An immunochromatographic reagent strip and detection method for small molecules in tobacco tar

Abstract

The application discloses an immunochromatography reagent strip for small molecule substances in tobacco tar and a detection method. A specific free nicotine antibody is added to a label pad of the reagent strip. A double-layer sample pad is used in the reagent strip, which comprises sample pad I and sample pad II arranged in an upper and lower mode, and is treated by two different treatment liquids. Treatment liquid I comprises Tween-60 and NPO-10, and treatment liquid II comprises borax, sodium cholate and polyvinyl alcohol (PVA). The application further provides a detection method for small molecule substances in tobacco tar by using the immunochromatography reagent strip. The small molecule substances are extracted by using an extraction liquid, the influence of alcohol on the immunochromatography product is reduced, and the above-mentioned immunochromatography detection reagent strip is used for detection, so that the preliminary screening of target drug small molecule substances can be quickly completed, and the detection result is correct and effective.

Description

Technical Field

[0001] This invention relates to the field of immunoassay detection technology, and in particular to an immunochromatographic reagent strip and detection method for small molecule substances in e-liquid. Background Technology

[0002] Electronic cigarettes are a type of tobacco product that has gained popularity in recent years. They consist of an electronic cigarette device and e-liquid. The device converts the e-liquid into an aerosol for the user to inhale. The main components of the e-liquid include nicotine, propylene glycol, glycerin, flavorings, and volatile organic compounds. Nicotine, also known as tobacco alkaloid, is a highly toxic chemical with addictive and dependent properties; excessive intake can be life-threatening. Propylene glycol is miscible with water, ethanol, and many organic solvents, exhibiting good solubility and low toxicity and irritation. It is widely used in cosmetics and the food industry, carrying most of the e-liquid flavor in electronic cigarettes and producing a small amount of vapor and throat hit. Glycerin has a sweeter taste than propylene glycol and is mainly used to produce a larger amount of vapor. Propylene glycol and glycerin can be combined according to specific needs; by adjusting their ratio, a balance between throat hit and vapor production can be achieved. Excessive intake of either can irritate and damage the respiratory tract and kidneys.

[0003] To date, the safety of e-cigarettes has not been fully scientifically proven. There is no systematic safety assessment data for e-cigarettes, either domestically or internationally. At present, it is impossible to determine what potential health risks e-cigarettes may pose to users.

[0004] There are many methods for detecting drug abuse, including gas chromatography-mass spectrometry (GC / MS), high-performance liquid chromatography-mass spectrometry (HPLC / MS), high-performance capillary electrophoresis, enzyme-linked immunosorbent assay (ELISA), and immunochromatography. GC / MS and LC / MS are currently the gold standard for drug detection and identification, but they are expensive and have long testing cycles, making them unsuitable for initial screening. Immunochromatography plays a significant role in rapid drug abuse screening, but the components of e-liquid itself can adversely affect immunochromatographic detection, leading to erroneous or abnormal results. Therefore, a sensitive and accurate method for detecting the components of e-liquid is urgently needed. Summary of the Invention

[0005] The first objective of this invention is to address the issue that the composition of e-liquid (including e-cigarette liquid, e-liquid powder, etc., hereinafter the same) itself can cause errors or abnormal detection results in immunochromatographic assays. This invention provides an immunochromatographic reagent strip for small molecules in e-liquid, ensuring that the detection results are accurate and valid.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] An immunochromatographic reagent strip for small molecules in e-liquid includes a base plate, absorbent paper, nitrocellulose membrane, labeling pad, and dual sample pads; the dual sample pads include sample pad I and sample pad II, which are arranged vertically and vertically, and the sample pad I and sample pad II have different lengths.

[0008] The sample pad I is treated with treatment solution I, which is prepared using the following method:

[0009] The treatment solution I was prepared by mixing Tween-60 and NPO-10, adding pure water and stirring until homogeneous.

[0010] Preferably, the concentration of Tween-60 in the treatment solution I is 5-15 mg / ml, the concentration of NPO-10 is 1-3 mg / ml, and the mass ratio of Tween-60 to NPO-10 is (3-5):1.

