CN113917025B - Kit for quantitatively detecting psychotropic drugs in biological samples and application of kit - Google Patents
Kit for quantitatively detecting psychotropic drugs in biological samples and application of kit Download PDFInfo
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- CN113917025B CN113917025B CN202111177824.2A CN202111177824A CN113917025B CN 113917025 B CN113917025 B CN 113917025B CN 202111177824 A CN202111177824 A CN 202111177824A CN 113917025 B CN113917025 B CN 113917025B
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- 229940001470 psychoactive drug Drugs 0.000 title claims abstract description 41
- 239000004089 psychotropic agent Substances 0.000 title claims abstract description 41
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- 239000012224 working solution Substances 0.000 claims abstract description 101
- 238000001514 detection method Methods 0.000 claims abstract description 90
- 239000000126 substance Substances 0.000 claims abstract description 70
- 239000002207 metabolite Substances 0.000 claims abstract description 48
- 239000003814 drug Substances 0.000 claims abstract description 38
- 238000000034 method Methods 0.000 claims abstract description 37
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- 238000012360 testing method Methods 0.000 claims abstract description 21
- 238000003556 assay Methods 0.000 claims abstract description 13
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- 238000000825 ultraviolet detection Methods 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 75
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- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 40
- 229960004431 quetiapine Drugs 0.000 claims description 40
- URKOMYMAXPYINW-UHFFFAOYSA-N quetiapine Chemical compound C1CN(CCOCCO)CCN1C1=NC2=CC=CC=C2SC2=CC=CC=C12 URKOMYMAXPYINW-UHFFFAOYSA-N 0.000 claims description 40
- 239000007864 aqueous solution Substances 0.000 claims description 39
- 229960004170 clozapine Drugs 0.000 claims description 39
- 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
- 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 36
- 239000000523 sample Substances 0.000 claims description 36
- 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
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 claims description 34
- 238000007865 diluting Methods 0.000 claims description 34
- 229960005017 olanzapine Drugs 0.000 claims description 34
- KVWDHTXUZHCGIO-UHFFFAOYSA-N olanzapine Chemical compound C1CN(C)CCN1C1=NC2=CC=CC=C2NC2=C1C=C(C)S2 KVWDHTXUZHCGIO-UHFFFAOYSA-N 0.000 claims description 34
- 239000012488 sample solution Substances 0.000 claims description 34
- 229960001785 mirtazapine Drugs 0.000 claims description 33
- RONZAEMNMFQXRA-UHFFFAOYSA-N mirtazapine Chemical compound C1C2=CC=CN=C2N2CCN(C)CC2C2=CC=CC=C21 RONZAEMNMFQXRA-UHFFFAOYSA-N 0.000 claims description 33
- 229960001534 risperidone Drugs 0.000 claims description 33
- RAPZEAPATHNIPO-UHFFFAOYSA-N risperidone Chemical compound FC1=CC=C2C(C3CCN(CC3)CCC=3C(=O)N4CCCCC4=NC=3C)=NOC2=C1 RAPZEAPATHNIPO-UHFFFAOYSA-N 0.000 claims description 33
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- 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 32
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- NTJOBXMMWNYJFB-UHFFFAOYSA-N amisulpride Chemical compound CCN1CCCC1CNC(=O)C1=CC(S(=O)(=O)CC)=C(N)C=C1OC NTJOBXMMWNYJFB-UHFFFAOYSA-N 0.000 claims description 32
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- 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 31
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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 29
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- 229960003991 trazodone Drugs 0.000 claims description 28
- PHLBKPHSAVXXEF-UHFFFAOYSA-N trazodone Chemical compound ClC1=CC=CC(N2CCN(CCCN3C(N4C=CC=CC4=N3)=O)CC2)=C1 PHLBKPHSAVXXEF-UHFFFAOYSA-N 0.000 claims description 28
- 229960001076 chlorpromazine Drugs 0.000 claims description 25
- ZPEIMTDSQAKGNT-UHFFFAOYSA-N chlorpromazine Chemical compound C1=C(Cl)C=C2N(CCCN(C)C)C3=CC=CC=C3SC2=C1 ZPEIMTDSQAKGNT-UHFFFAOYSA-N 0.000 claims description 25
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- SVWLIIFHXFGESG-UHFFFAOYSA-N formic acid;methanol Chemical group OC.OC=O SVWLIIFHXFGESG-UHFFFAOYSA-N 0.000 claims description 5
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- ABFPKTQEQNICFT-UHFFFAOYSA-M 2-chloro-1-methylpyridin-1-ium;iodide Chemical compound [I-].C[N+]1=CC=CC=C1Cl ABFPKTQEQNICFT-UHFFFAOYSA-M 0.000 description 3
- 229940005529 antipsychotics Drugs 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 229960005197 quetiapine fumarate Drugs 0.000 description 3
- GBBSUAFBMRNDJC-MRXNPFEDSA-N (5R)-zopiclone Chemical compound C1CN(C)CCN1C(=O)O[C@@H]1C2=NC=CN=C2C(=O)N1C1=CC=C(Cl)C=N1 GBBSUAFBMRNDJC-MRXNPFEDSA-N 0.000 description 2
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- 238000012544 monitoring process Methods 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 230000004799 sedative–hypnotic effect Effects 0.000 description 2
- 238000004885 tandem mass spectrometry Methods 0.000 description 2
- 229940126585 therapeutic drug Drugs 0.000 description 2
- 229960000820 zopiclone Drugs 0.000 description 2
- AKUVRZKNLXYTJX-UHFFFAOYSA-N 3-benzylazetidine Chemical compound C=1C=CC=CC=1CC1CNC1 AKUVRZKNLXYTJX-UHFFFAOYSA-N 0.000 description 1
- 101710179738 6,7-dimethyl-8-ribityllumazine synthase 1 Proteins 0.000 description 1
- 101710179734 6,7-dimethyl-8-ribityllumazine synthase 2 Proteins 0.000 description 1
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- 206010012239 Delusion Diseases 0.000 description 1
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- 101710122908 Lipoyl synthase 2, chloroplastic Proteins 0.000 description 1
- 101710101072 Lipoyl synthase 2, mitochondrial Proteins 0.000 description 1
- 208000020114 Schizophrenia and other psychotic disease Diseases 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- VREFGVBLTWBCJP-UHFFFAOYSA-N alprazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NN=C2CN=C1C1=CC=CC=C1 VREFGVBLTWBCJP-UHFFFAOYSA-N 0.000 description 1
- 229960004538 alprazolam Drugs 0.000 description 1
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- 229960001657 chlorpromazine hydrochloride Drugs 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 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
- 229960003120 clonazepam Drugs 0.000 description 1
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- 231100000868 delusion Toxicity 0.000 description 1
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- 229960002301 trazodone hydrochloride Drugs 0.000 description 1
- OHHDIOKRWWOXMT-UHFFFAOYSA-N trazodone hydrochloride Chemical compound [H+].[Cl-].ClC1=CC=CC(N2CCN(CCCN3C(N4C=CC=CC4=N3)=O)CC2)=C1 OHHDIOKRWWOXMT-UHFFFAOYSA-N 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
Classifications
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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
-
- 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
-
- 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
-
- 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
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
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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
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
-
- 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
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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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
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention provides a method for simultaneously and quantitatively detecting psychotropic drugs or metabolites thereof in biological samples by adopting a UPLC-UV detection method, which comprises the following steps: s1 chromatographic conditions; s2, preparing a working solution; s3 assay. Respectively obtaining retention time t R and peak area integral of the psychotropic drug and a metabolite standard substance thereof and a test substance, establishing a standard curve of the concentration in the mixed standard substance solution and the corresponding peak area/internal standard area ratio thereof, and obtaining the concentration of the test substance solution according to the peak area integral of the test substance solution and the standard curve; the detection kit prepared by the method is also disclosed; the invention adopts UPLC-UV method to realize separation of the common mental medicine in biological sample, and prepares the common mental medicine into a kit, which can rapidly, simply, conveniently and accurately quantitatively detect the in vivo concentration of the common clinical mental medicine, and has good academic and commercial value.
Description
Technical Field
The invention relates to the technical field of psychiatric drug detection kits, in particular to a kit for quantitatively detecting psychotropic drugs in biological samples and application thereof.
