Sample pretreatment method applicable to detection of ketamine analogues in biological detection material and detection method of ketamine analogues in biological detection material

Abstract

The sample pretreatment method comprises the following steps: adding a salting-out agent into the biological detection material to be detected, oscillating and uniformly mixing, adding ammonia water to adjust the pH value, then adding acetonitrile, and continuing oscillating treatment; and then freezing and centrifuging, separating supernate, and filtering to obtain a finished product. The invention establishes a sample pretreatment method for nine ketamine analogues in biological detection materials simultaneously suitable for UPLC-MS / MS and GC-MS / MS, one-time preparation of the sample can be realized, simultaneous sample injection detection of UPLC-MS / MS and GC-MS / MS can be realized, and detection results are mutually verified. The method can realize rapid qualitative and quantitative analysis of nine ketamine analogues in blood, urine and liver, and provides powerful technical support for poisoning diagnosis, clinical treatment and treatment of related cases of common ketamine analogues.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical detection, in particular to a sample pretreatment method and a detection method thereof suitable for detecting ketamine analogs in biological samples. Background Art

[0002] Ketamine is the new psychoactive substance with the most countries and regions reporting to the United Nations Office on Drugs and Crime, and its abuse is severe. Since ketamine has been listed as a controlled drug in countries around the world, in order to evade legal sanctions, criminals have been constantly seeking new ketamine analogs by modifying their chemical structures while retaining the aromatic cycloethylamine core structure of ketamine, so as to sell them as "legal ketamine". For each new ketamine analog, it is necessary to establish a new analytical method to detect these compounds in biological matrices and interpret the results. Therefore, it is very necessary to develop detection methods for ketamine analogs.

[0003] Common detection methods for ketamine analogs include immunoassay, chromatography, mass spectrometry, chromatography-mass spectrometry, etc. Among them, chromatography-mass spectrometry (gas chromatography-mass spectrometry and liquid chromatography) can effectively separate and purify the substance to be tested through the chromatographic part, and use the mass spectrometer as a detector to provide more material fragment information, which solves the problem of difficult qualitative analysis of the chromatography itself and greatly enhances the accuracy of the measurement. At the same time, the introduction of tandem mass spectrometry further reduces the interference of non-target ion fragments and enhances the sensitivity of the test. However, the current detection methods for common ketamine analogs are still based on the current situation of detecting a single or a small number of substances with a single instrument, and cannot effectively detect multiple common ketamine analogs at the same time.

[0004] Moreover, due to the differences in the injection methods and separation principles of the current gas chromatography-mass spectrometry and liquid chromatography methods, the sample pretreatment and injection solvents of the two methods are different. For example, liquid chromatography-tandem mass spectrometry generally uses acetonitrile to precipitate proteins, and then uses acetonitrile or methanol and other solvents with higher polarity for direct injection; while gas chromatography-tandem mass spectrometry generally uses ethyl acetate for liquid-liquid extraction, and the supernatant needs to be concentrated and evaporated to a fixed volume, and then ethyl acetate, propanol and other solvents with relatively lower polarity are used for injection. Therefore, there are some differences in the sample pretreatment methods and injection solvents of the two methods, that is, when mutual verification of two test results is required, such as when the analyte needs to be tested by gas chromatography-mass spectrometry and liquid chromatography at the same time, different pretreatment methods are often required for the analyte.

[0005] Therefore, how to achieve the simultaneous detection of multiple common ketamine analogs, find a sample pretreatment method that is suitable for both gas chromatography-mass spectrometry and liquid chromatography detection, and further improve the qualitative and quantitative detection effect and efficiency of common ketamine analogs in biological samples are technical issues that need to be further resolved. Summary of the invention

[0006] In view of the technical problems existing in the prior art, the present invention provides a sample pretreatment method and a detection method suitable for the inspection of ketamine analogues in biological samples. The method can realize the simultaneous detection of 9 common ketamine analogues, and can be simultaneously applicable to the detection of ultra-high performance liquid chromatography tandem mass spectrometry and gas chromatography tandem mass spectrometry.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] The present invention relates to a sample pretreatment method suitable for testing ketamine analogs in biological samples, comprising the following steps:

[0009] Add a salting-out agent to the biological sample to be tested, shake and mix, add ammonia water to adjust the pH, then add acetonitrile, continue shaking treatment; then freeze centrifuge, separate the supernatant, filter, and obtain.

[0010] Preferably, the ketamine analogue contained in the biological sample to be tested is any one or more combinations of ketamine, norketamine, deschloroketamine, fluamine, phencyclidine, tiletamine, methoxitamine, 2-oxo-PCE, and 3-MeO-PCE.

[0011] Preferably, the biological sample to be tested is liquid or solid; the liquid is blood or urine; the solid is liver.

[0012] The method of the present invention is applicable to the detection of blood, that is, whole blood, thus avoiding the problem that the existing method is only applicable to serum and plasma.

[0013] Preferably, when the biological sample to be tested is a liquid, the volume ratio of the amount of the salting-out agent to the biological sample to be tested is 1-10 mmol:1 mL; the volume ratio of the biological sample to be tested to acetonitrile is 1:2-4;

[0014] More preferably, when the biological sample to be tested is a liquid, the volume ratio of the amount of the salting-out agent to the biological sample to be tested is 2 mmol:1 mL; the volume ratio of the biological sample to be tested to acetonitrile is 1:2;

[0015] Preferably, when the biological sample to be tested is solid, the mass ratio of the amount of the salting-out agent to the mass of the biological sample to be tested is 1-10 mmol: 1 g; the mass ratio of the biological sample to be tested to the volume of acetonitrile is 1 g: 2-4 mL;

[0016] More preferably, when the biological sample to be tested is solid, the mass ratio of the amount of the salting-out agent to the mass of the biological sample to be tested is 2mmol:1g; the mass ratio of the biological sample to be tested to the volume ratio of acetonitrile is 1g:2mL;

[0017] Wherein, in the salting-out auxiliary liquid-liquid extraction system, the concentration of salting-out agent is too low, and organic phase and aqueous phase cannot realize complete stratification, and the concentration of salting-out agent is too high, which will affect the analysis accuracy, and also will cause certain damage to the instrument. The ratio of extraction solvent (acetonitrile) and blood has a very important impact on the extraction of components to be measured in blood, and higher extraction ratio can make recovery rate and matrix effect better, but also can cause the raising of its detection limit. Therefore, the inventive method optimizes the consumption of salting-out agent and acetonitrile, in order to obtain best extraction efficiency.

[0018] Preferably, the salting-out agent is sodium chloride, magnesium sulfate, potassium carbonate, ammonium formate or ammonium acetate;

[0019] More preferably, the salting-out agent is solid ammonium formate;

[0020] Among them, since the addition of water has a significant impact on the recovery rate of the substance to be measured, the method of the present invention selects to add solid salt. Furthermore, the selection and optimization of the salting-out agent of the present invention is conducive to the separation of the aqueous phase and the organic phase, improves the extraction efficiency, and while achieving phase separation, there is no need for concentration and redissolution treatment, which is more time-saving and labor-saving.

