Method for determining 15 new psychoactive substances in saliva through SLE-UPLC-MS / MS

Abstract

The invention discloses a method for determining 15 new psychoactive substances in saliva by combining solid-phase supported liquid-liquid extraction (SLE) with ultra-high liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), which specifically comprises the following steps: by taking a mixed solution of methyl tertiary butyl ether and normal hexane as an elution solvent, carrying out pretreatment on a saliva sample by adopting the solid-phase supported liquid-liquid extraction method; and then detecting the collected eluent by adopting an ultrahigh liquid chromatography-tandem mass spectrometry method so as to realize qualitative and quantitative determination of the new psychoactive substances in the saliva. The method has the advantages of rapidness, accuracy, high sensitivity, simplicity in operation and the like, the detection limit of the method is 0.2-0.5 ng / mL, the limit requirements at home and abroad can be met, and technical support is provided for monitoring and investigation of the new psychoactive substances in saliva.

Description

Technical Field

[0001] The present invention belongs to the field of analytical technology, and specifically relates to a method for determining 15 new psychoactive substances in saliva by solid supported liquid-liquid extraction (SLE) combined with ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS / MS). Background Art

[0002] Saliva is a colorless, thin liquid secreted by the oral cavity. It is composed of salivary gland secretions, oral mucosal exudates, and gingival sulcus fluid. It contains a variety of components, such as water, mucin, amylase, lysozyme, inorganic salts, drug residues and metabolites. There are two sources of drug molecules in saliva. One is oral drug residues, and the other is free drug molecules in the blood entering the salivary glands through epithelial cells. Compared with traditional biological samples such as blood, urine, and hair, saliva has the advantages of non-invasive sampling, no site restrictions, and a low probability of cross-infection. It is an important research object for current technical law enforcement.

[0003] New psychoactive substances are developing rapidly in the drug market due to their low cost, diverse structure and easy evasion of control. Currently, the most severely abused new psychoactive substances in the world are synthetic cannabinoids, cathinones, ketamines and fentanyls, while Chinese drug addicts mainly abuse synthetic cannabinoids, ketamines, cathinones and tryptamines.

[0004] Existing research on drugs in saliva mainly focuses on traditional drugs, involving few new psychoactive substances and a single type, which cannot meet the current rapidly growing detection needs of new psychoactive substances. In addition, the sample pretreatment methods mostly use protein precipitation, liquid-liquid extraction (LLE), solid phase extraction (SPE) and dispersed solid phase extraction (dSPE). The matrix effect of protein precipitation and liquid-liquid extraction is more obvious; the solid phase extraction method has cumbersome steps and is time-consuming, which is difficult to adapt to the current detection needs of efficient law enforcement and large-scale screening. Therefore, it is urgent to establish a rapid and efficient detection method for new psychoactive substances in saliva.

[0005] Solid supported liquid-liquid extraction (SLE) has simple steps and can effectively remove components such as proteins and phospholipids in biological samples, significantly reduce matrix effects and prevent emulsification. However, there are no reports on the use of solid supported liquid-liquid extraction columns for the enrichment and purification of new psychoactive substances in saliva samples. The present invention uses SLE to pre-treat saliva samples, and combines it with ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS / MS) to establish a detection method for the 10 most numerous and most popular synthetic cannabinoids and 5 cathinones among new psychoactive substances, which can provide a reference for the rapid detection and combating of new psychoactive substances. Summary of the invention

[0006] The purpose of the present invention is to provide a method for determining 15 new psychoactive substances in saliva by solid supported liquid-liquid extraction (SLE) combined with ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS / MS). This method has the advantages of rapidity, accuracy, high sensitivity and simple operation, can meet the limit requirements at home and abroad, and provides technical support for the monitoring and investigation of new psychoactive substances in saliva.

