A method for rapid screening and detection of illegal addition of drugs in food claiming to be alcoholism

Abstract

The present application relates to the technical field of food and drug detection, in particular to a method for rapidly screening and detecting illegally added drugs in food claiming to relieve alcoholism. The detection method mainly comprises: preparing a standard solution of illegally added drugs in food claiming to relieve alcoholism and drawing a standard curve; obtaining a sample solution after extracting and purifying the sample to be detected; and qualitatively and quantitatively detecting the sample solution by using optimized liquid chromatography tandem mass spectrometry detection conditions. The present application can simultaneously detect a plurality of possible added drugs in food claiming to relieve alcoholism, more quickly screen illegally added drugs in food, and the detection limit is 50 μg / kg, the correlation coefficient R 2 in the range of 50 μg / kg-500 μg / kg is greater than 0.990, the linear relationship is good, and the qualitative and quantitative results are reliable.

Description

Technical Field

[0001] This invention relates to the field of food and drug testing technology, specifically to a method for rapid screening and detection of illegally added drugs in foods that claim to cure hangovers. Background Technology

[0002] To meet consumer demand, various foods claiming to relieve hangovers have appeared on the market. Among these foods, many are suspected of containing added drugs, and their safety, harmfulness, and dosage have not been verified. According to the relevant provisions of the Food Safety Law, it is prohibited to add drugs to food, and adding drugs to food is an illegal act.

[0003] To date, there are no effective drugs to completely cure or prevent intoxication. Currently, commonly added hangover remedies fall into three main categories: first, drugs that promote mental clarity, such as naloxone; second, traditional Chinese medicine sedatives such as borneol; and third, diuretics that promote alcohol excretion. Currently, there is no method for detecting hangover remedies in food. To combat this food safety violation and deter the addition of drugs to food, a detection method for hangover remedies in food is needed. Existing technology CN112858516A provides a method for detecting diuretics in animal-derived foods, using pH-neutral acetonitrile extraction and HLB solid-phase extraction column purification. This method can detect diuretics in simple agricultural products such as meat, eggs, and dairy products, but its extraction recovery rate and anti-interference ability are unsatisfactory in complex matrices containing various traditional Chinese medicines and numerous polar compounds. Therefore, this patent provides a method that can simultaneously detect drugs that promote mental clarity, sedatives, and diuretics in foods claiming to cure hangovers. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention provides the following technical solution:

[0005] A method for rapid screening and detection of illegally added drugs in foods claiming to cure hangovers includes the following steps:

[0006] (1) Prepare standard solutions of illegally added drugs in foods that claim to cure hangovers and plot standard curves;

[0007] (2) The sample to be tested is extracted and purified to obtain the sample solution;

[0008] (3) The sample solution was qualitatively and quantitatively analyzed by liquid chromatography-tandem mass spectrometry. The liquid chromatography conditions were as follows: the column was a C18 column, the mobile phase A was 5 mol / L ammonium acetate containing 0.1% formic acid aqueous solution, the mobile phase B was acetonitrile, and the elution program was as follows: by volume fraction, in the first 0-5 min, mobile phase A was 90% and B was 10%; in the first 5-8 min, mobile phase A was 15% and B was 85%; in the first 8-10 min, mobile phase A was 90% and B was 10%.

[0009] The liquid chromatography-mass spectrometry (LC-MS) conditions were as follows: ion source type: electrospray ionization (ESI); electrospray voltage: positive source 5000V, negative source -4500V; mass spectrometry scanning mode: multiple reaction monitoring (MRM).

[0010] Preferably, in step (3), the C18 chromatographic column is 100mm*2.1mm, 2.5μm, the column temperature is 40℃, the injection volume is 5μL, and the flow rate is 0.4mL / min.

[0011] Preferably, the illegally added drugs in foods claiming to cure hangovers include drugs that promote mental alertness, sedatives, and diuretics.

[0012] Preferably, the drugs illegally added to the food claiming to relieve hangovers are amiloride, benzylfluthiazide, bumetanide, canrone, naloxone, naltrexone, tolvaptan, verapamil hydrochloride, indapamide, ethacrylic acid, sulfanilamide, buthiazine, cyclopenthiazine, furosemide, and chlorothiazide.

