CN114414707B

CN114414707B - Method and kit for detecting 19 drugs and metabolites thereof in blood by liquid chromatography tandem mass spectrometry

Info

Publication number: CN114414707B
Application number: CN202210321558.4A
Authority: CN
Inventors: 贾永娟; 刘春冉; 刘杏立; 张�杰; 倪君君
Original assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Current assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Priority date: 2022-03-30
Filing date: 2022-03-30
Publication date: 2022-07-22
Anticipated expiration: 2042-03-30
Also published as: CN114414707A

Abstract

The invention belongs to the technical field of drug detection, and particularly relates to a method and a kit for detecting 19 drugs and metabolites thereof in blood by liquid chromatography-tandem mass spectrometry. The substance to be detected comprises sulpiride, pentafluridol, mianserin, buspirone, tandospirone, hydroxyzine, diazepam, venlafaxine, moclobemide, imipramine, paroxetine, reboxetine, amitriptyline, sertraline, digoxin, clonazepam, clopidogrel, tolbutamide, glimepiride, 1-pyrimidinylpiperazine, desvenlafaxine, 6-hydroxybuspirone, desipramine, desmetazoline, desmedipam, clopidogrel metabolites; the detection method comprises the following steps: calibrating a standard solution, processing a sample to be detected, and detecting the sample to be detected by adopting high performance liquid chromatography-mass spectrometry. The embodiment of the invention can quickly and accurately measure the content, and the sample processing method is simple and easy to implement, high in sensitivity and accurate in quantification.

Description

Method and kit for detecting 19 drugs and metabolites thereof in blood by liquid chromatography tandem mass spectrometry

Technical Field

The invention belongs to the technical field of drug detection, and particularly relates to a method and a kit for detecting 19 drugs and metabolites thereof in blood by liquid chromatography-tandem mass spectrometry.

Background

Therapeutic Drug Monitoring (TDM) is a pharmaceutical clinical discipline for studying individualized drug treatment mechanisms, techniques, methods and clinical standards and transforming research results into clinical treatments to maximize rational drug administration. The key point is individualized drug treatment, TDM has clinical significance in optimizing drug treatment scheme, improving drug curative effect, reducing toxic and side effects, and simultaneously, drug treatment cost can be saved through reasonable medication maximization. The TDM monitoring is needed to be carried out on a plurality of types of medicines, in order to better monitor the medicine concentration, a plurality of hospital/third-party detection organizations develop a one-needle multi-detection method, most of the existing one-needle multi-detection methods put the same type of medicines into one method bag, but the actual situation is that the situation that a plurality of medicines are taken simultaneously for the same disease is less, but the situation that different types of medicines are taken simultaneously for the old is more and more common. In order to better monitor the concentration content of the taken medicine and better guide a clinician to effectively adjust the medication scheme, thereby obtaining a better treatment scheme and relieving the pain of a patient in the shortest time.

Patients with antipsychotic drugs, anxiolytic drugs, antidepressant drugs, cardiac glycosides, antiepileptic drugs, platelet aggregation inhibitors, hypoglycemic drugs and other drugs can take the drugs at the same time, different drugs play roles in different diseases, all the drugs need to be monitored in blood concentration, and individual drugs still have critical values and can cause poisoning after exceeding specific concentrations.

Therefore, there is an urgent need to find a method for detecting the above drugs and their metabolites in blood, which has simple and easy pretreatment operation, high sensitivity, and accurate quantification.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method and a kit for detecting 19 drugs and metabolites thereof in blood by liquid chromatography-tandem mass spectrometry.

The invention provides a method for detecting 19 drugs and metabolites thereof in liquid chromatography-tandem mass spectrometry blood, wherein substances to be detected in the detection method comprise the 19 drugs and the 7 metabolites; the 19 medicines comprise sulpiride, pentafluridol, mianserin, buspirone, tandospirone, hydroxyzine, diazepam, venlafaxine, moclobemide, imipramine, paroxetine, reboxetine, amitriptyline, sertraline, digoxin, clonazepam, clopidogrel, tolbutamide, glimepiride; the 7 metabolites comprise 1-pyrimidinylpiperazine, desvenlafaxine, 6-hydroxybuspirone, desipramine, desmetidine, desmedipam, clopidogrel MP derivatives; the detection method at least comprises the following steps:

s1, calibrating a standard solution: preparing a standard working solution containing the 19 medicaments and the 7 metabolites by using a methanol aqueous solution; preparing an internal standard working solution by adopting a methanol aqueous solution; obtaining standard curve equations of 19 drugs and 7 metabolites by using the standard working solution and the internal standard working solution;

s2, processing the sample to be detected: adding an extracting agent containing the internal standard working solution into the anticoagulated blood sample, extracting to obtain supernatant, adding a complex solution, uniformly mixing, and centrifuging to obtain a sample to be detected;

the extraction agent is selected from methyl tert-butyl ether, and the complex solution is selected from methanol with the volume ratio of 1: mixing the solution with water;

and S3, detecting the sample to be detected by adopting high performance liquid chromatography-mass spectrometry.

Optionally, the standard working solution contains the following standard substances: clopidogrel bisulfate standards, clopidogrel MP derivative standards, 1- (2-pyrimidine) piperazine standards, tandospirone standards, mianserin standards, imipramine hydrochloride standards, hydroxyzine hydrochloride standards, desipramine standards, penfluridol standards, digoxin standards, tolbutamide standards, paroxetine standards, reboxetine standards, venlafaxine standards, desvenlafaxine standards, glimepiride standards, buspirone standards, 6-hydroxybuspirone standards, clonazepam standards, sertraline standards, moclobemide standards, sulpiride standards, diazepam standards, nordiazepam standards, amitriptyline standards, noramitriptyline standards; the standard working solution comprises 8 grades of concentrations of L1, L2, L3, L4, L5, L6, L7 and L8;

preferably, methanol is used for each of the levels: the volume ratio of water is 7:3, diluting the mixed solution from the intermediate solution; the intermediate solution adopts methanol: the volume ratio of water is 7:3 is obtained by diluting the mother liquor; and the mother liquor is obtained by dissolving the standard substance by adopting methanol or a methanol water solution.

Optionally, the internal standard working solution contains the following internal standard substances: clopidogrel-D3, clopidogrel metabolite derivatives-13C 6, 1- (2-pyrimidine) piperazine-D8, tandospirone-D8, mianserin-D3, imipramine-D3, hydroxyzine-D8, desipramine-D3, pentafluridol-D7, amitriptyline-D3, nomitriptyline-D3, buspirone hydrochloride-D8, 6-hydroxybuspirone-D8, clonazepam-D4, digoxin-D3, venlafaxine-D6, glimepiride-D5, moclobemide-D8, levosulpiride-D3, diazepam-D5.

Preferably, the internal standard working solution contains the following internal standard substances in concentration: clonazepam-D4: 100 ng/mL; digoxin-D3: 100 ng/mL; clopidogrel-D3: 40 ng/mL; clopidogrel metabolite-C6: 500 ng/mL; glimepiride-D5: 100 ng/mL; pentaflurido-D7: 3200 ng/mL; imipramine-D3: 2000 ng/mL; desipramine-D3: 2000 ng/mL; mianserin-D3: 1000 ng/mL; tandospirone-D8: 20 ng/mL; 1-pyrimidinylpiperazine-D8: 10 ng/mL; buspirone-D8: 20 ng/mL; 6-hydroxybuspirone-D8: 20 ng/mL; hydroxyzine-D8: 40 ng/mL; venlafaxine-D6: 2000 ng/mL; moclobemide-D8: 1000 ng/mL; amitriptyline-D3: 500 ng/mL; noramitriptyline-D3: 2000 ng/mL; diazepam-D5: 400 ng/mL; levosulpiride-D3: 1000 ng/mL.

Optionally, the volume ratio of the serum/plasma sample to the extractant is 1: 5-1: 15, preferably 1: 10; preferably, the volume ratio of the serum/plasma sample to the internal standard working solution is 10: 1; more preferably, the supernatant is dried and then added into the reconstituted solution, and the volume ratio of the supernatant to the reconstituted solution is 9: 1-10: 1, preferably 9.5: 1.

optionally, the processing the sample to be tested includes: adding an internal standard working solution and an extracting agent into a serum/plasma sample, performing vortex for 4-8 minutes, preferably 5 minutes, and centrifuging for 8-12 minutes, preferably 10 minutes, taking a supernatant, drying, adding the complex solution, performing vortex for 0.5-2 minutes, preferably 1 minute, and centrifuging for 4-7 minutes, preferably 5 minutes to obtain a supernatant which is the sample to be detected;

the rotational speed of the vortex is 2000 rpm; the centrifugal speed was 14000 rpm. Optionally, when the hplc-ms is AB SCIEX;

the chromatographic column is SHIMADZU Shim-pack Shim-pack Velock C18, 2.1X 100mm, 2.7 μm; the column temperature is 50 ℃; mobile phase: the phase A is water containing formic acid and ammonium formate, and the phase B is methanol containing formic acid and ammonium formate; preferably, A is water containing 0.1 v/v% formic acid and 5mM ammonium formate; b is methanol containing 0.1 v/v% formic acid and 5mM ammonium formate;

gradient elution: 0-1 minute: 60% of A and 40% of B; 2.00-2.5 min: 40% of A and 60% of B; 3.50-5 minutes: 20% of A and 80% of B; 5.01-6.50 minutes: 60% of A and 40% of B; the flow rate is: 0.3 mL/min;

preferably, the sample injection amount of the standard working solution of L1, L2, L3, L4, L5 and L6 is 5 mu L, and the sample injection amount of the standard working solution of L7 and L8 is 1 mu L; the sample injection amount of the sample to be detected is 5 mu L; more preferably, the analysis time is 6.5 minutes;

the mass spectrum parameters are as follows: ion source temperature: 350 ℃; ion source high pressure: 5500V; air curtain air: 20L/min; collision gas: 8L/min; drying gas 1: 50L/min; drying gas 2: 50L/min.

