Mixed measurement method for nicotine content in zero-nickel matrix

Abstract

The invention provides a mixed detection method for the content of nicotine in a nickelous matrix. Comprising the following steps: determining the number of samples to be mixed based on the following conditions: (1) a control upper limit of nicotine content in a zero-nickel product and a detection limit of single detection of a detection system, (2) a minimum measuring range of a measuring tool to be used during detection, and (3) a single-batch sampling number of a production line; (4) the historical positive detection rate of the zero-nickel product and the maximum sample mixing quantity determined based on the Poisson distribution principle according to the maximum detection efficiency principle; based on the number of the to-be-mixed samples, respectively measuring sample liquids with preset volumes from the corresponding multiple sampling samples, and mixing to prepare matrix mixed to-be-detected samples; the matrix mixed sample to be detected is loaded and detected, and if the detection result is negative or the nicotine content does not exceed a positive threshold value corresponding to the control upper limit, the corresponding sample meets the requirement; and if the total content of nicotine in the detection result is greater than a positive threshold value corresponding to the control upper limit, respectively loading and detecting each sample corresponding to the matrix mixed sample to be detected.

Description

Technical Field

[0001] This application relates to the field of analytical testing technology, and in particular to a mixed method for the determination of nicotine content in a zero-nicotine matrix. Background Technology

[0002] Currently, e-cigarette bases are divided into two categories: nicotine-containing bases and nicotine-free bases (also known as zero-nicotine bases). To meet relevant requirements, nicotine-containing bases must have their nicotine content accurately quantified, and zero-nicotine bases also need to be accurately tested to ensure that there are no nicotine components or that the nicotine content is extremely low (for example, it generally needs to be controlled at the level of 1μg / mL, although different manufacturers may have some differences in their requirements).

[0003] To ensure that the nicotine content in the final zero-nicotine product meets the requirements, sampling tests are required at each stage, including incoming materials, finished products, and matrix infusion. This introduces a practical problem: multiple samples need to be tested for each production batch, resulting in high testing costs and low efficiency, and placing high demands on testing resource allocation (such as personnel and equipment). Meanwhile, the probability of actual product contamination with nicotine is very low, and positive nicotine cases are extremely rare. Overall, the effectiveness of the entire testing process is very low.

[0004] Therefore, given the large number of samples to be tested and the low detection rate, it is necessary to develop a more efficient testing method to improve the effectiveness of zero-nitric acid testing. Summary of the Invention

[0005] Based on this, this application provides a mixed determination method for nicotine content in zero-nicotine matrix to improve the detection efficiency of nicotine in zero-nicotine products.

[0006] This application provides a method for the mixed determination of nicotine content in a zero-nicotine matrix, comprising the following steps:

[0007] The quantity of samples to be mixed is determined based on the following conditions: ① the upper limit of nicotine content in zero-nicotine products and the detection limit of a single test of the detection system; ② the minimum range of the measuring instrument to be used during testing; ③ the number of samples taken per batch on the production line; ④ the historical positive detection rate of zero-nicotine products and the maximum quantity of samples to be mixed based on the principle of maximum detection efficiency according to the Poisson distribution principle.

[0008] Based on the number of samples to be mixed, a preset volume of sample liquid is measured from the corresponding multiple sampling samples and mixed to prepare a matrix-mixed test sample;

[0009] The nicotine content in the matrix mixture test sample is detected. If the test result is negative or the nicotine content does not exceed the positive threshold corresponding to the control upper limit, the corresponding sampled sample meets the requirements. If the total nicotine content in the test result is greater than the positive threshold corresponding to the control upper limit, the nicotine content in each sampled sample corresponding to the matrix mixture test sample is detected.

[0010] In some embodiments, the number of samples to be mixed is less than the ratio of the control upper limit to the detection limit.

[0011] In some embodiments, the number of samples to be mixed is less than the preset volume divided by the minimum range and rounded down.

[0012] In some embodiments, the number of samples to be mixed is not less than the number of samples taken.

[0013] In some embodiments, the number of samples to be mixed is not greater than the maximum number of samples to be mixed.

[0014] In some embodiments, determining the maximum pooled sample quantity based on the principle of maximum detection efficiency according to the Poisson distribution principle includes the following steps:

[0015] Substituting the historical positive detection rate into the formula for the relationship between the number of sample loading and detection based on the Poisson distribution principle, and considering the negative correlation between the maximum detection efficiency and the number of detections, the maximum number of mixed samples corresponding to the minimum number of sample loading and detections is determined as the maximum number of mixed samples.

