C13 isotope labeling internal standard substance of MAMP and metabolite of MAMP, preparation method and application of C13 isotope labeling internal standard substance
By preparing carbon-13 labeled MAMP and its metabolite internal standards, the problem of detection inaccuracy caused by the structural difference between the internal standard and the target substance in the existing technology was solved, realizing high sensitivity and high accuracy of MAMP detection in wastewater, and improving the scientific nature and legal effectiveness of drug control law enforcement.
Patent Information
- Application Number
- CN202511046468.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-29
- Publication Date
- 2025-11-07
AI Technical Summary
In existing technologies, the detection methods for MAMP and its metabolites suffer from insufficient isotope dilution due to the large structural differences between the internal standard and the target analyte, making it difficult to achieve stable quantification in complex biological matrices and affecting the accuracy and reliability of the detection, especially when the concentration of the target analyte in wastewater is extremely low.
Using carbon-13 labeled MAMP and its metabolite internal standards, azacyclopropane intermediates were constructed through a reductive cyclization reaction to ensure that the internal standards and target analytes had complete matching chemical properties, chromatographic retention times, and fragment ion spectra. Detection was performed using liquid chromatography-mass spectrometry.
It achieves highly sensitive detection of MAMP and its metabolites in wastewater, with a detection limit of 0.1 pg, a quantitation limit of ≤0.3 pg, a retention time consistency rate between internal standard and target analyte of less than 0.1%, and a spike recovery rate of 90-110%, thereby improving the accuracy of detection and the reliability of judicial evidence.
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Abstract
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 MAMP and its metabolites, a preparation method and application. BACKGROUND
[0002] In the trace analysis of new drugs such as methamphetamine (MAMP) and its metabolite amphetamine (AMP), matrix effect and insufficient internal standard matching 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 standard to correct the extraction efficiency and ionization interference of the target. However, the existing internal standard is mostly a chemical structure analog (such as D5-MAMP or D5-diazepam), which has a significant difference in molecular structure from the target, resulting in insufficient isotope dilution effect and difficulty in completely offsetting the interference in complex biological matrix (such as sewage, blood), affecting 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 resistance to matrix interference. This method uses isotope-labeled compounds as internal standards, and the physicochemical properties of the internal standard need to be highly consistent with the target analyte, with only a difference in molecular weight. In mass spectrometry analysis, the internal standard and the target have nearly the same ionization efficiency and chromatographic behavior, especially the retention time needs to be highly consistent with the target, which can correct the pretreatment loss and instrument fluctuation in real time, significantly improving the detection accuracy. Studies have shown that the structural homology of the internal standard and the target is the key to reducing matrix effect, and the current MAMP / AMP detection lacks high-quality specific isotope internal standards, and it is urgent to develop structure-matched labeled compounds.
[0004] Sewage has an irreplaceable advantage in drug detection practice as 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 the target in sewage is extremely low (usually in pg level), and there are interference factors such as pigment binding and exogenous pollution, which are strict in the sensitivity and specificity of the analysis method. The existing technology is difficult to achieve stable quantification of MAMP / AMP 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 MAMP and its metabolites, a preparation method and application.
[0006] To achieve the above object, the first aspect of the present application provides a carbon 13 isotope labeled internal standard of MAMP and its metabolite, which is mainly characterized by the chemical structural formula as shown in the following:
[0007]
[0008] wherein 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 AMP, which is mainly characterized by comprising 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 phenyl nucleophile to generate the carbon 13 isotope labeled internal standard of AMP 13 C-C3-phenylalanine.
[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 MAMP, which is mainly characterized by comprising the following steps:
[0015] The carbon 13 isotope labeled internal standard of AMP is used 13 C-C3-phenylalanine is subjected to a methylation reaction to generate the carbon 13 isotope labeled internal standard of MAMP 13 C-C3-methylphenylalanine, the carbon 13 isotope labeled internal standard of AMP 13 C-C3-phenylalanine 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 MAMP and its metabolite or the carbon 13 isotope labeled internal standard of AMP generated by the preparation method in detecting the content of AMP in sewage.
[0017] The fifth aspect of the present application provides the use of the carbon 13 isotope labeled internal standard of the MAMP and its metabolite or the MAMP carbon 13 isotope labeled internal standard prepared by the preparation method in detecting the content of MAMP in sewage.
[0018] In the above use, the method for detecting the content of MAMP and its metabolite 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 MAMP, C-C3-MAMP, AMP and C-C3-AMP standard solutions are added, after the same pretreatment steps as the sample to be detected, LC-MS / MS detection is carried out, and the peak area ratio and concentration of MAMP and C-C3-MAMP, AMP and C-C3-AMP are used to draw a standard curve; 13 13 C-C3-AMP standard solutions are added, after the same pretreatment steps as the sample to be detected, LC-MS / MS detection is carried out, and the peak area ratio and concentration of MAMP and C-C3-MAMP, AMP and C-C3-AMP are used to draw a standard curve; 13 13 C-C3-AMP standard solutions are added, after the same pretreatment steps as the sample to be detected, LC-MS / MS detection is carried out, and the peak area ratio and concentration of MAMP and C-C3-MAMP, AMP and C-C3-AMP 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 carried out, 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 method 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°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.
