Method for detecting concentration of arecoline and method for evaluating consumption of arecoline

Abstract

The invention relates to the technical field of arecoline concentration detection, in particular to an arecoline concentration detection method and an arecoline consumption evaluation method. The arecoline concentration detection method provided by the invention comprises the following steps: S1, adding an internal standard substance into a to-be-detected liquid, and acidifying to obtain the to-be-detected solution; s2, extracting the to-be-detected solution by adopting a solid-phase extraction method to obtain extract liquor; and S3, determining the concentration of arecoline in the extract liquor by adopting an ultra-high performance liquid chromatography-mass spectrometry method. According to the method disclosed by the invention, the to-be-detected solution is extracted and is matched with the ultra-high performance liquid chromatography-mass spectrometry, so that the arecoline concentration with an accurate result can be rapidly and efficiently detected, and the detection sensitivity on the arecoline concentration is high. Meanwhile, on the basis of a solid-phase extraction pretreatment method, the concentration of arecoline is detected in combination with an ultra-high performance liquid chromatography-mass spectrometer, the urban per capita consumption is calculated on the basis of sewage epidemiology (WBE), and the result is accurate.

Description

Technical Field

[0001] The invention relates to the technical field of arecoline concentration detection, and in particular to a method for detecting arecoline concentration and a method for evaluating arecoline consumption. Background Art

[0002] Betel nut is the seed of the betel nut tree (Areca catechu L.), which is widely distributed in most parts of the tropical Pacific, parts of Asia and East Africa. Chewing betel nut can cause a variety of adverse effects on human health. Arecoline is the main active ingredient of betel nut, an important indicator for evaluating betel nut consumption, a natural alkaloid, and a key chemical component that causes betel nut addictiveness and carcinogenicity. Therefore, the large-scale use of betel nut will not only cause human health problems, but also because it is widely present in various surface water bodies, has pseudo-persistence and ecotoxicity, can produce toxic effects on a variety of aquatic organisms such as some fish and snails, and pose a potential threat to the health of aquatic ecosystems and threaten the safety of the ecological environment. Since the concentration of arecoline in the environment is low (generally at the nanogram / liter level), and there are many interfering substances in the water environment, such as perfluorinated compounds or human life organic matter and other pollutants, which will interfere with and affect the test results, it is urgent to develop a method to accurately measure the concentration of arecoline in the water environment to provide a technical handle for further assessment of its ecological risks.

[0003] The prior art discloses a method for detecting arecoline in betel nut, which uses a solvent extraction gas chromatography-mass spectrometry method to determine the arecoline in betel nut. However, the determined arecoline concentration ranges from 4.12 to 5.25 mg / g, and the sensitivity is low. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defect of low sensitivity of the arecoline concentration detection method in the prior art, thereby providing a method for detecting the arecoline concentration.

[0005] Another technical problem to be solved by the present invention is to overcome the defect in the prior art that the betel nut consumption of a population cannot be evaluated by the concentration of arecoline in the water environment, thereby providing a method for evaluating the betel nut consumption.

[0006] To this end, on the one hand, the present invention provides a method for detecting the concentration of arecoline, comprising the following steps: S1, adding an internal standard to a liquid to be tested, acidifying, and obtaining a solution to be tested; S2, extracting the solution to be tested by solid phase extraction to obtain an extract; S3, determining the concentration of arecoline in the extract by ultra performance liquid chromatography-mass spectrometry.

[0007] In some embodiments, the ultra-high performance liquid chromatography conditions are: 0.1-0.5 vol% formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B,

[0008] The elution gradient was 0-0.5 min, 90-98 Vol% mobile phase A; 0.5-4 min, 30-40 Vol% mobile phase A; 4-5.1 min, 1-10 Vol% mobile phase A; 5.1-6.5 min, 90-98 Vol% mobile phase A.

[0009] In some of the embodiments, the filler of the chromatographic column of the ultra-high performance liquid chromatography is a reverse phase filler, preferably octadecylsilane bonded silica gel, the specification of the chromatographic column is 2.1-4.6mm×50-100mm, the column temperature is 35-45°C, and the flow rate is 0.3-0.5mL / min.

[0010] In some embodiments, the mass spectrometry conditions include an electrospray positive ion source, an ion source temperature of 500-600° C., a quantitative ion mass-to-charge ratio of 156-157, a qualitative ion mass-to-charge ratio of 44-45 or 127-128, and a multiple reaction monitoring mode.

[0011] In some embodiments, step S3 also includes the step of establishing a standard curve, and the specific steps are to prepare a target solution with a gradient concentration, obtain the ratio of the peak area of ​​the target and the product of the corresponding internal standard concentration and the corresponding internal standard peak area by ultra-performance liquid chromatography-mass spectrometry, and establish a standard curve.