[0016] In some embodiments, the quantity of samples to be mixed simultaneously satisfies the following conditions A to D:

[0017] A. Less than the ratio of the control upper limit to the detection limit;

[0018] B. A value less than the preset volume divided by the minimum range and then rounded down;

[0019] C. Not less than the stated sampling quantity;

[0020] D. Not greater than the maximum number of samples to be mixed.

[0021] In some embodiments, the number of samples to be mixed is the maximum value that simultaneously satisfies conditions A through D.

[0022] In some embodiments, the mixed testing method is used for mixed testing of nominally homologous and / or nominally heterologous matrices.

[0023] In some embodiments, the sample loading and detection are performed using a gas chromatography-mass spectrometry (GC-MS) system.

[0024] In some embodiments, the sampling samples include samples taken from the matrix supply, packaging, filling, and finished product stages.

[0025] The above-mentioned mixed testing method for nicotine content in zero-nicotine matrix can quickly and efficiently handle a large number of test samples in various processes such as incoming materials, finished products and filling of zero-nicotine products, significantly improving testing efficiency and meeting production and other related needs. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 A schematic diagram of a gas chromatography-mass spectrometry system provided in one embodiment. Detailed Implementation

[0028] To facilitate understanding of the present invention, a more complete description of this application will be provided below with reference to relevant embodiments. Preferred embodiments of the present application are given below. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that a thorough and complete understanding of the disclosure of this application will be achieved.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0030] As used herein, the terms "and / or," "or / and," and "and / or" encompass any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that, in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR."

[0031] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0032] In this application, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0033] This document only specifically discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, just as any upper limit can be combined with any other upper limit to form an unspecified range. Furthermore, each individually disclosed point or single value can itself serve as a lower or upper limit and be combined with any other point or single value or with other lower or upper limits to form an unspecified range.

[0034] Unless otherwise specified, the temperature parameters in this application may be either constant temperature processing or processing within a certain temperature range. The constant temperature processing allows temperature fluctuations within the precision range controlled by the instrument, such as ±5°C, ±4°C, ±3°C, ±2°C, or ±1°C.

[0035] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0036] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0037] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0038] In the description of the application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0039] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions. Unless otherwise specified, all technical features and optional technical features of this application can be combined to form new technical solutions.

[0040] Unless otherwise specified, all steps of this application may be performed sequentially or randomly, but sequentially is preferred.

[0041] To ensure that the nicotine content in the final zero-nicotine product meets the requirements, sampling tests are required at each stage, including incoming materials, finished products, and matrix infusion. This introduces a practical problem: multiple samples need to be tested for each production batch, resulting in high testing costs and low efficiency, and placing high demands on testing resource allocation (such as personnel and equipment). Meanwhile, the probability of actual product contamination with nicotine is very low, and positive nicotine cases are extremely rare. Overall, the effectiveness of the entire testing process is very low.

[0042] Therefore, given the large number of samples to be tested and the low detection rate, it is necessary to develop a more efficient testing method to improve the effectiveness of zero-nitric acid testing.

[0043] When using gas chromatography-mass spectrometry (GC-MS) to detect nicotine content in a zero-nicotine matrix, this application comprehensively determines an appropriate number of mixed samples, and then mixes multiple matrix samples for detection, thereby achieving the purpose of mixed testing of multiple matrix samples and thus improving testing efficiency.

[0044] One or more embodiments of this application provide a method for mixed testing of nicotine content in a zero-nicotine matrix, comprising the following steps: determining the quantity of samples to be mixed based on the following conditions: ① the upper limit of control of nicotine content in the zero-nicotine product and the detection limit of a single test by the detection system; ② the minimum range of the measuring instrument to be used during testing; ③ the number of samples taken per batch in the production line; ④ the historical positive detection rate of the zero-nicotine product and the maximum number of samples to be mixed based on the principle of maximum detection efficiency according to the Poisson distribution principle; based on the quantity of samples to be mixed, measuring a preset volume of sample liquid from the corresponding multiple sampling samples and mixing them to prepare a matrix mixed test sample; loading the matrix mixed test sample and testing the nicotine content therein; if the test result is negative or the nicotine content does not exceed the positive threshold corresponding to the upper limit of control, then the corresponding sampling product meets the requirements; if the total nicotine content in the test result is greater than the positive threshold corresponding to the upper limit of control, then loading each sampling sample corresponding to the matrix mixed test sample and testing the nicotine content therein.

