Method for determination of 15 new psychoactive substances in saliva by SLE-UPLC-MS / MS

Abstract

The application discloses a method for determining 15 new psychoactive substances in saliva by solid phase supported liquid-liquid extraction (SLE) combined with ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS / MS), which specifically uses a mixed solution of methyl tert-butyl ether and n-hexane as an elution solvent, adopts solid phase supported liquid-liquid extraction to pretreat saliva samples, and then adopts UPLC-MS / MS to detect the collected eluent, so as to realize qualitative and quantitative determination of the new psychoactive substances in saliva. The method has the advantages of rapidness, accuracy, high sensitivity and simple operation, the detection limit of the method is 0.2-0.5 ng / mL, can meet the domestic and foreign limit requirements, and provides technical support for monitoring and investigation of the new psychoactive substances in saliva.

Description

Technical Field

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

[0002] Saliva is a colorless, thin fluid secreted in the oral cavity, composed of salivary gland secretions, oral mucosal exudate, and gingival crevicular fluid. It contains various components, such as water, mucin, amylase, lysozyme, inorganic salts, drug residues, and metabolites. Drug molecules in saliva originate from two sources: oral drug residues and free drug molecules from the blood that enter the salivary glands through epithelial cells. Compared to traditional biological samples such as blood, urine, and hair, saliva offers advantages such as non-invasive and convenient sampling, no location restrictions, and a low probability of cross-infection, making it an important research subject for current technical law enforcement.

[0003] New psychoactive substances have seen rapid growth in the drug market due to their low cost, varied structures, and ease of circumventing regulations. Currently, the most abused new psychoactive substances globally include synthetic cannabinoids, cathinones, ketamine, and fentanyl-related substances. In my country, drug users primarily abuse synthetic cannabinoids, ketamine, cathinones, and tryptophan-related substances.

[0004] Current research on drugs in saliva mainly focuses on traditional drugs, with limited and limited coverage of new psychoactive substances, failing to meet the rapidly growing detection needs for these substances. Furthermore, sample pretreatment methods primarily employ protein precipitation, liquid-liquid extraction (LLE), solid-phase extraction (SPE), and dispersive solid-phase extraction (dSPE). Protein precipitation and LLE exhibit significant matrix effects, while SPE is cumbersome and time-consuming, making it unsuitable for the demands of efficient law enforcement and large-scale screening. Therefore, there is an urgent need to establish rapid and efficient methods for detecting new psychoactive substances in saliva.

[0005] Solid-phase supported liquid-liquid extraction (SLE) is a simple procedure that effectively removes proteins, phospholipids, and other components from biological samples, significantly reducing matrix effects and preventing emulsification. However, there are currently no research reports on the use of SLE columns for the enrichment and purification of new psychoactive substances in saliva samples. This invention employs SLE for saliva sample pretreatment and combines it with ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) to establish a method for detecting 10 synthetic cannabinoids and 5 cathinones, which are the most abundant and widely prevalent new psychoactive substances. This method can provide a reference for the rapid detection and combating of new psychoactive substances. Summary of the Invention

[0006] The purpose of this invention is to provide a method for determining 15 new psychoactive substances in saliva using solid-phase supported liquid-liquid extraction (SLE) combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). This method has the advantages of being rapid, accurate, highly sensitive, and simple to operate, and can meet domestic and international limit requirements, providing technical support for the monitoring and investigation of new psychoactive substances in saliva.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for determining 15 new psychoactive substances in saliva using solid-phase supported liquid-liquid extraction (SLE) combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) includes the following steps:

[0009] 1) Sample pretreatment:

[0010] Take 0.5 mL of blank saliva sample and 0.5 mL of saliva sample respectively, load them onto a solid-phase support liquid-liquid extraction column, apply positive pressure using a solid-phase extraction device to disperse the sample onto the packing material, let stand for 5 minutes, then add 2 mL of elution solvent in two portions, let stand for another 5 minutes, and collect the eluent at a flow rate of 1 mL / min. Then, blow the collected eluent to near dryness with nitrogen at 40 °C, redissolve it with 0.5 mL of acetonitrile, filter it through a 0.22 μm microporous membrane to obtain blank saliva matrix and sample solution;

[0011] 2) Preparation of mixed standard solutions:

[0012] Fifteen new psychoactive substances were dissolved in methanol and diluted to a final volume to prepare mixed standard stock solutions with a concentration of 1.0 μg / mL. Before use, an appropriate amount was measured, diluted with the prepared blank saliva matrix, and prepared into a series of mixed standard solutions with a mass concentration of 0-100 ng / mL.

