Method for detecting benzene ring standing substance by using two-dimensional nuclear magnetic resonance

Abstract

The invention belongs to the technical field of detection and analysis of novel drugs, and particularly relates to a method for detecting benzene ring standing substances by using two-dimensional nuclear magnetic resonance. According to the 1H-13C HSQC 2D NMR method provided by the invention, related parameters of an aromatic ring group selective pulse sequence and a carbonyl group selective pulse sequence are set, and two key structures, namely an aromatic ring structure and a cyclohexanone structure, of PCP can be specifically detected by the method through the two selective pulse sequences; therefore, the existence of PCP can be confirmed without relying on a complete chemical structure, and quantitative analysis is carried out. Besides, the method can realize qualitative and quantitative analysis of PCP and compounds with similar structures in complex matrixes, such as beverages, tobacco tar and the like, so as to carry out nondestructive and in-situ detection on related PCP drugs in food. In addition, the method has the function of early warning novel drugs with similar chemical structures.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection and analysis of new drugs, and specifically relates to a method for detecting phenylcyclidine-type substances using two-dimensional nuclear magnetic resonance. Background Art

[0002] New toxic substances (NPS) refer to synthetic hallucinogens and stimulants that act directly on the central nervous system and can lead to dependence with long-term use. Phencyclidine-like substances (PCP), a type of NPS, have become prevalent in the market in recent years, often covertly added to beverages, snacks, or e-cigarettes, posing a serious threat to public health. Currently, commonly used techniques for detecting NPS include high-resolution mass spectrometry (HR-MS), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS). However, mass spectrometry methods rely on databases of known compounds, and some newly synthesized substances are not yet included in these databases, making targeted detection methods ineffective. Furthermore, while GC-MS and LC-MS offer high sensitivity, they typically require complex pretreatment steps and are primarily used for biological sample testing. However, for PCP detection, the amount of PCP added is relatively large, requiring less sensitive samples. Furthermore, the wide variety of matrices makes pretreatment methods specific to each matrix time-consuming and labor-intensive, making existing detection methods unsuitable.

[0003] In contrast, nuclear magnetic resonance (NMR) technology can effectively perform in situ analysis, facilitating the identification and quantification of PCPs in mixtures. 1 H-NMR can detect NPS, but when detecting complex mixtures, not only does it require a pretreatment step, but the obtained one-dimensional spectrum signals also have serious overlap, making it impossible to specifically detect PCP. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for detecting phenylcyclidine-type substances using two-dimensional nuclear magnetic resonance. The method provided by the present invention can specifically detect the two key structures of PCP without requiring any pretreatment steps, thereby confirming the presence of PCP without relying on a complete chemical structure.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for detecting phenylcyclidine-type substances using two-dimensional nuclear magnetic resonance, comprising the following steps:

[0007] Mixing the sample to be tested, the internal standard solution and the phosphate buffer to obtain a test solution;

[0008] The test liquid is subjected to 1 H- 13C HSQC 2D NMR detection provides aromatic ring selective spectra and carbonyl selective spectra of phenylcyclidines;

[0009] described 1 H- 13 The selective pulse sequences for C HSQC 2D NMR detection include aromatic ring group selective pulse sequence and carbonyl group selective pulse sequence;

[0010] The aromatic ring group selective pulse sequence is a band-selective shaped pulse "Q3" with the F2 dimension (H spectrum) centered at 4.67 ppm, the F1 dimension (C spectrum) centered at 130 ppm, the J coupling parameter at 145 Hz, and the spectral width at 60 ppm.

[0011] The carbonyl-selective pulse sequence was a band-selective shaped pulse "Q5" with the F1 dimension (H spectrum) centered at 4.67 ppm, the F2 dimension (C spectrum) centered at 205 ppm, a J-coupling parameter of 10 Hz, and a spectral width of 30 ppm;

[0012] The phenylcyclidine substances are qualitatively identified by comparing the aromatic ring selectivity spectrum and the carbonyl selectivity spectrum of the phenylcyclidine substances with the spectra of predetermined standard substances of the phenylcyclidine substances.

[0013] Preferably, the phenylcyclidine-type substance includes one or more of fluamine, 2-(ethylamino)-2-(3-methoxyphenyl)cyclohexane-1-one, 2-amino-2-(2-fluorophenyl)cyclohexane-1-one, methylamine, ethylnorketamine, ethylnorfluamine, norketamine, bromamine, 2-(ethylamino)-2-(3-methoxyphenyl)-1-cyclohexanone, 2-(ethylamino)-2-(3-methylphenyl)-cyclohexane-1-one, ketamine and tiletamine.

[0014] Preferably, the band selective shape pulse "Q3" is a 180° pulse in the y direction of the C core in the polarization transfer stage.

[0015] Preferably, in the aromatic ring group selective pulse sequence, the recirculation waiting time is 1.5s, τ is 1.42ms; the number of scans is 8, the number of F1-dimensional sampling points is 64, and the acquisition time is 3ms.