[0045] It should be noted that the mixed determination method for nicotine content in zero-nicotine matrix described in this article can be used to determine the nicotine content in zero-nicotine e-liquid.

[0046] The term "mixed sample quantity" as used in the context refers to the number of matrix samples mixed together.

[0047] When determining the quantity of samples to be mixed, considering the upper limit of the nicotine content corresponding to the matrix in the zero-nicotine product and the detection limit of the detection system for a single test can reduce the occurrence of missed detections. Considering the minimum range of the measuring instrument to be used during testing helps to keep the sampling error during the matrix mixing and sample preparation process within a controllable range.

[0048] Understandably, the above-mentioned mixed testing method for nicotine content in zero-nicotine matrix can quickly and efficiently handle a large number of test samples in various processes such as matrix raw materials, finished products and filling of zero-nicotine products, significantly improving testing efficiency and meeting production and other related needs.

[0049] For a mixture of n matrix samples, in the extreme case (where one of the n samples contains nicotine), the limit of detection (LOD) and limit of quantitation (LOQ) of the pooled analysis are n times the original values, posing a risk of missed detection. While meeting analytical requirements, appropriately increasing the total amount of analytical matrix and optimizing the analytical process can improve the overall LOD and LOQ, effectively reducing the LOD and LOQ in the extreme case and controlling the risk of missed detection.

[0050] To ensure detection sensitivity and avoid false negatives, the efficacy of pooled testing depends on the positive rate and the number of samples in the pool. For example, assuming only a two-step test is performed, the first step tests the pooled group, and the positive threshold corresponding to the total nicotine content in the test results is set for the group, and the corresponding samples are then tested separately in the second step.

[0051] Based on the Poisson distribution, the expected number of tests for a single sample is E(X), and the detection efficiency is F = 1 / E(X). Therefore, the fewer the number of tests for a single sample, the higher the detection efficiency. The detection efficiency F is maximized when E(X) is minimized. The Poisson distribution formula is as follows:

[0052]

[0053] Where k is the number of mixed samples and p is the historical positive detection rate.

[0054] Based on the Poisson distribution formula, for a given historical positive detection rate p, the minimum value of E(X) corresponds to the maximum number of mixed samples determined based on the principle of maximum detection efficiency according to the Poisson distribution principle.

[0055] Based on the Poisson distribution formula, when the positive detection rate is 5%, theoretically, pooling 5 zero-nylon products yields the highest efficiency, i.e., a 5-sample pool is most efficient. When the positive detection rate is 1%, theoretically, pooling 10 zero-nylon products yields the highest efficiency, i.e., a 10-sample pool is most efficient. When the positive detection rate is 0.1%, theoretically, pooling 30 zero-nylon products yields the highest efficiency, i.e., a 30-sample pool is most efficient.

[0056] It is important to emphasize that when actually mixing samples, factors such as the upper limit of nicotine content in the matrix of zero-nicotine products, the operability of the testing process, the number of samples per batch on the production line, the resolution of instruments and methods, and the efficiency of mixed testing should be comprehensively considered to reasonably set the number of mixed samples.

[0057] In some implementations, the number of samples to be mixed is less than the ratio of the upper control limit to the detection limit, thereby reducing the occurrence of missed detections.

[0058] As one possible implementation, the quantity of samples to be mixed is less than the preset volume divided by the minimum range and then rounded down. This helps to keep the sampling error during the preparation of the matrix-mixed test sample within a controllable range.

[0059] It should be noted that the "preset volume" mentioned in the context refers to the pre-set sampling volume.

[0060] The "round down" mentioned in the context refers to taking an integer value that is no greater than the result of dividing the preset volume by the minimum range.

[0061] In some implementations, the number of samples to be mixed is not less than the number of samples taken.

[0062] As one possible implementation method, the number of samples to be mixed should not exceed the maximum number of samples to be mixed.

[0063] In some implementations, determining the maximum pooled sample size based on the Poisson distribution principle and the principle of maximum detection efficiency includes the following steps:

[0064] Substituting the historical positive detection rate into the expected relationship of the number of sample loading tests based on the Poisson distribution principle, and considering the negative correlation between the maximum detection efficiency and the number of detections, the maximum number of mixed samples corresponding to the minimum number of sample loading tests is determined as the maximum number of mixed samples.