[0011] The sample pad II is treated with treatment solution II, which is prepared using the following method:

[0012] The treatment solution II was prepared by mixing borax, sodium cholate and polyvinyl alcohol (PVA), adding pure water and stirring until homogeneous.

[0013] Preferably, the concentration of borax in the treatment solution II is 0.05M, the concentration of sodium cholate is 3-5mg / ml, the concentration of PVA is 5-8mg / ml, and the mass ratio of borax, sodium cholate and PVA is (4-5):1:(1.5-2).

[0014] Preferably, a specific free nicotine antibody is added to the labeling pad.

[0015] The primary function of sample pad I in the dual sample pad assembly is to emulsify esters and alcohols in the coarsely filtered extract, while sample pad II primarily functions to adjust the pH and ion concentration of the extract to be tested, thereby reducing individual differences between samples. In addition to the antibody (original)-labeled conjugate of the analyte, the labeling pad also contains specific free nicotine antibodies to neutralize nicotine substances in the e-liquid before the immune response occurs, reducing interference with the immune reaction.

[0016] The second objective of this invention is to provide a method for detecting small molecule substances in e-liquid, comprising the following steps:

[0017] Step (1): Mix NaCl (7.5-9 mg / ml), Na2HPO4 (2.0-6.0 mg / ml), NaH2PO4 (0.1-0.3 mg / ml), and HCl (0.5 M), add pure water and stir evenly to obtain an extract; mix the extract with e-liquid to extract small molecules from the e-liquid;

[0018] Step (2): The small molecule substances in the e-liquid extracted in step (1) are detected using the above-mentioned immunochromatographic reagent strips.

[0019] Preferably, the volume ratio of e-liquid to extract in step (1) is 1:(2-5).

[0020] The extract prepared by this invention can convert propylene glycol, the main component of e-liquid, into esters. When using immunochromatographic reagent strips for detection, the dual sample pads can filter out some esters and alcohols, while adjusting the differences between samples to ensure the stability of the analyte and the progress of the immunoassay.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) This invention proposes an immunochromatographic detection reagent strip for small molecules in e-liquid. The double-layer sample pad structure can emulsify and filter ester substances and adjust the differences between samples to ensure the stability of the small molecules to be tested and the immunochromatographic reaction. Specific free nicotine antibodies are added to the labeling pad to neutralize the nicotine components in the e-liquid and reduce interference with the immune reaction.

[0023] (2) This invention proposes a method for detecting small molecules in e-liquid. First, an extract of small molecules in e-liquid is prepared. Then, an ester is generated by reacting an inorganic acid with a diol (propylene glycol). This reduces the influence of alcohols on immunochromatographic products and allows the product to be directly used for the detection of immunochromatographic reagents. It also ensures the stable preservation of the target small molecules. Then, the above-mentioned immunochromatographic detection reagent strips are used for detection, which can quickly and effectively complete the initial screening of the target drug small molecules. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments.

[0025] In a first aspect, the present invention provides an immunochromatographic reagent strip for small molecule substances in e-liquid, comprising a base plate, absorbent paper, a nitrocellulose membrane, a labeling pad, and a dual sample pad; the dual sample pad includes sample pad I and sample pad II, with sample pad I disposed on top of sample pad II, and sample pad I and sample pad II having different lengths; the formulation of the treatment solution for sample pad I in the immunochromatographic reagent strip is: Tween-60 5-15 mg / ml, NPO-10 1-3 mg / ml, with the remainder being pure water, and the mass ratio of Tween-60 to NPO-10 is (3-5):1; the formulation of the treatment solution for sample pad II is: borax 0.05M, sodium cholate 3-5 mg / ml, PVA 5-8 mg / ml, with the remainder being pure water, and the mass ratio of borax, sodium cholate, and PVA is (4-5):1:(1.5-2). Specific free nicotine antibodies are added to the labeling pad in the immunochromatographic reagent strip.