[0021] Preferably, after adding ammonia water, the pH of the system is adjusted to 7-10;

[0022] More preferably, after adding ammonia water, the pH of the system is adjusted to 10;

[0023] Among them, since the aqueous solutions of aromatic cycloethylamine compounds such as ketamine analogs are generally weakly alkaline with a pH between 7 and 10, the most suitable extraction system for extracting ketamine analogs from biological samples is weakly alkaline.

[0024] Preferably, after adjusting the pH, the oscillation treatment speed is 1800-2000 r / min, and the time is 10-20 min;

[0025] Preferably, the temperature of the refrigerated centrifugation treatment is -4°C, the centrifugal speed is 8000-10000 r / min, and the time is 10-20 min;

[0026] Preferably, during filtration, the supernatant is filtered through a 0.22 μm organic membrane.

[0027] Preferably, the biological sample treated by the sample pretreatment method is suitable for detection by both ultra-high performance liquid chromatography tandem mass spectrometry and gas chromatography tandem mass spectrometry.

[0028] Among them, the present invention applies salting-out assisted liquid-liquid extraction to the pretreatment of ketamine analogs in biological samples, and establishes a pretreatment method for ketamine analogs in biological samples that is applicable to both gas chromatography tandem mass spectrometry and ultra-high performance liquid chromatography tandem mass spectrometry. It can achieve one-time pretreatment, UPLC-MS / MS and GC-MS / MS simultaneous sampling and detection, and the detection results can be mutually verified.

[0029] Preferably, the present invention provides a detection method suitable for testing ketamine analogs in biological samples, wherein the biological samples are treated with the sample pretreatment method, and the samples are detected using ultra-high performance liquid chromatography tandem mass spectrometry or gas chromatography tandem mass spectrometry.

[0030] Preferably, when the sample is detected by ultra-high performance liquid chromatography tandem mass spectrometry, the ultra-high performance liquid chromatography conditions are: Eclipse Plus C18 RRHD column; mobile phase: phase A is an aqueous solution containing 0.5‰ (v / v) ammonia and 2mM ammonium formate, and phase B is acetonitrile; flow rate: 0.4mL / min; column temperature 40°C; injection volume 1μL, and the total running time of the analytical method is 8.0min;

[0031] Elution program: 0-1min: 75% phase A + 25 (v / v)% phase B; 1-2min: 75 (v / v)%-30 (v / v)% phase A + 25 (v / v)%-70 (v / v)% phase B; 2-7min: 30 (v / v)%-5 (v / v)% phase A + 70 (v / v)%-95 (v / v)% phase B; 7-7.1min: 5 (v / v)%-75 (v / v)% phase A + 95 (v / v)%-25 (v / v)% phase B; 7.1-8min: 75 (v / v)% phase A + 25 (v / v)% phase B;

[0032] Mass spectrometry conditions: electrospray ion source positive ion mode, multiple reaction monitoring; ion source temperature 550°C, spray voltage 5.5 kV, air curtain gas pressure 20 psi, nebulizer gas pressure 40 psi, auxiliary heating gas pressure 50 psi.

[0033] Preferably, when the sample is detected by gas chromatography tandem mass spectrometry, programmed temperature injection is used;

[0034] Programmable temperature injection port parameters: constant linear velocity mode, linear velocity: 45.1 cm / s; purge flow rate: 3 mL / min; injection mode: split injection, split ratio: 20:1; initial injection port temperature: 65 °C, maintained for 1 min, increased to 280 °C at 200 °C / min, maintained for 6 min;

[0035] Chromatographic conditions: Agilent DB-5MS column, carrier gas: high-purity helium (99.999%), column heating program: initial temperature 60°C, hold for 2 min, increase to 280°C at 10°C / min, hold for 5 min;

[0036] Mass spectrometry conditions: electron bombardment ion source, ion source temperature: 230°C, interface temperature: 320°C, solvent delay time 2 min, scanning mode is multiple reaction monitoring mode, scanning interval is 0.3 s, collision gas is high-purity argon (99.999%).

[0037] Among them, since acetonitrile generally cannot be directly introduced into the gas chromatography, the present invention uses the PTV injection technology to inject the sample into the cold liner tube, gasify the acetonitrile by program temperature increase, and use a large venting gas flow rate to eliminate the acetonitrile gas; then quickly increase the gasification temperature to gasify and inject the substance to be tested. The influence of acetonitrile on the gas chromatography column and the detector is reduced, and the acetonitrile injection of the gas chromatography is realized.

[0038] The technical solution of the present invention achieves the following beneficial technical effects:

[0039] The sample pretreatment method of the present invention adjusts the type and water content of the salting-out agent and further adjusts the pH of the aqueous phase system to better achieve phase separation; and reasonably adjusts the dosage ratio of the biological sample to be tested, the salting-out agent (ammonium formate) and acetonitrile to improve the extraction efficiency. The conditions during UPLC-MS / MS and GC-MS / MS detection are reasonably adjusted. In particular, when applied to gas chromatography tandem mass spectrometry detection, a programmed temperature injection method is adopted, so that the sample pretreatment method of the present invention can be simultaneously applied to UPLC-MS / MS and GC-MS / MS detection, and can achieve simultaneous detection of 9 ketamine analogs.

[0040] The present invention takes blood matrix as the research object, optimizes the sample pretreatment method based on salting-out assisted liquid-liquid extraction, and combines ultra-high performance liquid chromatography tandem mass spectrometry with gas chromatography tandem mass spectrometry to obtain a sample pretreatment method that can detect 9 ketamine analogs in biological samples. The method was then applied to urine matrix and liver matrix. UPLC-MS / MS and GC-MS / MS analysis showed that under the pretreatment conditions, the recovery rate and matrix effect of 9 ketamine analogs were between 85% and 115%, which is also suitable for the pretreatment of 9 ketamine analogs in urine matrix and liver matrix. That is, the sample pretreatment method of the present invention can simultaneously detect 9 ketamine analogs in three biological samples (blood, liver and urine).