[0007] To achieve the above object, the present invention adopts the following technical solution: A method for determining 15 new psychoactive substances in saliva by solid supported liquid-liquid extraction (SLE) combined with ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS / MS) comprises the following steps: 1) Sample pretreatment: Take 0.5 mL of blank saliva sample and saliva sample respectively, load them onto solid phase supported liquid-liquid extraction cartridges, apply positive pressure with solid phase extraction device to disperse the sample onto the filler, let it stand for 5 minutes, then add elution solvent twice, 2 mL each time, let it stand for another 5 minutes, collect the eluate at a flow rate of 1 mL / min, then blow the collected eluate to near dryness with nitrogen at 40 °C, re-dissolve with 0.5 mL of acetonitrile, and filter through a 0.22 μm microporous filter membrane to obtain blank saliva matrix and sample solution; 2) Preparation of mixed standard solution: The 15 new psychoactive substance standards were dissolved in methanol and then fixed to volume to prepare mixed standard stock solutions with a concentration of 1.0 μg / mL. Before use, an appropriate amount was measured and diluted with the prepared blank saliva matrix to prepare a series of mixed standard solutions with a mass concentration of 0-100 ng / mL. 3) Drawing of standard curve: The sample solution obtained in step 1) and the series of mixed standard solutions obtained in step 2) were measured by UPLC-MS / MS, respectively. Then, standard curves were drawn with the mass concentration of 15 new psychoactive substances in the series of mixed standards as the horizontal axis and the peak area as the vertical axis. The types and contents of new psychoactive substances contained in the sample solutions were determined based on the sample peak results combined with the obtained standard curves.

[0008] Furthermore, the 15 new psychoactive substances are specifically pentyl-3-(4-methoxybenzoyl)indole (RCS-4), 1-butyl-3-(1-naphthoyl)indole (JWH 073), 1-pentyl-3-(4-ethyl-1-naphthoyl)indole (JWH-210), 1-hexyl-3-(1-naphthoyl)indole (JWH-019), 1-pentyl-3-(1-naphthoyl)indole (JWH-018), N-(1-carbamoyl-2-methylpropyl)-1-(cyclohexylmethyl)indazole-3-carboxamide (AB-CHMINACA), 1-pentyl-3-(4-methyl-1-naphthoyl)indole (JWH-122), 2-(2-methoxyphenyl)-1-(1- Pentyl-1H-indol-3-yl)ethanone (JWH-250), 1-(5-fluoropentyl)-3-(1-naphthoyl)-1H-indole (AM2201), N-(1-carbamoyl-2,2-dimethylpropyl)-1-pentylindazole-3-carboxamide (ADB-PINACA), 4-methyl-α-pyrrolidinobutyrophenone (MPBP), promazine hydrochloride (Promazine), 4-fluoromethcathinone (4-FMC), 4'-methyl-α-pyrrolidinobutyrophenone (PV-4), ethyl hydrochloride (MDEC).

[0009] Furthermore, the elution solvent in step 2) is a mixture of methyl tert-butyl ether and n-hexane in a volume ratio of 2:1.

[0010] Furthermore, the ultra high performance liquid chromatography conditions used in step 3) are as follows: chromatographic column: Welch Ultimate® PFP (2.1 mm×100 mm, 5 μm); column temperature: 40°C; injection volume: 10 μL; mobile phase A is a 0.1 vol% formic acid aqueous solution containing 20 mmol / L ammonium acetate, and mobile phase B is acetonitrile; flow rate is 0.20 mL / min; gradient elution program is: 0~1.0 min, 70%A; 1.0~2.0 min, 70%~30%A; 2.0~4.0min, 30%A; 4.0~5.0min, 30%~70%A; 5.0~6.0min, 70%A.

[0011] Furthermore, the mass spectrometry conditions used in step 3) are: electrospray ion source; positive ion mode; multiple reaction monitoring; capillary voltage: 3 kV; ion source temperature: 350 °C; nebulizer gas: nitrogen; nebulizer gas flow rate: 1000 L / h; collision gas: argon; collision gas flow rate: 50 L / h.