Optionally, when the hplc-ms is AB SCIEX; the internal standard of sulpiride is levosulpiride-D3, the internal standard of desvenlafaxine is venlafaxine-D6, the internal standard of moclobemide is moclobemide-D8, the internal standard of 6-hydroxybuspirone is 6-hydroxybuspirone-D8, the internal standard of tandospirone is tandospirone-D8, the internal standard of 1-pyrimidine piperazine is 1-pyrimidine piperazine-D8, the internal standard of buspirone is buspirone-D8, the internal standard of venlafaxine is venlafaxine-D6, the internal standard of mianserin is mianserin-D3, the internal standard of reboxetine is desmiramide-D3, the internal standard of paroxetine is clonazepam-D4, the internal standard of clonazepam is clonazepam-D4, the internal standard of imipramine is imipramine-D3, the internal standard of desipramipezine-D3, and the internal standard of amitriptyline-D3, the internal standard of the hydroxyzine is hydroxyzine-D8, the internal standard of the noramitriptyline is noramitriptyline-D3, the internal standard of the digoxin is digoxin-D3, the internal standard of the sertraline is mepimizine-D3, the internal standard of the tolbutamide is glimepiride-D5, the internal standard of the nordiazepam is diazepam-D5, the internal standard of the diazepam is diazepam-D5, the internal standard of the pentafluridol is pentafluridol-D7, the internal standard of the glimepiride is glimepiride-D5, the internal standard of the clopidogrel metabolite is clopidogrel metabolite-C6, and the internal standard of the clopidogrel is clopidogrel-D3.

Optionally, when the high performance liquid chromatography-mass spectrometer is Agilent LC1260/MS6470A, the chromatographic column is SHIMADZU Shim-pack Shim-pack Velock C18, and the parameters are 2.1 × 100mm and 2.7 μm; the column temperature is 50 ℃;

mobile phase: the phase A is water containing formic acid and ammonium formate, and the phase B is methanol containing formic acid and ammonium formate; preferably, A is water containing 0.1 v/v% formic acid and 5mM ammonium formate; b is methanol containing 0.1 v/v% formic acid and 5mM ammonium formate;

gradient elution: 0-1.00 min: 60% of A and 40% of B; 1.50-3.5 minutes: 30% of A and 70% of B; 4.00-5.50 minutes: a0%, B100%; 5.51-7.5 minutes: 60% of A and 40% of B; the flow rate is: 0.3 mL/min;

preferably, the sample injection amount of the standard working solution of L1, L2, L3, L4, L5 and L6 is 5 mu L, and the sample injection amount of the standard working solution of L7 and L8 is 2 mu L; the sample injection amount of the sample to be detected is 5 mu L; more preferably, the analysis time is 7.5 minutes;

the mass spectrum parameters are as follows: an acquisition mode: ESI (+); MRM; temperature of atomized gas: 200 ℃; atomizing airflow: 3L/min; temperature of sheath gas: 200 ℃; sheath gas flow: 11L/min; atomizing gas pressure: 45 psi; capillary voltage: 5000V; Delta-EMV: 100V.

Optionally, the internal standard of sulpiride is 1-pyrimideperazine-D8, the internal standard of desvenlafaxine is venlafaxine-D6, the internal standard of moclobemide is moclobemine-D8, the internal standard of 6-hydroxybuspirone is 6-hydroxybuspirone-D8, the internal standard of tandospirone is tandospirone-D8, the internal standard of 1-pyrimideperazine-D8, the internal standard of buspirone is buspirone-D8, the internal standard of venlafaxine is venlafaxine-D6, the internal standard of mianserin is mianserin-D3, the internal standard of reboxetine is hydroxyzine-D8, the internal standard of paroxetine is clonazepam-D4, the internal standard of clonazepam is clonazepam-D4, the internal standard of imipramipezine is imipramipezine-D3, the internal standard of desipramipezine-D3, and the internal standard of amitriptyline D3, the internal standard of the hydroxyzine is hydroxyzine-D8, the internal standard of the noramitriptyline is noramitriptyline-D3, the internal standard of the digoxin is digoxin-D3, the internal standard of the sertraline is clonazepam-D4, the internal standard of the tolbutamide is amitriptyline-D3, the internal standard of the nordiazepam is amitriptyline-D3, the internal standard of the diazepam is noramitriptyline-D3, the internal standard of the penfluridol is pentafluridol-D7, the internal standard of the glimepiride is glimepiride-D5, the internal standard of the clopidogrel metabolite is clopidogrel metabolite-C6, and the internal standard of the clopidogrel is clopidogrel-D3.

The invention also provides a kit for 19 medicines and metabolites thereof in blood, wherein the 19 medicines comprise sulpiride, pentafluridol, mianserin, buspirone, tandospirone, hydroxyzine, diazepam, venlafaxine, moclobemide, imipramine, paroxetine, reboxetine, amitriptyline, sertraline, digoxin, clonazepam, clopidogrel, tolbutamide, glimepiride; the metabolites include 1-pyrimidinylpiperazine, desvenlafaxine, 6-hydroxybuspirone, desipramine, desmirazone, desmedipam, clopidogrel metabolites;

the kit comprises the following reagents:

(1) internal standard working solution: (2) a standard working fluid; (3) extracting agent: methyl tert-butyl ether;

(4) compounding solution: the volume ratio is 1:1 methanol: mixing the solution with water; (5) positive and negative controls;

the internal standard working solution contains the following internal standards in concentration: clonazepam-D4: 100 ng/mL; digoxin-D3: 100 ng/mL; clopidogrel-D3: 40 ng/mL; clopidogrel metabolite-C6: 500 ng/mL; glimepiride-D5: 100 ng/mL; pentaflurido-D7: 3200 ng/mL; imipramine-D3: 2000 ng/mL; desipramine-D3: 2000 ng/mL; mianserin-D3: 1000 ng/mL; tandospirone-D8: 20 ng/mL; 1-pyrimidinylpiperazine-D8: 10 ng/mL; buspirone-D8: 20 ng/mL; 6-hydroxybuspirone-D8: 20 ng/mL; hydroxyzine-D8: 40 ng/mL; venlafaxine-D6: 2000 ng/mL; moclobemide-D8: 1000 ng/mL; amitriptyline-D3: 500 ng/mL; noramitriptyline-D3: 2000 ng/mL; diazepam-D5: 400 ng/mL; levosulpiride-D3: 1000 ng/mL.

The standard working solution contains the following standard substances: clopidogrel bisulfate standards, clopidogrel active metabolite derivative standards, 1- (2-pyrimidine) piperazine standards, tandospirone standards, mianserin standards, imipramine hydrochloride standards, hydroxyzine hydrochloride standards, desipramine standards, pentafluridol standards, digoxin standards, tolbutamide standards, paroxetine standards, reboxetine standards, venlafaxine standards, desvenlafaxine standards, glimepiride standards, buspirone standards, 6-hydroxybuspirone standards, clonazepam standards, sertraline standards, moclobemide standards, sulpiride standards, diazepam standards, desdiazepam standards, amitriptyline standards, noramitriptyline standards; preferably, the method also comprises a chromatographic column mobile phase; the phase A is water containing formic acid and ammonium formate, and the phase B is methanol containing formic acid and ammonium formate; more preferably, A is water containing 0.1 v/v% formic acid and 5mM ammonium formate; b is methanol containing 0.1 v/v% formic acid and 5mM ammonium formate; the positive control is added with a standard quality control product; the volume ratio of the negative control substance is 7:3 methanol: and (3) water.

Compared with the prior art, the technical scheme provided by the embodiment of the invention has the following advantages:

the embodiment of the invention provides a liquid quality analysis method for detecting the contents of 19 drugs and metabolites thereof in blood, which can rapidly and accurately detect the contents of the 19 drugs and the metabolites thereof, has the advantages of simple and easy sample processing method, high sensitivity, accurate quantification and wide application. The detection method has the advantages that the analysis time is 6.5 minutes or 7.5 minutes, 26 substances are sprayed by one needle, 10 samples can be analyzed in 1 hour, and the analysis time is short.

Drawings

FIG. 1 is a chromatogram for detecting a working solution with a certain concentration by using Agilent 6470 in an embodiment of the present invention;

FIG. 2 is a chromatogram of a quality control sample detected by Agilent 6470 according to an embodiment of the present invention;

FIG. 3 is a chromatogram for detecting a working solution with a certain concentration by using AB4500MD according to an embodiment of the present invention;

FIG. 4 is a chromatogram of a quality control sample detected by AB4500MD according to an embodiment of the present invention;

FIG. 5 is a chromatogram of the tailing of mianserin in a comparative example of the present invention;

FIG. 6 is a chromatogram of a reboxetine tail in a comparative example of the present invention;

FIG. 7 is a chromatogram of a tail of paroxetine in a comparative example of the present invention;

FIG. 8 is a chromatogram of the imipramine tail in a comparative example of the present invention;

FIG. 9 is a chromatogram showing improved anserin tailing in an example of the present invention;

FIG. 10 is a chromatogram showing improved reboxetine tailing in an embodiment of the present invention;

FIG. 11 is a chromatogram showing improved tailing of paroxetine in the examples of the present invention;

FIG. 12 is a chromatogram showing improved tailing of imipramine in an example of the present invention;

FIG. 13 is a chromatogram showing inseparability of an internal standard of a clopidogrel metabolite from impurities in a comparative example of the present invention;

fig. 14 is a chromatogram for separating an internal standard of a clopidogrel metabolite from impurities in an example of the present invention.

Detailed Description

In order that the above objects, features and advantages of the present disclosure may be more clearly understood, aspects of the present invention will be further described below. It should be noted that the embodiments and features of the embodiments of the present invention may be combined with each other without conflict.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the invention may be practiced otherwise than as described herein; it is to be understood that the embodiments described in this specification are only some embodiments of the invention, and not all embodiments.

