A method for simultaneously detecting 20 kinds of amphetamines, piperazines and benzodiazepines psychoactive substances in water body

Abstract

The application belongs to the field of environmental sample detection, and discloses a method for simultaneously detecting 20 kinds of benzene amphetamine, piperazine and benzodiazepine psychoactive substances in water bodies. The method simultaneously extracts and detects 20 kinds of benzene amphetamine, piperazine and benzodiazepine psychoactive substances in water bodies by using dispersion liquid liquid microextraction and high performance liquid chromatography mass spectrometry. The average recovery rate of the 20 target compounds is 71.5% to 108%, the standard deviation is 0.7% to 4.8%, the detection limit is 1.5 to 10.5 ng / L, and the quantitative limit is 6.0 to 42.2 ng / L. The method has high recovery rate, high sensitivity and good repeatability, and can be used for detecting benzene amphetamine, piperazine and benzodiazepine psychoactive substances in water bodies.

Description

Technical Field

[0001] This invention belongs to the field of environmental sample detection, specifically relating to a method for simultaneously detecting 20 kinds of amphetamine, piperazine and benzodiazepine psychoactive substances in water. Background Technology

[0002] Psychoactive substances are chemical substances that can cross the blood-brain barrier and act directly on the central nervous system, affecting brain function and temporarily altering an individual's perception, mood, consciousness, and behavior. These substances produce various psychoactive effects by regulating the balance of neurotransmitters in the brain and interfering with neuronal communication. Based on their pharmacological properties and chemical structure, psychoactive substances can be classified into several major categories: central nervous system depressants (such as barbiturates, alcohol, and benzodiazepines), central nervous system stimulants (such as caffeine, cocaine, and amphetamines), cannabis, hallucinogens (such as LSD and psilocybin), opioids (such as heroin, morphine, and methadone), and volatile solvents. Furthermore, to circumvent existing drug control laws, new psychoactive substances such as piperazines have emerged in recent years, posing a new public health challenge.

[0003] Although the addictive potential of various psychoactive substances differs, they typically produce pleasurable or reinforcing effects by influencing the brain's reward system (especially the dopamine pathway), leading to repeated use and dependence. Long-term use can cause increased tolerance and psychological dependence, and once discontinued, severe and painful withdrawal symptoms may occur, even endangering life.

[0004] These substances and their metabolites can enter the aquatic environment through human excretion, illegal discharge, or incomplete wastewater treatment, migrating and spreading with water bodies, posing a potential threat to aquatic organisms and human health. The concentration of psychoactive substances in environmental water bodies is extremely low, the matrix is ​​complex, and multiple types of substances often coexist. Furthermore, the limited coverage of standard spectral libraries presents a severe challenge to their accurate detection and analysis. Currently used analytical methods generally include pretreatment steps such as solid-phase extraction or liquid-liquid extraction for enrichment and purification, followed by qualitative and quantitative analysis using gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS / MS). Establishing analytical methods with high recovery rates, high sensitivity, and high selectivity for water bodies is an important prerequisite for in-depth research on the environmental behavior, ecological fate, and health risks of psychoactive substances. Summary of the Invention

[0005] To address the shortcomings and deficiencies in existing technologies, the present invention aims to provide a method for simultaneously detecting 20 amphetamine, piperazine, and benzodiazepine psychoactive substances in water. Specifically, it employs dispersive liquid-liquid microextraction and high-performance liquid chromatography-mass spectrometry to quantitatively determine the content of amphetamine, benzodiazepine, and piperazine psychoactive substances in water.

[0006] The objective of this invention is achieved through the following technical solution: A method for simultaneously detecting 20 amphetamine, piperazine, and benzodiazepine psychoactive substances in water includes the following steps: (1) Sample pretreatment: Take the water sample to be tested into a screw-capped centrifuge tube, add ammonia to adjust the pH to 10-12, add dichloromethane as the extractant and sodium hyaluronate aqueous solution as the dispersant, and shake until an emulsion is formed; after centrifuging the emulsion, transfer all the lower layer solution to another centrifuge tube, add pyridine and shake well, and a precipitate will appear; centrifuge to remove the precipitate, blow the remaining solution with nitrogen to near dryness, add methanol and 0.1% formic acid aqueous solution to reconstitute, filter, and obtain the sample solution; (2) Preparation of standard series solutions: Mix the standard solutions of the target compound to prepare a mixed standard solution, and then dilute it with methanol and 0.1% formic acid aqueous solution to prepare a standard series solution containing at least 5 non-zero concentration gradients, 1 mL each. (3) The standard series solutions of each concentration gradient in step (2) are measured using high performance liquid chromatography-mass spectrometry, and a standard working curve is established using the external standard method; the sample solution obtained in step (1) is measured using high performance liquid chromatography-mass spectrometry under the same conditions, and the chromatographic peak area of ​​the target compound measured in the sample solution is substituted into the standard working curve to obtain the mass concentration of the target compound in the sample solution. Then, the mass concentration of the target compound in the water sample to be tested is calculated according to the calculation formula (1). Formula (1) is In the formula r The mass concentration of the target compound in the water sample to be tested. c The mass concentration of the target compound in the sample solution. V To adjust the volume of the sample solution, V s This represents the volume of the water sample to be tested.