[0012] In some embodiments, step S2 further includes a step of stripping the extract, and the stripping step is to mix the acetonitrile solution with the extract to obtain a stripping solution.

[0013] Preferably, the concentration of the acetonitrile aqueous solution in the stripping solution is 5 to 15 vol%.

[0014] In some embodiments, the pH value of the solution to be tested after acidification in step S1 is 2-3.

[0015] In some embodiments, the internal standard is at least one of ketamine-d4, diazepam, and α-pinene.

[0016] Preferably, the volume ratio of the liquid to be tested to the internal standard solution is 8-12:1, and the concentration of the internal standard in the internal standard solution is 50-150 ppb.

[0017] In some embodiments, the solid phase extraction method in step S2 includes passing the test solution through an activated solid phase extraction column and eluting, and the target liquid obtained is the extract, wherein the eluent is a methanol solution containing ammonia water, and the volume ratio of ammonia water to methanol is 5-10:90-95.

[0018] In some embodiments, step S2 further comprises the step of concentrating the extract, and the volume of the obtained concentrated solution is 1.5%-3.5% of the volume of the extract.

[0019] In some embodiments, the solutions for activating the solid phase extraction column are methanol and ultrapure water in sequence.

[0020] In some embodiments, the concentration is carried out in a water bath with a constant temperature, and the concentration temperature is 35-45°C.

[0021] On the other hand, the present invention also provides a method for evaluating the consumption of arecoline, wherein the consumption of arecoline is calculated using formula (1):

[0022]

[0023] Wherein, M is the consumption of arecoline, mg / 1000 people / day, C is the detected concentration of arecoline obtained by the above-mentioned arecoline concentration detection method, ng / L, I is the influent flow rate of the sewage treatment plant, L / day, P is the number of people served by the sampling point, and f is the human metabolite correction factor of arecoline.

[0024] The human metabolite correction factor of arecoline reflects the proportion of the target substance excreted from the human body after oral administration to the total intake, and its accurate proportion is generally determined by toxicokinetic studies. The value of f is 0.8-1.2, preferably, f is 1, wherein, when the metabolic rate of arecoline is very low, the human metabolite correction factor of arecoline is 1 or 1.2, and when the metabolic rate of arecoline is high, the human metabolite correction factor of arecoline is 0.8.

[0025] At the same time, the present invention provides the application of the above-mentioned arecoline concentration detection method or an arecoline consumption assessment method in monitoring arecoline concentration in water environment, predicting arecoline consumption and predicting the occurrence of diseases related to betel nut consumption.

[0026] The diseases related to betel nut consumption include at least one of oral diseases, digestive system diseases, cardiovascular and cerebrovascular diseases, nervous system diseases, respiratory system diseases, and addictive dependence diseases.

[0027] The technical solution of the present invention has the following advantages:

[0028] 1. A method for detecting the concentration of arecoline provided by the present invention comprises the following steps: S1, adding an internal standard to a liquid to be tested, acidifying, and obtaining a solution to be tested; S2, extracting the solution to be tested by solid phase extraction to obtain an extract; S3, determining the concentration of arecoline in the extract by ultra-high performance liquid chromatography-mass spectrometry. The present invention uses solid phase extraction to concentrate and enrich the solution to be tested, and can separate arecoline from the solution to be tested by ultra-high performance liquid chromatography-mass spectrometry, and can quickly and efficiently perform qualitative and quantitative analysis of arecoline, and the detection of arecoline concentration is accurate and efficient.

[0029] 2. The detection method of arecoline concentration provided by the present invention, wherein step S2 further comprises a step of stripping the extract, wherein the stripping step comprises mixing an acetonitrile solution with the extract to obtain a stripping solution. The present invention adds a stripping step on the basis of the solid phase extraction method, and can further purify the sample, so that the arecoline concentration in the obtained liquid to be tested is more accurate.

[0030] 3. The method for detecting the concentration of arecoline provided by the present invention, the step of solid phase extraction in step S2 comprises passing the solution to be tested through an activated solid phase extraction column, and eluting, and the obtained target solution is an extract, wherein the eluent is a methanol solution containing ammonia water, and the volume ratio of ammonia water to methanol is 5-10:90-95. The present invention uses a methanol solution containing ammonia water as an eluent, improves the solubility of arecoline, helps to elute arecoline, and improves the detection accuracy of the arecoline concentration result.

[0031] 4. The present invention provides a method for evaluating the consumption of arecoline, wherein the consumption of arecoline is calculated using formula (1).

