Isotope drug-labeled compound and preparation method and use thereof

Abstract

The present invention relates to an isotope-labeled drug compound, its preparation method, and its use. The isotope-labeled drug compound provided by the present invention can be used as an internal standard for determining the content of the drug tramadol in samples such as sewage and wastewater. Tramadol can be qualitatively detected based on its retention time and ion pair matching, and specifically, the corresponding compound can be quantitatively detected based on its chromatographic peak area. Using this isotope-labeled drug compound as an internal standard can reduce the matrix effect of the sample and has excellent application prospects in forensic identification and other aspects.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation and application of psychoactive substance standard substances, and specifically relates to an isotope psychoactive substance labeled compound and a preparation method and application thereof. Background Art

[0002] For the detection of trace substances in sample samples, isotope dilution mass spectrometry (LDMS) using liquid chromatography-mass spectrometry (LC-MS) coupled with an isotope-labeled reference material as an internal standard is the most common and efficient method. Using an isotope-labeled reference material as an internal standard, the isotope-labeled reference material and the analyte are chemically identical, except for their molecular weight. Therefore, isotope dilution mass spectrometry achieves high detection accuracy and sensitivity, making it an indispensable tool for drug detection in wastewater.

[0003] The detection of drugs and related metabolites in sewage can provide a comprehensive and intuitive assessment of a city's drug prevalence. Combined with relevant data from drug enforcement and anti-drug efforts, it is a key tool for assessing a city's overall drug situation and conducting criminal investigations. It plays a crucial role in assessing and combating drug abuse. The detection of drugs and related metabolites in sewage can reveal a subject's long-term drug exposure, making it an essential testing tool for drug addiction assessment and community-based drug rehabilitation programs.

[0004] Currently, the method for detecting trace amounts of the drug tramadol in environmental or biological samples is generally liquid chromatography-mass spectrometry. Compounds with similar chemical properties to tramadol are selected as internal standards, and the tramadol content in the sample is determined using a standard curve. The internal standards selected are generally methoxyphenamine, D4-ketamine, or D3-diazepam.

[0005] Therefore, providing an isotope drug-labeled compound and using it to improve the accuracy and sensitivity of drug detection in samples is one of the problems that technical personnel in this field urgently need to solve. Summary of the Invention

[0006] In order to solve the problem of low accuracy and sensitivity of drug detection in the prior art, the present invention provides an isotope drug-labeled compound, the structural formula of which is as follows:

[0007]

[0008] Wherein, at least one of R1, R2, R3, and R4 is D, and the drug contains tramadol.

[0009] Preferably, the structural formula of the isotope drug-labeled compound is:

[0010]

[0011] A second aspect of the present invention provides a method for preparing the above-mentioned isotope drug-labeled compound, the preparation method comprising the following steps:

[0012] (1) Synthesis of intermediate D4-m-methoxybromobenzene using D4-m-nitrobromobenzene as raw material

[0013]

[0014] (2) D4-m-methoxybromobenzene is synthesized into D4-tramadol via Grignard reaction:

[0015]

[0016] The third aspect of the present invention provides the use of the above-mentioned isotope drug-labeled compound in detecting the content of the drug tramadol in biological samples or sewage.

[0017] In the above-mentioned use, the method for detecting the content of the drug tramadol in biological samples or sewage using the above-mentioned isotope drug-labeled compound comprises the following steps:

[0018] (1) Setting liquid chromatography-mass spectrometry detection conditions;

[0019] (2) Drawing of the standard curve: Different proportions of tramadol and D4-tramadol standard solutions were added to the negative sample corresponding to the sample to be tested. After the same pretreatment steps as the sample to be tested, LC-MS / MS detection was performed to draw a standard curve for the peak area ratio and concentration of tramadol and D4-tramadol; wherein the internal standard was the isotope drug-labeled compound. The pretreatment steps were as follows: the sample was filtered through filter paper, methanol (containing 10 ng / ml D4-tramadol) was added, and the sample was loaded through the SPE column. After loading, the sample was first rinsed with methanol and finally eluted with 5% ammonia-acetonitrile solution. The eluate was evaporated in a water bath under air flow, re-dissolved with methanol, and filtered through a membrane.

[0020] (3) Pretreatment and determination of the sample to be tested: The isotope drug-labeled compound is added to the sample to be tested as an internal standard, and after the same pretreatment steps as those used in drawing the standard curve, the sample is subjected to LC-MS / MS detection to obtain the quantitative parameters of the sample to be tested and the internal standard. The content of the sample to be tested can be calculated using the formula of the internal standard method standard curve.