Background
Antipsychotics (Antipsychotic drugs), also known as neuroleptics or nerve blockers (Neuroleptic), are a group of drugs used in the treatment of schizophrenia and other psychotic disorders. The traditional therapeutic dose does not affect the intelligence and consciousness of the patient, but can effectively control the mental symptoms of mental motion excitation, illusion, delusions, hostile emotion, thinking disorder, abnormal behavior and the like of the patient. The patients usually have sleep disorder, and often take the tranquilization medicines at the same time, so that the two medicines are required to be separated during detection to obtain a stable blood concentration detection value of the antipsychotics, and the interference of the rest clinical combined medicines is avoided. The clinical therapeutic drugs commonly used for psychiatric patients are: olanzapine, 9-OH risperidone, clozapine, norclozapine, risperidone, quetiapine fumarate, chlorpromazine hydrochloride, trazodone hydrochloride, mirtazapine, amisulpride, aripiprazole and dehydroaripiprazole are used together, and sedative hypnotics such as alprazolam, clonazepam, zopiclone and esmolam are usually used together for matched treatment, if the detection method cannot realize simultaneous separation of the combined drugs, the concentration of the psychotropic drugs is not accurate, so that the simultaneous realization of on-line separation of sedative hypnotics and antipsychotics has 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 administered drug therapy. The method for monitoring the clinical blood concentration in China at the present stage comprises the following steps: there are mainly High Performance Liquid Chromatography (HPLC), radioimmunoassay (RIA), gas Chromatography (GC), liquid tandem mass spectrometry (LC-MS), and the like. Since clinical patients often take a plurality of mental basic disease drugs at the same time, the detection methods of the drugs with different properties are generally different, and the detection devices used are different. Therefore, when in detection, equipment is required to be continuously switched or mobile phase is required to be replaced, and an instrument is balanced after the mobile phase is replaced, 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 medicine, is easy to interfere, and affects the quantitative result; the high performance liquid chromatography tandem mass spectrometry uses a mass spectrometer as a detection means, integrates high separation capacity of the high performance liquid chromatograph, high sensitivity and high selectivity of the mass spectrometer into a whole, and can accurately quantify the mass spectrum as the detection means, but on one hand, equipment is expensive, and on the other hand, isotope internal standard consumable is very expensive and is not friendly to the environment. Therefore, a detection method for simultaneously realizing online separation of a plurality of psychiatric drugs with short separation time and good effect is needed in the field, and the preparation of the detection method into a kit is beneficial to clinical detection.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention adopts the following technical scheme:
the first aspect of the present invention provides a method for simultaneously and quantitatively detecting a psychotropic drug or a metabolite thereof in a biological sample, wherein the method adopts an ultra-high performance liquid chromatography-ultraviolet detection method, and the method comprises the following steps:
S1 chromatographic conditions: (1) chromatography column: a C 18 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;
s2, preparing a working solution:
preparation of S21 internal standard solution:
S211, recovering the internal standard working solution of the gastric recovery: precisely absorbing a proper amount of the gastric re-installation standard substance, dissolving with a small amount of methanol, then fixing the volume with a methanol-water solution to prepare a gastric re-installation standard substance solution, and then diluting with a methanol-water solution to fix the volume to obtain an internal standard 1-gastric re-installation working solution;
s212 carbamazepine internal standard working solution: precisely absorbing a proper amount of carbamazepine standard substance, dissolving with a small amount of methanol, then fixing the volume with a methanol-water solution, preparing Cheng Kama's standard substance solution, and then diluting with the methanol-water solution to the fixed volume to obtain an internal standard 2-carbamazepine working solution;
s22, preparation of mixed standard solution:
S221, preparing a mixed standard mother solution: respectively precisely weighing a proper amount of common medicines in psychiatric department or metabolites such as 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 to constant volume with methanol-water solution to prepare mixed standard mother liquor; further, the preparation of the mixed standard mother solution comprises the following steps: respectively precisely weighing a proper amount of common medicines of the Shenke or metabolites thereof, namely amisulpride, clozapine, norclozapine, aripiprazole, dehydroaripiprazole, olanzapine, mirtazapine, risperidone, 9-OH risperidone, trazodone, chlorpromazine and quetiapine, 1-30 mg, mixing, dissolving with 1-30 mL of solvent, diluting with 40-70% v/v methanol-water solution to constant volume to prepare mixed standard mother liquor containing amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole with the concentration of 400 mug/mL, containing olanzapine, mirtazapine, risperidone and 9-OH risperidone with the concentration of 100 mug/mL, containing trazodone, chlorpromazine and quetiapine with the concentration of 200 mug/mL;
S222, preparing a quantitative mixed standard curve working solution: diluting the mixed standard mother solution with 40-70% v/v methanol-water solution according to a certain proportion to prepare 7 standard curve solutions of the medicines, wherein the solutions respectively contain amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole, the concentrations of the amipiprazole are 100, 50, 20, 10, 4, 2 and 1 mug/mL, the concentrations of olanzapine, mirtazapine, risperidone and 9-OH risperidone are 25, 12.5, 5, 2.5, 1, 0.5 and 0.25 mug/mL, and the concentrations of the trazodone, chlorpromazine and quetiapine are 50, 25, 10, 5, 2 and 1 and 0.5 mug/mL;
S223, preparation of mixed standard substance solution: respectively precisely sucking 10-40 mu L of the quantitative mixed standard curve working solution, precisely adding 10-40 mu L of the internal standard 1-gastric reset working solution and 10-40 mu L of the internal standard 2-carbamazepine working solution, drying, adding a proper amount of blank blood, plasma, serum or saliva and methyl tertiary butyl ether, performing first vortex, adding 100-500 mu L of 1-3mol/L sodium hydroxide aqueous solution and methyl tertiary butyl ether, performing second vortex and centrifugation to obtain a supernatant, drying the supernatant, and then adding methanol-aqueous solution containing formic acid for re-dissolution, vortex and filtration to obtain the finished product;
the preparation of the S23 test solution comprises the following steps:
S231 preparation of a blood, plasma, serum or saliva test sample solution: respectively precisely sucking 10-40 mu L of the internal standard 1-gastric reset working solution and 10-40 mu L of the internal standard 2-carbamazepine working solution prepared in S21, drying, mixing with a proper amount of blood, plasma, serum or saliva to be tested, performing first vortex, adding 100-500 mu L of sodium hydroxide aqueous solution and methyl tertiary butyl ether for performing second vortex, centrifuging after vortex to obtain a supernatant, drying the supernatant, adding methanol-aqueous solution containing formic acid for redissolution, vortex and filtering to obtain the final product;
Or S232 preparation of hair test sample solution: respectively precisely sucking 10-40 mu L of the internal standard 1-stomach compound safety working solution and 10-40 mu L of the internal standard 2-carbamazepine working solution prepared in S21, drying, mixing with a proper amount of hair sample to be tested, carrying out first vortex, adding 100-500 mu L of sodium hydroxide aqueous solution, carrying out ultrasonic treatment or standing, adding methyl tertiary butyl ether for carrying out second vortex, centrifuging after vortex to obtain a supernatant, drying the supernatant, adding methanol-aqueous solution containing formic acid, carrying out redissolution, vortex and filtering to obtain the hair sample;
S3 assay: injecting the mixed standard solution prepared in the step S22 and the sample solution prepared in the step S23 into an ultra-high performance liquid chromatograph respectively, adopting a gradient elution program to obtain 7 concentration mixed standard solution chromatograms and sample solution chromatograms respectively, respectively obtaining retention time t R and peak area integral of the psychotropic drug and the metabolite standard and the sample thereof, establishing a standard curve of the concentration in the mixed standard solution and the corresponding peak area/internal standard area ratio thereof, and then obtaining the concentration of the sample solution according to the peak area integral of the sample 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 blood plasma, blood serum and hair;
further, in the chromatographic conditions described in S1: mobile phase: phase A is 0.05v/v% formic acid-water solution; and phase B is 0.05v/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 scan;
Still 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 ℃; the sample injection amount of the working solution is 1.0-20.0 mu L;
Further, in step S21, the preparation of the internal standard solution specifically includes:
s211, recovering the internal standard working solution of the gastric recovery: precisely weighing 1-30 mg of the standard substance for the gastric recovery, dissolving with 1-30 mL of methanol, then fixing the volume with 40-70% v/v of methanol-water solution to prepare a stock solution for the gastric recovery with the concentration of 0.1-0.5 mg/mL, and then diluting with 40-70% v/v of methanol-water solution to 10-40 mug/mL to obtain an internal standard 1-gastric recovery working solution;
S212 carbamazepine internal standard working solution: precisely weighing 1-30 mg carbamazepine standard, dissolving with 1-30 mL of methanol, then fixing the volume with 40-70% v/v of 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 of methanol-water solution to 1-5 mug/mL to obtain an internal standard 2-carbamazepine working solution;
Further, the preparation of the S231 blood, plasma, serum or saliva test sample solution comprises the following steps: respectively precisely sucking 10-40 mu L of the internal standard 1-gastric reset working solution and 10-40 mu L of the internal standard 2-carbamazepine working solution prepared in S21, drying by nitrogen, mixing with 400-1000 mu L of melted frozen blood, plasma, serum or saliva samples to be tested into a 7-10 mL centrifuge tube, carrying out first vortex for 0.5-2 min, adding 100-500 mu L of 1-3 mol/L sodium hydroxide aqueous solution and 2-7 mL of methyl tertiary butyl ether, carrying out second vortex for 2-5 min, drying the centrifuged supernatant by nitrogen at the speed of 2500-5000 r/S for 4-10 min, adding 60-250 mu L of methanol-aqueous solution containing 0.05-0.15 v/v% formic acid and the concentration of 10-20 v/v% for redissolving, carrying out vortex for 0.5-2 min, and filtering by a nylon filter membrane to obtain the product; further preferably, the preparation of the S231 blood, plasma, serum or saliva sample solution comprises the following steps: respectively precisely sucking 20 mu L of the internal standard 1-gastric reset working solution and 20 mu L of the internal standard 2-carbamazepine working solution prepared in the step S21, drying by nitrogen, mixing with 1000 mu L of melted frozen serum sample to be tested into a 7mL centrifuge tube, performing first vortex for 1min, adding 200 mu L of 2mol/L sodium hydroxide aqueous solution, adding 3mL of methyl tertiary butyl ether, performing second vortex for 3min, performing further centrifugation for 5min, taking the supernatant after centrifugation, drying by nitrogen, adding 200 mu L of methanol-aqueous solution containing 0.1v/v% formic acid and 15% v/v, performing vortex for 1min, and performing filtration by a filter membrane to obtain the compound preparation;
