Method for simultaneously detecting 11 psychiatric drugs or metabolites thereof in blood and application

Abstract

The invention establishes a rapid, accurate and efficient high performance liquid chromatography-mass spectrometry detection method for detecting common antipsychotic drugs in blood, and can rapidly detect the concentration of 11 common antipsychotic drugs in a blood sample. The method is suitable for screening and quantitative detection of clinical common medicine types, clinical treatment medicine monitoring, poisoning screening and large-scale biological sample detection work in clinical and scientific research experiments of the psychiatry department, and has very remarkable significance in clinical use.

Description

Technical Field

[0001] This invention relates to the field of biological detection, specifically to a method and application for simultaneously detecting 11 psychiatric drugs or their metabolites in blood. Background Technology

[0002] Antipsychotic drugs, also known as potent tranquilizers or neuroleptics, are a group of medications used to treat schizophrenia and other psychotic mental disorders. At typical therapeutic doses, they do not affect a patient's intelligence or consciousness, but effectively control psychomotor agitation, hallucinations, delusions, hostility, thought disorders, and abnormal behavior. Patients often experience sleep disturbances and frequently take sedative-sedative medications concurrently. To monitor the blood concentration of these medications, rapid dissociation of these drugs is often necessary. Trazodone is a tetracyclic atypical antidepressant with antidepressant effects, central sedation, and mild muscle relaxant effects, but no anticonvulsant or central nervous system stimulant effects. It can improve sleep, significantly shorten the sleep latency in patients with depression, prolong overall sleep time, and improve sleep quality. Clozapine is a second-generation antipsychotic, primarily indicated for schizophrenia. It is commonly used as a second-line treatment for some refractory cases where other medications are ineffective or unsatisfactory. Clozapine can also be used to treat bipolar disorder and manic episodes. It can also be effective in treating altered consciousness, confusion, and amnesia in some elderly individuals. However, because clozapine's effects are complex and it produces relatively many adverse reactions, its widespread clinical use is limited, and it is generally used as a second-line drug. Norclozapine, a metabolite of clozapine, is also used as an important indicator for monitoring concentrations in clinical practice. Quetiapine is an atypical antipsychotic that interacts with multiple neurotransmitter receptors. It is primarily used for schizophrenia. It is well absorbed orally and completely metabolized; its effective dose may be lower than in the general population. Its safety profile is similar to other antipsychotics, and its use in the elderly should be approached with extreme caution. Therefore, rapid detection and simultaneous online separation of these antipsychotic drugs have significant clinical application value.

[0003] However, because clinical patients often take multiple medications for basic mental illnesses simultaneously, high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection methods frequently fail to achieve complete chromatographic separation of the drugs, leading to interference and affecting quantitative results. HPLC-tandem mass spectrometry (HPLC-MS / MS), using a mass spectrometer as the detection method, is a powerful analytical tool that combines the high separation capability of HPLC with the high sensitivity and selectivity of mass spectrometry. Therefore, there is an urgent need in this field for a detection method employing HPLC-tandem mass spectrometry to improve the accuracy and sensitivity of detection methods. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, the present invention adopts the following technical solution:

[0005] A method for simultaneously detecting 11 psychiatric drugs or their metabolites in blood, the method being high performance liquid chromatography-tandem mass spectrometry, wherein the drugs to be detected are aripiprazole, dehydroaripiprazole, paliperidone, risperidone, quetiapine, olanzapine, mirtazapine, amisulpride, trazodone, clozapine, and norclozapine, and the target substance is serum or plasma;

[0006] Furthermore, the detection method includes the following steps:

[0007] S1 chromatographic conditions:

[0008] The S11 chromatographic column is C 18 Chromatographic column;

[0009] S12 mobile phase: Phase A is a 20mM aqueous solution of formic acid amine containing 0.005 v / v% formic acid, and Phase B is a 0.1 v / v% formic acid-acetonitrile solution;

[0010] S13 needle washing solvent: an aqueous solution containing 0.05 v / v% formic acid;

[0011] S14 pretreatment column: Anavo @ Split-type 96-well HMR-P purification tube, containing 10mg of packing material, specification: 1mL;

[0012] Preparation of S2 working solution:

[0013] Preparation of S21 reference solution:

[0014] Accurately pipette 600 μL of aripiprazole, dehydroaripiprazole, and amisulpride solution (100 μg / mL), quetiapine, clozapine, and norclozapine standard solutions, 40 μL of paliperidone, risperidone, mirtazapine, and olanzapine standard solutions (100 μg / mL), and 500 μL of trazodone (100 μg / mL). Add these solutions to a 50 v / v% methanol-water solution containing 1 mM vitamin C and dilute to a 5 mL volumetric flask to prepare a mixed standard stock solution.