The embodiment of the invention provides a method for detecting 19 drugs and metabolites thereof in blood, wherein substances to be detected in the detection method comprise the 19 drugs and the 7 metabolites as shown in table 1:

TABLE 1

The detection method of the embodiment of the invention at least comprises the following steps:

s1, calibrating a standard solution: preparing a standard working solution containing the 19 medicines and the standard substance of 7 metabolites by using a methanol aqueous solution; preparing an internal standard working solution by adopting a methanol aqueous solution; obtaining a standard curve equation of 19 medicaments and 7 metabolites by using the standard working solution and the internal standard working solution;

s2, processing the sample to be detected: adding an extracting agent containing an internal standard working solution into a serum/plasma sample, extracting to obtain a supernatant, adding a complex solution into the supernatant, uniformly mixing, and centrifuging to obtain a sample to be detected;

wherein the extracting agent is selected from methyl tert-butyl ether, and the redissolution is selected from methanol with the volume ratio of 1: mixing the solution with water;

The embodiment of the invention provides a liquid quality analysis method for detecting the contents of 19 drugs and metabolites thereof in blood, which can rapidly and accurately detect the contents of the 19 drugs and the metabolites thereof, has the advantages of simple and easy sample processing method, high sensitivity, accurate quantification and wide application. The extraction agent is methyl tert-butyl ether which is selected for considering the properties of 19 medicines and 26 detection substances, and the methyl tert-butyl ether is finally selected because the polarities are different, so that the extraction effect of the substances to be detected can be ensured, the interference of impurities can be minimized, and the sample analysis can be completed in a short analysis time. In the conventional experimental operation, the redissolution is generally selected from pure organic solvents, such as methanol, acetonitrile, or organic solvents and water, and is mixed according to a certain ratio, the redissolution selected in the present application is methanol: water =1:1, the re-solution is determined to match pre-analysis conditions, so that the solvent effect can be effectively avoided, the sensitivity of responding low substances can be ensured, and the quantitative requirement can be met.

In S1 of the embodiment of the present invention, the method includes

S11, preparing various standard working solutions and internal standard working solutions:

the standard working solution of the embodiment of the invention contains the following standard substances: clopidogrel bisulfate standards, clopidogrel active metabolite derivative standards, 1- (2-pyrimidine) piperazine standards, tandospirone standards, mianserin standards, imipramine hydrochloride standards, hydroxyzine hydrochloride standards, desipramine standards, pentafluridol standards, digoxin standards, tolbutamide standards, paroxetine standards, reboxetine standards, venlafaxine standards, desvenlafaxine standards, glimepiride standards, buspirone standards, 6-hydroxybuspirone standards, clonazepam standards, sertraline standards, moclobemide standards, sulpiride standards, diazepam standards, desdiazepam standards, amitriptyline standards, noramitriptyline standards; the standard working solution comprises 8 grades of concentrations of L1, L2, L3, L4, L5, L6, L7 and L8. Specifically, the concentrations of the substances in each standard working solution are shown in table 2:

TABLE 2

Wherein, all levels of concentration adopt methanol: the volume ratio of water is 7:3, diluting the mixed solution from the intermediate solution; the intermediate solution adopts methanol: the volume ratio of water is 7:3, diluting the mixed solution from the mother solution; the mother liquor is obtained by dissolving a standard substance in absolute methanol or methanol water solution. Methanol is adopted: water = 7:3, the technical advantages of preparing the standard working solution and the intermediate solution by using the mixed solution are as follows: the standard substance is fully dissolved, and the storage time of the intermediate and working solution can be ensured to be long and stable.

In the preparation of mother liquor, some standard products are prepared by adopting anhydrous methanol, some standard products are prepared by adopting a methanol aqueous solution, and the ratio of methanol in the methanol aqueous solution is as follows: the volume ratio of water is 5-7: 3-5, preferably 1:1 or 7: 3; for example, the standard substance of buspirone, 6-hydroxy buspirone and clonazepam adopts methanol: preparing mother liquor by water =1:1, and preparing the glimepiride and moclobemide standard substance by adopting methanol: water = 7: and 3, preparing a mother solution, wherein the rest standard products can be prepared into the mother solution by adopting anhydrous methanol. The preparation method has the advantages that: the standard substance is fully dissolved and stored for a long time, and the mother liquor is still stable for 12 months at present, which is very critical for practical detection.

Specifically, the standard working solution can be prepared by the method of example 1 of the present invention.

The internal standard working solution of the embodiment of the invention contains the following internal standard substances: clopidogrel-D3, clopidogrel metabolite derivative-13C 6, 1- (2-pyrimidine) piperazine-D8, tandospirone-D8, mianserin-D3, imipramine-D3, hydroxyzine-D8, desipramine-D3, pentafluridol-D7, amitriptyline-D3, nomitriptyline-D3, buspirone hydrochloride-D8, 6-hydroxybuspirone-D8, clonazepam-D4, digoxin-D3, venlafaxine-D6, glimepiride-D5, moclobemide-D8, levosulpiride-D3, diazepam-D5.

In a preferred embodiment, the concentrations of the respective substances in the internal standard working solution are shown in table 3:

TABLE 3

	Internal standard substance	Concentration (ng/mL)
			1	clonazepam-D4	100
2	digoxin-D3	100
			3	clopidogrel-D3	40
4	Clopidogrel metabolite-C6	500
			5	Glimepiride-D5	100
6	Pentaflurido-D7	3200
			7	imipramine-D3	2000
8	desipramine-D3	2000
			9	mianserin-D3	1000
10	Tandospirone-D8	20
			11	1-pyrimidinylpiperazine-D8	10
12	buspirone-D8	20
			13	6-hydroxybuspirone-D8	20
14	hydroxyzine-D8	40
			15	venlafaxine-D6	2000
16	moclobemide-D8	1000
			17	amitriptyline-D3	500
18	Noramitriptyline-D3	2000
			19	diazepam-D5	400
20	levosulpiride-D3	1000

Specifically, the method of example 2 of the present invention can be used to prepare the internal standard working solution.

S12, obtaining standard curve equations of 19 drugs and 7 metabolites by using the standard working solution and the internal standard working solution.

The specific method comprises the following steps:

at least three different concentrations of standard working solution 10 μ L, internal standard working solution 10 μ L and methanol were first pipetted: 80 mu L of mixed solution with the water volume ratio of 1:1 is respectively put into a 1.5mL centrifuge tube to be mixed into at least three standard solutions, after the standard solutions are respectively uniformly mixed by vortex at the rotating speed of 2000rpm for 30S, a liquid chromatography-mass spectrometry instrument (the specific parameters are the same as S3) is used for detecting the standard solutions to obtain at least three standard solutions, 19 medicines and metabolites thereof and an internal standard chromatogram, taking the ratio of the peak area of each substance of the at least three standard solutions to the peak area of the corresponding internal standard substance as the ordinate y of the standard curve chart, taking the ratio of the concentration of each substance in the standard working solution to the corresponding internal standard concentration as the abscissa x of a standard curve graph, performing linear regression on at least three groups of data obtained by detection, fitting to obtain a standard curve equation of y = a x + b, and obtaining weight coefficients a and b; the relative concentration x of 26 substances in total of 19 drugs and metabolites thereof in the blood sample to be detected is obtained through calculation, and the concentration of the internal standard substance working solution is known, so that the concentration of 26 substances in total of 19 drugs and metabolites thereof in the blood to be detected in the sample can be calculated.

S2 of the embodiment of the present invention includes:

s21, obtaining a serum/plasma sample, wherein the specific method comprises the following steps: collecting at least 2mL of serum to be detected by using a serum collection tube/EDTA collection tube/heparin lithium collection tube, centrifuging at a centrifugal speed of 3500rpm for 10 minutes, taking supernatant to obtain serum/EDTA plasma/heparin lithium plasma, and freezing the serum or plasma at-20 ℃ for storage until the serum or plasma is reserved before analysis.

S22, adding internal standard working solution and methyl tert-butyl ether into the serum/plasma sample, and extracting to obtain a supernatant; the embodiment of the invention has the advantages that the extraction effect of all substances is better and the interference of impurities is less because the extraction agent is methyl tert-butyl ether. The volume ratio of serum/plasma sample to methyl tert-butyl ether was 1: 5-1: 15, preferably 1: 10. the digoxin signal is low, so if the extraction amount is too small, the extraction effect is poor, and the digoxin signal is not enough; if too much extractant is present, the nitrogen purge time is too long. The volume ratio of the anticoagulant liquid sample to the internal standard working solution is 10: 1;

the specific process parameters are as follows: adding an internal standard working solution and an extracting agent into a serum/plasma sample, performing vortex for 4-8 minutes, preferably 5 minutes, and then centrifuging for 8-12 minutes, preferably 10 minutes to obtain a supernatant;

s23, drying the supernatant, and adding a compound solution, wherein the compound solution is a methanol water solution with the volume ratio of 1: 1; the re-solution can be matched with a pre-analysis method, so that the solvent effect is avoided, and the response of low-concentration substances is ensured; volume ratio of supernatant to reconstituted solution 9: 1-10: 1, preferably 9.5: 1; if the addition amount of the complex solution is too large, the digoxin response is too low, and the requirement of the quantitative limit cannot be met, and if the addition amount of the complex solution is too small, the signal saturation of a part of substances with high response is serious.

The specific process parameters are as follows: drying the supernatant, preferably blowing the supernatant with nitrogen at room temperature;

s24, carrying out vortex mixing and centrifugation to obtain a sample to be detected;

the specific process parameters are as follows: vortexing for 0.5-2 min, preferably 1 min, and centrifuging for 4-7 min, preferably 5 min.

In S22-S24, the rotation speed of the vortex is 2000 rpm; the centrifugal speed was 14000 rpm.

S3 of the embodiment of the present invention includes:

the sample to be detected is detected by adopting high performance liquid chromatography-mass spectrometry, and the high performance liquid chromatography-mass spectrometry combination instrument comprises two types of high performance liquid chromatography mass spectrometry instruments, namely AB SCIEX and Agilent LC1260/MS6470A, and can be selected according to detection equipment in the actual detection process, so that the detection method provided by the embodiment of the invention has wider applicability.

The specific analysis and detection conditions are as follows:

when the high performance liquid chromatography-mass spectrometer is AB SCIEX;

the chromatographic column is SHIMADZU Shim-pack Shim-pack Velock C18, 2.1 × 100mm, 2.7 μm; the column temperature is 50 ℃;

gradient elution: 0-1 minute: 60% of A and 40% of B;

2.00-2.5 min: 40% of A and 60% of B;

3.50-5 minutes: 20% of A and 80% of B;

5.01-6.50 minutes: 60% of A and 40% of B;

the flow rate is: 0.3 mL/min;

preferably, the sample injection amount of the L1, L2, L3, L4, L5 and L6 standard working solutions is 5 mu L, and the sample injection amount of the L7 and L8 standard working solutions is 1 mu L; the sample volume of the sample to be tested is 5. mu.L.

More preferably, the analysis time is 6.5 minutes;

the mass spectrum parameters are as follows: ion source temperature: 350 ℃; ion source high pressure: 5500V; air curtain air: 20L/min; collision gas: 8L/min; drying gas 1: 50L/min; and (3) drying gas 2: 50L/min.