Or further, the preparation of the S232 hair test sample solution comprises the following steps: sequentially cleaning hair to be tested by using acetone and water, cutting after drying, precisely sucking 10-40 mu L of an internal standard 1-gastric reset working solution and 10-50 mg of an internal standard 2-carbamazepine working solution prepared by S21 respectively, drying by nitrogen, mixing with 10-50 mg of hair sample to be tested into a 7-10 mL centrifuge tube, adding 100-500 mu L of 1-3 mol/L sodium hydroxide aqueous solution, carrying out first vortex for 0.5-2 min, carrying out ultrasonic treatment for 1-4 h, adding 2-7 mL of methyl tert-butyl ether, carrying out second vortex for 2-5 min, drying the centrifuged supernatant by nitrogen at a speed of 2500-5000 r/separation core for 4-10 min, adding 60-250 mu L of methanol-aqueous solution containing 0.05-0.15 v/v% formic acid and having a concentration of 10-20 v/v% for redissolving, carrying out vortex for 0.5-2 min, and carrying out nylon filter membrane filtration to obtain the hair-washing liquid; further preferably, the preparation of the S232 hair test solution includes the following steps: sequentially cleaning hair to be tested with acetone and water, drying, shearing, precisely sucking 20 mu L of an internal standard 1-gastric reset working solution and 20 mu L of an internal standard 2-carbamazepine working solution prepared in S21 respectively, drying with nitrogen, mixing with 20mg of hair sample to be tested into a 7mL centrifuge tube, adding 200 mu L of a sodium hydroxide aqueous solution with the concentration of 2mol/L, carrying out first vortex for 1min, carrying out ultrasonic treatment for 2h, adding 4mL of methyl tertiary butyl ether, carrying out second vortex for 3min, separating the core of the centrifuge tube at the speed of 3000 revolutions per minute for 5min, taking 200 mu L of methanol-aqueous solution with the concentration of 15v/v% and containing 0.1v/v% formic acid after drying the supernatant after centrifuging, carrying out vortex for 1min, and filtering with a nylon filter membrane to obtain the hair to be tested;
Further, the gradient elution procedure described in the 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;
In a second aspect, the present invention provides 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 reagent;
(II) a TDM detection tube for detecting the sample solution, and the preparation method comprises the following steps: adding the internal standard solution into the TDM detection tube, and drying to obtain the TDM detection tube;
(III) complex solution: a methanol-water solution containing formic acid;
(IV) instruction book: the specification describes methods of use thereof;
and the kit is stored at the temperature of minus 20 ℃ to minus 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 blood plasma, blood serum and hair;
Further, the preparation method of the mixed standard solution TDM detection tube comprises the following steps:
S1, preparing an internal standard solution, wherein the internal standard solution comprises the following steps:
S11, recovering the internal standard working solution of the gastric recovery: precisely absorbing a proper amount of the gastric re-installation standard substance, dissolving with a small amount of methanol, then fixing the volume with a methanol-water solution to prepare a gastric re-installation standard substance solution, and then diluting with a methanol-water solution to fix the volume to obtain an internal standard 1-gastric re-installation working solution; further, the preparation method of the gastroscope internal standard working solution comprises the following steps: precisely weighing 1-30 mg of the standard substance for the gastric recovery, dissolving with 1-30 mL of methanol, then fixing the volume with 40-70% v/v of methanol-water solution to prepare a stock solution for the gastric recovery with the concentration of 0.1-0.5 mg/mL, and then diluting with 40-70% v/v of methanol-water solution to 10-40 mug/mL to obtain an internal standard 1-gastric recovery working solution;
And
S12 carbamazepine internal standard working solution: precisely absorbing a proper amount of carbamazepine standard substance, dissolving with a small amount of methanol, then fixing the volume with a methanol-water solution, preparing Cheng Kama's standard substance solution, and then diluting with the methanol-water solution to the fixed volume to obtain an internal standard 2-carbamazepine working solution; further, the preparation method of the carbamazepine internal standard working solution comprises the following steps: precisely weighing 1-30 mg carbamazepine standard, dissolving with 1-30 mL of methanol, then fixing the volume with 40-70% v/v of 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 of methanol-water solution to 10-40 mug/mL to obtain an internal standard 2-carbamazepine working solution;
S2, preparing a mixed standard solution TDM detection tube:
s21, preparing a mixed standard mother solution: respectively precisely weighing a proper amount of common medicines of the Shenke or metabolites thereof, namely amisulpride, clozapine, norclozapine, aripiprazole, dehydroaripiprazole, olanzapine, mirtazapine, risperidone, 9-OH risperidone, trazodone, chlorpromazine and quetiapine, 1-30 mg of the solvent is firstly used for dissolving after mixing, 40-70% v/v methanol-water solution is used for diluting and fixing volume to prepare mixed standard mother liquor containing amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole which are 400 mu g/mL, and mixed standard mother liquor containing olanzapine, mirtazapine, risperidone and 9-OH risperidone which are 100 mu g/mL and containing trazodone, chlorpromazine and quetiapine which are 200 mu g/mL;
S22, preparing a mixed standard curve working solution: diluting the mixed standard mother solution with 40-70 v/v% methanol-water solution according to a certain proportion to obtain amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole with concentrations of 100, 50, 20, 10, 4, 2 and 1 mug/mL respectively, with olanzapine, mirtazapine, risperidone and 9-OH risperidone with solubilities of 25, 12.5, 5, 2.5, 1, 0.5 and 0.25 mug/mL respectively, and with trazodone, chlorpromazine and quetiapine with concentrations of 50, 25, 10, 5, 2, 1 and 0.5 mug/mL respectively;
S23, preparation of a TDM detection tube of the mixed standard solution: respectively precisely sucking 10-40 mu L of the quantitative mixed standard curve working solution into a TDM detection tube, then adding 10-40 mu L of the internal standard 1-stomach resetting working solution and 10-40 mu L of the internal standard 2-carbamazepine working solution respectively, and drying by nitrogen to obtain the compound;
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 with nitrogen to obtain the product;
Further, the compound 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 substance solution: taking the mixed standard solution TDM detection tube, adding 100-1000 mu s 2L blank blood, plasma, serum or saliva and 2-7 mL methyl tertiary butyl ether, swirling for 1-5 min for the first time, adding 100-500 mu L sodium hydroxide aqueous solution with concentration of 1-3 mol/L and 2-7 mL methyl tertiary butyl ether, swirling for the second time, centrifuging for 2500-5000 r/min for 4-10 min to obtain supernatant, drying the supernatant, adding 100-150 mu L complex solution, and filtering with microporous filter membrane to obtain the final 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 1-3 mol/L, swirling for 0.5-2 min, adding 2-7 mL of methyl tertiary butyl ether, swirling for 1-5 min, centrifuging for 4-10 min at 2500-5000 r/min, transferring supernatant into the tube, drying with nitrogen, adding 100-150 mu L of complex solution, and filtering with a microporous filter membrane to obtain the serum;
(2) Hair sample detection liquid: taking 20mg of hair sample into a sample solution TDM detection tube, adding 100-500 mu L of 1-3 mol/L sodium hydroxide aqueous solution, carrying out first vortex for 0.5-2 min, carrying out ultrasonic treatment for 1-4 h, adding 2-7 mL of methyl tertiary butyl ether, carrying out second vortex for 2-5 min, separating a centrifuge tube at a speed of 2500-5000 r/m for 4-10 min, transferring supernatant into the test tube, drying with nitrogen, adding 100-150 mu L of complex solution, and filtering with a microporous filter membrane to obtain the hair sample;
S3, measuring: injecting 5-10 mu L of the standard substance solution prepared in the step S1 and the sample detection solution prepared in the step S2 into an ultra-high performance liquid chromatograph respectively, adopting a gradient elution program to obtain 7 concentration mixed standard substance solution chromatograms and test substance solution chromatograms respectively, obtaining retention time t R and peak area integral of the psychotropic substance and the metabolite standard substance thereof and the test substance thereof respectively, establishing a standard curve of the concentration in the mixed standard substance solution and the corresponding peak area/internal standard area ratio thereof, and obtaining the concentration of the test substance solution according to the peak area integral of the test substance solution and the standard curve; the gradient elution program is ：0min 5％v/v B、2min 10％v/v B、4min20％v/v B、6min 22％v/v B、12min 35％v/v B、15min 36％v/v B、18.5min40％v/v B、20min 50％v/v B、21min 90％v/v B、23min 90％v/v B、24min5％v/v B and 28min 5% v/v B;
In a third aspect, the present invention provides an application of any one of the above-mentioned kits in preparing 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 the hair mix standard solution of example 1
FIG. 3 is a standard graph of each drug in the serum mixed standard solution of example 1
FIG. 4 is a graph showing the standard curve of each drug in the serum mixed standard solution of example 1
FIG. 5 is a chromatogram of a serum sample of a patient taking clozapine, quetiapine fumarate, as described in example 2
FIG. 6 is a detection chromatogram of a serum sample from a patient taking amisulpride of example 3
FIG. 7 is a detection chromatogram of serum samples from patients taking clozapine drug of example 4
FIG. 8 is a detection chromatogram of serum samples from patients taking aripiprazole drug substance of example 5
FIG. 9 is a detection chromatogram of a hair sample of a patient taking olanzapine, trazodone, risperidone drugs of example 6
FIG. 10 is a detection chromatogram of a hair sample from a patient taking olanzapine, mirtazapine drug of example 7
FIG. 11 is a detection chromatogram of a hair sample from a patient taking quetiapine drug of example 8
Advantageous effects
1. The invention adopts UPLC-UV method to realize quantitative separation of human serum or psychiatric drugs in hair in biological sample, and prepares the kit to rapidly, simply, conveniently and accurately quantitatively detect in vivo concentration of clinical psychiatric drugs;
2. According to the method, the internal standard substances are added into the standard substance solution and the sample solution to be tested, and 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 by only adopting less than 10min, and the detection time is greatly shortened;
3. The invention adopts the economical ultraviolet detector to replace mass spectrum for detection, and has obvious academic significance and economic value for guiding clinical medication and analyzing samples;
4. According to the invention, the rapid and complete separation of the sample in a short time is realized 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 regulated by adding NaOH solution, the hair matrix is digested, the medicine components in the hair matrix are extracted by methyl tertiary butyl ether, the detection limit is obviously improved, expensive solid-phase extraction consumable materials are not needed, and the chromatographic column is more durable by sample injection after filtration, so that 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 medicine in the sample can be accurately measured;