[0012] The significant advantages of the present invention are: Compared with the traditional liquid-liquid extraction method, the present invention uses solid-phase supported liquid-liquid extraction for processing, which can significantly reduce the matrix effect of saliva samples. Combining it with ultra-high performance liquid chromatography tandem mass spectrometry, the established determination method has the advantages of being fast, accurate, highly sensitive and easy to operate. The detection limit of this method is 0.2~0.5 ng / mL, which can meet the domestic and international limit requirements, and provides technical support for the monitoring and investigation of new psychoactive substances in saliva. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a comparative diagram of the recovery rates of 15 new psychoactive substances using different elution solvents in the examples.

[0014] Figure 2 This is a comparison of the matrix effects of 15 new psychoactive substances using different elution solvents in the examples.

[0015] Figure 3 The recovery rates of 15 new psychoactive substances using mixed elution solvents in different ratios in the examples are shown.

[0016] Figure 4 This is the matrix effect of mixed elution solvents in different proportions on 15 new psychoactive substances in the examples.

[0017] Figure 5 Graph showing the total ion currents of 15 new psychoactive substances obtained in the examples.

[0018] Figure 6 The extracted chromatogram is the actual sample measured in the example. DETAILED DESCRIPTION

[0019] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto. Example

[0020] 1 Experimental part 1.1 Instruments and reagents Xero TQD liquid chromatography-tandem mass spectrometer (Waters, USA); ST16R high-speed centrifuge (Thermofisher, USA); SBHCONC / 1 nitrogen blowdown apparatus (Stuart, Germany); MTS240C multi-tube vortex oscillator (Shanghai Damu Industrial Co., Ltd.); Milli-Q ultrapure water machine (Millipore, USA).

[0021] Pentyl-3-(4-methoxybenzoyl)indole (RCS-4), 1-butyl-3-(1-naphthoyl)indole (JWH073), 1-pentyl-3-(4-ethyl-1-naphthoyl)indole (JWH-210), 1-hexyl-3-(1-naphthoyl)indole (JWH-019), 1-pentyl-3-(1-naphthoyl)indole (JWH-018), N-(1-carbamoyl-2-methylpropyl)-1-(cyclohexylmethyl)indazole-3-carboxamide (AB-CHMINACA), 1-pentyl-3-(4-methyl-1-naphthoyl)indole (JWH-122 )、2-(2-methoxyphenyl)-1-(1-pentyl-1H-indol-3-yl)ethanone (JWH-250), 1-(5-fluoropentyl)-3-(1-naphthoyl)-1H-indole (AM2201), N-(1-carbamoyl-2,2-dimethylpropyl)-1-pentylindazole-3-carboxamide (ADB-PINACA), 4-methyl-α-pyrrolidinophenone (MPBP), promazine hydrochloride (Promazine), 4-fluoromethcathinone (4-FMC), 4'-methyl-α-pyrrolidinophenone (PV-4), ethyl hydrochloride (MDEC), diphenylpentyl diamino ester (SKF 525A) (content 100 μg / mL, Tianjin Alta Technology Co., Ltd.); n-hexane, ethyl acetate, dichloromethane and methyl tert-butyl ether (analytical grade, CNW, Germany); methanol, acetonitrile (chromatographic grade, Merck, Germany); formic acid (chromatographic grade, Shandong Xiya Company); ammonium acetate (chromatographic grade, Shanghai Aladdin Company); 2 mL solid phase supported liquid extraction cartridge (Biotage, Sweden); other reagents were domestic analytical grade or premium grade; the experimental water was deionized water.

[0022] Blank saliva was provided by healthy adult volunteers with no history of drug abuse.

[0023] 1.2 Experimental methods 1.2.1 Preparation of standard solution Take appropriate amount of 15 new psychoactive substances standard solutions, dissolve them with methanol and make up to volume in a 10mL volumetric flask to prepare mixed standard stock solutions with a concentration of 1.0 μg / mL for later use.