[0041] The present invention establishes a sample pretreatment method for salting-out assisted liquid-liquid extraction of 9 ketamine analogs in biological samples that is simultaneously applicable to UPLC-MS / MS and GC-MS / MS, which can achieve one-time sample preparation, simultaneous sampling and detection of UPLC-MS / MS and GC-MS / MS, and mutual verification of detection results. However, existing detection methods are usually unable to achieve simultaneous detection of 9 ketamine analogs, and the sample pretreatment methods are not simultaneously applicable to UPLC-MS / MS and GC-MS / MS. Compared with the existing method of using a single instrument to detect a single or a small number of substances, this method can not only be simultaneously applicable to the detection of 9 substances and 2 instruments, but also has better detection indicators. Among them, for UPLC-MS / MS, the present invention has a correlation coefficient greater than 0.995, a detection limit of 0.05 ng / mL, a quantification limit of 0.1 ng / mL, a recovery rate greater than 88%, and a matrix effect between 85% and 115%; for GC-MS / MS, the present invention has a correlation coefficient greater than 0.998, a detection limit of 0.5 ng / mL, a quantification limit of 1 ng / mL, a recovery rate greater than 88%, and a matrix effect between 85% and 115%; it can realize rapid qualitative and quantitative analysis of 9 ketamine analogs in blood, urine and liver, and provides strong technical support for the diagnosis of common ketamine analog poisoning, clinical treatment and handling of related cases. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Changes in recovery rates at different water contents in Example 4 of the present invention (UPLC-MS / MS);

[0043] Among them, the data corresponding to the value of the vertical axis × 100% is the corresponding recovery rate (%), the same as the following figure;

[0044] Figure 2 Changes in recovery rates at different water contents in Example 4 of the present invention (GC-MS / MS);

[0045] Figure 3Recovery rates (UPLC-MS / MS) at different extraction solvent ratios in Example 5 of the present invention;

[0046] Among them, the horizontal axes are: Ketamine, Norketamine, Deschloroketamine (DCK), Fluoramine (2-FDCK), Phencyclidine (PCP), Tiletamine (Tiletamine), Methoxetamine (MEX), 2-oxo-PCE, 3-MeO-PCE, the same as the following figure;

[0047] Figure 4 Matrix effect (UPLC-MS / MS) at different extraction solvent ratios in Example 5 of the present invention; wherein the data corresponding to the value on the ordinate multiplied by 100% is the corresponding matrix efficiency (%), the same as in the following figure;

[0048] Figure 5 Recovery rates (GC-MS / MS) at different extraction solvent ratios in Example 5 of the present invention;

[0049] Figure 6 Matrix effect (GC-MS / MS) at different extraction solvent ratios in Example 5 of the present invention;

[0050] Figure 7 Recovery rates (UPLC-MS / MS) under different salting-out agents in Example 6 of the present invention;

[0051] Figure 8 Matrix effect (UPLC-MS / MS) under different salting-out agents in Example 6 of the present invention;

[0052] Fig. 9 Recovery rates (GC-MS / MS) under different salting-out agents in Example 6 of the present invention;

[0053] Fig.10 Matrix effect (GC-MS / MS) under different salting-out agents in Example 6 of the present invention;

[0054] Fig.11 Recovery rate of different ammonium formate concentrations in blood in Example 7 of the present invention (UPLC-MS / MS);

[0055] Fig.12 Example 7 of the present invention Matrix effect at different ammonium formate concentrations in blood (UPLC-MS / MS);

[0056] Fig.13 Recovery rate (GC-MS / MS) at different ammonium formate concentrations in blood in Example 7 of the present invention;

[0057] Fig.14Example 7 of the present invention Matrix effect at different ammonium formate concentrations in blood (GC-MS / MS);

[0058] Fig.15 Recovery rate (UPLC-MS / MS) at different ammonia concentrations in blood in Example 8 of the present invention;

[0059] Fig.16 Example 8 of the present invention: Matrix effect at different ammonia concentrations in blood (UPLC-MS / MS);

[0060] Fig.17 Recovery rate (GC-MS / MS) at different ammonia concentrations in blood in Example 8 of the present invention;

[0061] Fig.18 Example 8 of the present invention: Matrix effect (GC-MS / MS) at different ammonia concentrations in blood. DETAILED DESCRIPTION

[0062] In order to make the purpose, technical scheme and technical effect of the invention of this application clearer, the technical scheme in the embodiment of this application will be clearly and completely described below in conjunction with the drawings in the embodiment of this application. Obviously, the described embodiment is a part of the embodiment of this application, not all the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0063] The following is a detailed description of a sample pretreatment method and a detection method thereof suitable for testing ketamine analogs in biological samples according to the present invention through specific embodiments.

[0064] ① Some instruments and reagents used in the embodiments of the present invention:

[0065] LC-30A ultra-high performance liquid chromatograph (Shimadzu, Japan); QTRAP 5500 tandem quadrupole mass spectrometer (AB Sciex, USA); Eclipse Plus C18 (RRHD) chromatographic column (2.1 mm × 100 mm × 1.8 μm, Agilent, USA); GCMS-TQ8050 NX triple quadrupole gas chromatograph-mass spectrometer (Shimadzu, Japan); DB-5MS chromatographic column (30 m × 0.25 mm × 0.25 μm, Agilent, USA);

[0066] Ketamine, norketamine, dechloroketamine (DCK), fluramine (2-fluorodechloroketamine, 2-FDCK), phencyclidine (PCP), tiletamine (Tiletamine), methoxitamine (MEX), 2-oxo-PCE, 3-MeO-PCE and ketamine-D4 (Ketamine-D4) standard solutions were purchased from Tianjin Alta Company, with a concentration of 100 μg / mL.

[0067] Blood matrix (whole blood): from healthy volunteers who have not used the above 9 substances;

[0068] Urine matrix: from healthy volunteers who have not used the above 9 substances;

[0069] Liver matrix: from healthy pig liver that has not been treated with the above 9 substances.

[0070] 2. Preparation of standard solution used in the embodiment of the present invention:

[0071] A mixed standard working solution of ketamine analogs containing 9 ketamine analogs was prepared with methanol: the concentration of each ketamine analog in the mixed standard working solution of ketamine analogs was 10 μg / mL, and it was stored at -20°C; the standard solution of ketamine-D4 was diluted with methanol to obtain a 10 μg / mL standard working solution of ketamine-D4, and it was stored at -20°C. When used, it was diluted step by step with methanol to prepare a standard solution of the required concentration, and it was stored at -20°C.

[0072] Example 1 Sample pretreatment method

[0073] A sample pretreatment method suitable for testing ketamine analogs in biological samples comprises the following steps:

[0074] Transfer the biological sample to be tested (0.5mL blood, 0.5mL urine or 0.5g liver) into a 15mL plastic centrifuge tube, add 63mg (1mmol) ammonium formate and vortex to mix evenly, then add 5μL 25% (v / v) ammonia water (adjust the pH of the system to 10) and 1mL acetonitrile, oscillate at 1800r / min for 10min, use a high-speed refrigerated centrifuge to maintain the temperature at -4℃, and centrifuge at 8000r / min for 10min. Take the supernatant and filter it through a 0.22μm organic membrane before sampling and testing.

[0075] The ketamine analogs contained in the biological samples include any one or more combinations of ketamine, norketamine, deschloroketamine, fluamine, phencyclidine, tiletamine, methoxitamine, 2-oxo-PCE, and 3-Meo-PCE.

[0076] The above sample pretreatment method can be applied to the detection of ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS / MS) and gas chromatography tandem mass spectrometry (GC-MS / MS).

