Crystalline form of a compound of formula (i) and processes for preparing the same

Abstract

The application discloses a three-item combined detection kit for methamphetamine analogues in hair, a preparation method and a detection method, and is characterized in that the three-item combined detection kit comprises a quality control disc, a sample tube and a detection working solution, and is used for realizing detection of the methamphetamine analogues; the quality control disc comprises blank quality control, negative control and threshold control; and the detection working solution comprises mobile phase A and mobile phase B. The application relates to the field of forensic science toxic substance analysis, in particular to the three-item combined detection kit for methamphetamine analogues in hair, the preparation method and the detection method. The application aims to provide the three-item combined detection kit for methamphetamine analogues in hair, the preparation method and the detection method, and has the advantages of high detection efficiency, convenient operation and accurate results.

Description

Technical Field

[0001] This invention relates to the field of forensic toxicology analysis, specifically to a reagent kit for the combined detection of three methamphetamine analogues in hair, its preparation method, and its detection method. Background Technology

[0002] Methamphetamine, the main component of crystal meth, is the most common amphetamine-type drug and one of the most widely abused drugs globally in the 21st century. Pure methamphetamine is a tasteless or slightly bitter, transparent, oily liquid. For ease of transport, illegally trafficked methamphetamine is usually produced in the form of hydrochloride or sulfate, resembling rock candy or ice in shape, and is a granular or powdery solid, mostly white in color, hence the common name "ice." Methamphetamine use severely damages internal organs and brain tissue, and in severe cases can lead to kidney failure, mental disorders, and even death.

[0003] Methamphetamine, also known as desoxyephedrine, is derived from the chemical structure of ephedrine. While small doses may provide temporary stimulant and anti-fatigue effects, long-term use can cause varying degrees of physical and mental harm to abusers. Therefore, the production, sale, and use of desoxyephedrine were subsequently banned by the government. With the continuous development of economic globalization and social informatization, drugs have become a global social scourge, and the problems of drug manufacturing, trafficking, and abuse have become more prominent, with an increasing variety of drugs available. As one of the most abused second-generation drugs, methamphetamine has become one of the most harmful drugs in the world today.

[0004] The manufacture, sale, and trafficking of drugs have always been a major concern for anti-drug departments, and determining the source of drugs is crucial for solving drug-related cases. Some criminals frequently adulterate methamphetamine with substances that resemble drugs in appearance, such as N-isopropylbenzylamine, to increase the weight of the drug and thus obtain higher profits. Currently, the most mature methamphetamine detection kits are colloidal gold assay kits. These kits are used to detect amphetamine-type drugs in blood, urine, and saliva samples. However, the colloidal gold method is an immunoassay method, which is easily affected by interference from methamphetamine structural analogues, leading to false positive results. Therefore, the results cannot be used as a basis for forensic identification. Furthermore, the sensitivity of the colloidal gold method is low and cannot meet the threshold requirements stipulated in the national standards for hair drug detection; therefore, it cannot be applied to the detection of methamphetamine in hair.

[0005] Liquid chromatography-mass spectrometry (LC-MS / MS) refers to the process where components in a sample are separated by high-performance liquid chromatography (HPLC), and then sequentially introduced into a mass spectrometer through a suitable interface. First, the components are ionized by an ion source into fragment ions with a specific mass-to-charge ratio. These fragment ions are then separated and detected by a mass analyzer. Finally, computer processing yields a mass spectrum of a single component composed of these fragment ions, which is then used to identify the structural composition of that component. LC-MS combines the effective separation capabilities of HPLC with the strong component identification capabilities of mass spectrometry, and is widely used in fields such as biology, medicine, chemical engineering, agriculture, and the environment. The forensic identification industry technical specification "Liquid Chromatography-Tandem Mass Spectrometry Examination Method for 13 Narcotic Drugs and Metabolites in Hair" (SF / Z JD01070252018) describes a method for detecting methamphetamine, but lacks methods for detecting common methamphetamine adulterants or interfering substances such as N-isopropylbenzylamine. Meanwhile, there are currently no commercially available methamphetamine detection kits based on liquid chromatography-mass spectrometry (LC-MS), which hinders the reduction of inter-laboratory testing discrepancies and impedes the standardization of forensic identification. Therefore, in public security and forensic identification work involving grassroots case investigations and drug screening of specific individuals, there is an urgent need for a commercially available multi-analysis kit that can accurately identify and differentiate methamphetamine and its structural analogues. This kit would accurately identify and characterize methamphetamine, its adulterants, and detection interfering substances in methamphetamine, helping anti-drug departments quickly determine the source of drugs, shorten case investigation time, and is of great significance for solving drug cases. It would also provide direct evidence for the legislative management and sentencing of drugs. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a three-item combined detection kit for methamphetamine analogues in hair, as well as a preparation method and a detection method, which has high detection efficiency, convenient operation and accurate results.

