Method for detecting narcotics in urine through ionic liquid extraction-high performance liquid chromatography

Abstract

The invention provides a method for detecting narcotics in urine by ionic liquid extraction-high performance liquid chromatography, which comprises the following steps of: extracting the urine by using water-soluble imidazole salt ionic liquid as a dispersing agent and water-insoluble imidazole salt ionic liquid as an extracting agent; carrying out high performance liquid chromatography analysis on a precipitated phase obtained by extraction to obtain the content of drugs in the urine; the dispersing agent is selected from at least one of [C4MIM] BF4, [C6MIM] BF4 and [C4MIM-SH] Br, and the extracting agent is selected from at least one of [C4MIM] PF6, [C6MIM] PF6 and [C8MIM] PF6; the drugs comprise synthetic cathinone drugs and psychoactive drugs. The micro-extraction method developed by the invention has the advantages of being simple and convenient to operate, time-saving, efficient, green and environment-friendly. And the method has the advantages of wide linear range, good reproducibility and low detection limit, and is suitable for detection and analysis of the drug compounds in the urine matrix.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, specifically to a method for detecting drugs in urine using ion liquid extraction-high performance liquid chromatography. Background Technology

[0002] The abuse of new types of drugs has become a major threat to global public health and safety. This includes the illicit use of synthetic cathinones, such as ethyl fluoroquinolone (2-FXE) and methyl ketamine (2-MDCK), as well as psychoactive substances like dextromethorphan (DXM), tefenoxate (DIP), and etomidate (ET). These new drugs are not only highly hallucinogenic and addictive, but their metabolites also have short half-lives and exist in various forms in the human body, placing higher demands on biological sample detection technologies. Urine, as a primary sample for drug testing, contains a large number of endogenous interfering substances (such as urea, creatinine, and proteins) in its complex matrix, posing a significant challenge to traditional detection methods in the enrichment and separation of trace targets (ng / mL level).

[0003] Traditional sample pretreatment techniques such as liquid-liquid extraction (LLE) and solid-phase extraction (SPE) have significant technical bottlenecks: LLE typically requires 50-200 mL of organic solvents (such as dichloromethane, n-hexane, etc.), generating toxic waste and prone to emulsification during operation, leading to fluctuations in recovery rates; while SPE reduces solvent consumption, its column packing material is expensive, the activation-loading-elution process takes 2-3 hours, and its adsorption efficiency for polar metabolites is generally below 60%. More importantly, the enrichment factor of existing methods is usually only 10-50 times, which is insufficient to meet the pg-level detection limit requirements of ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). A novel ionic liquid-assisted dispersion-liquid microextraction (DLLME-IL) combined with LC-MS / MS achieves a detection sensitivity of 0.05-0.2 ng / mL and a relative standard deviation (RSD) of less than 5%. This technological breakthrough not only provides a reliable analytical method for forensic toxicology but also promotes the development of green analytical chemistry—reducing the amount of organic solvent used in a single detection from 20 mL to 0.2 mL. It boasts advantages such as simple operation, high concentration, and environmental friendliness, which has significant practical implications for drug investigation and emergency treatment. Methods for detecting new types of drugs in urine using DLLME technology are rarely reported. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the existing technology and to provide a method for detecting drugs in urine by ion liquid extraction-high performance liquid chromatography.

[0005] The technical solution adopted in this invention is as follows: a method for detecting drugs in urine by ionic liquid extraction-high performance liquid chromatography, wherein the urine is extracted by using an imidazole salt ionic liquid that is easily soluble in water as a dispersant and an imidazole salt ionic liquid that is poorly soluble in water as an extractant, and the content of drugs in the urine is obtained by high performance liquid chromatography analysis of the precipitate obtained by extraction.

[0006] The dispersant is selected from at least one of [C4MIM]BF4, [C6MIM]BF4, and [C4MIM-SH]Br, and the extractant is selected from at least one of [C4MIM]PF6, [C6MIM]PF6, and [C8MIM]PF6;

[0007] The drugs mentioned include synthetic cathinones and psychoactive drugs.

[0008] Preferably, the drug includes ethyl fluoroquinolone, methyl fluoroquinolone, dextromethorphan, tefenoxate, and etomidate.

