Phenethylamine psychoactive substance detection method

Abstract

The application relates to the technical field of compound detection, in particular to a phenethylamine psychoactive substance detection method. The detection method is detected by using a liquid chromatograph or liquid chromatography-mass spectrometry, the phenethylamine psychoactive substance includes at least one of 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe and 2C-P; the elution mode is gradient elution, wherein the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is a mixed solution of ammonium acetate and formic acid, wherein the concentration of ammonium acetate is 5-15 mmol / L, the concentration of formic acid is 0.05%-0.15%, the mobile phase B is acetonitrile, and elution is carried out according to a gradient elution program. The detection method realizes good separation among the six phenethylamine psychoactive substances through a specific gradient elution program, the peak type of the characteristic peaks in the chromatogram is good, the separation degree between adjacent characteristic peaks is good, the detection precision is high, the sensitivity is high, and the repeatability is good.

Description

Technical Field

[0001] This application belongs to the field of compound detection technology, and in particular relates to a method for detecting phenylethylamine psychoactive substances. Background Technology

[0002] Phenethylamine is a potent psychotropic drug with strong hallucinogenic and psychoactive effects. It can significantly alter a person's perception, emotions, and thought processes. After use, adverse reactions such as hallucinations, delusions, confusion, and increased burden on the cardiovascular system may occur. Long-term or high-dose use can easily lead to tolerance, dependence, and severe mental disorders, posing a high risk of abuse and public safety hazards.

[0003] Although some of these substances are strictly controlled as narcotics, some derivatives may still be illegally added to food or beverages. These substances can significantly affect the central nervous system even at low doses, producing hallucinogenic, stimulant, or mood-regulating effects. This not only seriously threatens consumers' physical and mental health, but their covert addition also undermines the basic principles of food safety. Current technologies, using liquid chromatography and liquid chromatography-mass spectrometry (LC-MS) combined with sample processing, can rapidly and accurately detect various phenylethylamine-like psychoactive substances in different foods.

[0004] However, phenethylamine psychoactive substances such as 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P have the same phenethylamine core, differing only in the substituents or side chain modifications on the benzene ring. Their molecular polarity and hydrophobicity are extremely similar, resulting in overlapping retention times during chromatographic separation. This makes baseline separation difficult, easily leading to false negative or false positive results and poor accuracy. Summary of the Invention

[0005] The purpose of this application is to provide a method for detecting phenethylamine psychoactive substances, so as to solve the technical problem of poor accuracy of detection results of phenethylamine psychoactive substances in the prior art.

[0006] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0007] In a first aspect, embodiments of this application provide a method for detecting phenethylamine-type psychoactive substances. The detection method of this application embodiment employs liquid chromatography or liquid chromatography-mass spectrometry, wherein the phenethylamine-type psychoactive substances include 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P; The elution method is gradient elution. The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is a mixed solution of ammonium acetate and formic acid, where the concentration of ammonium acetate is 5–15 mmol / L and the concentration of formic acid is 0.05%–0.15%. Mobile phase B is acetonitrile. Elution is performed using the gradient elution method shown below: .

[0008] The detection method of this application achieves good separation among six phenethylamine psychoactive substances (2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P) through a specific gradient elution program. The characteristic peaks in the chromatogram exhibit good peak shapes and excellent separation between adjacent peaks, resulting in high precision, high sensitivity, and good repeatability for the detection of phenethylamine psychoactive substances. This detection method can be used not only for the qualitative detection of phenethylamine psychoactive substances but also for their quantitative detection. Attached Figure Description

[0009] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0010] Figure 1 The chromatogram and mass spectrum of the primary ion extraction of 2,5-dimethoxy-4-chlorophenylethylamine are shown. Figure 1 In the figure, 'a' represents the primary ion extraction chromatogram of 2,5-dimethoxy-4-chlorophenylethylamine. Figure 1 b in the spectrum is the secondary mass spectrum of 2,5-dimethoxy-4-chlorophenylethylamine; Figure 2 The chromatogram and mass spectrum of N-(2-methoxybenzyl)-2-(2,5-dimethoxy-4-chlorophenyl)ethylamine are shown. Figure 2 In the figure, 'a' represents the primary ion extraction chromatogram of N-(2-methoxybenzyl)-2-(2,5-dimethoxy-4-chlorophenyl)ethylamine; Figure 2 b in the spectrum is the secondary mass spectrum of N-(2-methoxybenzyl)-2-(2,5-dimethoxy-4-chlorophenyl)ethylamine; Figure 3 The chromatogram and mass spectrum of the primary ion extraction of 2,5-dimethoxy-4-ethylphenylethylamine are shown. Figure 3 In the figure, 'a' represents the primary ion extraction chromatogram of 2,5-dimethoxy-4-ethylphenethylamine. Figure 3b in the image represents the secondary mass spectrum of 2,5-dimethoxy-4-ethylphenethylamine; Figure 4 The chromatogram and mass spectrum of the primary ion extraction of 2,5-dimethoxy-4-ethylphenylethylamine are shown. Figure 4 In the figure, 'a' represents the primary ion extraction chromatogram of 2,5-dimethoxy-4-ethylphenethylamine. Figure 4 b in the image represents the secondary mass spectrum of 2,5-dimethoxy-4-ethylphenethylamine; Figure 5 The chromatogram and mass spectrum of N-(2-methoxybenzyl)-2-(2,5-dimethoxy-4-iodophenyl)ethylamine are shown. Figure 5 In the figure, 'a' represents the primary ion extraction chromatogram of N-(2-methoxybenzyl)-2-(2,5-dimethoxy-4-iodophenyl)ethylamine; Figure 5 b in the spectrum is the secondary mass spectrum of N-(2-methoxybenzyl)-2-(2,5-dimethoxy-4-iodophenyl)ethylamine; Figure 6 The chromatogram and mass spectrum of the primary ion extraction of 2,5-dimethoxy-4-propylphenylethylamine are shown. Figure 6 In the figure, 'a' represents the primary ion extraction chromatogram of 2,5-dimethoxy-4-propylphenylethylamine. Figure 6 b in the spectrum is the secondary mass spectrum of 2,5-dimethoxy-4-propylphenylethylamine; Figure 7 Extraction ion chromatograms of six phenylethylamine psychoactive substances: 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P. Figure 8 This is a schematic diagram showing the recovery results of three different sample purification methods: direct injection, solid phase extraction (SPE), and dispersive solid phase extraction (dSPE). Figure 9 This is a schematic diagram of the mobile phase changes during the gradient elution process of Comparative Examples 1 to 4. Detailed Implementation

[0011] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0012] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0013] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can both mean: a, b, c, a~b (i.e., a and b), a~c, b~c, or a~b~c, where a, b, and c can be single or multiple.