[0007] The present invention achieves its objective by employing the following technical solution:

[0008] A three-item combined detection kit for methamphetamine analogues in hair, characterized in that it includes:

[0009] The quality control tray, sample tubes, and working solution are used to detect methamphetamine analogues.

[0010] The quality control panel includes blank quality control, negative control, and threshold control.

[0011] The detection working solution includes mobile phase A and mobile phase B.

[0012] As a further limitation of this technical solution, the inner packaging of the quality control plate and the sample tube is PP cryogenic grinding tube, and the inner packaging of the detection working solution is a brown glass reagent bottle.

[0013] As a further limitation of this technical solution, the blank quality control includes 20mg of negative blank hair;

[0014] The negative control consisted of 20 mg of negative blank hair and 1 ng of diphenylpentanediamine.

[0015] The threshold control standards include 20 mg of negative blank hair, 1 ng of diphenylpentanediamine, 4 ng of methamphetamine, 4 ng of N-isopropylbenzylamine, and 4 ng of phentermine.

[0016] The sample tube contained 1 ng of diphenylpentanediamine;

[0017] The liquid mobile phase A consists of an aqueous solution of 20 mmol / L ammonium acetate and 1% formic acid;

[0018] The liquid mobile phase B is composed of methanol.

[0019] As a further limitation of this technical solution, the negative blank hair refers to human negative hair.

[0020] As a further limitation of this technical solution, the methamphetamine analogues are methamphetamine, N-isopropylbenzylamine, and phentermine.

[0021] A method for preparing a three-item combined detection kit for methamphetamine analogues in hair, characterized by comprising the following steps:

[0022] S11: Preparation of human negative hair;

[0023] S111: Take human negative hair, wash twice with purified water, wash twice with acetone, and air dry;

[0024] S112: Cut the treated human hair into 0.1cm pieces for later use;

[0025] S12: Blank quality control preparation;

[0026] Five zirconium oxide grinding beads were added to the first set of PP cryo-grinding tubes, and 20 mg of human negative hair prepared by S11 was added. The tube cap was then tightened.

[0027] S13: Preparation of negative control;

[0028] S131: Add 10 μL of 100 ng / mL diphenylpentanediamine methanol solution to the second group of PP cryogenic grinding tubes, place them in a lyophilizer, and freeze-dry using the programmed drying method;

[0029] S132: Add 5 zirconium oxide grinding beads to the second set of PP cryogenic grinding tubes, put in 20mg of human negative hair prepared by S11, and tighten the tube cap;

[0030] S14: Preparation of threshold reference standard;

[0031] S141: Add 10 μL of 100 ng / mL diphenylpentanediamine methanol solution, 40 μL of 100 ng / mL methamphetamine methanol solution, 40 μL of 100 ng / mL N-isopropylbenzylamine methanol solution, and 40 μL of 100 ng / mL phentermine methanol solution to the third group of PP cryogenic grinding tubes, place them in a lyophilizer, and freeze-dry using the programmed process.

[0032] S142: Add 5 zirconium oxide grinding beads to the third PP cryogenic grinding tube, put in 20mg of human negative hair prepared by S11, and tighten the tube cap;

[0033] S15: Sample Tube Preparation

[0034] S151: Add 10 μL of 100 ng / mL diphenylpentanediamine methanol solution to the fourth group of PP cryogenic grinding tubes, place them in a freeze dryer, and freeze dry to a certain extent;

[0035] S152: Add 5 zirconium oxide grinding beads to the fourth PP cryogenic grinding tube and tighten the tube cap;

[0036] S16: Preparation of mobile phase A for detection working fluid;

[0037] S161: Weigh 1.54g of ammonium acetate, place it in a beaker, add 200mL of water to dissolve it, and then transfer the whole amount to a 1000mL volumetric flask. Add 200mL of water to the original beaker, rinse the beaker, and then transfer the whole amount to the above 1000mL volumetric flask. Add 1mL of formic acid to the 1000mL volumetric flask, and add water to make up to 1000mL.

[0038] S162: Dispense into brown glass reagent bottles;

[0039] S17: Preparation of mobile phase B for detection working fluid;

[0040] Measure 1000 mL of methanol solution and dispense it into brown glass reagent bottles.

[0041] As a further limitation of this technical solution, the freeze-drying process is as follows: pre-freeze at -40℃ for 3 hours, vacuum, then raise the pre-temperature to -20℃ and keep it warm for 3 hours, then raise it to -10℃ and keep it warm for 3 hours, then raise it to 0℃ and keep it warm for 3 hours, then raise it to 10℃ and keep it warm for 5 hours, then raise it to 20℃ and keep it warm for 5 hours to complete the freeze-drying.

[0042] A detection method for a three-item combined test kit for methamphetamine analogues in hair, characterized by comprising the following steps:

[0043] S21: Pretreatment of hair samples to be tested;

[0044] Take the hair sample to be tested, wash it twice with purified water and twice with acetone, air dry it, cut it into 0.1cm pieces, weigh 20mg and put it into a sample tube, add 1mL of methanol, tighten the tube cap and shake well.