[0009] Preferably, it includes the following steps:

[0010] (1) Preparation of standard solution: Take drug standard, prepare single stock solution or mixed standard stock solution, and dilute to obtain standard solution;

[0011] (2) Sample extraction: Take a urine sample into a container, add a dispersant and mix well, then add an extractant and mix, centrifuge to obtain a precipitate, dissolve the precipitate in the mobile phase to obtain the sample to be tested;

[0012] (3) High performance liquid chromatography analysis: The standard solution and the sample to be tested are detected and analyzed using a high performance liquid chromatograph;

[0013] (4) Plot the standard curve and calculate the results.

[0014] Preferably, in step (2), the volume ratio of the urine sample, dispersant, and extractant is 10000:(20-50):(70-100).

[0015] Preferably, in step (2), the extractant is added and mixed, and then sonicated for 0-14 minutes.

[0016] Preferably, in step (2), the cooling time at -10℃ to 10℃ is 0-12 minutes before centrifugation.

[0017] Preferably, in step (2), the centrifugation is performed at 4000 rpm for 0-12 minutes.

[0018] Preferably, in step (3), the chromatographic separation column is a Waters Acquity UPLC HSS T3 column (2.1 mm × 150 mm, 1.7 μm); the column temperature is 40 °C; the chromatographic mobile phase is 0.1% formic acid / water for mobile phase A and 0.1% formic acid acetonitrile for mobile phase B; the flow rate is 0.4 mL / min.

[0019] The elution method was gradient elution, and the gradient elution conditions were as follows: gradient elution times were 0 minutes, 2.8 minutes, 8 minutes, 8.1 minutes, 10 minutes, 10.1 minutes, and 15 minutes, and mobile phases A and B were set to 92% and 8%, 92% and 8%, 70% and 30%, 20% and 80%, 20% and 80%, 92% and 8%, and 92% and 8%, respectively.

[0020] Preferably, in step (1), the solvent used for preparation and dilution is methanol.

[0021] Preferably, in step (2), the urine sample is centrifuged at 3000 rpm for 5 minutes before being added to the container and filtered through a 0.22 μm microporous membrane.

[0022] The beneficial effects of this invention are as follows:

[0023] (1) The microextraction method developed in this invention has the advantages of being simple to operate, time-saving, efficient, and environmentally friendly. Furthermore, the method has a wide linear range, good reproducibility, and a low detection limit, making it suitable for the detection and analysis of narcotic compounds in urine matrices.

[0024] (2) In one embodiment of the present invention, [C4MIM-SH]Br serves as a pH adjuster, simplifying the pH adjustment steps in the experimental process; it also exhibits excellent hydrophilicity, making it a very good dispersant, environmentally friendly, and significantly reducing the amount of organic reagents required; it can produce obvious stratification, facilitating separation; and it provides lower LOD and higher recovery rate. Considering the multifunctional characteristics of this ionic liquid, it has great potential for development in chromatographic and spectroscopic analysis applications.

[0025] (3) By selecting the type of dispersant, the type of extractant, the volume of dispersant, the volume of extractant, the ultrasonic time, the cooling time, and the centrifugation time, the present invention optimizes the extraction conditions and obtains an optimal microextraction technology condition based on this type of ionic liquid. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0027] Figure 1 The [C4MIM-SH]Br prepared in this embodiment 1 H NMR;

[0028] Figure 2 The infrared spectrum of [C4MIM-SH]Br obtained in this embodiment;

[0029] Figure 3 The mass spectrum of [C4MIM-SH]Br obtained in this embodiment;

[0030] Figure 4 This is a curve showing the relationship between dispersant volume and pH.

[0031] Figure 5 This is the extraction operation process in this embodiment;

[0032] Figure 6 The extraction recovery rate of the target compound using different dispersants in this embodiment;

[0033] Figure 7 The extraction recovery rates of the target compounds using different extractants in this embodiment are shown.

[0034] Figure 8 The extraction recovery rates of the target compounds were obtained by using different cooling times in this embodiment.

[0035] Figure 9 The extraction recovery rates of the target compounds were obtained by using different centrifugation times in this embodiment.

