Carbon 13 isotope labeling internal standard substance of PMMA (polymethyl methacrylate) and metabolite thereof, preparation method and application

Abstract

The invention relates to a carbon 13 isotope labeling internal standard substance of PMMA (polymethyl methacrylate) and metabolite thereof. The invention further provides a corresponding preparation method and application. The invention fills the technical blank of PMMA / PMA exclusive isotope internal standard, promotes the mental active substance detection to develop towards the direction of higher sensitivity, lower error and stronger judicial credibility through the precise design and application of the carbon 13 labeled standard substance, and has great social value for public safety and health management.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of compound detection, in particular to a carbon 13 isotope labeled internal standard of PMMA and its metabolites, a preparation method and application. BACKGROUND

[0002] In recent years, the concealment and diversity of the abuse of psychoactive substances have increased significantly, and higher requirements have been put forward for accurate detection technology. Especially in the trace analysis of new drugs such as methylenedioxymethamphetamine (PMMA) and its metabolite paramethoxyamphetamine (PMA), the matrix effect and the lack of internal standard matching have become the core bottleneck restricting the detection accuracy. The current mainstream detection method is liquid chromatography-tandem mass spectrometry (LC-MS / MS), which relies on internal standards to correct the extraction efficiency and ionization interference of target substances. However, the existing internal standards are mostly chemical structural analogues (such as D3-PMMA or D5-diazepam), which have significant differences in molecular structure from the target substances, resulting in insufficient isotope dilution effect and difficulty in completely offsetting the interference in complex biological matrix (such as sewage, blood), which affects the reliability of quantitative results.

[0003] Isotope dilution mass spectrometry (IDMS) has become the gold standard for trace detection of psychoactive substances due to its ultra-high sensitivity and anti-matrix interference ability. This method uses isotopically labeled substances as internal standards, and the physicochemical properties of the internal standards need to be highly consistent with those of the target analytes, with only differences in molecular weight. In mass spectrometry analysis, the internal standard and the target substance have nearly the same ionization efficiency and chromatographic behavior, especially the retention time needs to be highly consistent with that of the target substance, which can correct the pretreatment loss and instrument fluctuations in real time, significantly improving the detection accuracy. Studies have shown that the structural homology of the internal standard and the target substance is the key to reducing matrix effects, and the current PMMA / PMA detection lacks high-quality special isotope internal standards, and it is urgent to develop structure-matched labeled compounds.

[0004] Sewage has an irreplaceable advantage in drug abuse practice as a biological evidence: it can trace drug exposure for up to 6 months, providing objective evidence for drug addiction determination, community detoxification effectiveness evaluation, and relapse monitoring. However, the concentration of target substances in sewage is extremely low (usually in pg level), and there are interference factors such as pigment binding and exogenous pollution, which have high requirements for the sensitivity and specificity of the analysis method. The existing technology is difficult to achieve stable quantification of PMMA / PMA in sewage matrix due to the lack of internal standard adaptability, which restricts the scientificity and legal effectiveness of drug control judicial identification. SUMMARY

[0005] The main purpose of the present application is to solve the above problems, and to provide a carbon 13 isotope labeled internal standard of PMMA and its metabolites, a preparation method and application.

[0006] In order to achieve the above-mentioned purpose, the first aspect of the present application provides a carbon 13 isotope labeled internal standard of PMMA and its metabolites, the main feature of which is that the chemical structure of the internal standard is as follows:

[0007]

[0008] In the formula, R is H or methyl.

[0009] The second aspect of the present application provides a preparation method of the carbon 13 isotope labeled internal standard of PMA, the main feature of which is that the preparation method comprises the following steps:

[0010] (1) 13 C-C3-alanine is generated under transition metal catalysis 13 C-C3-aziridine;

[0011] (2) 13 C-C3-aziridine reacts with a p-methoxy phenyl nucleophile to generate the carbon 13 isotope labeled internal standard of PMA 13 C-C3-p-methoxy phenylamine.

[0012] Preferably, in step (1), the transition metal catalyst is Ir(acac)(CO)2.