[0007] If the mass concentration of the target compound in the sample solution exceeds the upper limit of the linear range of the standard working curve in step (3), the amount of water sample to be tested in step (1) is reduced, and the pretreatment is repeated to obtain the sample solution, which is then quantitatively determined by high performance liquid chromatography-mass spectrometry.

[0008] In step (1), the volume of the water sample to be tested is 20 mL; the volume of dichloromethane added is 400 μL; the mass concentration of the sodium hyaluronate aqueous solution is 0.5%~1%, and its added volume is 400~800 μL; the volume ratio of pyridine to sodium hyaluronate aqueous solution is 3:1; the volume ratio of methanol to formic acid aqueous solution with a volume percentage concentration of 0.1% is 1:1, and the total volume of the two is 200 μL.

[0009] The shaking time in step (1) is 1 min; the centrifugation is carried out at 6000-8000 rpm for 5-10 min.

[0010] The target compounds mentioned in step (2) are 7 amphetamine psychoactive substances, 5 piperazine psychoactive substances, and 8 benzodiazepine psychoactive substances; among which the 7 amphetamine psychoactive substances are amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, p-methoxymethamphetamine, N,N-dimethyl-3,4-methylenedioxymethamphetamine, and 4-methylthioamphetamine; the 5 piperazine psychoactive substances are 1-piperidinepiperazine, benzylpiperazine, 1-(4-methoxyphenyl)piperazine, 1-(3-chlorophenyl)piperazine, and 1-(3-trifluoromethylphenyl)piperazine; and the 8 benzodiazepine psychoactive substances are nordiazepam, nitrazepam, fennalazepam, flunitrazepam, alprazolam, clonazepam, estazolam, and diazepam.

[0011] The conditions for the high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) determination described in step (3) are as follows: Chromatographic conditions: The column was an Agilent SB-AQ C18 column with a diameter of 1.8 μm and a volume of 2.1 × 150 mm; mobile phase A was an aqueous solution containing 0.1% formic acid by volume, and mobile phase B was methanol; the flow rate was 0.3 mL / min; the column temperature was 35℃; the injection volume was 10 μL; the gradient elution program was as follows: 0–6 min 5%–20% mobile phase B, 6–8 min 20%–39% mobile phase B, 8–12 min 39%–42% mobile phase B, 12–15 min 42%–100% mobile phase B, 15–18 min 100% mobile phase B, 18–18.1 min 100%–5% mobile phase B, and 5% mobile phase B was used until 23 min.

[0012] Mass spectrometry conditions: Ion source was electrospray ionization (ESI); scanning mode was positive ion mode; detection mode was multiple reaction detection (MRM); interface temperature was 300 °C, ion source temperature was 150 °C, desolvation gas temperature was 526 °C, and DL tube temperature was 250 °C. The mass spectrometry parameters of the target compound are shown in Table 1; Table 1 Mass Spectrometry Parameters of the Target Compound

[0013] This invention combines dispersive liquid-liquid microextraction with high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) for qualitative and quantitative analysis of trace psychoactive substances in water. This invention has the following advantages and beneficial effects compared to existing technologies: (1) This invention utilizes dispersive liquid-liquid microextraction coupled with high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) to simultaneously extract and detect 20 amphetamine, piperazine, and benzodiazepine psychoactive substances in water. The average recoveries of the 20 target compounds ranged from 71.5% to 108%, with standard deviations of 0.7% to 4.8%, limits of detection of 1.5 to 10.5 ng / L, and limits of quantitation of 6.0 to 42.2 ng / L. The method exhibits high recovery, high sensitivity, and good repeatability, and can be used for the detection of amphetamine, piperazine, and benzodiazepine psychoactive substances in water.

[0014] (2) Compared with the more commonly used solid phase extraction technology, the dispersion liquid-liquid microextraction used in this invention has the advantages of simple operation and less solvent. After optimizing the extraction conditions, the extraction effect is excellent. There is no need to use internal standard to correct the results, and the external standard method can obtain a satisfactory recovery rate.