[0015] Preparation of S22 isotope internal standard mixed solution:

[0016] Accurately pipette 300 μL of a 100 μg / mL aripiprazole-d8, dehydroaripiprazole, and amisulpride standard solutions; 500 μL of 100 μg / mL quetiapine, trazodone, clozapine, and norclozapine standard solutions; and 100 μL of paliperidone, risperidone, mirtazapine, and olanzapine standard solutions. Add 50 v / v% methanol-water solution containing 1 mM vitamin C and dilute to a 100 mL volumetric flask.

[0017] Preparation of the working solution for the S23 standard curve:

[0018] Accurately pipette the mixed standard stock solution prepared in S21 and serially dilute it with 50 v / v% methanol-water solution to prepare a series of linear working solutions containing aripiprazole, dehydroaripiprazole, amisulpride, quetiapine, clozapine, and norclozapine at concentrations of 12.0, 6.0, 3.0, 1.5, 0.75, 0.375, 0.1875, and 0.09375 μg / mL; trazodone at concentrations of 10.0, 5.0, 2.5, 1.25, 0.625, 0.5, 0.25, and 0.125 μg / mL; and paliperidone, risperidone, mirtazapine, and olanzapine at concentrations of 4, 2, 1, 0.5, 0.4, 0.25, 0.2, and 0.0625 μg / mL.

[0019] Preparation of S24 plasma or serum test solution:

[0020] Add an appropriate amount of the isotope internal standard mixed solution prepared by S22 to the plasma or serum sample to be tested, and then directly add it to Anavo. @ The 96-well HMR-P purification tube is split, and an appropriate amount of protein precipitant is added and mixed by blowing. It is allowed to stand for 10-20 minutes, then placed under positive pressure and slowly eluted. This positive pressure elution is repeated once more. The collected liquid is dried under nitrogen, and an appropriate amount of reconstituted solvent is added and shaken to reconstitute before direct injection or analysis. Further, the protein precipitant is an acetonitrile solution containing 0.05 v / v% formic acid; further, the reconstituted solvent is an acetonitrile-water solution containing 0.05 v / v% formic acid, wherein the acetonitrile:water volume ratio is 20:80; further, the positive pressure is 2-5 psi.

[0021] S3 mass spectrometry conditions:

[0022] Ion source: Electrospray ionization source (i.e., ESI) + Scanning mode: Multiple reaction monitoring (MRM); Carrier gas temperature: 345℃; Carrier gas flow rate: 10L / min; Nebulizer pressure: 35psi; Capillary voltage: 4000V; MRM mode scan;

[0023] S4 determination method: Inject the mixed standard solution prepared in S21 and the test solution prepared in S22 into a high performance liquid chromatograph, respectively, and use a gradient elution program to obtain chromatograms. Analyze the results based on the mass spectrometry detection results to obtain the detection results.

[0024] Furthermore, in the chromatographic conditions described in S4: the mobile phase gradient elution program is as follows: 0.0–1.0 min 15 v / v% B, mobile phase flow rate 0.1–0.5 mL / min; 1.3–4.2 min 22 v / v% B, mobile phase flow rate 0.1–0.5 mL / min; 4.5–5.0 min 30 v / v% B, mobile phase flow rate 0.05 mL / min; 6.0–6.5 min 50 v / v% B–90 v / v% B, mobile phase flow rate 0.4–0.5 mL / min; 8.5–9.0 min 90 v / v% B–15 v / v% B, mobile phase flow rate 0.4–0.5 mL / min; 10 min 15 v / v% B.