When the HPLC-MS is AB SCIEX, the internal standard corresponding to the substance to be detected is shown in Table 4:

TABLE 4

Substance to be measured	Internal standard substance	Corresponding abbreviations for internal standard substances
			Sulpiride	levosulpiride-D3	ZSBL-D3
Desvenlafaxine	venlafaxine-D6	WLFX-D6
			Moclobemide	moclobemide-D8	MLBA-D8
6-hydroxybuspirone	6-hydroxybuspirone-D8	6-OH-BUS-d8
			Tandospirone	Tandospirone-D8	TDR-D8
1-pyrimidinylpiperazines	1-pyrimidinylpiperazine-D8	1-PP-D8
			Buspirone	buspirone-D8	BUS-d8
Venlafaxine	venlafaxine-D6	WLFX-D6
			Mianserin	mianserin-D3	MASR-D3
Reboxetine	Noramitriptyline-D3	NTL-D3
			Paroxetine	clonazepam-D4	Lxxp-D4
Clonazepam	clonazepam-D4	Lxxp-D4
			Imipramine	imipramine-D3	IPM-D3
Desipramine	desipramine-D3	DPM-D3
			Amitriptyline	amitriptyline-D3	AMTL-D3
Hydroxyoxazines	hydroxyzine-D8	HOZ-D8
			Noramitriptyline	Noramitriptyline-D3	NTL-D3
Digoxin	digoxin-D3	Digoxin-D3
			Sertraline	desipramine-D3	DPM-D3
Tolbutamide	Glimepiride-D5	GLMN-D5
			Nordiazepam	diazepam-D5	DXP-D5
Diazepam	diazepam-D5	DXP-D5
			Pentafluridol	Pentaflurido-D7	PFRD-D7
Glimepiride	Glimepiride-D5	GLMN-D5
			Clopidogrel metabolites	Clopidogrel metabolite-C6	CAMD-13C6
Clopidogrel	clopidogrel-D3	CLP-D3

According to the above table, the invention completes the detection of 26 substances through 20 internal standard substances by screening the chromatographic mass spectrum conditions, and the common conditions of the internal standard substances are shown in table 5:

TABLE 5

According to the embodiment of the invention, under the condition of ensuring the detection accuracy, the detection method is greatly simplified, the detection efficiency is improved and the detection cost is reduced by sharing the internal standard substance.

Secondly, when the high performance liquid chromatography-mass spectrometer is Agilent LC1260/MS6470A,

the chromatographic column is SHIMADZU Shim-pack Shim-pack Velock C18,

the parameters are as follows: 2.1X 100mm, 2.7 μm;

the column temperature is 50 ℃;

gradient elution:

0-1.0 min: 60% of A and 40% of B;

1.50-3.50 minutes: 30% of A and 70% of B;

4.00-5.50 minutes: a0%, B100%;

5.51-7.5 min: 60% of A and 40% of B;

the flow rate is: 0.3 mL/min;

preferably, the sample injection amount of the standard working solution of L1, L2, L3, L4, L5 and L6 is 5 mu L, and the sample injection amount of the standard working solution of L7 and L8 is 2 mu L; the sample volume of the sample to be tested is 5. mu.L.

More preferably, the analysis time is 7.5 minutes;

the mass spectrum parameters are as follows: an acquisition mode: ESI (+); the temperature of MRM atomizing gas is 200 ℃; atomizing airflow: 3L/min; temperature of sheath gas: 200 ℃; sheath gas flow: 11L/min; nebulizer: 45 psi; capillary voltage: 5000V; Delta-EMV: 100V.

When the high performance liquid chromatography-mass spectrometer is Agilent LC1260/MS6470A, the internal standard corresponding to the substance to be detected is shown in Table 6:

TABLE 6

Substance to be measured	Internal standard substance	Corresponding abbreviations for internal standard substances
			Sulpiride	1-pyrimidinepiperazine-D8	1-PP-D8
Desvenlafaxine	venlafaxine-D6	WLFX-D6
			The interior label of the moclobemide is	moclobemide-D8	MLBA-D8
6-hydroxybuspirone	6-hydroxybuspirone-D8	6-OH-BUS-d8
			Tandospirone	tandospirone-D8	TDR-D8
1-pyrimidinylpiperazines	1-pyrimidinylpiperazine-D8	1-PP-D8
			Buspirone	buspirone-D8	BUS-d8
Venlafaxine	venlafaxine-D6	WLFX-D6
			Mianserin	mianserin-D3	MASR-D3
Reboxetine	hydroxyzine-D8	HOZ-D8
			Paroxetine	clonazepam-D4	Lxxp-D4
Clonazepam	clonazepam-D4	Lxxp-D4
			Imipramine	imipramine-D3	IPM-D3
Desipramine	desipramine-D3	DPM-D3
			Amitriptyline	amitriptyline-D3	AMTL-D3
Hydroxyzine	hydroxyzine-D8	HOZ-D8
			Noramitriptyline	Noramitriptyline-D3	NTL-D3
Digoxin	digoxin-D3	Digoxin-D3
			Sertraline	clonazepam-D4	Lxxp-D4
Tolbutamide	amitriptyline-D3	AMTL-D3
			Nordiazepam	amitriptyline-D3	AMTL-D3
Diazepam	Noramitriptyline-D3	NTL-D3
			Pentafluridol	Pentaflurido-D7	PFRD-D7
Glimepiride	Glimepiride-D5	GLMN-D5
			Clopidogrel metabolites	Clopidogrel metabolite-C6	CAMD-13C6，
Clopidogrel	clopidogrel-D3	CLP-D3

According to the above table, the invention completes the detection of 26 substances through 18 internal standard substances by screening the chromatographic mass spectrum conditions, and the common conditions of the internal standard substances are shown in table 7:

TABLE 7

According to the embodiment of the invention, under the condition of ensuring the detection accuracy, the detection method is greatly simplified, the detection efficiency is improved and the detection cost is reduced by sharing the internal standard substance. The detection method provided by the embodiment of the invention is respectively 6.5min and 7.5min, 26 substances are output by one needle, 10 samples can be analyzed within 1 hour, the analysis time is short, and in addition, 26 substance detections can be completed by 2 instruments with different models.

Example 1

This example illustrates one embodiment of the formulation of a standard working fluid:

1. clopidogrel: 1.56 mg of clopidogrel hydrogen sulfate standard substance is accurately weighed and placed in a 2mL freezing tube, 2mL of methanol is added for dissolution and volume fixing is carried out, thus obtaining a mother solution with the concentration of 597.8 mug/mL.

2. Clopidogrel metabolite: 1mL of methanol was added to a sample bottle containing 1mg of clopidogrel active metabolite derivative to dissolve, thereby obtaining a mother liquor having a concentration of 1000. mu.g/mL.

3. 1- (2-pyrimidine) piperazine: accurately weighing 5.78 mg of 1- (2-pyrimidine) piperazine standard substance to a 2mL freezing tube, adding 2mL methanol for dissolving, and uniformly mixing to obtain a mother solution with the concentration of 2832 mu g/mL.

4. Tandospirone: precisely weighing 2.13 mg of tandospirone standard substance into a 2mL freezing storage tube, adding 2mL of methanol for dissolving, and uniformly mixing to obtain mother liquor with the concentration of 1044 mu g/mL.

5. And (3) mianserin: the standard was purchased at a known concentration, volume 1mL, and solvent methanol, concentration 1000. mu.g/mL.

6. Imipramine: 4.2 mg of imipramine hydrochloride standard substance is precisely weighed to be a frozen tube of 2mL, 2mL of methanol is added for dissolving, and mother liquor with the concentration of 1858 mu g/mL is obtained after uniform mixing.

7. Hydroxyzine: 2.27 mg of the hydroxyzine hydrochloride standard substance is precisely weighed to be a 2mL freezing storage tube, 2mL methanol is added for dissolving, and the mother solution with the concentration of 937 mu g/mL is obtained after uniform mixing.

8. Desipramine: the standard was purchased at a known concentration, with a volume of 1mL, and the solvent was methanol, at a concentration of 1000. mu.g/mL.

9. Pentafluridol: 2.82 mg to 2mL of the pentafluridol standard substance are precisely weighed and stored in a freezing tube, 2mL of methanol is added for dissolving, and mother liquor with the concentration of 1368 mug/mL is obtained after uniform mixing.

10. Digoxin: weighing 2.00 mg of digoxin standard substance accurately by a balance, placing the digoxin standard substance in a 2mL freezing tube, adding 2mL of methanol for dissolving, and fully and uniformly mixing and dissolving to obtain mother liquor with the concentration of 1000 mu g/mL.

11. Tolbutamide: 2.812 mg of the standard was accurately weighed, placed in a 2mL cryopreservation tube, and dissolved by adding 2mL of methanol to obtain a mother solution with a concentration of 1406. mu.g/mL.

12. Paroxetine: 4.224 mg of the standard was weighed out accurately, placed in a 2mL freezer, and dissolved in 2mL of methanol to give a 1808. mu.g/mL mother liquor.

13. Reboxetine: 2.617 mg of the standard was weighed out accurately, placed in a 2mL freezer, and dissolved in 2mL of methanol to give a 961. mu.g/mL mother liquor.

14. Venlafaxine: 4.864 mg of standard substance is accurately weighed and placed in a 2mL freezing tube, and 2mL of methanol is added for dissolution, so that a mother solution with the concentration of 2145 mu g/mL is obtained.

15. Desvenlafaxine: the standard was purchased at a known concentration, with a volume of 1mL, and the solvent was methanol, at a concentration of 1000. mu.g/mL.

16. Glimepiride: accurately weigh 5.00mg of standard, place in a 15 mL cryovial, add 10 mL methanol: water = 7:3 to obtain a mother liquor with the concentration of 500 mu g/mL.

17. Buspirone: accurately weigh 4.36mg of standard, place in 2mL cryovial, add 2mL methanol: water =1:1 was dissolved to give a mother liquor having a concentration of 1985.53. mu.g/mL.

18. 6-hydroxybuspirone: 2.38 mg of the standard was accurately weighed, placed in a 2mL cryovial, and 2mL of methanol: water =1:1 to give a mother liquor with a concentration of 1131. mu.g/mL.

19. Clonazepam: 2.11mg of the standard was weighed accurately, first with 1mL of methanol solution, then methanol: water =1:1 to 2mL, giving a mother liquor with a concentration of 1055 μ g/mL.