[0026] Secondly, the present invention provides a method for detecting small molecule substances in e-liquid, comprising the following steps:

[0027] (1) Extraction of small molecules in e-liquid: Mix e-liquid and extractant at a ratio of 1:2 to 1:5 and let stand for 5-10 minutes; The extractant formula is: NaCl 7.5-9 mg / ml, Na2HPO4 2.0-6.0 mg / ml, NaH2PO4 0.1-0.3 mg / ml, 0.5 MHCL, and the remainder is pure water.

[0028] (2) Detection of small molecules in e-liquid: Add the processed e-liquid sample extract to the immunochromatographic test strip, start timing at the same time as adding the sample, observe whether the control line and the test line are clear and whether there is any tailing, stop timing after 5 minutes and analyze the results.

[0029] Example 1

[0030] A method for extracting, preparing, and detecting small molecule pharmaceutical substances in e-liquid, comprising the following steps:

[0031] (1) Extraction of small molecules in e-liquid: Mix e-liquid and extract at a volume ratio of 1:4 and let stand for 5 minutes; The extract formula is: NaCl 9mg / ml, Na2HPO4 3.0mg / ml, NaH2PO4 0.25mg / ml, 0.5M HCl, and the remainder is pure water.

[0032] (2) Detection of small molecule substances in e-liquid: The processed e-liquid sample extract was added to the immunochromatographic test strip. Timing was started simultaneously with sample addition. The control line and test line were observed to be clear and free of tailing. Timing was stopped after 5 minutes, and the results were analyzed. The immunochromatographic test strip contains two sample pads: Sample Pad I and Sample Pad II. The sample pad I treatment solution consisted of: 4g of Tween-60 at a concentration of 6mg / ml, 1g of NPO-10 at a concentration of 2mg / ml, and the remainder being pure water. The sample pad II treatment solution consisted of: 4g of borax at a concentration of 0.05M, 1g of sodium cholate at a concentration of 4mg / ml, 2g of PVA at a concentration of 8mg / ml, and the remainder being pure water. Specific free nicotine antibodies were added to the label pad of the immunochromatographic test strip.

[0033] Twenty-two etomidate-positive e-cigarette liquids and thirty etomidate-negative e-cigarette liquids were collected for etomidate detection, with GC / MS results used as a reference. The results showed that the positive concordance rate, negative concordance rate, and overall sample concordance rate were 95.5%, 96.7%, and 96.2%, respectively.

[0034] Table 1. Detection results of 30 etomidate-negative e-cigarette liquids and 22 etomidate-positive e-cigarette liquids.

[0035]

[0036] Comparative Example 1 (The same e-liquid sample as Example 1, except that the e-liquid was not extracted).

[0037] A method for extracting, preparing, and detecting small molecule pharmaceutical substances in e-liquid, comprising the following steps:

[0038] Add the e-liquid sample directly to the immunochromatographic test strip, starting the timer simultaneously. Observe whether the control line and test line are clear and free of trailing. Stop the timer after 5 minutes and analyze the results. The immunochromatographic test strip includes two sample pads: Sample Pad I and Sample Pad II. The sample pad I treatment solution is formulated as follows: 4g of Tween-60 at a concentration of 6mg / ml, 1g of NPO-10 at a concentration of 2mg / ml, and the remainder is pure water. The sample pad II treatment solution is formulated as follows: 4g of borax at a concentration of 0.05M, 1g of sodium cholate at a concentration of 4mg / ml, 2g of PVA at a concentration of 8mg / ml, and the remainder is pure water. Specific free nicotine antibodies are added to the label pad of the immunochromatographic test strip.

[0039] Etomidate was detected using the same e-liquid sample as in Example 1, with GC / MS results used as a reference. The results showed that the positive concordance rate, negative concordance rate, and total sample concordance rate were 86.4%, 93.3%, and 90.4%, respectively. Compared to the results of Example 1, the false positive rate increased, while the negative concordance rate and total sample concordance rate decreased.

[0040] Table 2. Detection results of 30 etomidate-negative e-cigarette liquids and 22 etomidate-positive e-cigarette liquids.