[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 method 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 MAMP and its metabolites in the biological material or the sewage is 10 -9 %-10%.
[0028] The carbon 13 isotope-labeled internal standard of MAMP 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 in 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 MAMP / AMP, the retention time is closer to the target than the deuterium internal standard, and the retention time consistency is more optimal; there is no isotope exchange effect of carbon 13, which is better than 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 MAMP and its metabolites can be applied to IDMS, and the following can be achieved:
[0030] Sensitivity is improved: the detection limit (LOD) is 0.1 pg (sewage matrix), and the quantitative limit (LOQ) is less than or equal to 0.3 pg;
[0031] Consistency is improved: the retention time consistency rate of the internal standard and the target is reduced to less than 0.1%;
[0032] Accuracy is optimized: the standard addition recovery rate (E%) is 90-110%, which is significantly better than 90-220% of the existing alternative internal standard;
[0033] The effectiveness of judicial evidence is strengthened: traceable data support conforming to the ISO / IEC 17025 standard is provided for drug suppression law enforcement and construction of a court evidence chain.
[0034] The application fills the technical gap of MAMP / AMP 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
[0035] Figure 1 For example 1 13 C-C3-MAMP high-resolution mass spectrum.
[0036] Figure 2 The H-NMR spectrum of C-C3-MAMP in Example 1 13 The H-NMR spectrum of C-C3-MAMP in Example 1 1 The H-NMR spectrum of C-C3-MAMP in Example 1
[0037] Figure 3 The H-NMR spectrum of C-C3-MAMP in Example 1 13 The H-NMR spectrum of C-C3-MAMP in Example 1 13 The H-NMR spectrum of C-C3-MAMP in Example 1
[0038] Figure 4 The H-NMR spectrum of C-C3-MAMP in Example 1 13 The H-NMR spectrum of C-C3-MAMP in Example 1
[0039] Figure 5 The H-NMR spectrum of C-C3-MAMP in Example 1 13 The H-NMR spectrum of C-C3-MAMP in Example 1 1 The H-NMR spectrum of C-C3-MAMP in Example 1
[0040] Figure 6 The H-NMR spectrum of C-C3-MAMP in Example 1 13 The H-NMR spectrum of C-C3-MAMP in Example 1 13 The H-NMR spectrum of C-C3-MAMP in Example 1
[0041] Figure 7 The H-NMR spectrum of C-C3-MAMP in Example 1
[0042] Figure 8 The H-NMR spectrum of C-C3-MAMP in Example 1
[0043] Figure 9 The H-NMR spectrum of C-C3-MAMP in Example 1 13 The H-NMR spectrum of C-C3-MAMP in Example 1
[0044] Figure 10 The H-NMR spectrum of C-C3-MAMP in Example 1 13 The H-NMR spectrum of C-C3-MAMP in Example 1
[0045] Figure 11 The H-NMR spectrum of C-C3-MAMP in Example 1
[0046] Figure 12 The H-NMR spectrum of C-C3-MAMP in Example 1 DETAILED DESCRIPTION
[0047] 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.
[0048] In the description of the present application, "matrix effect" refers to the influence and interference of substances other than the measured MAMP and its metabolites in the test material on the analysis process and detection results.
[0049] In the description of this invention, the mass concentration of MAMP and its metabolites in wastewater is 10. -9 %-10%.
[0050] The isotope-labeled internal standard provided by this invention 13 C-C3-Amphetamine and 13 The chemical structural formula of C-C3-methylamphetamine is as follows:
[0051]
[0052] The present invention will be further described below with reference to specific embodiments.
[0053] Example 1
[0054] Preparation by reduction-ring-opening reaction 13 C-C3-Amphetamine ( 13 Preparation method of C-C3-AMP.
[0055]
[0056] 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 phenyl magnesium bromide solution (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 ammonium chloride solution 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-MAMP), a colorless liquid, yields are shown in Table 1.
[0057] 1H NMR (600 MHz, cd3od) δ 7.35 (t, J = 7.5 Hz, 2H), 7.30 - 7.23 (m, 3H), 3.68-3.62 (m, 0.5H), 3.43 - 3.37 (m, 0.5H), 3.15-3.10 m, 0.5H), 2.95-2.87 (m, 1H), 2.72-2.67 (m, 0.5H), 1.36 (dt, J = 6.5, 4.4 Hz, 1.5H), 1.14 (dt, J = 6.5, 4.4 Hz, 1.5H).