[0016] Preferably, in the carbonyl-selective pulse sequence, the recirculation waiting time is 1 s, τ is 24.7 ms, the number of scans is 64, the number of F1-dimensional sampling points is 32, and the acquisition time is 3 ms.

[0017] Preferably, the internal standard solution is a deuterium aqueous solution of 4,4-dimethyl-4-silapentane-1-sulfonic acid.

[0018] Preferably, the concentration of the deuterium aqueous solution of 4,4-dimethyl-4-silapentane-1-sulfonic acid is 2 mg / mL.

[0019] The present invention provides a method for quantitatively analyzing phenylcyclidines using two-dimensional nuclear magnetic resonance, comprising the following steps:

[0020] Mixing the sample to be tested, the internal standard solution and the phosphate buffer to obtain a test solution;

[0021] The test liquid is subjected to 1 H- 13 C HSQC 2D NMR detection provides aromatic ring selective spectra and carbonyl selective spectra of phenylcyclidines;

[0022] described 1 H- 13 The selective pulse sequences for C HSQC 2D NMR detection include aromatic ring group selective pulse sequence and carbonyl group selective pulse sequence;

[0023] The aromatic ring group selective pulse sequence is a band-selective shaped pulse "Q3" with the F2 dimension (H spectrum) centered at 4.67 ppm, the F1 dimension (C spectrum) centered at 130 ppm, the J coupling parameter at 145 Hz, and the spectral width at 60 ppm.

[0024] The carbonyl-selective pulse sequence was a band-selective shaped pulse "Q5" with the F1 dimension (H spectrum) centered at 4.67 ppm, the F2 dimension (C spectrum) centered at 205 ppm, a J-coupling parameter of 10 Hz, and a spectral width of 30 ppm;

[0025] quantifying the phenylcyclidines based on the characteristic peak areas of the phenylcyclidines in the aromatic ring selectivity spectrum or the carbonyl selectivity spectrum of each phenylcyclidine and a predetermined standard curve;

[0026] The predetermined standard curve is a linear relationship between the concentration of phenylcyclidine in the sample to be tested and the characteristic peak area of phenylcyclidine

[0027] The present invention provides a method for detecting phenylcyclidine-type substances by using two-dimensional nuclear magnetic resonance, comprising the following steps: mixing a sample to be tested, an internal standard solution and a phosphate buffer solution to obtain a test solution; 1 H- 13 CHSQC 2D NMR detection, to obtain aromatic ring selective spectrum and carbonyl selective spectrum of phenylcyclidine substances; 1 H- 13The selective pulse sequences for C HSQC 2DNMR detection include an aromatic ring-selective pulse sequence and a carbonyl-selective pulse sequence; the aromatic ring-selective pulse sequence is a band-selective shaped pulse "Q3" with an F2-dimension (H spectrum) center of 4.67 ppm, an F1-dimension (C spectrum) center of 130 ppm, a J coupling parameter of 145 Hz, and a spectral width of 60 ppm; the carbonyl-selective pulse sequence is a band-selective shaped pulse "Q5" with an F1-dimension (H spectrum) center of 4.67 ppm, an F2-dimension (C spectrum) center of 205 ppm, a J coupling parameter of 10 Hz, and a spectral width of 30 ppm; the phenylcyclidines are qualitatively identified by comparing their aromatic ring-selective and carbonyl-selective spectra with the spectra of predetermined standard samples of the phenylcyclidines. The main structure of PCP is composed of a benzene ring (or thiazole ring) and cyclohexanone. These two parts have a great influence on toxicity and are easy to change. The selective pulse sequence set by the present invention only detects the signals related to C and H on the aromatic ring and cyclohexanone, filtering out the interference of other structures. At the same time, since the carbonyl carbon atom has no directly connected H, the pulse sequence uses the H on the adjacent C to interact with the carbonyl C. 2 J CH These two selective pulse sequences enable the specific detection of two key structures of PCP: the aromatic ring structure and the cyclohexanone structure, thus confirming the presence of PCP without relying on a complete chemical structure.

[0028] The results of the examples show that the present invention provides 1 H- 13 The C HSQC 2D NMR method can directly detect PCP in matrices such as beverages, chocolate, and e-liquids. Moreover, this method can not only quickly determine whether it belongs to the PCP-type drug through the target chemical characteristics, but also effectively identify the specific structure of new drugs by combining the mutual verification of carbonyl and aromatic ring selective spectra. In addition, this method shows good early warning capabilities when dealing with unknown compounds, providing a scientific basis for quickly locking potential drugs with similar structures. This study further confirms the reliability and sensitivity of selective nuclear magnetic resonance detection, provides strong technical support for the identification of PCP-type drugs and their derivatives in complex matrices, and provides an important reference for the dynamic monitoring and control of new drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 This is a schematic diagram of the structural formula of pure PCP;

[0031] Figure 2 It is a comparison of the one-dimensional proton spectrum of tiletamine and its Red Bull matrix;

[0032] Figure 3 For the standard of levitra 1 H- 13 C HSQC full spectrum;

