Isotope drug labeled compound as well as preparation method and application thereof

Abstract

The invention relates to an isotope drug labeling compound and a preparation method and application thereof, the isotope drug labeling compound provided by the invention can be used as an internal standard for determining the content of drug tramadol in detection materials such as sewage, and can be used for qualitatively detecting tramadol according to the retention time and ion pair matching of the isotope drug labeling compound, and particularly, the isotope drug labeling compound can be used for detecting the content of drug tramadol. And quantitatively detecting the corresponding compound according to the chromatographic peak area. When the isotope drug labeling compound is used as an internal standard substance, the matrix effect of a detected material can be reduced, and the isotope drug labeling compound has a good application prospect in the aspects of judicial expertise and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation and application of psychoactive substance standard materials, 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 the sample, the most common and efficient detection method is the isotope dilution mass spectrometry with liquid chromatography-mass spectrometry and isotope-labeled standard substance as internal standard. With isotope-labeled standard substance as internal standard, the chemical properties of isotope-labeled internal standard and the compound to be tested are completely consistent except for the molecular weight. Therefore, isotope dilution mass spectrometry can achieve high detection accuracy and sensitivity, and is an indispensable tool for drug detection in sewage.

[0003] The detection of drugs and related metabolites in sewage can comprehensively and intuitively evaluate the drug situation in a city. Combined with the comparison of relevant data on drug control and anti-drug work, it is one of the important means of evaluating the overall drug situation in the city and criminal investigation. It plays a key role in evaluating the drug situation in the city and focusing on crackdowns. The detection of drugs and related metabolites in sewage can reflect the long-term exposure to drugs of the tested subjects, and is an indispensable detection method for drug addiction determination and community drug rehabilitation.

[0004] At present, the method for detecting trace amounts of the drug tramadol in environmental or biological samples is generally liquid chromatography-mass spectrometry, which selects compounds with similar chemical properties to tramadol as internal standards and determines the content of tramadol in the samples through the standard curve method. The internal standards selected are generally the more common 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 view of the problems 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] Among them, 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] The 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-metamethoxybromobenzene 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 standard curve: Add different proportions of tramadol and D4-tramadol standard solution to the negative sample corresponding to the sample to be tested, and after the same pretreatment steps as the sample to be tested, perform LC-MS / MS detection, and make 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. The pretreatment steps are: filter the sample through filter paper, add methanol (containing 10ng / ml D4-tramadol), load the sample through the SPE column, and after loading, first rinse with methanol, and finally elute with 5% ammonia-acetonitrile solution, evaporate the eluent under air flow in a water bath, re-dissolve with methanol, and filter the 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 standard curve of the internal standard method.

[0021] Preferably, in step (1), the detection conditions of the liquid chromatography-mass spectrometry detection are: mobile phase: A: acetonitrile (0.01% formic acid), B: water (0.01% formic acid, 5% ammonium formate), gradient: 1min 80% A, 4min 95% A, 10min 95% A; chromatographic column: Poroshell120 PFP 3.0x100mm 1.9um; column temperature: 50°C; flow rate: 0.5mL / min; injection volume: 5μL; ion source: electrospray ion source, positive mode (ESI+); spray voltage 3500V; 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] Wherein, 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 biological sample or the sewage is 10 -7 %~10%.

[0028] In another aspect, the present invention provides a method for detecting the content of the drug tramadol in biological samples or sewage, the method comprising 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 the pretreatment, the isotope drug-labeled compound is added to the sample to be tested as an internal standard, and then 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 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 determining the content of the drug tramadol in sewage and biological samples, can reduce the matrix effect of the samples, and has a good application prospect in forensic identification and the like. 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 D4-tramadol in Example 1 of the present invention 1 H-NMR spectrum.

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

[0037] Figure 3 It 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 easy to understand, the present invention is further explained below with reference to specific diagrams.

[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-Method for preparing tramadol.

[0050]