[0077] Example 2 Method for detecting ketamine analogs using ultra-high performance liquid chromatography tandem mass spectrometry

[0078] The sample pretreatment method of Example 1 was used to treat the biological sample to be tested, and after the treatment, ultra-high performance liquid chromatography-tandem mass spectrometry was used for sample injection and detection.

[0079] UPLC conditions: Eclipse Plus C18 RRHD column (2.1 mm × 100 mm, 1.8 μm); mobile phase: phase A is an aqueous solution containing 0.5‰ (v / v) ammonia and 2 mM ammonium formate, and phase B is acetonitrile; flow rate: 0.4 mL / min; column temperature 40 °C; injection volume 1 μL, total running time of the analytical method is 8.0 min; gradient elution conditions are shown in Table 1.

[0080] Table 1 Gradient elution program of ultra-high performance liquid chromatography

[0081] Time (min) 0 1 2 7 7.1 8 Phase A (v / v, %) 75 75 30 5 75 75 Phase B (v / v, %) 25 25 70 95 25 25

[0082] Mass spectrometry conditions: electrospray ionization (ESI) positive ion mode, multiple reaction monitoring (MRM); ion source temperature 550°C, spray voltage 5.5 kV, curtain gas pressure (CUR) 20 psi, nebulizer gas pressure (GS1) 40 psi, auxiliary heating gas pressure (GS2) 50 psi.

[0083] Example 3 Method for detecting ketamine analogs using gas chromatography tandem mass spectrometry

[0084] The sample pretreatment method of Example 1 was used to treat the biological sample to be tested. After the treatment, the sample was tested by gas chromatography tandem mass spectrometry detection. The program temperature rise sample injection treatment was used during the detection.

[0085] Programmed temperature injection port (PTV) parameters: constant linear velocity mode, linear velocity: 45.1 cm / s; purge flow: 3 mL / min; injection mode: split injection, split ratio: 20:1; initial injection port temperature: 65 °C, hold for 1 min, increase to 280 °C at 200 °C / min, hold for 6 min.

[0086] Chromatographic conditions: Agilent DB-5MS column (30m×0.25mm×0.25μm), carrier gas is high-purity helium (99.999%), column heating program: initial temperature 60°C, hold for 2min, increase to 280°C at 10°C / min, hold for 5min.

[0087] Mass spectrometry conditions: electron impact ion source (EI source), ion source temperature: 230°C, interface temperature: 320°C, solvent delay time 2min, scanning mode is multiple reaction monitoring mode (MRM), scanning interval is 0.3s, and collision gas is high-purity argon (99.999%).

[0088] Example 4 Effect of water content of salting-out agent on extraction efficiency of ketamine analogs

[0089] The blood matrix was used as the biological sample to be tested, and the biological sample to be tested was treated with reference to the sample pretreatment method of Example 1, and the peak area was determined with reference to the detection method of Examples 2-3 to explore whether the salting-out agent contained water and the influence of water content: In this experiment, the fixed aqueous phase system (i.e., blood + water content of the salting-out agent) had a salting-out agent (NaCl) concentration of 2 mol / L and blood of 0.5 mL. The conditions of adding water of 0 mL, 0.25 mL, 0.5 mL, 1 mL, 1.5 mL, and 2 mL were investigated. [i.e., solid salt (1 mmol / L sodium chloride + 0 mL H 2 O) and salt solutions with different water addition amounts (1.5mmol NaCl+0.25mL H 2 O, 2mmol NaCl + 0.5mL H 2 O, 3mmol NaCl + 1mL H 2 O, 4mmol NaCl + 1.5mL H 2 O, 5mmol NaCl + 2mL H 2 O)], on the extraction efficiency of ketamine analogs in blood.

[0090] Preparation of blood matrix containing ketamine analogs: adding a mixed standard solution of ketamine analogs to the blood matrix (the concentration of each ketamine analog in the mixed standard solution is 10 μg / mL); the concentration of each ketamine analog in the obtained blood matrix containing ketamine analogs is 50 ng / mL;

[0091] The sample pretreatment method of this embodiment includes the following steps: 0.5 mL of blood matrix containing ketamine analogs is transferred into a 15 mL plastic centrifuge tube, and a salting-out agent (1 mmol sodium chloride + 0 mL H 2 O, 1.5mmol NaCl + 0.25mL H 2 O, 2mmol NaCl + 0.5mL H2 O, 3mmol NaCl + 1mL H 2 O, 4mmol NaCl + 1.5mL H 2 O, or 5mmol NaCl + 2mL H 2 O, that is, keep the concentration of the salting-out agent (NaCl) in the aqueous phase system at 2mol / L) and vortex to mix evenly, then add 5μL 25% (v / v) ammonia water (adjust the pH of the system to 10) and 2mL acetonitrile, oscillate at 1800r / min for 10min, use a high-speed refrigerated centrifuge to keep the temperature at -4°C, and centrifuge at 8000r / min for 10min. Take the supernatant and filter it through a 0.22μm organic membrane, inject it for detection; record the peak area, and the result is calculated as A.

[0092] In addition, according to the above-mentioned sample pretreatment method, 0.5 mL of blank blood matrix (i.e., no ketamine analogues were added) was treated (the salting-out agent added was 1 mmol of sodium chloride), and after centrifugation, the ketamine analogue mixed standard solution was added to all the supernatants (so that the concentration of each ketamine analogue in the supernatant was 50 ng / mL), vortexed and mixed, and then filtered through a 0.22 μm organic membrane, and sampled for detection; the peak area was recorded, and the result was calculated as B.

[0093] According to the above sample pretreatment method, the blank blood matrix (i.e., no ketamine analogues were added) was treated (the salting-out agent added was 1 mmol of sodium chloride), and after centrifugation, the ketamine analogue mixed standard solution was added to 0.5 mL of the supernatant (so that the concentration of each ketamine analogue in the supernatant was 25 ng / mL), vortexed and mixed, and then filtered through a 0.22 μm organic membrane, and sampled for detection; the peak area was recorded, and the result was calculated as C.

[0094] Add the mixed standard solution of ketamine analogs to 0.5 mL of acetonitrile (so that the concentration of each ketamine analog in the solution is 25 ng / mL), mix well, and then inject for detection; record the peak area, and the result is calculated as D.

[0095] The extraction efficiency of ketamine analogs was evaluated by recovery rate and matrix effect. Each group of experiments was repeated 6 times, and the mean of the peak area was calculated. Recovery rate (%) = (A / B) × 100%; Matrix effect (%) = (C / D) × 100%.