[0036] Figure 10 This example illustrates the relationship between the actual and predicted values ​​simulated by the CCD.

[0037] Figure 11 The three-dimensional surface plot and contour plot show the effect of dispersant volume and extractant volume on the average extraction recovery of drug compounds when the ultrasonic time is constant.

[0038] Figure 12 Three-dimensional surface plot and contour plot showing the effect of extractant volume and ultrasonic time on the average extraction recovery rate of drug compounds when the dispersant volume is constant.

[0039] Figure 13The three-dimensional surface plot and contour plot show the effect of dispersant volume and ultrasonic time on the average extraction recovery rate of drug compounds when the extractant volume remains constant. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0041] 1. Preparation of standard solutions: Measure a certain amount of ethyl fluorometholone (2-FXE), methyl methacrylate (2-MDCK), dextromethorphan (DXM), tefenoxate (DIP), and etomidate (ET) standards, and prepare a 1 mg / L mixed standard intermediate solution with methanol. Dilute the mixed standard intermediate solution stepwise to prepare standard solutions with multiple concentration gradients.

[0042] 2. Urine sample pretreatment: Take 5 mL of urine sample, centrifuge at 3000 rpm for 5 minutes, and filter it through a 0.22 μm microporous membrane. The resulting solution is used for the microextraction process.

[0043] The water used in this embodiment is ultrapure water (resistivity ≥ 18.22 MΩ·cm).

[0044] 3. Instruments used: Shimadzu high-performance liquid chromatography-triple quadrupole system (LCMS8050, Japan), consisting of an autosampler (SIL20), a binary high-pressure pump (LC20), a column oven (CTO20), and a mass spectrometer detector (LCMS8050); column specifications; electronic balance (Sartorius BSA, Sartorius Scientific Instruments (Beijing) Co., Ltd.); ultrapure water system (Milli-Q, Millipore).

[0045] 4. Chromatographic conditions: After microextraction pretreatment, LCMS was used for analysis of drug compound content. Chromatographic separation column: Waters Acquity UPLC HSS T3 column (2.1 mm × 150 mm, 1.7 μm); column temperature: 40℃; mobile phase: 0.1% formic acid / water (mobile phase A), 0.1% formic acid acetonitrile (mobile phase B); flow rate: 0.4 mL / min. Gradient elution was used for chromatographic separation, and the gradient elution program is shown in Table 1.

[0046] Table 1 Gradient elution program

[0047]

[0048] MS parameters: An ESI source was selected as the mass spectrometer ion source. The ion source parameters were as follows: scanning mode was positive ion mode; the scan mass range during optimization was 50-500 m / z; heating gas flow rate was 10 mL / min; nebulizer gas flow rate was 2.8 mL / min; interface temperature was 300℃; desolvation temperature was 526℃; DL tube temperature was 250℃; heating block temperature was 400℃; dryer flow rate was 10 mL / min; and high-purity argon was used as the collision gas. Multiple reaction monitoring (MRM) mode was used for qualitative and quantitative analysis. To obtain the highest response value of the target compounds on the instrument, the built-in optimization program was used to optimize the optimal ion pairs and collision voltages for the five drugs. The optimized MRM acquisition parameters for the five compounds are shown in Table 2.

[0049] Table 2. MRM optimization parameters for 5 types of drugs

[0050]

[0051] 5. Taking [C4MIM-SH]Br as an example to illustrate [C n Synthesis process of MIM-SH]Br

[0052] (1) Synthesis of 4-bromobutyl ethyl thioester

[0053] First, accurately weigh 3.21 g of 1,4-dibromobutane "a" (MW = 213.90 g·mol⁻¹). -1 0.015 mol of potassium thioacetate was added and dissolved in a flask containing 10 mL of tetrahydrofuran. Then, 1.53 g of potassium thioacetate "b" (MW = 113.95 gmol⁻¹, 0.013 mol) was added. The mixture was stirred under reflux at 61 °C and gradually changed from colorless to pale yellow. The reaction was monitored by thin-layer chromatography. When the reaction was complete, the precipitated solid was filtered, and the solvent was removed by rotary evaporation to obtain the crude product "c", which was a pale yellow liquid. Finally, product "c" was purified by column chromatography (petroleum ether: dichloromethane = 2:1) to obtain a yellow viscous liquid with a yield of approximately 52.1%.