[0025] The chromatographic conditions described in S1 also include: (4) column temperature of 35-45℃; (5) injection volume of working solution of 1.0-10.0μL; further, the chromatographic conditions described in S1 also include: (4) column temperature of 40℃; (5) injection volume of working solution of 2μL.

[0026] Furthermore, in S4, the retention time, detection ion pairs, and collision energies of the 11 drugs are as follows:

[0027]

[0028]

[0029] Beneficial effects:

[0030] This invention employs HPLC-MS / MS to simultaneously detect commonly used psychiatric drugs or their metabolites in biological samples, offering the following advantages:

[0031] 1. The method of the present invention uses acetonitrile as the B phase of the mobile phase, which has stronger elution ability and lower column pressure.

[0032] 2. The extended rinsing time ensures more thorough column rinsing and effectively avoids residue.

[0033] 3. The needle washing solvent has been improved, solving the problem of needle residue.

[0034] 4. The total analysis time of this invention is shorter, with the entire detection and rinsing process taking only about 10 minutes;

[0035] 5. The pretreatment method of this detection method is simpler and faster, and it can effectively remove the matrix in the blood, avoiding the matrix residue in the instrument after a large number of samples are injected, which may cause the retention time or data instability. The method is also more accurate and stable. Attached Figure Description

[0036] Figure 1 The image shows chromatograms of 11 commonly used antipsychotic drugs or their active metabolites in plasma. The drugs are: 2. Amisulpride, 4. Olanzapine, 6. Mirtazapine, 8. Paliperidone, 10. Risperidone, 12. Trazodone, 14. Norclozapine, 16. Clozapine, 18. Quetiapine, 20. Dehydroaripiprazole, and 21. Aripiprazole. Detailed Implementation

[0037] Example 1

[0038] S1: Liquid chromatography conditions:

[0039] S11 column: Waters ACQUITY BEH C 18 Chromatographic column (50×2.1mm, 1.7μm);

[0040] S12 mobile phase: Phase A is a 20mM aqueous solution of formic acid amine containing 0.005 v / v% formic acid, and Phase B is a 0.1 v / v% formic acid-acetonitrile solution;

[0041] S13 needle washing solvent: an aqueous solution containing 0.05 v / v% formic acid;

[0042] S14 pretreatment column: Anavo @ Split-type 96-well HMR-P purification tube, containing 10mg of packing material, specification: 1mL;

[0043] S15 mobile phase flow rate: 0.4 mL / min;

[0044] S16 column temperature: 40℃;

[0045] Preparation of S2 working solution:

[0046] Preparation of S21 reference solution: Accurately pipette 600 μL of aripiprazole, dehydroaripiprazole, and amisulpride solution with a concentration of 100 μg / mL, quetiapine, clozapine, and norclozapine standard solution, 40 μL of paliperidone, risperidone, mirtazapine, and olanzapine standard solution with a concentration of 100 μg / mL, and 500 μL of trazodone with a concentration of 100 μg / mL. Add 50 v / v% methanol-water solution containing 1 mM vitamin C to a 5 mL volumetric flask to prepare a mixed standard stock solution.

[0047] Preparation of S22 isotope internal standard mixed solution:

[0048] Accurately pipette 300 μL of a 100 μg / mL aripiprazole-d8, dehydroaripiprazole, and amisulpride standard solutions; 500 μL of 100 μg / mL quetiapine, trazodone, clozapine, and norclozapine standard solutions; and 100 μL of paliperidone, risperidone, mirtazapine, and olanzapine standard solutions. Add 50 v / v% methanol-water solution containing 1 mM vitamin C and dilute to a 100 mL volumetric flask.

[0049] Preparation of the working solution for the S23 standard curve:

[0050] Accurately pipette the mixed standard stock solution prepared in S21 and serially dilute it with 50 v / v% methanol-water solution to prepare a series of linear working solutions containing aripiprazole, dehydroaripiprazole, amisulpride, quetiapine, clozapine, and norclozapine at concentrations of 12.0, 6.0, 3.0, 1.5, 0.75, 0.375, 0.1875, and 0.09375 μg / mL; trazodone at concentrations of 10.0, 5.0, 2.5, 1.25, 0.625, 0.5, 0.25, and 0.125 μg / mL; and paliperidone, risperidone, mirtazapine, and olanzapine at concentrations of 4, 2, 1, 0.5, 0.4, 0.25, 0.2, and 0.0625 μg / mL.

