CN113917025A - Kit for quantitatively detecting psychotropic drugs in biological sample and application thereof - Google Patents
Kit for quantitatively detecting psychotropic drugs in biological sample and application thereof Download PDFInfo
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- CN113917025A CN113917025A CN202111177824.2A CN202111177824A CN113917025A CN 113917025 A CN113917025 A CN 113917025A CN 202111177824 A CN202111177824 A CN 202111177824A CN 113917025 A CN113917025 A CN 113917025A
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- 229940001470 psychoactive drug Drugs 0.000 title claims abstract description 40
- 239000004089 psychotropic agent Substances 0.000 title claims abstract description 40
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- 238000001514 detection method Methods 0.000 claims abstract description 79
- 239000012086 standard solution Substances 0.000 claims abstract description 77
- 238000002360 preparation method Methods 0.000 claims abstract description 71
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- 238000000034 method Methods 0.000 claims abstract description 46
- 239000003814 drug Substances 0.000 claims abstract description 41
- 229940079593 drug Drugs 0.000 claims abstract description 39
- 239000000523 sample Substances 0.000 claims abstract description 38
- 239000012085 test solution Substances 0.000 claims abstract description 35
- 238000003556 assay Methods 0.000 claims abstract description 8
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- 230000010354 integration Effects 0.000 claims abstract description 5
- 238000000825 ultraviolet detection Methods 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 125
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 75
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- FFGPTBGBLSHEPO-UHFFFAOYSA-N carbamazepine Chemical compound C1=CC2=CC=CC=C2N(C(=O)N)C2=CC=CC=C21 FFGPTBGBLSHEPO-UHFFFAOYSA-N 0.000 claims description 42
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- QZUDBNBUXVUHMW-UHFFFAOYSA-N clozapine Chemical compound C1CN(C)CCN1C1=NC2=CC(Cl)=CC=C2NC2=CC=CC=C12 QZUDBNBUXVUHMW-UHFFFAOYSA-N 0.000 claims description 39
- 229960004431 quetiapine Drugs 0.000 claims description 36
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- JNNOSTQEZICQQP-UHFFFAOYSA-N N-desmethylclozapine Chemical compound N=1C2=CC(Cl)=CC=C2NC2=CC=CC=C2C=1N1CCNCC1 JNNOSTQEZICQQP-UHFFFAOYSA-N 0.000 claims description 35
- 229960005017 olanzapine Drugs 0.000 claims description 35
- KVWDHTXUZHCGIO-UHFFFAOYSA-N olanzapine Chemical compound C1CN(C)CCN1C1=NC2=CC=CC=C2NC2=C1C=C(C)S2 KVWDHTXUZHCGIO-UHFFFAOYSA-N 0.000 claims description 35
- PMXMIIMHBWHSKN-UHFFFAOYSA-N 3-{2-[4-(6-fluoro-1,2-benzoxazol-3-yl)piperidin-1-yl]ethyl}-9-hydroxy-2-methyl-6,7,8,9-tetrahydropyrido[1,2-a]pyrimidin-4-one Chemical compound FC1=CC=C2C(C3CCN(CC3)CCC=3C(=O)N4CCCC(O)C4=NC=3C)=NOC2=C1 PMXMIIMHBWHSKN-UHFFFAOYSA-N 0.000 claims description 33
- CEUORZQYGODEFX-UHFFFAOYSA-N Aripirazole Chemical compound ClC1=CC=CC(N2CCN(CCCCOC=3C=C4NC(=O)CCC4=CC=3)CC2)=C1Cl CEUORZQYGODEFX-UHFFFAOYSA-N 0.000 claims description 33
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- CDONPRYEWWPREK-UHFFFAOYSA-N 7-[4-[4-(2,3-dichlorophenyl)piperazin-1-yl]butoxy]-1h-quinolin-2-one Chemical compound ClC1=CC=CC(N2CCN(CCCCOC=3C=C4NC(=O)C=CC4=CC=3)CC2)=C1Cl CDONPRYEWWPREK-UHFFFAOYSA-N 0.000 claims description 31
- 229960003036 amisulpride Drugs 0.000 claims description 31
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- DGBIGWXXNGSACT-UHFFFAOYSA-N clonazepam Chemical compound C12=CC([N+](=O)[O-])=CC=C2NC(=O)CN=C1C1=CC=CC=C1Cl DGBIGWXXNGSACT-UHFFFAOYSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
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- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention provides a method for simultaneously and quantitatively detecting psychotropic drugs or metabolites thereof in a biological sample by using a UPLC-UV detection method, which comprises the following steps: s1 chromatographic conditions; s2 preparation of working solution; s3 assay. Respectively obtaining the spiritual classRetention time t of drug and its metabolite standard and sampleRIntegrating peak areas, establishing a standard curve according to the concentration in the mixed standard solution and the peak area/internal standard area ratio corresponding to the concentration, and then obtaining the concentration of the test solution according to the peak area integration of the test solution and the standard curve; also discloses a detection kit prepared by the method; the invention adopts the UPLC-UV method to realize the separation of the psychiatric usual drugs in the biological sample, and prepares the biological sample into the kit, thereby rapidly, simply, conveniently and accurately quantitatively detecting the in vivo concentration of the clinical usual psychiatric drugs, and having good academic and commercial values.
Description
Technical Field
The invention relates to the technical field of psychiatric drug detection kits, in particular to a kit for quantitatively detecting psychiatric drugs in a biological sample and application thereof.
Background
Antipsychotics (antipathotic drugs), also known as strong tranquilizers or nerve blockers (neuroleptics), are a group of drugs used in the treatment of schizophrenia and other psychotic disorders. The normal therapeutic dose does not affect the intelligence and consciousness of the patient, but can effectively control the mental symptoms of psychomotor excitation, hallucination, delusion, hostile emotion, thought disorder, abnormal behaviors and the like of the patient. Patients usually have sleep disorder, and usually take sedative and tranquilizer medicines simultaneously, so the two medicines need to be separated at the time of detection to obtain a stable blood concentration detection value of the antipsychotic medicine, and the interference of the rest clinical combined medicines is avoided. Clinical treatment drugs commonly used by psychiatric patients are: olanzapine, 9-OH risperidone, clozapine, norclozapine, risperidone, quetiapine fumarate, chlorpromazine hydrochloride, trazodone hydrochloride, mirtazapine, amisulpride, aripiprazole, and dehydroaripiprazole, and in order to coordinate with treatment, patients usually meet and use sedative-hypnotic drugs such as alprazolam, clonazepam, zopiclone, estazolam, and the like, and if the detection method cannot realize simultaneous separation of the concomitant medication, the concentration quantification of the psychology drugs is inaccurate, so that the simultaneous online separation of the sedative-hypnotic drugs and the antipsychotic drugs has very important clinical application value.
Therapeutic drug concentration monitoring refers to measuring the concentration of a particular drug in a patient's blood at specified time intervals during a clinically conducted drug therapy. The method for monitoring the clinical blood concentration at present comprises the following steps: mainly includes High Performance Liquid Chromatography (HPLC), Radioimmunoassay (RIA), Gas Chromatography (GC), liquid tandem mass spectrometry (LC-MS), and the like. Because clinical patients often take a plurality of basic mental disease drugs at the same time, the detection methods of the drugs with different properties are generally different, and the used detection equipment is different. Therefore, when the detection is carried out, equipment needs to be switched or the mobile phase needs to be replaced continuously, and the instrument needs to be balanced after replacement, so that the detection time is prolonged, and the operation of clinical detection personnel is not facilitated.