[0065] In some implementations, the quantity of samples to be mixed simultaneously meets the following conditions A to D: A. less than the ratio of the upper control limit to the detection limit; B. less than the preset volume divided by the minimum range and rounded down; C. not less than the sampling quantity; D. not greater than the maximum mixed quantity.

[0066] As one possible implementation method, the number of samples to be mixed is the maximum value that simultaneously satisfies conditions A to D.

[0067] In some alternative implementations, the mixed testing method is used for mixed testing of nominally homologous and / or nominally heterologous matrices.

[0068] The method for determining nicotine content in a zero-nicotine matrix presented in this application can be used for the determination of nominally homogeneous matrices or nominally heterogeneous matrices.

[0069] Nominally, "same matrix" refers to samples taken from different stages of the same process, such as incoming materials, packaging, filling, and finished products. The purpose of the analysis is to confirm that the incoming materials are zero-nicotine products and that no nicotine contamination was introduced during the e-cigarette production process. It is easy to understand that since the matrix used in each stage is the same, pooled testing will not cause significant changes during GC-MS analysis. Provided that the instrument's testing requirements are met, its resolution, detection limit, and quantitation limit remain unchanged.

[0070] Nominally, heterogeneous matrices refer to matrices with different formulations. Based on the specificity of nicotine mass spectra, mixed assays can also be achieved through optimized pretreatment and mixing of heterogeneous matrices.

[0071] For nominally homogeneous matrices, optimized sample pretreatment mixing significantly improved testing performance, reduced reagent and consumable usage, and lowered testing costs. Similarly, for nominally heterogeneous matrices, based on the specificity of nicotine mass spectra, optimized sample pretreatment mixing also significantly improved testing performance, reduced reagent and consumable usage, and lowered testing costs.

[0072] Whether nominally the same matrix or a different matrix, the mixing optimization is achieved during the sample pretreatment process, which has no impact on existing GC-MS testing instruments or testing procedures (test parameters, standard curves, etc.).

[0073] In some implementations, sample loading and detection are performed using gas chromatography-mass spectrometry.

[0074] In some embodiments, a schematic diagram of the gas chromatography-mass spectrometry (GC-MS) instrument is shown below. Figure 1 As shown, when using gas chromatography-mass spectrometry (GC-MS) for testing, the sample is injected into the high-temperature vaporization chamber through the injection port, changing from a liquid to a gaseous state. It is then carried into the chromatographic column by the carrier gas. Within the column, the various components are separated through adsorption-desorption (cooling-heating) cyclic separation. The separated compounds enter the mass spectrometer sequentially according to time. In the mass spectrometer, the neutral molecules of each component are ionized into ions. These ions are separated under the combined action of electric and magnetic fields, ultimately yielding mass spectra and other spectra, enabling qualitative and quantitative analysis of the sample.

[0075] As one possible implementation, the sampling samples include those from the matrix supply, packaging, filling, and finished product stages.

[0076] In some embodiments, the step of preparing a matrix-mixed test sample by measuring a preset volume of sample liquid from multiple corresponding sampling samples based on the number of samples to be mixed includes: measuring a preset volume of sample liquid from multiple corresponding sampling samples based on the number of samples to be mixed and adding it to a sample bottle, then adding a solvent and an internal standard solution, quantifying, and preparing a matrix-mixed test sample.

[0077] As one possible implementation method, the mixed determination method for nicotine content in a zero-nicotine matrix further includes: preparing a series of standard concentration solutions and constructing a concentration standard curve based on the series of standard concentration solutions.

[0078] The above-mentioned method for the mixed determination of nicotine content in zero-nicotine matrix was systematically validated, and the specific process is as follows:

[0079] 1. Upper limit of nicotine content (U) in the matrix of zero-nicotine products c The detection limit (LOD) for a single test is set to 5000 ng / ml, and the upper limit of control (U) is set to 25 ng / ml. c The ratio of the limit of detection (LOD) to the limit of detection (LOD) is 2000, and the number of samples to be mixed must be less than 2000.

[0080] 2. The minimum volume R of the pipette used during the test is 5 μl. The preset volume measured when preparing the matrix to mix the sample to be tested is 320 μl. The preset volume divided by the minimum volume R is 64. The number of samples to be mixed must be less than 64.

