Method for detecting amount of drug based on solid phase microextraction and real-time analysis mass spectrometry

Abstract

The application discloses a detection method for detecting drug amount based on solid phase microextraction and real-time analysis mass spectrum, which comprises the following steps: preparing a solid phase microextraction head by using a coating material, preparing a standard sample to form a standard curve solution, detecting the standard curve solution by solid phase microextraction and real-time direct analysis mass spectrum, obtaining a mass spectrum diagram of the standard curve solution, and then obtaining a standard curve equation of the standard curve solution; obtaining a peak area value of a to-be-detected sample by solid phase microextraction and real-time direct analysis mass spectrum, and substituting the peak area value into the standard curve equation to obtain the concentration of corresponding drugs. By setting the method process, the detection steps of the application are obviously simplified, the constant volume, detection and calculation operations can be carried out on site according to actual needs, the operation mode and operation site are flexible, and the beneficial effect of improving the detection efficiency can be achieved.

Description

A method for detecting drug dosage based on solid-phase microextraction and real-time analytical mass spectrometry Technical Field

[0001] This invention relates to the technical field of drug detection methods, specifically to a detection method for drug quantity based on solid-phase microextraction and real-time analytical mass spectrometry. Background Technology

[0002] Currently, the most common drugs are methamphetamine (ammoniacein), heroin, marijuana, cocaine, and ketamine (morphine). In recent years, the emergence of new drugs such as methcathinone has posed new challenges to existing conventional detection technologies.

[0003] In the field of trace drug analysis and detection, laboratory drug detection methods mainly include gas chromatography (GC), high-performance liquid chromatography (HPLC), capillary electrophoresis, gas chromatography-mass spectrometry (GC / MS), high-performance liquid chromatography-mass spectrometry (HPLC / MS), and mass spectrometry (MS). Although these drug detection methods have high sensitivity and specificity, they usually require complex pretreatment before testing. Taking SPE as an example, limitations such as sample size, liquid level, and packing tightness increase the technical difficulty of operation. Furthermore, activation, purification, and elution are required, undoubtedly lengthening the processing time. Therefore, the pretreatment of these detection methods is cumbersome, time-consuming, and inefficient.

[0004] In recent years, although chromatography and mass spectrometry have become increasingly common methods for qualitative and quantitative analysis of samples, mass spectrometry requires stringent detection conditions. The ion source, limited by its own structure and operating environment (typically employing a closed ionization method or operating in a relative vacuum), struggles to meet the demands of real-time, in-situ, and in vivo analysis of actual samples, especially biological samples. Furthermore, the large size of the detection equipment often limits its use to laboratory settings, making it inconvenient for on-site, accurate, and rapid detection and analysis of narcotics in samples. Summary of the Invention

[0005] In order to overcome the shortcomings of the existing technology, the purpose of this invention is to provide a detection method for drug content based on solid phase microextraction and real-time analytical mass spectrometry. This detection method can simplify the detection steps, realize on-site detection and analysis, and improve detection flexibility and work efficiency.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for detecting drug levels based on solid-phase microextraction and real-time analytical mass spectrometry includes the following steps:

[0008] Solid-phase microextraction coating preparation: According to the sample to be tested and the type of drug to be detected, the corresponding coating solution is applied to the glass rod to make a solid-phase microextraction head;

[0009] Standard sample preparation and processing: Obtain standard curve solutions through standard sample preparation;

[0010] Solid-phase microextraction and real-time direct analysis mass spectrometry: The prepared solid-phase microextraction head is pushed into the standard curve solution for extraction, and then the extracted solid-phase microextraction head is analyzed by real-time direct analysis mass spectrometry.

[0011] Standard curve plotting: Obtain the mass spectrum of the standard curve solution, and then obtain the standard curve equation of the standard curve solution;

[0012] Sample testing: A liquid sample is obtained, and a solid-phase microextraction head is inserted into the sample for extraction. Then, the extracted solid-phase microextraction head is subjected to real-time direct mass spectrometry analysis to obtain the mass spectrum of the sampled solution. Based on the mass spectrum of the sampled solution and the standard curve equation, the content of drug components in the sampled solution is obtained.

[0013] Furthermore, in the process of preparing the coating solution and the solid-phase microextraction head, PDMS is selected as the coating material to prepare the coating solution, which includes the following steps:

[0014] (1) Using the PDMS material preparation kit, magnetic grapefruit peel biochar was used as component A, and a mixture of C18 and SCX was used as component B. Component A and component B were mixed in a weight ratio of 20:1 and then added to a mixed solution of dichloromethane and n-hexane in a weight ratio of 5:1, so that the final concentration of the mixture of component A and component B was 0.10 g / mL.

[0015] (2) Under vacuum conditions, heat the prepared glass rod coated with the above solution to 75-85°C and hold for two hours, then heat to 110-125°C and hold for three hours, and finally heat to 240-260°C and hold for eight hours to complete the preparation of the solid phase microextraction head.

[0016] Furthermore, the standard sample preparation process includes the preparation of standard stock solutions, the preparation of mixed standard solutions ① and ②, and the preparation of standard curve solutions.

