Method and kit for detecting antipsychotic drugs and metabolites thereof in sample

Abstract

The invention discloses a method for detecting antipsychotic drugs and metabolites thereof in a sample and a kit thereof, and relates to the technical field of biological detection.The kit comprises calibrators, quality control products, internal standard solutions and protein precipitators of 13 to-be-detected substances, and the protein precipitators comprise a zinc sulfate aqueous solution containing methanol, 13 analytes can be simultaneously detected, the separation degree of the to-be-detected substance is high, the detection time is short, the required sample size is small, and the method is suitable for TDM high-throughput detection of common antipsychotic drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and in particular, to a method for detecting antipsychotic drugs and their metabolites in a sample and a kit therefor. Background Art

[0002] Mental illness is a type of disease mainly manifested by abnormal behaviors, mental fluctuations, emotional disorders, thinking disorders, and cognitive disorders. At present, the treatment of mental illness still mainly relies on drug treatment. However, the plasma concentrations of some antipsychotic drugs have large individual differences, low therapeutic indices, narrow therapeutic windows, and are prone to cause adverse reactions, and the drug adverse reactions are closely related to their doses. In addition, as a result of individualized antipsychotic drug treatment, many patients have insufficient response to monotherapy and need additional drugs of other pharmacological categories. However, the interactions caused by blind combination of drugs will reduce the clinical efficacy or increase the side effects. Therefore, therapeutic drug monitoring (TDM) is widely used in clinical diagnosis to accurately quantify drug concentrations, achieve individualized medication, and achieve the purpose of optimized treatment.

[0003] TDM detects the concentrations of drugs and metabolites in body fluids (mainly blood) through advanced analytical methods, and combines pharmacokinetic principles and data analysis to individualize the clinical drug administration plan, so as to improve the efficacy, avoid or reduce toxic and side effects, and at the same time provide an important basis for the diagnosis and treatment of drug overdose poisoning and the medication compliance of patients. At present, among the chemical detection methods of TDM, liquid chromatography tandem mass spectrometry (LC-MS / MS) method has gradually become the first choice in drug concentration analysis due to its high selectivity, high accuracy, high sensitivity, short analysis time, and the ability to be used for the determination of almost all antipsychotic drugs and their metabolites.

[0004] In the prior art and existing products, certain developments have also been made in the detection of antipsychotic drugs by LC-MS / MS method. For example: In the literature "Determination of the concentrations of aripiprazole and dehydroaripiprazole in plasma of Chinese schizophrenia patients by UPLC-MS / MS method", an HPLC-MS / MS method for efficiently determining the concentrations of aripiprazole and its main active metabolite dehydroaripiprazole in human serum was established. However, its application range is narrow and it cannot meet the requirements for simultaneous detection and screening of multiple drugs. Chinese Patent Application (Application No. 201910722507.0) discloses "A method and kit for detecting 5 antipsychotic drugs and their main metabolites in blood". This method can simultaneously detect 5 antipsychotic drugs and their metabolites, but the drugs and their metabolites are not separated chromatographically, and there is a risk of isotope or isomer interference in quantification. Another example is that Chinese Patent Application (Application No. 201910058868.X) discloses a "Method and kit for simultaneously determining 35 antipsychotic drugs by high-performance liquid chromatography-tandem mass spectrometry". The quantification method of this method is the external standard method, and the accuracy is greatly affected by factors such as experimental extraction efficiency and instrument stability. In addition, the calibrator and quality control product in the detection kit are prepared immediately before use, the protein precipitant is used in a large amount or the precipitation is incomplete, the number of drug types is too large, and the drug separation degree is not good, which is not applicable in large-scale clinical detections and causes great waste of resources. Chinese Patent Application (Application No. 201810878794.X) discloses a "Kit and application for detecting antipsychotic drugs in serum and plasma by liquid chromatography-tandem mass spectrometry" for simultaneously detecting 11 antipsychotic drugs in serum and plasma. However, the large number of internal standards used will directly lead to a high cost of the entire detection process, and this method requires a large sample volume of 100 μL. Chinese Patent Application (Application No. 202010474881.6) discloses a "Kit for simultaneously detecting multiple antipsychotic drugs in serum" for simultaneously detecting 16 antipsychotic drugs in serum and plasma. However, multiple drugs are unstable and difficult to preserve in vitro, which increases the instability of experimental results; both the calibrator solution and the quality control product need to be prepared before use, and the operation is cumbersome during pretreatment and the experimental error is increased.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a method and kit for detecting antipsychotic drugs and their metabolites in a sample.