[0033] Figure 4 It is an aromatic ring and carbonyl selective HSQC pulse sequence;

[0034] Figure 5 This is a spectral comparison of fluamine standard and Red Bull matrix;

[0035] Figure 6 Comparison of chromatograms of bromamine, fluamine and ketamine standards and bromamine, fluamine and ketamine in matrix;

[0036] Figure 7 Comparison of the spectra of fluamine, bromamine, and ketamine in Red Bull, chocolate, and e-liquid matrices;

[0037] Figure 8 Linear regression model for the quantitative analysis of tiletamine by hydrogen solution NMR spectroscopy;

[0038] Figure 9 Linear regression model for the quantitative analysis of bromamine by hydrogen solution NMR spectroscopy;

[0039] Figure 10 Linear regression model for the quantitative analysis of ketamine by hydrogen solution NMR spectroscopy;

[0040] Figure 11 Linear regression model for the quantitative analysis of fluamine by hydrogen solution NMR spectroscopy;

[0041] Figure 12 Comparison of the spectra of methamphetamine standard and methamphetamine in tea matrix. DETAILED DESCRIPTION

[0042] The present invention provides a method for detecting phenylcyclidine-type substances using two-dimensional nuclear magnetic resonance, comprising the following steps:

[0043] Mixing the sample to be tested, the internal standard solution and the phosphate buffer to obtain a test solution;

[0044] The test liquid is subjected to 1 H- 13 C HSQC 2D NMR detection provides aromatic ring selective spectra and carbonyl selective spectra of phenylcyclidines;

[0045] described 1 H- 13 Selective pulse sequences for C HSQC 2D NMR detection include aromatic ring-selective pulse sequences and carbonyl-selective pulse sequences:

[0046] The aromatic ring group selective pulse sequence is a band-selective shaped pulse "Q3" with the F2 dimension (H spectrum) centered at 4.67 ppm, the F1 dimension (C spectrum) centered at 130 ppm, the J coupling parameter at 145 Hz, and the spectral width at 60 ppm.

[0047] The carbonyl-selective pulse sequence was a band-selective shaped pulse "Q5" with the F1 dimension (H spectrum) centered at 4.67 ppm, the F2 dimension (C spectrum) centered at 205 ppm, a J-coupling parameter of 10 Hz, and a spectral width of 30 ppm;

[0048] The phenylcyclidine substances are qualitatively identified by comparing the aromatic ring selectivity spectrum and the carbonyl selectivity spectrum of the phenylcyclidine substances with the spectra of predetermined standard substances of the phenylcyclidine substances.

[0049] As an embodiment of the present invention, the phenylcyclidine-type substances include one or more of fluamine, 2-(ethylamino)-2-(3-methoxyphenyl)cyclohexane-1-one, 2-amino-2-(2-fluorophenyl)cyclohexane-1-one, methylamine, ethylnorketamine, ethylnorfluamine, norketamine, bromamine, 2-(ethylamino)-2-(3-methoxyphenyl)-1-cyclohexanone, 2-(ethylamino)-2-(3-methylphenyl)-cyclohexane-1-one, ketamine and tiletamine.

[0050] Figure 4 is an aromatic ring and carbonyl selective HSQC pulse sequence, where Figure 4 A in the middle upper part is the aromatic ring selective pulse sequence, and B is the carbonyl selective pulse sequence.

[0051] As an embodiment of the present invention, in the aromatic ring group selective pulse sequence, the recirculation waiting time is 1.5s, τ is 1.42ms; the number of scans is 8, the number of F1-dimensional sampling points is 64, and the acquisition time is 3ms.

[0052] As an embodiment of the present invention, in the carbonyl-selective pulse sequence, the recirculation waiting time is 1 s, τ is 24.7 ms, the number of scans is 64, the number of F1-dimensional sampling points is 32, and the acquisition time is 3 ms.

[0053] Since the carbonyl C in cyclohexanone is not directly connected to H, the present invention uses a carbonyl selective pulse sequence to excite the H on the adjacent C, and uses the reaction of the proton with the carbonyl C to generate a 2 J CHCoupling is used to indirectly detect carbonyl C atoms. At the same time, the 13C band-selective pulse is used to filter the signals of other 13C nuclei to avoid interference from matrix signals.

[0054] As an embodiment of the present invention, 1 H- 13 After the C HSQC 2D NMR detection, the obtained nuclear magnetic resonance spectrum is preprocessed and baseline corrected. The baseline correction can be performed manually in stages on the nuclear magnetic resonance spectrum.

[0055] In an embodiment of the present invention, the pre-processing and baseline correction software is specifically TopSpin4.3.0.