[0013] 3) Plotting the standard curve:

[0014] The sample solution obtained in step 1) and the series of mixed standard solutions obtained in step 2) were respectively determined by UPLC-MS / MS. Then, standard curves were plotted with the mass concentration of 15 new psychoactive substances in the series of mixed standards as the abscissa and the peak area as the ordinate. Based on the sample peak results and the obtained standard curves, the types and contents of new psychoactive substances contained in the sample solution were determined.

[0015] Further, 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- 1-(5-fluoropentyl)-3-(1-naphthoyl)-1H-indole (AM2201), 1-(1-carbamoyl-2,2-dimethylpropyl)-1-pentylindazole-3-carboxamide (ADB-PINACA), 4-methyl-α-pyrrolidine phenylbutanone (MPBP), Promazine hydrochloride, 4-fluoromethcathinone (4-FMC), 4'-methyl-α-pyrrolidine phenylhexanone (PV-4), and ethyl hydrochloride (MDEC).

[0016] Further, the elution solvent mentioned in step 2) is a mixture of methyl tert-butyl ether and n-hexane in a volume ratio of 2:1.

[0017] Further, the ultra-high performance liquid chromatography (UHPLC) conditions used in step 3) are as follows: column: Welch Ultimate® PFP (2.1 mm × 100 mm, 5 μm); column temperature: 40 ℃; 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: 0.20 mL / min; gradient elution program: 0~1.0 min, 70%A; 1.0~2.0 min, 70%~30%A; 2.0~4.0 min, 30%A; 4.0~5.0 min, 30%~70%A; 5.0~6.0 min, 70%A.

[0018] Furthermore, the mass spectrometry conditions used in step 3) are as follows: electrospray ionization source; positive ion mode; multiple reaction monitoring; 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.

[0019] The significant advantages of this invention are:

[0020] Compared with traditional liquid-liquid extraction methods, this invention uses solid-phase supported liquid-liquid extraction, which can significantly reduce the matrix effect of saliva samples. Combining this with ultra-high performance liquid chromatography-tandem mass spectrometry, the established assay method has the advantages of being rapid, accurate, highly sensitive, and simple to operate. Moreover, the detection limit of this method is 0.2~0.5 ng / mL, which meets the domestic and international limit requirements, providing technical support for the monitoring and investigation of new psychoactive substances in saliva. Attached Figure Description

[0021] Figure 1 This is a graph showing the comparison of the recovery rates of 15 new psychoactive substances using different elution solvents in the examples.

[0022] Figure 2 This is a graph showing the matrix effect comparison of 15 new psychoactive substances using different elution solvents in the examples.

[0023] Figure 3 The recovery rates of 15 new psychoactive substances were achieved using different ratios of mixed elution solvents in the examples.

[0024] Figure 4 The matrix effect of different ratios of mixed elution solvents on 15 new psychoactive substances is shown in the examples.

[0025] Figure 5 The image shows the total ion chromatogram of 15 new psychoactive substances obtained in the examples.

[0026] Figure 6 This is the extraction chromatogram of the actual sample measured in the examples. Detailed Implementation

[0027] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto. Example

[0028] 1. Experimental Section

[0029] 1.1 Instruments and Reagents

[0030] Xero TQD liquid chromatography-tandem mass spectrometry system (Waters Corporation, USA); ST16R high-speed centrifuge (Thermofisher Corporation, USA); SBHCONC / 1 nitrogen evaporator (Stuart GmbH, Germany); MTS240C multi-tube vortex mixer (Shanghai Dam Industrial Co., Ltd.); Milli-Q ultrapure water system (Millipore Corporation, USA).

