A method for the quantitative determination of ethyl [2-(1H-indol-3-yl)ethyl]methylamine in a sample by means of nuclear magnetic resonance

Abstract

The present application relates to a kind of methods for quantitatively determining the content of ethyl [2-(1H-indol-3-yl)ethyl] methylamine in sample using nuclear magnetic resonance, the method comprises the following steps: to be measured sample is dissolved in deuterated solvent, to obtain sample solution;The internal standard is dissolved in deuterated solvent, to obtain internal standard solution;Add internal standard solution to the sample solution, to obtain detection solution;The detection solution is carried out 1 H-magnetic resonance detection, obtain 1 H-NMR spectrum;According to the 1 C in the H-NMR spectrum 13 H 18 The peak area ratio of the characteristic quantitative peak of N2 and the characteristic peak of internal standard, the content of ethyl [2-(1H-indol-3-yl)ethyl] methylamine in detection solution is calculated, the present application adopts quantitative nuclear magnetic resonance (qNMR) technology, realizes absolute quantification through the characteristic proton signal in ethyl [2-(1H-indol-3-yl)ethyl] methylamine molecule, does not need to rely on the control sample of measured substance, avoids the restriction brought by difficult or high cost of standard sample.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, specifically to a method for quantitatively determining the content of ethyl[2-(1H-indol-3-yl)ethyl]methylamine in a sample using nuclear magnetic resonance. Background Technology

[0002] Tryptamine derivatives are a class of organic compounds with important physiological activities, among which ethyl[2-(1H-indol-3-yl)ethyl]methylamine (molecular formula C) 13 H 18 Nitrosamine (N2), a typical tryptophan-based psychoactive substance, is classified as a controlled substance in most countries worldwide due to its central nervous system effects. The illegal abuse and circulation of this substance pose a serious threat to public health and social security. Simultaneously, it also holds significant research value in neuropharmacology, forensic toxicology analysis, and clinical poisoning diagnosis. Therefore, establishing efficient, sensitive, and accurate detection methods is both a pressing practical need and of scientific importance.

[0003] Currently, the detection techniques for tryptamines mainly rely on traditional methods such as chromatography-mass spectrometry (GC-MS, LC-MS / MS), high-performance liquid chromatography (HPLC), and immunoassay. Among these, GC-MS is considered the "gold standard" due to its high sensitivity and specificity, but it has limitations such as expensive equipment, complex pretreatment procedures, long detection cycles, and difficulty in achieving rapid on-site detection. While immunoassay is simple to operate and fast, it is susceptible to cross-reaction interference, lacks specificity, and is difficult to perform simultaneous quantitative analysis of multiple components.

[0004] For isomers like ethyl[2-(1H-indol-3-yl)ethyl]methylamine, which have structures highly similar to dimethyltryptamine (DMT) and diethyltryptamine (DET), existing detection technologies still have significant shortcomings. On the one hand, conventional chromatographic separation conditions are insufficient to achieve effective baseline separation from structural analogs, easily leading to qualitative misjudgments. On the other hand, in complex matrices (such as biological fluids, environmental samples, and seized drug mixtures), matrix inhibition effects significantly reduce detection sensitivity, making it difficult to meet the needs of trace detection.

[0005] In recent years, quantitative nuclear magnetic resonance (qNMR) technology has demonstrated unique advantages in the quantitative analysis of small molecule compounds. It can achieve absolute quantification without relying on reference standards, and its pretreatment steps are simple and it is highly tolerant to complex matrices. However, there are currently no reports on the specific application of quantitative NMR technology to the determination of ethyl[2-(1H-indol-3-yl)ethyl]methylamine, especially when the sample may contain common dopants such as propylene glycol and glycerol. How to achieve rapid, accurate, and interference-resistant quantification of ethyl[2-(1H-indol-3-yl)ethyl]methylamine using qNMR remains a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] Therefore, it is necessary to provide a method for quantitatively determining the content of ethyl[2-(1H-indol-3-yl)ethyl]methylamine in a sample using nuclear magnetic resonance, in order to solve the problems existing in the prior art.

