oral cavity and the oral sustained-release environment, thus preserving the natural balance of nicotine in the oral cavity.

3. The detection method according to claim 1, characterized in that, In step S2, the pretreatment is performed using a 0.22 μm organic microporous membrane, and the filtrate is used directly as the test solution.

4. The detection method according to claim 1, characterized in that, In step S2, the pretreatment process does not adjust the pH of the system, add acid or base reagents, use organic solvents for extraction, perform sample derivatization, or dilute the sample.

5. The detection method according to claim 1, characterized in that, In step S3, the free nicotine standard solution is prepared using a sodium hydroxide solution with a pH ≥ 9.5, and the protonated nicotine standard solution is prepared using a dilute acetic acid aqueous solution.

6. The detection method according to claim 1, characterized in that, In step S3, the concentration gradient of the standard solution is 0.1–100 μg / mL; the correlation coefficient R² of the established standard curve is ≥0.

999.

7. The detection method according to claim 1, characterized in that, The UPLC chromatographic detection conditions in step S4 are as follows: a BEHC18 column with dimensions of 2.1 mm × 100 mm and a particle size of 1.7 μm is used; the column temperature is 33–37 °C, the flow rate is 0.28–0.32 mL / min, the detection wavelength is 260 nm, the injection volume is 2 μL, and the isocratic elution time is 10 min.

8. The detection method according to claim 7, characterized in that, In step S4, the buffer mobile phase system is an acetonitrile-20mM acetic acid-ammonium acetate buffer solution with a volume ratio of 35:65, and the pH of the buffer solution is precisely adjusted to 5.2±0.

1.

9. The detection method according to claim 1, characterized in that, In step S4, the resolution R of the protonated nicotine and free nicotine chromatographic peaks is ≥1.

5.

10. The detection method according to claim 1, characterized in that, The nicotine spiked recovery rate of this method was 95.0%–105.0%, and the relative standard deviation of the detection results was ≤2.0%.

Images