[0007] The present invention is implemented as follows:

[0008] In a first aspect, an embodiment of the present invention provides a kit, which includes: a calibrator for an analyte, a quality control product for the analyte, an internal standard solution, and any one or a combination of a plurality of protein precipitants; the analyte includes: any one or more of olanzapine, clozapine, N-desmethylclozapine, risperidone, 9-hydroxyrisperidone, aripiprazole, dehydroaripiprazole, quetiapine, N-desalkylquetiapine, chlorpromazine, amisulpride, ziprasidone, and sulpiride; the protein precipitant includes an aqueous zinc sulfate solution containing methanol.

[0009] In a second aspect, an embodiment of the present invention provides an application of the kit as described in the foregoing embodiment in detecting antipsychotic drugs and their metabolites in a sample.

[0010] In a third aspect, an embodiment of the present invention provides a method for detecting antipsychotic drugs and their metabolites in a sample, which includes: using the kit as described in the foregoing embodiment to detect a sample to be tested.

[0011] The present invention has the following beneficial effects:

[0012] The detection method and the kit provided by the present invention can achieve simultaneous detection of 13 analytes, have a high resolution for the analyte to be detected, and good detection accuracy; the analysis time for each sample of this method and kit is short, only 4.5 minutes, which significantly improves the detection efficiency; the required sample volume is small, only 20 μL of sample volume can meet the detection requirements. The present invention provides a new way for the effective detection of antipsychotic drugs and their metabolites. Description of the Drawings

[0013] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, so they should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0014] Figure 1 It is a schematic diagram of the concentration detection process of 13 antipsychotic drugs and their metabolites in serum;

[0015] Figure 2 It is a resolution map of the analyte to be detected (TIC map of channel 1);

[0016] Figure 3 It is a resolution map of the analyte to be detected (TIC map of channel 2). Detailed Embodiments

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments not indicated by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0018] Regarding the deficiencies of the above technologies, the present invention provides an HPLC-MS / MS method for simultaneously detecting 13 common antipsychotic drugs and their active metabolites in human serum, a kit for supporting use, and a preparation method thereof. The 13 analytes include 9 antipsychotic drugs and their active metabolites, specifically including: olanzapine (OLA), clozapine (CLO), N-desmethylclozapine (N-CLO), risperidone (RIS), 9-hydroxyrisperidone (PAL), aripiprazole (ARI), dehydroaripiprazole (ND-ARI), quetiapine (QTP), N-desalkylquetiapine (ND-QTP), chlorpromazine (CPZ), amisulpride (AMI), ziprasidone (ZIP), and sulpiride (SUL).

[0019] The detection method provided by the embodiments of the present invention can accurately quantify the concentrations of various antipsychotic drugs and active metabolites in human serum, has high sensitivity, good separation of analytes to be measured, short detection time, and is suitable for high-throughput TDM of common antipsychotic drugs.

[0020] On the one hand, the embodiments of the present invention provide a kit, which includes: any one or a combination of calibration products of analytes to be measured, quality control products of analytes to be measured, internal standard solutions, and protein precipitants;

[0021] The analytes to be measured include: any one or more of olanzapine, clozapine, N-desmethylclozapine, risperidone, 9-hydroxyrisperidone, aripiprazole, dehydroaripiprazole, quetiapine, N-desalkylquetiapine, chlorpromazine, amisulpride, ziprasidone, and sulpiride;

[0022] The protein precipitant includes an aqueous zinc sulfate solution containing methanol. Using the protein precipitant of the present invention can cause the proteins in the sample to precipitate and denature under the influence of different forces, improve the efficiency of protein precipitation, and thus better achieve the effective detection of analytes.

[0023] In some embodiments, in the protein precipitant, the volume fraction of methanol is 80% - 95%, specifically any one or the range between any two of 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, and 95%.

[0024] In some embodiments, in the zinc sulfate aqueous solution, the concentration of zinc sulfate is 0.4 - 0.8 M, specifically any one of 0.4, 0.5, 0.6, 0.7, and 0.8 M or the range between any two of them.

[0025] In some embodiments, the calibrator is a lyophilized product obtained by lyophilizing the calibrator working solution; the quality control sample is a lyophilized product obtained by lyophilizing the quality control sample working solution; the calibrator working solution and the quality control sample working solution are solutions with concentration gradients prepared by diluting the standard solution of the analyte with known concentration with a matrix. Among them, the matrix includes bovine serum and a stabilizer, the stabilizer includes ascorbic acid, and the final concentration of the stabilizer in the matrix is 1 - 10 mg / mL, specifically any one of 1, 2, 4, 6, 8, and 10 mg / mL or the range between any two of them.