In addition, the high performance liquid chromatography ultraviolet detection method often cannot completely realize the chromatographic separation of the drugs, and is easy to interfere, thereby influencing the quantitative result; the high performance liquid chromatography tandem mass spectrometry is a powerful analysis and detection tool which integrates high separation capacity of a high performance liquid chromatograph, high sensitivity and high selectivity of the mass spectrometer, and the mass spectrometry can be accurately quantified as a detection means, but on one hand, equipment is expensive, and on the other hand, isotope internal standard consumables are also very expensive and are not friendly to the environment. Therefore, there is a need in the art for a detection method that can simultaneously realize online separation of multiple psychiatric drugs, has short separation time and good effect, and can be prepared into a kit for clinical detection.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention adopts the following technical scheme:
the invention provides a method for simultaneously and quantitatively detecting psychotropic drugs or metabolites thereof in a biological sample, which adopts an ultra-high performance liquid chromatography-ultraviolet detection method and comprises the following steps:
s1 chromatographic conditions: (1) a chromatographic column: c18A chromatographic column; (2) mobile phase: the phase A is formic acid-water solution, and the phase B is formic acid-methanol solution; (3) the detector is an Ultraviolet (UV) detector;
preparing a working solution of S2:
preparation of internal standard solution of S21:
s211 metoclopramide internal standard working solution: precisely absorbing a proper amount of the metoclopramide standard substance, dissolving with a small amount of methanol, diluting with methanol-water solution to constant volume to prepare metoclopramide standard substance solution, and diluting with methanol-water solution to constant volume to obtain an internal standard 1-metoclopramide working solution;
s212 carbamazepine internal standard working solution: precisely absorbing a proper amount of carbamazepine standard, dissolving by using a small amount of methanol, then diluting to constant volume by using a methanol-water solution to prepare a carbamazepine standard solution, and then diluting to constant volume by using the methanol-water solution to obtain an internal standard 2-carbamazepine working solution;
preparation of S22 mixed standard solution:
s221 standard mixing mother liquor preparation: accurately weighing a proper amount of commonly used psychiatric drugs or metabolites of the commonly used drugs respectively, namely amisulpride, clozapine, norclozapine, aripiprazole, dehydroaripiprazole, olanzapine, mirtazapine, risperidone, 9-OH risperidone, trazodone, chlorpromazine and quetiapine, mixing, dissolving with a proper amount of solvent, and diluting with methanol-water solution to a constant volume to prepare a mixed standard mother solution; further, the preparation of the mixed standard mother solution comprises the following steps: accurately weighing a proper amount of commonly used neurologic drugs or metabolites of the commonly used neurologic drugs, such as amisulpride, clozapine, norclozapine, aripiprazole, dehydroaripiprazole, olanzapine, mirtazapine, risperidone, 9-OH risperidone, trazodone, chlorpromazine and quetiapine, 1-30 mg respectively, dissolving the mixture with 1-30 mL of solvent, diluting the solution with 40-70% v/v of methanol-water solution to a constant volume to prepare a mixed mother liquor containing amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole with the concentration of 400 mu g/mL, the concentration of olanzapine, mirtazapine, risperidone and 9-OH risperidone of 100 mu g/mL and the concentration of trazodone, chlorpromazine and quetiapine of 200 mu g/mL;
s222, preparation of a quantitative mixed standard curve working solution: diluting the mixed standard mother liquor by using 40-70% v/v methanol-water solution with a certain concentration according to a certain proportion, and respectively preparing standard curve solutions with 7 concentrations of the medicines, wherein the solutions respectively contain amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole with the concentrations of 100, 50, 20, 10, 4, 2 and 1 mu g/mL, contain olanzapine, mirtazapine, risperidone and 9-OH risperidone with the concentrations of 25, 12.5, 5, 2.5, 1, 0.5 and 0.25 mu g/mL, and contain trazodone, chlorpromazine and quetiapine with the concentrations of 50, 25, 10, 5, 2,1 and 0.5 mu g/mL;
preparation of S223 mixed standard solution: respectively and precisely absorbing 10-40 mu L of the quantitative mixed standard curve working solution, precisely adding 10-40 mu L of each of the internal standard 1-metoclopramide working solution and the internal standard 2-carbamazepine working solution, drying, adding a proper amount of blank blood, plasma, serum or saliva and methyl tert-butyl ether, carrying out primary vortex, adding 100-500 mu L of sodium hydroxide aqueous solution with the concentration of 1-3mol/L and methyl tert-butyl ether, carrying out secondary vortex and centrifugation to obtain supernatant, drying the supernatant, and adding methanol-aqueous solution containing formic acid for redissolution, vortex and filtering to obtain the product;
the preparation of the test solution of S23 includes:
s231 preparation of blood, plasma, serum or saliva test solutions: respectively and precisely absorbing 10-40 mu L of each of the internal standard 1-metoclopramide working solution and the internal standard 2-carbamazepine working solution prepared by S21, drying, mixing with a proper amount of blood, plasma, serum or saliva to be detected, carrying out primary vortex, adding 100-500 mu L of sodium hydroxide aqueous solution and methyl tert-butyl ether for secondary vortex, carrying out centrifugation after vortex to obtain supernatant, drying the supernatant, adding methanol-aqueous solution containing formic acid for redissolving, vortex and filtering to obtain the oral liquid;
or S232 preparation of hair test solution: respectively and precisely absorbing 10-40 mu L of each of the internal standard 1-metoclopramide working solution and the internal standard 2-carbamazepine working solution prepared by S21, drying, mixing with a proper amount of hair sample to be detected, carrying out primary vortex, adding 100-500 mu L of sodium hydroxide aqueous solution, carrying out ultrasonic treatment or standing, adding methyl tert-butyl ether for secondary vortex, carrying out centrifugation after vortex to obtain supernatant, drying the supernatant, adding methanol-aqueous solution containing formic acid for redissolving, vortex, and filtering to obtain the product;
s3 assay: respectively injecting the mixed standard solution prepared in S22 and the test solution prepared in S23 into an ultra high performance liquid chromatograph, respectively obtaining mixed standard solution chromatograms with 7 concentrations and a test solution chromatogram by adopting a gradient elution procedure, and respectively obtaining the retention time t of the psychotropic drugs, the metabolite standard products thereof and the test solutionsRIntegrating peak areas, establishing a standard curve according to the concentration in the mixed standard solution and the peak area/internal standard area ratio corresponding to the concentration, and then obtaining the concentration of the test solution according to the peak area integration of the test solution and the standard curve;
further, the biological sample is selected from any one or more of blood, plasma, serum, saliva and hair; further preferably, the biological sample is selected from any one or more of plasma, serum and hair;
further, in the chromatographic conditions described in S1: mobile phase: phase A is 0.05 v/v% formic acid-water solution; and phase B is 0.05 v/v% formic acid-methanol solution;
further, in the chromatographic conditions described in S1: the ultraviolet detector is a VWD or DAD detector; further preferably, the detection wavelength of the ultraviolet detector is 254nm, 285nm or full wavelength scanning;
further, the chromatographic conditions of S1 further include: (4) the flow rate of the mobile phase is 0.1-0.5 mL/min; (5) the column temperature is 35-45 ℃; (6) the sampling amount of the working solution is 1.0-20.0 mu L;
further, in step S21, the preparing of the internal standard solution specifically includes:
s211 metoclopramide internal standard working solution: precisely weighing 1-30 mg of metoclopramide standard substance, dissolving with 1-30 mL of methanol, then fixing the volume with 40-70% v/v methanol-water solution to prepare metoclopramide stock solution with the concentration of 0.1-0.5 mg/mL, and then diluting with 40-70% v/v methanol-water solution to 10-40 mu g/mL to obtain internal standard 1-metoclopramide working solution;
s212 carbamazepine internal standard working solution: accurately weighing 1-30 mg of carbamazepine standard, dissolving with 1-30 mL of methanol, then fixing the volume with 40-70% v/v methanol-water solution to prepare a carbamazepine stock solution with the concentration of 0.1-0.5 mg/mL, and then diluting with 40-70% v/v methanol-water solution to 1-5 mu g/mL to obtain an internal standard 2-carbamazepine working solution;
further, the preparation of the S231 blood, plasma, serum or saliva test solution comprises the following steps: respectively and precisely absorbing 10-40 mu L of each of an internal standard 1-metoclopramide working solution and an internal standard 2-carbamazepine working solution prepared by S21, drying by blowing nitrogen, mixing with 400-1000 mu L of a melted frozen blood, plasma, serum or saliva sample to be detected into a 7-10 mL centrifuge tube, carrying out primary vortex for 0.5-2 min, adding 100-500 mu L of a sodium hydroxide aqueous solution with the concentration of 1-3mol/L and 2-7 mL of methyl tert-butyl ether, carrying out secondary vortex for 2-5 min, carrying out secondary vortex for 4-10 min on the centrifuged centrifuge tube at the speed of 2500-5000 r/m, carrying out blow-drying on centrifuged supernatant nitrogen, adding 60-250 mu L of a methanol-water solution containing 0.05-0.15 v/v% formic acid and with the concentration of 10-20 v/v%, carrying out re-dissolution, carrying out vortex for 0.5-2 min, and filtering by a nylon filter membrane to obtain the oral liquid; further preferably, the preparation of the S231 blood, plasma, serum or saliva test solution comprises the following steps: respectively and precisely absorbing 20 mu L of each of the internal standard 1-metoclopramide working solution and the internal standard 2-carbamazepine working solution prepared by S21, drying by blowing nitrogen, mixing the dried internal standard 1-metoclopramide working solution and 1000 mu L of the melted frozen serum sample to be detected into a 7mL centrifuge tube, carrying out primary vortex for 1min, adding 200 mu L of a 2mol/L sodium hydroxide aqueous solution, adding 3mL of methyl tert-butyl ether, carrying out secondary vortex for 3min, carrying out secondary centrifugation for 5min, taking the centrifuged supernatant, drying by blowing nitrogen, adding 200 mu L of a 15% v/v methanol-aqueous solution containing 0.1 v/v% formic acid for redissolving, carrying out vortex for 1min, and filtering by using a filter membrane to obtain the oral liquid;
or further, the preparation of the S232 hair test solution comprises the following steps: sequentially cleaning hair to be tested with acetone and water, drying, shearing, respectively and precisely absorbing 10-40 mu L of each of the internal standard 1-metoclopramide working solution and the internal standard 2-carbamazepine working solution prepared by S21, mixing with 10-50 mg of hair sample to be tested into a 7-10 mL centrifuge tube after drying with nitrogen, adding 100-500 mu L of sodium hydroxide solution with the concentration of 1-3mol/L, performing primary vortex for 0.5-2 min, performing ultrasonic treatment for 1-4 h, adding 2-7 mL of methyl tert-butyl ether, performing secondary vortex for 2-5 min, re-dissolving the centrifuge tube after vortex at the speed of 2500-5000 r/m for 4-10 min, drying supernatant after centrifugation with nitrogen, adding 60-250 mu L of methanol-water solution containing 0.05-0.15 v/v% formic acid and having the concentration of 10-20 v/v%, performing vortex for 0.5-2 min, filtering with nylon filter membrane to obtain; further preferably, the preparation of the S232 hair test solution comprises the following steps: sequentially cleaning hair to be detected with acetone and water, drying, shearing, precisely absorbing 20 mu L of each of an internal standard 1-metoclopramide working solution and an internal standard 2-carbamazepine working solution prepared by S21, respectively, blowing dry with nitrogen, mixing with 20mg of a hair sample to be detected into a 7mL centrifuge tube, adding 200 mu L of a 2mol/L sodium hydroxide aqueous solution, carrying out primary vortex for 1min, carrying out ultrasonic treatment for 2h, adding 4mL of methyl tert-butyl ether, carrying out secondary vortex for 3min, centrifuging the centrifuged centrifuge tube at a speed of 3000r/min for 5min, blowing dry supernatant after centrifugation with nitrogen, adding 200 mu L of a methanol-aqueous solution containing 0.1 v/v% formic acid and having a concentration of 15 v/v%, carrying out redissolution with vortex for 1min, and filtering with a nylon filter membrane to obtain the hair conditioner;