[0007] To achieve the above objectives, the present invention provides a technical solution: A method for quantitatively determining the content of ethyl[2-(1H-indol-3-yl)ethyl]methylamine in a sample using nuclear magnetic resonance, characterized by comprising the following steps: Dissolve the sample to be tested in a deuterated solvent to obtain a sample solution; The internal standard was dissolved in a deuterated solvent to obtain an internal standard solution; An internal standard solution is added to the sample solution to obtain a detection solution; The detection solution was subjected to 1 H-NMR detection, to obtain 1 H-NMR spectrum; According to the above 1 The content of ethyl[2-(1H-indol-3-yl)ethyl]methylamine in the detection solution was calculated by comparing the peak area ratio of the characteristic quantitative peak of ethyl[2-(1H-indol-3-yl)ethyl]methylamine with that of the internal standard in the 1H-NMR spectrum.

[0008] Preferably, the deuterated solvent is deuterated dimethyl sulfoxide.

[0009] Preferably, the internal standard is dimethyl terephthalate.

[0010] Preferably, the 1 In the H-NMR spectrum, the peak at chemical shift δH 2.23 ppm was used as the quantitative peak of ethyl[2-(1H-indol-3-yl)ethyl]methylamine, and the peak at chemical shift δH 8.08 ppm was used as the internal standard peak.

[0011] Preferably, the sample to be tested is a mixture containing ethyl[2-(1H-indol-3-yl)ethyl]methylamine, wherein the mixture contains propylene glycol and / or glycerol as dopants.

[0012] Preferably, the mass ratio of the internal standard to the sample to be tested is 1:6.

[0013] Preferably, the 1 The testing conditions for H-NMR detection include: The zg30 pulse sequence was used, with a spectral width of 11904.8 Hz, a relaxation time of 1 s, a pulse width of 14.90 μs, and 32 scans.

[0014] Preferably, in the step of calculating the content of ethyl[2-(1H-indol-3-yl)ethyl]methylamine in the detection solution based on the peak area ratio of the quantitative peak to the internal standard peak, the specific steps include: The peak area ratio of the quantitative peak to the internal standard peak is denoted as Y, and the mass ratio of ethyl[2-(1H-indol-3-yl)ethyl]methylamine to the internal standard in the detection solution is denoted as X. Substituting these values ​​into the pre-established linear regression equation Y = 0.5595X - 0.0043, X is calculated, and thus the content of ethyl[2-(1H-indol-3-yl)ethyl]methylamine is obtained.

[0015] Preferably, the linear regression equation is established in advance through the following steps: A standard solution of ethyl[2-(1H-indol-3-yl)ethyl]methylamine and a standard solution of dimethyl terephthalate (internal standard) were prepared using deuterated dimethyl sulfoxide. Two standard solutions were mixed in different proportions to prepare a series of working solutions with different mass ratios of ethyl[2-(1H-indol-3-yl)ethyl]methylamine and internal standard. The series of working solutions were respectively subjected to 1 H-NMR detection was used to obtain the results for each working solution. 1 H-NMR spectra, record the peak area ratio of the quantitative peak at δH 2.23 ppm to the internal standard peak at δH 8.08 ppm in each spectrum; A linear regression was performed with the mass ratio as the x-axis and the peak area ratio as the y-axis to obtain the linear regression equation.

[0016] Preferably, the sample to be tested is a tryptamine-based psychoactive substance sample in the form of an oil or lubricant.

[0017] Preferably, the ethyl[2-(1H-indol-3-yl)ethyl]methylamine has the molecular formula C 13 H 18 N2.

[0018] The beneficial effects of this invention are: 1. Quantitative analysis without the need for standards: This invention uses quantitative nuclear magnetic resonance (qNMR) technology to achieve absolute quantification through the characteristic proton signal in the ethyl[2-(1H-indol-3-yl)ethyl]methylamine molecule, without relying on the reference standard of the analyte, thus avoiding the limitations caused by the difficulty or high cost of obtaining standards.