[0014] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0015] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms "a" and "the" as used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0016] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass in the embodiments of this application can be a well-known unit of mass in the chemical industry, such as µg, mg, g, or kg.

[0017] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0018] To address the technical problem of poor accuracy in the detection results of phenethylamine-type psychoactive substances in existing technologies, this application proposes the following technical solution.

[0019] In a first aspect, embodiments of this application provide a method for detecting phenethylamine-type psychoactive substances. The detection method of this application embodiment employs liquid chromatography or liquid chromatography-mass spectrometry, wherein the phenethylamine-type psychoactive substances include 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P; The elution method is gradient elution. The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is a mixed solution of ammonium acetate and formic acid, where the concentration of ammonium acetate is 5–15 mmol / L and the concentration of formic acid is 0.05%–0.15%. Mobile phase B is acetonitrile. Elution is performed using the gradient elution method shown below: .

[0020] In this application, 2C-C refers to 2,5-dimethoxy-4-chlorophenethylamine (4-Chloro-2,5-dimethoxyphenethylamine, 2C-C); 2C-C-NBOMe refers to N-(2-methoxybenzyl)-2-(2,5-dimethoxy-4-chlorophenyl)ethylamine [2-(4-Chloro-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine, 2C-C-NBOMe]; 2C-D refers to 2,5-dimethoxy-4-methylphenethylamine (4-Methyl-2,5-dimethoxyphenethylamine, 2C-D); 2C-E refers to 2,5-dimethoxy-4-ethylphenethylamine (4-Ethyl-2,5-dimethoxyphenethylamine, 2C-E); 2C-I-NBOMe It refers to N-(2-methoxybenzyl)-2-(2,5-dimethoxy-4-iodophenyl)ethylamine [2-(4-Iodo-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine, 2C-I-NBOMe]; 2C-P refers to 2,5-dimethoxy-4-propylphenethylamine (4-Propyl-2,5-dimethoxyphenethylamine, 2C-P).

[0021] The method for detecting phenethylamine-like psychoactive substances proposed in this application constructs an efficient gradient elution program through systematic optimization of liquid chromatography conditions, successfully separating structurally highly similar compounds such as 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P. This method exhibits excellent selectivity and separation efficiency, with sharp and symmetrical peaks or ion peaks and good separation between adjacent peaks, fundamentally avoiding co-elution and effectively reducing the risk of qualitative misjudgment. Furthermore, this method combines high sensitivity, high precision, and good reproducibility, achieving a detection limit of 0.005 mg / L, enabling trace detection of target substances and meeting the detection requirements for low concentrations of phenethylamine-like psychoactive substances in complex matrices. This method is not only suitable for accurate qualitative identification of phenethylamine-like substances but can also achieve precise quantitative analysis by combining information such as the concentration of the sample solution. Thus, multiple target substances can be simultaneously detected qualitatively and quantitatively in a single injection, significantly improving analytical efficiency and result accuracy.

[0022] In some embodiments, when the detection method of this application uses liquid chromatography-mass spectrometry (LC-MS) for detection, it may include the following steps: Step S10: Prepare the test solution and the standard working solution, wherein the standard working solution contains 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe and 2C-P standards; Step S20: Inject the standard working solution and the test solution into the liquid chromatography-mass spectrometry instrument for detection; Step S30: Establish a high-resolution mass spectrometry library based on the spectral information of the standard working solution; Step S40: Determine the types of phenethylamine psychoactive substances contained in the sample based on the spectral information of the test solution and the high-resolution mass spectrometry library; The high-resolution mass spectrometry library includes the retention times, primary precursor ion and secondary fragment ion masses of 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe and 2C-P.

[0023] The detection method described in this application, using liquid chromatography-mass spectrometry (LC-MS), establishes a high-resolution mass spectral library by determining the retention time, primary precursor ion, and characteristic secondary fragment ion mass of each compound (2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P). Combined with LC conditions, this allows for the effective separation of 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P in the LC system. This enables the simultaneous detection of multiple phenethylamine-related psychoactive substances, significantly improving detection efficiency, shortening the analysis cycle, and reducing detection costs. Furthermore, the detection results are highly accurate and reliable, making it suitable for detecting trace components in complex matrices. In addition, when a calibration curve is established based on standard working solutions, the detected target analytes in the sample can be quantitatively calculated, resulting in highly accurate and stable analytical results.

[0024] Step S10: Test solutions were prepared using the sample to be tested, and standard solutions were prepared using 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P for use in subsequent detection steps.

[0025] In some embodiments, the test sample used to prepare the test solution can be a food product such as a beverage or cola.

[0026] In some embodiments, when the sample to be tested is cola, acetonitrile can be used to extract phenylethylamine psychoactive substances from cola. In an exemplary example, when the sample to be tested is cola, the preparation method of the test solution may include the following steps: mixing cola with acetonitrile and sodium chloride, performing a first separation treatment, collecting the supernatant and mixing it with octadecylsilane-bonded silica gel, performing a second separation treatment, and taking the supernatant to obtain the test solution. In another exemplary example, the preparation method of the cola test solution may specifically be as follows: accurately pipetting 1 mL of cola into a 50 mL centrifuge tube, adding 10 mL of acetonitrile and 1 g of sodium chloride, using sodium chloride to separate the organic and aqueous phases. Ultrasonic extraction for 10 min, centrifuging at 9500 r / min for 5 min, and taking 6 mL of the supernatant to a container containing 50 mg C 18 Filter the extract (octadecylsilane-bonded silica gel with a particle size of 40 μm to 60 μm) into a 15 mL centrifuge tube using a filter membrane, and use the filtrate as the test solution. It should be noted that the extracted solution can be diluted as needed by those skilled in the art.