further, the gradient elution procedure described in the S3 assay was: 0min 5% v/v B, 2min 10% v/v B, 4min 20% v/v B, 6min 22% v/v B, 12min 35% v/v B, 15min 36% v/v B, 18.5min 40% v/v B, 20min 50% v/v B, 21min 90% v/v B, 23min 90% v/v B, 24min 5% v/v B, and 28min 5% v/v B;
in a second aspect of the present invention, there is provided a kit for quantitatively detecting a psychotropic drug or a metabolite thereof in a biological sample, comprising:
the preparation method of the mixed standard solution TDM detection tube comprises the following steps: adding the internal standard solution and the mixed standard solution into a TDM detection tube, and drying to obtain the product;
(II) a TDM detection tube for detecting sample solution, wherein the preparation method comprises the following steps: adding an internal standard solution into the TDM detection tube, and drying to obtain the TDM detection tube;
(III) compounding solution: methanol-water solution containing formic acid;
(IV) the description: the specification describes methods of use thereof;
and the kit is stored at-20 to-15 ℃;
further, the biological sample is selected from any one or more of blood, plasma, serum, saliva and hair; further preferably, the biological sample is selected from any one or more of plasma, serum and hair;
further, the preparation method of the mixed standard solution TDM detection tube comprises the following steps:
s1 preparation of internal standard solution:
s11 metoclopramide internal standard working solution: precisely absorbing a proper amount of the metoclopramide standard substance, dissolving with a small amount of methanol, diluting with methanol-water solution to constant volume to prepare metoclopramide standard substance solution, and diluting with methanol-water solution to constant volume to obtain an internal standard 1-metoclopramide working solution; further, the preparation method of the metoclopramide internal standard working solution comprises the following steps: precisely weighing 1-30 mg of metoclopramide standard substance, dissolving with 1-30 mL of methanol, then fixing the volume with 40-70% v/v methanol-water solution to prepare metoclopramide stock solution with the concentration of 0.1-0.5 mg/mL, and then diluting with 40-70% v/v methanol-water solution to 10-40 mu g/mL to obtain internal standard 1-metoclopramide working solution;
and the number of the first and second groups,
s12 carbamazepine internal standard working solution: precisely absorbing a proper amount of carbamazepine standard, dissolving by using a small amount of methanol, then diluting to constant volume by using a methanol-water solution to prepare a carbamazepine standard solution, and then diluting to constant volume by using the methanol-water solution to obtain an internal standard 2-carbamazepine working solution; further, the preparation method of the carbamazepine internal standard working solution comprises the following steps: accurately weighing 1-30 mg of carbamazepine standard, dissolving with 1-30 mL of methanol, then fixing the volume with 40-70% v/v methanol-water solution to prepare a carbamazepine stock solution with the concentration of 0.1-0.5 mg/mL, and then diluting with 40-70% v/v methanol-water solution to 10-40 mu g/mL to obtain an internal standard 2-carbamazepine working solution;
s2 preparation of mixed standard solution TDM detection tubes:
preparing S21 mixed standard mother liquor: accurately weighing a proper amount of commonly used neurologic drugs or metabolites of the commonly used neurologic drugs, such as amisulpride, clozapine, norclozapine, aripiprazole, dehydroaripiprazole, olanzapine, mirtazapine, risperidone, 9-OH risperidone, trazodone, chlorpromazine and quetiapine, 1-30 mg respectively, dissolving the mixture with 1-30 mL of solvent, diluting the solution with 40-70% v/v methanol-water solution to a constant volume to prepare a mixed standard mother solution containing amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole at 400 mu g/mL, olanzapine, mirtazapine, risperidone and 9-OH risperidone at 100 mu g/mL and trazodone, chlorpromazine and quetiapine at 200 mu g/mL;
preparation of S22 mixed standard curve working solution: diluting the mixed standard mother liquor by using 40-70 v/v% methanol-water solution according to a certain proportion to obtain a mixed standard mother liquor containing amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole with the concentrations of 100, 50, 20, 10, 4, 2 and 1 mu g/mL respectively, the concentrations of olanzapine, mirtazapine, risperidone and 9-OH risperidone with the concentrations of 25, 12.5, 5, 2.5, 1, 0.5 and 0.25 mu g/mL respectively, and the concentrations of trazodone, chlorpromazine and quetiapine with the concentrations of 50, 25, 10, 5, 2,1 and 0.5 mu g/mL respectively;
preparation of a TDM detection tube for S23 mixed standard solution: respectively and precisely absorbing 10-40 mu L of the working solution of the quantitative mixed standard curve into a TDM detection tube, then adding 10-40 mu L of each of the internal standard 1-metoclopramide working solution and the internal standard 2-carbamazepine working solution, and drying by blowing with nitrogen gas to obtain the product;
further, the preparation method of the sample solution TDM detection tube comprises the following steps: adding the internal standard solution prepared in the step S1 into a TDM detection tube, and drying by blowing nitrogen to obtain the internal standard solution;
further, the complex solution is 0.05-0.15 v/v% formic acid and 10-20% v/v methanol-water solution;
further, the use method of the kit comprises the following steps:
s1, preparing a standard solution: taking the mixed standard substance solution TDM detection tube, adding 100-1000 mu [ s2] L of blank blood, plasma, serum or saliva and 2-7 mL of methyl tert-butyl ether, performing primary vortex for 1-5 min, then adding 100-500 mu L of 1-3mol/L sodium hydroxide aqueous solution and 2-7 mL of methyl tert-butyl ether, performing secondary vortex for 4-10 min at a speed of 2500-5000 r/min, obtaining supernatant, drying the supernatant, then adding 100-150 mu L of re-solution, and performing microfiltration membrane filtration to obtain the product;
s2, preparing a sample detection solution:
(2) serum sample detection solution: placing 100-1000 mu L of serum sample into a sample solution TDM detection tube, adding 100-500 mu L of sodium hydroxide aqueous solution with the concentration of 1-3mol/L, performing vortex for 0.5-2 min, adding 2-7 mL of methyl tert-butyl ether, performing vortex for 1-5 min, centrifuging for 4-10 min at the speed of 2500-5000 r/min, transferring supernatant into a test tube, performing nitrogen blow drying, adding 100-150 mu L of redissolution, and performing microfiltration membrane filtration to obtain the serum sample;
(2) hair sample detection solution: taking a 20mg hair sample into a sample solution TDM detection tube, adding 100-500 mu L of sodium hydroxide aqueous solution with the concentration of 1-3mol/L, carrying out primary vortex for 0.5-2 min, then carrying out ultrasonic treatment for 1-4 h, adding 2-7 mL of methyl tert-butyl ether, carrying out secondary vortex for 2-5 min, centrifuging the centrifuged tube at the speed of 2500-5000 r/separation for 4-10 min, transferring supernatant into a test tube, carrying out nitrogen blow-drying, adding 100-150 mu L of redissolution, and filtering with a microporous filter membrane to obtain the hair dye;
s3, determination method: respectively injecting 5-10 μ L of the standard solution prepared in S1 and the sample detection solution prepared in S2 into an ultra high performance liquid chromatograph, respectively obtaining mixed standard solution chromatograms and test sample solution chromatograms with 7 concentrations by adopting a gradient elution procedure, and respectively obtaining retention time t of the psychotropic drugs, the metabolite standard products and the test samplesRIntegrating peak areas, establishing a standard curve according to the concentration in the mixed standard solution and the peak area/internal standard area ratio corresponding to the concentration, and then obtaining the concentration of the test solution according to the peak area integration of the test solution and the standard curve; the gradient elution procedure is as follows: 0min 5% v/v B, 2min 10% v/v B, 4min 20% v/v B, 6min 22% v/v B, 12min 35% v/v B, 15min 36% v/v B, 18.5min 40% v/v B, 20min 50% v/v B, 21min 90% v/v B, 23min 90% v/v B, 24min 5% v/v B, and 28min 5% v/v B;
the third aspect of the invention provides a use of any one of the above-mentioned kits in the preparation of a detection reagent for quantitatively detecting a psychotropic drug or a metabolite thereof in a biological sample.
Drawings
FIG. 1 is a chromatogram of a serum-mixed standard solution of example 1
FIG. 2 is a chromatogram of a mixed standard solution for hair of example 1
FIG. 3 is a standard curve chart of each drug in the serum-mixed standard solution of example 1
FIG. 4 is a standard curve chart of each drug in the serum-mixed standard solution of example 1
FIG. 5 is a chromatogram of a serum sample from a patient administered clozapine and quetiapine fumarate, according to example 2
FIG. 6 is a chromatogram for measuring the serum sample of a patient administered amisulpride in example 3
FIG. 7 is a chromatogram for measuring serum samples of patients administered clozapine in example 4
FIG. 8 is a chromatogram for measuring the serum sample of a patient administered with aripiprazole of example 5
FIG. 9 is a chromatogram showing the detection of hair samples from the patients of example 6 who took olanzapine, trazodone and risperidone
FIG. 10 is a chromatogram showing the detection of hair samples of the patient of example 7 who took olanzapine or mirtazapine
FIG. 11 is a chromatogram of the examination of a hair sample from a patient of example 8 taking quetiapine as a drug
Advantageous effects
1. The invention adopts the UPLC-UV method to realize the quantitative separation of the psychiatric usual drugs in human serum or hair in the biological sample, and prepares the human serum or hair into a kit, thereby rapidly, simply, conveniently and accurately quantitatively detecting the in vivo concentration of the clinical usual psychiatric drugs;
2. according to the invention, by adding the internal standard substance into the standard solution and the sample solution to be tested and injecting the sample simultaneously, the peak positions of the samples with different retention times are accurately positioned in a short time, so that interference is prevented, the separation of various detection medicines is realized only within 10min, and the detection time is greatly shortened;
3. the invention adopts an economical ultraviolet detector to replace mass spectrometry for detection, and has significant academic significance and economic value for guiding clinical medication and analyzing samples;
4. the invention realizes the rapid and complete separation of the sample in a short time by further optimizing the separation conditions, such as increasing the flow rate and optimizing the elution gradient program;
5. the liquid-liquid extraction method is adopted, the pH value is adjusted by adding NaOH solution, the hair matrix is digested, the medicinal components in the hair matrix are extracted by methyl tert-butyl ether, the detection limit is obviously improved, expensive solid phase extraction consumables are not needed, the chromatographic column is more durable by sample injection after filtration, and the method is suitable for clinical detection of a large number of samples for a long time and has obvious economic value;
6. a specific quantitative calculation method is provided, so that the concentration of the drug in the sample can be accurately measured;
7. and a specific redissolution is selected for redissolving, so that the measurement accuracy is further improved.