[0013] Preferably, in step (2), the extraction method of the sample to be tested is as follows: adjust the pH value of anhydrous ethanol to a suitable range, use it as the extraction solution, and extract with ultrasound for 15±5 min; for amiloride, tolvaptan, indapamide, and sulfanilamide, adjust the pH value of anhydrous ethanol to 10.2±0.5; for benzylfluthiazide, bumetanide, ethacrynic acid, furosemide, canrone, chlorothiazide, naloxone, naltrexone, verapamil hydrochloride, buthiazine, and cyclopenthiazine, adjust the pH value of anhydrous ethanol to 4.5±0.5.

[0014] Preferably, in step (2), the purification method of the sample to be tested is as follows: after centrifugation of the extracted solution, the supernatant is added to the purification agent, vortexed, allowed to stand, and then the supernatant is filtered through a membrane; the purification agent is neutral alumina (ALN), C18, ethylenediamine-N-propylsilane (PSA) and magnesium sulfate (MgSO4), and their mass ratio is (3-10):(3-10):(3-10):10.

[0015] More preferably, the mass ratio of the purifying agent, neutral alumina (ALN), C18, ethylenediamine-N-propylsilane (PSA), and magnesium sulfate (MgSO4), is 5:5:3:10.

[0016] Preferably, the standard curve in step (1) is plotted as follows: the standard solution is measured according to the conditions described in step (3) to obtain the chromatographic peak area of ​​the corresponding standard solution, and the standard curve is plotted with the concentration of the standard working solution as the abscissa and the peak area of ​​the chromatographic peak of the quantitative ion as the ordinate.

[0017] Preferably, the detection limit of the method is ≤50μg / kg; the quantitation limit is ≤150mg / kg.

[0018] Preferably, the qualitative and quantitative ion pair parameters of the illegally added drug are as follows:

[0019]

[0020] Compared with the prior art, the advantages of the present invention are:

[0021] (1) The present invention can use a liquid phase tandem triple quadrupole mass spectrometer to simultaneously detect multiple possible additives in foods that claim to cure hangovers, and screen for illegal additives in food more quickly.

[0022] (2) The detection method provided by this invention has a detection limit of 50 μg / kg, and a correlation coefficient R in the range of 50 μg / kg to 500 μg / kg. 2 All values ​​are greater than 0.990, indicating that the linear relationship of this method is good and the qualitative and quantitative results are reliable.

[0023] (3) At present, various traditional Chinese medicines are added to hangover relief foods or hangover relief medicines. Most of them are plant ingredients with a wide variety of polar substances. The method of the present invention can extract and detect a variety of illegally added drugs from complex matrices and has a high extraction recovery rate and anti-interference ability. Attached Figure Description

[0024] Figure 1 This is the mass spectrum of amiloride.

[0025] Figure 2 This is the mass spectrum of benzylfluthiazide.

[0026] Figure 3 This is the mass spectrum of bumetanide.

[0027] Figure 4 This is the mass spectrum of canridine.

[0028] Figure 5 This is the mass spectrum of naloxone.

[0029] Figure 6 This is the mass spectrum of naltrexone.

[0030] Figure 7This is the mass spectrum of Tolvaptan.

[0031] Figure 8 This is the mass spectrum of verapamil hydrochloride.

[0032] Figure 9 This is the mass spectrum of indapamide.

[0033] Figure 10 This is the mass spectrum of ethacrylic acid.

[0034] Figure 11 This is the mass spectrum of sulfanilamide.

[0035] Figure 12 This is the mass spectrum of buthiazide.

[0036] Figure 13 This is the mass spectrum of cyclopentathiazide.

[0037] Figure 14 This is the mass spectrum of furosemide.

[0038] Figure 15 This is the mass spectrum of chlorothiazide. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0040] The reagents and materials used in this invention are as follows: water, grade I as specified in GB / T 6682; acetonitrile (CH3CN): mass spectrometry grade; methanol (CH3OH): analytical grade; formic acid (HCOOH): mass spectrometry grade; ammonium acetate (CH3COONH4): mass spectrometry grade; disposable microporous filter head: with 0.22μm microporous filter membrane, organic system.