[0096] The results showed that under different water addition conditions, the matrix effect was basically the same, while the recovery rate decreased with the increase of water addition. The UPLC-MS / MS results were as follows: Figure 1 The GC-MS / MS test results are shown in Figure 2As shown. Analyzing the reasons, as the proportion of the aqueous phase in the entire extraction system continues to increase, the stratification effect of the aqueous phase and the organic phase becomes worse, the acetonitrile dissolved in the aqueous phase continues to increase, and the supernatant gradually decreases. When the amount of water added is greater than 1.5mL, the aqueous phase and the organic phase cannot be effectively stratified and effectively extracted. Therefore, since the addition of water has a significant effect on the recovery rate of the substance to be measured (it continues to decrease with the increase in the amount of water added), the sample pretreatment method of the present invention selects the addition of solid salt.

[0097] Example 5 Effect of the ratio of extraction solvent to blood on the extraction efficiency of ketamine analogs

[0098] The blood matrix was used as the biological sample to be tested. The biological sample to be tested was treated with reference to the sample pretreatment method of Example 1, and the peak area was determined with reference to the detection method of Examples 2-3. The effect of the ratio of extraction solvent to blood (volume ratio of blood: acetonitrile = 1:2, 1:3, 1:4) on the extraction efficiency of the extract (ketamine analogue) was investigated.

[0099] Preparation of a blood matrix containing ketamine analogs: adding a mixed standard solution of ketamine analogs to the blood matrix; the concentration of each ketamine analog in the obtained blood matrix containing ketamine analogs is 50 ng / mL;

[0100] The sample pretreatment method of this embodiment includes the following steps: 0.5 mL of blood matrix containing ketamine analogs is transferred into a 15 mL plastic centrifuge tube, a salting-out agent (1 mmol sodium chloride) is added and vortexed to mix evenly, and then 5 μL of 25% (v / v) ammonia water (the pH of the system is adjusted to 10) and acetonitrile (1 mL, 1.5 mL or 2 mL) are added, and the mixture is shaken at 1800 r / min for 10 min, and a high-speed refrigerated centrifuge is used to maintain the temperature at -4°C and centrifuged at 8000 r / min for 10 min. The supernatant is filtered through a 0.22 μm organic membrane and then sampled for detection.

[0101] Referring to the method of Example 4, the extraction efficiency of ketamine analogs was evaluated by recovery rate and matrix effect. Among them, the UPLC-MS / MS test results are as follows Figure 3 , Figure 4 As shown; GC-MS / MS test results are as follows Figure 5 , 6 The results show that with the increase of acetonitrile dosage, the recovery rate and matrix effect do not change much. In order to reduce the detection limit of the substance to be tested and reduce the use of organic reagents, the sample pretreatment method of the present invention uses blood: acetonitrile = 1:2 for extraction.

[0102] Example 6 Effect of salting-out agent type on extraction efficiency of ketamine analogs

[0103] The blood matrix was used as the biological sample to be tested, and the biological sample to be tested was treated with reference to the sample pretreatment method of Example 1. The peak area was determined with reference to the detection method of Examples 2-3, and the effect of the type of salting-out agent (sodium chloride, magnesium sulfate, potassium carbonate, ammonium formate, ammonium acetate) on the extraction efficiency of the extract (ketamine analogue) was explored.

[0104] Preparation of a blood matrix containing ketamine analogs: adding a mixed standard solution of ketamine analogs to the blood matrix; the concentration of each ketamine analog in the obtained blood matrix containing ketamine analogs is 50 ng / mL;

[0105] The sample pretreatment method of this embodiment includes the following steps: 0.5 mL of blood matrix containing ketamine analogs is transferred into a 15 mL plastic centrifuge tube, 1 mmol of salting-out agent (sodium chloride, magnesium sulfate, potassium carbonate, ammonium formate or ammonium acetate) is added respectively, vortexed and mixed evenly, and then 5 μL of 25% (v / v) ammonia water (the pH of the system is adjusted to 10) and 2 mL of acetonitrile are added respectively, oscillated at 1800 r / min for 10 min, and centrifuged at 8000 r / min for 10 min using a high-speed refrigerated centrifuge to maintain the temperature at -4°C. The supernatant is filtered through a 0.22 μm organic membrane and then sampled for detection.

[0106] Referring to the method of Example 4, the extraction efficiency of ketamine analogs was evaluated by recovery rate and matrix effect. UPLC-MS / MS results are as follows Figure 7 , Figure 8 As shown; GC-MS / MS test results are as follows Fig. 9 , Fig.10 As shown, ammonium formate has the best overall extraction effect, followed by potassium carbonate. At the same time, compared with other types of salts, ammonium formate and ammonium acetate are volatile and have the least impact on the electrospray system and ion source of the mass spectrometer. Therefore, the sample pretreatment method of the present invention selects ammonium formate as the salting-out agent.

[0107] Example 7 Effect of salting-out agent concentration in blood on extraction efficiency of ketamine analogs

[0108] The blood matrix was used as the biological sample to be tested. The biological sample to be tested was treated with reference to the sample pretreatment method of Example 1, and the peak area was determined with reference to the detection method of Examples 2-3. The effect of the concentration of the salting-out agent (ammonium formate) in the blood (1 mol / L, 2 mol / L, 5 mol / L, 10 mol / L) on the extraction efficiency of the extract (ketamine analogue) was investigated.

[0109] Preparation of a blood matrix containing ketamine analogs: adding a mixed standard solution of ketamine analogs to the blood matrix; the concentration of each ketamine analog in the obtained blood matrix containing ketamine analogs is 50 ng / mL;

[0110] The sample pretreatment method of this embodiment includes the following steps: 0.5 mL of blood matrix containing ketamine analogs is transferred into a 15 mL plastic centrifuge tube, and the salting-out agent ammonium formate (0.5 mmol, 1 mmol, 2.5 mmol or 5 mmol) is added respectively, and vortexed and mixed evenly, and then 5 μL of 25% (v / v) ammonia water (the pH of the system is adjusted to 10) and 2 mL of acetonitrile are added respectively, and the mixture is shaken at 1800 r / min for 10 min, and the temperature is maintained at -4°C using a high-speed refrigerated centrifuge, and centrifuged at 8000 r / min for 10 min. The supernatant is filtered through a 0.22 μm organic membrane and then sampled for detection.

[0111] Referring to the method of Example 4, the extraction efficiency of ketamine analogs was evaluated by recovery rate and matrix effect. When the concentration of ammonium formate in the blood was 1 mol / L, the organic phase and the aqueous phase were not completely separated, and the volume of the organic phase was about 1.5 mL, which was less than the amount of acetonitrile added in the preparation stage (2 mL), so it was not sampled. The UPLC-MS / MS test results are as follows: Fig.11 , Fig.12 As shown in the figure, when the concentration of ammonium formate in the blood is 2 mol / L, the recovery rate is good, and the matrix effects of the three are not much different; the GC-MS / MS test results are as follows Fig.13 , Fig.14 It is shown that when the concentration of ammonium formate in the blood is 2 mol / L and 5 mol / L, the recovery rate and matrix effect are better and the extraction efficiency is high. In order to be applicable to both instruments at the same time and to reduce the salt content in the sample input by the instrument and reduce the damage to the instrument, the sample pretreatment method of the present invention selects the concentration of ammonium formate in the blood to be 2 mol / L.