Detailed Description
Example 1: detection method of complete mixed standard substance
A method for simultaneously detecting psychotropic drugs or metabolites thereof in vivo, which adopts an ultra-high performance liquid chromatography-ultraviolet detection method and comprises the following steps:
s1 chromatographic conditions: (1) a chromatographic column: c18Chromatographic column, ACQUITY UPLC BEH C18Diameter 2.1mm 50mm, filler particle size 1.7 μm; (2) mobile phase: phase A is 0.05 v/v% formic acid-water solution; phase B is 0.05 v/v% formic acid-methanol solution; (3) the detector is an Ultraviolet (UV) detector, and the detection wavelength of the UV detector is scanned at full wavelength; (4) the flow rate of the mobile phase is 0.4 mL/min; (5) the column temperature was 40 ℃; (6) the sampling amount of the working solution is 10.0 mu L;
the preparation of the S2 working solution comprises the following steps:
preparation of S20 needle washing solution: measuring 700mL of water by using a measuring cylinder, adding 300mL of methanol into a liquid phase washing bottle, and carrying out mixed ultrasonic treatment for 20min to obtain a methanol-water needle washing solution;
preparation of internal standard solution of S21:
s211 metoclopramide internal standard working solution: precisely weighing 20mg metoclopramide standard substance in a 100mL volumetric flask, dissolving with 10mL methanol, fixing the volume with 50% v/v methanol-water solution to prepare metoclopramide stock solution with the concentration of 0.2mg/mL, and diluting with 50% v/v methanol-water solution to 20 mu g/mL to obtain internal standard 1-metoclopramide working solution;
s212 carbamazepine internal standard working solution: precisely weighing a 20mg carbamazepine standard substance, dissolving 10mL of methanol into a 100mL volumetric flask, fixing the volume by using 50% v/v methanol-water solution to prepare a carbamazepine stock solution with the concentration of 0.2mg/mL, and diluting the carbamazepine stock solution to 2 mu g/mL by using 50% v/v methanol-water solution to obtain an internal standard 2-carbamazepine working solution;
preparation of S22 mixed standard solution:
s221 preparation of standard mixing mother liquor: precisely weighing 20mg of each mental disease medicament or metabolite standard thereof in a 100mL volumetric flask respectively, except aripiprazole and dehydroaripiprazole, dissolving the other medicines (containing amisulpride, clozapine, norclozapine, olanzapine, mirtazapine, risperidone, 9-OH risperidone, trazodone, chlorpromazine and quetiapine) in 10mL of methanol, dissolving aripiprazole and dehydroaripiprazole in 1% formic acid-containing methanol solution, completely dissolving, then diluting to constant volume by using 50% v/v methanol-water solution to prepare a standard mother solution containing amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole at 400 mu g/mL, olanzapine, mirtazapine, risperidone and 9-OH risperidone at 100 mu g/mL, and trazodone, chlorpromazine and quetiapine at 200 mu g/mL;
s222 preparation of quantitative mixed standard curve solution: diluting the mixed standard mother liquor by using 40-70% v/v methanol-water solution with concentration according to a certain proportion, and respectively preparing 7 standard curve solutions with concentrations of the medicines, wherein the solutions respectively contain amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole, the concentrations of the mixed standard curves LS-1, LS-2, LS-3, LS-4, LS-5, LS-6 and LS-7 solutions with concentrations of 50, 25, 10, 5, 2,1 and 0.5 μ g/mL and the concentrations of the mixed standard curves LS-1, LS-2, LS-3, LS-4, LS-5, LS-6 and LS-7 with concentrations of olanzapine, mirtazapine, risperidone and 9-OH risperidone of 25, 12.5, 5, 2.5, 1, 0.5 μ g/mL;
preparation of S223 mixed standard solution: respectively and precisely absorbing 20 mu L of the mixed standard curve solution into a 7mL centrifuge tube, then adding 20 mu L of each of the internal standard 1-metoclopramide working solution and the internal standard 2-carbamazepine working solution, drying by nitrogen, adding a proper amount of blank serum and methyl tert-butyl ether, performing primary vortex, then adding 200 mu L of 2mol/L sodium hydroxide aqueous solution and 3mL of methyl tert-butyl ether, performing secondary vortex for 3min, performing secondary vortex for 5min, taking the centrifuged supernatant, drying by nitrogen, adding 150 mu L of 15 v/v% methanol-water solution containing 0.1 v/v% formic acid and performing redissolution, performing vortex for 1min, and filtering by using a 0.25 mu m nylon filter membrane to obtain the compound standard curve solution;
s3 assay: injecting the mixed standard substance solution into an ultra-high performance liquid chromatograph, and adopting the following gradient elution procedure: 0min 5% v/v B, 2min 10% v/v B, 4min 20% v/v B, 6min 22% v/v B, 12min 35% v/v B, 15min 36% v/v B, 18.5min 40% v/v B, 20min 50% v/v B, 21min 90% v/v B, 23min 90% v/v B, 24min 5% v/v B and 28min 5% v/v B to obtain mixed standard solution, and obtaining retention time (t) of the psychotropic drug and metabolite thereofR) Obtaining the product; the retention time t of the psychotropic drugs and metabolites thereofRRespectively 3.179min for olanzapine, 3.932min for amisulpride, 4.347 for metoclopramide (internal standard 1), 4.863min for zopiclone, 5.416min for mirtazapine, 7.373min for 9-OH risperidone, 7.927min for trazodone, 8.306min for risperidone, 8.708min for norclozapine, 9.578min for clozapine, 11.135min for quetiapine, 13.199min for carbamazepine (internal standard 2);
here, the retention time (t) of the psychotropic drug and its metabolites is obtained by taking as an example only a mixed standard curve containing amisulpride, clozapine, norclozapine, aripiprazole, dehydroaripiprazole all at 100. mu.g/mL, olanzapine, mirtazapine, risperidone, 9-OH risperidone all at 25. mu.g/mL, and trazodone, chlorpromazine, quetiapine all at 50. mu.g/mL, and a quantitative curve LSS-1 chromatogram in serum thereof is shown in FIG. 1R) Obtaining the product; the retention time t of the psychotropic drugs and metabolites thereofRRespectively 3.179min for olanzapine, 3.935min for amisulpride, 4.363 for metoclopramide (internal standard 1), 5.434min for mirtazapine, 7.355min for 9-OH risperidone, 7.901min for trazodone, 8.299min for risperidone, 8.730min for norclozapine, 9.584min for clozapine, 11.121min for quetiapine, 13.213min for carbamazepine (internal standard 2), 17.397min for dehydroaripiprazole, 18.035min for aripiprazole, and 19.419min for chlorpromazine; the quantitative curve LSH-1 in hair is shown in figure 2, and chromatogram shows that the retention time (t) of the psychotropic drug and its metabolites is obtainedR) Obtaining the product; the retention time t of the psychotropic drugs and metabolites thereofRRespectively 3.177min for olanzapine, 3.924min for amisulpride, 4.351 for metoclopramide (internal standard 1), 5.431min for mirtazapine, 7.344min for 9-OH risperidone, 7.911min for trazodone, 8.300min for risperidone, 8.762min for norclozapine, 9.639min for clozapine, 11.15 min for quetiapine5min, carbamazepine 13.206min (internal standard 2), dehydroaripiprazole 17.439min, aripiprazole 18.058min, chlorpromazine 19.447 min.