[0017] Furthermore, the preparation of the standard stock solution includes: weighing various drug component solid standards into a volumetric flask, dissolving them in methanol and diluting to the mark to obtain a 1000 mg / L standard stock solution.

[0018] Furthermore, the preparation of the mixed standard solution ① and the mixed standard solution ② includes: extracting the standard stock solution into a volumetric flask, diluting and making up to volume with methanol to obtain a 10 mg / L mixed standard solution ①; extracting a portion of the mixed standard solution ①, diluting and making up to volume with methanol to obtain a 1 mg / L mixed standard solution ②.

[0019] Furthermore, the preparation of the standard curve solution includes: diluting the mixed standard solution ② with methanol aqueous solution to prepare multiple standard curve solutions of different concentrations, wherein the concentration of the isotope tracer in each standard curve solution is 100 μg / L.

[0020] Furthermore, when the solid-phase microextraction head is pushed into the standard curve solution for extraction, a magnetic stirrer is used to stir the standard curve solution, the solid-phase microextraction head is inserted 0.8-1.5 cm below the surface of the standard curve solution, and the extraction time is 15-30 minutes.

[0021] Furthermore, during the real-time direct analysis mass spectrometry analysis of the extracted solid-phase microextraction head, the solid-phase microextraction head, after extraction in the standard curve solution, is removed and inserted into the injection port of the real-time direct analysis mass spectrometry device, and real-time direct analysis mass spectrometry analysis is performed using the real-time direct analysis mass spectrometry device.

[0022] Furthermore, in the process of obtaining the standard curve equation of the standard curve solution: after performing real-time direct analysis mass spectrometry analysis using a real-time direct analysis mass spectrometry device, the mass spectrum of the standard curve solution is obtained. The peak area value of each standard curve solution is obtained through the mass spectrum. The drug concentration of each standard curve solution is used as the abscissa, and the peak area of ​​each standard curve solution is used as the ordinate. Linear regression analysis is performed to obtain the standard curve equation of the standard curve solution.

[0023] Furthermore, during sample testing, after obtaining the mass spectrum of the sample through real-time direct analysis mass spectrometry using a real-time direct analysis mass spectrometry device, the peak area value of the sample is obtained from the mass spectrum. This peak area value is then substituted into the standard curve equation to finally obtain the content of the drug component contained in the sample.

[0024] The present invention has the following beneficial effects:

[0025] 1. A method for detecting drug dosage based on solid-phase microextraction (SPE) and real-time direct mass spectrometry (RTMS) aims to simplify detection steps, enable on-site detection and analysis, and improve detection flexibility and work efficiency. The method involves preparing a solid-phase microextraction (SPE) head by coating a glass rod with a solution. Then, a standard curve solution is prepared using standard samples. In practice, multiple standard curve solutions of different concentrations can be prepared by controlling the increase or decrease of the standard sample mass. During SPE and RMS detection, the SPE head, coated with the solution, is inserted into the standard curve solution for extraction. Real-time direct mass spectrometry analysis is then performed on the SPE head to obtain the mass spectrum of the standard curve solution, from which the standard curve equation is derived.

[0026] 2. The standard curve equation mainly includes the concentration of the standard sample (drug component) and the corresponding parameter values ​​of the standard curve solution in the mass spectrum. Therefore, using this standard curve equation as a reference standard for detection, in subsequent actual detection of trace drugs (sample detection), the sample to be tested can be directly subjected to solid-phase microextraction and real-time direct analysis mass spectrometry to obtain the mass spectrum of the sample to be tested. The corresponding parameter values ​​are obtained from the mass spectrum of the sample to be tested, and these parameter values ​​are substituted into the standard curve equation to obtain the concentration value of the corresponding drug in the sampling solution. In this process, the extraction, volume adjustment, detection, and calculation operations for obtaining the standard curve equation before detection are mainly involved. In the actual detection operation, the same volume adjustment, detection, and calculation operations are performed. Compared with the operation steps before detection in the prior art, the detection steps of this invention are significantly simplified, and the volume adjustment, detection, and calculation operations can be performed on-site according to actual needs, thereby achieving the beneficial effects of flexible operation methods and operation sites, and improving detection efficiency. Attached Figure Description

[0027] Figure 1 is a flowchart of the method of the present invention.

[0028] Figure 2 is a bar chart comparing the extraction effects of three coating materials: PDMS, PA, and PEG.

[0029] Figure 3 is a scatter plot of the extraction effect of two drug sample solutions with respect to salinity.

[0030] Figure 4 is a scatter plot of the extraction effect of two drug sample solutions with respect to pH value.

[0031] Figure 5 is a scatter plot of the extraction effect of two drug sample solutions with respect to extraction time.

[0032] Figure 6 is a scatter plot of the extraction effect of two drug sample solutions with respect to stirring rate.

[0033] Figure 7 shows the mass spectrum of the methamphetamine sample solution after solid-phase microextraction and real-time direct analysis mass spectrometry.