[0032]

[0033] Wherein, M is the consumption of arecoline, mg / 1000 people / day, C is the concentration of arecoline detected by the above-mentioned detection method of arecoline concentration, ng / L, I is the inlet flow of the sewage treatment plant, L / day, P is the number of people served by the sampled sewage treatment plant, f is the human metabolite correction factor of arecoline, and the value range of f is 0.8-1.2. The present invention is based on sewage epidemiology (WBE), and estimates the consumption pattern of drugs and compounds at the community level by analyzing biomarkers in sewage. This method shows great potential in assessing the use of illegal or controlled substances, public health crises (such as drug abuse) and monitoring social trends. The present invention is based on the method of sewage epidemiology, and conducts a survey and evaluation of the population consumption of arecoline, which has the advantages of strong objectivity, non-invasiveness, real-time, comprehensiveness, low cost and high efficiency. At the same time, the evaluation method provided by the present invention can timely understand the consumption trend of arecoline by calculating the consumption of arecoline, and predict the health risks of oral cancer or other diseases related to betel nut consumption that may be brought. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 It is a chromatogram of arecoline extracted in Example 1 of the present invention;

[0036] Figure 2 It is the mass spectrum of arecoline extracted in Example 1 of the present invention. DETAILED DESCRIPTION

[0037] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0038] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0039] Hainan Province is one of the main areas for the production and chewing of fresh betel nuts in China, where more than 2 million people are mainly engaged in betel nut processing and consumption industries. The typical cities of Hainan Province, Haikou City and Sanya City, are selected as the research area to be typical, and the domestic sewage used in the embodiment is taken from the sewage treatment plant in Haikou City and the sewage treatment plant in Sanya City.

[0040] In the examples and comparative examples of the present invention, the standard substance is arecoline (purity 99.9%); the internal standard substance is ketamine-d4 (KET-d4) (purity 99.9%); the reagents are hydrochloric acid (analytical grade), acetonitrile, methanol and formic acid are all chromatographically pure, ammonia water (analytical grade), and the water used is ultrapure water prepared by a pure water system (Ecopur);

[0041] Consumables: 0.45 μm glass fiber filter (Jin Teng), Oasis MCX solid phase extraction column (the embodiment of the present invention takes 60 mg, 3 mL of filler as an example, but the filler size and amount of the technical solution of the present invention are not limited to this amount, nor is the filler size) (Waters, USA), 10 mL glass centrifuge tube, 0.22 μm nylon filter, 1 mL disposable syringe, 2 mL liquid chromatography injection bottle, pipette, pipette needle, rubber-tipped dropper, etc.;

[0042] The detection instruments are high performance liquid chromatography-mass spectrometry (UPLC-MS / MS), SPE-1000 fully automatic solid phase extraction instrument (Labtech), and M32 fully automatic parallel concentrator (Labtech).

[0043] Example 1

[0044] This embodiment provides a method for detecting the concentration of arecoline, and the specific steps and parameters are as follows:

[0045] (1) Pure arecoline was prepared into a sample with a concentration of 1 μg / L. After adding the isotope internal standard solution to the sample and mixing, the volume ratio of the sample to the isotope internal standard solution was 10:1. The concentration of the internal standard in the isotope internal standard solution was 100 ppb (the solvent in the isotope internal standard solution was methanol). The internal standard was ketamine-d4 (KET-d4). The pH value of the sample was adjusted to 3 with hydrochloric acid and then stored in a refrigerator at 4°C for subsequent analysis. The sample was processed within 48 hours.

[0046] (2) 5 mL of methanol and 5 mL of ultrapure water were used to activate and balance the MCX solid phase extraction column;

[0047] The sample after pretreatment in step (1) was passed through the column at a flow rate of 1 mL / min, and then eluted with 6 mL of methanol solution containing aqueous ammonia (in the methanol solution containing aqueous ammonia, the volume ratio of aqueous ammonia to methanol was 5:95), and the eluate was collected in a 10 mL glass centrifuge tube and concentrated by a parallel concentrator to obtain a concentrated solution with a volume of 1.5 vol% of the eluate, wherein the condition of the parallel concentrator was a constant temperature of 40°C in a water bath.

[0048] A 10 vol% acetonitrile aqueous solution was added to the concentrate to make the concentrate volume to 1 mL, and the concentrate was filtered through a 0.22 μm nylon filter membrane to obtain a sample solution for standby use.

[0049] (3) 1 mg of the standard substance arecoline was accurately weighed on a 1 / 100,000 balance and prepared into a stock solution with a concentration of 0.1 g / L using methanol. The first-level stock solution was taken and prepared into a series of standard working solutions with different concentration gradients of 0 μg / L, 0.2 μg / L, 0.5 μg / L, 1 μg / L, 2 μg / L, 5 μg / L, 10 μg / L, and 20 μg / L using methanol as the solvent;

[0050] The parameters of the standard working solution were determined by high performance liquid chromatography-mass spectrometry. The target concentration was used as the horizontal axis, and the ratio of the peak area of ​​the target and the corresponding internal standard concentration to the peak area of ​​the corresponding internal standard was used as the vertical axis to establish a calibration curve. The R 2 =0.99.