[0013] In step (2), the reaction catalyst is CuBr or CuI; the reaction is stirred for 2 hours at temperature t2, and then the temperature is raised to t3 and the reaction is carried out at room temperature for T2 hours, t2 is -35℃-25℃, t3 is 0℃-25℃, and T2 is 1-4h.

[0014] The third aspect of the present application provides a preparation method of the carbon 13 isotope labeled internal standard of PMMA, the main feature of which is that the preparation method comprises the following steps:

[0015] The carbon 13 isotope labeled internal standard of PMA is used 13 C-C3-p-methoxy phenylamine is subjected to a methylation reaction to generate the carbon 13 isotope labeled internal standard of PMMA 13 C-C3-p-methoxy methyl phenylamine, the carbon 13 isotope labeled internal standard of PMA 13 C-C3-p-methoxy phenylamine is generated by the preparation method.

[0016] The fourth aspect of the present application provides the use of the carbon 13 isotope labeled internal standard of PMMA and its metabolites or the carbon 13 isotope labeled internal standard of PMA generated by the preparation method in detecting the content of PMA in sewage.

[0017] The fifth aspect of the present application provides the use of the PMMA and its metabolite carbon 13 isotope labeled internal standard or the PMMA carbon 13 isotope labeled internal standard prepared by the preparation method in the detection of the PMMA content in sewage.

[0018] In the above use, the method for detecting the PMMA and its metabolite content in biological samples or sewage by using the carbon 13 isotope labeled internal standard comprises the following steps:

[0019] (1) Setting the liquid chromatography-mass spectrometry detection conditions;

[0020] (2) Drawing a standard curve: in the negative sample corresponding to the sample to be detected, different proportions of PMMA, 13 C-C3-PMMA, PMA and 13 C-C3-PMA standard solution are added, after the same pretreatment steps as the sample to be detected, LC-MS / MS detection is performed, and the peak area ratio and concentration of PMMA and 13 C-C3-PMMA, PMA and 13 C-C3-PMA are used to draw a standard curve;

[0021] (3) Pretreatment and determination of the sample to be detected: the sample to be detected is added with the carbon 13 isotope labeled internal standard, after the same pretreatment steps as used in drawing the standard curve, LC-MS / MS detection is performed, the quantitative parameters of the sample to be detected and the internal standard are obtained, and the content of the sample to be detected can be calculated by using the internal standard curve formula.

[0022] Preferably, in step (1), the mobile phase of the liquid chromatography-mass spectrometry detection is: A: acetonitrile (0.01% formic acid), B: water (0.01% formic acid, 5% ammonium formate), gradient: 1min 10% A, 2min 50% A, 4min 90% A, 6min 95% A; chromatographic column: Poroshell120 PFP 3.0x100mm 1.9um; column temperature: 30℃; flow rate: 0.5mL / min; injection volume: 5μL; ion source: electrospray ion source, positive mode (ESI+); spray voltage 3500V; ion source temperature: 340℃; collision gas: nitrogen.

[0023] Preferably, in step (2), the mass concentration of the internal standard in the mixed standard solution is 0.1%-99.9%.

[0024] Preferably, in step (2), the mass concentration of the internal standard in the sample to be detected is 0.1%-99.9%.

[0025] Preferably, in step (3), the internal standard formula calculation is: X=(Y-b0) / b1;

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

[0027] Preferably, the mass content of the psychoactive substance PMMA and its metabolites in the biological material or the sewage is 10 -9 %-10%.

[0028] The carbon 13 isotope-labeled internal standard of PMMA and its metabolites, the preparation method and the application of the application adopt carbon 13-labeled amino acids as a carbon 13 source, construct an aziridine as a key intermediate through a reduction cyclization reaction, the intermediate can be directly used for subsequent reactions without purification, and the amphetamine structure is directly constructed through ring opening reaction with a metal organic reagent; the carbon 13 label is located at key positions of a benzene ring and an alkyl chain, ensures complete matching of chemical properties, chromatographic retention time and fragment ion spectrum with natural PMMA / PMA, the retention time is closer to the target than the deuterium internal standard, and the retention time consistency is more optimal; the carbon 13 has no isotope exchange effect, is superior to hydrogen-deuterium exchange that may occur in long-term storage or pretreatment of the deuterium internal standard, and the recovery rate is closer to 100% in an alkaline environment.