[0045] S22 quality control plate reconstitution;

[0046] Add 1 mL of methanol to each of the blank control, negative control, and threshold control, tighten the cap, and shake well.

[0047] S23 grinding and extraction;

[0048] Place the blank control, negative control, threshold control, and sample tubes into a cryo-grinder for cryo-grinding, then centrifuge them in a high-speed centrifuge, and filter the supernatant.

[0049] S24: Detection and analysis;

[0050] Take 50 μL of each of the filtrates and analyze them using liquid chromatography-mass spectrometry.

[0051] As a further limitation of this technical solution, the detection and analysis includes setting liquid chromatography conditions and mass spectrometry conditions, selecting characteristic ion pairs, comparing the retention time of the chromatographic peaks of the test sample and the abundance ratio of characteristic ion pairs for qualitative analysis, and using the external standard method of peak area for quantitative analysis.

[0052] As a further limitation of this technical solution, the liquid chromatography conditions include the use of a C18 column;

[0053] The liquid chromatography conditions also include elution mobile phase A and mobile phase B, and the elution method is gradient elution;

[0054] The mass spectrometry conditions included a flow rate of 0.35 mL / min, a column temperature of 25 °C, electrospray ionization positive ion mode, multiple reaction monitoring mode, a nitrogen nebulizer gas of 3.0 L / min, a nitrogen desiccator gas of 10.0 L / min, a heating gas of 10.0 L / min, an injection volume of 5 μL, an injection port temperature of 280 °C, an interface temperature of 300 °C, an interface voltage of 4 kV, a heating module temperature of 400 °C, and a DL temperature of 250 °C. Selected characteristic ion pairs included methamphetamine with m / z 150.10 > 91.05 and m / z 150.10 > 119.10, N-isopropylbenzylamine with m / z 150.10 > 91.05 and m / z 150.10 > 65.05, and phentermine with m / z 150.10 > 91.05 and m / z 150.10 > 65.05. 150.10 > 133.10 is considered a characteristic ion pair.

[0055] Compared with the prior art, the advantages and positive effects of the present invention are:

[0056] 1. The three-item combined detection kit and detection method for methamphetamine analogues in hair provided by this invention can perform qualitative and quantitative detection of methamphetamine, N-isopropylbenzylamine and phentermine in hair, and can meet the national standard detection threshold of 0.2 ng / mg. It has good specificity and high sensitivity, and will play an important role, especially in the blind screening detection of methamphetamine target substances.

[0057] 2. The three-item combined detection kit for methamphetamine analogues in hair provided by this invention includes a quality control tray, which integrates the preparation of blank quality control, negative control, and threshold-added control samples, as well as internal standards and standard substances, into the finished kit. This reduces the number of steps required by testing personnel, minimizes differences in standard substances, and reduces the introduction of factors that could lead to uncertainty. The kit results in high accuracy, low detection limit, and a wide linear range. The widespread application of this finished kit can significantly reduce inter-laboratory testing variability within the forensic science industry, facilitating standardization of drug testing and mutual recognition of test results between laboratories. Attached Figure Description

[0058] Figure 1 This is the LC MS / MS spectrum of the blank quality control in Example 1 of the present invention;

[0059] Figure 2 This is the LC MS / MS chromatogram of the negative control in Example 1 of this invention. SKF is shown in the figure. 252A It is an internal standard for diphenylpentanediamine;

[0060] Figure 3 This is the LC MS / MS chromatogram of the threshold reference standard in Example 1 of the present invention. Compound 1, compound 2, and compound 3 in the figure are methamphetamine, N-isopropylbenzylamine, and phentermine, respectively.

[0061] Figure 4 For the present invention Figure 3 Magnified tiled LC MS / MS spectrum of the intermediate threshold reference standard;

[0062] Figure 5 The LC MS / MS spectra of the three mixed standards in Example 2 of this invention;

[0063] Figure 6 This is an LC MS / MS chromatogram of hair samples obtained using the kit and detection method of this invention in the experiments of this invention. Detailed Implementation

[0064] The following detailed description of a specific embodiment of the present invention is provided in conjunction with the accompanying drawings. However, it should be understood that the scope of protection of the present invention is not limited to the specific embodiment.