[0034] Figure 8 shows the mass spectrum of the ketamine sample solution after solid-phase microextraction and real-time direct analysis mass spectrometry.

[0035] Figure 9 shows the mass spectrum of the morphine sample solution after solid-phase microextraction and real-time direct analysis mass spectrometry. Detailed Implementation

[0036] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. Terms such as “upper,” “inner,” “middle,” “left,” “right,” and “one” used in this specification are merely for clarity of description and are not intended to limit the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention.

[0037] To enable readers to understand solid-phase microextraction, solid-phase microextraction fiber coating, real-time direct analysis mass spectrometry, and real-time direct analysis mass spectrometry combined with solid-phase microextraction, and thus to better understand the specific technical solutions described in this invention, solid-phase microextraction, solid-phase microextraction fiber coating, real-time direct analysis mass spectrometry, and real-time direct analysis mass spectrometry combined with solid-phase microextraction will be introduced first before introducing the specific embodiments and experimental examples of this invention.

[0038] 1. Solid-phase microextraction (SPE): Developed from solid-phase extraction (SPE), SPE is a simple and effective sample adsorption / desorption pretreatment technique that integrates sampling, extraction, concentration, and injection. It offers advantages such as ease of operation, short processing time, rapid and efficient determination, and the elimination of the need for any organic solvents. Furthermore, this technique requires simple equipment, operates quickly without additional auxiliary equipment, and boasts high analytical sensitivity, with detection limits reaching μg / L to ng / L. It also requires small sample volumes and does not necessitate complete extraction or equilibration. Currently, in drug analysis, SPE is widely used for the detection of amphetamines, methcathinone, cocaine and its derivatives, cannabis, ketamine, and other abused drugs and illicit substances in urine, blood, saliva, hair, and other biological samples.

[0039] 2. Solid-phase microextraction fiber coating: The properties and preparation methods of coating materials are the core content of solid-phase microextraction technology research. The structure and properties of the materials directly affect the selectivity and application range of solid-phase microextraction, and to a large extent determine the accuracy and sensitivity of the entire analysis results.

[0040] 3. Real-time direct mass spectrometry: This is a thermal desorption and ionization technique. Under atmospheric pressure, a neutral or inert gas (such as nitrogen or helium) generates excited-state atoms through discharge. These excited-state atoms are then rapidly heated and accelerated by an electric field, causing them to desorb and instantly ionize the marker compounds or analyte compounds on the surface of the sample. Mass spectrometry or tandem mass spectrometry is then used for detection, thereby enabling real-time direct analysis of the sample.

[0041] 4. Real-time Direct Analysis Mass Spectrometry Combined with Solid Phase Microextraction: The analytical method combining solid phase microextraction (SPME) with real-time direct analysis mass spectrometry (SPME-DART-MS) is a novel approach both domestically and internationally. This method maximizes the advantages of both technologies, enabling high-throughput, in-situ, and online analysis of real samples with efficient and rapid sample pretreatment. Furthermore, it is not limited by the working environment (e.g., vacuum environment) and can be performed normally under atmospheric pressure. Currently, the application of real-time direct analysis mass spectrometry combined with solid phase microextraction in various fields is still in its early exploratory stage.

[0042] Based on the above introduction to solid-phase microextraction, solid-phase microextraction fiber coating, real-time direct analysis mass spectrometry, and real-time direct analysis mass spectrometry combined with solid-phase microextraction, the specific embodiments and experimental examples of the present invention are described in detail below:

[0043] Example 1

[0044] A method for detecting drug levels based on solid-phase microextraction and real-time analytical mass spectrometry includes the following steps:

[0045] Solid-phase microextraction coating preparation: According to the sample to be tested and the type of drug to be detected, the corresponding coating solution is applied to the glass rod to make a solid-phase microextraction head;

[0046] Standard sample preparation and processing: A standard curve solution is obtained through standard sample preparation, wherein the standard samples are various drug components;

[0047] Solid-phase microextraction and real-time direct analysis mass spectrometry: The prepared solid-phase microextraction head is pushed into the standard curve solution for extraction, and then the extracted solid-phase microextraction head is analyzed by real-time direct analysis mass spectrometry.

[0048] Standard curve plotting: Obtain the mass spectrum of the standard curve solution, and then obtain the standard curve equation of the standard curve solution;

[0049] Sample testing: A liquid sample is obtained, and a solid-phase microextraction head is inserted into the sample for extraction. Then, the extracted solid-phase microextraction head is subjected to real-time direct mass spectrometry analysis to obtain the mass spectrum of the sampled solution. Based on the mass spectrum of the sampled solution and the standard curve equation, the content of drug components in the sampled solution is obtained.

[0050] Specifically, this invention aims to simplify the detection process, enable on-site detection and analysis, and improve detection flexibility and work efficiency. Therefore, a solid-phase microextraction (SPE) head is prepared by coating a glass rod with a solution. Then, a standard curve solution is prepared using standard samples. In practice, multiple standard curve solutions of different concentrations can be prepared by controlling the increase or decrease in the mass of the standard samples. Subsequently, during SPE and real-time direct mass spectrometry analysis, the SPE head, coated with the solution, is inserted into the standard curve solution for extraction. Real-time direct mass spectrometry analysis is then performed on the SPE head to obtain the mass spectrum of the standard curve solution, from which the standard curve equation is derived.