[0024] 1.2.2 Sample pretreatment 1.2.2.1 Solid Support Liquid Extraction Take 0.5 mL of blank saliva sample and saliva sample respectively, load them onto 2 mL solid phase supported liquid extraction cartridges, apply positive pressure with solid phase extraction device to disperse the sample onto the filler, let it stand for 5 minutes, then add 2×2 ml of elution solvent (methyl tert-butyl ether: n-hexane = 2:1, V / V), let it stand for 5 minutes, collect the eluate at a flow rate of 1 mL / min, and apply positive pressure to discharge the remaining eluate after the elution solvent drips off. The collected eluate is blown to near dryness with nitrogen at 40 °C, re-dissolved with 0.5 ml of acetonitrile, and filtered through a 0.22 μm microporous filter membrane to obtain blank saliva matrix and sample solution, which are ready for determination on the machine.

[0025] 1.2.2.2 Liquid-liquid extraction Take 0.5 ml of saliva sample, add 1 mL of boric acid buffer with pH 9.2, extract with 2 mL of ether, vortex, and centrifuge. Volatilize the supernatant in a 60°C water bath, re-dissolve with 0.5 ml of acetonitrile by vortexing, pass through a 0.22 μm microporous filter, and wait for the determination on the machine.

[0026] 1.2.3 UPLC-MS / MS analysis 1.2.3.1 Chromatographic conditions Chromatographic column: Welch Ultimate® PFP (2.1 mm×100 mm, 5 μm), mobile phase A is 0.1% formic acid aqueous solution containing 20 mmol / L ammonium acetate, mobile phase B is acetonitrile, flow rate is 0.20 mL / min, column temperature: 40 ℃, injection volume: 10 μL, gradient elution program: 0~1.0 min, 70% A; 1.0~2.0 min, 70 %~30%A; 2.0~4.0min, 30%A; 4.0~5.0min, 30%~70%A; 5.0~6.0min, 70%A.

[0027] 1.2.3.2 Mass spectrometry conditions 5 electrospray ion source; positive ion mode (ESI+); multiple reaction monitoring (MRM); capillary voltage: 3 kV; ion source temperature: 350 ℃; nebulizer gas: nitrogen; nebulizer gas flow rate: 1000 L / h; collision gas: argon; collision gas flow rate: 50 L / h; The mass spectrometry parameters of 15 new psychoactive substances are shown in Table 1.

[0028] Table 1 Relevant information and analytical parameters of 15 new psychoactive substances and internal standards

[0029] 1.3 Data processing The Masslynx data processing system provided by the instrument was used for experimental data acquisition, analysis and processing, and charts were drawn using Excel and Origin 9.0 software.

[0030] 2 Results and analysis 2.1 Comparison of pre-treatment methods Two pretreatment methods, liquid-liquid extraction and solid-phase supported liquid-liquid extraction, were investigated in the experiment. The specific operation was to take a blank saliva sample, prepare it into a 10 ng / mL target substance spiked sample, and use the methods under 1.2.2.1 and 1.2.2.2 for pretreatment respectively (prepare 3 parallel samples for each concentration), and evaluate it with the recovery rate of the compound as an indicator. The results are shown in Table 2.

[0031] Table 2 Comparison of the effects of different pre-treatment methods

[0032] The results in Table 2 show that when solid-phase supported liquid-liquid extraction was used for pretreatment, the recoveries of 15 new psychoactive substances were between 88.3% and 113.8%, which was significantly better than the 41.2% to 70.4% of the liquid-liquid extraction method.

[0033] 2.2 Optimization of solid phase supported liquid-liquid extraction conditions The solid phase supported liquid-liquid extraction column uses diatomaceous earth treated by a special process as filler, so the elution solvent needs to be a solution that is immiscible with water, and the choice of solvent depends on the physical and chemical properties of the target, such as the solubility and polarity. Since the compounds to be detected are mostly weakly polar or medium-polar substances, the extraction effects of five kinds of solvents, namely n-hexane, methyl tert-butyl ether, dichloromethane, ether and ethyl acetate, were investigated in the experiment (three parallel experiments were set for each solvent), and the recovery rate of the compound was used as an indicator for evaluation. The results are shown in Figure 1 .