[0027] Cola contains a complex composition, including sugars, acids, pigments, and caffeine, which can easily interfere with the detection of target analytes. Acetonitrile extraction and octadecylsilane-bonded silica gel purification, combined with liquid chromatography-mass spectrometry (LC-MS), can effectively extract and enrich trace amounts of phenylethylamine in the sample while removing matrix interference, significantly improving the method's selectivity and sensitivity. Under optimized chromatographic conditions, six target analytes, including 2C-C and 2C-C-NBOMe, can still achieve good separation in the cola matrix, with stable retention times and high characteristic ion response intensity, effectively meeting the need for accurate detection of extremely low concentrations of components in actual samples. It should be noted that the cola in this application can be sugary cola, low-sugar cola, or sugar-free cola; the specific type, flavor, and brand are not limited. This test solution preparation method is not only applicable to the detection of phenylethylamine psychoactive substances in cola but also to the detection of phenylethylamine psychoactive substances in complex food matrices such as tea beverages and energy drinks, demonstrating broad applicability.

[0028] In some embodiments, a standard working solution may contain one phenylethylamine psychoactive substance, or two or more phenylethylamine psychoactive substances, without specific limitation. In one embodiment, the standard working solution may contain 2C-C standard; in another embodiment, the same standard working solution may contain 2C-C standard and 2C-C-NBOMe standard; and in yet another embodiment, the same standard working solution may contain six standards: 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P.

[0029] In some embodiments, the concentrations of the 2C-C standard, 2C-C-NBOMe standard, 2C-D standard, 2C-E standard, 2C-I-NBOMe standard, and 2C-P standard in the standard working solution can range from 0.5 to 100 μg / L. Typical but non-limiting concentrations in exemplary examples include 0.5 μg / L, 5 μg / L, 10 μg / L, 50 μg / L, and 100 μg / L. Controlling the concentration of the working solution within this range helps to ensure the detection signal remains within the linear response range, avoids overloading of the mass spectrometry ion source or a decrease in sensitivity, and also meets the signal-to-noise ratio requirements at low concentration points. In actual measurements, an appropriate standard curve concentration gradient can be selected based on the estimated concentration range of the sample to improve the accuracy of the detection results.

[0030] Step S20: The test solution and standard solution are injected into a liquid chromatography-mass spectrometry (LC-MS) instrument for detection, and the chromatograms and mass spectrometry information are recorded.

[0031] In some embodiments, when using liquid chromatography-mass spectrometry (LC-MS) to detect standard working solutions and test solutions, the mobile phase of the LC can be a mixture of ammonium acetate and formic acid (mobile phase A) and acetonitrile (mobile phase B), wherein the concentration of ammonium acetate in mobile phase A can be 5–15 mmol / L, and the concentration of formic acid can be 0.05%–0.15%. In exemplary cases, the concentration of ammonium acetate in mobile phase A can be typical but not limiting concentrations such as 5 mmol / L, 8 mmol / L, 10 mmol / L, 12 mmol / L, and 15 mmol / L, or any concentration between any two concentrations; the concentration of formic acid can be typical but not limiting concentrations such as 0.05%, 0.08%, 0.1%, 0.12%, and 0.15%, or any concentration between any two concentrations.

[0032] In some embodiments, the liquid chromatography may use a mixed solution of 10 mmol / L ammonium acetate and 0.1% formic acid as mobile phase A, and employ a gradient elution method as shown below: .

[0033] Elution using this gradient program, combined with chromatographic conditions such as column and column temperature, achieves excellent separation among CC, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P, with sharp and symmetrical peaks and stable retention times. This provides high-purity chromatographic peaks with low matrix interference for subsequent mass spectrometry detection, significantly improving the qualitative confirmation and quantitative repeatability of target analytes. After 13.1 min, the system rapidly returns to initial conditions, promoting column reequilibration and providing favorable separation conditions for the next sample injection.

[0034] In some embodiments, the column may be a column packed with octadecylsilane-bonded silica gel. In an exemplary example, the column may be an Agilent Poroshell 120 EC-C. 18 The chromatographic column has a specification of 2.7 μm and a diameter of 2.1 μm. 100 mm. Using this column enables baseline separation of each target analyte in a shorter time, while reducing column pressure, improving detection accuracy, sensitivity, and peak repeatability.

[0035] In some embodiments, the column temperature can be 23~27 °C. In exemplary cases, the column temperature can be a typical but not limiting temperature such as 23 °C, 24 °C, 25 °C, 26 °C, or 27 °C, or any temperature between any two. Controlling the column temperature within this range can effectively improve separation efficiency and reduce peak broadening, while reducing retention time drift caused by temperature fluctuations, thereby further improving the accuracy and repeatability of the detection results.

[0036] In some embodiments, the flow rate can be 0.25–0.35 mL / min. Typical but not limiting flow rates in exemplary examples include 0.25 mL / min, 0.26 mL / min, 0.27 mL / min, 0.28 mL / min, 0.29 mL / min, 0.31 mL / min, 0.32 mL / min, 0.33 mL / min, 0.34 mL / min, and 0.35 mL / min. Controlling the flow rate within this range can, on the one hand, further improve the separation of phenethylamine-type psychoactive substances and shorten the analysis time, thereby increasing detection efficiency. On the other hand, controlling the flow rate within this range can also reduce the impact of system pressure fluctuations on retention time stability, thus improving the robustness of the method and the stability of instrument operation. Furthermore, a suitable flow rate can reduce peak broadening effects, improve the response efficiency of the mass spectrometry interface, enhance detection sensitivity, and improve the accuracy of detection results.

[0037] In some embodiments, the injection volume can be 1–10 μL. In exemplary cases, typical but not limiting injection volumes include 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, and 10 μL. Controlling the injection volume within this range can improve the signal intensity of the target analyte while reducing peak broadening or column overload caused by excessive injection volume. This ensures that each analyte maintains good response values ​​and peak symmetry even at low concentrations, further enhancing the method's sensitivity and linear range.