[0051] The standard curve equation mainly includes the concentration of the standard sample (drug component) and the corresponding parameter values ​​of the standard curve solution in the mass spectrum. Therefore, using this standard curve equation as a reference standard for detection, in subsequent actual detection of trace drugs (sample detection), the sample to be tested can be directly subjected to solid-phase microextraction and real-time direct analysis mass spectrometry to obtain the mass spectrum of the sample to be tested. The corresponding parameter values ​​are obtained from the mass spectrum of the sample to be tested, and these parameter values ​​are substituted into the standard curve equation to obtain the concentration value of the corresponding drug in the sampled solution.

[0052] The process mainly involves extraction, volume determination, detection, and calculation to obtain the standard curve equation before detection. In the actual detection process, the same volume determination, detection, and calculation operations are performed. Compared with the operation steps before detection in the prior art, the detection steps of the present invention are significantly simplified, and the volume determination, detection, and calculation operations can be performed on-site according to actual needs. This achieves the beneficial effects of flexible operation methods and operation sites, and improved detection efficiency.

[0053] Furthermore, in the preparation process of the coating solution, different coating materials need to be selected to fabricate the extraction membrane (fiber coating) based on the selection of the sample to be tested and the properties of different drugs and their metabolites. The following describes the preparation operation of the coating solution: Selecting PDMS as the coating material to prepare the coating solution includes the following steps:

[0054] (1) Using the PDMS material preparation kit, magnetic grapefruit peel biochar was used as component A, and a mixture of C18 and SCX was used as component B. Component A and component B were mixed in a weight ratio of 20:1 and then added to a mixed solution of dichloromethane and n-hexane in a weight ratio of 5:1, so that the final concentration of the mixture of component A and component B was 0.10 g / mL.

[0055] Among them, component A, magnetic pomelo peel-derived biochar, is a high-quality adsorbent; in component B, C18 is an abbreviation for octadecylsilane bonded phase, with a purity of 80%; and SCX is a benzenesulfonic acid-based filler in silica gel matrix, with a purity of 20%.

[0056] (2) Apply the prepared solution evenly to the surface of the glass rod;

[0057] (3) Under vacuum conditions, heat the prepared glass rod coated with the above solution to 80°C and keep it for two hours, then heat it to 120°C and keep it for three hours, and finally heat it to 250°C and keep it for eight hours to complete the preparation of the solid phase microextraction head.

[0058] Furthermore, in order to disclose the process of preparing standard samples, the standard sample preparation process includes the preparation of standard stock solutions, the preparation of mixed standard solutions ① and ②, and the preparation of standard curve solutions.

[0059] The preparation of the standard stock solution includes: weighing 10 mg (±0.01 mg) of each solid standard of various drug components into a 10 mL volumetric flask, dissolving in methanol, and diluting to the mark. The concentration of the standard stock solution should be controlled at approximately 1000 mg / L. If the properties of the drug component standard affect the concentration (e.g., excessive viscosity), the weighing amount, volume, or final concentration may be adjusted accordingly. The standard stock solution should be stored in a refrigerator at -18℃±4℃ and has a shelf life of one year. Each time a new stock solution is prepared, an interim check is required to ensure its stability. The shelf life of the stock solution must not exceed the shelf life of the standard reference material used to prepare it.

[0060] The preparation of the mixed standard solution ① and mixed standard solution ② includes: transferring an appropriate amount of the above standard stock solution into a 10 mL volumetric flask, diluting it with methanol and making up to the mark to obtain a 10 mg / L mixed standard solution 1. Mixed standard solution ① (10 mg / L) is stored in a refrigerator at -4℃±4℃ and has a shelf life of 3 months. Accurately transfer 1.0 mL of mixed standard solution ① (10 mg / L) into a 10 mL volumetric flask, diluting it with methanol and making up to the mark to obtain a 1 mg / L mixed standard solution ②, which is stored in a refrigerator at -4℃±4℃ and has a shelf life of 1 month.

[0061] The preparation of the standard curve solutions includes: diluting the above-mentioned mixed standard solution ② (1 mg / L) with methanol-water (1:1) solution to prepare ten standard curve solutions with different concentrations: 0.1 μg / L, 0.5 μg / L, 1.0 μg / L, 5.0 μg / L, 10.0 μg / L, 50.0 μg / L, 100.0 μg / L, 500.0 μg / L, 1000 μg / L, and 5000 μg / L. The concentration of the isotope tracer in each standard curve solution is 100 μg / L. It is worth noting that the standard curve solutions should be prepared immediately before use.

[0062] Furthermore, when the solid-phase microextraction head is pushed into the standard curve solution for extraction, a magnetic stirrer is used to stir the standard curve solution, the solid-phase microextraction head is inserted 0.8-1.5 cm below the surface of the standard curve solution, and the extraction time is 15-30 minutes.