[0031] 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-indole-3-yl) ethyl ketone (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-pyrrolidine phenylbutanone (MPBP), promethazine hydrochloride (Promazine), 4-fluoromethcathinone (4-FMC), 4'-methyl-α-pyrrolidine phenylhexanone (PV-4), ethyl hydrochloride (MDEC), diphenylpentanediamine (SKF) 525A (content 100 μg / mL, Tianjin Alta Technology Co., Ltd.); n-hexane, ethyl acetate, dichloromethane, and methyl tert-butyl ether (analytical grade, CNW GmbH, Germany); methanol and acetonitrile (chromatographic grade, Merck GmbH, Germany); formic acid (chromatographic grade, Shandong Xiya Company); ammonium acetate (chromatographic grade, Shanghai Aladdin Company); 2 mL solid-phase support liquid-liquid extraction column (Biotage AG, Sweden); other reagents were domestically produced analytical grade or superior grade; the experimental water was deionized water.

[0032] Blank saliva samples were provided by healthy adult volunteers with no history of drug use.

[0033] 1.2 Experimental Methods

[0034] 1.2.1 Preparation of standard solutions

[0035] Take an appropriate amount of the standard solutions of 15 new psychoactive substances, dissolve them in methanol, and dilute to 10 mL in a volumetric flask to prepare a mixed standard stock solution with a concentration of 1.0 μg / mL for later use.

[0036] 1.2.2 Sample Pretreatment

[0037] 1.2.2.1 Solid-phase supported liquid-liquid extraction

[0038] Take 0.5 mL each of blank saliva sample and saliva sample, and load them onto a 2 mL solid-phase support liquid-liquid extraction column. Apply positive pressure using a solid-phase extraction device to disperse the samples onto the packing material. Let stand for 5 minutes, then add elution solvent (methyl tert-butyl ether: n-hexane = 2:1, V / V) at a rate of 2 × 2 mL. After standing for 5 minutes, collect the eluent at a flow rate of 1 mL / min. After the eluent has been completely added, apply positive pressure to drain the remaining eluent. The collected eluent is purged to near dryness with nitrogen at 40 °C, redissolved with 0.5 mL of acetonitrile, and filtered through a 0.22 μm microporous membrane to obtain the blank saliva matrix and sample solution, ready for instrumental analysis.

[0039] 1.2.2.2 Liquid-liquid extraction

[0040] Take 0.5 ml of saliva sample, add 1 mL of borate buffer (pH 9.2), extract with 2 mL of diethyl ether, vortex, and centrifuge. Evaporate the supernatant to dryness in a 60°C water bath, reconstitute with 0.5 mL of acetonitrile via vortexing, filter through a 0.22 μm microporous membrane, and prepare for instrumental analysis.

[0041] 1.2.3 UPLC-MS / MS Analysis

[0042] 1.2.3.1 Chromatographic conditions

[0043] Chromatographic column: Welch Ultimate® PFP (2.1 mm × 100 mm, 5 μm), mobile phase A was 0.1% formic acid aqueous solution containing 20 mmol / L ammonium acetate, mobile phase B was acetonitrile, flow rate: 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; 2.0~4.0 min, 30% A; 4.0~5.0 min, 30%~70% A; 5.0~6.0 min, 70% A.

[0044] 1.2.3.2 Mass Spectrometry Conditions

[0045] 5. Electrospray ionization source; positive ion mode (ESI+); multiple reaction monitoring (MRM); capillary voltage: 3 kV; ion source temperature: 350 ℃; nebulizing gas: nitrogen; nebulizing gas flow rate: 1000 L / h; collision gas: argon; collision gas flow rate: 50 L / h; mass spectrometry parameters of 15 new psychoactive substances are shown in Table 1.

[0046] Table 1. Relevant information and analytical parameters of 15 new psychoactive substances and internal standards.