[0112] Example 8 Effect of the Alkalinity of the Extraction System on the Extraction Efficiency of Ketamine Analogs

[0113] The blood matrix was used as the biological sample to be tested, and the biological sample to be tested was treated with reference to the sample pretreatment method of Example 1, and the peak area was determined with reference to the detection method of Examples 2-3, to explore the effect of the weak alkaline conditions of the extraction system (the percentage of the volume of added ammonia water (volume concentration: 25%) to the original blood volume was 2% (i.e., the pH of the system was adjusted to 11), 1% (i.e., the pH of the system was adjusted to 10), 0.1% (i.e., the pH of the system was adjusted to 9), 0.01% (i.e., the pH of the system was adjusted to 8), and when no ammonia water was added (i.e., the pH of the system was 7), on the extraction efficiency of the extract (ketamine analogue).

[0114] Preparation of a blood matrix containing ketamine analogs: adding a mixed standard solution of ketamine analogs to the blood matrix; the concentration of each ketamine analog in the obtained blood matrix containing ketamine analogs is 50 ng / mL;

[0115] The sample pretreatment method of this embodiment includes the following steps: 0.5 mL of blood matrix containing ketamine analogs is transferred into a 15 mL plastic centrifuge tube, 1 mmol of salting-out agent ammonium formate is added and vortexed to mix evenly, 25% (v / v) ammonia water (10 μL, 5 μL, 0.5 μL, 0.05 μL or 0 μL) is added respectively, pH is adjusted to 11, 10, 9, 8, 7 respectively, 2 mL of acetonitrile is added, oscillated at 1800 r / min for 10 min, a high-speed refrigerated centrifuge is used to maintain the temperature at -4°C, and centrifuged at 8000 r / min for 10 min. The supernatant is filtered through a 0.22 μm organic membrane and then sampled for detection.

[0116] Referring to the method of Example 4, the extraction efficiency of ketamine analogs was evaluated by recovery rate and matrix effect. Fig.15 , 16 When using liquid chromatography-mass spectrometry for detection, the best extraction effect is achieved when the concentration of ammonia in the blood is 1% and 0.01%; the results of gas chromatography-tandem mass spectrometry are shown in Fig.17 , Fig.18 As shown, the extraction effect is best when the concentration of ammonia water in the blood is 1%. Because the pretreatment method must take into account the sampling detection of two chromatography-tandem mass spectrometry, the sample pretreatment method of the present invention selects the concentration of ammonia water in the blood to be 1% (that is, after adding ammonia water, the pH of the system is adjusted to 10).

[0117] Example 9 Applicability of the sample pretreatment method of the present invention to urine matrix

[0118] The urine matrix was used as the biological sample to be tested, and the biological sample to be tested was treated with reference to the sample pretreatment method of Example 1, and the peak area was determined with reference to the detection method of Examples 2-3, to explore the applicability of the sample pretreatment method of the present invention to the urine matrix.

[0119] Preparation of urine matrix containing ketamine analogs: adding a mixed standard solution of ketamine analogs to the urine matrix (the concentration of each ketamine analog in the mixed standard solution is 10 μg / mL); the concentration of each ketamine analog in the obtained urine matrix containing ketamine analogs is 50 ng / mL;

[0120] The sample pretreatment method of this embodiment includes the following steps: 0.5 mL of urine matrix containing ketamine analogs is transferred into a 15 mL plastic centrifuge tube, 1 mmol of salting-out agent ammonium formate is added, vortexed and mixed evenly, 5 μL of 25% (v / v) ammonia water (the pH of the system is adjusted to 10) and 2 mL of acetonitrile are added, oscillated at 1800 r / min for 10 min, and centrifuged at 8000 r / min for 10 min using a high-speed refrigerated centrifuge to maintain the temperature at -4°C. The supernatant is filtered through a 0.22 μm organic membrane and then sampled for detection.

[0121] Referring to the method of Example 4, the applicability of the sample pretreatment method of the present invention to the urine matrix was evaluated by recovery rate and matrix effect. Among them, the results obtained by UPLC-MS / MS are shown in Table 2: the recovery rates of 9 ketamine analogs are between 90.2% and 108.6%, and the matrix effect is between 89.3% and 104.1%. The results obtained by GC-MS / MS are shown in Table 3: the recovery rates of 9 ketamine analogs are between 92.52% and 104.11%, and the matrix effect is between 88.20% and 111.39%. It shows that the sample pretreatment method of the present invention is suitable for the extraction of 9 ketamine analogs in urine.

[0122] Table 2 Recovery and matrix effect of 9 ketamine analogs in urine matrix (UPLC-MS / MS)

[0123] 2-F-DCK MEX Ketamine DCK 3-Meo-PCE PCP Norketamine Tiletamine 2-oxo-PCE Recovery rate 92.96％ 100.33％ 97.78％ 97.42％ 89.69％ 94.18％ 96.12％ 108.68％ 96.82％ Matrix Effects 89.56％ 92.85％ 96.99％ 90.43％ 104.11％ 97.52％ 90.91％ 89.32％ 90.68％

[0124] Table 3 Recovery and matrix effect of 9 ketamine analogs in urine matrix (GC-MS / MS)

[0125]

[0126] Example 10 Applicability of the sample pretreatment method of the present invention to liver matrix

[0127] The liver matrix was used as the biological sample to be tested, and the biological sample to be tested was treated with reference to the sample pretreatment method of Example 1, and the peak area was determined with reference to the detection method of Examples 2-3 to explore the applicability of the sample pretreatment method of the present invention to the liver matrix.

[0128] Preparation of liver matrix containing ketamine analogs: take the homogenized liver matrix, add the ketamine analog mixed standard solution (the concentration of each ketamine analog in the mixed standard solution is 10 μg / mL), mix and set aside; based on the mass of the liver matrix, the mass of each ketamine analog contained in each gram of liver matrix containing ketamine analogs is 50 ng, recorded as 50 ng / g;

[0129] The sample pretreatment method of this embodiment includes the following steps: 0.5 g of liver matrix containing ketamine analogs is transferred into a 15 mL plastic centrifuge tube, 1 mmol of salting-out agent ammonium formate is added, vortexed and mixed evenly, 5 μL of 25% (v / v) ammonia water and 2 mL of acetonitrile are added, oscillated at 1800 r / min for 10 min, and centrifuged at 8000 r / min for 10 min using a high-speed refrigerated centrifuge to maintain the temperature at -4°C. The supernatant is filtered through a 0.22 μm organic membrane and then sampled for detection.