[0021] Preferably, in step (1), the detection conditions of the liquid chromatography-mass spectrometry detection are as follows: mobile phase: A: acetonitrile (0.01% formic acid), B: water (0.01% formic acid, 5% ammonium formate), gradient: 1 min 80% A, 4 min 95% A, 10 min 95% A; chromatographic column: Poroshell120 PFP 3.0x100mm 1.9um; column temperature: 50°C; flow rate: 0.5 mL / min; injection volume: 5 μL; ion source: electrospray ionization source, positive mode (ESI+); spray voltage 3500 V; ion source temperature: 340°C; collision gas: nitrogen.

[0022] Preferably, in step (2), the mass concentration of the internal standard in the mixed standard solution is 0.1% to 99.9%.

[0023] Preferably, in step (3), the mass concentration of the internal standard in the sample to be tested is 0.1% to 99.9%.

[0024] Preferably, in step (3), the formula of the internal standard method standard curve is:

[0025]

[0026] Where X is the concentration of the sample to be tested, Y is the peak area obtained by liquid chromatography-mass spectrometry, b0 is the intercept of the standard curve, and b1 is the slope of the standard curve.

[0027] Preferably, the mass content of the drug tramadol in the above biological sample or the sewage is 10 -7 %~10%.

[0028] Another aspect of the present invention provides a method for detecting the content of the drug tramadol in biological samples or sewage, which includes using an isotope drug-labeled compound as an internal standard to detect the drug tramadol.

[0029] Preferably, the above method for detecting the content of the drug tramadol in biological samples or sewage comprises the following steps:

[0030] (1) Setting liquid chromatography-mass spectrometry detection conditions;

[0031] (2) preparing a mixed standard solution containing the sample to be tested and an internal standard, performing LC-MS / MS detection, and making a standard curve for the peak area ratio and concentration of tramadol and D4-tramadol; wherein the internal standard is the isotope drug-labeled compound.

[0032] (3) Pretreatment and determination of the sample to be tested: After pretreatment, the isotope drug-labeled compound is added to the sample to be tested as an internal standard, and then subjected to LC-MS / MS detection to obtain quantitative parameters of the sample to be tested and the internal standard. The content of the sample to be tested can be calculated using the internal standard method formula.

[0033] The isotope drug-labeled compound D4-tramadol provided by the present invention can be used as an internal standard for the determination of the drug tramadol content in sewage and biological samples, can reduce the matrix effect of the samples, and has good application prospects in forensic identification and other aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the high-resolution mass spectrum of D4-tramadol in Example 1 of the present invention.

[0035] Figure 2A is the D4-tramadol in Example 1 of the present invention 1 H-NMR spectrum.

[0036] Figure 2B is the D4-tramadol in Example 1 of the present invention 13 C-NMR spectrum.

[0037] Figure 3 This is the extracted ion chromatogram of D4-tramadol in Example 1 of the present invention.

[0038] Figure 4 The chromatogram for detecting tramadol in sewage sample in Example 2 of the present invention Figure 1 .

[0039] Figure 5 This is chromatogram 2 of the detection of tramadol sample in sewage in Example 2 of the present invention.

[0040] Figure 6 This is the standard curve of tramadol in sewage in Example 2 of the present invention. DETAILED DESCRIPTION

[0041] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below with reference to specific illustrations.

[0042] In the description of the present invention, "biological samples" refer to human tissue samples, such as sewage, blood, urine, etc.

[0043] In the description of the present invention, "matrix effect" refers to the influence and interference of substances other than the analyte tramadol in the sample on the analysis process and test results.

[0044] In the description of the present invention, the mass concentration of tramadol in the biological sample or the sewage is 10 -7 %~10%.

[0045] The isotope drug-labeled compound D4-tramadol provided by the present invention has the following structural formula:

[0046]

[0047] The present invention is further described below with reference to specific embodiments.

[0048] Example 1

[0049] D4- Tramadol preparation method.

[0050]