[0047]

[0048] 1.3 Data Processing

[0049] Experimental data were collected, analyzed, and processed using the instrument's built-in Masslynx data processing system, and charts were generated using Excel and Origin 9.0 software.

[0050] 2 Results and Analysis

[0051] 2.1 Comparison of Pretreatment Methods

[0052] Two pretreatment methods, liquid-liquid extraction and solid-phase support liquid-liquid extraction, were investigated in the experiment. Specifically, blank saliva samples were taken and prepared into 10 ng / mL target compound additive samples. The pretreatment was carried out using the methods under sections 1.2.2.1 and 1.2.2.2 (three parallel samples were prepared for each concentration), and the recovery rate of the compound was used as the evaluation index. The results are shown in Table 2.

[0053] Table 2 Comparison of the effects of different pretreatment methods

[0054]

[0055] The results in Table 2 show that when solid-phase supported liquid-liquid extraction was used for pretreatment, the recovery rates of the 15 new psychoactive substances ranged from 88.3% to 113.8%, which was significantly better than the 41.2% to 70.4% recovery rate of liquid-liquid extraction.

[0056] 2.2 Optimization of Solid-Phase Support Liquid-Liquid Extraction Conditions

[0057] Solid-phase supported liquid-liquid extraction columns use diatomaceous earth processed with a special technique as packing material. Therefore, the elution solvent must be a solution immiscible with water, and the choice of solvent depends on the solubility and polarity of the target analyte. Since the compounds to be detected are mostly weakly or moderately polar substances, the extraction effects of five solvents—n-hexane, methyl tert-butyl ether, dichloromethane, diethyl ether, and ethyl acetate—were investigated in the experiment (three parallel experiments were set up for each solvent). The recovery rate of the compounds was used as the evaluation index, and the results are shown in [Figure number missing]. Figure 1 .

[0058] like Figure 1 As shown, the extraction effects of the five solvents varied considerably. Among them, the recovery rate of n-hexane was 23.5%~41.2%, with a low elution efficiency. The recovery rates of methyl tert-butyl ether, dichloromethane, diethyl ether, and ethyl acetate were relatively high. Among them, methyl tert-butyl ether had the most ideal extraction effect, with a recovery rate ranging from 82.6% to 94.5%.

[0059] As the polarity of the elution solvent increases, the amount of eluted salivary matrix also increases, resulting in a stronger matrix effect. The matrix effects of the five elution solvents are shown in [reference needed]. Figure 2 .

[0060] like Figure 2 As shown, the moderately polar solvents diethyl ether and ethyl acetate exhibit a strong matrix inhibition effect, while the matrix inhibition effect is weaker when dichloromethane and methyl tert-butyl ether are used. Therefore, methyl tert-butyl ether is preferred as the elution solvent.

[0061] Considering that n-hexane has minimal chromatographic peak interference when used as an elution solvent, and that it is chemically stable, does not readily react with the stationary phase, and is volatile, the elution capacity and matrix effect of mixed elution solvents prepared with methyl tert-butyl ether and n-hexane in different ratios were further investigated. The results are shown in the figures below. Figure 3 , 4 .

[0062] Depend on Figure 3 , 4 It is evident that when the ratio of methyl tert-butyl ether to n-hexane is 2:1, the recoveries of 15 new psychoactive substances range from 88.3% to 113.8%, indicating a weak matrix effect. However, increasing the content of methyl tert-butyl ether in the eluent does not significantly improve the recovery rate, and the recoveries of two synthetic cannabinoids, ADB-PINCA and AB-CHMINACA, decrease, indicating a high matrix effect. Furthermore, increasing the content of n-hexane in the eluent significantly reduces the recovery rates of most compounds. Therefore, after comprehensive consideration, a volume ratio of methyl tert-butyl ether to n-hexane of 2:1 was selected as the eluent.