[0130] Referring to the method of Example 4, the applicability of the sample pretreatment method of the present invention to the liver matrix was evaluated by recovery rate and matrix effect. Among them, the UPLC-MS / MS test results are shown in Table 4: the recovery rates of 9 ketamine analogs are between 91.37% and 103.33%, and the matrix effects are between 89.86% and 110.68%. The GC-MS / MS test results are shown in Table 5: the recovery rates of 9 ketamine analogs are between 89.07% and 108.97%, and the matrix effects are between 89.91% and 108.74%. It shows that the sample pretreatment method of the present invention is suitable for the extraction of 9 ketamine analogs in the liver.

[0131] Table 4 Recovery and matrix effects of 9 ketamine analogs in liver matrix (UPLC-MS / MS) Table 5 Recovery and matrix effects of 9 ketamine analogs in liver matrix (GC-MS / MS)

[0132]

[0133] Methodological validation

[0134] Methodological validation was performed on three biological samples: blood, urine and liver: linear range, limit of detection (LOD), limit of quantification (LOQ), matrix effect, recovery rate, intra-day precision and inter-day precision.

[0135] ① With the mass concentration of each target substance (x) as the abscissa and the ratio of the peak area of ​​the target substance to the peak area of ​​ketamine-D4 (y) as the ordinate, linear fitting was performed on each substance to obtain the regression curve, linear range and correlation coefficient (R 2 ); the detection limit (LOD) and quantification limit (LOQ) of each target were calculated at the addition levels corresponding to 3-fold and 10-fold signal-to-noise ratio (S / N), respectively.

[0136] Wherein, the preparation of a series of matrices (blood, urine and liver) of different concentrations of ketamine analogs is as follows: a series of volumes of mixed standard solutions of ketamine analogs are taken and mixed with blood matrix, urine matrix and liver matrix respectively; the concentrations of each ketamine analog in the obtained series of blood matrices are 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 200, 500 ng / mL respectively; the concentrations of each ketamine analog in the series of urine matrices are 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 200, 500 ng / mL respectively; the content of each ketamine analog in the series of liver matrices is 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 200, 500 ng / g (i.e., the amount of each ketamine analog contained in each gram of liver matrix).

[0137] The results showed that: blood matrix (UPLC-MS / MS): 9 ketamine analogs had good linearity within the corresponding linear range, and the correlation coefficient (R 2 )>0.995, the detection limit was 0.05ng / mL, and the quantification limit was 0.1ng / mL; blood matrix (GC-MS / MS): 9 ketamine analogs had good linearity within the corresponding linear range, and the correlation coefficient (R 2 )>0.998, the detection limit was 0.5ng / mL, and the quantification limit was 1ng / mL. Urine matrix (UPLC-MS / MS): The 9 ketamine analogs had good linearity within the corresponding linear range, and the correlation coefficient (R 2 )>0.995, the detection limit was 0.05ng / mL, and the quantification limit was 0.1ng / mL; urine matrix (GC-MS / MS): 9 ketamine analogs had good linearity within the corresponding linear range, and the correlation coefficient (R 2 )>0.998, the detection limit was 0.5ng / mL, and the quantification limit was 1ng / mL. Liver matrix (UPLC-MS / MS): The 9 ketamine analogs had good linearity within the corresponding linear range, and the correlation coefficient (R 2 )>0.995, the detection limit was 0.05ng / g, and the quantification limit was 0.1ng / g; Liver matrix (GC-MS / MS): 9 ketamine analogs had good linearity within the corresponding linear range, and the correlation coefficient (R 2 )>0.998, the detection limit is 0.5ng / g, and the quantification limit is 1ng / g. That is, the sensitivity and linear range of this method can meet the needs of forensic toxicology testing.

[0138] ② Determine the matrix effect and recovery rate of blood and urine samples with different concentrations (0.1ng / mL, 5ng / mL, 200ng / mL), and liver samples (0.1ng / g, 5ng / g, 200ng / g).

[0139] The results showed that: blood matrix (UPLC-MS / MS): the recoveries at three spiked concentrations (high, medium and low (200 ng / mL, 5 ng / mL, 0.1 ng / mL)) were between 89.04% and 113.2%, and the matrix effect was between 90.45% and 112.27%; blood matrix (GC-MS / MS): the recoveries at three spiked concentrations (high, medium and low) were between 91.91% and 110.92%, and the matrix effect was between 91.91% and 109.57%. Urine matrix (UPLC-MS / MS): The recoveries at high, medium and low spike concentrations were between 89.54% and 111.23%, and the matrix effect was between 90.64% and 112.45%; Urine matrix (GC-MS / MS): The recoveries at high, medium and low spike concentrations were between 90.24% and 111.96%, and the matrix effect was between 88.36% and 108.99%. Liver matrix (UPLC-MS / MS): The recoveries at high, medium and low spike concentrations were between 90.22% and 109.81%, and the matrix effect was between 88.92% and 108.03%; Liver matrix (GC-MS / MS): The recoveries at high, medium and low spike concentrations were between 89.33% and 110.53%, and the matrix effect was between 88.09% and 109.93%. This indicates that the recovery rate of this method is good, the matrix has little effect on the detection results of the target, and it can meet the detection requirements.

[0140] ③ Six groups of blood and urine samples (0.1 ng / mL, 5 ng / mL, 200 ng / mL) and liver samples (0.1 ng / g, 5 ng / g, 200 ng / g) of different concentrations were prepared in parallel. The above samples were injected and tested at three different times of the day: morning, noon, and evening. The relative standard deviation of the peak area of ​​9 ketamine analogs at different concentrations was calculated to obtain the intra-day precision; the same operation was performed for 5 consecutive days to obtain the inter-day precision. The results are shown in Tables 6-11 below.

[0141] The results showed that: blood matrix (UPLC-MS / MS): the intra-day RSD (n=6) of the nine ketamine analogs under this method was between 1.72% and 9.64%, and the inter-day RSD (n=5) was between 4.35% and 11.30%; blood matrix (GC-MS / MS): the intra-day RSD (n=6) was between 2.03% and 11.57%, and the inter-day RSD (n=5) was between 2.34% and 11.45%. Urine matrix (UPLC-MS / MS): intra-day RSD (n=6) was between 1.71% and 10.29%, and inter-day RSD (n=5) was between 4.35% and 11.30%; urine matrix (GC-MS / MS): intra-day RSD (n=6) was between 2.42% and 11.78%, and inter-day RSD (n=5) was between 1.76% and 12.38%. Liver matrix (UPLC-MS / MS): intra-day RSD (n=6) was between 2.35% and 10.23%, and inter-day RSD (n=5) was between 3.00% and 10.83%; liver matrix (GC-MS / MS): intra-day RSD (n=6) was between 0.37% and 9.62%, and inter-day RSD (n=5) was between 0.97% and 11.26%. This indicates that this method has good intra-day and inter-day precision and can meet the detection requirements.