[0019] 2. High specificity and good anti-interference ability: This invention targets common dopants (propylene glycol, glycerol) in actual samples of ethyl[2-(1H-indol-3-yl)ethyl]methylamine, and compares them with... 1 The H-NMR spectrum was used to select the non-interfering characteristic peak at δH 2.23 ppm as the quantitative peak, which effectively avoided cross-reaction and matrix inhibition effects. It can accurately distinguish MET from its isomers (such as DMT and DET) and solve the problem of baseline separation in chromatography.

[0020] 3. Simple pretreatment and rapid detection: This invention does not require complex derivatization or cumbersome chromatographic separation conditions. After the sample is dissolved, an internal standard is added and the sample can be detected on the instrument. The detection cycle is short and high-throughput analysis can be achieved.

[0021] 4. Low cost and easy operation: This invention does not require expensive consumables such as chromatographic columns and mass spectrometry ion sources, has low instrument maintenance costs, standardized testing conditions, and is easy to promote.

[0022] 5. High accuracy and good reliability: After comparison with HPLC, there was no statistically significant difference in the results of the two methods (p>0.05). The relative error of the method of this invention is between -4.01% and +3.23%, with an average relative error of only 2.92%, which shows good accuracy. Attached Figure Description

[0023] Figure 1 The spectrum is 1H-qNMR. Figure 2 To obtain the linear regression equation graph; Figure 3 For Y1 1 HNMR spectrum; Figure 4 For Y2 1 HNMR spectrum; Figure 5 For Y3 1 HNMR spectrum. Detailed Implementation

[0024] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0025] In the embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified. Example 1

[0026] Ethyl[2-(1H-indol-3-yl)ethyl]methylamine, dimethyl terephthalate, propylene glycol, and glycerol were dissolved separately in deuterated dimethyl sulfoxide (DMSO-d6) for further processing. 1 H-NMR detection. Detection conditions: ZG30 pulse sequence, spectral width 11904.8 Hz, relaxation time 1 s, pulse width 14.90 μs, 32 scans. Data processing was performed using MestReNova software. 1 H-qNMR spectrum as shown Figure 1 As shown. Depend on Figure 1 It can be seen that dimethyl terephthalate has a clear single peak at δH 8.08 ppm, and it does not overlap with the peaks of MET and other dopants. Therefore, δH 8.08 ppm was selected as the internal standard peak.

[0027] The MET signal peak at δH 2.23 ppm did not interfere with the peaks of propylene glycol, glycerol, and the internal standard. Therefore, δH 2.23 ppm was selected as the quantitative peak for MET.

[0028] Example 2: Establishing a Linear Relationship (1) Solution preparation: A 10 mg / mL MET solution and a 10 mg / mL dimethyl terephthalate internal standard solution were prepared using DMSO-d6.

[0029] Mix according to the proportions shown in Table 1 to prepare a series of working solutions with MET concentrations of 0.05 mg / mL, 0.1 mg / mL, 0.26 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, and 8 mg / mL. The internal standard solution was added to each working solution in a volume of 50 μL, and the total volume was made up to 500 μL with DMSO-d6.

[0030] Table 1: Preparation of a series of working solutions (2) Detection and regression equation: The above series of working solutions were transferred into 5 mm NMR tubes, and then processed according to Example 1. 1Detection was performed under 1H-NMR conditions. The peak area ratio Y of δH 2.23 ppm (quantitative peak of ethyl[2-(1H-indol-3-yl)ethyl]methylamine) and δH 8.08 ppm (internal standard peak) in each spectrum was recorded. Linear regression was performed with the mass ratio X of MET to internal standard as the x-axis and the peak area ratio Y as the y-axis. The linear regression equation is as follows: Figure 2 As shown Depend on Figure 2 We can obtain: Y = 0.5595X - 0.0043, correlation coefficient R. 2 = 0.9991, which indicates that MET has a good linear relationship in the concentration range of 0.05 to 8 mg / mL.