[0051] Preparation of S24 test solution

[0052] Accurately pipette 100 μL of plasma / serum sample into Anavo. @ Anavo @ Place the split-type 96-well HMR-P purification tube on a 96-well receiving plate, add 20 μL of isotope internal standard mixed solution, let stand for 10–20 minutes, add 400 μL of acetonitrile solution containing 0.05 v / v% formic acid, mix well by pipetting, place under positive pressure, apply 3 PSI pressure, and completely press the liquid into the 96-well receiving plate. Repeat the process of adding 400 μL of acetonitrile solution containing 0.1 v / v% formic acid and pressing it into the 96-well receiving plate under positive pressure. Dry the receiving plate under nitrogen blowing, add 100 μL of acetonitrile-water solution containing 0.05 v / v% formic acid (volume ratio 20:80), shake to reconstitute, and then directly inject or transfer to a vial with an inner tube for analysis.

[0053] S3 mass spectrometry conditions:

[0054] Ion source: Electrospray ionization source (i.e., ESI) + Scanning mode: Multiple reaction monitoring (MRM); Carrier gas temperature: 345℃; Carrier gas flow rate: 10L / min; Nebulizer pressure: 35psi; Capillary voltage: 4000V; MRM mode scan;

[0055] S4 determination method:

[0056] The working solutions prepared by S21 were injected into a high-performance liquid chromatograph (HPLC) and subjected to a gradient elution program to obtain chromatograms. The chromatograms were then analyzed based on the mass spectrometry results. Injection volume: 2.0 μL for each working solution. Gradient elution program: The mobile phase gradient elution program was as follows: 0.0–1.0 min 15 v / v%B, mobile phase flow rate 0.1–0.5 mL / min; 1.3–4.2 min 22 v / v%B, mobile phase flow rate 0.1–0.5 mL / min; 4.5–5.0 min 30 v / v%B, mobile phase flow rate 0.05 mL / min; 6.0–6.5 min 50 v / v%B–90 v / v%B, mobile phase flow rate 0.4–0.5 mL / min; 8.5–9.0 min 90 v / v% B to 15 v / v% B, mobile phase flow rate of 0.4 to 0.5 mL / min, 10 min 15 v / v% B;

[0057] 3.3 Test Results

[0058]

[0059]

[0060] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various modifications and alterations without departing from the spirit and scope of the present invention, and all such modifications and alterations shall be within the protection scope of the present invention.

Claims

1. A method for simultaneously detecting 11 psychiatric drugs or their metabolites in blood, characterized in that, The method is high performance liquid chromatography-tandem mass spectrometry, the detection drugs are aripiprazole, dehydroaripiprazole, paliperidone, risperidone, quetiapine, olanzapine, mirtazapine, amisulpride, trazodone, clozapine, and norclozapine, and the detection object is serum or plasma.