20. Sertraline: the standard was purchased at a known concentration, with a volume of 1mL, and the solvent was methanol, at a concentration of 1000. mu.g/mL.

21. Moclobemide: molobemide standard 1.47 mg was weighed out accurately and placed in a 2mL freezer tube and dissolved by adding 1mL of diluent (methanol: water = 7: 3) to yield a 1470. mu.g/mL stock solution.

22. Sulpiride: 2 mg of the standard substance is accurately weighed and placed in a 2mL freezing tube, and 2mL of methanol is added for dissolution to obtain a mother solution with the concentration of 1000 mug/mL.

23. Diazepam: 2.02 mg of the standard substance was accurately weighed, placed in a 2mL cryopreservation tube, and dissolved in 2mL of methanol to obtain a mother liquor having a concentration of 1000. mu.g/mL.

24. Diazepam: the standard was purchased at a known concentration, volume 1mL, and solvent methanol, concentration 1000. mu.g/mL.

25. Amitriptyline: 4.34 mg of the standard was weighed out accurately, placed in a 2mL cryopreservation tube, and dissolved in 2mL of methanol to give a mother liquor having a concentration of 1918. mu.g/mL.

26. Noramitriptyline: 4.24 mg of the standard was weighed out accurately, placed in a 2mL cryopreservation tube, and dissolved in 2mL of methanol to obtain a mother liquor having a concentration of 1863. mu.g/mL.

With methanol: water = 7:3, diluting the mother liquor to obtain substance intermediate solutions, wherein the concentrations of the intermediate solutions are shown in a table 8:

TABLE 8

Serial number	Name of substance	Concentration of intermediate solution (μ g/mL)
			1	Sulpiride	200
2	1-pyrimidinylpiperazines	40
			3	Desvenlafaxine	100
4	Moclobemide	400
			5	6-hydroxybuspirone	40
6	Tandospirone	40
			7	Buspirone	40
8	Venlafaxine	400
			9	Mianserin	400
10	Reboxetine	400
			11	Paroxetine	400
12	Imipramine	400
			13	Desipramine	400
14	Clonazepam	100
			15	Amitriptyline	400
16	Hydroxyoxazines	400
			17	Noramitriptyline	400
18	Digoxin	20
			19	Sertraline	400
20	Nordiazepam	1000
			21	Tolbutamide	100
22	Diazepam compounds	1000
			23	Pentafluridol	320
24	Glimepiride	100
			25	Clopidogrel	20
26	Clopidogrel metabolites	100

With methanol: water = 7:3, diluting the intermediate solution to obtain a standard working solution, wherein the concentration of the working solution is shown in a table 9:

TABLE 9

The standard dilution process is shown in table 10:

TABLE 10

Marking points	Dilution process	Diluent (methanol: water 7: 3)
			L7	Taking L8500. mu.L	500μL
L6	Collecting L7400 μ L	600μL
			L5	Taking L6500 μ L	500μL
L4	Taking L5500 μ L	500μL
			L3	Take L4400. mu.L	600μL
L2	Collecting L3500 μ L	500μL
			L1	Collecting L2500 μ L	500μL

The concentrations of the substances of each level in the obtained standard working solution are shown in Table 2.

Example 2

This example illustrates a specific embodiment of the preparation of an internal standard working solution:

1. clopidogrel-D3: 1.79 mg of clopidogrel-D3 sulfate standard substance is accurately weighed, placed in a 2mL freezing tube, and dissolved by adding 2mL of methanol to obtain a mother solution with the concentration of 687.4 mug/mL.

2. Clopidogrel active metabolite derivative-13C 6: 1mg is purchased, 1.059 mg is actually purchased, and 1mL of methanol is directly added into the sample bottle for dissolution, so as to obtain a mother liquor with a concentration of 978.45 mug/mL.

3. 1- (2-pyrimidine) piperazine-D8: the standard substance specification is 2.5 mg, 1mL of methanol is directly added into a sample bottle to be dissolved, then the sample bottle is transferred into a 15 mL centrifuge tube, 1mL of methanol is added into the sample bottle for dissolving 4 times, the dissolved solution is transferred into the 15 mL centrifuge tube, and mother liquor with the concentration of 500 mu g/mL is obtained after uniform mixing.

4. tandospirone-D8: 2.35 mg of tandospirone-D8 standard substance is precisely weighed into a 2mL freezing tube, 2mL of methanol is added for dissolution, and mother liquor with the concentration of 1175 mu g/mL is obtained after uniform mixing.

5. mianserin-D3: the standard was purchased at a known concentration, volume 1mL, and solvent methanol, concentration 100. mu.g/mL.

6. imipramine-D3: the standard was purchased at a known concentration, volume 1mL, and solvent methanol, concentration 100. mu.g/mL.

7. hydroxyzine-D8: unsealing 1mg of hydroxyzine-D8 standard substance, accurately adding 1mL of methanol, and uniformly mixing to obtain mother liquor with the concentration of 1000 mug/mL.

8. desipramine-D3: the standard was purchased at a known concentration, with a volume of 1mL, and the solvent was methanol, at a concentration of 100. mu.g/mL.

9. pentaflurido-D7: the purchase specification is 10 mg, the actual specification is 10.067 mg, 2mL of ethanol is directly and accurately added into a sample bottle for dissolving, and the mother solution with the concentration of 4978 mu g/mL is obtained after uniform mixing.

10. amitriptyline-D3: the standard was purchased at a known concentration, volume 1mL, and solvent methanol, concentration 100. mu.g/mL.

11. noramitriptyline-D3: 1.58 mg of the standard noramitriptyline hydrochloride-D3 is accurately weighed by a balance to be frozen in a 2mL tube, and 1.58 mL of methanol is added to be dissolved to obtain a mother solution with the concentration of 1 mg/mL.

12. Buspirone hydrochloride-D8: buspirone hydrochloride-D8 internal standard 1mg dissolved in 5mL of methanol: water =1:1, the concentration of the mother liquor was 168.714. mu.g/mL.

13. 6-hydroxybuspirone-D8: 1mg of internal standard 6-hydroxybuspirone-D8, dissolved in 10 mL of methanol: water =1:1, the concentration of the mother liquor in the solution is 100 mug/mL.

14. clonazepam-D4: the standard was purchased at a known concentration, with a volume of 1mL, and the solvent was methanol, at a concentration of 1000. mu.g/mL. .

15. digoxin-D3: the standard substance specification is 2.50 mg, 1mL of methanol is accurately added into a sample bottle directly for dissolving, the dissolved solution is transferred into a 2mL freezing tube, and then 1mL of methanol is added: water =1:1 and transferred to the same vial to give a mother liquor at a concentration of 1250 μ g/mL.

16. venlafaxine-D6: the standard was purchased at a known concentration, with a volume of 1mL, and the solvent was methanol, at a concentration of 100. mu.g/mL.

17. glimepiride-D5: the standard, 1mg, was dissolved in 2mL of acetonitrile to give a mother liquor having a concentration of 475. mu.g/mL.

18. moclobemide-D8: the standard sample was 1mg, and dissolved in 2mL of methanol to obtain a mother liquor having a concentration of 490. mu.g/mL.

19. levosulpiride-D3: the purchasing specification of the standard substance is 1mg, the actual specification is 1.03 mg, 500 mu L of methanol is accurately added into a sample bottle directly for dissolving, the dissolved solution is transferred into a 2mL freezing tube, and 500 mu L of the dissolved solution is added for 3 times and transferred into the same freezing tube, so that mother solution with the concentration of 515 mu g/mL is obtained.

20. diazepam-D5: the purchasing specification of the standard substance is 1mg, the actual specification is 1.00 mg, 1mL of methanol is accurately added into the sample bottle directly for dissolving, and the dissolved solution is transferred to a 2mL freezing tube to obtain a mother solution with the concentration of 1000 mug/mL.

With methanol: water = 7: and 3, diluting the internal standard mother liquor to obtain internal standard intermediate liquor, wherein the concentrations of the internal standard mother liquor and the intermediate liquor of each substance are shown in a table 11:

TABLE 11

Serial number	Name of substance	Concentration of intermediate or mother liquor (. mu.g/mL)
			1	clonazepam-D4	10
2	digoxin-D3	1
			3	clopidogrel-D3	1
4	Clopidogrel metabolite-C6	10
			5	Glimepiride-D5	10
6	Pentaflurido-D7	320
			7	imipramine-D3	100
8	desipramine-D3	100
			9	mianserin-D3	10
10	tandospirone-D8	1
			11	1-pyrimidinepiperazine-D8	1
12	buspirone-D8	2
			13	6-hydroxybuspirone-D8	2
14	hydroxyzine-D8	1
			15	venlafaxine-D6	100
16	moclobemide-D8	100
			17	amitriptyline-D3	100
18	Noramitriptyline-D3	100
			19	diazepam-D5	10
20	levosulpiride-D3	50

An internal standard working solution was prepared according to the procedure shown in table 12:

TABLE 12

The concentrations of the respective substances in the internal standard working solution are shown in table 3.

Example 3

This example illustrates a specific implementation of sample processing and detection:

1. at least three different concentrations of standard working solution 10 μ L, internal standard working solution 10 μ L and methanol were first pipetted: 80 mu L of mixed solution with the water volume ratio of 1:1 is respectively put into a 1.5mL centrifuge tube to be mixed into at least three standard solutions, after the standard solutions are respectively uniformly mixed by vortex at the rotating speed of 2000rpm for 30S, a liquid chromatography-mass spectrometry instrument (the specific parameters are the same as S3) is used for detecting the standard solutions to obtain at least three standard solutions, 19 medicines and metabolites thereof and an internal standard chromatogram, taking the ratio of the peak area of each substance of the at least three standard solutions to the peak area of the corresponding internal standard substance as the ordinate y of the standard curve chart, taking the ratio of the concentration of each substance in the standard working solution to the corresponding internal standard concentration as the abscissa x of a standard curve graph, performing linear regression on at least three groups of data obtained by detection, fitting to obtain a standard curve equation of y = a x + b, and obtaining weight coefficients a and b; the relative concentration x of 26 substances in total of 19 drugs and metabolites thereof in the blood sample to be detected is obtained through calculation, and the concentration of the internal standard substance working solution is known, so that the concentration of 26 substances in total of 19 drugs and metabolites thereof in the blood to be detected in the sample can be obtained through calculation.