[0031] Example 3: Method accuracy verification (compared with HPLC) (1) Sample preparation: Prepare 10 mg / mL solutions of ethyl[2-(1H-indol-3-yl)ethyl]methylamine, propylene glycol, glycerol, and internal standard (dimethyl terephthalate) using DMSO-d6. Prepare three simulated samples according to the following formulations: Sample 1: Take 300 μL of ethyl[2-(1H-indol-3-yl)ethyl]methylamine solution + 300 μL of propylene glycol solution, and mix well; Sample 2: Take 300 μL of ethyl[2-(1H-indol-3-yl)ethyl]methylamine solution + 300 μL of glycerol solution, and mix well; Sample 3: Take 200 μL of ethyl[2-(1H-indol-3-yl)ethyl]methylamine solution + 200 μL of propylene glycol solution + 200 μL of glycerol solution, and mix well.

[0032] Two replicates were prepared for each sample; one was used for HPLC determination, and the other was prepared by adding 100 μL of internal standard solution. 1 Determined by H-qNMR method.

[0033] (2) HPLC determination conditions: An Agilent 1260 high-performance liquid chromatograph with a C18 column was used. Mobile phase A was chromatographic acetonitrile, and mobile phase B was H3PO4-triethylamine buffer solution (4.12 mL of concentrated H3PO4 was diluted with water to 200 mL, 5.56 mL of triethylamine was added, and then water was added to bring the volume to 1000 mL. The solution was filtered through a 0.45 μm filter membrane and degassed by sonication). Isocratic elution, 20% A; Flow rate: 1 mL / min; Injection volume: 5 μL; Detection wavelength: 266 nm; Running time: 15 min.

[0034] (3) Comparison of results: use 1 The content of ethyl[2-(1H-indol-3-yl)ethyl]methylamine in the three samples was determined by 1H-qNMR and HPLC respectively. The relative error was calculated with the theoretical preparation value as the standard. The results are shown in Table 2.

[0035] Table 2 Comparison of qNMR and HPLC results for ethyl[2-(1H-indol-3-yl)ethyl]methylamine As shown in Table 2, paired t-tests of the measurement results from the two methods revealed no statistically significant difference (p>0.05), indicating that the method established in this invention... 1 The H-qNMR quantitative method has good accuracy and high consistency with conventional HPLC methods.

[0036] Example 4: Measurement of actual samples (1) Preparation of internal standard solution: Dissolve dimethyl terephthalate in DMSO-d6 to prepare a 10 mg / mL internal standard solution.

[0037] (2) Sample solution preparation: Take three actual samples to be tested, numbered Y1, Y2 and Y3, and dissolve them in DMSO-d6 to prepare sample solutions.

[0038] (3) Preparation of detection solution: Add 100 μL of internal standard solution to each of the above sample solutions Y1, Y2 and Y3, mix well to obtain the detection solution.

[0039] (4) 1 H-qNMR determination: Transfer the entire detection solution to a 5 mm NMR tube, and proceed as described in Example 1. 1 Detection was performed under H-NMR conditions to obtain 1 H-NMR spectrum; Y1 1 H-NMR spectrum as shown Figure 3 As shown; Y2 1 H-NMR spectrum as shown Figure 4 As shown; Y3 1 H-NMR spectrum as shown Figure 5 As shown.

[0040] (5) Content calculation: Read the peak area ratio Y of the quantitative peak (δH 2.23 ppm) and the internal standard peak (δH 8.08 ppm) of ethyl[2-(1H-indol-3-yl)ethyl]methylamine from the spectrum.

[0041] The Y-values ​​for samples Y1, Y2, and Y3 were 1.7019, 1.7284, and 1.0699, respectively. The added internal standard mass was 1 mg for each sample.

[0042] Substituting the Y value into the linear regression equation Y = 0.5595X - 0.0043, the mass ratio X of ethyl[2-(1H-indol-3-yl)ethyl]methylamine to the internal standard was calculated, and then the content of ethyl[2-(1H-indol-3-yl)ethyl]methylamine was calculated: Sample Y1: X = (1.7019 + 0.0043) / 0.5595 = 3.0459, MET content = 3.0459 mg; Sample Y2: X = (1.7284 + 0.0043) / 0.5595 = 3.0968, MET content = 3.0968 mg; Sample Y3: X = (1.0699 + 0.0043) / 0.5595 = 1.9199, MET content = 1.9199 mg.