[0051] Under the same conditions, the sample solution obtained in step (2) is injected into a high performance liquid chromatography-mass spectrometer for measurement, the concentration of the target compound in the solution is calculated using a standard working curve, and then the content of the target compound in the sample is calculated based on the sample volume;

[0052] The liquid chromatography conditions were as follows:

[0053] Chromatographic column:

[0054] Liquid chromatography column: ACQUITY UPLC BEHC18, 1.7 μm, 50 mm × 2.1 mm, Waters, MA, USA);

[0055] Injection volume: 5 μL;

[0056] The flow rate was 0.4 mL / min.

[0057] Column temperature: 40°C;

[0058] Mobile phase: A, 0.1% formic acid aqueous solution, B, acetonitrile;

[0059] The elution gradient is: 0-0.5 min, 98 vol% mobile phase A, 0.5-4.0 min, 35 vol% mobile phase A; 4.0-5.1 min, 2 vol% mobile phase A; 5.1-6.5 min, 98 vol% mobile phase A.

[0060] The mass spectrometry conditions were as follows:

[0061] Mass spectrometer: triple quadrupole mass spectrometer;

[0062] Ion source: electrospray positive ion source;

[0063] Ion source temperature: 550°C;

[0064] The monitoring mode was MRM;

[0065] The quantitative ion pairs (parent ion and daughter ion) and collision voltage and cone voltage of arecoline and internal standard are shown in Table 1.

[0066] Table 1 Main mass spectrometry parameters of arecoline and internal standard

[0067]

[0068] The above steps were repeated 3 times, and the recovery rate of arecoline spiked and the relative standard deviation (%) were recorded in Table 2.

[0069] Table 2 Arecoline spike recovery and relative standard deviation (%)

[0070]

[0071] It can be seen from Table 2 that the detection method of arecoline concentration provided in this embodiment is accurate and reliable.

[0072] The chromatogram obtained by ultra-high performance liquid chromatography of the sample spiked for the first time in this example is shown in Figure 1 The mass spectra obtained by mass spectrometry are shown in Figure 2 .

[0073] Example 2

[0074] This embodiment provides a method for detecting the concentration of arecoline, and the specific steps and parameters are as follows:

[0075] (1) Pure arecoline was prepared into a sample with a concentration of 1 μg / L. After adding the isotope internal standard solution to the sample and mixing, the volume ratio of the sample to the isotope internal standard solution was 8:1. The concentration of the internal standard in the isotope internal standard solution was 150 ppb (the solvent in the isotope internal standard solution was methanol). The internal standard was diazepam. The pH value of the sample was adjusted to 2 with hydrochloric acid and then stored in a refrigerator at 4°C for subsequent analysis. The sample was processed within 48 hours.

[0076] (2) 5 mL of methanol and 5 mL of ultrapure water were used to activate and balance the MCX solid phase extraction column;

[0077] The sample after pretreatment in step (1) was passed through the column at a flow rate of 1 mL / min, and then eluted with 6 mL of methanol solution containing aqueous ammonia (in the methanol solution containing aqueous ammonia, the volume ratio of aqueous ammonia to methanol was 10:90), and the eluate was collected in a 10 mL glass centrifuge tube and concentrated by a parallel concentrator to obtain a concentrated solution with a volume of 3.5 vol% of the eluate, wherein the condition of the parallel concentrator was a constant temperature of 35°C in a water bath.

[0078] A 15 vol% acetonitrile aqueous solution was added to the concentrate to make the concentrate volume to 1 mL, and the concentrate was filtered through a 0.22 μm nylon filter membrane to obtain a sample solution for standby use.

[0079] (3) 1 mg of the standard substance arecoline was accurately weighed on a 1 / 100,000 balance and prepared into a stock solution with a concentration of 0.1 g / L using methanol. The first-level stock solution was taken and prepared into a series of standard working solutions with different concentration gradients of 0 μg / L, 0.2 μg / L, 0.5 μg / L, 1 μg / L, 2 μg / L, 5 μg / L, 10 μg / L, and 20 μg / L using methanol as the solvent;

[0080] The parameters of the standard working solution were determined by high performance liquid chromatography-mass spectrometry. The target concentration was used as the horizontal axis, and the ratio of the peak area of ​​the target and the corresponding internal standard concentration to the peak area of ​​the corresponding internal standard was used as the vertical axis to establish a calibration curve. The R 2 =0.99.