2. Centrifugation for testing blood

Taking at least 2mL of blood of the blood collection tube/EDTA blood collection tube/heparin lithium blood collection tube to be detected, centrifuging at a centrifugal speed of 3500rpm for 10min, taking supernatant to obtain blood serum/EDTA blood plasma/heparin lithium blood plasma, and freezing the blood serum or blood plasma at-20 ℃ for storage before analysis.

3. Treatment of samples to be tested

Using a liquid transfer gun to transfer 10 mu L of internal standard working solution prepared in example 2 into a 1.5mL centrifuge tube, then adding 100 mu L of serum/plasma, adding 1000 mu L of methyl tertiary butyl ether, vortex and shake for 5min at the rotating speed of 2000rpm, centrifuging at a high speed of 14000 r/min for 10min, transferring 950 mu L of supernatant to a 1.5mL plastic centrifuge tube, blowing nitrogen to dry at room temperature, and adding 100 mu L of methanol: water =1:1,2000 r/min, vortex and mixing uniformly for 1 min, 14000 r/min high-speed centrifugation for 5min, taking 90 mu L of supernatant, and detecting the supernatant by using a high performance liquid chromatography triple four-level rod tandem mass spectrometer with the sample size of 5 mu L.

4. Detection of a sample to be tested

Transferring 90 mu L of the sample to be detected in the step, detecting the sample to be detected by using a liquid chromatography-mass spectrometer, wherein the sample injection amount is 5 mu L, and obtaining chromatograms of 19 drugs, metabolites thereof and internal standard in the sample to be detected; substituting the ratio y of the peak areas of the 19 drugs and the metabolites thereof in the chromatogram to the peak area of the internal standard substance into the standard curve equation y = a x + b in the step 1, and calculating to obtain the ratio x of the concentrations of the 19 drugs and the metabolites thereof in the sample to be detected to the corresponding internal standard concentration, wherein the concentration of each substance in the internal standard working solution is known, so that the concentrations of the 19 drugs and the metabolites thereof in the blood to be detected in the sample are calculated.

The detection parameters are as follows:

1. when the HPLC-MS is AB SCIEX, the specific parameters are as follows;

the instrument model specification is: AB SCIEX Jasper HPLC MS TRIPLE QUAD 4500MD (AB 4500MD for short);

the high performance liquid chromatography-mass spectrometer comprises: agilent LC1260/MS 6470A; specific instrument parameters are shown in tables 13-15:

watch 13

TABLE 14

Parameter(s)	Set value (ESI +)
		Ion Source Temperature (TEM)	350℃
Ion source high pressure (IS)	5500 V
		Air curtain gas (CUR)	20 L/min
Collision gas (CAD)	8 L/min
		Drying gas 1 (GS 1)	50 L/min
Drying gas 2 (GS 2)	50 L/min

Watch 15

Name of substance	Retention time/min	Parent ion	Daughter ions	DP	CE	EP	CXP
								SBL	0.66	342.1	112	50	80	6	10
LSBL-D3	0.66	345.1	112	50	80	6	10
								N-WLFX	0.96	264.2	107.1	60	78	6	10
MLBA	1.00	269	138.9	69	85	6	10
								MLBA-D8	1.00	277	138.9	69	85	6	10
6-OH-BUS	1.09	402.4	122	120	65	6	10
								6-OH-BUS-D8	1.09	410.4	122	120	65	6	10
TDR	1.12	384.6	122.1	110	50	6	10
								TDR-D8	1.12	392.2	122.2	110	50	6	10
1-PP	0.67	165.3	122	60	40	6	10
								1-PP-D8	0.67	173.4	127	60	40	6	10
BUS	1.46	386.2	122	115	56	6	10
								BUS-D8	1.46	394.3	122	135	56	6	10
WLFX	1.91	278.3	121	60	78	6	10
								WLFX-D6	1.91	284.3	121	60	78	6	10
MASR	2.57	265.1	58.1	100	72	6	10
								MASR-D3	2.57	268.1	60.7	100	72	6	10
RBXT	3.16	314.2	176.1	70	48	6	10
								PLXT	3.32	330.1	123.1	70	48	6	10
LXXP	3.42	316.2	270.1	65	48	6	10
								LXXP-D4	3.42	320.2	274.1	65	48	6	10
IPM	3.39	281.4	58	50	20	6	10
								IPM-D3	3.39	284.4	60.9	50	20	6	10
DPM	3.43	267.1	72.1	35	82	6	10
								DPM-D3	3.43	270.1	74.8	35	82	6	10
AMTL	3.51	278.1	91	65	16	6	10
								AMTL-D3	3.51	281.1	105	65	18	6	10
HOZ	3.52	375.2	201.2	58	62	6	10
								HOZ-D8	3.52	383	201	58	48	6	10
NIL	3.58	264.1	91	30	16	6	10
								NIL-D3	3.58	267.1	191.1	30	18	6	10
Digoxin	3.88	798.5	651.4	40	23	6	10
								Digoxin-D3	3.88	801.5	654.4	40	23	6	10
Ser	3.91	306	275	35	34	6	10
								JBHDN	3.6	271.1	91	55	37	6	10
N-DXP	4.22	271	165	55	70	6	10
								DXP	4.42	284.9	154	135	68	6	10
DXP-D5	4.42	289.9	154	135	58	6	10
								PFRD	4.58	524.1	109	80	38	6	10
PFRD-D8	4.58	531.1	111	80	34	6	10
								GLMN	4.91	491.3	352.1	95	18	6	10
GLMN-D5	4.91	496.3	357.1	95	18	6	10
								CAMD	5.03	504.2	154.9	75	62	6	10
CAMD-13C6	5.03	510.2	155	75	62	6	10
								CLP	5.2	322.2	212	75	26	6	10
CLP-D3	5.2	325.2	215	75	26	6	10

Wherein, because the material kind is more, propose the collection mode to be the segmentation collection. The internal standards corresponding to the substances to be tested are shown in tables 4 and 5. The chromatogram for detecting the working solution with a certain concentration by using AB4500MD is shown in figure 3, the chromatogram for detecting the quality control sample is shown in figure 4, and the quality control sample is a positive control sample.

2. Specific parameters of Agilent LC1260/MS6470A (Agilent 6470 for short) are shown in tables 16-18:

TABLE 16

TABLE 17

Parameter(s)	Set value
		Acquisition mode	ESI(﹢)；MRM
Temperature of atomizing Gas (Gas Temp)	200℃
		Atomization Gas Flow (Gas Flow)	3 L/min
Sheath Gas temperature (Sheath Gas Temp)	200℃
		Sheath Gas Flow (Sheath Gas Flow)	11 L/min
Atomization air pressure (Nebulizer)	45 psi
		Capillary Voltage (Capillary) (+)	5000 V
Delta-EMV（+）：	100 V

Watch 18

Name of substance	Retention time/min	Parent ion	Daughter ions	Dwell	Fragmentor	CE	Cell Accelerator Voltage
								SBL	0.74	342	112	10	100	26	5
N-WLFX	0.98	264.2	107.1	10	100	52	5
								MLBA	1.02	269	139	10	120	64	5
MLBA-D8	1.02	277	139	10	120	64	5
								6-OH-BUS	1.07	402.3	121.9	10	200	30	5
6-OH-BUS-d8	1.07	410.3	121.9	10	200	35	5
								TDR	1.09	384.6	122.2	10	180	57	5
TDR-D8	1.09	392.6	122.3	10	180	57	5
								1-PP	1.77	165.3	122.1	10	140	17	5
1-PP-D8	1.77	173.4	127.1	10	140	17	5
								BUS	1.36	386.2	121.9	10	170	30	5
BUS-d8	1.36	394.3	121.9	10	200	35	5
								WLFX	1.79	278.2	121.2	10	100	40	5
WLFX-D6	1.79	284.2	121.2	10	100	40	5
								MASR	2.48	265.1	58.1	10	130	56	5
MASR-D3	2.48	268.1	60.7	10	130	56	5
								RBXT	3.44	314.2	176.1	10	100	30	5
PLXT	3.59	330.2	123.1	10	135	34	5
								Lxxp	3.61	316.2	270.1	10	160	25	5
Lxxp-D4	3.61	320.2	274.1	10	160	25	5
								IPM	3.65	281.3	57.9	10	180	100	5
IPM-D3	3.65	284.3	60.9	10	180	100	5
								DPM	3.70	267.3	72.1	10	135	47	5
DPM-D3	3.70	270.3	74.8	10	135	47	5
								AMTL	3.76	278.1	90.7	10	135	28	5
AMTL-D3	3.76	281.1	104.8	10	135	24	5
								HOZ	3.77	375.3	201.2	10	100	40	5
HOZ-D8	3.77	383	201	10	100	40	5
								NIL	3.82	264.1	91	10	100	25	5
NTL-D3	3.82	267.1	191.1	10	100	24	5
								Digoxin	3.90	798.5	651.4	10	100	11	5
Digoxin-D3	3.90	801.5	654.4	10	100	11	5
								Ser	4.00	306.1	275	10	80	22	5
JBHDN	4.19	271.1	91.1	10	145	66	5
								N-DXP	4.20	271	165.1	10	140	36	5
DXP	4.40	285	154.1	10	135	46	5
								PFRD	4.68	524.5	109.4	10	180	82	5
PFRD-D7	4.68	531.5	111.2	10	180	82	5
								GLMN	5.33	491.3	352.3	10	110	10	5
GLMN-D5	5.33	496.3	357.3	10	110	10	5
								CAMD	5.70	504	154.9	10	85	45	5
CAMD-13C6	5.70	510	155	10	85	45	5
								CLP	5.78	322.1	212	10	100	15	5
CLP-D3	5.78	325.1	215	10	100	15	5

Wherein, because the material kind is more, propose the collection mode to be the segmentation collection. The internal standards corresponding to the substances to be tested are shown in tables 6 and 7. The chromatogram for detecting the working solution with a certain concentration by using Agilent 6470 is shown in figure 1, the chromatogram for detecting the quality control sample is shown in figure 2, and the quality control sample is a positive control substance.