[0026] By adding a stabilizer to the calibrator and the quality control sample and then performing lyophilization, the stability of the calibrator and the quality control sample can be significantly improved, which is convenient for their transportation and storage. When in use, only reconstitution with water is required, and the operation is simple and convenient.

[0027] In some embodiments, the standard solution of the analyte is obtained by diluting the standard with methanol.

[0028] In some embodiments, in the calibrator working solution and / or the quality control sample working solution, the volume ratio of the standard solution of the analyte to the matrix is 1:10 - 30, specifically any one of 1:10, 1:15, 1:16, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, and 1:30 or the range between any two of them.

[0029] In some embodiments, the matrix further includes a preservative.

[0030] In some embodiments, the volume fraction of the preservative in the matrix is 0.01% - 5%, specifically any one of 0.01%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 3%, 4%, and 5% or the range between any two of them.

[0031] In some embodiments, the preservative includes Proclin300.

[0032] In some embodiments, the lyophilization procedure includes the following steps: cooling to -50 - -40 °C for pre-freezing for 6 - 10 h; heating to -20 - -10 °C for the first sublimation for 13 - 17 h; heating to -10 - 10 °C for the second sublimation for 8 - 12 h; heating to 30 - 40 °C for analytical drying for 8 - 12 h.

[0033] In some embodiments, the freeze-drying procedure includes the following steps: cooling to -48 to -43 °C for pre-freezing for 7 to 9 hours; heating to -17 to -13 °C for the first sublimation for 14 to 16 hours; heating to -5 to 5 °C for the second sublimation for 9 to 11 hours; heating to 33 to 37 °C for analytical drying for 9 to 11 hours.

[0034] In some embodiments, the cooling rate of pre-freezing, the heating rate of the first sublimation, the heating rate of the second sublimation, and the heating rate of analytical drying can independently be 0.1 to 5 °C / min, specifically any one or the range between any two of 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 3, 4, and 5 °C / min.

[0035] The kit provided by the embodiments of the present invention includes an internal standard solution, and the corresponding quantification method is the internal standard method. The internal standard method can correct the matrix effect and reduce the deviation caused by different operation levels of the testers and different experimental conditions.

[0036] In some embodiments, the internal standard solution includes an internal standard mixed solution, and the internal standards in the internal standard mixed solution include any one or a combination of more than one of the following: N-desmethylclozapine-d 8 , risperidone-d 4 , 9-hydroxyrisperidone-d 4 , quetiapine-d 8 , chlorpromazine hydrochloride-d 6 , ziprasidone-d 8 , and amisulpride-d 5 . Deuterated internal standards of analytes to be measured with similar polarity, similar chemical structure, and similar concentration response were selected for sharing. Only 7 deuterated internal standards of analytes to be measured were used for 13 analytes, which not only met the requirements of the matrix effect but also reduced the detection cost.

[0037] In some embodiments, in the internal standard mixed solution, the molar ratio of N-desmethylclozapine-d 8 , risperidone-d 4 , 9-hydroxyrisperidone-d 4 , quetiapine-d 8 , chlorpromazine hydrochloride-d 6 , ziprasidone-d 8 , and amisulpride-d 5 is 3 to 5:1:1 to 3:2 to 4:18 to 22:4 to 6:0.5 to 3. Specifically, the ratio of N-desmethylclozapine-d 8 and risperidone-d 4 can be 3:1, 4:1, or 5:1; the ratio of risperidone-d 4and 9-hydroxy risperidone-d 4 The ratio of 4 and quetiapine-d 8 can be 1:1, 1:2 or 1:3; the ratio of risperidone-d 4 and chlorpromazine hydrochloride-d 6 can be 1:18, 1:19, 1:20, 1:21 or 1:22; the ratio of risperidone-d 4 and ziprasidone-d 8 can be 1:4, 1:5 or 1:6; the ratio of risperidone-d 4 and amisulpride-d 5 can be 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5 or 1:3. In some embodiments, the internal standard solution contains the protein precipitant.

[0038] In some embodiments, the volume ratio of the internal standard mixed solution to the protein precipitant is 1:15 to 30, specifically any one or the range between any two of 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:28 and 1:30.

[0039] In some embodiments, the kit further includes mobile phase A and mobile phase B.

[0040] In some embodiments, mobile phase A includes an aqueous solution containing ammonium acetate, and the final concentration of sodium acetate is 1 to 10 mmol / L, specifically any one or the range between any two of 1, 2, 4, 6, 8 and 10 mmol / L.