[0041] The reagents used in this invention are prepared as follows:

[0042] Methanol-water solution (1:1): Mix methanol and water in equal volumes;

[0043] 5 mmol / L ammonium acetate solution (containing 0.1% formic acid): Weigh 0.385 g ammonium acetate, add 1 mL formic acid, and dilute to 1 L with water;

[0044] Standard substances: Amiloride, benzylfluthiazide, bumetanide, canrone, naloxone, naltrexone, tolvaptan, verapamil hydrochloride, indapamide, ethacrylic acid, sulfanilamide, buthiazine, cyclopenthiazine, furosemide, chlorothiazide;

[0045] Preparation of standard stock solution: Accurately weigh an appropriate amount of standard substance and prepare a stock solution with methanol at a concentration of 100 μg / mL. Store the stock solution at ≤-18℃.

[0046] Preparation of mixed standard intermediate solution: Take 1 mL of standard stock solution and dilute to 100 mL with methanol to obtain a concentration of 1000 ng / mL. Store the mixed standard solution below -18℃.

[0047] Preparation of working solution: Dilute an appropriate amount of mixed standard intermediate solution with methanol aqueous solution (1:1) to a concentration of 50 ng / mL.

[0048] The instruments and equipment used in this invention are as follows:

[0049] Liquid chromatography-tandem quadrupole mass spectrometer: equipped with an electrospray ionization (ESI) source; ultrasonic cleaner; centrifuge: speed ≥ 4000 r / min; analytical balance: sensitivity 0.00001 g and 0.01 g.

[0050] Example 1

[0051] Step 1: Identification of Illegal Additives

[0052] Currently, no drugs specifically for relieving hangovers have been found in various literatures. Most reported hangover remedies are traditional Chinese medicine preparations, none of which contain clearly effective active ingredients. Through years of research and testing on this type of food, it has been found that three main categories of drugs may be added: Western medicines for mental alertness, traditional Chinese medicines for promoting wakefulness, and diuretics. This project developed detection methods for 15 commonly used drugs in this type of food: amiloride, benzfluthiazide, bumetanide, canrone, naloxone, naltrexone, tolvaptan, verapamil hydrochloride, indapamide, ethacrynic acid, sulfanilamide, buthiazine, cyclopenthiazine, furosemide, and chlorothiazide.

[0053] Step 2: Research on extraction methods:

[0054] Different extraction methods were designed to address the stability of the aforementioned 15 drugs under different environments. Furthermore, considering that the purpose of adding these drugs is to alleviate hangovers, the presence of alcohol is generally considered. Based on these factors, and after multiple experiments, this project adopted the following extraction method:

[0055] Extraction Method 1 (Applicable to amiloride, tolvaptan, indapamide, and sulfanilamide):

[0056] Take anhydrous ethanol and adjust the pH to 10.2 with concentrated ammonia as the extraction solution. Mix the solid or semi-solid samples thoroughly and grind them finely. Shake the liquid sample thoroughly and weigh 1g (accurate to 0.01g) into a 10mL volumetric flask. Add an appropriate amount of extraction solution and extract by sonication for 15min. Cool to room temperature and dilute to the mark with ethanol. Transfer to a centrifuge tube and centrifuge at 20000r / min for 3min. Take 1.5mL of the supernatant into a purification tube containing 50mg of neutral alumina (ALN), 50mg of C18, 30mg of ethylenediamine-N-propylsilane (PSA), and 100mg of magnesium sulfate (MgSO4). Vortex for 1min and let stand for 5min. Filter the supernatant through a 0.22μm microporous membrane.

[0057] Extraction Method 2 (applicable to benzylfluthiazide, bumetanide, ethacrynic acid, furosemide, canrone, chlorothiazide, naloxone, naltrexone, verapamil hydrochloride, buthiazine, cyclopenthiazine): Take anhydrous ethanol and adjust the pH to 4.5 with phosphoric acid as the extraction solution. Mix the solid or semi-solid sample thoroughly and grind it finely. Shake the liquid sample thoroughly and weigh 1g (accurate to 0.01g) into a 10mL volumetric flask. Add an appropriate amount of extraction solution and extract by sonication for 15min. Cool to room temperature and make up to the mark with ethanol. Transfer to a centrifuge tube and centrifuge at 20000r / min for 3min. Take 1.5mL of the supernatant into a purification tube containing 50mg ALN, 50mg C18, 30mg PSA and 100mg MgSO4. Vortex for 1min and let stand for 5min. Filter the supernatant through a 0.22μm microporous membrane.