Example 4

This example presents a kit for detecting 19 drugs and their metabolites in blood by liquid chromatography tandem mass spectrometry. The composition of the kit is shown in table 19:

watch 19

Numbering	Name (R)	Make up of	1 part by volume
				1	Internal standard working fluid	Shown in Table 3	/
2	Standard working fluid	Shown in Table 2	/
				3	Extracting agent	Methyl tert-butyl ether	1000μL
4	Compound solution	The volume ratio is 1:1 methanol: water mixed solution	100μL
				5	Positive and negative control	The positive control is added with a standard quality control product; the negative control is methanol with a volume ratio of 7: 3: water (I)	/
6	Chromatographic column mobile phase	A is water containing 0.1 v/v% formic acid and 5mM ammonium formate; b is methanol containing 0.1 v/v% formic acid and 5mM ammonium formate	/

Experimental example 1

First, the linear relationship and quantitative limits of the method

The standard working solution of 19 drugs and metabolites thereof with each concentration of 10 μ L prepared in example 1 and example 2 is added with 10 μ L of internal standard working solution, 90 μ L of blank serum/plasma is added for uniform injection, the concentration is measured from low to high according to the measuring conditions of example 3, and a standard curve is obtained by plotting the peak area-concentration of quantitative chromatogram, and the results show that the linear range and the quantitative limit of the 19 drugs and metabolites thereof are as follows:

1. limit of detection (LOD) and limit of quantitation (LOQ):

HPLC MS TRIPLE limit of detection (LOD) and limit of quantitation (LOQ) of QUAD 4500MD are shown in Table 20:

watch 20

Name of substance	Limit of quantitation (ng/mL)	Detection limit (ng/mL)
			Sulpiride	1.25	0.42
Desvenlafaxine	0.50	0.17
			Moclobemide	2.00	0.67
6-hydroxybuspirone	0.10	0.03
			Tandospirone	0.10	0.03
1-pyrimidinylpiperazines	0.10	0.03
			Buspirone	0.05	0.02
Venlafaxine	2.50	0.83
			Mianserin	0.20	0.07
Reboxetine	1.00	0.33
			Paroxetine	0.50	0.17
Clonazepam	0.50	0.17
			Imipramine	1.00	0.33
Desipramine	1.00	0.33
			Amitriptyline	0.50	0.17
Hydroxyzine	0.10	0.03
			Noramitriptyline	0.50	0.17
Digoxin	0.10	0.03
			Sertraline	0.40	0.13
Tolbutamide	0.50	0.17
			Nordiazepam	2.00	0.67
Diazepam compounds	2.00	0.67
			Pentafluridol	0.80	0.27
Glimepiride	0.05	0.02
			Clopidogrel metabolites	0.10	0.03
Clopidogrel	0.02	0.01

The detection Limits (LOD) and quantitation Limits (LOQ) of Agilent LC1260/MS6470A are shown in Table 21:

TABLE 21

Name of substance	Limit of quantitation (ng/mL)	Detection limit (ng/mL)
			Sulpiride	1.25	0.42
Desvenlafaxine	0.02	0.007
			Moclobemide	2.00	0.67
6-hydroxybuspirone	0.03	0.01
			Tandospirone	0.03	0.01
1-pyrimidinylpiperazines	0.02	0.007
			Buspirone	0.045	0.015
Venlafaxine	0.63	0.21
			Mianserin	0.36	0.12
Reboxetine	1.36	0.45
			Paroxetine	0.65	0.22
Clonazepam	0.40	0.13
			Imipramine	1.32	0.44
Desipramine	1.42	0.47
			Amitriptyline	1.06	0.35
Hydroxyzine	0.24	0.08
			Noramitriptyline	0.96	0.32
Digoxin	0.03	0.01
			Sertraline	0.68	0.23
Tolbutamide	0.02	0.007
			Nordiazepam	1.45	0.48
Diazepam	1.56	0.52
			Pentafluridol	0.12	0.04
Glimepiride	0.03	0.01
			Clopidogrel metabolites	0.12	0.04
Clopidogrel	0.03	0.01

(3) Linear range:

sulpiride: 10ng/mL to 2000 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

1-pyrimidinylpiperazine: 0.2ng/mL to 40.0 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Desvenlafaxine: 5ng/mL to 1000 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Moclobemide: 20ng/mL to 4000 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

6-hydroxybuspirone: 0.2ng/mL to 40.0 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Tandospirone: 0.2ng/mL to 40.0 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Buspirone: 0.2ng/mL to 40.0 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Venlafaxine: 10ng/mL to 2000 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Mianserin: 2ng/mL to 400 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Reboxetine: 10ng/mL to 2000 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Paroxetine: 2ng/mL to 400 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Imipramine: 10ng/mL to 2000 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Desipramine: 10ng/mL to 2000 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Clonazepam: 1ng/mL to 200 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Amitriptyline: 10ng/mL to 2000 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Hydroxyzine: 2ng/mL to 400 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Noramitriptyline: 10ng/mL to 2000 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Digoxin: 0.1ng/mL to 20.0 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Sertraline: 2ng/mL to 400 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Diazepam: 20ng/mL to 4000 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Tolbutamide: 0.5ng/mL to 100.0 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Pentafluridol: 1.6ng/mL to 320.0 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Glimepiride: 0.5ng/mL to 100.0 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Clopidogrel: 0.1ng/mL to 20.0 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Clopidogrel metabolite: 0.5ng/mL to 100.0 ng/mL; the linearity is good, and the correlation coefficient R2 is more than 0.9900.

Second, the recovery rate and precision of the method

19 medicines and metabolite standard working solutions thereof are prepared into high, medium and low 3 concentrations to carry out sample adding recovery rate experiments and precision experiments, the determination is carried out according to the method of the

embodiment

3, 3 batches of analysis and determination are repeated, and the recovery rate and the precision are respectively shown in the following tables 22 and 23. The average recovery rate of the 19 drugs and metabolites thereof in the range of 3 addition levels of low, medium and high is 85% -105%, and the relative standard deviation is 0.00% -8.33%.

TABLE 22 HPLC MS TRIPLE QUAD 4500MD recovery results

TABLE 23 Agilent LC1260/MS6470A recovery results

By integrating the verification tests, the technical indexes such as detection limit, recovery rate and precision of the method meet the requirements, the method for detecting the concentrations of 19 drugs and metabolites thereof in blood has good reproducibility and high sample recovery rate, and the accuracy of the detection result is improved.

Comparative example 1

This comparative example illustrates the technical effect of an extractant being methyl tert-butyl ether.

Experiment design: the samples were tested as in example 3, with the following differences: the pretreatment mode adopts liquid-liquid extraction instead of protein precipitation. The experimental results were obtained: direct protein precipitation, digoxin signal too low, can not reach the quantitative requirements.

Comparative example 2

This comparative example serves to illustrate the technical effect of mobile phase and gradient elution conditions.

Experiment design: the samples were tested as in example 3, with the following differences:

1. the Agilent 6470 gradient elution conditions are as follows:

0-1.0 min: 50% of A and 50% of B;

1.50-3.50 minutes: 20% of A and 80% of B;

4.00-5.50 minutes: a0%, B100%;

5.51-7.5 minutes: 50% of A and 50% of B;

the flow rate is: 0.3 mL/min.

The obtained test results are shown in fig. 5 to 8. As shown in fig. 5, mianserin tailing; as shown by fig. 6, reboxetine tail; as shown in fig. 7, paroxetine tails; as shown in fig. 8, imipramine smears.

The test results obtained by using the gradient elution conditions of the present invention are shown in fig. 9 to 12. The improved mianserin tail was shown in fig. 9, reboxetine tail was shown in fig. 10, paroxetine tail was shown in fig. 11, and imipramine tail was shown in fig. 12.

2. AB4500MD gradient elution conditions were:

0-1 minute: 60% of A and 40% of B;

1.01-2.5 minutes: 40% of A and 60% of B;

2.51-5 minutes: a0%, B100%;

5.01-6 minutes: 60% of A and 40% of B;

the flow rate is: 0.3 mL/min.

The test results obtained are shown in fig. 13. As can be seen from fig. 13, under this elution condition: the internal standard of the clopidogrel metabolite can not be separated from impurities.

The test results obtained with the gradient elution conditions of the present invention are shown in fig. 14, and it can be seen from fig. 14 that the clopidogrel metabolite internal standard and impurities can be effectively separated under the elution conditions.

3. Mobile phase:

when the high performance liquid chromatography mass spectrometer is Agilent LC1260/MS6470A, the modified mobile phase is: the same as in the present example except for water (0.1% formic acid +4mM ammonium formate)/methanol (0.1% formic acid +4mM ammonium formate) gave the test results as shown in Table 24:

watch 24

As can be seen from Table 24, there was a slight change in the buffer salt concentration, and the recovery rate of tolbutamide was 120%, which was not satisfactory. This phenomenon occurs due to the low concentration plus scalar quantity, which determines that the tolbutamide recovery results are not acceptable at the above buffer salt concentration.

The foregoing are merely exemplary embodiments of the present disclosure, which enable those skilled in the art to understand or practice the present disclosure. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the disclosure. Thus, the present disclosure is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting 19 drugs and metabolites thereof in blood by liquid chromatography-tandem mass spectrometry is characterized in that,

the substances to be tested of the method comprise 19 drugs and 7 metabolites;

the 19 medicines comprise sulpiride, pentafluridol, mianserin, buspirone, tandospirone, hydroxyzine, diazepam, venlafaxine, moclobemide, imipramine, paroxetine, reboxetine, amitriptyline, sertraline, digoxin, clonazepam, clopidogrel, tolbutamide, glimepiride; the 7 metabolites comprise 1-pyrimidinylpiperazine, desvenlafaxine, 6-hydroxybuspirone, desipramine, nomitriptyline, desidiazepam and clopidogrel MP derivatives;

the method comprises at least the following steps:

s2, processing the sample to be detected: adding an extracting agent containing the internal standard working solution into the anticoagulated blood sample, extracting to obtain a supernatant, drying the supernatant, adding a complex solution, uniformly mixing, and centrifuging to obtain a sample to be detected; the volume ratio of the anticoagulant liquid sample to the extractant is 1: 5-1: 15; the volume ratio of the anticoagulant liquid sample to the internal standard working solution is 10: 1; the volume ratio of the supernatant to the double solution is 9: 1-10: 1; the extraction agent is selected from methyl tert-butyl ether, and the complex solution is selected from methanol with the volume ratio of 1: mixing the solution with water;

s3, detecting the sample to be detected by adopting high performance liquid chromatography-mass spectrometry;

when the high performance liquid chromatography-mass spectrometer is AB SCIEX;

the chromatographic column is SHIMADZU Shim-pack Shim-pack Velox C18, 2.1 × 100mm, 2.7 μm;

mobile phase: a is water containing 0.1 v/v% formic acid and 5mM ammonium formate; b is methanol containing 0.1 v/v% formic acid and 5mM ammonium formate;

gradient elution: 0-1 minute: 60% of A and 40% of B;