[0063] Furthermore, in order to perform real-time direct analysis mass spectrometry analysis on the extracted solid-phase microextraction head, the solid-phase microextraction head, after extraction in the standard curve solution, is removed and inserted into the injection port of the real-time direct analysis mass spectrometry device, and then the real-time direct analysis mass spectrometry device is used for real-time direct analysis mass spectrometry analysis.

[0064] When performing real-time direct analysis mass spectrometry on a solid-phase microextraction head using a real-time direct analysis mass spectrometry device, the measurement conditions are as follows:

[0065] 1) DART ion source and triple quadrupole mass spectrometry were used;

[0066] 2) Ion mass scan range: m / z 40~500;

[0067] 3) Detection mode: Data acquisition in positive ion mode;

[0068] 4) Ion transport tube temperature: 300℃;

[0069] 5) The ionizing gas is high-purity helium, and the standby gas is high-purity nitrogen;

[0070] 6) The grid voltage range is 100-400V, and the air pressure is 0.2-0.5MPa;

[0071] 7) Detection method: Multiple Response Monitoring (MRM);

[0072] 8) The collision voltage is 0-200eV and the collision pressure is 0-5mTorr.

[0073] Furthermore, in the process of obtaining the standard curve equation of the standard curve solution: after performing real-time direct analysis mass spectrometry analysis using a real-time direct analysis mass spectrometry device, the mass spectrum of the standard curve solution is obtained. The peak area value of each standard curve solution is obtained through the mass spectrum. The drug concentration of each standard curve solution is used as the abscissa, and the peak area of ​​each standard curve solution is used as the ordinate. Linear regression analysis is performed to obtain the standard curve equation of the standard curve solution.

[0074] Furthermore, during sample testing, after obtaining the mass spectrum of the sample through real-time direct analysis mass spectrometry using a real-time direct analysis mass spectrometry device, the peak area value of the sample is obtained from the mass spectrum. This peak area value is then substituted into the standard curve equation to finally obtain the content of the drug component contained in the sample.

[0075] To ensure the accuracy of sample testing, multiple samples of 2-5 mL are taken in parallel during testing. In this embodiment, three samples are used. These samples can be of the same type, such as all sewage, or of different types, such as urine, saliva, and sewage. The three samples are placed in three separate sampling bottles, labeled Experimental Group 1, Experimental Group 2, and Experimental Group 3. No pretreatment is required before extraction. Three solid-phase microextraction heads are then inserted into the samples of Experimental Group 1, Group 2, and Group 3, respectively, for extraction. Real-time direct mass spectrometry (RTMS) is then used to obtain the mass spectra of the samples corresponding to Experimental Group 1, Group 2, and Group 3. The peak areas of different experimental groups are obtained from these mass spectra. These peak area values ​​are then substituted into the standard curve equation to determine the content of different drug components in each experimental group.

[0076] Based on the above disclosure of a method for detecting drug levels based on solid-phase microextraction and real-time analytical mass spectrometry, the design principle of this invention will be further described below:

[0077] This method combines real-time direct mass spectrometry with solid-phase microextraction (SPE). SPE utilizes SPE technology, synthesizing different extraction heads with probe coatings of various materials. Complex samples undergo simple pretreatment. The probe is then inserted into the sample and extracted after a certain time – a rapid, efficient, and pollution-free process that completes the entire operation of sampling, extraction, concentration, and injection. This overcomes the problems of cumbersome operations, long processing times, and complex reagent requirements in traditional sample pretreatment methods, significantly improving the analytical efficiency for trace drugs in complex biological samples.

[0078] The analytical detection section employs real-time direct mass spectrometry, allowing the entire analytical process to be performed at atmospheric pressure without the need for a vacuum system. Under atmospheric pressure, the analyte is instantly ionized through continuous nitrogen / helium blowing and bombardment by a high-temperature electric field. Compared to conventional chromatography-mass spectrometry, the ion source section does not require a vacuum system, sample pretreatment is simplified, chromatographic separation is eliminated, and mass spectrometry contamination is reduced. It also offers high sensitivity, low sample loss, ease of operation, short sample analysis time, and enables real-time qualitative analysis.

[0079] Based on the above introduction of the design principles of this invention, the following beneficial effects of the overall solution of this invention compared to the prior art are obtained:

[0080] (1) Pioneering Application of Real-Time Direct Mass Spectrometry Combined with Solid-Phase Microextraction in Trace Drug Analysis and Detection. The research and application of real-time direct mass spectrometry combined with solid-phase microextraction as a novel and efficient analytical method in various fields is still relatively slow. This method is pioneering in the research and application of trace drug analysis and detection, which is of great significance for the accurate analysis and rapid detection of drug components. The pretreatment technology of this method integrates sampling, extraction, concentration, and injection, making it convenient to operate, time-saving, and fast and efficient. It requires no organic solvents, representing true solid-phase extraction and avoiding secondary environmental pollution. The instrument is simple, requiring no auxiliary equipment, suitable for on-site analysis, and easy to operate. It has high sensitivity, enabling ultra-trace analysis, reaching the nanogram per gram (ng / g) level of detection.