7. The specific redissolution is selected for redissolution, so that the measurement accuracy is further improved.
Detailed Description
Example 1: detection method of complete mixed standard
A method for simultaneously detecting in vivo psychotropic drugs or metabolites thereof, the method adopts an ultra-high performance liquid chromatography-ultraviolet detection method, and the method comprises the following steps:
s1 chromatographic conditions: (1) chromatography column: a C 18 column, ACQUITY UPLC BEH C 18 diameter 2.1mm 50mm, packing size 1.7 μm; (2) mobile phase: phase A is 0.05v/v% formic acid-water solution; phase B is 0.05v/v% formic acid-methanol solution; (3) The detector is an Ultraviolet (UV) detector that detects a full wavelength scan of wavelengths; (4) the mobile phase flow rate is 0.4mL/min; (5) column temperature is 40 ℃; the sample injection amount of the working solution is 10.0 mu L;
the preparation of the S2 working solution comprises the following steps:
s20, preparing a needle washing solution: measuring 700mL of water into a liquid-phase washing bottle by using a measuring cylinder, adding 300mL of methanol, mixing and carrying out ultrasonic treatment for 20min to obtain a methanol-water washing needle solution;
preparation of S21 internal standard solution:
S211, recovering the internal standard working solution of the gastric recovery: precisely weighing 20mg of the Weifuan standard substance in a 100mL volumetric flask, dissolving with 10mL of methanol, then fixing the volume with 50% v/v of methanol-water solution to prepare a Weifuan stock solution with the concentration of 0.2mg/mL, and diluting with 50% v/v of methanol-water solution to 20 mug/mL to obtain an internal standard 1-Weifuan working solution;
S212 carbamazepine internal standard working solution: precisely weighing 20mg carbamazepine standard, dissolving 10mL of methanol in a 100mL volumetric flask, then 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 then diluting to 2 mug/mL by using 50% v/v methanol-water solution to obtain an internal standard 2-carbamazepine working solution;
s22, preparation of mixed standard solution:
S221, preparation of mixed standard mother liquor: respectively precisely weighing 20mg of each psychotic drug or metabolite standard substance thereof in a 100mL volumetric flask, dissolving the rest drugs (amisulpride, clozapine, norclozapine, olanzapine, mirtazapine, risperidone, 9-OH risperidone, trazodone, chlorpromazine and quetiapine) in 10mL of methanol, dissolving the aripiprazole and the dehydroaripiprazole in a 1% formic acid-containing methanol solution, and then fixing the volume by using 50% v/v methanol-water solution to prepare a standard substance mother solution containing amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole at 400 mug/mL, olanzapine, mirtazapine, risperidone and 9-OH risperidone at 100 mug/mL, and containing trazodone, chlorpromazine and quetiapine at 200 mug/mL;
s222, preparation of quantitative mixed standard curve solution: diluting the mixed standard mother solution with 40-70% v/v methanol-water solution according to a certain proportion to prepare 7 standard curve solutions of the medicines, wherein the solutions respectively contain mixed standard curves LS-1, LS-2, LS-3, LS-4, LS-5, LS-6 and LS-7 with the concentrations of amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole of 100, 50, 20, 10, 4, 2 and 1 mug/mL, the concentrations of olanzapine, mirtazapine, risperidone and 9-OH risperidone of 25, 12.5, 5, 2.5, 1, 0.5 and 0.25 mug/mL, and the concentrations of trazodone, chlorpromazine and quetiapine of 50, 25, 10, 5, 2 and 1 and 0.5 mug/mL;
S223, preparation of mixed standard substance solution: respectively precisely sucking 20 mu L of the mixed standard curve solution to 7mL of a centrifuge tube, then adding 20 mu L of the internal standard 1-gastric reset working solution and 20 mu L of the internal standard 2-carbamazepine working solution, blowing dry by nitrogen, adding a proper amount of blank serum and methyl tertiary butyl ether, performing first vortex, adding 200 mu L of sodium hydroxide aqueous solution with the concentration of 2mol/L and 3mL of methyl tertiary butyl ether, performing second vortex for 3min, performing further centrifugation for 5min, taking supernatant after centrifugation, blowing dry by nitrogen, adding 150 mu L of methanol-aqueous solution with the concentration of 15v/v% and 0.1v/v% formic acid, performing re-dissolution by nitrogen, performing vortex for 1min, and filtering by using a nylon filter membrane with the concentration of 0.25 mu m to obtain the compound standard curve;
s3 assay: injecting the mixed standard solution into an ultra-high performance liquid chromatograph, and adopting the following gradient elution program ：0min 5％v/v B、2min 10％v/v B、4min 20％v/v B、6min22％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 the retention time (t R) of the psychotropic drug and the metabolite thereof; the psychotropic drug and the metabolite thereof have retention time t R of olanzapine 3.179min, amisulpride 3.932min, trovaquone 4.347 (internal standard 1), zopiclone 4.863min, mirtazapine 5.416min, 9-OH risperidone 7.373min, trazodone 7.927min, risperidone 8.306min, norclozapine 8.708min, clozapine 9.578min, quetiapine 11.135min, carbamazepine 13.199min (internal standard 2), respectively;
Here, only take amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole as 100 mug/mL, olanzapine, mirtazapine, risperidone and 9-OH risperidone as 25 mug/mL, and the mixed standard curve containing trazodone, chlorpromazine and quetiapine as 50 mug/mL, the quantitative curve LSS-1 chromatogram in serum is shown in figure 1, and the psychotropic drug and the metabolite retention time (t R) are obtained; the psychotropic drug and the metabolite retention time t R thereof are respectively olanzapine 3.179min, amisulpride 3.935min, trovamine 4.363 (internal standard 1), mirtazapine 5.434min, 9-OH risperidone 7.355min, trazodone 7.901min, risperidone 8.299min, norclozapine 8.730min, clozapine 9.584min, quetiapine 11.121min, carbamazepine 13.213min (internal standard 2), dehydroaripiprazole 17.397min, aripiprazole 18.035min and chlorpromazine 19.419min; the hair quantitative curve LSH-1 is shown in figure 2, and the retention time (t R) of the psychotropic drug and its metabolite is obtained; the psychotropic drugs and the metabolites thereof have retention time t R min of olanzapine 3.177min, amisulpride 3.924min, trovamine 4.351 (internal standard 1), mirtazapine 5.431min, 9-OH risperidone 7.344min, trazodone 7.911min, risperidone 8.300min, norclozapine 8.762min, clozapine 9.639min, quetiapine 11.155min, carbamazepine 13.206min (internal standard 2), dehydroaripiprazole 17.439min, aripiprazole 18.058min, chlorpromazine 19.447min.