[0056] The present invention also provides a method for quantitatively analyzing phenylcyclidines using two-dimensional nuclear magnetic resonance, comprising the following steps:

[0057] Mixing the sample to be tested, the internal standard solution and the phosphate buffer to obtain a test solution;

[0058] The test liquid is subjected to 1 H- 13 C HSQC 2D NMR detection provides aromatic ring selective spectra and carbonyl selective spectra of phenylcyclidines;

[0059] described 1 H- 13 Selective pulse sequences for C HSQC 2D NMR detection include aromatic ring-selective pulse sequences and carbonyl-selective pulse sequences:

[0060] The aromatic ring group selective pulse sequence is a band-selective shaped pulse "Q3" with the F2 dimension (H spectrum) centered at 4.67 ppm, the F1 dimension (C spectrum) centered at 130 ppm, the J coupling parameter at 145 Hz, and the spectral width at 60 ppm.

[0061] The carbonyl-selective pulse sequence was a band-selective shaped pulse "Q5" with the F1 dimension (H spectrum) centered at 4.67 ppm, the F2 dimension (C spectrum) centered at 205 ppm, a J-coupling parameter of 10 Hz, and a spectral width of 30 ppm;

[0062] quantifying the phenylcyclidines based on the characteristic peak areas of the phenylcyclidines in the aromatic ring selectivity spectrum or the carbonyl selectivity spectrum of each phenylcyclidine and a predetermined standard curve;

[0063] The predetermined standard curve is a linear relationship between the concentration of the phencyclidine-like substance in the sample to be tested and the characteristic peak area of the phencyclidine-like substance.

[0064] In order to further illustrate the present invention, the scheme of the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be understood as limiting the scope of protection of the present invention.

[0065] Materials involved in the examples

[0066] Materials: PCP pure product (structural formula see Figure 1 ): Fluoroketamine, 2-(ethylamino)-2-(3-methoxyphenyl)cyclohexan-1-one (MXE), 2-amino-2-(2-fluorophenyl)cyclohexan-1-one, methylamine (2-MDCK), ethylnorketamine (NENK), ethylnorfluamine (2-FXE), norketamine, bromoketamine, 2-(ethylamino)-2-(3-methoxyphenyl)-1-cyclohexanone (2-Oxo-PCE), 2-(ethylamino)-2-(3-methylphenyl)-cyclohexan-1-one (DMXE), ketamine, tiletamine.

[0067] Reagents: phosphate buffer, ultrapure water, deuterated water, 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS);

[0068] Commercially available products: iced tea (Tea), Red Bull (RedBull), chocolate (COCO), e-liquid (E-liquid, a relatively viscous yellow liquid, the main component of which is glycerol, which is well soluble in water);

[0069] GraphPad Prism 10.1.2 and Microsoft Excel 2021 were used to construct a linear regression model related to PCP concentration and peak integration.

[0070] Comparative Example 1

[0071] The one-dimensional proton spectra of tiletamine standard and its in Red Bull matrix were tested using the following test conditions: presaturated water peak suppression pulse sequence noesygppr1d test, presaturation was achieved by applying a 25 Hz radio frequency (RF) field, spectral width: 16 ppm; relaxation delay (D1): 30 s; transmitter frequency offset (O1P): 5 ppm, number of scans (NS): 32, analysis time: 15 minutes, and test results are shown in Figure 2. Figure 2 ( Figure 2 for tiletamine and its one-dimensional proton spectrum in Red Bull matrix), from Figure 2It can be seen that in the chemical shift range of 2-4 ppm, the signals of the tiletamine standard and the matrix overlap to a great extent and are difficult to distinguish.

[0072] Comparative Example 2

[0073] HSQC pulse sequence was used to test the properties of tiletamine standard and its concentration in Red Bull matrix. 1 H- 13 CHSQC full spectrum, test results see Figure 3 ( Figure 3 For the standard of levitra 1 H- 13 C HSQC full spectrum), from Figure 3 It can be seen that there are many spectral signals, the required testing time is long, and there are a large number of matrix interference signals in the Red Bull matrix in this range, making specific detection difficult.

[0074] Example 1

[0075] (1) Test conditions:

[0076] The aromatic ring group-selective pulse sequence was a band-selective shaped pulse "Q3" with an F2-dimension (H spectrum) center of 4.67 ppm, an F1-dimension (C spectrum) center of 130 ppm, a J-coupling parameter of 145 Hz, and a spectral width of 60 ppm. The recirculation wait time was 1.5 s, and τ was 1.42 ms. The number of scans was 8, the number of F1-dimension sampling points was 64, and the acquisition time was 3 ms.

[0077] The carbonyl-selective pulse sequence was a band-selective shaped pulse "Q5", with the F1 dimension (H spectrum) center at 4.67 ppm, the F2 dimension (C spectrum) center at 205 ppm, the J coupling parameter at 10 Hz, and the spectral width at 30 ppm; the recirculation waiting time was 1 s, and τ was 24.7 ms; the number of scans was 64, the number of F1 dimension sampling points was 32, and the acquisition time was 3 ms.

[0078] (2) Preparation of internal standard solution:

[0079] 1.2 mg of 4,4-dimethyl-4-silapentane-1-sulfonic acid was added to deuterated water to prepare a 2 mg / mL internal standard solution.