[0034] like Figure 1 As shown in the figure, the extraction effects of the five solvents are quite different. The recovery rate of n-hexane is 23.5%~41.2%, and the elution efficiency is low. The recovery rates of methyl tert-butyl ether, dichloromethane, ether, and ethyl acetate are relatively high, among which methyl tert-butyl ether has the most ideal extraction effect, with a recovery rate ranging from 82.6% to 94.5%.

[0035] As the polarity of the elution solvent increases, the amount of saliva matrix eluted also increases accordingly, resulting in a stronger matrix effect. The matrix effects of the five elution solvents are shown in Figure 2 .

[0036] like Figure 2 As shown, the medium polar solvents diethyl ether and ethyl acetate exhibited a stronger matrix suppression effect, while when dichloromethane and methyl tert-butyl ether were used, the matrix suppression effect was weaker. Therefore, methyl tert-butyl ether was preferably selected as the elution solvent.

[0037] Considering that n-hexane has less interference with chromatographic peaks when used as elution solvent, and that n-hexane has stable chemical properties, is not easy to react with the stationary phase, and is volatile, the elution ability and matrix effect of preparing mixed elution solvents with methyl tert-butyl ether and n-hexane in different ratios were further investigated. The results are shown in Table 1. Figure 3 , 4 .

[0038] Depend on Figure 3 , 4 It can be seen that when the ratio of methyl tert-butyl ether to n-hexane is 2:1, the recovery rate of 15 new psychoactive substances is 88.3%~113.8%, and the matrix effect is weak. When the content of methyl tert-butyl ether in the elution solvent increases, the recovery rate does not increase significantly, and the recovery rates of two synthetic cannabinoids, ADB-PINCA and AB-CHMINACA, decrease, and the matrix effect is high; when the content of n-hexane in the elution solvent increases, the recovery rate of most compounds decreases significantly. Therefore, after comprehensive consideration, methyl tert-butyl ether and n-hexane were selected as the elution solvent in a volume ratio of 2:1.

[0039] On this basis, the amount of elution solvent used was further optimized. The results showed that when 2 ml of elution solvent (methyl tert-butyl ether: n-hexane = 2:1, V / V) was used for elution each time, the content of the target substance eluted each time showed a decreasing trend. By the third elution, the content of the target substance was lower than the instrument detection limit. Therefore, it was finally chosen to elute twice with 2 ml of elution solvent.

[0040] 2.3 Optimization of chromatographic conditions 2.3.1 Determination of chromatographic column The experiment investigated Welch Ultimate® PFP (2.1 mm × 100 mm, 5 μm), ACQUITY UPLC® HSS T3 (2.1 mm × 50 mm, 1.8 μm), ACQUITY UPLC® BEHC 18 (2.1 mm×50 mm, 1.7 μm) Separation effects of three chromatographic columns.

[0041] The results show that using C 18 When using the T3 column, the two synthetic cannabinoids ADB-PINCA and AB-CHMINACA showed peak tailing and many interfering peaks, resulting in poor separation. This may be because the synthetic cannabinoids and cathinones used are a series of derivatives obtained by chemical modification on the basis of the original substances (mainly by introducing weak polar groups on the benzene ring), and their polarity is similar, resulting in C 18 The separation effect of the T3 column is not good; the bonding of the PFP column has good selectivity for such substances, so the separation effect is good, the peak shape is symmetrical, and the overall performance is better than that of the C18 and T3 columns.

[0042] 2.3.2 Determination of organic phase in mobile phase The effects of acetonitrile and methanol on the separation effect were investigated in the experiment. The results showed that acetonitrile had a better separation effect on synthetic cannabinoids and cathinones than methanol, and its peak shape was sharp and symmetrical. Therefore, acetonitrile was selected as the organic phase in the mobile phase.