[0065] To address the technical problems of side-chain isomers and interfering substances affecting the accuracy of existing methamphetamine detection methods in hair samples, and to solve the problem of cumbersome procedures affecting repeatability, this invention provides a three-item simultaneous detection kit for methamphetamine analogues in hair, along with its preparation and detection methods. Accurate simultaneous determination of methamphetamine and its isomer analogues in hair is challenging primarily because the sample is human hair, and the sample pretreatment process is complex. This invention provides a kit with built-in sample tubes for processing the hair sample, solidifying the extraction method of the target analytes and simplifying the extraction steps. Secondly, the target analytes themselves are structurally similar side-chain isomers, and traditional detection methods such as colloidal gold assays and immunoassays have poor resolution, leading to false positives. This invention provides a kit detection method based on liquid chromatography-mass spectrometry (LC-MS). LC-MS can effectively separate the side-chain isomers of methamphetamine, and combined with mass spectrometry... Spectroscopic techniques pinpoint characteristic parent-daughter ion pairs of the target analyte, resolving the technical problem of mutual interference in isomer determination. Finally, to simultaneously process three methamphetamine analogues from human hair, the detection instrument needs to accurately and sensitively detect the analyte. Considering the compatibility of reagents and detection instruments, the overall detection procedure, and the determination of various operating parameters, a significant amount of work is required. This invention provides a pre-made reagent kit that pre-defines the required reagent composition, proportions, and detection method for the reaction system. Client-side testing laboratories can directly set relevant detection parameters and use the reagent kit provided by this invention for detection according to its user manual. Furthermore, the reagent kit provided by this invention includes a quality control disc containing the internal standard and standard substances required for detection. The detection process only requires adding the sample hair and methanol reconstitution solution, greatly simplifying the detection operation.

[0066] This invention includes:

[0067] The quality control tray, sample tubes, and working solution are used to detect methamphetamine analogues.

[0068] The quality control panel includes blank quality control, negative control, and threshold control.

[0069] The detection working solution includes mobile phase A and mobile phase B.

[0070] The quality control tray and the inner packaging of the sample tube are both PP cryogenic grinding tubes, and the inner packaging of the detection working solution is a brown glass reagent bottle.

[0071] The blank control consisted of 20 mg of negative blank hair samples;

[0072] The negative control consisted of 20 mg of negative blank hair and 1 ng of diphenylpentanediamine.

[0073] The threshold control standards include 20 mg of negative blank hair, 1 ng of diphenylpentanediamine, 4 ng of methamphetamine, 4 ng of N-isopropylbenzylamine, and 4 ng of phentermine.

[0074] The sample tube contained 1 ng of diphenylpentanediamine;

[0075] The liquid mobile phase A consists of an aqueous solution of 20 mmol / L ammonium acetate and 1% formic acid;

[0076] The liquid mobile phase B is composed of methanol.

[0077] The aforementioned diphenylpentanediamine is an internal standard. Using an internal standard and limiting its concentration in each test sample solution can ensure the sensitivity of the internal standard detection, offset operational errors, and assist in judging the efficiency of the pretreatment process and whether the instrument and other detection conditions are normal.

[0078] The concentration of each standard substance added in the threshold reference standard mentioned above is 0.2 ng / mg, which corresponds to the threshold concentration in the national standard. It can be used to compare the detection results of the test sample. By comparing the peak area of ​​the target ion pair peak with the internal standard method, the detection result of the sample can be directly identified. For example, if the retention time and characteristic ion pair abundance ratio of methamphetamine in the test sample both meet the qualitative requirements, and the ratio of the peak area of ​​the quantitative ion pair of methamphetamine in the test sample to the peak area of ​​the diphenylpentanediamine internal standard is greater than the corresponding peak area ratio of methamphetamine in the threshold reference standard, then the detection result of methamphetamine in the test sample is positive; otherwise, it is negative.

[0079] The negative blank hair refers to human negative hair.

[0080] The methamphetamine analogues mentioned are methamphetamine, N-isopropylbenzylamine, and phentermine, with the following structural formulas:

[0081]

[0082] A method for preparing a three-item combined detection kit for methamphetamine analogues in hair, characterized by comprising the following steps:

[0083] S11: Preparation of human negative hair;

[0084] S111: Take human negative hair, wash twice with purified water, wash twice with acetone, and air dry;

[0085] S112: Cut the treated human hair into 0.1cm pieces for later use;

[0086] S12: Blank quality control preparation;

[0087] Five zirconium oxide grinding beads were added to the first set of PP cryo-grinding tubes, and 20 mg of human negative hair prepared by S11 was added. The tube cap was then tightened.

[0088] S13: Preparation of negative control;

[0089] S131: Add 10 μL of 100 ng / mL diphenylpentanediamine methanol solution to the second group of PP cryogenic grinding tubes, place them in a lyophilizer, and freeze-dry using the programmed drying method;

[0090] S132: Add 5 zirconium oxide grinding beads to the second set of PP cryogenic grinding tubes, put in 20mg of human negative hair prepared by S11, and tighten the tube cap;

[0091] S14: Preparation of threshold reference standard;

[0092] S141: Add 10 μL of 100 ng / mL diphenylpentanediamine methanol solution, 40 μL of 100 ng / mL methamphetamine methanol solution, 40 μL of 100 ng / mL N-isopropylbenzylamine methanol solution, and 40 μL of 100 ng / mL phentermine methanol solution to the third group of PP cryogenic grinding tubes, place them in a lyophilizer, and freeze-dry using the programmed process.