[0081] Under the same conditions, the sample solution obtained in step (2) is injected into a high performance liquid chromatography-mass spectrometer for measurement, the concentration of the target compound in the solution is calculated using a standard working curve, and then the content of the target compound in the sample is calculated based on the sample volume;

[0082] The liquid chromatography conditions were as follows:

[0083] Chromatographic column:

[0084] Liquid chromatography column: ACQUITY UPLC BEHC18, 1.7 μm, 50 mm × 2.1 mm, Waters, MA, USA);

[0085] Injection volume: 5 μL;

[0086] The flow rate was 0.3 mL / min.

[0087] Column temperature: 35°C;

[0088] Mobile phase: A, 0.5 vol% formic acid aqueous solution, B, acetonitrile;

[0089] The elution gradient is: 0-0.5 min, 90 vol% mobile phase A; 0.5-4.0 min, 30 vol% mobile phase A; 4.0-5.1 min, 10 vol% mobile phase A; 5.1-6.5 min, 90 vol% mobile phase A.

[0090] The mass spectrometry conditions were as follows:

[0091] Mass spectrometer: triple quadrupole mass spectrometer;

[0092] Ion source: electrospray positive ion source;

[0093] Ion source temperature: 550°C;

[0094] The monitoring mode was MRM;

[0095] The quantitative ion pairs (parent ion and daughter ion) and collision voltage and cone voltage of arecoline and internal standard are shown in Table 1.

[0096] Example 3

[0097] This embodiment provides a method for detecting the concentration of arecoline, and the specific steps and parameters are as follows:

[0098] (1) Pure arecoline was prepared into a sample with a concentration of 1 μg / L. After adding the isotope internal standard solution to the sample and mixing, the volume ratio of the sample to the isotope internal standard solution was 12:1. The concentration of the internal standard in the isotope internal standard solution was 50 ppb (the solvent in the isotope internal standard solution was methanol). The internal standard was diazepam. The pH value of the sample was adjusted to 3 with hydrochloric acid and then stored in a refrigerator at 4°C for subsequent analysis. The sample was processed within 48 hours.

[0099] (2) 5 mL of methanol and 5 mL of ultrapure water were used to activate and balance the MCX solid phase extraction column;

[0100] The sample after pretreatment in step (1) was passed through the column at a flow rate of 1 mL / min, and then eluted with 6 mL of methanol solution containing aqueous ammonia (in the methanol solution containing aqueous ammonia, the volume ratio of aqueous ammonia to methanol was 8:92), and the eluate was collected in a 10 mL glass centrifuge tube and concentrated by a parallel concentrator to obtain a concentrated solution with a volume of 2.5 vol% of the eluate, wherein the condition of the parallel concentrator was a constant temperature of 35°C in a water bath.

[0101] A 5 vol% acetonitrile aqueous solution was added to the concentrate to make the concentrate volume to 1 mL, and the concentrate was filtered through a 0.22 μm nylon filter membrane to obtain a sample solution for standby use.

[0102] (3) 1 mg of the standard substance arecoline was accurately weighed on a 1 / 100,000 balance and prepared into a stock solution with a concentration of 0.1 g / L using methanol. The first-level stock solution was taken and prepared into a series of standard working solutions with different concentration gradients of 0 μg / L, 0.2 μg / L, 0.5 μg / L, 1 μg / L, 2 μg / L, 5 μg / L, 10 μg / L, and 20 μg / L using methanol as the solvent;

[0103] The parameters of the standard working solution were determined by high performance liquid chromatography-mass spectrometry. The target concentration was used as the horizontal axis, and the ratio of the peak area of ​​the target and the corresponding internal standard concentration to the peak area of ​​the corresponding internal standard was used as the vertical axis to establish a calibration curve. The R 2 =0.99.

[0104] Under the same conditions, the sample solution obtained in step (2) is injected into a high performance liquid chromatography-mass spectrometer for measurement, the concentration of the target compound in the solution is calculated using a standard working curve, and then the content of the target compound in the sample is calculated based on the sample volume;

[0105] The liquid chromatography conditions were as follows:

[0106] Chromatographic column:

[0107] Liquid chromatography column: ACQUITY UPLC BEHC18, 1.7 μm, 50 mm × 2.1 mm, Waters, MA, USA);

[0108] Injection volume: 5 μL;

[0109] The flow rate was 0.4 mL / min.

[0110] Column temperature: 45°C;

[0111] Mobile phase: A, 0.2 vol% formic acid aqueous solution, B, acetonitrile;

[0112] The elution gradient is: 0-0.5 min, 90 vol% mobile phase A; 0.5-4.0 min, 40 vol% mobile phase A; 4.0-5.1 min, 1 vol% mobile phase A; 5.1-6.5 min, 90 vol% mobile phase A.