S4, standard curve making and calculation method:
s41: establishing a standard curve by using the concentration of the linear working solution in the serum and the ratio of the corresponding peak area to the internal standard area, and calculating the r value by using a correlation coefficient correl function in Excel, specifically calculating in the example:
a. the linear range of olanzapine is: 5-500ng/mL, wherein the linear equation is that y is 0.0111X-0.2065, C is X1.0 mL/sample volume (mL) to be detected, and r is 0.9987;
b. the linear range of amisulpride is: 20-2000ng/mL, wherein a linear equation is that y is 0.0026X +0.0023, C is 1.0 mL/sample volume (mL), and r is 0.9999;
c. the linear range of mirtazapine is: 5-500ng/mL, wherein the linear equation is that y is 0.0019X +0.0076, C is X1.0 mL/sample volume (mL), and r is 0.9998;
d.9-OH Risperidone has a linear range of: 5-500ng/mL, wherein the linear equation is that y is 0.0014X-0.011, C is 1.0 mL/sample volume (mL) to be detected, and r is 0.9997;
e. the linear range of trazodone is: 10-1000ng/mL, wherein the linear equation is that y is 0.0048X-0.0065, C is 1.0 mL/sample volume (mL), and r is 1.0000;
f. the linear range of risperidone is: 5-500ng/mL, wherein the linear equation is that y is 0.0022X-0.0199, C is 1.0 mL/sample volume (mL) to be detected, and r is 0.9998;
g. the linear range of norclozapine is: 20-2000ng/mL, wherein the linear equation is that y is 0.0078X-0.197, C is X is 1.0 mL/sample volume (mL), and r is 0.9997;
h. the linear range of clozapine is: 20-2000ng/mL, linear equation is that y is 0.0108X-0.1535, C is 1.0 mL/sample volume (mL),
r＝0.9999；
i. the linear range of quetiapine is: 10-1000ng/mL, linear equation is y ═ 0.013X-0.0848, C ═ X1.0 mL/sample volume to be measured (mL),
r＝1.0000；
j. the linear range of dehydroaripiprazole is: 20-2000ng/mL, wherein the linear equation is that y is 0.0024X-0.0332, C is 1.0 mL/sample volume (mL), and r is 0.9991;
k. the linear range of aripiprazole is: 20-2000ng/mL, linear equation is y is 0.003X-0.0447, C is X1.0 mL/sample volume (mL),
r＝0.9996；
the linear range of chlorpromazine is: 10-500ng/mL, linear equation is that y is 0.0058X-0.0473, C is 1.0 mL/sample volume (mL),
r＝0.9998；
the standard curves are shown in FIGS. 3 a-k;
s41: establishing a standard curve by using the concentration of the linear working solution in the hair and the ratio of the corresponding peak area to the internal standard area, and calculating a r value by using a correlation coefficient correl function in Excel, wherein the r value is calculated in the specific example:
a. the linear range of olanzapine is: 0.25-12.5 mug/mg, linear equation is y-0.1831 x-0.0812; c ═ x 20/hair weight to be measured (mg), r ═ 0.9991;
b. the linear range of amisulpride is: 1-50 μ g/mg, linear equation y ═ 0.0524x + 0.0143; c ═ x 20/hair weight to be measured (mg), r ═ 0.9997;
c. the linear range of mirtazapine is: 0.25-12.5 mug/mg, linear equation is y ═ 0.0299 x-0.0039; c ═ x 20/hair weight to be measured (mg), r ═ 0.9992;
d.9-OH Risperidone has a linear range of: 0.25-12.5 mug/mg, linear equation is that y is 0.0283x + 0.0014; c ═ x 20/hair weight to be measured (mg), r ═ 1.0000;
e. the linear range of trazodone is: 0.5-25 mug/mg, linear equation is y ═ 0.085x + 0.0049; c ═ x 20/hair weight to be measured (mg); r is 0.9997;
f. the linear range of risperidone is: 0.25-12.5 mug/mg, linear equation is y ═ 0.0406x-0.0122, C ═ x 20/hair weight to be measured (mg); r is 0.9998;
g. the linear range of norclozapine is: 1-50 mug/mg, linear equation is y-0.1530 x-0.0602, C-x 20/hair weight to be measured (mg), r-0.9998;
h. the linear range of clozapine is: 1-50 mug/mg, linear equation is y-0.1882 x-0.0374; c ═ x 20/hair weight to be measured (mg), r ═ 1.0000;
i. the linear range of quetiapine is: 0.5-25 mug/mg, linear equation is that y is 0.2328 x-0.0316; c ═ x 20/hair weight to be measured (mg), r ═ 0.9998;
j. the linear range of dehydroaripiprazole is: 1-50 mug/mg, and the linear equation is that y is 0.0448 x-0.0076; c ═ x 20/hair weight to be measured (mg), r ═ 0.9999;
k. the linear range of aripiprazole is: 1-50 μ g/mg, linear equation y-0.0617 x-0.0069; c ═ x 20/hair weight to be measured (mg), r ═ 0.9996;
the linear range of chlorpromazine is: 0.5-25 mug/mg, linear equation is y ═ 0.1112 x-0.0141; c ═ x 20/hair weight to be measured (mg), r ═ 0.9987;
the standard curves are shown in FIGS. 4 a-k;
EXAMPLE 2 detection of serum samples from patients taking clozapine and quetiapine fumarate
The chromatographic conditions of S1 and the preparation method of the S22 mixed standard solution are the same as those of example 1;
s231 serum test solution preparation: respectively and precisely absorbing 20 mu L of each of the internal standard 1-metoclopramide working solution and the internal standard 2-carbamazepine working solution prepared by S21, mixing the internal standard 1-metoclopramide working solution and 500 mu L of the melted frozen serum sample to be detected into a 7mL centrifugal tube after drying nitrogen, performing primary vortex for 1min, adding 200 mu L of sodium hydroxide aqueous solution with the concentration of 2mol/L and 3mL of methyl tert-butyl ether, performing secondary vortex for 3min, performing secondary vortex for 5min at the speed of 3000r/min on the centrifugal tube after vortex, drying the centrifuged supernatant nitrogen, adding 150 mu L of methanol-water solution containing 0.1 v/v% formic acid and the concentration of 15 v/v% for redissolution, performing vortex for 1min, and filtering by using a 0.25 mu m nylon filter membrane to obtain the oral liquid;
s3 assay: injecting the sample solution into ultra high performance liquid chromatograph, performing gradient elution procedure (same as example 1) to obtain chromatogram, and obtaining retention time (t) of the psychotropic drug and its metabolite as shown in FIG. 5R) Obtaining the product; the retention time t of the psychotropic drugs and metabolites thereofRRespectively as follows: internal standard 1 metoclopramide 4.348min, peak Area 19836; norclozapine 8.897min, peak Area 10031; clozapine 9.783min, peak Area 15801; quetiapine 11.262min, peak Area 6609; internal standard 2 carbamazepine 13.206min, peak Area 28121;
respectively substituting the peak area ratio of norclozapine, clozapine and quetiapine to the peak area ratio of internal standard 1 metoclopramide as y into the linear equation y of norclozapine of 0.0078X-0.197, and C of 1/0.5 mL; clozapine's linear equation y is 0.0108X-0.1535, C X1/0.5 mL; (ii) a The linear equation y of quetiapine is 0.013X-0.0848, and C is X1/0.5 mL; x is calculated to respectively obtain the blood concentration of the norclozapine of 180.2ng/mL, the blood concentration of the clozapine of 175.9ng/mL and the blood concentration of the quetiapine of 64.3 ng/mL.
Example 3 detection of serum samples from patients taking amisulpride
The chromatographic conditions of S1 and the preparation method of the S22 mixed standard solution are the same as those of example 1;
preparation of S231 sample solution and determination of S3 the same as in example 2:
the results are shown in FIG. 6, obtaining the retention time (t) of the psychotropic drug and its metabolitesR) Obtaining the product; retention time t of amisulprideR3.949min, peak Area 36114; internal standard 1 metoclopramide 4.361min, peak Area 25131; internal standard 2 carbamazepine 13.218min, peak Area 39927. The peak area ratio of the amisulpride/the peak area ratio of the internal standard 1 metoclopramide is calculated as y, and the linear equation y is substituted into 0.0026X +0.0023, and C is substituted into X1/0.5 mL, so that the amisulpride blood concentration C is calculated to be 1103.6 ng/mL.
EXAMPLE 4 testing of serum samples from patients taking the drug clozapine
The chromatographic conditions of S1, the internal standard solution of S21 and the mixed standard solution of S22 were prepared in the same manner as in example 1;
s231 preparation of test solution and S3 measurement method are the same as example 2;
as a result: as shown in FIG. 7, the retention time (t) of the psychotropic drug and its metabolites was obtainedR) Obtaining the product; internal standard 1 metoclopramide 4.354min, peak Area 23670; nor clozapine 8.896min, the peak Area is 13904; clozapine 9.769min, peak Area 32451; internal standard 2 carbamazepine 13.203min, peak Area 35948. Respectively substituting the ratios of the peak areas of norclozapine and clozapine to the peak area of internal standard 1 metoclopramide into the linear equation y of norclozapine of 0.0078X-0.197, and C of 1/0.5 mL; clozapine's linear equation y is 0.0108X-0.1535, C X1/0.5 mL; x is calculated to obtain the blood concentration of the norclozapine of 201.1ng/mL and the blood concentration of the clozapine of 282.3ng/mL respectively.
EXAMPLE 5 detection of serum samples from patients taking the drug Aripiprazole
The chromatographic conditions of S1, the internal standard solution of S21 and the mixed standard solution of S22 were prepared in the same manner as in example 1;
s231 preparation of test solution and S3 measurement method are the same as example 2;
as a result: as shown in FIG. 8, the retention time (t) of the psychotropic drug and its metabolites was obtainedR) And Area of peak (Area) to obtain; internal standard 1 metoclopramide 4.363min, peak area 19100; internal standard 2 carbamazepine 13.216min, peak area 27607; 17.614min for dehydroaripiprazole, peak area 1425; aripiprazole 18.208min, peak area 5573.
Respectively substituting the ratios of the peak areas of the dehydroaripiprazole and the aripiprazole to the peak area of the internal standard 2 carbamazepine into the linear equation y of the dehydroaripiprazole of 0.0024X-0.0332, C of X of 1/0.5mL, the linear equation y of the aripiprazole of 0.003X-0.0447 and C of X of 1/0.5mL/L, respectively calculating X to obtain the blood concentration of the dehydroaripiprazole of 70.7ng/mL and the blood concentration of the aripiprazole of 164.4 ng/mL.
EXAMPLE 6 testing of hair samples from patients taking olanzapine, trazodone, risperidone
The chromatographic conditions of S1, the internal standard solution of S21 and the mixed standard solution of S22 were prepared in the same manner as in example 1;
s231 preparation of test solution and S3 measurement method are the same as example 2;
the preparation method of the S232 hair test solution comprises the following steps:
rubbing hair at the back of a pillow of a patient by sticking the scalp, sequentially cleaning the hair with acetone and water, drying a section of hair which is close to the scalp by 1cm, cutting the hair into pieces for later use, precisely weighing 20.45mg to 7mL of hair to be measured, precisely absorbing 20 mu L of each of the internal standard 1-metoclopramide working solution and the internal standard 2-carbamazepine working solution prepared by S21, drying the hair with nitrogen, adding 200 mu L of sodium hydroxide aqueous solution with the concentration of 2mol/L, carrying out primary vortex for 1min, carrying out ultrasonic treatment for 2.5h, adding 3mL of methyl tert-butyl ether, carrying out secondary vortex for 3min, carrying out secondary vortex for 5min, drying the centrifuged centrifugal tube at the speed of 3000r/m, adding 200 mu L of methanol-aqueous solution containing 0.1 v/v% formic acid and the concentration of 15 v/v% to redissolve, vortex for 1min, filtering the hair with a 0.25 mu m nylon filter membrane, obtaining the product;
as a result: as shown in FIG. 9, the psychotropic drug and its metabolite retention time (t) was obtainedR) And peak areas (area) are: retention time (t) of internal standard 1 metoclopramideR) 4.367min, peak area 10395; 9-OH risperidone (t)R) 7.209min, peak area 122602; risperidone Retention time (t)R) 8.440min, peak area 41104; retention time (t) of quetiapineR) 11.23min, Peak area 40848, Retention time of internal standard 2 carbamazepine (t)R) 13.2,11min, peak area 20820.