[0081] (2) Compared with conventional chromatography-mass spectrometry, the analytical detection technology of this method can be carried out under normal pressure conditions, the ion source does not require a vacuum system, the sample pretreatment is simple, chromatographic separation is not required, and mass spectrometry contamination can be reduced; it has high sensitivity and low sample loss; it is easy to operate, the sample analysis time is short, and real-time qualitative analysis can be achieved.

[0082] (3) This method can achieve simultaneous analysis of multiple components; it does not require lengthy chromatographic separation, and the analysis speed is in the second range, which can effectively save time (the analysis time for a single sample is only a few seconds).

[0083] (4) The extraction membrane (fiber coating) can be synthesized according to requirements; it can be made into a vehicle-mounted device for convenient and quick results;

[0084] (5) This method can be used to detect complex matrices (such as blood, urine, saliva and other bodily fluids, as well as sewage, air, etc.).

[0085] Experimental Example 1 (Selection of Coating Material)

[0086] Solid-phase microextraction (SPE) heads were prepared using three coating materials: PDMS, PA, and PEG. Two of each type of SPE head were used, forming a group of three. These two groups of SPE heads were then inserted into solutions containing the same concentration of AMP (amphetamine) and MAMP (methamphetamine), respectively, for extraction. Each SPE head was inserted to the same depth and for the same extraction time. The six SPE heads were then inserted into the inlet of a real-time direct mass spectrometry (RTMS) device for analysis, yielding the corresponding mass spectrum for each SPE head. The peak area values ​​were then obtained from these mass spectra. A bar chart was constructed with the coating material as the x-axis and the peak area value as the y-axis, as shown in Figure 2. The bar chart in Figure 2 shows that all three coating materials exhibited extraction effects on different drugs, with PDMS showing the best extraction effect.

[0087] Experimental Example 2 (Selection of Ionization Gas Temperature)

[0088] Gas temperature significantly affects the ionization efficiency of real-time direct analysis mass spectrometry (RTMS). Too low a temperature leads to incomplete ionization, while too high a temperature can cause thermal decomposition of some sample components. This study investigated the ionization efficiency at different ionization temperatures (250℃, 300℃, 350℃, and 400℃) while keeping all RMS conditions constant. The results showed that the response was highest at 300℃. Therefore, 300℃ was selected as the ionization gas temperature.

[0089] Experimental Example 3 (Selection of Sample Solution Salinity)

[0090] The ionic strength of a sample solution affects the solubility of the analyte and its distribution between the liquid and gas phases. Therefore, two sets of sample solutions were prepared: one set containing the same concentration of amphetamine, and the other containing the same concentration of methamphetamine. Five aliquots were prepared for each set. Different concentrations (0%, 10%, 20%, 30%, and 40%) of NaCl were added to each of the five aliquots. Ten identical solid-phase microextraction (SPE) heads were then inserted into each of the ten aliquots for extraction. Each SPE head was inserted to the same depth below the liquid surface for the same extraction time. These ten SPE heads were then inserted into the inlet of a real-time direct mass spectrometry (RTMS) device for analysis. The corresponding mass spectrum for each SPE head was obtained, and the peak area values ​​were calculated to investigate the effect of ionic strength on the extraction rate.

[0091] Next, a scatter plot was constructed with the salinity of the sample solution as the x-axis and the peak area as the y-axis, as shown in Figure 3. The scatter plot in Figure 3 shows that the peak area is largest in the sample solution with 30% NaCl added, indicating the highest amount extracted by fiber extraction; therefore, 30% NaCl is optimal. Thus, 30% NaCl can be added during the preparation of the standard curve solution and simultaneously when obtaining the test sample, thereby maintaining the salinity of both the standard curve solution and the test sample at an optimal level, thus improving the detection effect.

[0092] Experiment Example 4 (Selection of pH value for sample solution)

[0093] The pH value of a sample solution affects the solubility of the analyte and its distribution between the liquid and gas phases. Therefore, two sample solutions containing amphetamine and methamphetamine, respectively, with a pH of 7, were used. NaOH was added to both solutions to adjust the pH. After each adjustment, two solid-phase microextraction (SPE) heads were inserted into the two sample solutions for extraction. Each SPE head was inserted to the same depth below the liquid surface for the same extraction time. The two SPE heads were then inserted into the inlet of a real-time direct mass spectrometry (RTMS) device for analysis. The corresponding mass spectra for each SPE head were obtained, and the peak area values ​​were calculated to investigate the effect of pH on extraction efficiency.

[0094] Next, a scatter plot was constructed with the pH value of the sample solution on the x-axis and the peak area value on the y-axis, as shown in Figure 4. The scatter plot in Figure 4 shows that the peak area is largest at pH 14, indicating the highest amount of fiber extraction; therefore, pH 14 is optimal. Thus, adjusting the pH to 14 during the preparation of the standard curve solution and simultaneously adjusting the pH to 14 when acquiring the test sample maintains the pH of both the standard curve solution and the test sample at their optimal state, thereby improving the detection effect.