[0058] Step 3: Sample purification

[0059] Since the target drugs are mostly oil-soluble, the primary task of purification is to remove interference from fats. ALN has a strong effect on removing oil interference. In addition, C18 can remove carbohydrates and enhance lipid removal. At the same time, PSA is used to remove pigments and organic acids, and magnesium sulfate is used for rapid dehydration of the sample, which can increase the sample loading and recovery rate. Therefore, a combination of these four packing materials was finally selected for sample purification. The purification effects of PSA, C18, ALN, and GCB, both individually and in combination, are compared as follows:

[0060] A. 30mgALN+50mgC18+50mgPSA+100mgMgSO4

[0061] B. 50mgALN+50mgC18+50mgPSA+100mgMgSO4

[0062] C. 50mgALN+100mgC18+100mgPSA+100mgMgSO4

[0063] D. 100mgALN+30mgC18+30mgPSA+100mgMgSO4

[0064] E. 50mgALN+50mgC18+30mgPSA+100mgMgSO4

[0065] The distribution of the effects of five different packing materials on the recovery rate of 15 drug residues in foods claiming to relieve hangovers is shown in Table 1.

[0066] Table 1. Distribution of Residue Recovery Rates of 15 Drugs with Different Purification Packing Materials

[0067]

[0068] Comparative experiments showed that using 50mgALN+50mgC18+30mgPSA+100mgMgSO4 as the dispersion extraction purification packing material resulted in the best purification effect and recovery rate.

[0069] Step 4: Determination of Mass Spectrometry Detection Method

[0070] Currently, there are no detection methods for hangover remedies, and no relevant literature has been found. After solving the extraction method problem, this patent develops mass spectrometry methods for each standard, identifies and optimizes the most suitable qualitative and quantitative ion pairs. The qualitative and quantitative ion pair data for 15 illegally added drugs are as follows:

[0071] Table 2. Mass spectrometry parameters of 15 hangover remedies

[0072]

[0073] Example 2

[0074] Step 1: Preparation of test solutions. Test solution 1: (for amiloride, tolvaptan, indapamide, and sulfanilamide):

[0075] Take anhydrous ethanol and adjust the pH to 10.2 with concentrated ammonia as the extraction solution. Mix the solid or semi-solid samples thoroughly and grind them finely. Shake the liquid sample thoroughly and weigh 1g (accurate to 0.01g) into a 10mL volumetric flask. Add an appropriate amount of extraction solution and extract by sonication for 15min. Cool to room temperature and dilute to the mark with ethanol. Transfer to a centrifuge tube and centrifuge at 20000r / min for 3min. Take 1.5mL of the supernatant into a purification tube containing 50mgALN, 50mgC18, 30mgPSA, and 100mgMgSO4. Vortex for 1min and let stand for 5min. Filter the supernatant through a 0.22μm microporous membrane.

[0076] Test solution 2: (Applicable to benzylfluthiazide, bumetanide, ethacrynic acid, furosemide, canrone, chlorothiazide, naloxone, naltrexone, verapamil hydrochloride, buthiazine, cyclopenthiazine): Take anhydrous ethanol, adjust the pH to 4.5 with phosphoric acid as the extraction solution. Mix the solid or semi-solid sample thoroughly and grind it finely; shake the liquid sample thoroughly, weigh 1g (accurate to 0.01g) and place it in a 10mL volumetric flask, add an appropriate amount of extraction solution, extract by sonication for 15min, cool to room temperature, make up to volume with ethanol, transfer to a centrifuge tube, centrifuge at 20000r / min for 3min, take 1.5mL of the supernatant into a purification tube containing 50mg ALN, 50mg C18, 30mg PSA, and 100mg MgSO4, vortex for 1min, let stand for 5min, and filter the supernatant through a 0.22μm microporous membrane.

[0077] Blank test: Without adding the sample, the blank solution is prepared by treating it in the same way as the sample.