[0051] At 0°C, dissolve D4-3-bromonitrobenzene (CAS. No. 81395-19-6, 177 mg, 1 mmol) and Pd-C (40%, 50 mg) in 20 mL of deuterated methanol. Degas the mixture with argon and then fill it with 10 atm of deuterium. After reacting at room temperature for 24 hours, filter and remove the catalyst. After removing the methanol by rotary evaporation, add 10 mL of 0.05 M sulfuric acid under an ice-salt bath, followed by dropwise addition of aqueous sodium nitrite (69 mg) and stir to form a homogeneous solution. Slowly heat the mixture to boiling, then cool to room temperature after 20 minutes. After complete conversion as monitored by TLC, extract the mixture five times with 200 mL of dichloromethane. Combine the organic phases, dry them over anhydrous magnesium sulfate, filter to remove the desiccant, and rotary evaporator to remove the solvent. Add 20 mL of tetrahydrofuran, 168 mg of sodium bicarbonate, and 142 mg of iodomethane. After reacting at room temperature for 24 hours, evaporate most of the solvent, dissolve in dichloromethane, wash with 2% aqueous sodium hydroxide solution and saturated brine, and dry over anhydrous magnesium sulfate. Remove the desiccant by filtration, and remove the solvent by rotary evaporation. Purification by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) afforded 180 mg of a colorless, transparent oil, which was dissolved in 50 mL of anhydrous, oxygen-free tetrahydrofuran. 40 mg of magnesium turnings was added, and the mixture was degassed with argon and heated to a slight boil to initiate the reaction. The mixture was then cooled to room temperature and filtered to remove excess magnesium under argon. The mixture was then added dropwise to a solution of 2-(N,N-dimethylaminomethyl)-1-hexanone (148 mg, 0.95 mmol) in tetrahydrofuran. The mixture was stirred at room temperature for 2 hours, then heated to reflux for 4 hours. Insoluble matter was removed by filtration, and the solvent was evaporated. The mixture was dissolved in dichloromethane and washed with water, saturated ammonium chloride solution, saturated brine, and water, respectively. The mixture was then extracted with dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate, filtered to remove the desiccant, and the solvent was removed by rotary evaporation. Purification by silica gel column chromatography (petroleum ether:ethyl acetate = 15:1, 5:1, 1:1) afforded 120 mg (45%) of D4-tramadol as a pale yellow solid.

[0052] 1H NMR(600MHz, Methanol-d4)δ3.80(s,3H),2.97(dd,J=13.3,9.2Hz,1H),2.83–2.51(m,7H),2.23(dddd,J=12.0,9.3,4.0,2.8Hz,1H),1.98–1.90(m,1H) ,1.90–1.85(m,1H),1.80(ddt,J=12.6,9.0,6.7Hz,2H),1.76–1.70(m,2H), 1.65–1.60(m,1H),1.54(dddd,J=16.8,13.0,8.4,3.8Hz,1H),1.29(s,1H).m 13C NMR (151MHz, cd3od) δ159.48, 148.54, 128.72, 116.26, 111.00, 110.35, 73.89, 60.02, 53.72, 41.00, 39.60, 25.15, 24.17, 20.48. MS (EI) m / z: Calcd. for C16H21D4NO2 [M] + 267.1; Found: 267.1. Spectrum see Figure 1 、 2A , 2B, 3.

[0053] Example 2

[0054] D4-tramadol was used as the internal standard to detect the content of tramadol in wastewater.

[0055] (1) Liquid chromatography-mass spectrometry detection conditions:

[0056] a) Instrument model: Agilent 1290-6470QQQ;

[0057] b) Chromatographic column: Poroshell 120 PFP 3.0x100mm 1.9um;

[0058] c) Column temperature: 50°C;

[0059] d) Mobile phase: A: acetonitrile (0.01% formic acid), B: water (0.01% formic acid, 5% ammonium formate), gradient: 1 min 80% A, 4 min 95% A, 10 min 95% A;

[0060] e) Flow rate: 0.5 mL / min;

[0061] f) Injection volume: 5 μL;

[0062] g) Ion source: electrospray ionization source, positive mode (ESI+);

[0063] h) Spray voltage 3500V;

[0064] i) Ion source temperature: 340°C;

[0065] j) Collision gas: nitrogen.

[0066] The ion pairs and corresponding conditions are shown in Table 1:

[0067] Table 1

[0068]

[0069] (2) Sample pretreatment and testing

[0070] The sewage sample was filtered through filter paper, 100 mL was taken, 2 mL of methanol (containing 10 ng / ml D4-tramadol) was added, and 50 mL of each portion was loaded on the SPE column at a speed of 5 mL / min. After loading, it was first rinsed with 5 mL of methanol and finally eluted with 5 mL of 5% ammonia-acetonitrile solution. The eluate was evaporated to dryness under air flow in a 45°C water bath, re-dissolved with 80 μL of methanol, filtered through a 0.22 μL filter membrane, and 5 μL was subjected to LC-MS / MS analysis to obtain the following: Figure 4 and 5 The quantification ion pair is 191.0 / 222.0, and the peak area ratios of tramadol and internal standard D4-tramadol in the two tests are 43526 / 846298, respectively.