[0113] The mass spectrometry conditions were as follows:

[0114] Mass spectrometer: triple quadrupole mass spectrometer;

[0115] Ion source: electrospray positive ion source;

[0116] Ion source temperature: 550°C;

[0117] The monitoring mode was MRM;

[0118] The quantitative ion pairs (parent ion and daughter ion) and collision voltage and cone voltage of arecoline and internal standard are shown in Table 1.

[0119] Example 4

[0120] This embodiment provides a method for detecting the concentration of arecoline, and the specific steps and parameters are as follows:

[0121] 1. Sample collection:

[0122] Sewage samples were collected from the inlet of Haikou Changliu Sewage Treatment Plant, Baishamen Sewage Treatment Plant Phase II, Baishamen Sewage Treatment Plant Phase I, Guilinyang Sewage Treatment Plant and Jinniuling Sewage Treatment Plant in March 2023 and July 2023 respectively. The sample collection method was to use an automatic sampler to collect 24-hour mixed samples. The 24-hour mixed samples were collected once every 2 hours, with a total of 500 mL, and 3 parallel samples were collected at one sampling point.

[0123] 2. Sample processing:

[0124] After adding the isotope internal standard solution to the collected sample and mixing, the volume ratio of the collected sample to the isotope internal standard solution is 10:1, the concentration of the internal standard in the isotope internal standard solution is 100 ppb (the solvent in the isotope internal standard solution is methanol), and the internal standard is ketamine-d4 (KET-d4). After adjusting the pH value of the sample to 3 with hydrochloric acid, it is placed in a refrigerator at 4°C for subsequent analysis. The sample is processed within 48 hours.

[0125] 3. Detection of Arecoline Concentration in Samples:

[0126] 3.1 Sample pre-treatment:

[0127] Take 500 mL of the sample obtained in step (2) and filter it through a 0.45 μm glass fiber filter to remove suspended matter and impurities in the water;

[0128] 3.2 Sample enrichment

[0129] The MCX solid phase extraction column was activated and equilibrated using 5 mL of methanol and 5 mL of ultrapure water in sequence;

[0130] The pretreated sample was passed through the column at a flow rate of 1 mL / min, and then eluted with 6 mL of methanol solution containing aqueous ammonia (in the methanol solution containing aqueous ammonia, the volume ratio of aqueous ammonia to methanol was 5:95). The eluate was collected in a 10 mL glass centrifuge tube and concentrated using a parallel concentrator to obtain a concentrate with a volume of 1.5 vol% of the eluate, wherein the condition of the parallel concentrator was a constant temperature of 40°C in a water bath.

[0131] Add 10 vol% acetonitrile aqueous solution to the concentrate, dilute the concentrate to 1 mL, and filter through a 0.22 μm nylon filter membrane to obtain the sample solution to be tested for later use.

[0132] 3.3 Sample separation and identification

[0133] Weigh 1 mg of the standard substance arecoline accurately on a 1 / 100,000 balance, prepare a stock solution with a concentration of 0.1 g / L with methanol, take the first-level stock solution, use methanol as solvent, prepare a series of standard working solutions with different concentration gradients of 0 μg / L, 0.2 μg / L, 0.5 μg / L, 1 μg / L, 2 μg / L, 5 μg / L, 10 μg / L, and 20 μg / L;

[0134] The parameters of the standard working solution were determined by high performance liquid chromatography-mass spectrometry. The target concentration was used as the horizontal axis, and the ratio of the peak area of ​​the target and the corresponding internal standard concentration to the peak area of ​​the corresponding internal standard was used as the vertical axis to establish a calibration curve. The R 2 =0.99.

[0135] Under the same conditions, the sample solution obtained in step 3.2 of this embodiment is injected into a high performance liquid chromatography-mass spectrometer for measurement, the concentration of the target compound in the solution is calculated using the standard working curve, and then the content of the target compound in the sample is calculated based on the sample volume;

[0136] The liquid chromatography conditions were as follows:

[0137] Chromatographic column: liquid chromatography column (ACQUITY UPLC BEHC18, 1.7 μm, 50 mm × 2.1 mm, Waters, MA, USA); injection volume 5 μL; flow rate 0.4 mL / min; column temperature 40 °C;

[0138] Mobile phase: A: 0.1 vol% formic acid aqueous solution, B: acetonitrile;

[0139] The elution gradient is: 0-0.5 min, 98 vol% mobile phase A; 0.5-4.0 min, 35 vol% mobile phase A; 4.0-5.1 min, 2 vol% mobile phase A; 5.1-6.5 min, 98 vol% mobile phase A.

[0140] The mass spectrometry conditions were as follows:

[0141] Mass spectrometer: triple quadrupole mass spectrometer;

[0142] Ion source: electrospray positive ion source;

[0143] Ion source temperature: 550°C;

[0144] The monitoring mode was MRM;

[0145] The quantitative ion pairs (parent ion and daughter ion) and collision voltage and cone voltage of arecoline and internal standard are shown in Table 1.