[0080] 1) The preparation methods of the three matrix solutions are as follows:

[0081] 425 μL of Master Kong iced black tea was added to a centrifuge tube to obtain the tea matrix;

[0082] Take 425 μL of Red Bull and add it to a centrifuge tube to obtain Red Bull matrix;

[0083] 100 μL of electronic cigarette oil and 325 μL of water were mixed to prepare a solution, which was then added to a centrifuge tube to obtain an electronic cigarette oil matrix.

[0084] Dissolve 100 mg of chocolate in 1 mL of 60°C hot water and sonicate for 5 minutes. After cooling, remove the supernatant and transfer it to a centrifuge tube. Centrifuge at 10,000 rpm for 3 minutes. Filter the supernatant through a 0.22 μm filter to obtain the chocolate matrix.

[0085] 1 mg of pure PCP was added to deionized water to prepare a 1 mg / mL standard solution.

[0086] 2) Preparation of test solution:

[0087] Seven kinds of PCP pure powder (bromamine, ketamine, ethylnorfluamine, ethylnorketamine, tiletamine, MXE, 2-oxo-PCE) were weighed at 1, 2, 5, 10, 15, and 20 mg respectively. The above samples were dissolved in diluent (the diluent consisted of 850 μL water, 50 μL buffer and 100 μL internal standard solution) to prepare seven standard solutions of 1, 2, 5, 10, 15, and 20 mg / mL.

[0088] 1, 2, 4, and 7 mg of fluramine were dissolved in diluent (the diluent consisted of 850 μL water, 50 μL phosphate buffer and 100 μL internal standard solution) to prepare 1, 2, 4, and 7 mg / mL fluramine standard solutions.

[0089] 2) Detection of standards and matrices

[0090] Weigh 0.5 mg of each PCP standard powder and dissolve the above samples in 850 μL of water, 50 μL of phosphate buffer, and 100 μL of internal standard solution to prepare a 0.5 mg / mL standard solution;

[0091] According to the "(1) detection conditions", the concentration of 0.5 mg / LPCP pure sample was tested, and the aromatic ring selectivity and carbonyl selectivity spectra were shown in Figure 5 .from Figure 5 It can be seen that the signal of carbonyl C in pure PCP is mainly concentrated between 209-212 ppm. Since the signal intensity will weaken when the sample signal approaches the lower boundary of the spectrum, the center of the C spectrum is set to 205 ppm and the spectrum width is 30 ppm, which can ensure that the signals of all standard products are clearly visible.

[0092] ② Weigh 1.5 mg of fluramine powder on an analytical balance and place it in a centrifuge tube. Add 75 μL of phosphate buffer and 150 μL of internal standard solution. Once completely dissolved, remove 75 μL and place it in a centrifuge tube. Then, add 425 μL of Red Bull to the tube and dissolve completely. Transfer 500 μL to an NMR tube to obtain the fluramine standard in Red Bull matrix.

[0093] The fluoroquinolone standard in Red Bull matrix was tested according to “(1) Test conditions”. The test results are shown in Figure 5 , Figure 5 Comparison of the spectra of a fluamine standard and a Red Bull matrix: A shows the aromatic ring-selective spectra of the fluamine standard and Red Bull matrix; B shows the carbonyl-selective spectra of the fluamine standard and Red Bull matrix. Comparison of the two spectra of the fluamine standard and matrix reveals no signal overlap in the aromatic ring-selective spectra. While the carbonyl-selective spectra exhibited some unfilterable impurity signals at 2.7 ppm, no signal overlap was observed. Analysis confirmed that this signal originated from citric acid. The chemical shift of C in all PCP samples differed significantly from that of the citric acid signal, thus preventing interference with the assay.

[0094] The carbonyl-selective spectrum showed some unfilterable impurity signals at 2.7 ppm, but no signal overlap was observed. Analysis confirmed that the signal originated from citric acid. The chemical shift of C in all PCP samples was significantly different from that of citric acid, thus preventing interference with the assay.

[0095] Example 2

[0096] Weigh 1.5 mg of fluramine powder on an analytical balance and add it to a centrifuge tube. Add 75 μL of phosphate buffer and 150 μL of internal standard solution. Once completely dissolved, divide the solution into three portions and add them to three centrifuge tubes. Add 425 μL of Red Bull, 425 μL of the aforementioned e-cigarette oil solution, and 425 μL of the aforementioned chocolate solution to each of the three centrifuge tubes. Once dissolved completely, 500 μL of each solution was transferred to three NMR tubes to obtain fluramine solutions in Red Bull, chocolate, and e-cigarette oil matrices.

[0097] Weigh 1.5 mg of ketamine powder on an analytical balance and add it to a centrifuge tube. Add 75 μL of phosphate buffer and 150 μL of internal standard solution. Once completely dissolved, divide the solution into three portions and add them to three centrifuge tubes. Add 425 μL of Red Bull, 425 μL of the aforementioned e-cigarette oil solution, and 425 μL of the aforementioned chocolate solution to each of the three centrifuge tubes. Once dissolved completely, 500 μL of each solution was transferred to three NMR tubes to obtain ketamine solutions in Red Bull, chocolate, and e-cigarette oil matrices, respectively.