[0054]

[0055] (2) Synthesis of (4-(acetylmercapto)butyl)-3-methylimidazolium bromide

[0056] Next, 0.46 g of N-methylimidazole "d" (MW = 82.05 g·mol⁻¹, 0.0056 mol) was added to the flask and dissolved in 15 mL of acetonitrile. Then, 1.42 g of "c" (MW = 209.97 g·mol⁻¹, 0.0068 mol) was added dropwise, and the mixture was refluxed at 81 °C for 3–4 hours. Throughout the reaction, the reaction was monitored by thin-layer chromatography until reactant "d" disappeared. Heating was then stopped, the solvent was removed, and the mixture was dissolved in methanol. Finally, the mixture was extracted several times with petroleum ether to obtain pure product "e" as a pale yellow viscous liquid, with a yield of 92.4%.

[0057]

[0058] (3) Preparation of 1-(4-mercapto)-butyl-3-methylimidazolium bromide [C4MIM-SH]Br

[0059] Finally, 1.81 g (MW = 292.02 g mol⁻¹, 0.0062 mol) of the product “e” obtained in step 2 was introduced into a flask and dissolved in 24 mL of water. 0.496 g of NaOH was added to the flask, and the reaction was carried out in an ice bath for 6 hours. The pH was then adjusted to 2 with HCl at 25 °C, and the reaction was continued for another 8 hours. When the reaction was complete, the mixture was diluted with water and extracted with ethyl acetate. The water was then discarded, and the ethyl acetate was removed by rotary evaporation to obtain the crude product “f”. The crude product “f” was dissolved in anhydrous ethanol to remove residual NaCl, the precipitate was filtered off, and the extraction solvent was removed to obtain the pure product “f”, a pale yellow viscous liquid, with a yield of approximately 95%.

[0060]

[0061] Characterization:

[0062] 1 H NMR spectrum and analysis

[0063] 1 H NMR(500MHz,D2O)δ8.64(s,1H),7.40(t,J=5.5Hz,1H),7.34(t,J=10.0Hz,1H),4.34(t,J =10.0Hz,2H),3.79(s,3H),2.64(t,J=5.0Hz,2H),1.92-1.85(m,2H),1.63-1.57(m,2H)( Figure 1For [C4MIM-SH]Br, the singlets and triplets at chemical shifts of 8.64, 7.40, and 7.34 ppm are attributed to hydrogen atoms on the imidazole ring; the triplet at chemical shift 4.34 ppm is attributed to hydrogen atoms on the -CH2- group attached to the N atom on the imidazole ring; the singlet at chemical shift 3.79 ppm is attributed to hydrogen atoms on the -CH3 group; and the multiplets at chemical shifts of 2.64 ppm, 1.92–1.85 ppm, and 1.63–1.57 ppm are attributed to hydrogen atoms on the alkyl chain. No proton shift was observed in the -SH group in this study. Because the hydrogen atoms in the thiol group are reactive, under conditions where deuterated water is used as a solvent, the hydrogen atoms in the thiol group are replaced by deuterated forms, resulting in the loss of the proton signal in the thiol group.

[0064] The infrared spectrum of [C4MIM-SH]Br is as follows: Figure 2 As shown, the absorption bands of the characteristic functional groups are assigned as follows: IR(KBr)υ / (cm⁻¹), 3090, 1631, 840 (CH=CH), 2473 (-SH), 1573 (C=N), 1168 (CN), 2939, 1384 (-CH₃), and 2860, 1455, 756 (-CH₂-). (3090, 1631, 840cm⁻¹) -1 The peak at 1573 cm⁻¹ is due to the absorption peak of the CH stretching vibration of the imidazole ring. -1 The peak at 2473 cm⁻¹ is due to the CN stretching vibration of the imidazole ring. -1 It is the absorption peak of the SH mercapto stretching vibration.