Respectively substituting the ratios of the peak areas of 9-OH risperidone, risperidone and quetiapine to the peak area of the internal standard 1 metoclopramide into the hair linear equation y of 9-OH risperidone of 0.0283x + 0.0014; c ═ x 20/hair weight to be measured (mg); the hair linear equation y of risperidone is 0.0406 x-0.0122; c is 20 x/hair weight (mg) to be measured, and the hair linear equation y of quetiapine is 0.2328 x-0.0316; c ═ x 20/hair weight to be measured (mg); c, respectively calculating the drug concentration of 9-OH risperidone in the hair to be 407.54ng/mg, the drug concentration of risperidone in the hair to be 99.89ng/mg and the drug concentration of quetiapine in the hair to be 16.64 ng/mg.
Example 7 testing of hair samples from patients taking olanzapine and mirtazapine drugs
The chromatographic conditions of S1, the internal standard solution of S21 and the mixed standard solution of S22 were prepared in the same manner as in example 1; s232 preparation method of hair sample solution, the determination method of S3 is the same as that of example 6; the actual weighed mass of hair to be tested was recorded as 20.35 mg.
As a result: as shown in FIG. 10, the retention time (t) of the psychotropic drug and its metabolites was obtainedR) Obtaining the product; measured retention time t of psychotropic drug and metabolites thereofR3.200min for olanzapine respectively, and the peak area is 4120; internal standard 1 metoclopramide 4.367min, peak area 9347; mirtazapine 5.397min, peak area 49598, internal standard 2 carbamazepine 13.217min, peak area 14505. Substituting the ratio of the olanzapine peak area/internal standard 1 Ganfean peak area as y into olanzapine in hair to obtain a linear equation y of 0.1831x-0.0812, C of x 20/hair weight (mg) to be detected to obtain the olanzapine content C of 2.85ng/mg, and substituting the ratio of the mirtazapine peak area to the internal standard 1 Ganfean peak area into mirtazapine in hair to obtain a linear equation y of 0.0299 x-0.0039; and C is 20 x/hair weight (mg) to be measured, so that the mirtazapine content C in the hair is 177.60 ng/mg.
Example 8: detection of hair samples from patients taking quetiapine drugs
The chromatographic conditions of S1, the internal standard solution of S21 and the mixed standard solution of S22 were prepared in the same manner as in example 1; s232 Hair test sample solution was prepared in the same manner as in example 5; the S3 assay was the same as in example 6; the actual weighed mass of hair to be tested was recorded as 20.45 mg.
As a result: as shown in FIG. 11, the psychotropic drugs and their metabolites retention times (t) were obtainedR) Obtaining the product; measured retention time t of psychotropic drug and metabolites thereofRRespectively as internal standard 1 metoclopramide 4.367min, peak area is 10395; quetiapine 11.230min, peak area 40848; internal standard 2 carbamazepine 13.211min, peak area 20820. Substituting the ratio of the peak area of quetiapine to the peak area of internal standard 1 metoclopramide as y into a linear equation of y being 0.2328 x-0.0316; and C is x 20/the weight (mg) of the hair to be measured, and the content C of the quetiapine in the hair is 16.64 ng/mg.
Example 9: detection kit for simultaneously detecting common psychiatric drugs
A kit for quantitatively detecting a psychotropic drug or a metabolite thereof in a biological sample, comprising:
the preparation method of the mixed standard solution TDM detection tube comprises the following steps: s1 preparation of internal standard solution:
s11 internal standard 1-metoclopramide working solution: precisely absorbing a proper amount of the metoclopramide standard substance, dissolving with a small amount of methanol, diluting with methanol-water solution to constant volume to prepare metoclopramide standard substance solution, and diluting with methanol-water solution to constant volume to obtain an internal standard 1-metoclopramide working solution; further, the preparation method of the metoclopramide internal standard working solution comprises the following steps: precisely weighing 1-30 mg of metoclopramide standard substance, dissolving with 1-30 mL of methanol, then fixing the volume with 40-70% v/v methanol-water solution to prepare metoclopramide stock solution with the concentration of 0.1-0.5 mg/mL, and then diluting with 40-70% v/v methanol-water solution to 10-40 mu g/mL to obtain internal standard 1-metoclopramide working solution;
and the number of the first and second groups,
2-carbamazepine working solution as internal standard of S12: precisely absorbing a proper amount of carbamazepine standard, dissolving by using a small amount of methanol, then diluting to constant volume by using a methanol-water solution to prepare a carbamazepine standard solution, and then diluting to constant volume by using the methanol-water solution to obtain an internal standard 2-carbamazepine working solution; further, the preparation method of the carbamazepine internal standard working solution comprises the following steps: accurately weighing 1-30 mg of carbamazepine standard, dissolving with 1-30 mL of methanol, then fixing the volume with 40-70% v/v methanol-water solution to prepare a carbamazepine stock solution with the concentration of 0.1-0.5 mg/mL, and then diluting with 40-70% v/v methanol-water solution to 10-40 mu g/mL to obtain an internal standard 2-carbamazepine working solution;
s2 preparation of mixed standard solutions:
preparing S21 mixed standard mother liquor: accurately weighing a proper amount of commonly used neurology medicines or metabolites of the commonly used neurology medicines, such as amisulpride, clozapine, norclozapine, aripiprazole, dehydroaripiprazole, olanzapine, mirtazapine, risperidone, 9-OH risperidone, trazodone, chlorpromazine and quetiapine, 1-30 mg respectively, dissolving the mixed medicines with 1-30 mL of solvent, diluting the dissolved medicines with 40-70% v/v of methanol-water solution to a constant volume to prepare a mixed standard mother solution with the concentration of amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole of 400 mu g/mL, the concentration of olanzapine, mirtazapine, risperidone and 9-OH risperidone of 100 mu g/mL and the concentration of trazodone, chlorpromazine and quetiapine of 200 mu g/mL;
preparation of S22 mixed standard curve working solution: diluting the mixed standard mother liquor by using 40-70 v/v% methanol-water solution according to a certain proportion to obtain a mixed standard mother liquor containing amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole with the concentrations of 100, 50, 20, 10, 4, 2 and 1 mu g/mL respectively, the concentrations of olanzapine, mirtazapine, risperidone and 9-OH risperidone with the concentrations of 25, 12.5, 5, 2.5, 1, 0.5 and 0.25 mu g/mL respectively, and the concentrations of trazodone, chlorpromazine and quetiapine with the concentrations of 50, 25, 10, 5, 2,1 and 0.5 mu g/mL respectively;
preparation of a TDM detection tube for S23 mixed standard solution: respectively and precisely absorbing 10-40 mu L of the working solution of the quantitative mixed standard curve into a TDM detection tube, then adding 10-40 mu L of the internal standard 1-metoclopramide working solution and 10-40 mu L of the internal standard 2-carbamazepine working solution, and drying by blowing with nitrogen gas to obtain the product;
(II) a TDM detection tube for detecting sample solution, wherein the preparation method comprises the following steps: adding the internal standard solution prepared in the step S1 into a TDM detection tube, and drying by blowing nitrogen to obtain the internal standard solution;
(III) compounding solution: methanol-water solution containing 0.1 v/v% formic acid and having a concentration of 15% v/v;
(IV) the description: the specification describes methods of use thereof:
s1, preparing a standard solution: taking the mixed standard substance solution TDM detection tube, adding 500 mu L of blank serum and 3mL of methyl tert-butyl ether, performing primary vortex for 1min, then adding 200 mu L of 2mol/L sodium hydroxide aqueous solution and 3mL of methyl tert-butyl ether, performing secondary vortex, performing 3000r/min centrifugation for 5min to obtain supernatant, drying the supernatant, then adding 100-150 mu L of redissolution, and performing microfiltration membrane filtration to obtain the product;
s2, preparing a sample solution:
(1) serum sample detection solution: putting 500 mu L of serum sample into a sample solution TDM detection tube, adding 200 mu L of sodium hydroxide aqueous solution with the concentration of 2mol/L, whirling for 1min, adding 3mL of methyl tert-butyl ether, whirling for 3min, centrifuging for 5min at 3000r/min, transferring supernatant into a 2mL test tube, drying by nitrogen, adding 100-150 mu L of redissolution, and filtering by a microporous filter membrane to obtain the serum sample;
(2) hair sample detection solution: taking a 20mg hair sample into a sample solution TDM detection tube, adding 200 mu L of a 2mol/L sodium hydroxide aqueous solution, carrying out primary vortex for 1min and ultrasonic treatment for 2h, adding 3mL methyl tert-butyl ether, carrying out secondary vortex for 3min, centrifuging a centrifugal tube after vortex for 5min at 3000r/min, transferring supernatant into a 2mL test tube, carrying out nitrogen blow-drying, adding 100-150 mu L redissolution, and filtering with a microporous filter membrane to obtain the hair sample;
s3, determination method: respectively injecting 5-10 μ L of the standard solution prepared in S1 and the sample detection solution prepared in S2 into an ultra high performance liquid chromatograph, respectively obtaining mixed standard solution chromatograms and test sample solution chromatograms with 7 concentrations by adopting a gradient elution procedure, and respectively obtaining retention time t of the psychotropic drugs, the metabolite standard products and the test samplesRIntegrating peak areas, establishing a standard curve according to the concentration in the mixed standard solution and the peak area/internal standard area ratio corresponding to the concentration, and then obtaining the concentration of the test solution according to the peak area integration of the test solution and the standard curve; the gradient elution procedure is as follows: 0min 5% v/v B, 2min 10% v/v B, 4min 20% v/v B, 6min 22% v/v B, 12min 35% v/v B, 15min 36% v/v B, 18.5min 40% v/v B, 20min 50% v/v B, 21min 90% v/v B, 23min 90% v/v B, 24min 5% v/v B, and 28min 5% v/v B;
and the kit is stored at-20 to-15 ℃.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made by one skilled in the art without departing from the spirit and scope of the invention.