[0038] In some embodiments, mass spectrometry conditions may include at least one of the following (1) to (6): (1) The ion source is an electrospray ion source; (2) The electrospray voltage is 5000~6000V; (3) The ion source temperature is 530~570℃; (4) The air curtain pressure is 0.23~0.27MPa; (5) The pressure of the atomizing gas is 0.35~0.4MPa; (6) The auxiliary gas pressure is 0.4~0.45MPa.

[0039] In the example, the mass spectrometry conditions can be as follows: Ion source: Electrospray ionization (ESI); Scanning method: Positive and negative ion scanning in Information Dependent Acquisition (IDA) mode; Electrospray voltage: 5500 V (positive mode); Ion source temperature: 550 °C; Air curtain pressure: 0.241 MPa; Atomizing gas pressure: 0.379 MPa; Auxiliary gas pressure: 0.414 MPa; TOF-MS parameters: Acquisition range 50–500 m / z; Declustering voltage 80 V; Collision energy 10 eV; TOF-MS / MS parameters: Acquisition range 50–500 m / z; Declustering voltage 80 V; Collision energy 20 eV, 35 eV, 50 eV.

[0040] Detection under these mass spectrometry conditions enables highly sensitive and selective detection of phenylethylamine-like substances, effectively avoiding matrix interference and improving the accuracy of qualitative and quantitative analysis of target analytes. Coordinated optimization of various parameters ensures long-term instrument stability, making it suitable for rapid screening and confirmatory analysis of trace active ingredients in complex samples.

[0041] Step S30: Based on the spectral information of the standard working solution, a high-resolution mass spectrometry library was established, which includes the retention times, primary precursor ions and secondary fragment ions of 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe and 2C-P.

[0042] A high-resolution mass spectrometry (HMS) library was established, creating a reference system for precise identification and confirmation. This library records the retention times and precise mass numbers of primary precursor ions of each target analyte under specific chromatographic conditions, as well as characteristic secondary fragment ions generated at different collision energies. The integration of multiple pieces of information from the HMS library allows detection to move beyond reliance on single features. When analyzing analytes, precise comparison of the spectral information of the analyte with this HMS library enables efficient matching of corresponding compounds. This allows for clear differentiation and identification of structurally similar compounds such as 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P, significantly reducing the risk of false positives and false negatives.

[0043] Step S40: The spectral information of the test solution is compared with a high-resolution mass spectrometry library to determine the types of phenethylamine psychoactive substances contained in the test solution. When the test solution does not contain 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P, the test sample is negative.

[0044] In some embodiments, when the spectral information of the test solution meets the following (1) to (3), it is determined that the test sample contains the target analyte: (1) The signal-to-noise ratio (S / N) of the target object's signal response is ≥3; (2) The retention time of the target analyte deviates from the retention time in the high-resolution mass spectrometry library by ≤0.2 min or the retention time of the target analyte deviates from the retention time in the high-resolution mass spectrometry library by ≤2.5% and does not exceed 0.5 min; (3) The primary precursor ion and at least one secondary fragment ion of the target analyte are matched with the high-resolution mass spectrometry database, and the mass accuracy deviation of the primary precursor ion is ≤5 ppm. When the m / z of the primary precursor ion is <200, its absolute deviation should be <1mDa, and the mass accuracy deviation of the secondary fragment ion is ≤10 ppm.

[0045] By combining multiple parameters such as signal-to-noise ratio, retention time, primary precursor ion, and secondary fragment ions, the presence of target analytes in the sample is determined, effectively improving the specificity and accuracy of detection and enhancing the reliability of trace component identification in complex matrices.

[0046] In some embodiments, when using liquid chromatography to detect phenethylamine-type psychoactive substances, the following steps may be included: Inject the test solution and the standard working solution of phenethylamine psychoactive substances into the liquid chromatograph and record the chromatogram; The types and / or contents of phenethylamine psychoactive substances in the test solution are determined based on the chromatographic peaks of the test solution and the phenethylamine psychoactive substances. Among them, the contents of 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe and 2C-P in the standard working solution of phenethylamine psychoactive substances are 0.5~100 μg / L, respectively; The detection wavelength is 287~291 nm, and can be selected from typical but not limited wavelengths such as 287 nm, 288 nm, 289 nm, 290 nm, and 291 nm, or any wavelength between any two wavelengths.

[0047] When using liquid chromatography for detection, the chromatographic conditions, the preparation methods of the test solution and the standard working solution are as described above, and will not be repeated here.

[0048] The detection method in this application optimizes chromatographic conditions, achieving effective separation between 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P. This allows for detection not only using liquid chromatography-mass spectrometry (LC-MS) but also by liquid chromatography alone. Liquid chromatography detection not only provides high accuracy but also significantly reduces equipment costs and simplifies operation; it eliminates the need for complex ion sources and vacuum systems, simplifying daily maintenance; and it has relatively lower requirements for laboratory conditions (such as power and space), making it easier to popularize and implement in routine analysis.

[0049] Based on the good linearity of six phenethylamine psychoactive substances (2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P) in the concentration range of 0.5–100 μg / L, liquid chromatography can be used for both qualitative and quantitative detection of phenethylamine psychoactive substances.

[0050] When using liquid chromatography (LC) for qualitative detection of phenethylamine-type psychoactive substances, if the retention time of the chromatographic peak in the test sample solution is consistent with that of the standard working solution, it can be preliminarily determined that the sample contains the target substance. Specifically, the retention time deviation between the two solutions should not exceed 0.2 minutes, or the relative deviation should not exceed 2.5% (and the deviation should not be greater than 0.5 minutes). To further confirm the results, wavelength scanning of the chromatographic peaks can be performed to compare the maximum absorption wavelengths of the test sample and the standard working solution at the corresponding chromatographic peaks, thereby enhancing the reliability of the qualitative judgment.

[0051] When performing quantitative detection, one approach is to use the external standard method, directly calculating the concentration of the target analyte by comparing its peak area in the test sample solution with that in the standard working solution, and then calculating the content of the target analyte in the sample. Another approach is to first establish a linear equation using the peak areas of the target analyte at different concentrations in the standard working solution, and then calculate the specific content based on this equation and the peak area of ​​the target analyte in the test sample.