[0063] Based on this, the amount of elution solvent used was further optimized. The results showed that when eluting with 2 ml of elution solvent (methyl tert-butyl ether: n-hexane = 2:1, V / V), the content of the target substance eluted each time showed a decreasing trend. By the third elution, the content of the target substance was already below the instrument detection limit. Therefore, it was finally decided to use 2 ml of elution solvent for elution twice.

[0064] 2.3 Optimization of chromatographic conditions

[0065] 2.3.1 Determination of chromatographic column

[0066] The experiments examined Welch Ultimate® PFP (2.1 mm × 100 mm, 5 μm), ACQUITY UPLC® HSS T3 (2.1 mm × 50 mm, 1.8 μm), and ACQUITY UPLC® BEHC. 18 Separation performance of three chromatographic columns (2.1 mm × 50 mm, 1.7 μm).

[0067] The results show that using C 18When using a T3 column, the synthetic cannabinoids ADB-PINCA and AB-CHMINACA exhibited peak tailing and numerous interfering peaks, resulting in poor separation. This may be because the synthetic cannabinoids and cathinones used are a series of derivatives obtained through chemical modification of the original substances (mainly by introducing weakly polar groups onto the benzene ring). Their similar polarity leads to C 18 The separation performance of the T3 column was poor; while the PFP column showed good selectivity for these substances, resulting in better separation, symmetrical peaks, and overall superior performance compared to the C column. 18 And T3 chromatographic column.

[0068] 2.3.2 Determination of the organic phase in the mobile phase

[0069] The effects of acetonitrile and methanol on the separation efficiency were investigated in the experiment. The results showed that acetonitrile was superior to methanol in the separation of synthetic cannabinoids and cathinones, with sharp and symmetrical peaks. Therefore, acetonitrile was chosen as the organic phase in the mobile phase.

[0070] 2.3.3 Determination of salt content in the mobile aqueous phase

[0071] Synthetic cannabinoids and cathinones are mostly weakly basic or neutral. Adding formic acid in positive ion mode can promote the ionization of the target analytes, while adding ammonium acetate can enhance their stability and improve separation efficiency. The separation effects of adding different amounts of salt to the aqueous phase were investigated in the experiment. The results showed that adding 20 mmol / L ammonium acetate to a 0.1% formic acid aqueous solution resulted in the best mass spectrometric response and peak shape for the target analytes.

[0072] 2.4 Methodological Validation

[0073] Take the blank saliva matrix prepared under section 1.2.2.1, add it to 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 instrument testing. Then, plot the 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.

[0074] Table 3. Linear range, linear equation, correlation coefficient, limit of detection and limit of quantitation for 15 new psychoactive substances.

[0075]

[0076] The results in Table 3 show that the 15 new psychoactive substances exhibited good linearity in the range of 0–100 ng / mL, with correlation coefficients (R0 and R0). 2The range was 0.995–0.999. The limits of detection (LOD) and quantitation (LOQ) were determined using a signal-to-noise ratio (S / N) of 3 and 10 times (S / N=10). The results showed that the LOD for 15 new psychoactive substances was 0.2–0.5 ng / mL, and the LOD was 1–2 ng / mL.

[0077] Accurately transfer 0.5 ml of blank saliva sample and add mixed standard working solutions of 15 new psychoactive substances at concentrations of 2, 10, and 50 ng / mL, respectively. Process the samples according to the pretreatment method in section 1.2.2.1. Perform six parallel determinations at each spiking level and calculate the recovery rate. The spiking recovery rates are shown in Table 4.

[0078] Table 4. Spiked recoveries and relative standard deviations of 15 new psychoactive substances (n=6)

[0079]

[0080] As shown in Table 4, the recoveries at the three spiked concentrations ranged from 85.7% to 113.7%, and the relative standard deviations (RSDs) ranged from 3.6% to 14.2%, indicating that the method has good spiked recoveries and stability.