[0029] The carbon 13 isotope-labeled internal standard of PMMA and its metabolites can be applied to IDMS, and the following can be achieved:

[0030] Sensitivity improvement: the detection limit (LOD) is 0.1 pg / mg (sewage matrix), and the quantitative limit (LOQ) is ≤0.3 pg / mg;

[0031] Precision optimization: the batch / intra-batch coefficient of variation (CV) is <3%, which is significantly superior to the existing alternative internal standard (CV 8-15%);

[0032] Strengthening of judicial evidence effectiveness: providing traceable data support in line with the ISO / IEC 17025 standard for drug suppression law enforcement and court evidence chain construction.

[0033] The application fills the technical gap of PMMA / PMA exclusive isotope internal standards, promotes the development of psychoactive substance detection in the direction of higher sensitivity, lower error and stronger judicial public credibility through precise design and application of carbon 13-labeled standard substances, and has great social value for public safety and health management. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The high-resolution mass spectrum of C-C3-PMMA in Example 1 13

[0035] Figure 2 The high-resolution mass spectrum of C-C3-PMMA in Example 1 13 ​C-C3-PMMA 1 H-NMR spectrum.

[0036] Figure 3 C-C3-PMMA of Example 1 13 C-C3-PMMA 13 C-NMR spectrum.

[0037] Figure 4 C-C3-PMMA of Example 2 13 C-C3-PMA high resolution mass spectrum.

[0038] Figure 5 C-C3-PMMA of Example 2 13 C-C3-PMA 1 H-NMR spectrum.

[0039] Figure 6 C-C3-PMA of Example 2 13 C-C3-PMA 13 C-NMR spectrum.

[0040] Figure 7 Sewage PMA standard curve of Example 3

[0041] Figure 8 Sewage PMA standard curve of Example 3

[0042] Figure 9 Sewage PMA of Example 3 13 C-C3-PMA sample detection chromatogram.

[0043] Figure 10 Sewage PMMA of Example 3 13 C-C3-PMMA sample detection chromatogram.

[0044] Figure 11 Sewage D5-PMA sample detection chromatogram of Example 4

[0045] Figure 12 Sewage D5-PMMA sample detection chromatogram of Example 4 DETAILED DESCRIPTION

[0046] In order to enable a clearer understanding of the technical content of the present application, the following examples are described in detail. However, it should be noted that these descriptions are only to further illustrate the features and advantages of the present application, and are not a limitation on the claims of the application.

[0047] In the description of the present application, "matrix effect" refers to the influence and interference of substances other than the measured PMMA and its metabolites in the test material on the analysis process and detection results.

[0048] In the description of this invention, the mass concentration of PMMA and its metabolites in wastewater is 10. -9 %-10%.

[0049] The isotope-labeled internal standard provided by this invention 13 C-C3-p-methoxyamphetamine and 13 The chemical structural formula of C-C3-p-methoxymethylamphetamine is as follows:

[0050]

[0051] The present invention will be further described below with reference to specific embodiments.

[0052] Example 1

[0053] Preparation by reduction-ring-opening reaction 13 C-C3-p-methoxyamphetamine ( 13 Preparation method of C-C3-PMA.

[0054]

[0055] The carbon-13 labeled amino acid (DL-alanine-) was synthesized under nitrogen protection. 13 C3 (CAS: 144476-54-0, 10 mmol) and catalyst C1 (14023-80-4) were dissolved in 50 mL of anhydrous and oxygen-free solvent S1, and hydrogen was introduced at 0 °C P1. After the reaction was complete, a 30% sulfuric acid-methanol solution was added, and the reaction was carried out at a specified temperature t1 at room temperature T1. The mixture was diluted with diethyl ether and separated into layers. The organic layer was dried with anhydrous magnesium sulfate, filtered, and concentrated. Under nitrogen protection, 5 mmol of catalyst C2 and 60 mmol of 1M solution of p-methoxyphenyl magnesium bromide (60 mL) were added to a two-necked flask in anhydrous tetrahydrofuran. The reaction mixture was cooled to t2 and stirred for about 30 minutes. A C13-labeled azacyclopropane solution dissolved in 160 mL of anhydrous tetrahydrofuran was slowly added dropwise using a syringe. Stirring was continued at temperature t2 for 2 hours, and the temperature was raised to t3 at room temperature T2 for 2 hours. The reaction was quenched with an aqueous solution of ammonium chloride and extracted three times with ethyl acetate (EtOAc) (40 mL each time). The combined organic phases were washed with saturated sodium chloride solution, and the organic layer was dried over anhydrous magnesium sulfate. The mixture was then filtered and concentrated. The crude product was purified by rapid alkaline alumina column chromatography (n-hexane / ethyl acetate, 80:20) to obtain the final product. 13 C-C3-PMMA), a colorless liquid, yields are shown in Table 1.