[0052] To enable those skilled in the art to clearly understand the above-described implementation details and operations of this application, and to demonstrate the significant advancements in the detection method for phenethylamine-type psychoactive substances in the embodiments of this application, the following examples illustrate the above technical solutions.

[0053] Example 1 This embodiment provides a method for detecting phenethylamine-type psychoactive substances. The detection method in this embodiment uses high-performance liquid chromatography-quadrupole tandem mass spectrometry (HPLC-QMS). The detection instrument in this embodiment is an HPLC-QMS / time-of-flight mass spectrometer with an electrospray ionization (ESI) source.

[0054] 1. Testing conditions The chromatographic conditions for the detection method in this embodiment are as follows: the chromatographic column is an Agilent Poroshell 120 EC-C. 18 (2.7 μm, 2.1) A 100 mm column (or a column with equivalent column efficiency) was used, with a column temperature of 25 °C, a flow rate of 0.3 mL / min, and an injection volume of 5 μL. Mobile phase A was a mixture of 10 mmol / L ammonium acetate and 0.1% formic acid, and mobile phase B was acetonitrile. Binary gradient elution was used, and the gradient elution program is shown in Table 1.

[0055] Table 1 Time / min Mobile phase A / % Mobile phase B / % 0.0 95 5 6.0 75 25 8.0 30 70 12.0 2 98 13.0 2 98 13.1 95 5 15.0 95 5 The mass spectrometry conditions for the detection method in this embodiment are as follows: the ion source is an electrospray ionization (ESI) source; the scanning mode is positive and negative ion scanning in information-dependent acquisition mode (IDA); the electrospray voltage is 5500 V (positive mode); the ion source temperature is 550°C; the curtain gas pressure is 0.241 MPa; the nebulizer gas pressure is 0.379 MPa; the auxiliary gas pressure is 0.414 MPa; the TOF-MS parameters are: acquisition range 50–500 m / z, declustering voltage 80 V, collision energy 10 eV; the TOF-MS / MS parameters are: acquisition range 50–500 m / z, declustering voltage 80 V, collision energy 20 eV, 35 eV, and 50 eV.

[0056] 2. Reagent preparation method (1) Preparation of mobile phase A: Measure an appropriate amount of water, add 0.77g of ammonium acetate, add 1mL of formic acid, transfer to a 1L volumetric flask, and dilute to the mark with water.

[0057] (2) Standard intermediate solutions: Six phenylethylamine psychoactive substances, namely 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P, are shown in Table 2. The concentrations of commercially available standard stock solutions of 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P are all 100 mg / L, and the solvent is methanol. Take 100 μL of each of the above standard stock solutions and place them in a 10 mL brown volumetric flask. Dilute to the mark with methanol to prepare standard intermediate solutions of each phenylethylamine psychoactive substance with a concentration of 1 mg / L. Prepare fresh solutions immediately before use.

[0058] (3) Standard working solutions: The above-mentioned intermediate solutions of phenylethylamine psychoactive substances with a concentration of 1 mg / L were diluted with acetonitrile to prepare a series of 2C-C standard working solutions, 2C-C-NBOMe standard working solutions, 2C-D standard working solutions, 2C-E standard working solutions, 2C-I-NBOMe standard working solutions and 2C-P standard working solutions.

[0059] (4) Mixed standard intermediate solution: Take 100 μL of each of the above standard stock solutions into a 10 mL brown volumetric flask, dilute with methanol to the mark, and prepare a mixed standard intermediate solution with a concentration of 1 mg / L. Prepare fresh before use.

[0060] (5) Mixed standard working solution: Accurately transfer appropriate amounts of the mixed standard intermediate solution and dilute it with acetonitrile to form a series of standard working solutions. Prepare the solution immediately before use.

[0061] Table 2

[0062] (6) Sample pretreatment methods The sample was cola. 1 mL of cola was placed in a 50 mL centrifuge tube, along with 10 mL of acetonitrile and 1 g of sodium chloride. The mixture was extracted by sonication for 10 min, then centrifuged at 9500 r / min for 5 min. 6 mL of the supernatant was collected and transferred to a container containing 50 mg of sodium chloride. 18 Filter the solution into a 15 mL centrifuge tube containing octadecylsilane-bonded silica gel with a particle size of 40 μm to 60 μm. Use a 13 mm × 0.22 μm (or equivalent) organic phase filter membrane. Take the filtrate and dilute it appropriately according to the actual concentration for analysis by liquid chromatography-quadrupole / time-of-flight mass spectrometry.

[0063] 3. Establishment of a high-resolution mass spectrometry library The standard working solutions of 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P were injected into a liquid chromatography-quadrupole / time-of-flight mass spectrometer for detection. The high-resolution mass spectrometry parameters of these six phenethylamine psychoactive substances are shown in Table 3. The primary ion extraction chromatogram and secondary mass spectrum are shown in Table 3. Figures 1 to 6 As shown.