[0015] (3) This invention uses an aqueous solution of sodium hyaluronate as a dispersant. Sodium hyaluronate, as a natural polymer, has the advantages of being non-toxic and having good water solubility. Through shaking, the extractant and the water sample can form a good emulsion effect, thereby improving the extraction efficiency. After extraction, adding 3 times the volume of pyridine to the extractant can cause a small amount of sodium hyaluronate dissolved in the extractant to precipitate out. On the other hand, it can also break the electrostatic interaction between the carboxyl group of sodium hyaluronate and the amino group of the target compound, so that more of the target compound dissolves in the extractant, thereby improving the recovery rate.

[0016] (4) At present, there are few monitoring technologies for various amphetamines, piperazines and benzodiazepines in water bodies. This invention provides a set of efficient, rapid and accurate analytical detection technologies from the aspects of pretreatment and instrumental analysis, which are applicable to the detection of psychoactive substances in actual water samples. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0018] The instruments and reagents used in the following examples are: Twenty target compounds, namely amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, p-methoxymethamphetamine, N,N-dimethyl-3,4-methylenedioxymethamphetamine, 4-methylthioamphetamine, 1-piperidinepiperazine, benzylpiperazine, 1-(4-methoxyphenyl)piperazine, 1-(3-chlorophenyl)piperazine, 1-(3-trifluoromethylphenyl)piperazine, nordiazepam, nitrazepam, fennalazepam, flunitrazepam, alprazolam, clonazepam, estazolam, and diazepam, were purchased from Cerilliant. The methanol, formic acid, and dichloromethane used in the experiment were all of chromatographic grade. The water used was Grade I water, and the ammonia and sodium hyaluronate were of analytical grade. All glassware used in the experiment was washed with tap water, rinsed with Grade I water, dried, and then baked in a muffle furnace at 300°C or above for 3-4 hours.

[0019] The detection instrument was a Shimadzu LCMS-8045 liquid chromatography-mass spectrometry system.

[0020] Example 1

[0021] (1) Sample pretreatment Take 20 mL of the water sample to be tested into a screw-capped centrifuge tube, add ammonia to adjust the pH to 10, add 400 μL of extraction solvent (dichloromethane) and 400 μL of dispersant (1% sodium hyaluronate aqueous solution), and shake for 1 min until the solution becomes an emulsion. Centrifuge the emulsion at 6000 rpm for 10 min, and use a disposable syringe to draw the lower layer solution into another centrifuge tube. Add 1.2 mL of pyridine and shake well, and a small amount of precipitate will appear. Centrifuge at 6000 rpm for 10 min to remove the precipitate, transfer the remaining upper layer solution to a test tube, blow it to near dryness with nitrogen, and add 200 μL of methanol and 0.1% formic acid aqueous solution (1:1 volume ratio) to reconstitute. Filter with a 0.44 μm filter to obtain the sample solution.

[0022] (2) Preparation of standard series solutions Prepare a mixed standard solution by taking standard solutions of 20 target compounds and diluting it with a mixed solution of methanol and formic acid aqueous solution with a volume ratio of 1:1 to prepare a series of standard solutions with concentration gradients of 0.5 μg / L, 1 μg / L, 5 μg / L, 10 μg / L, 20 μg / L and 50 μg / L, 1 mL of each solution.

[0023] (3) Instrument testing The standard series solutions of each concentration gradient in step (2) were measured using high performance liquid chromatography-mass spectrometry (HPLC-MS / MS). A standard working curve was established using the external standard method with the target compound concentration as the y-axis and the quantitative ion pair peak area as the x-axis. The sample solution obtained in step (1) was measured using HPLC-MS / MS under the same conditions. The retention time, ion pair abundance ratio, and other results of the target compound measured in the sample solution were compared with the results of the standard solution as a qualitative basis. The chromatographic peak area of ​​the target compound measured in the sample solution was substituted into the standard working curve to obtain the mass concentration of the target compound in the sample solution. Then, the mass concentration of the target compound in the water sample to be tested was calculated according to the calculation formula (1). If the content of the target compound in the sample solution exceeds the upper limit of the linear range of the standard working curve, the amount of water sample to be tested in step (1) was reduced, and the sample solution was obtained again through pretreatment. HPLC-MS / MS was then performed.

[0024] The formula (1) is In the formula r The mass concentration of the target compound in the water sample to be tested. c The mass concentration of the target compound in the sample solution. V To adjust the volume of the sample solution, V s This represents the volume of the water sample to be tested.