[0065] Mass spectrometry of [C4MIM-SH]Br, determined by electron spray ionization (ESI), showed [C4MIM-SH]... + The characteristic of the group with an m / z of 171.2 ( Figure 3 ).

[0066] Because [C4MIM-SH]Br has a thiol group, it possesses potential acidity. When 90 μL of extraction solvent was added to 10 mL of urine, and the dispersant [C4MIM-SH]Br was gradually added, the pH change of the system was observed. When the volume increased from 0 to 40 μL, the pH of the solution dropped sharply from 5.61 to 3.32. Figure 4 However, further additions of 40 to 100 μL resulted in a stable pH of the solution (final pH = 3.16). Because the extraction efficiency of the target substance is higher in the acidic pH range, this ionic liquid was also used as a pH adjuster.

[0067] Example 1

[0068] like Figure 5The above embodiment illustrates a pretreatment technique for Dispersive Liquid-Liquid Microextraction (DLLME) based on ionic liquids (ILs). The specific process is as follows:

[0069] First, 10 mL of pretreated urine sample was added to a 15 mL conical centrifuge tube. Then, 40 μL of dispersant [C4MIM] BF4 was added to the centrifuge tube and mixed thoroughly. Subsequently, 80 μL of extraction solvent [C4MIM] PF6 was added, and the mixture was sonicated for 5 min. The suspension was then cooled at 0 °C for 5 min and centrifuged at 4000 rpm for 5 min, allowing the extraction solvent to settle at the bottom of the centrifuge tube. Finally, the supernatant was discarded, and the precipitate was dissolved in 200 μL of mobile phase for LCMS analysis.

[0070] Example 2

[0071] The only difference between this embodiment and Embodiment 1 is that the dispersant is replaced with [C6MIM]BF6.

[0072] Example 3

[0073] The only difference between this embodiment and Example 1 is that the dispersant is replaced with [C4MIM-SH]Br.

[0074] Example 4

[0075] The only difference between this embodiment and Embodiment 1 is that the extractant is replaced with [C6MIM]PF6.

[0076] Example 5

[0077] The only difference between this embodiment and Embodiment 1 is that the extractant is replaced with [C8MIM]PF6.

[0078] Example 6

[0079] The only difference between this embodiment and Embodiment 1 is that the cooling time is 0 min.

[0080] Example 7

[0081] The only difference between this embodiment and Embodiment 1 is that the cooling time is 3 minutes.

[0082] Example 8

[0083] The only difference between this embodiment and Embodiment 1 is that the cooling time is 6 minutes.

[0084] Example 9

[0085] The only difference between this embodiment and Embodiment 1 is that the cooling time is 9 minutes.

[0086] Example 10

[0087] The only difference between this embodiment and Embodiment 1 is that the cooling time is 12 minutes.

[0088] Example 11

[0089] The only difference between this embodiment and Embodiment 1 is that the centrifugation time is 0 min.

[0090] Example 12

[0091] The only difference between this embodiment and Embodiment 1 is that the centrifugation time is 3 minutes.

[0092] Example 13

[0093] The only difference between this embodiment and Embodiment 1 is that the centrifugation time is 6 minutes.

[0094] Example 14

[0095] The only difference between this embodiment and Embodiment 1 is that the centrifugation time is 9 minutes.

[0096] Example 15

[0097] The only difference between this embodiment and Embodiment 1 is that the centrifugation time is 12 minutes.

[0098] Examples 1-3 selected imidazole salt ionic liquids with different carbon chain lengths [C n Using [MIM]BF4 (n=4,6) and [C4MIM-SH]Br as dispersants, the extraction efficiency of three ionic liquids for several drug compounds was studied. Experimental results show that the dispersing solvent has a significant impact on the recovery rate of the target substances, and is therefore key to optimization. Different carbon chain lengths have a considerable influence on the properties of ionic liquids (such as solubility, viscosity, density, etc.). Therefore, as a dispersant, it also has a significant impact on the extraction recovery rate of the substances. Figure 6 It can be seen that [C4MIM-SH]Br has the highest recovery rate, with extraction rates of 77.3% for [C4MIM]BF4, 75.33% for [C6MIM]BF4, and 83.1% for [C4MIM-SH]Br. Therefore, in the DLLME program, [C4MIM-SH]Br plays two roles: pH adjuster and dispersant.