2.00-2.5 min: 40% of A and 60% of B;

3.50-5.00 min: 20% of A and 80% of B;

5.01-6.50 minutes: 60% of A and 40% of B;

the internal standard of sulpiride is levosulpiride-D3, the internal standard of desvenlafaxine is venlafaxine-D6, the internal standard of moclobemide is moclobemide-D8, the internal standard of 6-hydroxybuspirone is 6-hydroxybuspirone-D8, the internal standard of tandospirone is tandospirone-D8, the internal standard of 1-pyrimidine piperazine is 1-pyrimidine piperazine-D8, the internal standard of buspirone is buspirone-D8, the internal standard of venlafaxine is venlafaxine-D6, the internal standard of mianserin is mianserin-D3, the internal standard of reboxetine is desmiramide-D3, the internal standard of paroxetine is clonazepam-D4, the internal standard of clonazepam is clonazepam-D4, the internal standard of imipramine is imipramine-D3, the internal standard of desipramipezine-D3, and the internal standard of amitriptyline-D3, the internal standard of the hydroxyzine is hydroxyzine-D8, the internal standard of the noramitriptyline is noramitriptyline-D3, the internal standard of the digoxin is digoxin-D3, the internal standard of the sertraline is mepimizine-D3, the internal standard of the tolbutamide is glimepiride-D5, the internal standard of the nordiazepam is diazepam-D5, the internal standard of the diazepam is diazepam-D5, the internal standard of the pentafluridol is pentafluridol-D7, the internal standard of the glimepiride is glimepiride-D5, the internal standard of the clopidogrel metabolite is clopidogrel metabolite-C6, and the internal standard of the clopidogrel is clopidogrel-D3;

when the high performance liquid chromatography-mass spectrometer is Agilent LC1260/MS6470A,

the chromatographic column is SHIMADZU Shim-pack Shim-pack Velox C18,

the parameters are 2.1 multiplied by 100mm and 2.7 mu m;

gradient elution:

0-1.00 min: 60% of A and 40% of B;

1.50-3.50 minutes: 30% of A and 70% of B;

4.00-5.50 minutes: a is 0 percent, B is 100 percent;

5.51-7.5 minutes: 60% of A and 40% of B;

the internal standard of sulpiride is 1-pyrimidylpiperazine-D8, the internal standard of desvenlafaxine is venlafaxine-D6, the internal standard of moclobemide is moclobemide-D8, the internal standard of 6-hydroxybuspirone is 6-hydroxybuspirone-D8, the internal standard of tandospirone is tandospirone-D8, the internal standard of 1-pyrimidylpiperazine is 1-pyrimidylpiperazine-D8, the internal standard of buspirone is buspirone-D8, the internal standard of venlafaxine is venlafaxine-D6, the internal standard of mianserin is mianserin-D3, the internal standard of reboxetine is hydroxyzine-D8, the internal standard of paroxetine is clonazepam-D4, the internal standard of clonazepam is clonazepam-D4, the internal standard of imipramiperine is imipramipeline-D3, the internal standard of desipramiperine-D3 and the internal standard of amitriptyline-D3, the internal standard of the hydroxyzine is hydroxyzine-D8, the internal standard of the noramitriptyline is noramitriptyline-D3, the internal standard of the digoxin is digoxin-D3, the internal standard of the sertraline is clonazepam-D4, the internal standard of the tolbutamide is amitriptyline-D3, the internal standard of the nordiazepam is amitriptyline-D3, the internal standard of the diazepam is noramitriptyline-D3, the internal standard of the penfluridol is pentafluridol-D7, the internal standard of the glimepiride is glimepiride-D5, the internal standard of the clopidogrel metabolite is clopidogrel metabolite-C6, and the internal standard of the clopidogrel is clopidogrel-D3.

2. The method of claim 1,

the standard working solution contains the following standard substances: clopidogrel bisulfate standards, clopidogrel MP derivative standards, 1- (2-pyrimidine) piperazine standards, tandospirone standards, mianserin standards, imipramine hydrochloride standards, hydroxyzine hydrochloride standards, desipramine standards, pentafluridol standards, digoxin standards, tolbutamide standards, paroxetine standards, reboxetine standards, venlafaxine standards, desvenlafaxine standards, glimepiride standards, buspirone standards, 6-hydroxybuspirone standards, clonazepam standards, sertraline standards, moclobemide standards, sulpiride standards, diazepam standards, desdiazepam standards, amitriptyline standards, noramitriptyline standards; the standard working solution comprises 8 grades of concentrations of L1, L2, L3, L4, L5, L6, L7 and L8.

3. The method of claim 2,

the concentration of each grade adopts methanol: the volume ratio of water is 7:3, diluting the mixed solution from the intermediate solution; the intermediate solution adopts methanol: the volume ratio of water is 7:3, diluting the mixed solution from the mother solution; and the mother liquor is obtained by dissolving the standard substance by adopting methanol or a methanol water solution.

4. The method of claim 1,

5. The method of claim 1, wherein the volume ratio of the anticoagulant fluid sample to the extractant is 1: 10;

drying the supernatant, and adding the complex solution, wherein the volume ratio of the supernatant to the complex solution is (9.5): 1.

6. the method of claim 1, wherein processing the sample to be tested comprises: adding an internal standard working solution and an extracting agent into an anticoagulated blood sample, performing vortex for 4-8 minutes, centrifuging for 8-12 minutes, taking a supernatant, drying, adding the complex solution, performing vortex for 0.5-2 minutes, and centrifuging for 4-7 minutes to obtain a supernatant, namely the sample to be detected;

the rotation speed of the vortex is 2000 rpm; the centrifugal speed was 14000 rpm.

7. The method according to any one of claims 1 to 6,

when the high performance liquid chromatography-mass spectrometer is AB SCIEX;

the column temperature is 50 ℃;

the flow rate is: 0.3 mL/min; the sample injection amount of the L1, L2, L3, L4, L5 and L6 standard working solutions is 5 mu L, and the sample injection amount of the L7 and L8 standard working solutions is 1 mu L; the sample injection amount of the sample to be detected is 5 mu L; the analysis time was 6.5 minutes;

the mass spectrum parameters are as follows:

ion source temperature: 350 ℃;

ion source high pressure: 5500V;

air curtain air: 20L/min;

collision gas: 8L/min;

drying gas 1: 50L/min;

drying gas 2: 50L/min.

8. The method according to any one of claims 1 to 6,

the column temperature is 50 ℃;

the flow rate is: 0.3 mL/min; the sample injection amount of the standard working solution of L1, L2, L3, L4, L5 and L6 is 5 mu L, and the sample injection amount of the standard working solution of L7 and L8 is 2 mu L; the sample injection amount of the sample to be detected is 5 mu L; the analysis time was 7.5 minutes;

the mass spectrum parameters are as follows:

an acquisition mode: ESI (+); MRM;

temperature of atomized gas: 200 ℃;

atomizing airflow: 3L/min;

temperature of sheath gas: 200 ℃;

sheath gas flow: 11L/min;

atomizing gas pressure: 45 psi;

capillary voltage: 5000V;

Delta-EMV：100 V。

9. a kit for 19 medicines and metabolites thereof in blood is characterized in that the 19 medicines comprise sulpiride, pentafluridol, mianserin, buspirone, tandospirone, hydroxyzine, diazepam, venlafaxine, moclobemide, imipramine, paroxetine, reboxetine, amitriptyline, sertraline, digoxin, clonazepam, clopidogrel, tolbutamide, glimepiride; the metabolites comprise 1-pyrimidinylpiperazine, desvenlafaxine, 6-hydroxybuspirone, desipramine, noramitriptyline, nordiazepam and clopidogrel metabolites;

the kit comprises the following reagents:

(1) internal standard working solution:

(2) a standard working fluid;

(3) extracting agent: methyl tert-butyl ether;

(4) compounding solution: the volume ratio is 1:1 methanol: mixing the solution with water;

(5) positive and negative controls;

the internal standard working solution contains the following internal standard substances in concentration: clonazepam-D4: 100 ng/mL; digoxin-D3: 100 ng/mL; clopidogrel-D3: 40 ng/mL; clopidogrel metabolite-C6: 500 ng/mL; glimepiride-D5: 100 ng/mL; pentaflurido-D7: 3200 ng/mL; imipramine-D3: 2000 ng/mL; desipramine-D3: 2000 ng/mL; mianserin-D3: 1000 ng/mL; tandospirone-D8: 20 ng/mL; 1-pyrimidinylpiperazine-D8: 10 ng/mL; buspirone-D8: 20 ng/mL; 6-hydroxybuspirone-D8: 20 ng/mL; hydroxyzine-D8: 40 ng/mL; venlafaxine-D6: 2000 ng/mL; moclobemide-D8: 1000 ng/mL; amitriptyline-D3: 500 ng/mL; noramitriptyline-D3: 2000 ng/mL; diazepam-D5: 400 ng/mL; levosulpiride-D3: 1000 ng/mL;

the standard working solution contains the following standard substances: clopidogrel bisulfate standards, clopidogrel active metabolite derivative standards, 1- (2-pyrimidine) piperazine standards, tandospirone standards, mianserin standards, imipramine hydrochloride standards, hydroxyzine hydrochloride standards, desipramine standards, pentafluridol standards, digoxin standards, tolbutamide standards, paroxetine standards, reboxetine standards, venlafaxine standards, desvenlafaxine standards, glimepiride standards, buspirone standards, 6-hydroxybuspirone standards, clonazepam standards, sertraline standards, moclobemide standards, sulpiride standards, diazepam standards, nordiazepam standards, amitriptyline standards, noramitriptyline standards;

the kit also contains a chromatographic column mobile phase; a is water containing 0.1 v/v% formic acid and 5mM ammonium formate; b is methanol containing 0.1 v/v% formic acid and 5mM ammonium formate; the positive reference substance is a standard quality control substance; the negative control is methanol with a volume ratio of 7: 3: and (3) water.