[0041] In some embodiments, mobile phase B includes a mixed solution of methanol and acetonitrile containing formic acid, wherein the volume fraction of formic acid is 0.001%-0.2%, specifically any one or the range between any two of 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.15% and 0.2%, and the volume ratio of methanol to acetonitrile is 1 to 3:1 to 3, specifically any one or the range between any two of 1:1, 1:2, 1:3, 2:1, 2:3, 3:1 and 3:2.

[0042] On the other hand, the embodiments of the present invention provide the use of the kit as described in any of the foregoing embodiments in detecting antipsychotic drugs and their metabolites in a sample.

[0043] In addition, an embodiment of the present invention further provides a method for detecting antipsychotic drugs and their metabolites in a sample, which includes: detecting a sample to be detected using the kit described in any of the foregoing embodiments.

[0044] In some embodiments, the detection method includes: preprocessing the sample, and performing liquid chromatography-tandem mass spectrometry detection on the preprocessed sample;

[0045] The preprocessing includes: adding a protein precipitant or an internal standard solution containing the protein precipitant to the sample, and after centrifugation, taking the supernatant for subsequent detection.

[0046] In some embodiments, the elution conditions of the chromatography are as follows:

[0047] From 0 to 1.2 min, the volume fraction of mobile phase B is maintained at 10% - 20%, and the volume fraction of mobile phase A is maintained at 80% - 90%;

[0048] From 1.2 to 2.5 min, the volume fraction of mobile phase B is increased to 85% - 95%, and the volume fraction of mobile phase A is decreased to 5% - 15%;

[0049] From 2.5 to 3.5 min, the volume fraction of mobile phase B is maintained at 85% - 95%, and the volume fraction of mobile phase A is maintained at 5% - 15%;

[0050] From 3.5 to 4.0 min, the volume fraction of mobile phase B is decreased to 10% - 20%, and the volume fraction of mobile phase A is increased to 80% - 90%;

[0051] From 4.0 to 4.5 min, the volume fraction of mobile phase B is maintained at 10% - 20%, and the volume fraction of mobile phase A is maintained at 80% - 90%.

[0052] In some embodiments, the elution conditions of the chromatography are as follows:

[0053] From 0 to 1.2 min, the volume fraction of mobile phase B is maintained at 13% - 17%, and the volume fraction of mobile phase A is maintained at 83% - 87%;

[0054] From 1.2 to 2.5 min, the volume fraction of mobile phase B is increased to 88% - 92%, and the volume fraction of mobile phase A is decreased to 8% - 12%;

[0055] From 2.5 to 3.5 min, the volume fraction of mobile phase B is maintained at 88% - 92%, and the volume fraction of mobile phase A is maintained at 8% - 12%;

[0056] From 3.5 to 4.0 min, the volume fraction of mobile phase B is decreased to 13% - 17%, and the volume fraction of mobile phase A is increased to 83% - 87%;

[0057] 4.0 - 4.5 min, the volume fraction of mobile phase B is maintained at 13% - 17%, and the volume fraction of mobile phase A is maintained at 83% - 87%.

[0058] In some embodiments, the chromatographic column used in the chromatography is a phenyl chromatographic column.

[0059] In some embodiments, the chromatographic column is a Phenyl - Hexyl LC Column with a particle size of 2.6 μm.

[0060] In some embodiments, the column temperature of the chromatographic column is 35 - 55 °C.

[0061] In some embodiments, the conditions of the mass spectrometry include: using an electrospray ionization source, the detection mode is positive ion mode, the scanning mode is multiple reaction monitoring, spray capillary voltage (Capliary): 2.5 - 3.5 V; ion source temperature: 120 - 150 °C; nebulizing gas temperature is 350 - 550 °C; nebulizing gas flow rate is 800 - 1000 L / h, and cone hole gas flow rate is 50 - 100 L / h.

[0062] In some embodiments, the detection method is not directly aimed at the diagnosis or treatment of diseases.

[0063] The features and properties of the present invention will be further described in detail below in conjunction with embodiments.

[0064] Example 1

[0065] (1) A kit for detecting antipsychotic drugs and their active metabolites.

[0066] The antipsychotic drugs and their active metabolites include: olanzapine (OLA), clozapine (CLO), N - desmethylclozapine (N - CLO), risperidone (RIS), 9 - hydroxyrisperidone (PAL), aripiprazole (ARI), dehydroaripiprazole (ND - ARI), quetiapine (QTP), N - dealkylquetiapine (ND - QTP), chlorpromazine (CPZ), amisulpride (AMI), ziprasidone (ZIP), sulpiride (SUL).