[0043] 2.3.3 Determination of the amount of salt in the mobile phase Synthetic cannabinoids and cathinones are mostly weakly alkaline or neutral. Adding formic acid in positive ion mode can promote the ionization of the target, and adding ammonium acetate can enhance the stability of the target and improve the separation effect. The separation effect of adding different amounts of salt to the aqueous phase was investigated in the experiment. The results showed that when 20 mmol / L ammonium acetate was added to the aqueous solution of 0.1% formic acid, the mass spectrometry response of the target was the best and the peak shape was the best.

[0044] 2.4 Methodological validation Take the blank saliva matrix prepared in 1.2.2.1, add the mixed standard stock solution to prepare a series of mixed standard solutions with mass concentrations of 0, 2, 5, 10, 20, 50, and 100 ng / mL for testing on the machine, and then draw a standard curve with the mass concentration of each component as the abscissa and the peak area as the ordinate, and perform linear fitting. The results are shown in Table 3.

[0045] Table 3 Linear range, linear equation, correlation coefficient, detection limit and quantification limit of 15 new psychoactive substances

[0046] The results in Table 3 show that the 15 new psychoactive substances have a good linear relationship in the range of 0-100 ng / mL, and the correlation coefficient (R 2 ) was in the range of 0.995~0.999. The detection limit and quantification limit of the method were determined by 3 times signal-to-noise ratio (S / N=3) and 10 times signal-to-noise ratio (S / N=10). The results showed that the detection limit of 15 new psychoactive substances was 0.2~0.5 ng / mL, and the quantification limit was 1~2ng / mL.

[0047] Accurately pipette 0.5 ml of blank saliva sample, add 2, 10, 50 ng / mL of the mixed standard working solution of 15 new psychoactive substances, respectively, and treat according to the pretreatment method under 1.2.2.1. Perform 6 parallel measurements for each spike level and calculate the recovery. The spike recovery is shown in Table 4.

[0048] Table 4 Spiked recoveries and relative standard deviations of 15 new psychoactive substances (n=6)

[0049] As shown in Table 4, the recoveries at the three spiked concentrations were 85.7%~113.7%, and the relative standard deviations (RSDs) were 3.6%~14.2%, indicating that the method had good spiked recoveries and stability.

[0050] 2.5 Actual sample analysis The method was used to detect the saliva samples of 10 drug addicts captured by the local police, and SKF 525A was used as the internal standard. The results showed that the recovery rate of the internal standard was (92.7±10.8)%, indicating that the analysis results were reliable. One positive sample of new psychoactive substances was detected in the saliva samples of 10 drug addicts. The detected substance was the synthetic cannabinoid ADB-PINCA, with a content of 19.8 ng / mL. Its chromatogram is shown in the figure below. Figure 6 This proves that the method established by the present invention can be used to verify actual cases.

[0051] 3 Conclusion The present invention establishes a method for determining 15 new psychoactive substances in saliva by solid-phase supported liquid-liquid extraction combined with ultra-high performance liquid chromatography tandem mass spectrometry. Compared with the traditional liquid-liquid extraction method, the solid-phase supported liquid-liquid extraction method significantly reduces the matrix effect of saliva samples and has the advantages of rapidity, accuracy, high sensitivity and simple operation. The detection limit of this method is 0.2~0.5 ng / mL, the quantitative limit is 1~2 μg / L, the average recovery rate of spiked is 85.7%~113.7%, and the relative standard deviation is 3.6%~14.2%. It is suitable for qualitative and quantitative detection of saliva samples and can meet the limit requirements at home and abroad, providing technical support for the monitoring and investigation of new psychoactive substances.