S4, manufacturing a standard curve and a calculation method:
S41: establishing a standard curve from the concentration of the linear working solution in 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, wherein the r value is calculated by the specific example:
a. The linear range of olanzapine is: 5-500ng/mL, the linear equation is y=0.0111X-0.2065, c=x 1.0 mL/sample volume to be measured (mL), r=0.9987;
b. the linear range of amisulpride is: 20-2000ng/mL, the linear equation is y=0.0026x+0.0023, c=x 1.0 mL/sample volume to be measured (mL), r=0.9999;
c. the linear range of mirtazapine is: 5-500ng/mL, the linear equation is y=0.0019x+0.0076, c=x 1.0 mL/sample volume to be measured (mL), r=0.9998;
The linear range of d.9-OH risperidone is: 5-500ng/mL, the linear equation is y=0.0014x-0.011, c=x×1.0 mL/sample volume to be measured (mL), r=0.9997;
e. The linear range of trazodone is: 10-1000ng/mL, linear equation y=0.0048x-0.0065, c=x 1.0 mL/sample volume to be measured (mL), r=1.0000;
f. The linear range of risperidone is: 5-500ng/mL, the linear equation is y=0.0022x-0.0199, c=x 1.0 mL/sample volume to be measured (mL), r=0.9998;
g. The linear range of norclozapine is: 20-2000ng/mL, linear equation y=0.0078X-0.197, c=x 1.0 mL/sample volume to be measured (mL), r=0.9997;
h. The linear range of clozapine is: 20-2000ng/mL, linear equation y=0.0108x-0.1535, c=x 1.0 mL/sample volume to be measured (mL),
r＝0.9999；
I. The linear range of quetiapine is: 10-1000ng/mL, linear equation y=0.013X-0.0848, c=x 1.0 mL/sample volume to be measured (mL),
r＝1.0000；
J. The linear range of dehydroaripiprazole is: 20-2000ng/mL, linear equation y=0.0024x-0.0332, c=x 1.0 mL/sample volume to be measured (mL), r= 0.9991;
k. The linear range of aripiprazole is: 20-2000ng/mL, linear equation y=0.003X-0.0447, c=x 1.0 mL/sample volume to be measured (mL),
r＝0.9996；
The linear range of chlorpromazine is: 10-500ng/mL, linear equation y=0.0058x-0.0473, c=x 1.0 mL/sample volume to be measured (mL),
r＝0.9998；
The standard curves are shown in fig. 3 a-k;
s41: establishing a standard curve by using the ratio of the concentration of the linear working solution in the hair to the corresponding peak area/internal standard area, and calculating the r value by using a function of a correlation coefficient correl in Excel, wherein the r value is calculated in the specific example:
a. The linear range of olanzapine is: 0.25-12.5 μg/mg, linear equation y= 0.1831x-0.0812; c=x×20 per hair weight to be measured (mg), r= 0.9991;
b. The linear range of amisulpride is: 1-50 μg/mg, linear equation y=0.0524x+0.0143; c=x×20 per hair weight to be measured (mg), r=0.9997;
c. the linear range of mirtazapine is: 0.25-12.5 μg/mg, linear equation y=0.0299 x-0.0039; c=x×20 per hair weight to be measured (mg), r= 0.9992;
the linear range of d.9-OH risperidone is: 0.25-12.5 μg/mg, linear equation y=0.0283x+0.0014; c=x×20 per hair weight to be measured (mg), r=1.0000;
e. the linear range of trazodone is: 0.5-25 μg/mg, linear equation y=0.085x+0.0049; c=x×20/weight of hair to be measured (mg); r=0.9997;
f. The linear range of risperidone is: 0.25-12.5 μg/mg, linear equation y=0.0406x-0.0122, c=x×20/weight of hair to be measured (mg); r=0.9998;
g. The linear range of norclozapine is: 1-50 μg/mg, linear equation y= 0.1530x-0.0602, c=x20 per hair weight to be measured (mg), r=0.9998;
h. The linear range of clozapine is: 1-50 μg/mg, linear equation y= 0.1882x-0.0374; c=x×20 per hair weight to be measured (mg), r=1.0000;
i. the linear range of quetiapine is: 0.5-25 μg/mg, linear equation y= 0.2328x-0.0316; c=x×20 per hair weight to be measured (mg), r=0.9998;
j. The linear range of dehydroaripiprazole is: 1-50 μg/mg, linear equation y=0.0448 x-0.0076; c=x×20 per hair weight to be measured (mg), r=0.9999;
k. the linear range of aripiprazole is: 1-50 μg/mg, linear equation y= 0.0617x-0.0069; c=x×20 per hair weight to be measured (mg), r= 0.9996;
The linear range of chlorpromazine is: 0.5-25 μg/mg, with linear equation y=0.1112 x-0.0141; c=x×20 per hair weight to be measured (mg), r=0.9987;
The standard curves are shown in fig. 4 a-k;
Example 2 detection of serum samples from patients taking clozapine, quetiapine fumarate
The S1 chromatographic conditions and the preparation method of the S22 mixed standard solution are the same as in example 1;
S231 serum test sample solution preparation: respectively precisely sucking 20 mu L of the internal standard 1-gastric reset working solution and 20 mu L of the internal standard 2-carbamazepine working solution prepared in S21, drying by nitrogen, mixing with 500 mu L of melted frozen serum sample to be tested into a 7mL centrifuge tube, performing first vortex for 1min, adding 200 mu L of 2mol/L sodium hydroxide aqueous solution and 3mL of methyl tertiary butyl ether, performing second vortex for 3min, separating the centrifuge tube at a speed of 3000 rpm for 5min after vortex, taking and drying the supernatant liquid after centrifugation by nitrogen, adding 150 mu L of methanol-aqueous solution containing 0.1v/v% formic acid and 15v/v% for redissolution, performing vortex for 1min, and filtering by a 0.25 mu m nylon filter membrane to obtain the compound liquid;
S3 assay: injecting the sample solution into an ultra-high performance liquid chromatograph, and adopting a gradient elution program (same as example 1) to obtain a chromatogram, wherein the chromatogram is shown in fig. 5, and the retention time (t R) of the psychotropic drug and the metabolite thereof is obtained; the retention time t R of the psychotropic drugs and the metabolites thereof is respectively as follows: internal standard 1 Weifuan 4.348min, peak Area 19836; the peak Area of the norclozapine is 10031 after 8.897 min; clozapine 9.783min, peak Area 15801; quetiapine 11.262min, peak Area 6609; internal standard 2 carbamazepine 13.206min, peak Area 28121;
Substituting the peak area ratio of norclozapine, clozapine and quetiapine to the peak area ratio of the internal standard 1 gastric reset into linear equation y=0.0078X-0.197 of norclozapine and C=x 1/0.5mL of quetiapine respectively; the linear equation y=0.0108x-0.1535 for clozapine, c=x1/0.5 mL; ; quetiapine's linear equation y=0.013X-0.0848, c=x 1/0.5mL; and respectively calculating the blood concentration of the norclozapine obtained by the X to be 180.2ng/mL, the blood concentration of the clozapine to be 175.9ng/mL and the blood concentration of the quetiapine to be 64.3ng/mL.
Example 3 detection of serum samples from patients taking amisulpride
The S1 chromatographic conditions and the preparation method of the S22 mixed standard solution are the same as in example 1;
S231 test solution preparation, S3 assay were as in example 2:
The results are shown in FIG. 6, and the psychotropic drug and its metabolites were obtained (t R); amisulpride retention time t R is 3.949min, peak Area is 36114; internal standard 1 Weifuan 4.361min, peak Area 25131; internal standard 2 carbamazepine 13.218min, peak Area 39927. The peak area ratio of amisulpride/internal standard 1 gastric reset is calculated as y, and is substituted into a linear equation y=0.0026x+0.0023, C=X is 1/0.5mL, so that the amisulpride blood concentration C is calculated as 1103.6ng/mL.
Example 4 detection of serum samples from patients taking the drug clozapine
The S1 chromatographic conditions, the S21 internal standard solution and the S22 mixed standard solution are the same as in example 1;
s231 sample solution preparation and S3 assay were the same as in example 2;
results: as shown in fig. 7, the psychotropic drug and its metabolite retention time (t R) were obtained; internal standard 1 Weifuan 4.354min, peak Area 23670; desmethylclozapine 8.896 minutes, peak Area 13904; clozapine 9.769min, peak Area 32451; internal standard 2 carbamazepine 13.203min, peak Area 35948. Substituting the ratios of peak areas of norclozapine and clozapine to peak areas of the internal standard 1 gastric reset into linear equations y=0.0078X-0.197 and C=X 1/0.5mL of norclozapine respectively; the linear equation y=0.0108x-0.1535 for clozapine, c=x1/0.5 mL; and respectively calculating the blood concentration of the norclozapine obtained by the X to be 201.1ng/mL, and the blood concentration of the clozapine to be 282.3ng/mL.
EXAMPLE 5 detection of serum samples from patients taking the drug aripiprazole
The S1 chromatographic conditions, the S21 internal standard solution and the S22 mixed standard solution are the same as in example 1;
s231 sample solution preparation and S3 assay were the same as in example 2;
Results: as shown in fig. 8, the psychotropic drug and its metabolite retention time (t R) and peak Area (Area) were obtained; internal standard 1 Weifuan 4.363min, peak area 19100; internal standard 2 carbamazepine 13.216min with peak area 27607; dehydroaripiprazole is present for 17.614min with a peak area of 1425; aripiprazole 18.208min with a peak area of 5573.
The ratios of the peak areas of the dehydroaripiprazole and the aripiprazole to the peak area of the internal standard 2 carbamazepine are respectively substituted into linear equation y=0.0024x-0.0332, C=x1/0.5 mL, linear equation y=0.003X-0.0447 and C=x1/0.5 mL of the aripiprazole, and the blood concentration of the dehydroaripiprazole is respectively calculated to be 70.7ng/mL and the blood concentration of the aripiprazole is respectively calculated to be 164.4ng/mL.