[0064] Table 3 Serial Number Chinese name English name CAS number Chemical formula Ionization mode Theoretical exact mass number Fragment Ion 1 Fragment Ion 2 Fragment Ion 3 Fragment Ion 4 Retention time 1 2,5-Dimethoxy-4-chlorophenylethylamine 4-Chloro-2,5-dimethoxyphenethylamine 88441-14-9 <![CDATA[C 10 H 14 ClNO2]]> M+H 216.0786 199.0524 184.0291 169.0055 164.0837 6.89 2 N-(2-Methoxybenzyl)-2-(2,5-Dimethoxy-4-chlorophenyl)ethylamine 2-(4-Chloro-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine 1227608-02-7 <![CDATA[C 18 H 22 ClNO3]]> M+H 336.1361 121.0643 93.0702 91.0540 / 8.47 3 2,5-Dimethoxy-4-methylphenylethylamine 4-Methyl-2,5-dimethoxyphenethylamine 24333-19-5 <![CDATA[C 11 H 17 NO2]]> M+H 196.1332 179.1071 164.0837 149.0602 91.0547 6.72 4 2,5-Dimethoxy-4-ethylphenylethylamine 4-Ethyl-2,5-dimethoxyphenethylamine 71539-34-9 <![CDATA[C 12 H 19 NO2]]> M+H 210.1489 193.1222 178.0989 163.0755 105.0702 7.82 5 N-(2-Methoxybenzyl)-2-(2,5-Dimethoxy-4-iodophenyl)ethylamine 2-(4-Iodo-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine 919797-19-6 <![CDATA[C 18 H 22 INO3]]> M+H 428.0717 272.1414 121.0643 93.0703 91.0542 8.62 6 2,5-Dimethoxy-4-propylphenylethylamine 4-Propyl-2,5-dimethoxyphenethylamine 207740-22-5 <![CDATA[C 13 H 21 NO2]]> M+H 224.1645 207.1381 192.1150 163.0756 105.0705 8.14 Based on the high-resolution mass spectrometry parameters of six phenethylamine psychoactive substances (2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P) shown in Table 3, a sample can be identified as a suspected positive sample if the following three conditions are met during sample detection: 1. The target analyte signal response S / N ≥ 3; 2. The retention time of the target analyte deviates from the retention time in the high-resolution mass spectrometry library by ≤ 0.2 min or ±2.5% (not exceeding 0.5 min); 3. Compared with the high-resolution mass spectrometry database, the target analyte can match a primary precursor ion and a secondary fragment ion, and the mass accuracy deviation of the primary precursor ion is ≤ 5 ppm (when the primary precursor ion m / z < 200, its absolute deviation should be < 1 mDa), and the mass accuracy deviation of the secondary fragment ion is ≤ 10 ppm.

[0065] 4. Sample testing Ten batches of commercially available cola were tested for phenylethylamine psychoactive substances using the detection method described in this embodiment, and no target substance was detected in any of them.

[0066] Ten batches of negative samples for phenylethylamine psychoactive substances were collected, and six phenylethylamine psychoactive substances, namely 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe and 2C-P, were added to the limit of detection (0.005 mg / L). After processing according to the sample pretreatment method, the samples were injected for detection. All 10 batches of spiked samples could effectively detect the six phenylethylamine psychoactive substances.

[0067] 5. Methodological Validation Preparation method of spiked sample: Take negative cola that does not contain phenylethylamine psychoactive substances, and add an appropriate amount of phenylethylamine psychoactive substance standard according to experimental requirements.

[0068] (1) Target object separation degree test The mixed standard working solution was injected into a liquid chromatography-quadrupole / time-of-flight mass spectrometer for detection, and the extracted ion chromatograms of the target analytes were examined. The results are as follows: Figure 7 As shown.

[0069] (2) Investigation of the influence of matrix effect Matrix effect (ME) refers to the significant enhancement or inhibition of the analytical process by interfering components within a sample, thus affecting the accuracy of the results. It is calculated as: ME(%) = |B / A-1|×100%, where A is the slope of the calibration curve for the sample matrix solution and B is the slope of the solvent calibration curve. |ME|<20% indicates a weak matrix effect, 20%≤|ME|<50% indicates a moderate matrix effect, and |ME|≥50% indicates a strong matrix effect. Using cola as a representative sample, corresponding blank matrix solutions were prepared to investigate the matrix effect of representative samples. The results are shown in Table 4.

[0070] In Table 4, "Linear equation (solvent)" refers to the calibration curve established using a mixed standard working solution (or individual substance standard solutions) in a pure solvent system, with target concentrations of 0.5 μg / L, 5 μg / L, 10 μg / L, 50 μg / L, and 100 μg / L, respectively. "Linear equation (matrix solution)" refers to the calibration curve established by adding a series of standard working solutions of the same concentration to a cola blank matrix solution, with the concentration gradient set consistent with the solvent system. The blank matrix solution is prepared as follows: Cola containing none of the six phenylethylamine psychoactive substances is treated according to the above "sample pretreatment method" to obtain the blank matrix solution.

[0071] Table 4 Serial Number English abbreviation Linear equation (solvent) Linear equation (matrix solution) |ME| / % 1 2C-C y=1187x-170 y=1387x-280 16.8 2 2C-C-NBOMe y = 17000x + 2780 y = 18200x + 2360 7.1 3 2C-D y=291x-133 y=305x-105 4.8 4 2C-E y=2505x+13 y=2781x+25 11.0 5 2C-I-NBOMe y = 17111x + 1527 y = 18201x + 1280 6.4 6 2C-P y=4100x+437 y=4500x+568 9.8 As shown in Table 4, the results indicate that the target substances exhibit varying degrees of matrix effect in cola, with |ME| all less than 20%. The matrix effect range falls within the weak-intensity interference range, indicating that the detection method in this embodiment, using a standard working solution prepared with a solvent, can meet the requirements of the detection work. By comparing the slopes of the two types of calibration curves, the degree of influence of the matrix on the target substance's response can be intuitively reflected, providing a basis for evaluating the applicability and reliability of the method.

[0072] (3) Specificity Twenty samples of different brands of cola were collected, and none of these 20 cola samples contained phenylethylamine-like psychoactive substances. One mL of each cola sample was placed in a 50 mL centrifuge tube, and 10 mL of acetonitrile and 1 g of sodium chloride were added. The tubes were extracted by sonication for 10 min, then centrifuged at 9500 r / min for 5 min. Six mL of the supernatant was collected and transferred to a container containing 50 mg of sodium chloride. 18 15 mL centrifuge tubes (octadecylsilane-bonded silica gel, particle size 40 μm–60 μm) were filtered through a 13 mm × 0.22 μm (or equivalent) organic phase filter membrane. The filtrate was then injected into a liquid chromatography-quadrupole / time-of-flight mass spectrometer for detection. The results showed that no interference with the six phenylethylamine psychoactive substances was detected in any of the 20 cola samples, indicating that the blank in the detection method for phenylethylamine psychoactive substances in cola in this application embodiment was free from interference.