[0142] Table 6 Intra-day and inter-day precision of 9 ketamine analogs in blood (UPLC-MS / MS)

[0143]

[0144] Table 7 Intra-day and inter-day precision of 9 ketamine analogs in blood (GC-MS / MS)

[0145]

[0146] Table 8 Intra-day and inter-day precision of 9 ketamine analogs in urine (UPLC-MS / MS)

[0147]

[0148] Table 9 Intra-day and inter-day precision of 9 ketamine analogs in urine (GC-MS / MS)

[0149]

[0150] Table 10 Intra-day and inter-day precision of 9 ketamine analogs in liver (UPLC-MS / MS)

[0151]

[0152] Table 11 Intra-day and inter-day precision of 9 ketamine analogs in liver (GC-MS / MS)

[0153]

[0154] Application Examples

[0155] In August 2021, criminal suspects Lin and Zhang, who were suspected of taking K powder, were arrested in Chaoyang District, Beijing.

[0156] Before the test, the dilution effect was investigated, and 9 ketamine analogs were prepared with a mass concentration of 1000 ng / mL. The samples were diluted 10 times with blank matrix, so that the mass concentration of the diluted samples was within the linear range of the standard curve (repeated preparation of 6 groups), and the coefficient of variation of the concentration of the diluted quality control samples was calculated. The results showed that the relative deviation was 5.6%, which was less than 15%, and met the requirements for biological sample determination.

[0157] The whole blood and urine of the suspect Lin were taken, diluted 10 times, and screened and tested using the methods of Examples 1-3. 2-FDCK and Ketamine were detected in the samples. The results of ultra-high performance liquid chromatography tandem mass spectrometry were: the concentration of 2-FDCK in the blood was 524ng / mL, and the concentration of Ketamine was 286ng / mL; the concentration of 2-FDCK in urine was 345ng / mL, and the concentration of Ketamine was 237ng / mL. The results of gas chromatography tandem mass spectrometry were: the concentration of 2-FDCK in the blood was 495ng / mL, and the concentration of Ketamine was 254ng / mL. The concentration of 2-FDCK in urine was: 302ng / mL, and the concentration of Ketamine was 219ng / mL.

[0158] The whole blood and urine of the suspect Zhang were taken, diluted 10 times, and screened and tested using the methods of Examples 1-3. 2-FDCK and Ketamine were detected in the samples. The results of ultra-high performance liquid chromatography tandem mass spectrometry were: the concentration of 2-FDCK in the blood was 629ng / mL, and the concentration of Ketamine was 372ng / mL; the concentration of 2-FDCK in urine was 547ng / mL, and the concentration of Ketamine was 289ng / mL. The results of gas chromatography tandem mass spectrometry were: the concentration of 2-FDCK in the blood was 594ng / mL, and the concentration of Ketamine was 309ng / mL. The concentration of 2-FDCK in urine was 478ng / mL, and the concentration of Ketamine was 221ng / mL.

[0159] According to GB / T 43449-2023 Quality Control Specifications for Forensic Toxicology Analysis Laboratories, if the deviation between the two detection methods is within 20%, the measurement is accurate. Therefore, the method of the present invention meets the requirements.

[0160] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A sample pretreatment method suitable for testing ketamine analogs in biological samples, characterized in that: The following steps are involved: Add a salting-out agent to the biological sample to be tested, shake and mix, add ammonia water to adjust the pH, then add acetonitrile, continue shaking treatment; then freeze centrifuge, separate the supernatant, filter, and obtain.

2. The sample pretreatment method according to claim 1, characterized in that: The ketamine analogs contained in the biological sample to be tested are any one or more combinations of ketamine, norketamine, deschloroketamine, fluamine, phencyclidine, tiletamine, methoxitamine, 2-oxo-PCE, and 3-MeO-PCE.

3. The sample pretreatment method according to claim 2, characterized in that: The biological sample to be tested is liquid or solid; the liquid is blood or urine; the solid is liver.

4. The sample pretreatment method according to claim 3, characterized in that: When the biological sample to be tested is a liquid, the volume ratio of the amount of the salting-out agent to the biological sample to be tested is 1-10 mmol:1 mL; the volume ratio of the biological sample to be tested to acetonitrile is 1:2-4; When the biological sample to be tested is solid, the mass ratio of the amount of the salting-out agent to the biological sample to be tested is 1-10 mmol: 1 g; the mass ratio of the biological sample to be tested to the volume of acetonitrile is 1 g: 2-4 mL.

5. The sample pretreatment method according to claim 1, characterized in that: The salting-out agent is sodium chloride, magnesium sulfate, potassium carbonate, ammonium formate or ammonium acetate; after adding ammonia water, the pH of the system is adjusted to 7-10.

6. The sample pretreatment method according to claim 5, characterized in that: The salting-out agent is solid ammonium formate; after adding ammonia water, the pH of the system is adjusted to 10.

7. The sample pretreatment method according to claim 1, characterized in that: After adjusting the pH, the oscillation treatment speed is 1800-2000r / min, and the time is 10-20min; The temperature of the frozen centrifugation treatment is -4°C, the centrifugal speed is 8000-10000 r / min, and the time is 10-20 min; when filtering, the supernatant is filtered through a 0.22 μm organic membrane.

8. A detection method suitable for testing ketamine analogs in biological samples, characterized in that: The biological sample is treated by the sample pretreatment method according to any one of claims 1 to 7, and the sample is detected by ultra-high performance liquid chromatography tandem mass spectrometry or gas chromatography tandem mass spectrometry.

9. The detection method according to claim 8, characterized in that: When the samples were detected by ultra-high performance liquid chromatography tandem mass spectrometry, the ultra-high performance liquid chromatography conditions were as follows: mobile phase: phase A was an aqueous solution containing 0.5‰ (v / v) ammonia and 2 mM ammonium formate, phase B was acetonitrile; flow rate was 0.4 mL / min; column temperature was 40°C; injection volume was 1 μL, and the total running time was 8.0 min; Mass spectrometry conditions: electrospray ion source positive ion mode, multiple reaction monitoring; ion source temperature 550°C, spray voltage 5.5 kV, air curtain gas pressure 20 psi, nebulizer gas pressure 40 psi, auxiliary heating gas pressure 50 psi.

10. The detection method according to claim 8, characterized in that: When the samples are tested by gas chromatography-tandem mass spectrometry, programmed temperature injection is used; Program temperature injection port parameters: constant linear velocity mode, linear velocity: 45.1 cm / s; Purge flow rate: 3 mL / min; injection mode: split injection, split ratio: 20:1; initial injection port temperature: 65 °C, maintained for 1 min, increased to 280 °C at 200 °C / min, maintained for 6 min; Chromatographic conditions: carrier gas was high-purity helium, column temperature program: initial temperature 60°C, maintained for 2 min, increased to 280°C at 10°C / min, maintained for 5 min; Mass spectrometry conditions: electron bombardment ion source, ion source temperature: 230°C, interface temperature: 320°C, solvent delay time 2 min, scanning mode is multiple reaction monitoring mode, scanning interval is 0.3 s, and collision gas is high-purity argon.

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