[0095] Experimental Example 5 (Selection of Extraction Time)

[0096] In solid-phase microextraction (SPE), there is a direct relationship between extraction yield and extraction time; however, SPE is an incomplete extraction technique. Therefore, achieving the maximum extraction yield is not necessary, and saving time and improving analytical efficiency are also important considerations. Two sample solutions containing amphetamine and methamphetamine, respectively, were taken. Multiple SPE heads were divided into two portions and inserted into the two sample solutions for extraction. Each SPE head was inserted to the same depth below the liquid surface. Every 10 minutes, one SPE head was removed and inserted into the inlet of a real-time direct analysis mass spectrometer (RTMS) for analysis. The corresponding mass spectrum for each SPE head was obtained, and the peak area value was derived from the mass spectrum.

[0097] Next, a scatter plot was created with time on the x-axis and peak area on the y-axis, as shown in Figure 5. The scatter plot in Figure 5 shows that the increase slows down after 30 minutes. Considering both the effectiveness of extraction and the time cost, 30 minutes is considered the optimal extraction time.

[0098] Experimental Example 6 (Selection of Stirring Rate)

[0099] Appropriate rotation speed can increase the dispersion rate of target molecules from the liquid phase to the gas phase. Therefore, two sample solutions containing amphetamine and methamphetamine were taken respectively. The stirring speed of the two sample solutions was adjusted, and two solid-phase microextraction (SPE) heads were inserted into the two sample solutions for extraction. The insertion depth of the SPE heads was controlled to be consistent, and the extraction time was the same. Then, the two SPE heads were inserted into the inlet of a real-time direct analysis mass spectrometer for real-time direct analysis mass spectrometry. The corresponding mass spectrum of each SPE head was obtained, and the corresponding peak area value was obtained from the mass spectrum.

[0100] Next, a scatter plot was constructed with stirring rate on the x-axis and peak area on the y-axis, as shown in Figure 6. The scatter plot in Figure 6 shows that the extraction yield increases significantly from 400 rpm to 600 rpm, but the increase slows down from 600 rpm to 700 rpm. Therefore, 600 rpm is the optimal stirring speed.

[0101] Experiment Example 7 (Linearity Reliability of Standard Curve Equation)

[0102] Taking common drugs methamphetamine, morphine, and ketamine as examples, standard curve solutions were prepared using the method described in Example 1. The standard curve equations were obtained through solid-phase microextraction and real-time direct mass spectrometry analysis, as shown in Table 1 below. This was used to examine the linearity, limit of detection, and limit of quantitation of the method.

[0103] Table 1. Standard curve equations and related parameters for standard curve solutions prepared from various drugs.

[0104]

[0105] As can be seen from the table above, the standard curve equations corresponding to the standard curve solutions prepared from common drugs show good linearity in the range of 0.1-100 μg / L, with a linear range of 0.9960-0.9974 and RSD <10%. The method has good reproducibility and can be used to determine common drugs in the matrix.

[0106] Experimental Example 8

[0107] In order to verify whether the sample concentrations of the three drugs in Experiment Example 7 are within the allowable relative standard deviation range compared with the theoretical concentrations obtained by the standard curve equation of Experiment Example 7, and further verify whether the drug concentration detection method of the present invention is applicable to the quantitative detection of common drugs in wastewater.

[0108] The quantitative results of methamphetamine, morphine, and ketamine in actual samples (taking wastewater samples as an example) were obtained using the external standard quantification method. Three drug standards were added to three groups of wastewater samples, with three samples in each group. The concentrations of the drug standards in the three samples of each group were 1 μg / L, 10 μg / L, and 100 μg / L, respectively.

[0109] The standard curve solution was prepared in the manner described in Example 1. It was then analyzed by real-time direct mass spectrometry using a solid-phase microextraction head and a real-time direct mass spectrometry device. Each wastewater sample was detected by the real-time direct mass spectrometry device to obtain the corresponding peak area value (as shown in Figures 7 to 9). The obtained peak area value was substituted into the corresponding standard curve equation for the same type of drug as in Example 7 to obtain the theoretical concentration of each wastewater sample. The corresponding relative standard deviation value was further calculated. The theoretical concentrations and standard deviations of the three sets of standard samples are shown in Table 2 below.

[0110] Table 2 Theoretical concentrations and relative standard deviations of various drugs

[0111]

[0112] As shown in Table 2, the RSD (relative standard deviation) of each sample is ≤11%, and the sample concentration calculated based on the standard curve is close to the theoretical value, indicating that this method is suitable for the quantitative detection of common drugs in wastewater.

[0113] The embodiments of the present invention are not limited thereto. Based on the above description of the present invention, and using common technical knowledge and conventional means in the field, the present invention can be modified, replaced or combined in various other forms without departing from the basic technical idea of ​​the present invention, and all such modifications, replacements or combinations fall within the scope of protection of the present invention.