Example 6 detection of hair samples from patients taking olanzapine, trazodone and risperidone
The S1 chromatographic conditions, the S21 internal standard solution and the S22 mixed standard solution are the same as in example 1;
s231 sample solution preparation and S3 assay were the same as in example 2;
s232 preparation of hair test solution comprises the following steps:
Cutting hair at the back of a pillow of a patient, rubbing the hair with acetone and water, drying a section close to the scalp for 1cm, cutting the dried section for later use, precisely weighing the hair to be tested into a centrifuge tube with a volume of 20.45mg to 7mL, precisely sucking 20 mu L of an internal standard 1-gastric reset working solution prepared by S21 and 20 mu L of an internal standard 2-carbamazepine working solution respectively, drying with nitrogen, adding 200 mu L of a sodium hydroxide aqueous solution with a concentration of 2mol/L, carrying out first vortex for 1min, carrying out ultrasonic treatment for 2.5h, adding 3mL of methyl tert-butyl ether, carrying out second vortex for 3min, drying the centrifuged supernatant with nitrogen at a speed of 3000 rpm for 5min, adding 200 mu L of methanol-aqueous solution with a concentration of 15v/v% containing formic acid, carrying out re-dissolution with 1min, and filtering with a nylon filter membrane with a concentration of 0.25 mu m;
Results: as shown in fig. 9, the retention time (t R) and peak area (area) of the psychotropic drug and its metabolite were obtained as: the retention time (t R) of the internal standard 1 gastric reset is 4.367min, and the peak area is 10395;9-OH risperidone (t R) 7.209min with peak area 122602; risperidone retention time (t R) 8.440min with peak area 41104; quetiapine retention time (t R) was 11.23min, peak area 40848, internal standard 2 carbamazepine retention time (t R) was 13.2,11min, peak area 20820.
Substituting the ratio of the peak areas of 9-OH risperidone, risperidone and quetiapine to the peak area of the internal standard 1-gastric reset into a hair linear equation y=0.0283x+0.0014 of 9-OH risperidone respectively; c=x×20/weight of hair to be measured (mg); hair linear equation y=0.0406x-0.0122 for risperidone; c=x×20 per hair weight (mg) to be measured, hair linear equation y= 0.2328x-0.0316 for quetiapine; c=x×20/weight of hair to be measured (mg); and (3) calculating the concentration of the 9-OH risperidone in the hair of 407.54ng/mg, the concentration of the risperidone in the hair of 99.89ng/mg and the concentration of the quetiapine in the hair of 16.64ng/mg respectively.
Example 7 detection of Hair samples from patients taking olanzapine and mirtazapine drugs
The S1 chromatographic conditions, the S21 internal standard solution and the S22 mixed standard solution are the same as in example 1; s232 hair test sample solution preparation method, S3 assay method is the same as example 6; the actual weighed mass of hair to be measured was recorded as 20.35mg.
Results: as shown in fig. 10, the psychotropic drug and its metabolite retention time (t R) were obtained; the measured retention time t R of the psychotropic drugs and the metabolites thereof was 3.200min each, and the peak area was 4120; internal standard 1 Weifuan 4.367min, peak area 9347; mirtazapine 5.397min, peak area 49598, internal standard 2 carbamazepine 13.217min, peak area 14505. Substituting the ratio of olanzapine peak area to internal standard 1 gastric complex An Feng area as y into olanzapine in hair to obtain linear equation y= 0.1831x-0.0812, wherein c=x is 20 per weight of hair to be measured (mg), obtaining olanzapine content C in hair to be 2.85ng/mg, substituting the ratio of mirtazapine peak area to internal standard 1 gastric complex An Feng area y into mirtazapine in hair to obtain linear equation y=0.0299 x-0.0039; c=x.20 per weight of hair to be measured (mg), giving a content of mirtazapine C in the hair of 177.60ng/mg.
Example 8: detection of hair samples from patients taking quetiapine
The S1 chromatographic conditions, the S21 internal standard solution and the S22 mixed standard solution are the same as in example 1; s232 preparation of hair test solution the same as in example 5; s3 assay was as in example 6; the actual weighed mass of hair to be measured was recorded as 20.45mg.
Results: as shown in fig. 11, the psychotropic drug and its metabolite retention time (t R) were obtained; the measured retention time t R of the psychotropic drug and the metabolite thereof is respectively 4.367min of internal standard 1 Weifuan, and the peak area is 10395; quetiapine 11.230min, peak area 40848; internal standard 2 carbamazepine 13.211min with a peak area of 20820. Substituting the ratio of quetiapine peak area to internal standard 1 gastric reset peak area as y into the linear equation in hair to be y= 0.2328x-0.0316; c=x.20 per weight of hair to be measured (mg) the content C of quetiapine in the hair is 16.64ng/mg.
Example 9: detection kit for simultaneously detecting common medicines in psychiatric department
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, preparing an internal standard solution, wherein the internal standard solution comprises the following steps:
S11 internal standard 1-Weifuan working solution: precisely absorbing a proper amount of the gastric re-installation standard substance, dissolving with a small amount of methanol, then fixing the volume with a methanol-water solution to prepare a gastric re-installation standard substance solution, and then diluting with a methanol-water solution to fix the volume to obtain an internal standard 1-gastric re-installation working solution; further, the preparation method of the gastroscope internal standard working solution comprises the following steps: precisely weighing 1-30 mg of the standard substance for the gastric recovery, dissolving with 1-30 mL of methanol, then fixing the volume with 40-70% v/v of methanol-water solution to prepare a stock solution for the gastric recovery with the concentration of 0.1-0.5 mg/mL, and then diluting with 40-70% v/v of methanol-water solution to 10-40 mug/mL to obtain an internal standard 1-gastric recovery working solution;
And
S12 internal standard 2-carbamazepine working solution: precisely absorbing a proper amount of carbamazepine standard substance, dissolving with a small amount of methanol, then fixing the volume with a methanol-water solution, preparing Cheng Kama's standard substance solution, and then diluting with the methanol-water solution to the fixed volume to obtain an internal standard 2-carbamazepine working solution; further, the preparation method of the carbamazepine internal standard working solution comprises the following steps: precisely weighing 1-30 mg carbamazepine standard, dissolving with 1-30 mL of methanol, then fixing the volume with 40-70% v/v of 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 of methanol-water solution to 10-40 mug/mL to obtain an internal standard 2-carbamazepine working solution;
S2, preparing a mixed standard substance solution:
S21, preparing a mixed standard mother solution: respectively precisely weighing a proper amount of common medicines of the Shenke or metabolites thereof, namely amisulpride, clozapine, norclozapine, aripiprazole, dehydroaripiprazole, olanzapine, mirtazapine, risperidone, 9-OH risperidone, trazodone, chlorpromazine and quetiapine, 1-30 mg of the solvent is firstly used for dissolving after mixing, 40-70% v/v methanol-water solution is used for diluting and fixing volume to prepare mixed standard mother liquor with concentration of amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole of 400 mu g/mL, and mixed standard mother liquor with concentration of olanzapine, mirtazapine, risperidone, 9-OH risperidone of 100 mu g/mL, trazodone, chlorpromazine and quetiapine of 200 mu g/mL;
S22, preparing a mixed standard curve working solution: diluting the mixed standard mother solution with 40-70 v/v% methanol-water solution according to a certain proportion to obtain amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole with concentrations of 100, 50, 20, 10, 4, 2 and 1 mug/mL respectively, with olanzapine, mirtazapine, risperidone and 9-OH risperidone with solubilities of 25, 12.5, 5, 2.5, 1, 0.5 and 0.25 mug/mL respectively, and with trazodone, chlorpromazine and quetiapine with concentrations of 50, 25, 10, 5, 2, 1 and 0.5 mug/mL respectively;
S23, preparation of a TDM detection tube of the mixed standard solution: respectively precisely sucking 10-40 mu L of each quantitative mixed standard curve working solution into a TDM detection tube, then adding 10-40 mu L of each internal standard 1-stomach resetting working solution and 10-40 mu L of each internal standard 2-carbamazepine working solution, and drying by nitrogen to obtain the finished product;
(II) a TDM detection tube for detecting the sample solution, and the preparation method comprises the following steps: adding the internal standard solution prepared in the step S1 into a TDM detection tube, and drying with nitrogen to obtain the product;
(III) complex solution: methanol-water solution containing 0.1v/v% formic acid and 15% v/v concentration;
(IV) instruction book: the specification describes the method of using the same:
S1, preparing a standard substance solution: taking the mixed standard solution TDM detection tube, adding 500 mu L of blank serum and 3mL of methyl tertiary butyl ether, carrying out primary vortex for 1min, then adding 200 mu L of sodium hydroxide aqueous solution with the concentration of 2mol/L and 3mL of methyl tertiary butyl ether, carrying out secondary vortex and 3000r/min for 5min, obtaining supernatant, drying the supernatant, then adding 100-150 mu L of complex solution, and carrying out microporous filter membrane filtration to obtain the mixed standard solution TDM;
s2, preparing a sample solution:
(1) Serum sample detection solution: taking 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, swirling for 1min, adding 3mL of methyl tertiary butyl ether, swirling for 3min, centrifuging for 5min at 3000r/min, transferring supernatant into a 2mL test tube, drying by nitrogen, adding 100-150 mu L of complex solution, and filtering by a microporous filter membrane to obtain the serum;
(2) Hair sample detection liquid: taking 20mg of hair sample into a sample solution TDM detection tube, adding 200 mu L of sodium hydroxide aqueous solution with the concentration of 2mol/L, carrying out first vortex for 1min, carrying out ultrasonic treatment for 2h, adding 3mL of methyl tertiary butyl ether, carrying out second vortex for 3min, centrifuging a centrifuge tube at 3000r/min for 5min after vortex, transferring supernatant into a 2mL test tube, drying by nitrogen, adding 100-150 mu L of complex solution, and filtering by a microporous filter membrane to obtain the hair sample;
S3, measuring: injecting 5-10 mu L of the standard substance solution prepared in the step S1 and the sample detection solution prepared in the step S2 into an ultra-high performance liquid chromatograph respectively, adopting a gradient elution program to obtain 7 concentration mixed standard substance solution chromatograms and test substance solution chromatograms respectively, obtaining retention time t R and peak area integral of the psychotropic substance and the metabolite standard substance thereof and the test substance thereof respectively, establishing a standard curve of the concentration in the mixed standard substance solution and the corresponding peak area/internal standard area ratio thereof, and obtaining the concentration of the test substance solution according to the peak area integral of the test substance solution and the standard curve; the gradient elution program is ：0min 5％v/v B、2min 10％v/v B、4min20％v/v B、6min 22％v/v B、12min 35％v/v B、15min 36％v/v B、18.5min40％v/v B、20min 50％v/v B、21min 90％v/v B、23min 90％v/v B、24min5％v/v B and 28min 5% v/v B;
and the kit is stored at the temperature of minus 20 ℃ to minus 15 ℃.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that the invention is not limited thereto, but various changes and modifications can be made by one skilled in the art without departing from the spirit and scope of the present invention.