2. The method for simultaneously detecting 11 psychiatric drugs or their metabolites in blood according to claim 1, characterized in that, The detection method includes the following steps: S1 chromatographic conditions: The S11 chromatographic column is C 18 Chromatographic column; S12 mobile phase: Phase A is a 20mM aqueous solution of formic acid amine containing 0.005 v / v% formic acid, and Phase B is a 0.1 v / v% formic acid-acetonitrile solution; S13 needle washing solvent: an aqueous solution containing 0.05 v / v% formic acid; S14 pretreatment column: Anavo @ Split-type 96-well HMR-P purification tube, containing 10mg of packing material, specification: 1mL; Preparation of S2 working solution: Preparation of S21 reference solution: Accurately pipette 600 μL of aripiprazole, dehydroaripiprazole, and amisulpride solution (100 μg / mL), quetiapine, clozapine, and norclozapine standard solutions, 40 μL of paliperidone, risperidone, mirtazapine, and olanzapine standard solutions (100 μg / mL), and 500 μL of trazodone (100 μg / mL). Add these solutions to a 50 v / v% methanol-water solution containing 1 mM vitamin C and dilute to a 5 mL volumetric flask to prepare a mixed standard stock solution. Preparation of S22 isotope internal standard mixed solution: Accurately pipette 300 μL of a 100 μg / mL aripiprazole-d8, dehydroaripiprazole, and amisulpride standard solutions; 500 μL of 100 μg / mL quetiapine, trazodone, clozapine, and norclozapine standard solutions; and 100 μL of paliperidone, risperidone, mirtazapine, and olanzapine standard solutions. Add 50 v / v% methanol-water solution containing 1 mM vitamin C and dilute to a 100 mL volumetric flask. Preparation of the working solution for the S23 standard curve: Accurately pipette the mixed standard stock solution prepared in S21 and serially dilute it with 50 v / v% methanol-water solution to prepare a series of linear working solutions containing aripiprazole, dehydroaripiprazole, amisulpride, quetiapine, clozapine, and norclozapine at concentrations of 12.0, 6.0, 3.0, 1.5, 0.75, 0.375, 0.1875, and 0.09375 μg / mL; trazodone at concentrations of 10.0, 5.0, 2.5, 1.25, 0.625, 0.5, 0.25, and 0.125 μg / mL; and paliperidone, risperidone, mirtazapine, and olanzapine at concentrations of 4, 2, 1, 0.5, 0.4, 0.25, 0.2, and 0.0625 μg / mL. Preparation of S24 plasma or serum test solution: Add an appropriate amount of the isotope internal standard mixed solution prepared by S22 to the plasma or serum sample to be tested, and then directly add it to Anavo. @ The 96-well HMR-P purification tube is split, and an appropriate amount of protein precipitant is added and mixed by blowing. It is allowed to stand for 10-20 minutes, then placed under positive pressure and slowly eluted. This positive pressure elution is repeated once more. The collected liquid is dried under nitrogen, and an appropriate amount of reconstituted solvent is added and shaken to reconstitute before direct injection or analysis. Further, the protein precipitant is an acetonitrile solution containing 0.05 v / v% formic acid; further, the reconstituted solvent is an acetonitrile-water solution containing 0.05 v / v% formic acid, wherein the acetonitrile:water volume ratio is 20:80; further, the positive pressure is 2-5 psi. S3 mass spectrometry conditions: Ion source: Electrospray ionization source (i.e., ESI) + Scanning mode: Multiple reaction monitoring (MRM); Carrier gas temperature: 345℃; Carrier gas flow rate: 10L / min; Nebulizer pressure: 35psi; Capillary voltage: 4000V; MRM mode scan; S4 determination method: Inject the mixed standard solution prepared in S21 and the test solution prepared in S22 into a high performance liquid chromatograph, respectively, and use a gradient elution program to obtain chromatograms. Analyze the results based on the mass spectrometry detection results to obtain the detection results.

3. The method for simultaneously detecting 11 psychiatric drugs or their metabolites in blood according to claim 1, characterized in that, In the chromatographic conditions described in S4: the mobile phase gradient elution program is 0.0–1.0 min 15 v / v% B, mobile phase flow rate is 0.1–0.5 mL / min; 1.3–4.2 min 22 v / v% B, mobile phase flow rate is 0.1–0.5 mL / min; 4.5–5.0 min 30 v / v% B, mobile phase flow rate is 0.05 mL / min; 6.0–6.5 min 50 v / v% B–90 v / v% B, mobile phase flow rate is 0.4–0.5 mL / min; 8.5–9.0 min 90 v / v% B–15 v / v% B, mobile phase flow rate is 0.4–0.5 mL / min; 10 min 15 v / v% B.

4. The method for simultaneously detecting 11 psychiatric drugs or their metabolites in blood according to claim 1, characterized in that, The chromatographic conditions described in S1 also include: (4) the column temperature is 35-45℃; (5) the injection volume of the working solution is 1.0-10.0μL; further, the chromatographic conditions described in S1 also include: (4) the column temperature is 40℃; (5) the injection volume of the working solution is 2μL.

5. The method for simultaneously detecting 11 psychiatric drugs or their metabolites in blood according to claim 1, characterized in that, In S4, the retention times, detection ion pairs, and collision energies of the 11 drugs are as follows: 。

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