[0093] S142: Add 5 zirconium oxide grinding beads to the third PP cryogenic grinding tube, put in 20mg of human negative hair prepared by S11, and tighten the tube cap;

[0094] S15: Sample Tube Preparation

[0095] S151: Add 10 μL of 100 ng / mL diphenylpentanediamine methanol solution to the fourth group of PP cryogenic grinding tubes, place them in a freeze dryer, and freeze dry to a certain extent;

[0096] S152: Add 5 zirconium oxide grinding beads to the fourth PP cryogenic grinding tube and tighten the tube cap;

[0097] S16: Preparation of mobile phase A for detection working fluid;

[0098] S161: Weigh 1.54g of ammonium acetate, place it in a beaker, add 200mL of water to dissolve it, and then transfer the whole amount to a 1000mL volumetric flask. Add 200mL of water to the original beaker, rinse the beaker, and then transfer the whole amount to the above 1000mL volumetric flask. Add 1mL of formic acid to the 1000mL volumetric flask, and add water to make up to 1000mL.

[0099] S162: Dispense into brown glass reagent bottles;

[0100] S17: Preparation of mobile phase B for detection working fluid;

[0101] Measure 1000 mL of methanol solution and dispense it into brown glass reagent bottles.

[0102] The freeze-drying process is as follows: pre-freeze at -40℃ for 3 hours, vacuum the system, then raise the pre-temperature to -20℃ and hold for 3 hours, then raise it to -10℃ and hold for 3 hours, then raise it to 0℃ and hold for 3 hours, then raise it to 10℃ and hold for 5 hours, then raise it to 20℃ and hold for 5 hours to complete the freeze-drying process.

[0103] A detection method for a three-item combined test kit for methamphetamine analogues in hair, characterized by comprising the following steps:

[0104] S21: Pretreatment of hair samples to be tested;

[0105] Take the hair sample to be tested, wash it twice with purified water and twice with acetone, air dry it, cut it into 0.1cm pieces, weigh 20mg and put it into a sample tube, add 1mL of methanol, tighten the tube cap and shake well.

[0106] S22 quality control plate reconstitution;

[0107] Add 1 mL of methanol to each of the blank control, negative control, and threshold control, tighten the cap, and shake well.

[0108] S23 grinding and extraction;

[0109] Place the blank control, negative control, threshold control, and sample tubes into a cryo-grinder for cryo-grinding, then centrifuge them in a high-speed centrifuge, and filter the supernatant.

[0110] S24: Detection and analysis;

[0111] Take 50 μL of each of the filtrates and analyze them using liquid chromatography-mass spectrometry.

[0112] The detection and analysis include setting liquid chromatography and mass spectrometry conditions, selecting characteristic ion pairs, comparing the retention time and characteristic ion pair abundance ratio of the chromatographic peaks of the test sample and the positive control sample for qualitative analysis, and using the external standard method of peak area for quantitative analysis.

[0113] The liquid chromatography conditions include the use of a C18 column;

[0114] The liquid chromatography conditions also include elution mobile phase A and mobile phase B, and the elution method is gradient elution;

[0115] The gradient elution procedure is shown in Table 1 below:

[0116] Table 1 Gradient elution program

[0117] Time / min 0 12 14 14.1 18 B / ％ 10 95 95 10 Stop

[0118] The mass spectrometry conditions included a flow rate of 0.35 mL / min, a column temperature of 25 °C, electrospray ionization positive ion mode (ESI+), multiple reaction monitoring (MRM) mode, a nitrogen nebulizer gas of 3.0 L / min, a nitrogen desiccator gas of 10.0 L / min, a heating gas of 10.0 L / min, an injection volume of 5 μL, an injection port temperature of 280 °C, an interface temperature of 300 °C, an interface voltage of 4 kV, a heating module temperature of 400 °C, and a DL temperature of 250 °C. Selected characteristic ion pairs included methamphetamine with m / z 150.10 > 91.05 and m / z 150.10 > 119.10, N-isopropylbenzylamine with m / z 150.10 > 91.05 and m / z 150.10 > 65.05, and phentermine with m / z 150.10 > 91.05 and m / z 150.10 > 65.05. 150.10 > 133.10 is considered a characteristic ion pair.

[0119] The parameters for multiple reaction monitoring (MRM) mode ion pairs are shown in Table 2 below:

[0120] Table 2. Ion pair parameters in Multiple Reaction Monitoring (MRM) mode

[0121]

[0122]

[0123] Example 1:

[0124] Quality control of the three-item combined detection kit for methamphetamine analogues in hair provided by this invention:

[0125] S22: Reconstitute the quality control tray;

[0126] Add 1 mL of methanol to each of the blank control, negative control, and threshold control, tighten the cap, and shake well.