Claims

1. A method for detecting drug levels based on solid-phase microextraction and real-time analytical mass spectrometry, characterized in that, Includes the following steps: Solid-phase microextraction coating preparation: According to the sample to be tested and the type of drug to be detected, the corresponding coating solution is applied to the glass rod to make a solid-phase microextraction head; In the preparation of the coating solution and the preparation of the solid-phase microextraction head, PDMS is selected as the coating material to prepare the coating solution, including the following steps: (1) Using a PDMS material preparation kit, magnetic grapefruit peel biochar is used as component A, and a mixture of C18 and SCX is used as component B. Component A and component B are mixed in a weight ratio of 20:1, and then added to a mixed solution of dichloromethane and n-hexane in a weight ratio of 5:1, so that the final concentration of the mixture of component A and component B is 0.10 g / mL; (2) Under vacuum conditions, the prepared glass rod coated with the above solution is heated to 75-85°C and held for two hours, then heated to 110-125°C and held for three hours, and finally heated to 240-260°C and held for eight hours, so that the final concentration of the mixture of component A and component B is 0.10 g / mL; The process includes: fabrication of a solid-phase microextraction (SPE) head; standard sample preparation: obtaining a standard curve solution through standard sample preparation; SPE and real-time direct mass spectrometry (MS / MS): extracting the prepared SPE head into the standard curve solution, followed by real-time direct MS / MS analysis of the extracted SPE head; standard curve plotting: obtaining the mass spectrum of the standard curve solution, and then obtaining the standard curve equation; sample detection: obtaining a liquid sample, extracting the sample with the SPE head, and then performing real-time direct MS / MS analysis of the extracted SPE head to obtain the mass spectrum of the sampled solution. Based on the mass spectrum of the sampled solution and the standard curve equation, the content of the drug component in the sampled solution is determined.

2. The method for detecting drug quantity based on solid-phase microextraction and real-time analytical mass spectrometry as described in claim 1, characterized in that: The standard sample preparation process includes the preparation of standard stock solutions, the preparation of mixed standard solutions ① and ②, and the preparation of standard curve solutions.

3. The method for detecting drug quantity based on solid-phase microextraction and real-time analytical mass spectrometry as described in claim 2, characterized in that: The preparation of the standard stock solution includes: weighing various drug component solid standards into a volumetric flask, dissolving them in methanol and diluting to the mark to obtain a 1000 mg / L standard stock solution.

4. The method for detecting drug quantity based on solid-phase microextraction and real-time analytical mass spectrometry as described in claim 3, characterized in that: The preparation of the mixed standard solution ① and mixed standard solution ② includes: extracting the standard stock solution into a volumetric flask, diluting and making up to volume with methanol to obtain a 10 mg / L mixed standard solution ①; extracting a portion of the mixed standard solution ①, diluting and making up to volume with methanol to obtain a 1 mg / L mixed standard solution ②.

5. The method for detecting drug quantity based on solid-phase microextraction and real-time analytical mass spectrometry as described in claim 4, characterized in that: The preparation of the standard curve solution includes: diluting the mixed standard solution ② with methanol aqueous solution to prepare multiple standard curve solutions of different concentrations, wherein the concentration of the isotope tracer in each standard curve solution is 100 μg / L.

6. The method for detecting drug quantity based on solid-phase microextraction and real-time analytical mass spectrometry as described in claim 1, characterized in that: When extracting the standard curve solution by pushing the solid-phase microextraction head into the solution, the solution is stirred with a stirrer. The solid-phase microextraction head is inserted 0.8-1.5 cm below the surface of the solution, and the extraction time is 15-30 minutes.

7. The method for detecting drug quantity based on solid-phase microextraction and real-time analytical mass spectrometry as described in claim 1, characterized in that: During the real-time direct analysis mass spectrometry (RTMS) analysis of the extracted solid-phase microextraction head, the solid-phase microextraction head, after extraction in the standard curve solution, is removed and inserted into the inlet of the real-time direct analysis mass spectrometry device for analysis.

8. The method for detecting drug quantity based on solid-phase microextraction and real-time analytical mass spectrometry as described in claim 7, characterized in that: In the process of obtaining the standard curve equation of the standard curve solution: after performing real-time direct analysis mass spectrometry analysis using a real-time direct analysis mass spectrometer, the mass spectrum of the standard curve solution is obtained. The peak area value of each standard curve solution is obtained from the mass spectrum. The drug concentration of each standard curve solution is used as the abscissa, and the peak area of ​​each standard curve solution is used as the ordinate. Linear regression analysis is performed to obtain the standard curve equation of the standard curve solution.

9. The method for detecting drug quantity based on solid-phase microextraction and real-time analytical mass spectrometry as described in claim 8, characterized in that: During sample testing, after obtaining the mass spectrum of the sample through real-time direct analysis mass spectrometry using a real-time direct analysis mass spectrometry device, the peak area value of the sample is obtained from the mass spectrum. This peak area value is then substituted into the standard curve equation to finally obtain the content of the drug component contained in the sample.

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