Claims (15)
1. A method for simultaneously and quantitatively detecting psychotropic drugs or metabolites thereof in a biological sample is characterized in that the method adopts an ultra-high performance liquid chromatography-ultraviolet detection method, and is characterized by comprising the following steps:
s1 chromatographic conditions: (1) a chromatographic column: c18A chromatographic column; (2) mobile phase: the A phase is formic acid-water solution, and the B phase is formic acid-methanolA solution; (3) the detector is an Ultraviolet (UV) detector;
preparing a working solution of S2:
preparation of internal standard solution of S21:
s211 internal standard 1-metoclopramide working solution: precisely absorbing a proper amount of the metoclopramide standard substance, dissolving with a small amount of methanol, diluting with methanol-water solution to constant volume to prepare metoclopramide standard substance solution, and diluting with methanol-water solution to constant volume to obtain an internal standard 1-metoclopramide working solution;
s212 internal standard 2-carbamazepine internal standard working solution: precisely absorbing a proper amount of carbamazepine standard, dissolving by using a small amount of methanol, then diluting to constant volume by using a methanol-water solution to prepare a carbamazepine standard solution, and then diluting to constant volume by using the methanol-water solution to obtain an internal standard 2-carbamazepine working solution;
preparation of S22 mixed standard solution:
s221 standard mixing mother liquor preparation: accurately weighing a proper amount of commonly used psychiatric drugs or metabolites of the commonly used drugs respectively, namely amisulpride, clozapine, norclozapine, aripiprazole, dehydroaripiprazole, olanzapine, mirtazapine, risperidone, 9-OH risperidone, trazodone, chlorpromazine and quetiapine, mixing, dissolving with a proper amount of solvent, and diluting with methanol-water solution to a constant volume to prepare a mixed standard mother solution; further, the preparation of the mixed standard mother solution comprises the following steps: accurately weighing a proper amount of commonly used neurologic drugs or metabolites of the commonly used neurologic drugs, such as amisulpride, clozapine, norclozapine, aripiprazole, dehydroaripiprazole, olanzapine, mirtazapine, risperidone, 9-OH risperidone, trazodone, chlorpromazine and quetiapine, 1-30 mg respectively, dissolving the mixture with 1-30 mL of solvent, diluting the solution with 40-70% v/v methanol-water solution to a constant volume to prepare a mixed standard mother solution containing amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole with the concentration of 400 mu g/mL, the concentration of olanzapine, mirtazapine, risperidone and 9-OH risperidone of 100 mu g/mL and the concentration of trazodone, chlorpromazine and quetiapine of 200 mu g/mL;
s222, preparation of a quantitative mixed standard curve working solution: diluting the mixed standard mother liquor by using 40-70% v/v methanol-water solution with a certain concentration according to a certain proportion, and respectively preparing 7 standard curve solutions containing the medicines, wherein the solutions respectively contain amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole with the concentrations of 100, 50, 20, 10, 4, 2 and 1 mu g/mL, contain olanzapine, mirtazapine, risperidone and 9-OH risperidone with the concentrations of 25, 12.5, 5, 2.5, 1, 0.5 and 0.25 mu g/mL, and contain trazodone, chlorpromazine and quetiapine with the concentrations of 50, 25, 10, 5, 2,1 and 0.5 mu g/mL;
preparation of S223 mixed standard solution: respectively and precisely absorbing 10-40 mu L of the quantitative mixed standard curve working solution, precisely adding 10-40 mu L of each of the internal standard 1-metoclopramide working solution and the internal standard 2-carbamazepine working solution, drying, adding a proper amount of blank blood, plasma, serum or saliva and methyl tert-butyl ether, performing primary vortex, adding 100-500 mu L of a sodium hydroxide aqueous solution with the concentration of 1-3mol/L and methyl tert-butyl ether, performing secondary vortex and centrifugation to obtain a supernatant, drying the supernatant, and adding a methanol-aqueous solution containing formic acid for redissolving, vortex and filtering to obtain the compound preparation;
the preparation of the test solution of S23 includes:
s231 preparation of blood, plasma, serum or saliva test solutions: respectively and precisely absorbing 10-40 mu L of each of the internal standard 1-metoclopramide working solution and the internal standard 2-carbamazepine working solution prepared by S21, drying, mixing with a proper amount of blood, plasma, serum or saliva to be detected, carrying out primary vortex, adding 100-500 mu L of sodium hydroxide aqueous solution and methyl tert-butyl ether for secondary vortex, carrying out centrifugation after vortex to obtain supernatant, drying the supernatant, adding methanol-aqueous solution containing formic acid for redissolving, vortex and filtering to obtain the oral liquid;
or S232 preparation of hair test solution: respectively and precisely absorbing 10-40 mu L of each of the internal standard 1-metoclopramide working solution and the internal standard 2-carbamazepine working solution prepared by S21, drying, mixing with a proper amount of hair sample to be detected, carrying out primary vortex, adding 100-500 mu L of sodium hydroxide aqueous solution, carrying out ultrasonic treatment or standing, adding methyl tert-butyl ether for secondary vortex, carrying out centrifugation after vortex to obtain supernatant, drying the supernatant, adding methanol-aqueous solution containing formic acid for redissolving, vortex, and filtering to obtain the product;
s3 assay: respectively injecting the mixed standard solution prepared in S22 and the test solution prepared in S23 into an ultra high performance liquid chromatograph, respectively obtaining mixed standard solution chromatograms with 7 concentrations and a test solution chromatogram by adopting a gradient elution procedure, and respectively obtaining the retention time t of the psychotropic drugs, the metabolite standard products thereof and the test solutionsRAnd integrating peak areas, establishing a standard curve according to the concentration in the mixed standard solution and the peak area/internal standard area ratio corresponding to the concentration, and then obtaining the concentration of the test solution according to the peak area integration of the test solution and the standard curve.
2. The method for simultaneously and quantitatively detecting the psychotropic drugs or the metabolites thereof in the biological samples according to claim 1, wherein the biological samples are selected from any one or more of blood, plasma, serum, saliva and hair; further preferably, the biological sample is selected from any one or more of plasma, serum and hair.
3. The method for simultaneously and quantitatively detecting psychotropic drugs or metabolites thereof in biological samples according to claim 1, wherein in the chromatographic conditions of S1: mobile phase: phase A is 0.05 v/v% formic acid-water solution; and phase B was 0.05 v/v% formic acid-methanol solution.
4. The method for simultaneously and quantitatively detecting psychotropic drugs or metabolites thereof in biological samples according to claim 1, wherein in the chromatographic conditions of S1: the ultraviolet detector is a VWD or DAD detector; further preferably, the detection wavelength of the ultraviolet detector is 254nm, 285nm or full wavelength scanning.
5. The method for simultaneously and quantitatively detecting psychotropic drugs or metabolites thereof in biological samples according to claim 1, wherein the chromatographic conditions of S1 further comprise: (4) the flow rate of the mobile phase is 0.1-0.5 mL/min; (5) the column temperature is 35-45 ℃; (6) the sampling amount of the working solution is 1.0-20.0 mu L.
6. The method for simultaneously and quantitatively detecting psychotropic drugs or metabolites thereof in a biological sample according to claim 1,
in step S21, the preparation of the internal standard solution specifically includes:
s211 metoclopramide internal standard working solution: precisely weighing 1-30 mg of metoclopramide standard substance, dissolving with 1-30 mL of methanol, then fixing the volume with 40-70% v/v methanol-water solution to prepare metoclopramide stock solution with the concentration of 0.1-0.5 mg/mL, and then diluting with 40-70% v/v methanol-water solution to 10-40 mu g/mL to obtain internal standard 1-metoclopramide working solution;
s212 carbamazepine internal standard working solution: precisely weighing 1-30 mg of carbamazepine standard, dissolving with 1-30 mL of methanol, then fixing the volume with 40-70% v/v methanol-water solution to prepare a carbamazepine stock solution with the concentration of 0.1-0.5 mg/mL, and then diluting with 40-70% v/v methanol-water solution to 1-5 mu g/mL to obtain an internal standard 2-carbamazepine working solution.
7. The method for simultaneously and quantitatively detecting psychotropic drugs or metabolites thereof in biological samples according to claim 1, wherein the preparation of the S231 blood, plasma, serum or saliva test solution comprises the following steps:
respectively and precisely absorbing 10-40 mu L of each of an internal standard 1-metoclopramide working solution and an internal standard 2-carbamazepine working solution prepared by S21, drying by blowing nitrogen, mixing with 400-1000 mu L of a melted frozen blood, plasma, serum or saliva sample to be detected into a 7-10 mL centrifuge tube, carrying out primary vortex for 0.5-2 min, adding 100-500 mu L of a sodium hydroxide aqueous solution with the concentration of 1-3mol/L and 2-7 mL of methyl tert-butyl ether, carrying out secondary vortex for 2-5 min, carrying out secondary vortex for 4-10 min on the centrifuged centrifuge tube at the speed of 2500-5000 r/m, drying the centrifuged supernatant by nitrogen, adding 60-250 mu L of a methanol-water solution containing 0.05-0.15 v/v% formic acid and with the concentration of 10-20 v/v%, carrying out redissolving, carrying out vortex for 0.5-2 min, and filtering by a nylon filter membrane to obtain the oral liquid.