[0127] S23: Grinding and extraction;

[0128] Place all the reconstituted quality control plates, including blank quality control, negative control and threshold control, into a cryogenic grinder. The cryogenic grinder is set at 60 Hz. Pre-cool for 1 min, then freeze and grind at -40 ℃ for 2 min. Repeat the grinding process 8 times with a 10 s interval. Then place the plates in a high-speed centrifuge and centrifuge at 13000 r / min for 5 min. Take the supernatant and filter it through a 0.22 μm hydrophilic PTFE membrane. Keep the filtrate for later use.

[0129] S24: Detection and analysis;

[0130] Take 50 μL of each of the filtrates and analyze them using liquid chromatography-mass spectrometry.

[0131] Liquid chromatography conditions:

[0132] Liquid Chromatography-Tandem Mass Spectrometry: Shimadzu LCMS-8045 Triple Quadrupole Liquid Chromatography-Mass Spectrometry System;

[0133] Column: Phenomenex Gemini NX-C18 (150mm×3mm×5μm);

[0134] Mobile phase A: Detection working fluid mobile phase A;

[0135] Mobile phase B: Detection working fluid mobile phase B;

[0136] Gradient elution, the gradient elution conditions are shown in Table 1;

[0137] Flow rate: 0.35 mL / min;

[0138] Column temperature: 25℃;

[0139] Injection volume: 5 μL.

[0140] Mass spectrometry conditions:

[0141] Ion source: Electrospray ionization positive ion mode (ESI+);

[0142] The atomizing gas is nitrogen at 3.0 L / min, the dryer gas is nitrogen at 10.0 L / min, and the heating gas is air at 10.0 L / min.

[0143] Inlet temperature 280℃, interface temperature 300℃, interface voltage 4kV, heating module temperature 400℃, DL temperature 250℃;

[0144] Scanning method: Multiple response monitoring (MRM) mode;

[0145] The detection ion pair parameters, retention times, characteristic ion pair abundance ratios, and allowable ranges for each compound are shown in Table 3 below.

[0146] Table 3. Detection parameters, retention times, characteristic ion pair abundance ratios, and permissible ranges for each compound.

[0147]

[0148] Quality control charts, such as Figures 1-4 As shown;

[0149] Figure 1 The LC-MS / MS chromatogram of the blank quality control shows that the target analytes were not detected in the blank quality control, indicating that the endogenous substances present in the blank hair will not interfere with the detection of the three target analytes and internal standards described in this invention. Figure 2The LC-MS / MS chromatogram of the negative control shows that the target analyte was not detected in the negative control, while the diphenylpentanediamine internal standard showed a peak with a good peak shape, indicating that the internal standard was effective and the detection system was under control. Figure 3 The LC-MS / MS chromatograms of the threshold reference standard show that all target analytes and diphenylpentanediamine internal standard were detected, with good peak shapes and signal-to-noise ratios (S / N) greater than 3, indicating that the detection limit of the detection system is not higher than the detection threshold specified in the national standard and that the detection system is under control. Figure 4 for Figure 3 The magnified LC-MS / MS plot of the medium threshold reference standard shows that the relative error between the peak elution time (retention time) of the characteristic ion pairs of each target analyte and the retention time given in the kit instructions is within ±2.5%, and the ion pair abundance ratio is within the allowable range given in the kit, which meets the qualitative requirements of the target analytes. This indicates that the detection system can be used for the detection and identification of three target analytes and the reporting of results.

[0150] The results of Example 1 show that the method of the present invention has high selectivity, specificity and detection accuracy.

[0151] Example 2

[0152] This embodiment presents a comparative analysis of gradient elution conditions under the liquid chromatography conditions described in Example 1.

[0153] The purpose of this embodiment is to compare and analyze the gradient elution conditions in the detection and analysis conditions of the three-item combined detection kit for methamphetamine analogues in hair provided by the present invention, mainly focusing on the establishment and effectiveness verification of the gradient elution conditions. The specific technical steps adopted are as follows:

[0154] A mixed standard of three detection targets for the three-item combined detection kit for methamphetamine analogues in hair provided by the present invention was prepared, with each standard having a concentration of 0.33 ng / mg;

[0155] The mixed standard was tested using the hair follicle methamphetamine analogue three-item detection kit provided by this invention. The gradient elution conditions in the liquid chromatography are shown in Table 4 below:

[0156] Table 4 Gradient elution conditions in liquid chromatography

[0157]

[0158] Figure 5 The LC-MS / MS chromatograms of the mixed standards obtained in Example 2 show that, under the conditions of Example 2, the retention times of methamphetamine, N-isopropylbenzylamine, and phentermine were 3.938 min, 3.700 min, and 4.109 min, respectively. There is partial overlap between the peaks of methamphetamine and N-isopropylbenzylamine. Figure 3Under the conditions of Example 1, the retention times of methamphetamine, N-isopropylbenzylamine, and phentermine were 6.511 min, 5.462 min, and 7.294 min, respectively. The peaks of each compound were independent and there was no cross-interference between them, indicating that the liquid chromatography separation effect under the gradient elution conditions of Example 1 (Table 1) was better than that of Example 2. Therefore, the gradient elution conditions shown in Table 1 are preferred for the three-item combined detection kit for methamphetamine analogues in hair provided by the present invention.