[0056] 1H NMR (600 MHz, cd3od) δ 7.26 - 7.14 (m, 2H), 7.00 - 6.79 (m, 2H), 3.77 (s, 3H), 3.62-3.54 (m, 0.5H), 3.38 - 3.31 (m, 0.5H), 3.06-2.97 (m, 0.5H), 2.88 - 2.77 (m, 1H), 2.67-2.59 (m, 0.5H), 1.35 (dt, J = 6.5, 4.4 Hz, 1.5H), 1.13 (dt, J = 6.5, 4.4 Hz, 1.5H).

[0057] 13 C NMR (151 MHz, cd3od) δ 160.52 (s), 131.44 (q), 129.11 (d), 115.40 (d), 55.76 (s), 50.47 (q), 40.97 (d), 18.29 (d). HR-MS (ESI / TOF) m / z: Calcd. for C713C3H16NO [M+H]+169.1333; Found: 169.1329. Spectra see Figures 1 to 3 .

[0058] wherein the above amino acid reduction-ring-opening reaction condition screening results are as shown in Table 1.

[0059] Table 1: Amino acid reduction-ring-opening reaction condition screening table

[0060]

[0061]

[0062] Example 2

[0063] 13 C-C3-p-methoxymethylamphetamine 13 C-C3-PMMA) preparation method.

[0064] 13 C-C3-p-methoxymethylamphetamine (169 mg, 1 mmol) was dissolved in ethyl formate (10 mL), heated to 100 °C and heated for 2 hours. After cooling, the solvent was removed by rotary evaporation, 5 mL of ether was added for dissolution, and then the solution was added dropwise to a suspension of lithium aluminum hydride (30 mg, 0.8 mmol) in ether. After heating to reflux for 5 hours, it was cooled to 0 °C, and water was added dropwise. The insoluble matter was removed by diatomite filtration, and the diatomite was washed with 10 mL of ether, and the organic phases were combined, washed with 5% sodium hydroxide aqueous solution, and dried over magnesium sulfate. The drying agent was removed by filtration, and the solvent was removed by rotary evaporation. Purification by silica gel column chromatography (petroleum ether: ethyl acetate = 4:1) gave 13C-C3-p-methoxymethylamphetamine, colorless liquid (73 mg, 39%).

[0065] 1 H NMR (600 MHz, cd3od) δ 7.18 (dd, J = 8.5, 4.0 Hz, 2H), 6.93 - 6.88 (m, 2H), 3.77 (s, 3H), 3.56-3.48 (m, 0.5H), 3.30 - 3.24 (m, 0.5H), 3.21-3.17 (m, 0.5H), 2.99-2.91 (m, 0.5H), 2.84-2.76 (m, 0.5H), 2.71 (d, J = 3.6 Hz, 3H), 2.63-2.55 (m, 0.5H), 1.33 (dt, J = 6.6, 4.4 Hz, 1.5H), 1.11 (dt, J = 6.6, 4.4 Hz, 1.5H).

[0066] 13C NMR (151 MHz, cd3od) δ 160.56 (s), 131.51 (q), 128.74 (d), 115.42 (d), 57.99 (d), 55.76 (s), 39.42 (d), 30.97 (s), 15.72 (d). HR-MS (ESI / TOF) m / z: Calcd. for C10H12D4NO [M+H]+183.1489; Found: 183.1486. See Figures 4 to 6 .

[0067] Example 3

[0068] The contents of p-methoxyamphetamine and p-methoxymethylamphetamine in sewage were detected with C-C3-p-methoxyamphetamine 13 C-C3-p-methoxyamphetamine 13 C-C3-PMA) and 13 C-C3-p-methoxymethylamphetamine 13 C-C3-PMMA) as internal standard.