[0073] (4) Sensitivity Cola was used as the matrix sample, and different volumes of mixed standard intermediate solution were added to obtain the spiked solution. The spiked solution was processed according to the sample pretreatment method to obtain the sensitivity detection solution, which was then detected by liquid chromatography-quadrupole / time-of-flight mass spectrometry. The results showed that at a concentration of 0.005 mg / L, the signal-to-noise ratio of the six phenethylamine psychoactive substances was greater than 3. As a unified detection method, the detection limit was set at 0.005 mg / L for each phenethylamine psychoactive substance.

[0074] Another 20 cola samples were taken for method applicability verification: a mixed standard intermediate solution was added to the cola to obtain a spiking solution. The spiking solution was treated according to the pretreatment method and injected into the instrument for data acquisition and analysis. The results showed that for each target ion in the 20 samples: S / N≥3; retention time difference≤0.1 min; primary precursor ion mass accuracy error≤5 ppm; secondary fragment ion mass accuracy deviation≤10 ppm.

[0075] The specificity and sensitivity verification of the cola detection method in this embodiment refers to GB 5009.295-2023 "National Food Safety Standard - General Rules for Validation of Chemical Analysis Methods" 3.1 Performance parameter selection, and the specificity and sensitivity verification of the qualitative method selection.

[0076] (5) Investigation of extraction solvent Phenethylamine psychoactive substances such as 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P are slightly soluble or sparingly soluble in water. Their water solubility is effectively improved upon salt formation. To enhance extraction efficiency in the detection process, a water-methanol or water-acetonitrile system was used to extract the hydrochloride and free forms of these six phenylethylamine psychoactive substances: 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P.

[0077] Methods: Spiked cola containing 0.050 mg / L phenethylamine-like psychoactive substances was used for the investigation. Sample pretreatment was performed using either a water-methanol or water-acetonitrile system. Results showed that the recoveries of phenethylamine-like psychoactive substances were similar using both water-methanol and water-acetonitrile methods. However, the water-acetonitrile system allows for the separation of the aqueous and organic phases through salting-out, which is beneficial for subsequent purification; therefore, acetonitrile was used as the extraction solvent.

[0078] (6) Investigation of purification conditions This study investigated the effects of three different sample purification methods—direct injection, solid-phase extraction (SPE), and dispersive solid-phase extraction (dSPE)—on spiked cola containing 0.050 mg / L phenylethylamine-like psychoactive substances. The methods were as follows: spiked cola was placed in centrifuge tubes, acetonitrile and sodium chloride were added, and the samples were extracted by sonication, centrifuged, and the supernatant was collected and purified using the following three methods: Method 1: direct injection detection; Method 2: solid-phase extraction (SPE) using an HLB column for sample purification; Method 3: dSPE using 50 mg of C18 adsorbent. Results are as follows: Figure 8 As shown.

[0079] like Figure 8 As shown, the three sample purification methods—direct injection, solid-phase extraction (SPE), and dSPE—all exhibited high recovery rates. Specifically, the recovery rates for the six target analytes after direct injection ranged from 86.2% to 96.2%, after SPE purification from 80.2% to 95.2%, and after dSPE purification from 84.2% to 96.2%. This application employs dSPE for sample purification, which enables rapid screening and further reduces matrix influence.

[0080] Example 2 This embodiment provides a method for detecting phenethylamine-type psychoactive substances. The detection method in this embodiment is liquid chromatography, and the chromatographic conditions are as follows: The chromatographic column was an Agilent Poroshell 120 EC-C. 18 (2.7 μm 2.1) A 100 mm column was used, with a column temperature of 25 °C, a flow rate of 0.3 mL / min, an injection volume of 5 μL, and a detection wavelength of 289 nm. Mobile phase A was a mixed solution of 10 mmol / L ammonium acetate and 0.1% formic acid, and mobile phase B was acetonitrile. Binary gradient elution was used, and the gradient elution program was the same as that in Example 1, as shown in Table 1.

[0081] The preparation methods for the test solution and the mixed standard working solution in this embodiment are the same as in Example 1. The test solution and the mixed standard working solution (or standard working solution) are injected separately. The presence of the six phenethylamine-type psychoactive substances in the test solution is determined based on the chromatograms of the mixed standard working solution and the test solution. If a chromatographic peak with a retention time comparable to that of the phenethylamine-type psychoactive substance in the mixed standard working solution is present in the test solution, it can be preliminarily determined that the sample contains the phenethylamine-type psychoactive substance.

[0082] The same detection method as in Example 1 was used to test 10 batches of cola samples and 10 batches of spiked samples. The test results were the same as those in Example 1.

[0083] Comparative Examples 1 to 4 Comparative Examples 1 to 4 each provide a method for detecting phenethylamine-type psychoactive substances. The detection methods of Comparative Examples 1 to 4 are basically the same as those in Example 1, except that the column temperature and elution gradient are different. The column temperature of Comparative Examples 1 to 4 is 30 °C, and the elution gradients of Comparative Examples 1 to 4 are as follows: Figure 9 As shown in Table 5: Table 5 / gradient Comparative Example 1 ①0~6.0 min, 5%-40%B; 6.0~8.0 min, 40%-70%B; 8.0~12.0 min, 70%-98%B; 12.0~13.0 min, 98%B; 13.0~13.1 min, 98%-5%B; 13.1~15.0 min, 5%B Comparative Example 2 ②0~6.0 min, 5%-35%B; 6.0~8.0 min, 35%-70%B; 8.0~12.0 min, 70%-98%B; 12.0~13.0 min, 98%B; 13.0~13.1 min, 98%-5%B; 13.1~15.0 min, 5%B Comparative Example 3 ③0~6.0 min, 5%-30%B; 6.0~8.0 min, 30%-70%B; 8.0~12.0 min, 70%-98%B; 12.0~13.0 min, 98%B; 13.0~13.1 min, 98%-5%B; 13.1~15.0 min, 5%B Comparative Example 4 ④0~6.0 min, 5%-25%B; 6.0~8.0 min, 25%-70%B; 8.0~12.0 min, 70%-98%B; 12.0~13.0 min, 98%B; 13.0~13.1 min, 98%-5%B; 13.1~15.0 min, 5%B The mixed standard working solution was injected and tested separately. The results showed that under the conditions of comparative examples 1 to 3, the six phenylethylamine psychoactive substances could not be separated well. Under the condition of comparative example 4, the six phenylethylamine psychoactive substances had a better separation degree. The results are shown in Table 6.