[0146] The arecoline concentrations obtained from the samples collected from each sampling point in March 2023 and July 2023 in this embodiment are shown in Table 3.

[0147] Table 3 Arecoline concentration test results at each sampling point

[0148]

[0149] It can be seen that the detection method provided in the embodiment of the present invention can detect extremely low concentrations of arecoline, achieving a sensitivity of nanograms per liter, and is suitable for environmental monitoring, especially for the detection of trace pollutants. At the same time, due to the presence of a large amount of organic matter and other chemical substances in the sewage environment, these substances will interfere with the detection results of arecoline. By adding an internal standard, the present application can correct the losses that occur during sample processing and analysis, thereby improving the reliability of the test results and ensuring the accuracy of the results.

[0150] Example 5

[0151] This embodiment provides a method for detecting the concentration of arecoline. The specific steps and parameters are the same as those of Example 4, except that the sample sampling point is Sanya City. The specific sampling point information and the detected arecoline concentration are shown in Table 4.

[0152] Table 4 Sample sampling point information and arecoline detection concentration

[0153]

[0154]

[0155] Application Example 1

[0156] Although betel nut is used as a traditional medicine or food in some areas, excessive intake can cause a variety of potential hazards to the human body and seriously threaten human health. Therefore, it is urgent to quickly and accurately assess the population's consumption of betel nut to provide data support for further assessment of its health risks. Traditional methods for investigating the consumption of betel nut in the population often rely on questionnaires or sales data. These methods have limitations such as strong subjectivity, limited coverage, organizational difficulties and long time consumption, resulting in inaccurate survey data, serious delays in survey results and high costs.

[0157] Sewage epidemiology is an emerging method for monitoring chemical substances. Since chemical compounds cannot be fully absorbed by the human body after consumption, they are usually excreted from the body through feces and urine in the form of parent compounds or their metabolites, and enter the sewage treatment plant with urban domestic sewage. By analyzing the concentration of biomarkers in the influent of the sewage treatment plant, the consumption patterns of target chemical substances by the population in the area are estimated based on parameters such as human metabolic mechanism, influent flow rate and service population, so as to prevent and control such chemical substances and improve the level of public health.

[0158] This application example selects arecoline as a biomarker of betel nut, and provides a method for evaluating the population consumption of arecoline based on the sewage epidemiology method. Combined with formula (2), the population consumption calculation formula for arecoline is obtained as shown in formula (1).

[0159]

[0160] Where, C is the concentration of arecoline detected (ng / L); I is the influent flow rate of the sewage treatment plant (L / day); f is the human metabolite correction factor of arecoline, which reflects the correction relationship between the target substance arecoline and the actual consumption substance, and f is taken as 1; P is the population served by the sewage treatment plant; x h is the percentage (%) of the parent compound taken into the human body through route h and excreted without being metabolized; R h is the proportion of arecoline taken into the human body through food intake; M is the per capita consumption of arecoline (mg / 1000 people / day).

[0161] According to the population served by the sewage treatment plant and the water inflow rate, which are data collected in real time and on-site during sampling, this application example takes the five sewage treatment plants provided in Example 4 as an example. The relevant parameters are shown in Table 3. Combined with formula (1), the consumption of arecoline in Haikou City is calculated, and the results are shown in Table 5.

[0162] Table 5 Sewage information and arecoline consumption at each sampling point

[0163]

[0164]

[0165] According to the data in Table 5, the arecoline consumption in various districts of Haikou City was 38.15 mg / 1000 people / day in March 2023, and 38.05 mg / 1000 people / day in July 2023.

[0166] Application Example 2

[0167] The population consumption of arecoline in Sanya was evaluated by using the population consumption evaluation method of arecoline provided in Application Example 1 according to the arecoline concentrations at each sampling point detected in Example 5. The results are shown in Table 6. The parameters of each sampling point in Sanya and the population consumption of arecoline are shown in Table 4.

[0168] Table 6 Sampling point parameters and arecoline consumption in Sanya

[0169]

[0170]

[0171] According to Table 6, the population consumption of arecoline in Sanya City obtained by the population consumption evaluation method of arecoline provided by the present invention is 51.91 mg / 1000 people / day.