[0081] 3. If a single batch of a production line involves approximately 8 processes, then the sampling quantity for a single batch of the production line is 8, and the quantity of samples to be mixed must be no less than 8.

[0082] 4. The historical positive detection rate is calculated as 0.1%. Substitute the historical positive detection rate into the expected relationship of the number of sample loading tests based on the Poisson distribution principle. According to the negative correlation between the maximum detection efficiency and the number of detections, the number of mixed samples corresponding to the minimum number of detections is determined to be 30. The number of mixed samples must not exceed the maximum number of mixed samples.

[0083] Based on the above four aspects, a sample quantity of 8-30 is sufficient to cover the testing requirements. Considering the current production control situation, a sample quantity of 8 is sufficient. However, taking efficiency into account, the sample quantity can be set to 16 or 24, with a maximum of 30.

[0084] After determining the number of samples to be mixed based on the above process, 50 batches of samples with a total of 8 samples were tested, and all were negative. One batch was tested as a human control, and it was positive after mixing, with a nicotine content of 0.63 ng / ml. Then, the 8 groups of samples in this batch were tested separately, and 7 groups were not detected, while 1 group was found to have a nicotine concentration of 5.03 ng / ml.

[0085] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0086] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for determining the nicotine content in a zero-nicotine matrix, characterized in that, Includes the following steps: The quantity of samples to be mixed is determined based on the following conditions: ① the upper limit of nicotine content in zero-nicotine products and the detection limit of a single test of the detection system; ② the minimum range of the measuring instrument to be used during testing; ③ the number of samples taken per batch on the production line; ④ the historical positive detection rate of zero-nicotine products and the maximum quantity of samples to be mixed based on the principle of maximum detection efficiency according to the Poisson distribution principle. Based on the number of samples to be mixed, a preset volume of sample liquid is measured from the corresponding multiple sampling samples and mixed to prepare a matrix-mixed test sample; The nicotine content in the matrix mixture test sample is detected. If the test result is negative or the nicotine content does not exceed the positive threshold corresponding to the control upper limit, the corresponding sampled sample meets the requirements. If the total nicotine content in the test result is greater than the positive threshold corresponding to the control upper limit, the nicotine content in each sampled sample corresponding to the matrix mixture test sample is detected.

2. The method for determining nicotine content in a zero-nicotine matrix as described in claim 1, characterized in that, The number of samples to be mixed is less than the ratio of the control upper limit to the detection limit.

3. The method for determining nicotine content in a zero-nicotine matrix as described in claim 1, characterized in that, The quantity of samples to be mixed is less than the preset volume divided by the minimum range and then rounded down.

4. The method for determining nicotine content in a zero-nicotine matrix as described in claim 1, characterized in that, The number of samples to be mixed is not less than the number of samples taken.

5. The method for determining nicotine content in a zero-nicotine matrix as described in claim 1, characterized in that, The number of samples to be mixed is not greater than the maximum number of samples to be mixed.

6. The method for determining nicotine content in a zero-nicotine matrix as described in claim 5, characterized in that, Determining the maximum pool size based on the Poisson distribution principle and the principle of maximum detection efficiency includes the following steps: Substituting the historical positive detection rate into the expected relationship of sample loading and detection times based on the Poisson distribution principle, and considering the negative correlation between the maximum detection efficiency and the number of detections, the maximum number of mixed samples corresponding to the minimum number of sample loading and detections is determined as the maximum number of mixed samples.

7. The method for determining nicotine content in a zero-nicotine matrix as described in claim 1, characterized in that, The quantity of samples to be mixed simultaneously meets the following conditions A~D: A. Less than the ratio of the control upper limit to the detection limit; B. A value less than the preset volume divided by the minimum range and then rounded down; C. Not less than the stated sampling quantity; D. Not greater than the maximum number of samples to be mixed.

8. The method for determining nicotine content in a zero-nicotine matrix as described in claim 7, characterized in that, The number of samples to be mixed is the maximum value that simultaneously satisfies conditions A through D.

9. The method for determining the nicotine content in a zero-nicotine matrix as described in any one of claims 1 to 8, characterized in that, The mixed assay method is used for mixed assays of nominally homologous and / or nominally heterologous matrices.

10. The method for determining nicotine content in a zero-nicotine matrix as described in any one of claims 1 to 8, characterized in that, The sample loading and detection were performed using gas chromatography-mass spectrometry; and / or The sampling samples include those from the matrix supply, packaging, filling, and finished product stages.

Images