[0058] 13 C NMR (151 MHz, cd3od) δ 135.97 (d), 128.96 (t), 128.56 (d), 126.95 (s), 48.88 (t), 40.35 (d), 16.86 (d).
[0059] HR-MS (ESI / TOF) m / z: Calcd. for C6 13 C3H 14 N[M+H] + 139.1227; Found: 139.1223. See Figure 1. Figures 1 to 3 .
[0060] wherein the above amino acid reduction-ring opening reaction condition screening results are as shown in Table 1.
[0061] Table 1: Amino acid reduction-ring opening reaction condition screening table
[0062]
[0063]
[0064] Example 2
[0065] 13 C-C3-methylamphetamine 13 (C-C3-MAMP) preparation method.
[0066] 13C-C3-phenylpropanol (139 mg, 1 mmol) was dissolved in ethyl formate (10 mL), heated to 100 °C for 2 hours. After cooling, the solvent was removed by rotary evaporation. The solution was then dissolved in 5 mL of diethyl ether and added dropwise to an ether suspension of lithium aluminum hydride (30 mg, 0.8 mmol). The mixture was heated to reflux for 5 hours, then cooled to 0 °C, and water was added dropwise. Insoluble matter was removed by diatomaceous earth filtration. The diatomaceous earth was washed with 10 mL of diethyl ether, and the organic phases were combined, washed with 5% sodium hydroxide aqueous solution, and dried over wastewater magnesium sulfate. The drying agent was removed by filtration, and the solvent was removed by rotary evaporation. The solution was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 4:1) to obtain... 13 C-C3-methylamphetamine, colorless liquid (76 mg, 55%).
[0067] 1 H NMR(600MHz,cd3od)δ7.35(dd,J=8.1,6.9Hz,2H),7.31–7.25(m,3H),3.62-3.56(m,0.5H),3.38–3.31(m,0.5H),3.29-3.21(m,0.5H),3.08-3 .00(m,0.5H),2.89-.2.82m,0.5H),2.72(d,J=3.6Hz,3H),2.65-2.60( m,0.5H),1.33(dt,J=6.7,4.3Hz,1.5H),1.12(dt,J=6.7,4.3Hz,1.5H).
[0068] 13 C NMR(151MHz,cd3od)δ137.14(d),130.47(t),130.05(d),128.48(s),57.82(t),40.24(d),30.97(s),15.77(d).
[0069] HR-MS(ESI / TOF)m / z:Calcd.for C7 13 C3H 16 N[M+H] + 153.1383; Found: 153.1379. See spectrum. Figures 4 to 6 .
[0070] Example 3
[0071] by 13 C-C3-Amphetamine ( 13 C-C3-AMP) and 13 C-C3-Methamphetamine ( 13 C-C3-MAMP) is used as an internal standard to detect the content of amphetamine and methamphetamine in wastewater.
[0072] (1) Liquid chromatography-mass spectrometry detection conditions:
[0073] a) Instrument model: Shimadzu LCMS-8050;
[0074] b) Column: Allure PFPP 2.1x 100mm 5um;
[0075] c) Column temperature: 40°C;
[0076] 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;
[0077] e) Flow rate: 0.4 mL / min;
[0078] f) Injection volume: 2 μL;
[0079] g) Ion source: electrospray ion source, positive mode (ESI+);
[0080] h) Spray voltage 4000V;
[0081] i) Ion source temperature: 300°C;
[0082] j) Collision gas: nitrogen.
[0083] Ion pairs and corresponding conditions are shown in Table 2:
[0084] Table 2
[0085]
[0086] (2) Sample pretreatment
[0087] The sewage sample was filtered through filter paper, 100 mL was taken, 2 mL of methanol (containing 10 ng / ml 13 C-C3-AMP and 10 ng / ml 13 C-C3-MAMP) was added, 50 mL 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 in a 40°C water bath under air flow, 80 μL of methanol was used for redissolution, and after passing through a 0.22 μL filter membrane, 2 μL was injected into the LC-MS / MS for analysis.
[0088] (3) Standard curve drawing
[0089] 50 μL of methanol (containing 50 ng / ml 13 C-C3-AMP and 50 ng / ml13 C-C3-MAMP), 20 μL of AMP and MAMP standard controls with concentrations of 1.25, 2.5, 5, 12.5, 25, 250 ng / ml were added, sewage addition samples with concentrations of 0.5, 1.0, 2.0, 5.0, 10, 100 ng / L were prepared in triplicate, vortexed for 3 min, soaked at room temperature for 30 min, and then treated according to the sample pretreatment process, and then detected by LC-MS / MS. The peak area ratios of AMP, 13 C-C3-AMP were plotted against concentrations to obtain a graph as shown in Figure 7 The standard curve formula was Y=(0.737142)X+(0.0873420), where X was the concentration of the sample to be tested, Y was the peak area obtained by liquid chromatography-mass spectrometry detection, b0=0.0873420 was the intercept of the standard curve, and b1=0.737142 was the slope of the standard curve.