[0099] In microextraction, the extractant under ideal conditions should meet the following requirements: poor water solubility, good stability, higher density than water, and good extraction ability for the target analyte. Examples 1, 4, and 5 selected imidazole salt ionic liquids [C4MIM]PF6, [C6MIM]PF6, and [C8MIM]PF6 with different carbon chain lengths as extractants to study the extraction efficiency of the three ionic liquids for several drug compounds. Experimental results showed that [C4MIM]PF6, [C6MIM]PF6, and [C8MIM]PF6... n The different carbon chain lengths of MIM]PF6 affect its solubility (solubilities are 18.8 mg / L). -1 7.5 mg L -1 2.0 mg L -1 This difference in extraction efficiency is due to the high water solubility of [C4MIM]PF6, which results in the failure to extract the target compound. Figure 7 The extraction recoveries of [C6MIM]PF6 and [C8MIM]PF6 were similar, with [C6MIM]PF6 at 79.2% and [C8MIM]PF6 at 80.1%. However, [C8MIM]PF6 had a higher viscosity than [C6MIM]PF6. Therefore, [C6MIM]PF6, with its lower viscosity, was the optimal solvent for extraction.

[0100] Examples 6-10 used [C4MIM]BF4 as a dispersant and [C6MIM]PF6 as an extractant to investigate the effect of different cooling times on the extraction efficiency of this system. Figure 8 As shown, the extraction rates were consistently good when the cooling time was between 9 and 12 minutes. When the cooling time increased from 0 minutes to 9 minutes, the average extraction recovery rate of the target substance increased from 70.0% to 86.1%. This is because cooling increases the viscosity of the ionic liquid, facilitating its separation from water and indirectly improving the extraction recovery rate of the target substance. With further increases in cooling time, the recovery rate of the target substance decreased from 86.1% to 84.3%. This may be due to excessively long cooling times, resulting in excessively high viscosity of the ionic liquid, which prevents complete separation of the ionic liquid from the aqueous phase during centrifugation, thus reducing the extraction recovery rate. Therefore, this method uses 9 minutes as the optimal cooling time in this embodiment.

[0101] Examples 11-15 used [C4MIM]BF4 as a dispersant and [C6MIM]PF6 as an extractant to investigate the effect of different centrifugation times on the extraction efficiency of this system. As an important factor in phase separation, if the centrifugation time is too short, the ionic liquid phase cannot be completely separated from the aqueous phase; however, if the centrifugation time is too long, some of the ionic liquid phase will adsorb onto the centrifuge tube wall, thereby reducing the recovery rate. Figure 9As shown, when the centrifugation time increased from 0 min to 6 min, the average extraction recovery rate of the target compound increased from 72.3% to 83.1%; however, when the centrifugation time increased from 6 min to 12 min, the extraction recovery rate of the target compound decreased slightly, from 83.1% to 80.8%. Therefore, in this embodiment, a centrifugation time of 6 min was selected as the optimal cooling time.

[0102] Through preliminary single-factor optimization, the dispersant volume, extractant volume, and ultrasonic time were determined to be the main factors in this microextraction pretreatment technology.

[0103] Table 3 CCD Experimental Design Table

[0104]

[0105]

[0106] Analysis of variance (ANOVA) of the CCD model equations and correlation coefficients revealed that the p-value < 0.0001, indicating a high correlation; the lack of fit was 0.8315, greater than 0.05, indicating differences between parallel groups. Therefore, this model can be applied to analyze these three factors. Figure 4 Given: A, B, C, AC, A 2 B 2 and C 2 The p-values ​​were all less than 0.05, indicating that the three factors directly affected the recovery rate of the compound. Equation (4) shows the contribution of each factor to the recovery rate:

[0107] Y=b0 +b1A+b2B+b3C+b4AB+b5AC+b6BC+b7A 2 +b8B 2 +b9C 2 (4)

[0108] b0=90.72; b1=5.03; b2=25.08; b3=-3.67; b4=-9.35; b5=7.55; b6=8.7;

[0109] b7=-5.5; b8=-20.67; b9=-3.7.