[0078] Step 2: Analyze the test solution using a liquid chromatography-tandem quadrupole mass spectrometer.

[0079] The liquid chromatography conditions are as follows:

[0080] a. Chromatographic column: C18 column, 100mm*2.1mm, 2.5μm.

[0081] b. Column temperature: 40℃.

[0082] c. Injection volume: 5 μL.

[0083] d. Flow rate: 0.4 mL / min.

[0084] e. Mobile phase: A is 5 mol / L ammonium acetate (containing 0.1% formic acid aqueous solution), B is acetonitrile, and the gradient elution program is shown in Table 3.

[0085] Table 3 Gradient elution program table

[0086] 0 90 10 2 90 10 5 15 85 8 15 85 8.1 90 10 10 90 10

[0087] The conditions for liquid chromatography-mass spectrometry are as follows:

[0088] a. Ion source type: Electrospray ionization (ESI);

[0089] b. Electrospray voltage: Positive source 5000V, negative source -4500V;

[0090] c. Mass spectrometry scanning mode: Multiple reaction monitoring (MRM).

[0091] Step 3: Qualitative Measurement and Analysis

[0092] a. Scan the sample using the instrument method and mass spectrometry parameters described in step two. If the target analyte does not respond, the sample is not detected and quantification is not required.

[0093] b. If the target analyte is positive after scanning in step a of step three, then the sample and standard working solution are measured according to the conditions in step two. The chromatographic retention time of each compound in the sample and standard solution is recorded. When a chromatographic peak with the same retention time as a certain standard peak is detected in the sample (within ±2.5%), and the deviation between the relative abundance ratio of the selected monitoring ion pair in the sample chromatogram and the relative abundance ratio (k) of the ion in the equivalent concentration standard solution does not exceed the range specified in Table 4, it can be determined that the corresponding compound is detected in the sample.

[0094] Table 4. Maximum permissible deviation of relative ion abundance during qualitative confirmation.

[0095] Maximum permissible deviation (%) ±20 ±25 ±30 ±50

[0096] Step 4: Quantitative Determination and Analysis

[0097] The standard working solution was measured under the instrument reference conditions described in step two to obtain the corresponding peak area of ​​the standard solution. A standard curve was plotted using the external standard method, with the concentration of the standard working solution on the x-axis and the peak area of ​​the quantitative ion on the y-axis.

[0098] If the target analyte is detected, calculate the content of the target analyte in the sample using the following formula:

[0099]

[0100] In the formula:

[0101] X—The content of each analyte in the sample, in milligrams per kilogram (mg / kg);

[0102] c—the concentration of the analyte, expressed in micrograms per liter (μg / L);

[0103] V—Final volume of the sample solution, in milliliters (mL);

[0104] m—the mass represented by the sample solution, in grams (g);

[0105] f—Dilution factor.

[0106] The calculation results should exclude blank values ​​and retain three significant figures.

[0107] The detection limit for the hangover relief test using this method is 50 μg / kg.

[0108] Example 3 Method Verification

[0109] 1. Linear range

[0110] A standard curve was plotted with the concentration of the mixed standard series working solutions as the x-axis and the peak area as the y-axis. The correlation coefficient R of the 15 drug residues in the range of 50 μg / kg to 500 μg / kg was calculated. 2 All values ​​are greater than 0.990, indicating a good linear relationship between the methods.

[0111] 2. Limit of detection and limit of quantitation

[0112] According to the pretreatment method, a certain concentration of standard solution was added to the blank matrix. The concentration with a signal-to-noise ratio of 3 was used as the detection limit, and the concentration with a signal-to-noise ratio of 10 was used as the quantitation limit. The results are shown in Table 5.

[0113] Table 5 Limit of Detection and Limit of Quantification

[0114]

[0115] 3. Method accuracy and reproducibility precision

[0116] Using a certain brand of mulberry hangover relief tablets and a certain brand of kudzu root and jujube hangover relief capsules (no positive components detected) as matrices, recovery and precision experiments were conducted at three spiking levels: 50 μg / kg, 150 μg / kg, and 500 μg / kg. Appropriate amounts of reference standards were added according to the sample preparation method, and six parallel determinations were performed using the optimized pretreatment and detection method. The recovery rates were calculated, and the results are shown in Table 6.