[0098] Weigh 1.5 mg of bromamine powder on an analytical balance and add it to a centrifuge tube. Add 75 μL of phosphate buffer and 150 μL of internal standard solution. Once completely dissolved, divide the solution into three portions and add them to three centrifuge tubes. Add 425 μL of Red Bull, 425 μL of the aforementioned e-cigarette oil solution, and 425 μL of the aforementioned chocolate solution to each of the three centrifuge tubes. Once dissolved completely, 500 μL of each solution was transferred to three NMR tubes to obtain bromamine solutions in Red Bull, chocolate, and e-cigarette oil matrices.

[0099] Ketamine, fluramine and bromamine were detected in three matrices: Red Bull, chocolate and e-cigarette oil according to “(1) Detection Conditions”. Figure 6 Comparison of the spectra of bromamine, fluamine and ketamine standards and bromamine, fluamine and ketamine in matrix. Figure 6 A is the comparison of aromatic ring selectivity spectra of bromamine standard with Red Bull, chocolate and e-liquid matrix; B is the comparison of aromatic ring selectivity spectra of fluamine standard with Red Bull, chocolate and e-liquid matrix; C is the comparison of aromatic ring selectivity spectra of ketamine standard with Red Bull, chocolate and e-liquid matrix; D is the comparison of carbonyl selectivity spectra of bromamine standard with Red Bull, chocolate and e-liquid matrix; E is the comparison of carbonyl selectivity spectra of fluamine standard with Red Bull, chocolate and e-liquid matrix; F is the comparison of carbonyl selectivity spectra of ketamine standard with Red Bull, chocolate and e-liquid matrix. Figure 6 From AC, we can see that ketamine in the matrix has three signals at 7.6ppm and 7.8ppm; fluamine in the matrix has four signals at 7.3ppm, 7.4ppm, 7.6ppm and 7.7ppm; bromamine in the matrix also has four signals at 7.5ppm, 7.6ppm and 7.8ppm. Figure 6 From the DF, we can see that in the Red Bull matrix, there is a strong signal at 2.7ppm, which is consistent with the experimental results of the blank matrix, indicating that the signal comes from the citric acid in the matrix, and the two signals of ketamine in the matrix at 2.5ppm and 3.3ppm come from the standard; the signals of fluamine in the matrix appear at 2.5ppm and 3.2ppm, and the signals of bromamine in the matrix appear at 2.6ppm and 3.3ppm, and the other signals come from the matrix. Figure 6 Results demonstrate that the aromatic ring- and carbonyl-selective pulse sequence provided by the present invention exhibits excellent sensitivity and selectivity when detecting PCP signals in real samples within a matrix. Through the design of this pulse sequence, the method provided by the present invention accurately extracts target signals, significantly improves detection sensitivity, and effectively reduces interference, providing reliable technical support for PCP analysis in complex samples.

[0100] Example 3

[0101] Weigh 1.5 mg of fluramine powder on an analytical balance and add it to a centrifuge tube. Add 75 μL of phosphate buffer and 150 μL of internal standard solution. Once completely dissolved, divide the solution into three portions and add them to three centrifuge tubes. Add 425 μL of Red Bull, 425 μL of the aforementioned e-cigarette oil solution, and 425 μL of the aforementioned chocolate solution to each of the three centrifuge tubes. Once dissolved completely, 500 μL of each solution was transferred to three NMR tubes to obtain fluramine solutions in Red Bull, chocolate, and e-cigarette oil matrices.

[0102] Weigh 1.5 mg of ketamine powder on an analytical balance and add it to a centrifuge tube. Add 75 μL of phosphate buffer and 150 μL of internal standard solution. Once completely dissolved, divide the solution into three portions and add them to three centrifuge tubes. Add 425 μL of Red Bull, 425 μL of the aforementioned e-cigarette oil solution, and 425 μL of the aforementioned chocolate solution to each of the three centrifuge tubes. Once dissolved completely, 500 μL of each solution was transferred to three NMR tubes to obtain ketamine solutions in Red Bull, chocolate, and e-cigarette oil matrices, respectively.

[0103] Weigh 1.5 mg of bromamine powder on an analytical balance and add it to a centrifuge tube. Add 75 μL of phosphate buffer and 150 μL of internal standard solution. Once completely dissolved, divide the solution into three portions and add them to three centrifuge tubes. Add 425 μL of Red Bull, 425 μL of the aforementioned e-cigarette oil solution, and 425 μL of the aforementioned chocolate solution to each of the three centrifuge tubes. Once dissolved completely, 500 μL of each solution was transferred to three NMR tubes to obtain bromamine solutions in Red Bull, chocolate, and e-cigarette oil matrices.