[0071] (3) Drawing of standard curve

[0072] 1 mL of methanol (containing 10 ng / mL D4-tramadol) was added to 50 mL of negative sewage sample, and tramadol standard reference substance was added to prepare sewage spiked samples with concentrations of 0.5, 1, 2, 5, 10, 20, and 50 ng / L. Two parallel copies of each concentration were prepared. After sample pretreatment according to the sample pretreatment process, LC-MS / MS was performed for detection. A standard curve was drawn for the peak area ratio and concentration of tramadol and D4-tramadol, as shown below. Figure 6 The standard curve formula is y = 0.004428 × x + 0.208958, where X is the concentration of the sample to be tested, Y is the peak area obtained by liquid chromatography-mass spectrometry, b0 = 0.208958 is the intercept of the standard curve, and b1 = 0.004428 is the slope of the standard curve.

[0073] According to the quantitative relationship between peak area ratio and concentration in the standard curve, the calculation formula is:

[0074]

[0075] According to the quantitative relationship between the peak area ratio and concentration in the standard curve, the content of tramadol in the test sample was calculated to be 6 ng / L.

[0076] When the isotope drug-labeled compound provided by the present invention is used to detect the drug tramadol in a sample, an appropriate amount of the isotope drug-labeled compound D4-tramadol is added to the sample, and after appropriate pretreatment according to detection requirements, liquid chromatography-mass spectrometry (LC-MS / MS) detection is performed. The isotope drug-labeled compound is used as an internal standard. In the multiple reaction monitoring (MRM) mode, the ratio of the peak area of ​​the target substance and the substance to be detected is compared to achieve qualitative and quantitative detection of the substance to be detected. The drug that can be detected is tramadol, and the specificity and sensitivity are high.

[0077] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. An isotope drug-labeled compound, characterized in that: The structural formula of the isotope drug-labeled compound is: 。 2. A method for preparing an isotope drug-labeled compound according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Synthesis of D4-m-methoxybromobenzene using D4-m-nitrobromobenzene as raw material; ； (2) D4-m-methoxybromobenzene is synthesized into D4-tramadol via Grignard reaction: 。 3. Use of the isotope drug-labeled compound according to claim 1 in detecting the content of the drug tramadol in biological samples or sewage.

4. The use according to claim 3, characterized in that The method for detecting the content of the drug tramadol in biological samples or sewage using the isotope drug-labeled compound according to claim 1 comprises the following steps: (1) Setting liquid chromatography-mass spectrometry detection conditions; (2) Drawing of a standard curve: Add different proportions of tramadol and D4-tramadol standard solutions to the negative test sample corresponding to the test sample, and after the same pretreatment steps as the test sample, perform LC-MS / MS detection, and draw a standard curve based on the peak area ratio and concentration of tramadol and D4-tramadol, wherein the internal standard is the isotope drug-labeled compound described in claim 1; (3) Pretreatment and determination of the sample to be tested: The isotope drug-labeled compound described in claim 1 is added to the sample to be tested as an internal standard. After the same pretreatment steps as those used in drawing the standard curve, the sample is subjected to LC-MS / MS detection to obtain the quantitative parameters of the sample to be tested and the internal standard. The content of the sample to be tested can be calculated using the formula of the internal standard method standard curve.

5. The use according to claim 4, characterized in that In the step (1), the detection conditions of the liquid chromatography-mass spectrometry detection are as follows: mobile phase: A: acetonitrile, 0.01% formic acid, B: water, 0.01% formic acid, 5% ammonium formate, gradient: 1 min 80% A, 4 min 95% A, 10 min 95% A; chromatographic column: Poroshell120 PFP 3.0 x100mm 1.9 um; column temperature: 50°C; flow rate: 0.5 mL / min; injection volume: 5 μL; ion source: electrospray ionization source, positive mode ESI+; spray voltage 3500 V; ion source temperature: 340°C; collision gas: nitrogen.

6. The use according to claim 4, characterized in that In the step (2), the mass concentration of the internal standard in the mixed standard solution is 0.1% to 99.9%; in the step (3), the mass concentration of the internal standard in the sample to be tested is 0.1% to 99.9%.

7. The use according to claim 4, characterized in that In the step (3), the formula of the internal standard method standard curve is: ； Where X is the concentration of the sample to be tested, Y is the peak area obtained by liquid chromatography-mass spectrometry, b0 is the intercept of the standard curve, and b1 is the slope of the standard curve.

8. The use according to claim 3, characterized in that The content of tramadol in the biological sample or sewage is 10 -7 %~10%.

9. A method for detecting the content of the drug tramadol in biological samples or sewage, the method comprising using the isotope drug-labeled compound according to claim 1 as an internal standard to detect the drug tramadol.

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