[0067] 1.1. Composition of the kit:

[0068] The kit includes calibration standards, quality control samples, internal standard solution and protein precipitant.

[0069] Calibrator: A standard solution with a concentration gradient obtained by diluting with methanol is mixed with a matrix in a volume ratio of 1:19 to obtain a calibrator working solution (100 μL). After freeze-drying, a calibrator (lyophilized powder) is obtained. When in use, it is reconstituted with 100 μL of pure water; the matrix is composed of fetal bovine serum added with ascorbic acid (stabilizer) and Proclin 300 (lyoprotectant), the final concentration of ascorbic acid is 5 mg / mL, and the final concentration of Proclin 300 is 1% (volume fraction).

[0070] Quality control sample: A standard solution with a concentration gradient obtained by diluting with methanol is mixed with a matrix in a volume ratio of 1:19 to obtain a quality control sample working solution (100 μL). After freeze-drying, a quality control sample (lyophilized powder) is obtained. When in use, it is reconstituted with 100 μL of pure water; the matrix is composed of fetal bovine serum added with ascorbic acid (stabilizer) and Proclin 300 (lyoprotectant), the final concentration of ascorbic acid is 5 mg / mL, and the final concentration of Proclin 300 is 1% (volume fraction).

[0071] Internal standard solution: It is obtained by mixing an internal standard mixed solution, methanol, zinc sulfate, and water in a volume ratio of 1:17:0.02:1.98. The internal standards in the internal standard mixed solution include: N-demethylclozapine-d 8 (N-CLO-d 8 )、Risperidone-d 4 (RIS-d 4 )、9-Hydroxyrisperidone-d 4 (PAL-d 4 )、Quetiapine-d 8 (QTP-d 8 )、Chlorpromazine hydrochloride-d 6 (CPZ-d 6 )、Ziprasidone-d 8 (ZIP-d 8 )、Amisulpride-d 5 (AMI-d 5 )。

[0072] Protein precipitant: An aqueous zinc sulfate solution containing methanol; among them, the concentration of zinc sulfate in the aqueous zinc sulfate solution is 0.6 M, and the volume ratio of methanol to the 0.6 M aqueous zinc sulfate solution is 9:1.

[0073] 1.2. Preparation of the kit

[0074] A. Prepare a stock solution of about 1 mg / ml for each of the 13 analyte standards;

[0075] B. According to the concentration of the stock solution, prepare high-concentration mixed solutions 1 and 2 of 13 analytes as required. The concentrations of the 13 analytes in mixed solution 1 and mixed solution 2 are as follows: olanzapine 9.60 μg / mL, 4.00 μg / mL; clozapine 30.00 μg / mL, 20.00 μg / mL; N-desmethylclozapine 30.00 μg / mL, 20.00 μg / mL; risperidone 3.60 μg / mL, 2.40 μg / mL; 9-hydroxyrisperidone 3.60 μg / mL, 2.40 μg / mL; aripiprazole 30.00 μg / mL, 20.00 μg / mL; dehydroaripiprazole 30.00 μg / mL, 20.00 μg / mL; quetiapine 30.00 μg / mL, 20.00 μg / mL; N-desalkylquetiapine 30.00 μg / mL, 20.00 μg / mL; chlorpromazine 24.00 μg / mL, 12.00 μg / mL; amisulpride 30.00 μg / mL, 12.80 μg / mL; ziprasidone 24.00 μg / mL, 8.00 μg / mL; sulpiride 30.00 μg / mL, 20.00 μg / mL.

[0076] C. Dilute mixed solution 1 and mixed solution 2 successively according to the dilution requirements to obtain standard solutions with concentration gradients. Mix the standard solutions with concentration gradients and the matrix evenly at a volume ratio of 1:19 to obtain calibration working solutions and quality control working solutions, and then lyophilize to obtain calibrators and quality control samples. The matrix is bovine serum containing 5 mg / mL ascorbic acid (stabilizer) and 1% Proclin 300 (lyoprotectant).

[0077] The lyophilization procedure is as follows:

[0078]

[0079] D. Prepare an internal standard stock solution using an internal standard reference substance and prepare an internal standard mixed solution as required; use the prepared protein precipitant to dilute it at a volume ratio of 1:25 for the internal standard mixed solution and the protein precipitant to obtain an internal standard solution. The concentrations of the analytes in the internal standard solution are as follows: N-desmethylclozapine-d 8 2.40 ng / mL; risperidone-d 4 0.60 ng / mL; 9-hydroxyrisperidone-d 4 1.20 ng / mL; quetiapine-d 8 1.80 ng / mL; chlorpromazine-d 6 12.00 ng / mL; amisulpride-d 5 0.90 ng / mL; ziprasidone-d 8 3.00 ng / mL.