[0081] 2.5 Analysis of actual samples

[0082] This method was used to test saliva samples from 10 drug users arrested by local police, with SKF 525A used as an internal standard. The results showed an internal standard recovery rate of (92.7±10.8)%, indicating reliable analytical results. One sample from the saliva of the 10 drug users tested positive for a new psychoactive substance, the synthetic cannabinoid ADB-PINCA, at a concentration of 19.8 ng / mL. Its chromatogram is shown below. Figure 6 This demonstrates that the method established in this invention can be tested in real-world cases.

[0083] 3. Conclusion

[0084] This invention establishes a method for determining 15 new psychoactive substances in saliva using solid-phase supported liquid-liquid extraction combined with ultra-high performance liquid chromatography-tandem mass spectrometry. Compared with the traditional liquid-liquid extraction method, solid-phase supported liquid-liquid extraction significantly reduces the matrix effect of saliva samples and has the advantages of being rapid, accurate, highly sensitive, and simple to operate. The detection limit of this method is 0.2–0.5 ng / mL, the quantitation limit is 1–2 μg / L, the average recovery rate is 85.7%–113.7%, and the relative standard deviation is 3.6%–14.2%. This method is suitable for qualitative and quantitative detection of saliva samples and meets domestic and international limit requirements, providing technical support for the monitoring and investigation of new psychoactive substances.

[0085] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for determining 15 new psychoactive substances in saliva using solid-phase supported liquid-liquid extraction combined with ultra-high performance liquid chromatography-tandem mass spectrometry, characterized in that, Includes the following steps: 1) Sample pretreatment: Take 0.5 mL of blank saliva sample and 0.5 mL of saliva sample respectively, load them onto a solid-phase support liquid-liquid extraction column, apply positive pressure to disperse the sample and let it stand for 5 minutes, then add 2 mL of elution solvent in two portions, let it stand for another 5 minutes, and collect the eluent at a flow rate of 1 mL / min. Blow the collected eluent to near dryness with nitrogen at 40 °C, redissolve it with 0.5 mL of acetonitrile, and filter it through a 0.22 μm microporous membrane to obtain blank saliva matrix and sample solution; 2) Preparation of mixed standard solutions: Fifteen new psychoactive substances were dissolved in methanol and diluted to a final volume to prepare mixed standard stock solutions with a concentration of 1.0 μg / mL. Before use, an appropriate amount was measured, diluted with the prepared blank saliva matrix, and prepared into a series of mixed standard solutions with a mass concentration of 0-100 ng / mL. 3) Plotting the 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. Then, standard curves were plotted with the mass concentration of 15 new psychoactive substances in the series of mixed standards as the abscissa and the peak area as the ordinate. Based on the sample peak results and the obtained standard curves, the types and contents of new psychoactive substances contained in the sample solution were determined. 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-indole-3-yl)acetone, 1-(5-fluoropentyl)-3-(1-naphthoyl)-1H-indole, N-(1-carbamoyl-2,2-dimethylpropyl)-1-pentylindazole-3-carboxamide, 4-methyl-α-pyrrolidine phenylbutanone, promethazine hydrochloride, 4-fluoromethcathinone, 4'-methyl-α-pyrrolidine phenylhexanone, ethyl hydrochloride; The ultra-high performance liquid chromatography (UHPLC) conditions used in step 3) were as follows: column: Welch Ultimate® PFP, 2.1 mm × 100 mm, 5 μm; Column temperature: 40 ℃; injection volume: 10 μL; mobile phase A was a 0.1 vol% formic acid aqueous solution containing 20 mmol / L ammonium acetate, and mobile phase B was acetonitrile; flow rate: 0.20 mL / min; gradient elution program: 0~1.0 min, mobile phase A volume percentage is 70%; 1.0~2.0 min, the volume percentage of mobile phase A decreases from 70% to 30%A; From 2.0 to 4.0 min, the volume percentage of mobile phase A remained at 30%; from 4.0 to 5.0 min, the volume percentage of mobile phase A increased from 30% to 70%A; from 5.0 to 6.0 min, the volume percentage of mobile phase A remained at 70%A. The mass spectrometry conditions used were: electrospray ionization source; positive ion mode; multiple reaction monitoring; 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.

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

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