[0069] (1) Liquid chromatography-mass spectrometry detection conditions:

[0070] a) Instrument model: Shimadzu LCMS-8050;

[0071] b) Chromatographic column: Allure PFPP 2.1x 100mm 5um;

[0072] c) Column temperature: 40°C;

[0073] d) Mobile phase: A: acetonitrile, B: water (0.1% formic acid), gradient: 0 min 5% A, 7 min 90% A, 9 min 90% A, 9.1 min 5% A, 11 min 5% A;

[0074] e) Flow rate: 0.4 mL / min;

[0075] f) Injection volume: 2 μL;

[0076] g) Ion source: electrospray ion source, positive mode (ESI+);

[0077] h) Spray voltage 4000 V;

[0078] i) Ion source temperature: 300 °C;

[0079] j) Collision gas: nitrogen.

[0080] The ion pairs and corresponding conditions are shown in Table 2:

[0081] Table 2

[0082]

[0083] (2) Sample pretreatment

[0084] The sewage sample was filtered through filter paper, 100 mL was taken, 2 mL of methanol (containing 10 ng / ml 13 C-C3-PMA and 10 ng / ml 13 C-C3-PMMA) was added, 50 mL of each was loaded on the SPE column at a speed of 5 mL / min, after loading, 5 mL of methanol was used for elution, and finally 5 mL of 5% ammonia water-acetonitrile solution was used for elution, the eluent was dried under air flow in a 40 °C water bath, 80 μL of methanol was used for reconstitution, and then it was passed through a 0.22 μL filter membrane, and 2 μL was injected into the LC-MS / MS for analysis.

[0085] (3) Standard curve drawing

[0086] In 50 mL of negative sewage sample, 50 μL of methanol (containing 50 ng / ml 13 C-C3-PMA and 50 ng / ml 13 C-C3-PMMA) was added, 20 μL of PMA and PMMA standard control with concentrations of 1.25, 2.5, 5, 12.5, 25, 250 ng / ml was added, and sewage addition samples with concentrations of 0.5, 1.0, 2.0, 5.0, 10, 100 ng / L were prepared, each concentration was prepared in triplicate, vortexed for 3 min, and soaked at room temperature for 30 min, then treated according to the sample pretreatment process, and then detected by LC-MS / MS, and the PMA, 13The peak area ratio of C-C3-PMA and the concentration were used to make a standard curve, and a graph as shown in Figure 7 The standard curve formula is Y=(0.500306)X+(0.142091), wherein X is the concentration of the sample to be tested, Y is the peak area obtained by liquid chromatography-mass spectrometry detection, b0=0.142091 is the intercept of the standard curve, and b1=0.500306 is the slope of the standard curve.

[0087] The peak area ratio of PMMA and 13 The peak area ratio of C-C3-PMA and the concentration were used to make a standard curve, and a graph as shown in Figure 8 The peak area ratio of C-C3-PMA and the concentration were used to make a standard curve, and a graph as shown in Figures 9 to 12 The peak area ratio of C-C3-PMA and the concentration were used to make a standard curve, and a graph as shown in 13 The peak area ratio of C-C3-PMA and the concentration were used to make a standard curve, and a graph as shown in 13 The peak area ratio of C-C3-PMA and the concentration were used to make a standard curve, and a graph as shown in

[0088] Example 4

[0089] Comparison of performance of carbon 13 labeled substances and deuterium standard substances

[0090] Under the same detection conditions as in Example 3, the urban domestic sewage samples under different pH conditions and different concentrations were detected, and the results of the retention time coincidence rate and the addition recovery rate were compared as follows:

[0091] The retention time coincidence rate calculation formula is:

[0092] F%=(t1-t0) / t0*100%

[0093] Wherein F% is the retention time coincidence rate, t1 is the retention time of the internal standard, t0 is the retention time of the target, and the smaller the F% value, the more similar the chromatographic behavior of the internal standard to the target, and the better the performance of the internal standard. The retention time coincidence rate comparison results are shown in Table 3.