[0084] Table 6 target Retention time / min Half-peak width / min Adjacent peaks Resolution R 2C-C 6.78 0.06 2C-D 1.48 2C-C-NBOMe 8.45 0.05 2C-I-NBOMe 1.89 2C-D 6.63 0.06 2C-C 1.48 2C-E 7.79 0.05 2C-P 4.01 2C-I-NBOMe 8.61 0.05 2C-C-NBOMe 1.89 2C-P 8.13 0.05 2C-E 4.01 Note: R = 1.18 × (t2-t1) / (W) h / 2 (1) + W h / 2(2)); t2, t1: Retention time of two adjacent peaks (later peak - earlier peak); W h / 2 (1) W h / 2 (2): Half-peak widths of the two peaks; R≥1.5 is considered complete separation, and R≥1.2 meets the quantitative requirements. The judgment rule comes from General Chapter 0512 "High Performance Liquid Chromatography" in Part IV of the Pharmacopoeia of the People's Republic of China (2020 Edition).

[0085] As shown in Table 6, although the only difference between the chromatographic conditions of Comparative Example 4 and those of Example 1 is the column temperature (the column temperature of Example 1 is 25°C, and the column temperature of Comparative Example 4 is 30°C), the resolution between the two adjacent peaks 2C-C and 2C-D in Comparative Example 4 is still less than 1.5.

[0086] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for detecting phenethylamine-type psychoactive substances, wherein the method employs liquid chromatography or liquid chromatography-mass spectrometry, characterized in that, The phenylethylamine psychoactive substances include 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe, and 2C-P; The elution method used in the chromatographic conditions is gradient elution. The mobile phase includes mobile phase A and mobile phase B. Mobile phase A is a mixed solution of ammonium acetate and formic acid, where the concentration of ammonium acetate is 5–15 mmol / L and the concentration of formic acid is 0.05%–0.15%. Mobile phase B is acetonitrile. Elution is performed using the gradient elution method shown below: 。 2. The detection method as described in claim 1, characterized in that, The concentration of ammonium acetate in mobile phase A is 10 mmol / L, and the concentration of formic acid is 0.1%. The elution method used in the chromatography is a gradient elution method as shown below: 。 3. The detection method as described in claim 1, characterized in that, Chromatographic conditions include at least one of the following (1) to (4): (1) The column temperature is 23~27 ℃; (2) The chromatographic column is a chromatographic column packed with octadecylsilane-bonded silica gel; (3) The flow rate is 0.25~0.35 mL / min; (4) The injection volume is 1~10 μL.

4. The detection method as described in claim 3, characterized in that, Chromatographic conditions include at least one of the following (1) to (4): (1) The column temperature is 25 ℃ (2) The chromatographic column was an Agilent Poroshell 120 EC-C. 18 Chromatographic column, specification 2.7 μm 2.1 100 mm; (3) The flow rate is 0.3 mL / min; (4) The injection volume is 5 μL.

5. The detection method according to any one of claims 1-4, characterized in that, The detection of phenethylamine-like psychoactive substances using liquid chromatography-mass spectrometry includes the following steps: Prepare the test solution and the standard working solution, wherein the standard working solution contains the 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe and 2C-P standards; The standard working solution and the test solution were injected into a liquid chromatography-mass spectrometry system for detection. A mass spectrometry library was established based on the spectral information of the standard working solution. The types of phenethylamine-like psychoactive substances contained in the test sample were determined based on the spectral information of the test sample solution and the mass spectrometry library. The mass spectrometry library includes the retention times, primary precursor ion and secondary fragment ion masses of 2C-C, 2C-C-NBOMe, 2C-D, 2C-E, 2C-I-NBOMe and 2C-P.

6. The detection method as described in claim 5, characterized in that, The concentrations of the 2C-C standard, 2C-C-NBOMe standard, 2C-D standard, 2C-E standard, 2C-I-NBOMe standard and 2C-P standard in the standard working solution are 0.5~100μg / L, respectively.

7. The detection method as described in claim 5, characterized in that, Mass spectrometry conditions include at least one of the following (1) to (6): (1) The ion source is an electrospray ion source; (2) The electrospray voltage is 5000~6000V; (3) The ion source temperature is 530~570℃; (4) The air curtain pressure is 0.23~0.27MPa; (5) The pressure of the atomizing gas is 0.35~0.4MPa; (6) The auxiliary gas pressure is 0.4~0.45MPa.

8. The detection method as described in claim 5, characterized in that, If the chromatographic information of the test sample solution meets the following (1) to (3), then it is determined that the test sample contains the target analyte: (1) The signal-to-noise ratio of the target object's signal response is ≥3; (2) The retention time of the target analyte deviates from the retention time in the mass spectrometry library by ≤0.2 min; Alternatively, the retention time of the target analyte deviates from the retention time in the mass spectrometry library by ≤2.5%, and does not exceed 0.5 min; (3) The primary precursor ion and at least one secondary fragment ion of the target ion match the mass spectrometry database, and the mass accuracy deviation of the primary precursor ion is ≤5 ppm and the mass accuracy deviation of the secondary fragment ion is ≤10 ppm.

9. The detection method as described in claim 5, characterized in that, The sample to be tested is cola, and the preparation method of the test solution includes the following steps: The cola was mixed with acetonitrile and sodium chloride for a first separation process. The supernatant was collected and mixed with octadecylsilane-bonded silica gel for a second separation process. The supernatant was collected to obtain the test solution.

10. The detection method as described in claim 1, characterized in that, The detection of phenethylamine-type psychoactive substances using liquid chromatography includes the following steps: Inject the test solution and the standard working solution of phenethylamine psychoactive substances into the liquid chromatograph and record the chromatogram; The types and / or contents of phenethylamine psychoactive substances in the test solution are determined based on the chromatographic peaks of the test solution and the phenethylamine psychoactive substances. The detection wavelength is 287~291 nm.

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