[0117] Table 6. Recovery rates of 15 drug residues (hangover relief tablets)

[0118]

[0119] The results showed that the parallelism of the sample detection was good, the RSD was less than or equal to RSD≤10%, and the recovery rate was 60%-120%, which met the relevant method validation requirements.

[0120] 4. Actual sample measurement

[0121] A batch each of turmeric-flavored hangover pills, hangover tablets, and hangover gummies were purchased from the market and tested according to the method. The detection results of 15 drug residues are shown in Table 7 (unit: μg / kg):

[0122] Table 7. Actual sample measurement data (n=3)

[0123]

[0124] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for rapid screening and detection of illegally added drugs in foods claiming to cure hangovers, characterized in that, The steps include the following: (1) Prepare standard solutions of illegally added drugs in foods that claim to cure hangovers and plot standard curves; (2) The sample to be tested is extracted and purified to obtain the sample solution; (3) The sample solution was qualitatively and quantitatively analyzed by liquid chromatography-tandem mass spectrometry. The liquid chromatography conditions were as follows: the column was a C18 column, the mobile phase A was 5 mol / L ammonium acetate containing 0.1% formic acid aqueous solution, the mobile phase B was acetonitrile, and the elution program was as follows: by volume fraction, in the first 0-5 min, mobile phase A was 90% and B was 10%; in the first 5-8 min, mobile phase A was 15% and B was 85%; in the first 8-10 min, mobile phase A was 90% and B was 10%. The liquid chromatography-mass spectrometry (LC-MS) conditions were as follows: ion source type: electrospray ionization (ESI); electrospray voltage: positive source 5000V, negative source -4500V; mass spectrometry scanning mode: multiple reaction monitoring (MRM). The drugs illegally added to the food products that claimed to cure hangovers were amiloride, benzylfluthiazide, bumetanide, canrone, naloxone, naltrexone, tolvaptan, verapamil hydrochloride, indapamide, ethacrylic acid, sulfanilamide, buthiazine, cyclopenthiazine, furosemide, and chlorothiazide. In step (2), the extraction method of the sample to be tested is as follows: adjust the pH value of anhydrous ethanol to a suitable range and use it as the extraction solution for ultrasonic extraction for 15±5 min; for amiloride, tolvaptan, indapamide, and sulfanilamide, adjust the pH value of anhydrous ethanol to 10.2±0.5; for benzylfluthiazide, bumetanide, ethacrynic acid, furosemide, canrone, chlorothiazide, naloxone, naltrexone, verapamil hydrochloride, buthiazine, and cyclopenthiazine, adjust the pH value of anhydrous ethanol to 4.5±0.

5. In step (2), the purification method of the sample to be tested is as follows: after centrifugation of the extracted solution, the supernatant is added to the purification agent, vortexed, allowed to stand, and then the supernatant is filtered through a membrane; the purification agent is neutral alumina, C18, ethylenediamine-N-propylsilane and magnesium sulfate, and the mass ratio is (3~10):(3~10):(3~10):10; In step (3), the C18 chromatographic column is 100 mm * 2.1 mm, 2.5 μm, and the column temperature is 40 °C.

2. The method according to claim 1, characterized in that, In step (3), the injection volume is 5 μL and the flow rate is 0.4 mL / min.

3. The method according to claim 1, characterized in that, The mass ratio of neutral alumina, C18, ethylenediamine-N-propylsilane, and magnesium sulfate in the purifying agent is 5:5:3:

10.

4. The method according to claim 1, characterized in that, The specific steps for plotting the standard curve in step (1) are as follows: the standard solution is measured according to the conditions described in step (3) to obtain the chromatographic peak area of ​​the corresponding standard solution. The standard curve is plotted with the concentration of the standard working solution as the abscissa and the peak area of ​​the chromatographic peak of the quantitative ion as the ordinate.

5. The method according to claim 1, characterized in that, The detection limit of the method is ≤50 μg / kg; the quantitation limit is ≤150 mg / kg.

6. The method according to claim 1, characterized in that, The qualitative and quantitative ion pair parameters of the illegally added drug are as follows, where * indicates a quantitative ion pair: 。

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