[0080] (2) A detection method for an antipsychotic drug and its active metabolite, comprising the following steps. The schematic diagram of the detection process can be referred to Figure 1 .

[0081] 2.1. Sample pretreatment

[0082] Respectively add the calibrator and the quality control product to pure water, dissolve and mix well to obtain the calibrator working solution and the quality control product working solution; take 20 μL of each sample (calibrator working solution, quality control product working solution and the sample to be tested) and add it to a 96-well plate or a centrifuge tube, add 180 μL of the internal standard solution, and obvious white flocculent precipitate appears in the sample (the impurity proteins in the sample are precipitated); shake and mix well; centrifuge at 5000 rpm for 10 minutes to remove the precipitated proteins; take 30 μL of the supernatant, add 120 μL of pure water and mix well, and then perform on-machine detection.

[0083] 2.2. Detection conditions

[0084] The ultra-high performance liquid chromatography conditions are as follows:

[0085] Mobile phase A: An aqueous solution containing 5 mmol / L ammonium acetate;

[0086] Mobile phase B: A mixed solution of methanol and acetonitrile containing formic acid, wherein the volume fraction of formic acid is 0.1%, and the volume ratio of methanol to acetonitrile is 1:1;

[0087] The chromatographic column model is 2.6 μm Phenyl-Hexyl LC Column; the column temperature is set at 40 °C;

[0088] The elution program is as follows:

[0089] 0 - 1.2 min, the volume fraction of mobile phase B is maintained at 15%; at 1.2 - 2.5 min, the volume fraction of mobile phase B is increased to 90%; 2.5 - 3.5 min, the volume fraction of mobile phase B is maintained at 90%; 3.5 - 4.5 min, the volume fraction of mobile phase B is decreased and maintained at 15%, and the collection time for each sample is 4.5 min; the flow rate is 0.5 ml / min; the injection volume is 25 μL.

[0090] Table 1 Elution program

[0091] Time (min) 0.01 1.2 1.21 2.5 3.5 3.51 4.0 4.01 4.5 Mobile phase B % 15 15 15 90 90 90 15 15 15 Flow rate mL / min: 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

[0092] After the sample is separated by liquid phase, it enters the mass spectrometer to be ionized, enters the quadrupole mass analyzer through the ion transmission system, is separated according to the mass-to-charge ratio, and reaches the detector for detection.

[0093] The mass spectrometry conditions are as follows.

[0094] Ion source: Electrospray ionization source (ESI), positive ion mode; Spray capillary voltage (Capliary): 3.0 V; Ion source temperature: 150 °C; Nebulizing gas temperature: 500 °C; Nebulizing gas flow rate: 1000 L / h, Cone gas flow rate: 80 L / h; Scanning mode: Multiple reaction monitoring; The mass spectrometry parameters of each target are as follows in the table.

[0095] Table 2 Mass spectrometry parameters

[0096]

[0097]

[0098] 2.3. Data processing

[0099] Record the ion count response values of the drug to be tested and the internal standard respectively. Use TargetLynx software. With the concentration ratio of the standard substance to the internal standard as the X-axis and the peak area ratio of the standard substance to the internal standard as the Y-axis, based on the calibration standards, establish the calibration curve for each analyte. Substitute the response value ratio of each drug to be tested in the sample obtained by detection to its corresponding internal standard into the calibration curve to calculate the concentrations of the analytes in the quality control samples and the samples to be tested.

[0100] Check the correlation coefficient of the linear fitting equation and the quality control recovery rate of the antipsychotic drugs within their respective concentration ranges. The linear r is greater than 0.9900, and the recovery rate of the quality control samples is between 85% and 115%. The quantitative linear ranges and the correlation coefficients of the standard curves of each analyte are as follows in the table.

[0101] Table 3 Quantitative linear ranges and correlation coefficients of the standard curves of each analyte

[0102]

[0103] The TIC diagrams of 13 antipsychotic drugs and their active metabolites are shown in Figures 2 to 3 .

[0104] Example 2

[0105] Verify the influence of different precipitants on the detection.

[0106] Based on the kit and detection method of Example 1, set up 3 experimental groups 1 - 3. Experimental group 3 uses the kit and detection method of Example 1. The kits and detection methods of experimental groups 1 - 2 are roughly the same as those of experimental group 3, except that the protein precipitants are different.