[0110] Where Y represents the average recovery rate of BPs, b0 is the intercept, b1-b9 are parameters, "+" indicates that the factor has a promoting effect on the recovery rate, and "-" indicates an inhibiting effect. The absolute value of b represents the degree of influence on the recovery rate, and the correlation coefficient is R. 2 =0.9898, the corrected correlation coefficient is R 2=0.9715, indicating that this equation can objectively reflect the relationship between various factors and the recovery rate.

[0111] Table 4. Variance Analysis of CCD Design

[0112]

[0113]

[0114] Figure 10 (Left) indicates that the actual and predicted values ​​are evenly distributed on both sides of the regression equation, which shows that this model can reflect the real optimization process. Figure 10 (Right) shows the random distribution of the remaining points, indicating that the recovery rate values ​​obtained from all experiments are stable and reliable.

[0115] To understand more about how the experimental factors influence each other, we used the controlled variable method, keeping one factor constant and observing the interaction between the other two factors. We then used software to create 3D surface plots and contour maps. Figure 11 It can be seen that, under the condition of maintaining ultrasonic time for 7 minutes, the effects of dispersant volume and extractant volume on the average extraction recovery rate of drug compounds are as follows: when the dispersant volume increases from 0 μL to 33 μL and the extractant volume increases from 10 μL to 87 μL, the average recovery rate of the five drug compounds gradually increases with the increase of dispersant and extractant volume; however, when the dispersant volume increases from 33 μL to 60 μL and the extractant volume increases from 87 μL to 120 μL, the average recovery rate of the five drug compounds decreases.

[0116] like Figure 12 As shown, when the dispersant volume remained constant at 30 μL, as the volume of the extractant and the ultrasonic time gradually increased, the average extraction recovery rate of the drug compound reached its maximum when the volume of the extractant was 87 μL and the ultrasonic time was 9 min.

[0117] When the extractant volume remains constant at 60 μL, the three-dimensional surface plot and contour plot of the effect of dispersant volume and ultrasonic time on the average extraction recovery of drug compounds are shown below. Figure 13 The average extraction recovery of the compound reached its peak when the dispersant volume was 33 μL and the sonication time was 9 min. With further increases in dispersant volume and sonication time, the average recovery began to decrease.

[0118] Through the study of the above three interacting factors, the optimal extraction conditions for this method were obtained, namely, 33 μL of dispersant, 87 μL of extractant, and 9 min of ultrasonic time.

[0119] Through single-factor screening and CCD condition optimization, under optimal conditions, the linear range (LR) and correlation coefficient (R²) were analyzed.2 The method established in this scheme is evaluated using indicators such as limit of detection (LOD) and precision (RSD). Table 5 shows that the correlation coefficient R of the linear equation... 2 The range was 0.9956–0.9993; the limit of detection (LOD) in urine was 0.156 μg / L. -1 2-FXE, 0.254 μg L -1 2-MDCK, 0.244 μg L -1 DXM, 0.25 μg L -1 DIP, 0.09 μg L -1 ET.

[0120] Table 5 Performance metrics of the In-situ DIME method (sample, equation, linearity, limit of detection, limit of quantitation)

[0121]

[0122] To verify the stability of the above-mentioned method, its intra-day / inter-day precision was tested. This was done at three concentration levels (10 μg / L). -1 20 μg L -1 50 μg L -1 The experiment was conducted with intraday precision measured every two hours for a total of six measurements, and the average value was taken. Inter-day precision was measured at 8:00 AM each day for a total of six measurements, and the average value was taken. The experimental results in Table 6 show that, at different concentration levels, the intraday precision of the target compound ranged from 1.42% to 3.90%, and the inter-day precision ranged from 1.47% to 4.72%. This indicates that the method has good stability and can meet the detection requirements.

[0123] Table 6. Intra-day and inter-day precision of the target compounds (n=6)

[0124]

[0125] Three detection concentrations (10 μg / L) were added to the actual sample. -1 20 μg L -1 50 μg L -1 After using the target compound standards, the recoveries of the target compound in actual samples ranged from 92.5-95.4% (2-FXE); 91.2-92.3% (2-MDCK); 93.6-96.2% (DXM); 94.0-96.0% (DIP); and 89.5-93.5% (ET) (as shown in Table 7). In summary, the method established in this embodiment is highly effective for detecting the target substance in actual samples.