Claims (15)
1. The method for simultaneously and quantitatively detecting the psychotropic drugs or the metabolites thereof in the biological sample is characterized by adopting an ultra-high performance liquid chromatography-ultraviolet detection method and comprising the following steps of:
S1 chromatographic conditions: (1) chromatography column: a C 18 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;
s2, preparing a working solution:
preparation of S21 internal standard solution:
S211 internal standard 1-gastric reset working solution: precisely absorbing a proper amount of the gastric re-installation standard substance, dissolving with a small amount of methanol, then fixing the volume with a methanol-water solution to prepare a gastric re-installation standard substance solution, and then diluting with a methanol-water solution to fix the volume to obtain an internal standard 1-gastric re-installation working solution;
S212 internal standard 2-carbamazepine internal standard working solution: precisely absorbing a proper amount of carbamazepine standard substance, dissolving with a small amount of methanol, then fixing the volume with a methanol-water solution, preparing Cheng Kama's standard substance solution, and then diluting with the methanol-water solution to the fixed volume to obtain an internal standard 2-carbamazepine working solution;
s22, preparation of mixed standard solution:
S221, preparing a mixed standard mother solution: respectively precisely weighing a proper amount of common medicines in psychiatric department or metabolites such as 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 to constant volume with methanol-water solution to prepare mixed standard mother liquor;
S222, preparing a quantitative mixed standard curve working solution: diluting the mixed standard mother solution with 40-70% v/v methanol-water solution according to a certain proportion to prepare 7 standard curve solutions containing the medicines, wherein the solutions respectively contain amisulpride, clozapine, norclozapine, aripiprazole, and dehydroaripiprazole, the concentrations of the amipiprazole are 100, 50, 20, 10, 4, 2 and 1 mug/mL, the concentrations of the olanzapine, mirtazapine, risperidone and 9-OH risperidone are 25, 12.5, 5, 2.5, 1, 0.5 and 0.25 mug/mL, and the concentrations of the trazodone, chlorpromazine and quetiapine are 50, 25, 10, 5, 2, 1 and 0.5 mug/mL;
S223, preparation of mixed standard substance solution: respectively precisely sucking 10-40 mu L of the quantitative mixed standard curve working solution, precisely adding 10-40 mu L of the internal standard 1-gastric reset working solution and 10-40 mu L of the internal standard 2-carbamazepine working solution, drying, adding a proper amount of blank blood, plasma, serum or saliva and methyl tertiary butyl ether, performing first vortex, adding 100-500 mu L of 1-3mol/L sodium hydroxide aqueous solution and methyl tertiary butyl ether, performing second vortex and centrifugation to obtain a supernatant, drying the supernatant, and then adding methanol-aqueous solution containing formic acid for re-dissolution, vortex and filtration to obtain the finished product;
the preparation of the S23 test solution comprises the following steps:
S231 preparation of a blood, plasma, serum or saliva test sample solution: respectively precisely sucking 10-40 mu L of the internal standard 1-gastric reset working solution and 10-40 mu L of the internal standard 2-carbamazepine working solution prepared in S21, drying, mixing with a proper amount of blood, plasma, serum or saliva to be tested, performing first vortex, adding 100-500 mu L of sodium hydroxide aqueous solution and methyl tertiary butyl ether for performing second vortex, centrifuging after vortex to obtain a supernatant, drying the supernatant, adding methanol-aqueous solution containing formic acid for redissolution, vortex and filtering to obtain the final product;
Or S232 preparation of hair test sample solution: respectively precisely sucking 10-40 mu L of the internal standard 1-stomach compound safety working solution and 10-40 mu L of the internal standard 2-carbamazepine working solution prepared in S21, drying, mixing with a proper amount of hair sample to be tested, carrying out first vortex, adding 100-500 mu L of sodium hydroxide aqueous solution, carrying out ultrasonic treatment or standing, adding methyl tertiary butyl ether for carrying out second vortex, centrifuging after vortex to obtain a supernatant, drying the supernatant, adding methanol-aqueous solution containing formic acid, carrying out redissolution, vortex and filtering to obtain the hair sample;
S3 assay: and (3) respectively injecting the mixed standard solution prepared in the step (S22) and the sample solution prepared in the step (S23) into an ultra-high performance liquid chromatograph, respectively obtaining 7 concentration mixed standard solution chromatograms and sample solution chromatograms by adopting a gradient elution program, respectively obtaining retention time t R and peak area integral of the psychotropic drugs and the metabolite standard substances thereof and the sample, respectively, establishing a standard curve of the concentration in the mixed standard solution and the corresponding peak area/internal standard area ratio thereof, and then obtaining the concentration of the sample solution according to the peak area integral of the sample solution and the standard curve.
2. The method for simultaneous quantitative detection of psychotropic drugs or metabolites thereof in a biological sample according to claim 1, wherein the preparing of the labeling mother liquor of S221 comprises: and respectively precisely weighing a proper amount of common medicines of the Shenke or metabolites thereof, namely amisulpride, clozapine, norclozapine, aripiprazole, dehydroaripiprazole, olanzapine, mirtazapine, risperidone, 9-OH risperidone, trazodone, chlorpromazine and quetiapine, 1-30 mg of the mixture is dissolved by 1-30 mL of solvent, and 40-70% v/v of methanol-water solution is adopted to dilute and fix the volume to prepare a mixed standard mother solution containing amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole, wherein the concentration of the olanzapine, mirtazapine, risperidone and 9-OH risperidone is 100 mug/mL, and the concentration of the trazodone, chlorpromazine and quetiapine is 200 mug/mL.
3. The method for simultaneous quantitative detection of psychotropic drugs or metabolites thereof according to claim 1, wherein said biological sample is selected from any one or more of blood, plasma, serum, saliva and hair.
4. The method for simultaneous quantitative detection of psychotropic drugs or metabolites thereof in a biological sample according to claim 1, wherein the chromatographic conditions of S1: mobile phase: phase A is 0.05v/v% formic acid-water solution; and phase B was 0.05v/v% formic acid-methanol solution.
5. The method for simultaneous quantitative detection of psychotropic drugs or metabolites thereof in a biological sample according to claim 1, wherein the chromatographic conditions of S1: the ultraviolet detector is a VWD or DAD detector.
6. The method for simultaneous quantitative detection of psychotropic drugs or metabolites thereof according to claim 5, wherein said ultraviolet detector has a detection wavelength of 254nm or 285nm.
7. The method for simultaneous quantitative detection of psychotropic drugs or metabolites thereof in a biological sample 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 ℃; the sample injection amount of the working solution is 1.0-20.0 mu L.
8. The method for simultaneous quantitative detection of psychotropic drugs or metabolites thereof according to claim 1,
In step S21, the preparation of the internal standard solution specifically includes:
s211, recovering the internal standard working solution of the gastric recovery: precisely weighing 1-30 mg of the standard substance for the gastric recovery, dissolving with 1-30 mL of methanol, then fixing the volume with 40-70% v/v of methanol-water solution to prepare a stock solution for the gastric recovery with the concentration of 0.1-0.5 mg/mL, and then diluting with 40-70% v/v of methanol-water solution to 10-40 mug/mL to obtain an internal standard 1-gastric recovery working solution;
S212 carbamazepine internal standard working solution: precisely weighing 1-30 mg carbamazepine standard, dissolving with 1-30 mL of methanol, then fixing the volume with 40-70% v/v of 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 of methanol-water solution to 1-5 mug/mL to obtain an internal standard 2-carbamazepine working solution.