8. The method for simultaneously and quantitatively detecting psychotropic drugs or metabolites thereof in biological samples according to claim 1, wherein the preparation of the S232 hair test solution comprises the steps of: sequentially cleaning hair to be tested with acetone and water, drying, shearing, respectively and precisely absorbing 10-40 mu L of each of the internal standard 1-metoclopramide working solution and the internal standard 2-carbamazepine working solution prepared by S21, mixing with 10-50 mg of hair sample to be tested into a 7-10 mL centrifuge tube after drying with nitrogen, adding 100-500 mu L of sodium hydroxide solution with the concentration of 1-3mol/L, performing primary vortex for 0.5-2 min, performing ultrasonic treatment for 1-4 h, adding 2-7 mL of methyl tert-butyl ether, performing secondary vortex for 2-5 min, re-dissolving the centrifuge tube after vortex at the speed of 2500-5000 r/m for 4-10 min, drying supernatant after centrifugation with nitrogen, adding 60-250 mu L of methanol-water solution containing 0.05-0.15 v/v% formic acid and having the concentration of 10-20 v/v%, performing vortex for 0.5-2 min, filtering with nylon filter membrane.
9. The method for simultaneously and quantitatively detecting psychotropic drugs or metabolites thereof in a biological sample according to claim 1, wherein said gradient elution procedure in said S3 assay is: 0min 5% v/v B, 2min 10% v/v B, 4min 20% v/v B, 6min 22% v/v B, 12min 35% v/v B, 15min 36% v/v B, 18.5min 40% v/v B, 20min 50% v/v B, 21min 90% v/v B, 23min 90% v/v B, 24min 5% v/v B, and 28min 5% v/v B.
10. A kit for quantitatively detecting a psychotropic drug or a metabolite thereof in a biological sample, comprising:
the preparation method of the mixed standard solution TDM detection tube comprises the following steps: adding the internal standard solution and the mixed standard solution into a TDM detection tube, and drying to obtain the product;
(II) a TDM detection tube for detecting sample solution, wherein the preparation method comprises the following steps: adding an internal standard solution into the TDM detection tube, and drying to obtain the TDM detection tube;
(III) compounding solution: methanol-water solution containing formic acid; preferably, the complex solution is a methanol-water solution containing 0.05-0.15 v/v% formic acid and having a concentration of 10-20% v/v;
(IV) the description: the instructions describe methods of use of the kit;
and the kit is stored at-20 to-15 ℃.
11. The kit according to claim 10, wherein the biological sample is selected from any one or more of blood, plasma, serum, saliva and hair; further preferably, the biological sample is selected from any one or more of plasma, serum and hair.
12. The kit according to claim 10, wherein the method for preparing the mixed standard solution TDM detection tube comprises the following steps:
s1 preparation of internal standard solution:
s11 internal standard 1-metoclopramide internal standard working solution: precisely absorbing a proper amount of the metoclopramide standard substance, dissolving with a small amount of methanol, diluting with methanol-water solution to constant volume to prepare metoclopramide standard substance solution, and diluting with methanol-water solution to constant volume to obtain an internal standard 1-metoclopramide working solution; further, the preparation method of the metoclopramide internal standard working solution comprises the following steps: precisely weighing 1-30 mg of metoclopramide standard substance, dissolving with 1-30 mL of methanol, then fixing the volume with 40-70% v/v methanol-water solution to prepare metoclopramide stock solution with the concentration of 0.1-0.5 mg/mL, and then diluting with 40-70% v/v methanol-water solution to 10-40 mu g/mL to obtain internal standard 1-metoclopramide working solution;
and the number of the first and second groups,
2-carbamazepine internal standard working solution of S12: precisely absorbing a proper amount of carbamazepine standard, dissolving by using a small amount of methanol, then diluting to constant volume by using a methanol-water solution to prepare a carbamazepine standard solution, and then diluting to constant volume by using the methanol-water solution to obtain an internal standard 2-carbamazepine working solution; further, the preparation method of the carbamazepine internal standard working solution comprises the following steps: accurately weighing 1-30 mg of carbamazepine standard, dissolving with 1-30 mL of methanol, then fixing the volume with 40-70% v/v methanol-water solution to prepare a carbamazepine stock solution with the concentration of 0.1-0.5 mg/mL, and then diluting with 40-70% v/v methanol-water solution to 10-40 mu g/mL to obtain an internal standard 2-carbamazepine working solution;
s2 preparation of mixed standard solution TDM detection tubes:
preparing S21 mixed standard mother liquor: accurately weighing a proper amount of commonly used neurologic drugs or metabolites of the commonly used neurologic drugs, such as amisulpride, clozapine, norclozapine, aripiprazole, dehydroaripiprazole, olanzapine, mirtazapine, risperidone, 9-OH risperidone, trazodone, chlorpromazine, quetiapine and 1-30 mg respectively, dissolving the mixture with 1-30 mL of solvent, diluting the solution with 40-70% v/v methanol-water solution to a constant volume to prepare a mixed standard mother solution containing the amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole at 400 mu g/mL, the content of the olanzapine, mirtazapine, risperidone and 9-OH risperidone is 100 mu g/mL, and the content of the trazodone, chlorpromazine and quetiapine is 200 mu g/mL;
preparation of S22 mixed standard curve working solution: diluting the mixed standard mother liquor by using 40-70 v/v% methanol-water solution according to a certain proportion to obtain a mixed standard mother liquor containing amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole with the concentrations of 100, 50, 20, 10, 4, 2 and 1 mu g/mL respectively, the concentrations of olanzapine, mirtazapine, risperidone and 9-OH risperidone with the concentrations of 25, 12.5, 5, 2.5, 1, 0.5 and 0.25 mu g/mL respectively, and the concentrations of trazodone, chlorpromazine and quetiapine with the concentrations of 50, 25, 10, 5, 2,1 and 0.5 mu g/mL respectively;
preparation of a TDM detection tube for S23 mixed standard solution: respectively and precisely absorbing 10-40 mu L of the working solution of the quantitative mixed standard curve into a TDM detection tube, then adding 10-40 mu L of each of the internal standard 1-metoclopramide working solution and the internal standard 2-carbamazepine working solution, and drying by blowing with nitrogen gas to obtain the product.
13. The kit according to claim 10, wherein the method for preparing the TDM detection tube for the sample solution comprises the following steps: and (4) respectively adding 10-40 mu L of the internal standard 1-metoclopramide working solution and the internal standard 2-carbamazepine working solution prepared in the step S1 into a TDM detection tube, and drying by using nitrogen to obtain the product.
14. The kit of claim 10, wherein the method of use comprises:
s1, preparing a standard solution: taking the mixed standard solution TDM detection tube, adding 100-1000 [ s1] mu L of blank blood, plasma, serum or saliva and 2-7 mL of methyl tert-butyl ether, performing primary vortex for 1-5 min, then adding 100-500 mu L of sodium hydroxide aqueous solution with the concentration of 1-3mol/L and 2-7 mL of methyl tert-butyl ether, performing secondary vortex for 4-10 min at the speed of 2500-5000 r/min, obtaining supernatant, drying the supernatant, then adding 100-150 mu L of redissolution, and performing microfiltration membrane filtration to obtain the product;
s2, preparing a sample detection solution:
(1) serum sample detection solution: placing 100-1000 mu L of serum sample into the sample solution TDM detection tube, adding 100-500 mu L of sodium hydroxide aqueous solution with the concentration of 1-3mol/L, performing vortex for 0.5-2 min, adding 2-7 mL of methyl tert-butyl ether, performing vortex for 1-5 min, performing centrifugation for 4-10 min at the speed of 2500-5000 r/min, transferring supernatant into a test tube, performing nitrogen blow drying, adding 100-150 mu L of redissolution, and performing microfiltration membrane filtration to obtain the serum sample;
or (2) a hair sample detection solution: taking a 20mg hair sample into the sample solution TDM detection tube, adding 100-500 mu L of a sodium hydroxide aqueous solution with the concentration of 1-3mol/L, carrying out primary vortex for 0.5-2 min, then carrying out ultrasonic treatment for 1-4 h, adding 2-7 mL of methyl tert-butyl ether, carrying out secondary vortex for 2-5 min, carrying out centrifugal tube after vortex for 4-10 min at the speed of 2500-5000 r/separation center, transferring supernatant into a test tube, carrying out nitrogen blow drying, adding 100-150 mu L of redissolution, and filtering with a microporous filter membrane to obtain the hair dye;
s3, determination method: respectively injecting 5-10 μ L of the standard solution prepared in S1 and the sample detection solution prepared in S2 into an ultra high performance liquid chromatograph, respectively obtaining mixed standard solution chromatograms and test sample solution chromatograms with 7 concentrations by adopting a gradient elution procedure, and respectively obtaining retention time t of the psychotropic drugs, the metabolite standard products and the test samplesRIntegrating peak areas, and establishing a standard curve by using the concentration in the mixed standard substance solution and the corresponding peak area/internal standard area ratioThen, obtaining the concentration of the test solution according to the peak area integral of the test solution and the standard curve; the gradient elution procedure is as follows: 0min 5% v/v B, 2min 10% v/v B, 4min 20% v/v B, 6min 22% v/v B, 12min 35% v/v B, 15min 36% v/v B, 18.5min 40% v/v B, 20min 50% v/v B, 21min 90% v/v B, 23min 90% v/v B, 24min 5% v/v B, and 28min 5% v/v B.
15. Use of the kit according to any one of claims 10 to 14 for the preparation of a test agent for the quantitative detection of psychotropic drugs or metabolites thereof in a biological sample.
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