[0126] Table 7. Extraction recoveries of the actual target compounds in the samples (mean ± SD, n = 3)

[0127]

[0128] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for detecting drugs in urine using ionic liquid extraction-high performance liquid chromatography, characterized in that: It uses water-soluble imidazole salt ionic liquid as a dispersant and water-insoluble imidazole salt ionic liquid as an extractant to extract urine. The precipitate obtained by extraction is analyzed by high performance liquid chromatography to obtain the drug content in urine. The dispersant is selected from at least one of [C4MIM]BF4, [C6MIM]BF4, and [C4MIM-SH]Br, and the extractant is selected from at least one of [C4MIM]PF6, [C6MIM]PF6, and [C8MIM]PF6; The drugs mentioned include synthetic cathinones and psychoactive drugs.

2. The method for detecting drugs in urine by ion liquid extraction-high performance liquid chromatography according to claim 1, characterized in that: The drugs mentioned include ethyl fluoroquinolone, methyl ketamine, dextromethorphan, tefenoxate, and etomidate.

3. The method for detecting drugs in urine by ion liquid extraction-high performance liquid chromatography according to claim 1 or 2, characterized in that, Includes the following steps: (1) Preparation of standard solution: Take drug standard, prepare single stock solution or mixed standard stock solution, and dilute to obtain standard solution; (2) Sample extraction: Take a urine sample into a container, add a dispersant and mix well, then add an extractant and mix, centrifuge to obtain a precipitate, dissolve the precipitate in the mobile phase to obtain the sample to be tested; (3) High performance liquid chromatography analysis: The standard solution and the sample to be tested are detected and analyzed using a high performance liquid chromatograph; (4) Plot the standard curve and calculate the results.

4. The method for detecting drugs in urine by ion liquid extraction-high performance liquid chromatography according to claim 3, characterized in that: In step (2), the volume ratio of the urine sample, dispersant, and extractant is 10000:(20-50):(70-100).

5. The method for detecting drugs in urine by ion liquid extraction-high performance liquid chromatography according to claim 3, characterized in that: In step (2), after adding the extractant and mixing, sonicate for 0-14 minutes.

6. The method for detecting drugs in urine by ionic liquid extraction-high performance liquid chromatography according to claim 3, characterized in that: In step (2), the cooling time is 0-12 minutes at -10℃ to -10℃ before centrifugation.

7. The method for detecting drugs in urine by ion liquid extraction-high performance liquid chromatography according to claim 3, characterized in that: In step (2), centrifuge at 4000 rpm for 0-12 minutes.

8. The method for detecting drugs in urine by ion liquid extraction-high performance liquid chromatography according to claim 3, characterized in that: In step (3), the chromatographic separation column was a Waters Acquity UPLC HSS T3 column (2.1 mm × 150 mm, 1.7 μm); the column temperature was 40 °C; the mobile phase was 0.1% formic acid / water for mobile phase A and 0.1% formic acid acetonitrile for mobile phase B; and the flow rate was 0.4 mL / min. The elution method was gradient elution, and the gradient elution conditions were as follows: gradient elution times were 0 minutes, 2.8 minutes, 8 minutes, 8.1 minutes, 10 minutes, 10.1 minutes, and 15 minutes, and mobile phases A and B were set to 92% and 8%, 92% and 8%, 70% and 30%, 20% and 80%, 20% and 80%, 92% and 8%, and 92% and 8%, respectively.

9. The method for detecting drugs in urine by ion liquid extraction-high performance liquid chromatography according to claim 3, characterized in that: In step (1), the solvent used for preparation and dilution is methanol.

10. The method for detecting drugs in urine by ion liquid extraction-high performance liquid chromatography according to claim 3, characterized in that: In step (2), the urine sample is centrifuged at 3000 rpm for 5 minutes before being added to the container and filtered through a 0.22 μm microporous membrane.

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