[0090] The peak area ratios of MAP and 13 C-C3-MAMP were plotted against concentrations to obtain a graph as shown in Figure 8 Figures 9 to 12 The sample detection chromatograms of 13 C-C3-AMP, 13 C-C3-MAMP, D5-AMP, and D5-MAMP in sewage were obtained. The standard curve formula was Y=(1.11262)X+(0.711321), where X was the concentration of the sample to be tested, Y was the peak area obtained by liquid chromatography-mass spectrometry detection, b0=0.711321 was the intercept of the standard curve, and b1=1.11262 was the slope of the standard curve.
[0091] Example 4
[0092] Comparison of performance of carbon-13 labeled markers and deuterium labeled standard substances
[0093] The same detection conditions as in Example 3 were used to detect urban domestic sewage samples under different pH conditions and different concentrations, and the results of the retention time coincidence rate and the addition recovery rate were compared as follows:
[0094] The retention time coincidence rate calculation formula was:
[0095] F%=(t1-t0) / t0*100%
[0096] Where F% was the retention time coincidence rate, t1 was the retention time of the internal standard, t0 was 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, indicating that the performance of the internal standard was better. The comparison results of the retention time coincidence rate are shown in Table 3.
[0097] Table 3 retention time coincidence rate comparison table
[0098] Target Internal Standard Retention Time Concordance AMP 13 C-C3-AMP]]> 0.06％ AMP D4-AMP 1.22％ MAMP 13 C-C3-MAMP 0.02％ MAMP D4-MAMP 0.97％
[0099] The calculation formula of the recovery rate is as follows:
[0100] E% = (A-B) / C*100%
[0101] Wherein A is the detection result after spiking, B is the detection result before spiking, C is the spiking amount, E% is the spiking recovery rate, 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 spiking recovery rate of the detection results of different internal standards under different conditions is shown in Table 4.
[0102] Table 4 spiking recovery rate of the detection results of different internal standards under different conditions
[0103] Internal Standard D4-AMP 13 C-C3-AMP]]> D4-MAMP 13 C-C3-MAMP Target AMP AMP MAMP MAMP Acidic Wastewater 114％ 101％ 109％ 103％ Alkaline Wastewater 218％ 103％ 194％ 106％ Oxidizing Wastewater 184％ 106％ 137％ 103％ Reducing Wastewater 127％ 105％ 122％ 102％
[0104] Acidic wastewater, alkaline wastewater, oxidizing wastewater and reducing wastewater: the matrix is configured by adding domestic wastewater, 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.
[0105] The isotopically labeled internal standard provided by the application can be added into the test material in an appropriate amount when detecting the psychoactive substance MAMP in the test material. 13 C-C3-MAMP, after appropriate pretreatment according to the detection requirements, liquid chromatography-mass spectrometry (LC-MS / MS) detection is carried out, so that the detection of the test material is realized. 13 C-C3-MAMP is the internal standard, in the multiple reaction monitoring (MRM) mode, by comparing the peak area ratio of the target substance and the test substance, the qualitative and quantitative detection of the test substance is realized, the psychoactive substance that can be detected is MAMP, the specificity is strong, and the sensitivity is high.
[0106] 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 a MAMP and metabolites thereof, characterized in that, The chemical structural formula of the internal standard is shown as follows: Wherein, R is H or methyl.
2. A method for preparing an AMP 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 phenyl nucleophiles to generate AMP carbon 13 isotope-labeled internal standards 13 C-C3-phenylalanine.
3. The method of preparing an AMP carbon 13 isotope-labeled internal standard of claim 2, wherein, 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 MAMP carbon 13 isotope labeled internal standard, characterized by, The preparation method comprises the following steps: Use of AMP carbon 13 isotope labeled internal standard 13 C-C3-phenylpropylamine is methylated to produce the MAMP carbon 13 isotope labeled internal standard 13 C-C3-methylphenylpropylamine, AMP carbon 13 isotope labeled internal standard 13 C-C3-phenylpropylamine is produced by the method of claim 2 or 3.
5. Use of the carbon 13 isotope-labeled internal standard of the MAMP and metabolite thereof of claim 1 or the carbon 13 isotope-labeled internal standard of the AMP prepared by the preparation method of claims 2-3 in detecting the content of AMP in sewage.
6. Use of the carbon 13 isotope-labeled internal standard of the MAMP and metabolite thereof of claim 1 or the carbon 13 isotope-labeled internal standard of the MAMP prepared by the preparation method of claim 4 in detecting the content of MAMP in sewage.