[0041]

[0042] Comparative Example 2 (the same e-cigarette oil sample as Example 1, except that the immunochromatographic reagent contained only a single-layer sample pad)

[0043] A method for extracting, preparing, and detecting small molecule pharmaceutical substances in e-liquid, comprising the following steps:

[0044] (1) Extraction of small molecules in e-liquid: Mix e-liquid and extract at a ratio of 1:4 and let stand for 5 minutes; The extract formula is: NaCl 9mg / ml, Na2HPO4 3.0mg / ml, NaH2PO4 0.25mg / ml, 0.5M HCl, and the remainder is pure water.

[0045] (2) Detection of small molecules in e-liquid: The processed e-liquid sample extract was added to the immunochromatographic test strip. Timing was started simultaneously with sample addition. The control line and test line were observed to be clear and free of tailing. Timing was stopped after 5 minutes, and the results were analyzed. The sample pad treatment solution in the immunochromatographic test strip consisted of: 4g of 0.05M borax, 1g of 4mg / ml sodium cholate, 2g of 8mg / ml PVA, and the remainder being pure water. Specific free nicotine antibodies were added to the label pad of the immunochromatographic test strip.

[0046] Etomidate was detected using the same e-liquid sample as in Example 1, with GC / MS results used as a reference. The results showed that the positive concordance rate, negative concordance rate, and total sample concordance rate were 90.9%, 96.6%, and 94.2%, respectively. Compared to the results of Example 1, the false positive rate increased, while the negative concordance rate and total sample concordance rate decreased.

[0047] Table 3. Detection results of 30 etomidate-negative e-cigarette liquids and 22 etomidate-positive e-cigarette liquids.

[0048]

[0049]

[0050] As can be seen from the data of Example 1 and Comparative Examples 1-2, only the solutions within the scope of the claims of this invention can meet the above requirements in all aspects and yield the optimal extract. Changes in the proportions, substitution / addition / subtraction of raw materials, or alterations in the order of addition will all have corresponding negative impacts.

[0051] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0052] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. An immunochromatographic reagent strip for small molecule substances in e-liquid, comprising a base plate, absorbent paper, a nitrocellulose membrane, a labeling pad, and a sample pad; the labeling pad overlaps one end of the nitrocellulose membrane, and the absorbent paper overlaps the other end of the nitrocellulose membrane; characterized in that, The sample pad overlaps the marking pad, and the sample pad is a double-layered sample pad, including sample pad I and sample pad II arranged vertically. The sample pad I is treated with treatment solution I, which is prepared using the following method: The treatment solution I is prepared by mixing Tween-60 and NPO-10, adding pure water and stirring evenly, wherein the concentration of Tween-60 is 5-15 mg / ml, the concentration of NPO-10 is 1-3 mg / ml, and the mass ratio of Tween-60 to NPO-10 is (3-5):

1. The sample pad II is treated with treatment solution II, which is prepared using the following method: Borax, sodium cholate, and polyvinyl alcohol (PVA) were mixed, and pure water was added and stirred evenly to prepare the treatment solution II. The concentration of borax was 0.05M, the concentration of sodium cholate was 3-5 mg / ml, the concentration of polyvinyl alcohol (PVA) was 5-8 mg / ml, and the mass ratio of borax, sodium cholate, and polyvinyl alcohol (PVA) was (4-5):1:(1.5-2). The small molecule is etomidate.

2. The immunochromatographic reagent strip according to claim 1, characterized in that, Specific free nicotine antibodies are added to the labeling pad.

3. A method for detecting small molecule substances in e-liquid, characterized in that, The detection method includes the following steps: Step (1): Mix NaCl with a concentration of 7.5-9 mg / ml, Na2HPO4 with a concentration of 2.0-6.0 mg / ml, NaH2PO4 with a concentration of 0.1-0.3 mg / ml and HCl with a concentration of 0.5 M, add pure water and stir evenly to obtain the extract; Step (2): Mix the extract with the e-liquid, let it stand, and then extract the small molecule substances in the e-liquid. Step (3): Use the immunochromatographic reagent strip described in any one of claims 1-2 to detect the small molecule substances in the e-liquid extracted in step (2).

4. The detection method according to claim 3, characterized in that, In step (2), the volume ratio of e-liquid to extract is 1:(2-5).

5. The detection method according to claim 3, characterized in that, The settling time in step (2) is 5-10 minutes.

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