[0025] The conditions for the determination using the high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) instrument are as follows: Chromatographic conditions: The column was an Agilent SB AQ C18 column with a diameter of 1.8 μm and a volume of 2.1 × 150 mm; mobile phase A was an aqueous solution containing 0.1% formic acid by volume, and mobile phase B was methanol; the flow rate was 0.3 mL / min; the column temperature was 35℃; the injection volume was 10 μL; the gradient elution program was as follows: 0–6 min 5%–20% mobile phase B, 6–8 min 20%–39% mobile phase B, 8–12 min 39%–42% mobile phase B, 12–15 min 42%–100% mobile phase B, 15–18 min 100% mobile phase B, 18–18.1 min 100%–5% mobile phase B, and 5% mobile phase B was used until 23 min.

[0026] Mass spectrometry conditions: the ion source was an electrospray ionization (ESI) source; the scanning mode was positive ion mode; the detection mode was multiple reaction detection (MRM); the interface temperature was 300 °C, the ion source temperature was 150 °C, the desolvation gas temperature was 526 °C, and the DL tube temperature was 250 °C.

[0027] The mass spectrometry parameters of the 20 target compounds are shown in Table 1.

[0028] Experimental results: A. Linear Range The 20 target compounds showed good linearity in the concentration range of 0.5–50 μg / L and can be used for quantitative analysis, as shown in Table 2.

[0029] B. Accuracy and Precision The accuracy of the method was verified using the standard addition method. Standard solutions were added to blank water samples and mixed thoroughly to obtain spiked concentrations of 50 ng / L and 200 ng / L. Three parallel samples were set up for each concentration point. After sample extraction, the theoretical concentrations of the target compounds in the test solutions were 5 μg / L and 20 μg / L. The average recoveries of the 20 target compounds ranged from 71.5% to 108%, with standard deviations ranging from 0.7% to 4.8%, indicating good accuracy and precision of the method.

[0030] C. Limit of detection and limit of quantitation Standard solutions were added to blank samples to achieve a target compound concentration of 20 ng / L. Seven parallel samples were prepared, extracted, and detected. The standard deviation of the results was calculated, and the limit of detection (LOD) was calculated according to the requirements of HJ 168 standard. The limit of quantitation (LOQ) was set at four times the LOD. As shown in Table 3, the LODs for the 20 target compounds ranged from 1.5 to 10.5 ng / L, and the LODs ranged from 6.0 to 42.2 ng / L.

[0031] Table 2 Standard curves of 20 target compounds

[0032] Table 3. Spike recoveries, limits of detection, and limits of quantitation for 20 target compounds.

[0033] Comparative Example 1 (1) Sample pretreatment Take 20 mL of blank water sample into a screw-capped centrifuge tube, add the mixed standard solution to prepare a sample with a spiked concentration of 200 ng / L. Adjust the pH to 10 with ammonia, add 400 μL of extraction solvent (dichloromethane), without adding sodium hyaluronate solution as dispersant, and shake for 1 min. Centrifuge the liquid at 6000 rpm for 10 min, and use a disposable syringe to transfer the lower layer solution to a test tube. Blow under nitrogen until nearly dry, and add 200 μL of a 1:1 (v / v) methanol and 0.1% (v / v) formic acid aqueous solution to redissolve the solution. Filter through a 0.44 μL filter to obtain the sample solution.

[0034] The other analytical steps are the same as in Example 1.

[0035] The sample containing only the extractant, without the sodium hyaluronate solution as a dispersant, failed to form a stable emulsion after shaking. The extractant droplets in water were relatively large, resulting in poor dispersion, and the extractants immediately aggregated at the bottom upon cessation of shaking. Compared to Example 1, the recovery rates of the 20 target compounds in Comparative Example 1 were significantly reduced, as shown in Table 4. This indicates that adding an aqueous sodium hyaluronate solution as a dispersant effectively improves the dispersion of the extractant in water, thereby increasing the recovery rate.

[0036] Table 4. Spike recoveries of the 20 target compounds in Comparative Example 1