[0052] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for determining 15 new psychoactive substances in saliva by solid phase supported liquid-liquid extraction combined with ultra-high performance liquid chromatography tandem mass spectrometry, characterized in that: The steps include: 1) Sample pretreatment: Take 0.5 mL of blank saliva sample and saliva sample respectively, load them onto solid phase supported liquid extraction cartridges, apply positive pressure to disperse the samples and let them stand for 5 minutes, then add elution solvent twice, 2 mL each time, let them stand for another 5 minutes, collect the eluate at a flow rate of 1 mL / min, blow the collected eluate to near dryness with nitrogen at 40 °C, re-dissolve it with 0.5 mL of acetonitrile, and filter it through a 0.22 μm microporous filter membrane to obtain blank saliva matrix and sample solution; 2) Preparation of mixed standard solution: The 15 new psychoactive substance standards were dissolved in methanol and then fixed to volume to prepare mixed standard stock solutions with a concentration of 1.0 μg / mL. Before use, appropriate amounts were taken and diluted with the prepared blank saliva matrix to prepare a series of mixed standard solutions with a mass concentration of 0-100 ng / mL. 3) Drawing of standard curve: The sample solution obtained in step 1) and the series of mixed standard solutions obtained in step 2) were respectively determined by ultra high performance liquid chromatography tandem mass spectrometry, and then standard curves were drawn with the mass concentration of 15 new psychoactive substances in the series of mixed standards as the horizontal axis and the peak area as the vertical axis; the types and contents of the new psychoactive substances contained in the sample solutions were then determined based on the sample peak results combined with the obtained standard curves.

2. The method according to claim 1, characterized in that The 15 new psychoactive substances are specifically pentyl-3-(4-methoxybenzoyl)indole, 1-butyl-3-(1-naphthoyl)indole, 1-pentyl-3-(4-ethyl-1-naphthoyl)indole, 1-hexyl-3-(1-naphthoyl)indole, 1-pentyl-3-(1-naphthoyl)indole, N-(1-carbamoyl-2-methylpropyl)-1-(cyclohexylmethyl)indazole-3-carboxamide, 1-pentyl-3-( 4-methyl-1-naphthoyl)indole, 2-(2-methoxyphenyl)-1-(1-pentyl-1H-indol-3-yl)ethanone, 1-(5-fluoropentyl)-3-(1-naphthoyl)-1H-indole, N-(1-carbamoyl-2,2-dimethylpropyl)-1-pentylindazole-3-carboxamide, 4-methyl-α-pyrrolidinophenone, promazine hydrochloride, 4-fluoromethcathinone, 4'-methyl-α-pyrrolidinophenone, ethyl hydrochloride.

3. The method according to claim 1, characterized in that The elution solvent in step 1) is a mixture of methyl tert-butyl ether and n-hexane in a volume ratio of 2:

1.

4. The method according to claim 1, characterized in that The ultra-high performance liquid chromatography conditions used in step 3) are as follows: chromatographic column: Welch Ultimate® PFP, specification: 2.1 mm × 100 mm, 5 μm; Column temperature: 40 ℃; injection volume: 10 μL; mobile phase A is 0.1 vol% formic acid aqueous solution containing 20 mmol / L ammonium acetate, and mobile phase B is acetonitrile; flow rate is 0.20 mL / min; gradient elution program is: 0-1.0 min, the volume proportion of mobile phase A is 70%; 1.0-2.0 min, the volume proportion of mobile phase A decreases from 70% to 30%A; From 2.0 to 4.0 min, the volume proportion of mobile phase A was maintained at 30%; from 4.0 to 5.0 min, the volume proportion of mobile phase A increased from 30% to 70%A; from 5.0 to 6.0 min, the volume proportion of mobile phase A was maintained at 70%A.

5. The method according to claim 1, characterized in that The mass spectrometry conditions used in step 3) are: electrospray ion source; positive ion mode; multiple reaction monitoring; capillary voltage: 3 kV; ion source temperature: 350 °C; nebulizer gas: nitrogen; nebulizer gas flow rate: 1000 L / h; collision gas: argon; collision gas flow rate: 50 L / h.

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