[0094] Table 3 Retention time coincidence rate comparison table

[0095] Target Internal standard Retention time concordance PMA 13 C-C3-PMA ​ 0.03％ PMA D4-PMA 0.71％ PMMA 13 C-C3-PMMA ​ 0.02％ PMMA D4-PMMA 0.70％

[0096] The addition recovery rate calculation formula is as follows:

[0097] E%=(A-B) / C*100%

[0098] Wherein A is the detection result after adding the standard, B is the detection result before adding the standard, C is the standard addition amount, E% is the recovery rate of the standard addition, and the value of E% closer to 100% indicates that the result of the detection system is more real, thereby indicating that the performance of the internal standard is better. The detection results of different internal standards under different conditions are shown in Table 4.

[0099] Table 4: Recovery rate of the detection results of different internal standards under different conditions

[0100]

[0101]

[0102] Acidic wastewater, alkaline wastewater, oxidizing wastewater and reducing wastewater: the matrix is configured by adding domestic sewage, wherein the acidic solution is adjusted to pH=2 by using hydrochloric acid, the strong alkaline is adjusted to pH=12 by using sodium hydroxide, the oxidizing solution is 0.1 mol / L sodium hypochlorite and peracetic acid, and the reducing solution is 0.1 mol / L sodium sulfite.

[0103] The isotopically labeled internal standard provided by the application can be added into the detection sample in an appropriate amount when the psychoactive substance PMMA in the detection sample is detected. 13 C-C3-PMMA, after appropriate pretreatment according to the detection requirements, liquid chromatography-mass spectrometry (LC-MS / MS) detection is performed, so that the detection sample can be detected. 13 C-C3-PMMA is the internal standard, in the multiple reaction monitoring (MRM) mode, by comparing the peak area ratio of the target substance and the to-be-detected substance, the qualitative and quantitative detection of the to-be-detected substance can be realized, the psychoactive substance that can be detected is PMMA, the specificity is strong, and the sensitivity is high.

[0104] In this specification, the application has been described with reference to its specific embodiments. However, it is obvious that various modifications and changes can be made without departing from the spirit and scope of the application. Therefore, the specification should be considered as illustrative rather than limiting.

Claims

1. A carbon 13 isotope labeled internal standard of PMMA and its metabolites, characterized in that, The chemical structural formula of the internal standard is shown as follows: Wherein, R is H or methyl.

2. A method of preparing a PMA carbon 13 isotope labeled internal standard, characterized by, The preparation method comprises the following steps: (1) 13 C-C3-Propionyl under transition metal catalysis 13 C-C3-Aziridine; (2) 13 C-C3-aziridine with p-methoxyphenyl nucleophile to produce PMA carbon 13 isotope-labeled internal standard 13 C-C3-p-methoxyphenylpropylamine.

3. The method for preparing the PMA carbon-13 isotope-labeled internal standard according to claim 2, characterized in that, In step (1), the transition metal catalyst is Ir(acac)(CO)2; In step (2), the reaction catalyst is CuBr or Cul; the reaction is stirred for 2 hours at temperature t2, and then the temperature is raised to t3 and the reaction is carried out at room temperature for T2 hours, t2 is -35℃-25℃, t3 is 0℃-25℃, and T2 is 1-4h.

4. A method of preparing a PMMA carbon 13 isotope labeled internal standard, characterized by, The preparation method comprises the following steps: Use of PMA carbon 13 isotope labeled internal standard 13 C-C3-p-methoxyphenylpropylamine is methylated to produce the PMA carbon 13 isotope labeled internal standard 13 C-C3-p-methoxymethylphenylpropylamine, PMA carbon 13 isotope labeled internal standard 13 C-C3-p-methoxyphenylpropylamine is produced by the method of claim 2 or 3.

5. Use of the carbon 13 isotope-labeled internal standard of PMMA and metabolites thereof in claim 1 or the PMMA carbon 13 isotope-labeled internal standard prepared by the preparation method in claims 2-3 in detecting the content of PMA in sewage.

6. Use of the carbon 13 isotope-labeled internal standard of PMMA and metabolites thereof in claim 1 or the PMMA carbon 13 isotope-labeled internal standard prepared by the preparation method in claim 4 in detecting the content of PMMA in sewage.

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