[0107] Experimental group 1 uses methanol as the precipitant, and the spike recovery rates of 13 analytes are as follows.

[0108] Table 4 Spike recovery rates of 13 analytes in experimental group 1

[0109]

[0110]

[0111] In Experiment Group 2, acetonitrile was used as the precipitant, and the spiked recovery rates of 13 analytes were as follows.

[0112] Table 5 Spiked Recovery Rates of 13 Analytes in Experiment Group 2

[0113]

[0114]

[0115] In Experiment Group 3, a mixed solution of methanol, zinc sulfate, and water was used as the precipitant, and the spiked recovery rates of 13 analytes were as follows.

[0116] Table 6 Spiked Recovery Rates of 13 Analytes in Experiment Group 3

[0117]

[0118] By comparison, when the precipitant was the aqueous solution of zinc sulfate containing methanol in Experiment Group 3 (volume ratio of methanol to 0.6 M aqueous zinc sulfate solution = 9:1), the accuracy of 13 analytes was within 85%-115%.

[0119] Example 3

[0120] Verify the influence of different product forms on detection.

[0121] Based on the kit and detection method provided in Example 1, Experiment Group 1 (calf serum), Experiment Group 2 (calf serum + stabilizer), and Experiment Group 3 (lyophilized calf serum + stabilizer) were set up. Experiment Group 3 used the kit and detection method provided in Example 1. Experiment Groups 1-2 were roughly the same as Experiment Group 3, except that the calibrators were different. The matrix of the calibrator in Experiment Group 1 did not contain the stabilizer compared with that in Experiment Group 3, and the calibrator in Experiment Group 2 did not undergo the lyophilization procedure compared with that in Experiment Group 3.

[0122] Based on Experiment Groups 1-3, the real-time stability of the product was detected, and the results were as follows.

[0123] Table 7 Stability Results

[0124]

[0125]

[0126]

[0127] It can be seen from the results that the relative deviation of the calibrator in Experimental Group 3 within 12 months of frozen storage is within 15%, that is, it can be stably stored frozen for 12 months.

[0128] Example 4

[0129] Verify the performance of the kit and the detection method provided in Example 1.

[0130] (1) Accuracy of the quality control sample

[0131] Table 8 Accuracy of the quality control sample

[0132]

[0133]

[0134] (2) Investigation results of upper and lower limit samples

[0135] Table 9 Investigation results of upper and lower limit samples

[0136]

[0137] (3) Investigation results of spike recovery

[0138] Table 10 Investigation results of spike recovery

[0139]

[0140]

[0141] (4) Investigation results of precision

[0142] Table 11 Investigation results of precision 1

[0143]

[0144] Table 12 Investigation results of precision 2

[0145]

[0146] Table 13 Investigation results of precision 3

[0147]

[0148]

[0149] Kits 1 to 3 are kits of different batches.

[0150] (5) Results of inter-batch precision

[0151] Table 14 Results of inter-batch precision

[0152]

[0153] (6) Residual investigation results

[0154] Table 15 Residual investigation results

[0155]

[0156]

[0157] Based on the above experiments, the technical indicators such as precision, recovery rate, and residue of the kit and detection method provided by the embodiments of the present invention all meet the requirements. The kit can simultaneously detect the concentrations of 13 antipsychotic drugs in human serum, with good reproducibility and good spike recovery rate, thus improving the accuracy of the detection results and eliminating systematic errors.

[0158] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A kit, characterized in that: It includes: Any one or more combinations of calibrators for the analyte, quality control products for the analyte, internal standard solutions and protein precipitants; The analyte comprises any one or more of olanzapine, clozapine, N-norclozapine, risperidone, 9-hydroxyrisperidone, aripiprazole, dehydroaripiprazole, quetiapine, N-desalkylquetiapine, chlorpromazine, amisulpride, ziprasidone and sulpiride; The protein precipitant includes an aqueous zinc sulfate solution containing methanol.

2. The kit according to claim 1, characterized in that In the protein precipitant, the volume fraction of methanol is 80% to 95%; Optionally, in the aqueous zinc sulfate solution, the concentration of zinc sulfate is 0.4 to 0.8 M; Optionally, in the aqueous zinc sulfate solution, the concentration of zinc sulfate is 0.5-0.7M.