[0172] According to the "Hainan Statistical Yearbook 2022", the production of betel nut in Hainan is 29.48 tons, and the permanent population of Hainan Province is 10.2702 million. According to the research results of the literature [1] Hou Qianyu et al. "LC-PDA determination of the content of four alkaloids in betel nut, burnt betel nut, and big belly peel." Chinese Modern Applied Pharmacy (2023).", the content of arecoline in 1g of betel nut is 2.21-7.41 mg / g. Based on the range of betel nut production and arecoline content, the theoretical maximum and minimum consumption of arecoline in Hainan Province in 2022 are calculated to be 58.27 mg / 1000 people / day and 17.38 mg / 1000 people / day, respectively. The data of Application Examples 1 and 2 of the present invention are within the scope of the above-mentioned investigation and research data, but the method provided in the literature requires large-scale sampling, population surveys and other tedious work to determine the consumption of regional arecoline. The present invention is based on the sewage epidemiological method combined with the arecoline concentration detection method provided by the present invention, and the arecoline consumption in Haikou and Sanya can be calculated, and the result data all fall within the above-mentioned range. Compared with estimating the arecoline consumption based on betel nut production, the method provided by the present invention can more accurately estimate the arecoline consumption in cities and regions, which has certain benefits for subsequent risk assessment, policy control, etc. in cities.

[0173] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for detecting arecoline concentration, characterized in that, The following steps are included: S1, adding an internal standard substance to the liquid to be tested, acidifying, and obtaining a solution to be tested; S2, extracting the solution to be tested by solid phase extraction to obtain an extract; S3, the concentration of arecoline in the extract was determined by ultra-performance liquid chromatography-mass spectrometry.

2. The method for detecting arecoline concentration according to claim 1, wherein The conditions of the ultra-high performance liquid chromatography are: 0.1-0.5 vol% formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, The elution gradient was, 0-0.5 min, 90-98 Vol% mobile phase A, 0.5-4 min, 30-40 Vol% mobile phase A, 4-5.1 min, 1-10 Vol% mobile phase A, 5.1-6.5 min, 90-98 Vol% mobile phase A.

3. The method for detecting arecoline concentration according to claim 2, wherein The filler of the chromatographic column of the ultra-high performance liquid chromatography is a reverse phase filler, preferably octadecylsilane bonded silica gel, the specification of the chromatographic column is 2.1-4.6 mm×50-100 mm, the column temperature is 35-45° C., and the flow rate is 0.3-0.5 mL / min; and / or, The mass spectrometry conditions include: an electrospray positive ion source, an ion source temperature of 500-600° C., a quantitative ion mass-to-charge ratio of 156-157, a qualitative ion mass-to-charge ratio of 44-45 or 127-128, and a multiple reaction monitoring mode.

4. The method for detecting arecoline concentration according to claim 1, wherein Step S3 also includes the step of establishing a standard curve, which specifically comprises preparing a target solution with a gradient concentration, obtaining the ratio of the peak area of ​​the target and the product of the corresponding internal standard concentration to the corresponding internal standard peak area by ultra-high performance liquid chromatography-mass spectrometry, and establishing a standard curve.

5. The method for detecting the concentration of arecoline according to claim 3, wherein Step S2 also includes a step of stripping the extract, wherein the stripping step comprises mixing the acetonitrile solution with the extract to obtain a stripping solution. The concentration of the acetonitrile aqueous solution in the stripping solution is 5-15 vol%.

6. The method for detecting the concentration of arecoline according to claim 5, wherein The pH value of the solution to be tested after acidification in step S1 is 2-3; and / or, The internal standard is at least one of ketamine-d4, diazepam, and α-pinene; and / or, The volume ratio of the liquid to be tested to the internal standard solution is 8-12:1, and the concentration of the internal standard in the internal standard solution is 50-150 ppb.

7. The method for detecting the concentration of arecoline according to claim 6, wherein The solid phase extraction method in step S2 includes passing the test solution through an activated solid phase extraction column and eluting, and the target solution obtained is the extract, wherein the eluent is a methanol solution containing ammonia water, and the volume ratio of ammonia water to methanol is 5-10:90-95; and / or, The step S2 also includes the step of concentrating the extract, and the volume of the obtained concentrated solution is 1.5%-3.5% of the volume of the extract.

8. The method for detecting the concentration of arecoline according to claim 7, wherein The solutions for activating the solid phase extraction column are methanol and ultrapure water in sequence; and / or, The concentration is carried out in a water bath at a constant temperature of 35-45°C.

9. A method for evaluating arecoline consumption, characterized in that: The consumption of arecoline was calculated using formula (1): Wherein, M is the consumption of arecoline, mg / 1000 people / day, C is the detected concentration of arecoline obtained by the arecoline concentration detection method described in any one of claims 1-8, ng / L, I is the influent flow rate of the sewage treatment plant, L / day, P is the number of people served by the sampling point, and f is the human metabolite correction factor of arecoline, and the value range of f is 0.8-1.

2.

10. Application of the method for detecting the arecoline concentration according to any one of claims 1 to 8 or the method for evaluating the arecoline consumption according to claim 9 in monitoring the arecoline concentration in aquatic environments, predicting the arecoline consumption, and predicting the occurrence of diseases related to betel nut consumption.

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