9. The method for simultaneous quantitative detection of psychotropic drugs or metabolites thereof in a biological sample according to claim 1, wherein said preparation of a sample solution of S231 blood, plasma, serum or saliva comprises the steps of:
Respectively precisely sucking 10-40 mu L of the internal standard 1-gastric reset working solution and the internal standard 2-carbamazepine working solution prepared by S21, blowing nitrogen to dry, mixing with 400-1000 mu L of melted frozen blood, plasma, serum or saliva samples to be tested into a 7-10 mL centrifuge tube, carrying out first vortex for 0.5-2 min, adding 100-500 mu L of 1-3 mol/L sodium hydroxide aqueous solution and 2-7 mL of methyl tertiary butyl ether, carrying out second vortex for 2-5 min, drying the centrifuged supernatant nitrogen at the speed of 2500-5000 rpm for 4-10 min, adding 60-250 mu L of methanol-aqueous solution containing 0.05-0.15 v/v% formic acid and the concentration of 10-20 v/v% for redissolving, carrying out filtration by a nylon filter membrane, and obtaining the vortex.
10. The method for simultaneous quantitative detection of psychotropic drugs or metabolites thereof in a biological sample according to claim 1, wherein said preparation of S232 hair test solution comprises the steps of: sequentially cleaning hair to be tested by using acetone and water, cutting after drying, respectively precisely sucking 10-40 mu L of an internal standard 1-gastric reset working solution prepared by S21 and 10-50 mg of an internal standard 2-carbamazepine working solution, drying by nitrogen, mixing with 10-50 mg of hair sample to be tested into a 7-10 mL centrifuge tube, adding 100-500 mu L of 1-3 mol/L sodium hydroxide aqueous solution, carrying out first vortex for 0.5-2 min, carrying out ultrasonic treatment for 1-4 h, adding 2-7 mL of methyl tert-butyl ether, carrying out second vortex for 2-5 min, carrying out rotation/separation on the centrifuge tube at a speed of 2500-5000 r/S for 4-10 min, taking and drying by using centrifuged supernatant nitrogen, adding 60-250 mu L of methanol-aqueous solution containing 0.05-0.15 v/v% formic acid and the concentration of 10-20 v/v% for redissolving, carrying out vortex for 0.5-2 min, and carrying out nylon filter membrane filtration.
11. The method of claim 1, wherein the gradient elution procedure in the S3 assay is ：0min 5％v/v B、2min 10％v/v B、4min 20％v/v B、6min 22％v/vB、12min 35％v/v B、15min 36％v/v B、18.5min 40％v/v B、20min50％v/v B、21min 90％v/v B、23min 90％v/v B、24min 5％v/v B and 28min 5% v/v B.
12. 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, preparing an internal standard solution:
S11 internal standard 1-Weifuan internal standard working solution: precisely absorbing a proper amount of the gastric re-installation standard substance, dissolving with a small amount of methanol, then fixing the volume with a methanol-water solution to prepare a gastric re-installation standard substance solution, and then diluting with a methanol-water solution to fix the volume to obtain an internal standard 1-gastric re-installation working solution; further, the preparation method of the gastroscope internal standard working solution comprises the following steps: precisely weighing 1-30 mg of the standard substance for the gastric recovery, dissolving with 1-30 mL of methanol, then fixing the volume with 40-70% v/v of methanol-water solution to prepare a stock solution for the gastric recovery with the concentration of 0.1-0.5 mg/mL, and then diluting with 40-70% v/v of methanol-water solution to 10-40 mug/mL to obtain an internal standard 1-gastric recovery working solution;
And
S12 internal standard 2-carbamazepine internal standard working solution: precisely absorbing a proper amount of carbamazepine standard substance, dissolving with a small amount of methanol, then fixing the volume with a methanol-water solution, preparing Cheng Kama's standard substance solution, and then diluting with the methanol-water solution to the fixed volume to obtain an internal standard 2-carbamazepine working solution; further, the preparation method of the carbamazepine internal standard working solution comprises the following steps: precisely weighing 1-30 mg carbamazepine standard, dissolving with 1-30 mL of methanol, then fixing the volume with 40-70% v/v of 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 of methanol-water solution to 10-40 mug/mL to obtain an internal standard 2-carbamazepine working solution;
S2, preparing a mixed standard solution TDM detection tube:
S21, preparing a mixed standard mother solution: respectively precisely weighing a proper amount of common medicines of the Shenke or metabolites thereof, namely amisulpride, clozapine, norclozapine, aripiprazole, dehydroaripiprazole, olanzapine, mirtazapine, risperidone, 9-OH risperidone, trazodone, chlorpromazine, quetiapine and 1-30 mg, dissolving the medicines in 1-30 mL of solvent after mixing, and diluting and fixing the volume by 40-70% v/v methanol-water solution to prepare mixed standard mother liquor containing amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole with 400 mug/mL, and mixed standard mother liquor containing olanzapine, mirtazapine, risperidone and 9-OH risperidone with 100 mug/mL and containing trazodone, chlorpromazine and quetiapine with 200 mug/mL;
S22, preparing a mixed standard curve working solution: diluting the mixed standard mother solution with 40-70 v/v% methanol-water solution according to a certain proportion to obtain amisulpride, clozapine, norclozapine, aripiprazole and dehydroaripiprazole with concentrations of 100, 50, 20, 10, 4, 2 and 1 mug/mL respectively, with olanzapine, mirtazapine, risperidone and 9-OH risperidone with solubilities of 25, 12.5, 5, 2.5, 1, 0.5 and 0.25 mug/mL respectively, and with trazodone, chlorpromazine and quetiapine with concentrations of 50, 25, 10, 5, 2, 1 and 0.5 mug/mL respectively;
S23, preparation of a TDM detection tube of the mixed standard solution: respectively precisely sucking 10-40 mu L of the quantitative mixed standard curve working solution into a TDM detection tube, then adding 10-40 mu L of the internal standard 1-stomach resetting working solution and 10-40 mu L of the internal standard 2-carbamazepine working solution respectively, and drying by nitrogen to obtain the compound;
(II) a TDM detection tube for detecting the sample solution, and the preparation method comprises the following steps: adding 10-40 mu L of the internal standard 1-Weifuan working solution and the internal standard 2-carbamazepine working solution prepared in the step S1 into a TDM detection tube respectively, and drying with nitrogen to obtain the compound;
(III) complex solution: the compound solution is methanol-water solution with the concentration of 0.05-0.15 v/v% formic acid of 10-20 v/v;
(IV) instruction book: the instructions describe methods of using the kit; and the kit is stored at the temperature of minus 20 ℃ to minus 15 ℃.
13. The kit according to claim 11, wherein the biological sample is selected from any one or more of blood, plasma, serum, saliva, hair.
14. The kit of claim 11, wherein the method of use comprises:
S1, preparing a standard substance solution: taking the mixed standard solution TDM detection tube, adding 100-1000 mu L of blank blood, plasma, serum or saliva and 2-7 mL of methyl tertiary butyl ether, carrying out primary vortex for 1-5 min, then adding 100-500 mu L of sodium hydroxide aqueous solution with the concentration of 1-3 mol/L and 2-7 mL of methyl tertiary butyl ether, carrying out secondary vortex, carrying out 2500-5000 r/min, centrifuging for 4-10 min, obtaining supernatant, drying the supernatant, then adding 100-150 mu L of complex solution, and carrying out microfiltration membrane filtration, thus obtaining the mixed standard solution;
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 1-3 mol/L, swirling for 0.5-2 min, adding 2-7 mL of methyl tertiary butyl ether, swirling for 1-5 min, centrifuging for 4-10 min at 2500-5000 r/min, transferring supernatant into the test tube, drying with nitrogen, adding 100-150 mu L of complex solution, and filtering with a microporous filter membrane to obtain the serum sample;
Or (2) hair sample detection liquid: taking 20mg of hair sample into the sample solution TDM detection tube, adding 100-500 mu L of 1-3 mol/L sodium hydroxide aqueous solution, carrying out first vortex for 0.5-2 min, carrying out ultrasonic treatment for 1-4 h, adding 2-7 mL of methyl tertiary butyl ether, carrying out second vortex for 2-5 min, separating a centrifuge tube at a speed of 2500-5000 r/min for 4-10 min, transferring supernatant into the test tube, carrying out nitrogen blow-drying, adding 100-150 mu L of complex solution, and carrying out microfiltration membrane filtration to obtain the hair sample;
S3, measuring: injecting 5-10 mu L of the standard substance solution prepared in the step S1 and the sample detection solution prepared in the step S2 into an ultra-high performance liquid chromatograph respectively, adopting a gradient elution program to obtain 7 concentration mixed standard substance solution chromatograms and test substance solution chromatograms respectively, obtaining retention time t R and peak area integral of the psychotropic substance and the metabolite standard substance thereof and the test substance thereof respectively, establishing a standard curve of the concentration in the mixed standard substance solution and the corresponding peak area/internal standard area ratio thereof, and obtaining the concentration of the test substance solution according to the peak area integral of the test substance solution and the standard curve; the gradient elution procedure was ：0min 5％v/v B、2min10％v/v B、4min 20％v/v B、6min 22％v/v B、12min 35％v/v B、15min36％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 a kit according to any one of claims 12 to 14 for the preparation of a reagent for quantitative detection of psychotropic drugs or their metabolites in a biological sample.
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| CN112305140A (en) * | 2020-09-25 | 2021-02-02 | 上海市精神卫生中心（上海市心理咨询培训中心） | Method for detecting psychotropic drugs and metabolites thereof in vivo and application |
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| CN112305140A (en) * | 2020-09-25 | 2021-02-02 | 上海市精神卫生中心（上海市心理咨询培训中心） | Method for detecting psychotropic drugs and metabolites thereof in vivo and application |
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