3. The kit according to claim 1, characterized in that The calibration product is a lyophilized product obtained by lyophilizing the calibration product working solution; the quality control product is a lyophilized product obtained by lyophilizing the quality control product working solution; The calibration substance working solution and the quality control substance working solution are solutions with a concentration gradient formed by diluting a standard solution of the analyte with a known concentration through a matrix; Wherein, the matrix comprises bovine serum and a stabilizer, the stabilizer comprises ascorbic acid, and the final concentration of the stabilizer in the matrix is ​​1 to 10 mg / mL; Optionally, the standard solution of the analyte is obtained by diluting a standard with methanol; Optionally, in the calibration working solution and / or the quality control working solution, the volume ratio of the standard solution of the analyte to the matrix is ​​1:10-30; Optionally, the matrix further comprises a preservative; Optionally, the volume fraction of the preservative in the matrix is ​​0.01% to 5%; Optionally, the preservative comprises Proclin300.

4. The kit according to claim 3, characterized in that The freeze-drying procedure includes the following steps: cooling to -50 to -40°C for pre-freezing for 6 to 10 hours; heating to -20 to -10°C for the first sublimation for 13 to 17 hours; heating to -10 to 10°C for the second sublimation for 8 to 12 hours; heating to 30 to 40°C for analytical drying for 8 to 12 hours.

5. The kit according to any one of claims 1 to 4, characterized in that The internal standard solution includes an internal standard mixed solution, and the internal standard in the internal standard mixed solution includes any one or more combinations of the following: N-desmethylclozapine-d8, risperidone-d4, 9-hydroxyrisperidone-d4, quetiapine-d8, chlorpromazine hydrochloride-d6, ziprasidone-d8 and amisulpride-d5; Optionally, in the internal standard mixed solution, the molar ratio of N-desmethylclozapine-d8, risperidone-d4, 9-hydroxyrisperidone-d4, quetiapine-d8, chlorpromazine hydrochloride-d6, ziprasidone-d8 and amisulpride-d5 is 3-5:1:1-3:2-4:18-22:4-6:0.5-3; Optionally, the internal standard solution contains the protein precipitant; Optionally, the volume ratio of the internal standard mixed solution to the protein precipitant is 1:15-30.

6. The kit according to any one of claims 1 to 4, characterized in that The kit also includes mobile phase A and mobile phase B; Optionally, the mobile phase A comprises an aqueous solution containing ammonium acetate, and the final concentration of sodium acetate is 1 to 10 mmol / L; Optionally, the mobile phase B comprises a mixed solution of methanol containing formic acid and acetonitrile, wherein the volume fraction of formic acid is 0.001%-0.2%, and the volume ratio of methanol to acetonitrile is 1-3:1-3.

7. Use of the kit according to any one of claims 1 to 6 in detecting antipsychotic drugs and their metabolites in a sample.

8. A method for detecting antipsychotic drugs and their metabolites in a sample, characterized in that: It includes: The test sample is tested using the kit described in any one of claims 1 to 6.

9. The detection method according to claim 8, characterized in that: The detection method comprises: pre-treating the sample, and performing liquid chromatography tandem mass spectrometry detection on the pre-treated sample; The pretreatment includes: adding a protein precipitant or an internal standard solution containing the protein precipitant to the sample, centrifuging the sample, and taking the supernatant for subsequent detection.

10. The detection method according to claim 8 or 9, characterized in that: The elution conditions of the chromatography are as follows: 0-1.2min, the volume fraction of mobile phase B is maintained at 10%-20%, and the volume fraction of mobile phase A is maintained at 80%-90%; 1.2-2.5 min, the volume fraction of mobile phase B increases to 85%-95%, and the volume fraction of mobile phase A decreases to 5%-15%; 2.5-3.5 min, the volume fraction of mobile phase B is maintained at 85%-95%, and the volume fraction of mobile phase A is maintained at 5%-15%; 3.5-4.0 min, the volume fraction of mobile phase B is reduced to 10%-20%, and the volume fraction of mobile phase A is increased to 80%-90%; 4.0-4.5 min, the volume fraction of mobile phase B is maintained at 10%-20%, and the volume fraction of mobile phase A is maintained at 80%-90%; Optionally, the chromatographic column used in the chromatography is a phenyl chromatographic column; Optionally, the chromatographic column is a Phenyl-Hexyl LC Column with a particle size of 2.6 μm; Optionally, the column temperature of the chromatographic column is 35 to 55°C; Optionally, the mass spectrometry conditions include: using an electrospray ion source, the detection mode is a positive ion mode, the scanning mode is multiple reaction monitoring, the spray capillary voltage is 2.5-3.5V; the ion source temperature is 120-150°C; the nebulizing gas temperature is 350-550°C; the nebulizing gas flow rate is 800-1000L / h, and the cone gas flow rate is 50-100L / h.

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