[0159] Example 3

[0160] This embodiment conducted a series of experiments to verify the detection method of the three-item combined detection kit for methamphetamine analogues in hair provided by the present invention. These experiments mainly included verifying the detection limit, quantitation limit, and linear range of the detection method described in this invention, as shown below:

[0161] Hair samples were added at different concentrations of the three target compounds described in this invention, extracted using an S23 grinding mill, and then analyzed by LC-MS / MS. The limits of detection (LOD) for all three target compounds described in this invention were 0.005 ng / mg, and the limits of quantitation (LOQ) for all three target compounds described in this invention were 0.025 ng / mg. See Table 5 below for details:

[0162] Table 5 Limits of Detection and Limits of Quantification for Target Substances

[0163]

[0164] Hair samples with different concentrations of the three target compounds described in this invention were extracted using S23 grinding and then analyzed by LC-MS / MS. The linear range of the detection methods for the three target compounds described in this invention was between 0.025 ng / mg and 10.000 ng / mg. Quantitative analysis using the external standard method showed good correlation. See Table 6 below for details:

[0165] Table 6 Linear Range of Target Detection Methods

[0166]

[0167]

[0168] Example 4

[0169] This embodiment uses the reagent kit and detection method provided by the present invention, and refers to the instrument and parameter settings in Example 1 to detect positive hair samples in the experiment. Figure 6The LC-MS / MS spectra of three target substances in hair samples were obtained using the kit and detection method provided by this invention in a screening experiment. The results showed that methamphetamine, N-isopropylbenzylamine, and phentermine were detected in the samples. The concentration of methamphetamine was greater than the threshold, and the detection result was positive. The concentrations of N-isopropylbenzylamine and phentermine were lower than the threshold, and the detection results were negative.

[0170] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0171] The above-disclosed embodiments are merely specific examples of the present invention. However, the present invention is not limited thereto, and any variations that can be conceived by those skilled in the art should fall within the protection scope of the present invention.

Claims

1. A three-item detection kit for methamphetamine analogues in hair, characterized in that, include: The quality control tray, sample tubes, and working solution are used to detect methamphetamine analogues. The quality control panel includes blank quality control, negative control, and threshold control. The detection working solution includes mobile phase A and mobile phase B; The blank control includes negative blank hairs; The negative control standards include negative blank hair and diphenylpentanediamine; The threshold controls include negative blank hair, diphenylpentanediamine, methamphetamine, N-isopropylbenzylamine, and phentermine; The sample tube contains diphenylpentanediamine; The liquid mobile phase A consists of an aqueous solution of 20 mmol / L ammonium acetate and 1% formic acid; The liquid mobile phase B is composed of methanol; The methamphetamine analogues mentioned are methamphetamine, N-isopropylbenzylamine, and phentermine.

2. The three-item combined detection kit for methamphetamine analogues in hair according to claim 1, characterized in that: The quality control tray and the inner packaging of the sample tube are both PP cryogenic grinding tubes, and the inner packaging of the detection working solution is a brown glass reagent bottle.

3. The three-item combined detection kit for methamphetamine analogues in hair according to claim 1, characterized in that: The blank control consisted of 20 mg of negative blank hair samples; The negative control consisted of 20 mg of negative blank hair and 1 ng of diphenylpentanediamine. The threshold control standards include 20 mg of negative blank hair, 1 ng of diphenylpentanediamine, 4 ng of methamphetamine, 4 ng of N-isopropylbenzylamine, and 4 ng of phentermine. The sample tube contained 1 ng of diphenylpentanediamine; The liquid mobile phase A consists of an aqueous solution of 20 mmol / L ammonium acetate and 1% formic acid; The liquid mobile phase B is composed of methanol.

4. The three-item combined detection kit for methamphetamine analogues in hair according to claim 3, characterized in that: The negative blank hair refers to human negative hair.