[0104] Ketamine, fluamine and bromamine were tested in three matrices, Red Bull, chocolate and e-cigarette oil, according to "(1) test conditions". The test results are shown in Figure 7 , Figure 7 The spectral comparison of fluamine, bromamine and ketamine in Red Bull, chocolate and e-liquid matrix. Figure 7 Figure A is the comparison of the aromatic ring selectivity spectra of fluamine, bromamine and ketamine in Red Bull, chocolate and e-liquid matrices; Figure B is the comparison of the carbonyl selectivity spectra of fluamine, bromamine and ketamine in Red Bull, chocolate and e-liquid matrices. Figure 7 As shown in the data, in the aromatic ring and carbonyl selectivity spectra, the signals of the three samples showed significant differences. These different signal characteristics can accurately distinguish ketamine, fluamine and bromamine, which fully proves that the detection method provided by the present invention shows excellent resolution and selectivity in distinguishing PCP substances with similar structures. This result provides strong technical support for the accurate analysis of PCP substances in complex matrices.

[0105] Example 4

[0106] PCP spectra collected from two sequences ( Figure 5 ) Comparative analysis showed that the aromatic ring selective pulse sequence had no signal filtering in the corresponding area and had a stronger signal than the carbonyl selective pulse sequence (carbonyl selective spectra usually require an accurate quantitative integration at around 5 mg / mL). Therefore, the aromatic ring selective pulse sequence was selected to establish the standard curve.

[0107] ① Establishment of standard curve

[0108] Five different concentrations of tiletamine, ketamine, and bromamine standard solutions (1, 2, 5, 10, and 20 mg / mL) and four different concentrations of fluamine solutions (1, 2, 4, and 7 mg / mL) were analyzed using an aromatic ring selective pulse sequence.

[0109] The standard curve was drawn with the concentration of the standard solution as the independent variable and the integrated area of all peak signals as the dependent variable (see Figures 8-11 ), Figure 8 is the linear regression model for the quantitative analysis of tiletamine by hydrogen solution NMR spectroscopy, where Figure 8 A is a linear regression model based on the characteristic signal of tiletamine at 7.73 ppm; B is a linear regression model based on the characteristic signal of tiletamine at 7.21 ppm; C is a linear regression model based on the characteristic signal of tiletamine at 7.22 ppm. Figure 9 is the linear regression model for the quantitative analysis of bromamine by hydrogen solution nuclear magnetic resonance spectroscopy, where Figure 9 A is a linear regression model based on the characteristic signal of bromamine at 7.87 ppm; B is a linear regression model based on the characteristic signal of bromamine at 7.83 ppm; C is a linear regression model based on the characteristic signal of bromamine at 7.51 ppm; D is a linear regression model based on the characteristic signal of bromamine at 7.66 ppm. Figure 10 is the linear regression model for the quantitative analysis of ketamine by hydrogen solution NMR spectroscopy, where Figure 10 A is a linear regression model based on the characteristic signal of ketamine at 7.88 ppm; B is a linear regression model based on the characteristic signal of ketamine at 7.62 ppm; C is a linear regression model based on the characteristic signal of ketamine at 7.61 ppm. Figure 11 is the linear regression model for the quantitative analysis of fluamine by hydrogen solution nuclear magnetic resonance spectroscopy; Figure 11 Figure A is a linear regression model based on the characteristic signal of fluamine at 7.76 ppm; Figure B is a linear regression model based on the characteristic signal of fluamine at 7.66 ppm; Figure C is a linear regression model based on the characteristic signal of fluamine at 7.48 ppm; and Figure D is a linear regression model based on the characteristic signal of fluamine at 7.32 ppm.

[0110] like Figures 8-11As shown, within the concentration range of 1 to 20 mg / mL, the correlation coefficients for the tiletamine standard curve at 7.73, 7.21, and 7.22 ppm were 0.9963, 0.9992, and 0.9983, respectively; the correlation coefficients for the bromamine standard curve at 7.87, 7.83, 7.51, and 7.66 ppm were 0.9904, 0.9932, 0.9951, and 0.9918, respectively; the correlation coefficients for the ketamine standard curve at 7.88, 7.62, and 7.61 ppm were 0.9903, 0.9903, and 0.9959, respectively; and the correlation coefficients for the fluamine standard curve at 7.76, 7.66, 7.48, and 7.32 ppm were 0.9930, 0.9901, 0.9858, and 0.9874, respectively. These correlation coefficients indicate that the analytical method has good linearity within this concentration range.

[0111] ②Detection limit and quantification limit

[0112] The limits of detection (LOD) and quantification (LOQ) of tiletamine, bromamine, ketamine, and fluamine were calculated based on the signal-to-noise ratios S / N=3 and S / N=10, respectively, as follows: the LOD of tiletamine was 0.20 mg / mL, and the LOQ was 0.67 mg / mL; the LOD of bromamine was 0.098 mg / mL, and the LOQ was 0.32 mg / mL; the LOD of ketamine was 0.075 mg / mL, and the LOQ was 0.25 mg / mL; and the LOD of fluamine was 0.050 mg / mL, and the LOQ was 0.17 mg / mL.