[0051] At 0°C, D4-3-bromonitrobenzene (CAS.No.81395-19-6, 177mg, 1mmol) and Pd-C (40%, 50mg) were dissolved in 20mL of deuterated methanol. After the mixture was degassed with argon, 10atm of deuterium gas was introduced, and the reaction was carried out at room temperature for 24 hours, and the catalyst was filtered out. After removing the methanol by rotary evaporation, 10mL of 0.05M sulfuric acid was added under an ice-salt bath, and sodium nitrite aqueous solution (69mg) was added dropwise, and stirred to form a homogeneous solution. The temperature was slowly raised to boiling, and the temperature was lowered to room temperature after 20 minutes. After the reaction conversion was completely monitored by TLC, it was extracted 5 times with 200mL of dichloromethane, the organic phases were combined and dried over anhydrous magnesium sulfate, the desiccant was filtered out, and the solvent was removed by rotary evaporation. Add 20 mL of tetrahydrofuran, 168 mg of sodium bicarbonate and 142 mg of iodomethane, react at room temperature for 24 hours, evaporate most of the solvent, dissolve with dichloromethane, wash with 2% sodium hydroxide aqueous solution and saturated brine, and dry with anhydrous magnesium sulfate. Filter to remove the desiccant, and remove the solvent by rotary evaporation. Purification by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) gave 180 mg of colorless transparent oil, which was dissolved in 50 mL of anhydrous and oxygen-free tetrahydrofuran, 40 mg of magnesium chips was added, and the mixture was degassed with argon and heated to a slight boiling point to initiate the reaction, then cooled to room temperature, filtered to remove excess magnesium under an argon environment, and then added dropwise to a tetrahydrofuran solution of 2-(N,N-dimethylaminomethyl)-1-hexanone (148 mg, 0.95 mmol), stirred at room temperature for 2 hours after the addition was completed, heated to reflux for 4 hours, filtered to remove insoluble matter, evaporated to remove the solvent, dissolved in dichloromethane, washed with water, saturated ammonium chloride solution, saturated brine, and water, respectively, extracted with dichloromethane, combined organic phases were dried with 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) eluted to obtain 120 mg (45%) of light yellow solid D4-tramadol.

[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 sewage.

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

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

[0057] b) Chromatographic column: Poroshell120 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 ion 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 eluent was evaporated under air flow in a 45°C water bath, re-dissolved with 80 μL of methanol, and after passing through a 0.22 μL filter membrane, 5 μL was subjected to LC-MS / MS analysis to obtain the following Figure 4 and 5 The chromatogram shown in FIG. 1 shows a quantitative ion pair of 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) Standard curve drawing

[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 samples were prepared in parallel for each concentration. After being processed according to the sample pretreatment process, LC-MS / MS was used for detection. A standard curve was made for the peak area ratio and concentration of tramadol and D4-tramadol, and the following was obtained: 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 the peak area ratio and concentration in the standard curve, according to the calculation formula:

[0074]

[0075] According to the quantitative relationship between the peak area ratio and the concentration in the standard curve, the content of tramadol in the sample to be tested 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 liquid chromatography-mass spectrometry (LC-MS / MS) detection is performed after appropriate pretreatment according to the detection requirements. The isotope drug-labeled compound is used as an internal standard, and in a 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 above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. An isotope drug-labeled compound, characterized in that: The structural formula of the compound is as follows: Among them, at least one of R1, R2, R3, and R4 is D, and the drug contains tramadol.

2. The isotope drug-labeled compound according to claim 1, characterized in that: The structural formula of the isotope drug-labeled compound is:

3. A method for preparing an isotope drug-labeled compound according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: (1) Synthesizing D4-m-methoxybromobenzene using D4-m-nitrobromobenzene as a raw material; (2) D4-metamethoxybromobenzene is synthesized into D4-tramadol via Grignard reaction:

4. Use of the isotope drug-labeled compound according to claim 1 or 2 in detecting the content of the drug tramadol in biological samples or sewage.

5. The use according to claim 4, 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 or 2 comprises the following steps: (1) Setting liquid chromatography-mass spectrometry detection conditions; (2) Drawing of a standard curve: adding tramadol and D4-tramadol standard solutions in different proportions to the negative test sample corresponding to the test sample, and after the same pretreatment steps as the test sample, LC-MS / MS detection is performed to draw a standard curve of the peak area ratio and concentration of tramadol and D4-tramadol, wherein the internal standard is the isotope drug-labeled compound according to claim 1 or 2; (3) Pretreatment and determination of the sample to be tested: The isotope drug-labeled compound described in claim 2 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 standard curve of the internal standard method.

6. The use according to claim 5, characterized in that In the step (1), the detection conditions of the liquid chromatography-mass spectrometry detection are: mobile phase: A: acetonitrile (0.01% formic acid), B: water (0.01% formic acid, 5% ammonium formate), gradient: 1min 80% A, 4min 95% A, 10min 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 ion source, positive mode (ESI+); spray voltage 3500 V; ion source temperature: 340°C; collision gas: nitrogen.

7. The use according to claim 5, 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%.

8. The use according to claim 5, characterized in that In the step (3), the formula of the internal standard method standard curve is: Wherein, 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.

9. The use according to claim 4, characterized in that: The content of tramadol in the biological sample or sewage is 10 -7 %~10%.

10. 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 or 2 as an internal standard to detect the drug tramadol.

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