5. The preparation method of the three-item combined detection kit for methamphetamine analogues in hair as described in claim 3, characterized in that, Includes the following steps: S11: Preparation of human negative hair; S111: Take human negative hair, wash twice with purified water, wash twice with acetone, and air dry; S112: Cut the treated human hair into 0.1cm pieces for later use; S12: Blank quality control preparation; Five zirconium oxide grinding beads were added to the first set of PP cryo-grinding tubes, and 20 mg of human negative hair prepared by S11 was added. The tube cap was then tightened. S13: Preparation of negative control; S131: Add 10 μL of 100 ng / mL diphenylpentanediamine methanol solution to the second group of PP cryogenic grinding tubes, place them in a lyophilizer, and freeze-dry using the programmed drying method; S132: Add 5 zirconium oxide grinding beads to the second set of PP cryogenic grinding tubes, put in 20mg of human negative hair prepared by S11, and tighten the tube cap; S14: Preparation of threshold reference standard; S141: Add 10 μL of 100 ng / mL diphenylpentanediamine methanol solution, 40 μL of 100 ng / mL methamphetamine methanol solution, 40 μL of 100 ng / mL N-isopropylbenzylamine methanol solution, and 40 μL of 100 ng / mL phentermine methanol solution to the third group of PP cryogenic grinding tubes, place them in a lyophilizer, and freeze-dry using the programmed process. S142: Add 5 zirconium oxide grinding beads to the third PP cryogenic grinding tube, put in 20mg of human negative hair prepared by S11, and tighten the tube cap; S15: Sample Tube Preparation S151: Add 10 μL of 100 ng / mL diphenylpentanediamine methanol solution to the fourth group of PP cryogenic grinding tubes, place them in a freeze dryer, and freeze dry to a certain extent; S152: Add 5 zirconium oxide grinding beads to the fourth PP cryogenic grinding tube and tighten the tube cap; S16: Preparation of mobile phase A for detection working fluid; S161: Weigh 1.54g of ammonium acetate, place it in a beaker, add 200mL of water to dissolve it, and then transfer the whole amount to a 1000mL volumetric flask. Add 200mL of water to the original beaker, rinse the beaker, and then transfer the whole amount to the above 1000mL volumetric flask. Add 1mL of formic acid to the 1000mL volumetric flask, and add water to make up to 1000mL. S162: Dispense into brown glass reagent bottles; S17: Preparation of mobile phase B for detection working fluid; Measure 1000 mL of methanol solution and dispense it into brown glass reagent bottles.

6. The preparation method of the three-item combined detection kit for methamphetamine analogues in hair according to claim 5, characterized in that: The freeze-drying process is as follows: pre-freeze at -40℃ for 3 hours, vacuum the system, then raise the pre-temperature to -20℃ and hold for 3 hours, then raise it to -10℃ and hold for 3 hours, then raise it to 0℃ and hold for 3 hours, then raise it to 10℃ and hold for 5 hours, then raise it to 20℃ and hold for 5 hours to complete the freeze-drying process.

7. The detection method using the three-item combined detection kit for methamphetamine analogues in hair as described in claim 3, characterized in that, Includes the following steps: S21: Pretreatment of hair samples to be tested; Take the hair sample to be tested, wash it twice with purified water and twice with acetone, air dry it, cut it into 0.1cm pieces, weigh 20mg and put it into a sample tube, add 1mL of methanol, tighten the tube cap and shake well. S22 quality control plate reconstitution; Add 1 mL of methanol to each of the blank control, negative control, and threshold control, tighten the cap, and shake well. S23 grinding and extraction; Place the blank control, negative control, threshold control, and sample tubes into a cryo-grinder for cryo-grinding, then centrifuge them in a high-speed centrifuge, and filter the supernatant. S24: Detection and analysis; Take 50 μL of each of the filtrates and perform detection and analysis using liquid chromatography-mass spectrometry; the detection and analysis includes setting liquid chromatography conditions and mass spectrometry conditions, the liquid chromatography conditions including using a C18 column; The liquid chromatography conditions also include elution mobile phase A and mobile phase B, with gradient elution as follows: ； The mass spectrometry conditions include electrospray ionization positive ion mode, and the detection method is multiple reaction monitoring (MRM) mode. The MRM ion pair parameters are as follows: 。 8. The detection method of the three-item combined detection kit for methamphetamine analogues in hair according to claim 7, characterized in that: The detection and analysis include setting liquid chromatography and mass spectrometry conditions, selecting characteristic ion pairs, comparing the retention time and characteristic ion pair abundance ratio of the chromatographic peaks of the test sample and the positive control sample for qualitative analysis, and using the external standard method of peak area for quantitative analysis.

9. The detection method of the three-item combined detection kit for methamphetamine analogues in hair according to claim 7, characterized in that: The liquid chromatography conditions include the use of a C18 column; The liquid chromatography conditions also include elution mobile phase A and mobile phase B, and the elution method is gradient elution; The mass spectrometry conditions included a flow rate of 0.35 mL / min, a column temperature of 25 °C, electrospray ionization positive ion mode, multiple reaction monitoring mode, a nitrogen nebulizer gas of 3.0 L / min, a nitrogen desiccator gas of 10.0 L / min, a heating gas of 10.0 L / min, an injection volume of 5 μL, an injection port temperature of 280 °C, an interface temperature of 300 °C, an interface voltage of 4 kV, a heating module temperature of 400 °C, and a DL temperature of 250 °C. Selected characteristic ion pairs included methamphetamine with m / z 150.10 > 91.05 and m / z 150.10 > 119.10, N-isopropylbenzylamine with m / z 150.10 > 91.05 and m / z 150.10 > 65.05, and phentermine with m / z 150.10 > 91.05 and m / z 150.10 > 65.

05. 150.10 > 133.10 is considered a characteristic ion pair.

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