[0113] Example 5

[0114] The above detection conditions were used to analyze a new drug in a certain brand of iced black tea, and its carbonyl selectivity and aromatic ring selectivity spectra were obtained (see Figure 12 ), Figure 12 The spectra of methamine standard and methamine in tea matrix are compared. A is the aromatic ring selective spectra of methamine standard and methamine in tea matrix; B is the carbonyl spectra of methamine standard and methamine in tea matrix; Figure 12 As shown, the substance has both carbonyl and aromatic ring structural characteristics. By comparing the chemical shift data in its two-dimensional nuclear magnetic resonance (2D NMR) spectrum with the established standard database, it was confirmed that the substance is methylamine ketone (2-MDCK).

[0115] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for detecting phenylcyclidines using two-dimensional nuclear magnetic resonance, characterized in that: The following steps are involved: Mixing the sample to be tested, the internal standard solution and the phosphate buffer to obtain a test solution; The test liquid is subjected to 1 H- 13 C HSQC 2D NMR detection provides aromatic ring selective spectra and carbonyl selective spectra of phenylcyclidines; described 1 H- 13 The selective pulse sequences for C HSQC 2D NMR detection include aromatic ring group selective pulse sequence and carbonyl group selective pulse sequence; The aromatic ring group-selective pulse sequence is a band-selective shaped pulse "Q3" with an F2 dimension (H spectrum) centered at 4.67 ppm, an F1 dimension (C spectrum) centered at 130 ppm, a J coupling parameter of 145 Hz, and a spectral width of 60 ppm. The carbonyl-selective pulse sequence was a band-selective shaped pulse "Q5" with the F1 dimension (H spectrum) centered at 4.67 ppm, the F2 dimension (C spectrum) centered at 205 ppm, a J coupling parameter of 10 Hz, and a spectral width of 30 ppm; The phenylcyclidine substances are qualitatively identified by comparing the aromatic ring selectivity spectrum and the carbonyl selectivity spectrum of the phenylcyclidine substances with the spectra of predetermined standard substances of the phenylcyclidine substances.

2. The method according to claim 1, wherein The phenylcyclidine substances include one or more of fluamine, 2-(ethylamino)-2-(3-methoxyphenyl)cyclohexane-1-one, 2-amino-2-(2-fluorophenyl)cyclohexane-1-one, methylamine, ethylnorketamine, ethylnorfluamine, norketamine, bromamine, 2-(ethylamino)-2-(3-methoxyphenyl)-1-cyclohexanone, 2-(ethylamino)-2-(3-methylphenyl)-cyclohexane-1-one, ketamine and tiletamine.

3. The method according to claim 1, wherein The band selective shaped pulse "Q3" is a 180° pulse in the y direction of the C core in the polarization transfer stage.

4. The method according to claim 1, wherein In the aromatic ring group selective pulse sequence, the recirculation waiting time is 1.5s, τ is 1.42ms, the number of scans is 8, the number of F1-dimensional sampling points is 64, and the acquisition time is 3ms.

5. The method according to claim 1, wherein In the carbonyl-selective pulse sequence, the recirculation waiting time is 1 s, τ is 24.7 ms, the number of scans is 64, the number of F1-dimensional sampling points is 32, and the acquisition time is 3 ms.

6. The method according to claim 1, wherein The internal standard solution is a deuterium aqueous solution of 4,4-dimethyl-4-silapentane-1-sulfonic acid.

7. The method according to claim 6, wherein The concentration of the deuterium aqueous solution of 4,4-dimethyl-4-silapentane-1-sulfonic acid is 2 mg / mL.

8. A method for quantifying phenylcyclidines using two-dimensional nuclear magnetic resonance, characterized in that: The following steps are involved: Mixing the sample to be tested, the internal standard solution and the phosphate buffer to obtain a test solution; The test liquid is subjected to 1 H- 13 C HSQC 2D NMR detection provides aromatic ring selective spectra and carbonyl selective spectra of phenylcyclidines; described 1 H- 13 The selective pulse sequences for C HSQC 2D NMR detection include aromatic ring group selective pulse sequence and carbonyl group selective pulse sequence; The aromatic ring group-selective pulse sequence is a band-selective shaped pulse "Q3" with an F2 dimension (H spectrum) centered at 4.67 ppm, an F1 dimension (C spectrum) centered at 130 ppm, a J coupling parameter of 145 Hz, and a spectral width of 60 ppm. The carbonyl-selective pulse sequence was a band-selective shaped pulse "Q5" with the F1 dimension (H spectrum) centered at 4.67 ppm, the F2 dimension (C spectrum) centered at 205 ppm, a J coupling parameter of 10 Hz, and a spectral width of 30 ppm; quantifying the phenylcyclidines based on the characteristic peak areas of the phenylcyclidines in the aromatic ring selectivity spectrum or the carbonyl selectivity spectrum of each phenylcyclidine and a predetermined standard curve; The predetermined standard curve is a linear relationship between the concentration of the phencyclidine-like substance in the sample to be tested and the characteristic peak area of the phencyclidine-like substance.

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