[0043] It should be noted that the specific parameters or reagents in the above embodiments are specific or preferred embodiments under the concept of the present invention, and not limitations thereof; those skilled in the art can make adaptive adjustments within the concept and protection scope of the present invention.

Claims

1. A method for quantitatively determining the content of ethyl[2-(1H-indol-3-yl)ethyl]methylamine in a sample using nuclear magnetic resonance, characterized in that, Includes the following steps: Dissolve the sample to be tested in a deuterated solvent to obtain a sample solution; The internal standard was dissolved in a deuterated solvent to obtain an internal standard solution; An internal standard solution is added to the sample solution to obtain a detection solution; The detection solution was subjected to 1 H-NMR detection, to obtain 1 H-NMR spectrum; According to the above 1 The content of ethyl[2-(1H-indol-3-yl)ethyl]methylamine in the detection solution was calculated by comparing the peak area ratio of the characteristic quantitative peak of ethyl[2-(1H-indol-3-yl)ethyl]methylamine with that of the internal standard in the 1H-NMR spectrum.

2. The method according to claim 1, characterized in that, The deuterated solvent is deuterated dimethyl sulfoxide.

3. The method according to claim 1, characterized in that, The internal standard is dimethyl terephthalate.

4. The method according to claim 1, characterized in that, The 1 In the H-NMR spectrum, the peak at chemical shift δH 2.23 ppm was used as the quantitative peak of ethyl[2-(1H-indol-3-yl)ethyl]methylamine, and the peak at chemical shift δH 8.08 ppm was used as the internal standard peak.

5. The method according to claim 1, characterized in that, The sample to be tested is a mixture containing ethyl[2-(1H-indol-3-yl)ethyl]methylamine, wherein the mixture contains propylene glycol and / or glycerol as dopants.

6. The method according to claim 1, characterized in that, The mass ratio of the internal standard to the sample to be tested is 1:

6.

7. The method according to claim 1, characterized in that, The 1 The testing conditions for H-NMR detection include: The zg30 pulse sequence was used, with a spectral width of 11904.8 Hz, a relaxation time of 1 s, a pulse width of 14.90 μs, and 32 scans.

8. The method according to claim 1, characterized in that, The specific steps in calculating the content of ethyl[2-(1H-indol-3-yl)ethyl]methylamine in the detection solution based on the peak area ratio of the quantitative peak to the internal standard peak include: The peak area ratio of the quantitative peak to the internal standard peak is denoted as Y, and the mass ratio of ethyl[2-(1H-indol-3-yl)ethyl]methylamine to the internal standard in the detection solution is denoted as X. Substituting these values ​​into the pre-established linear regression equation Y = 0.5595X - 0.0043, X is calculated, and thus the content of ethyl[2-(1H-indol-3-yl)ethyl]methylamine is obtained.

9. The method according to claim 7, characterized in that, The linear regression equation is established in advance through the following steps: A standard solution of ethyl[2-(1H-indol-3-yl)ethyl]methylamine and a standard solution of dimethyl terephthalate (internal standard) were prepared using deuterated dimethyl sulfoxide. Two standard solutions were mixed in different proportions to prepare a series of working solutions with different mass ratios of ethyl[2-(1H-indol-3-yl)ethyl]methylamine and internal standard. The series of working solutions were respectively subjected to 1 H-NMR detection was used to obtain the results for each working solution. 1 H-NMR spectra, record the peak area ratio of the quantitative peak at δH 2.23 ppm to the internal standard peak at δH 8.08 ppm in each spectrum; A linear regression was performed with the mass ratio as the x-axis and the peak area ratio as the y-axis to obtain the linear regression equation.

10. The method according to claim 1, characterized in that, The sample to be tested is a tryptamine-based psychoactive substance in oil or lubricant form.

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