[0037] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for simultaneously detecting 20 amphetamine, piperazine, and benzodiazepine psychoactive substances in water, characterized in that... Includes the following steps: (1) Sample pretreatment: Take the water sample to be tested into a screw-capped centrifuge tube, add ammonia to adjust the pH to 10-12, add dichloromethane as the extractant and sodium hyaluronate aqueous solution as the dispersant, and shake until an emulsion is formed; after centrifuging the emulsion, take all the lower layer solution into another centrifuge tube, add pyridine and shake well, and a precipitate will appear; Centrifugation was used to remove the precipitate. The remaining solution was then purged with nitrogen until nearly dry. Methanol and a 0.1% (v / v) formic acid aqueous solution were added to redissolve the solution. After filtration, the sample solution was obtained. (2) Preparation of standard series solutions: Mix the standard solutions of the target compound to prepare a mixed standard solution, and then dilute it with methanol and 0.1% formic acid aqueous solution to prepare a standard series solution containing at least 5 non-zero concentration gradients, 1 mL each. (3) The standard series solutions of each concentration gradient in step (2) are measured using high performance liquid chromatography-mass spectrometry, and a standard working curve is established using the external standard method; the sample solution obtained in step (1) is measured using high performance liquid chromatography-mass spectrometry under the same conditions, and the chromatographic peak area of ​​the target compound measured in the sample solution is substituted into the standard working curve to obtain the mass concentration of the target compound in the sample solution. Then, the mass concentration of the target compound in the water sample to be tested is calculated according to the calculation formula (1). Formula (1) is In the formula ρ The mass concentration of the target compound in the water sample to be tested. c The mass concentration of the target compound in the sample solution. V To adjust the volume of the sample solution, V s The volume is the water sample to be tested.

2. The method for simultaneously detecting 20 amphetamine, piperazine, and benzodiazepine psychoactive substances in water according to claim 1, characterized in that: If the mass concentration of the target compound in the sample solution exceeds the upper limit of the linear range of the standard working curve in step (3), the amount of water sample to be tested in step (1) is reduced, and the pretreatment is repeated to obtain the sample solution, which is then quantitatively determined by high performance liquid chromatography-mass spectrometry.

3. The method for simultaneously detecting 20 amphetamine, piperazine, and benzodiazepine psychoactive substances in water according to claim 1, characterized in that: In step (1), the volume of the water sample to be tested is 20 mL; the volume of dichloromethane added is 400 μL; the mass concentration of the sodium hyaluronate aqueous solution is 0.5%~1%, and its added volume is 400~800 μL; the volume ratio of pyridine to sodium hyaluronate aqueous solution is 3:1; the volume ratio of methanol to formic acid aqueous solution with a volume percentage concentration of 0.1% is 1:1, and the total volume of the two is 200 μL.

4. The method for simultaneously detecting 20 amphetamine, piperazine, and benzodiazepine psychoactive substances in water according to claim 1, characterized in that: The oscillation time in step (1) is 1 min; the centrifugation is carried out at 6000-8000 rpm for 5-10 min.

5. The method for simultaneously detecting 20 amphetamine, piperazine, and benzodiazepine psychoactive substances in water according to claim 1, characterized in that: The target compounds mentioned in step (2) are 7 amphetamine psychoactive substances, 5 piperazine psychoactive substances, and 8 benzodiazepine psychoactive substances; among which the 7 amphetamine psychoactive substances are amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, p-methoxymethamphetamine, N,N-dimethyl-3,4-methylenedioxymethamphetamine, and 4-methylthioamphetamine; the 5 piperazine psychoactive substances are 1-piperidinepiperazine, benzylpiperazine, 1-(4-methoxyphenyl)piperazine, 1-(3-chlorophenyl)piperazine, and 1-(3-trifluoromethylphenyl)piperazine; and the 8 benzodiazepine psychoactive substances are nordiazepam, nitrazepam, fennalazepam, flunitrazepam, alprazolam, clonazepam, estazolam, and diazepam.

6. The method for simultaneously detecting 20 amphetamine, piperazine, and benzodiazepine psychoactive substances in water according to claim 1, characterized in that: The conditions for the high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) determination described in step (3) are as follows: Chromatographic conditions: The column was an Agilent SB-AQ C18 column with a particle size of 1.8 μm and a diameter of 2.1 × 150 mm; mobile phase A was an aqueous solution containing 0.1% formic acid by volume, and mobile phase B was methanol; the flow rate was 0.3 mL / min; the column temperature was 35℃; the injection volume was 10 μL; the gradient elution program was as follows: 0–6 min 5%–20% mobile phase B, 6–8 min 20%–39% mobile phase B, 8–12 min 39%–42% mobile phase B, 12–15 min 42%–100% mobile phase B, 15–18 min 100% mobile phase B, 18–18.1 min 100%–5% mobile phase B, with 5% mobile phase B continuing until 23 min. Mass spectrometry conditions: Ion source was electrospray ionization (ESI); scanning mode was positive ion mode; detection mode was multiple reaction detection (MRM); interface temperature was 300 °C, ion source temperature was 150 °C, desolvation gas temperature was 526 °C, and DL tube temperature was 250 °C. The mass spectrometry parameters of the target compound are shown in Table 1; Table 1 Mass Spectrometry Parameters of the Target Compound 。

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