CN113933423B - Detection method for determining 23 kinds of psychotropic drugs and metabolites in human serum - Google Patents
Detection method for determining 23 kinds of psychotropic drugs and metabolites in human serum Download PDFInfo
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Abstract
The invention provides a detection method for measuring 23 kinds of psychotropic drugs and metabolites in human serum, which belongs to the technical field of psychotropic drugs and metabolites detection, mainly uses 9 kinds of stable isotope internal standards, and respectively selects a pair of quantitative ion pairs for each substance to be detected, takes the corresponding retention time as a qualitative basis, uses standard substances to make standard curve quantification, and adopts the accuracy and the effectiveness of quality control substance investigation methods with low, medium and high levels to effectively avoid matrix effects; the detection method provided by the invention has the characteristics of simple and convenient sample pretreatment, small required sample volume, high sensitivity, high accuracy and high flux.
Description
Technical Field
The invention belongs to the technical field of detection of psychotropic drugs and metabolites, and particularly relates to a method for simultaneously and quantitatively analyzing the concentration of 23 psychotropic drugs and metabolites in human serum based on an LC-MS/MS technology.
Background
There are about 200 drugs that can be used, which have been developed successively in the past 60 years in the psychiatric department, and these drugs have become effective and indispensable drugs for the treatment of mental disorders and mental symptoms. Therapeutic drug monitoring (Therapeutic drug monitoring, TDM) is a very effective method of tailored dosing regimens for individual patients. The main reason for using TDM to guide psychotropic drug therapy is the significant individual differences in pharmacokinetics of the patient. In the case of nearly identical doses, the homeostatic drug concentrations may differ by more than a factor of 20 from individual to individual, possibly due to differences in absorption, distribution, metabolism, excretion of the drug caused by differences in co-morbid, age, concomitant medication and genetic profile of the patient. Different dosage forms of the same medicine can also cause different in-vivo medicine concentrations due to different absorption degrees and absorption modes. TDM uses quantitative measurement of drug concentration in plasma or serum to perform dose titration of individual patients for optimal therapeutic effect, better tolerability, and reduced risk of poisoning. The TDM can also timely find out whether the patient stops taking medicine, reduces or takes medicine excessively in the treatment process, so that the patient can be helped to correctly know the taking medicine, and the dosage of the medicine can be adjusted according to the plasma concentration of a considerable amount of mental medicine in clinic at present.
The immune-based detection method is the most commonly used TDM method and has the characteristics of accuracy, rapidness and convenience. The method adopts the specific reaction of the antigen of the object to be detected and the antibody, and produces chemical reaction for detection by means of enzyme and a substrate or fluorescent label and the like to carry out quantitative determination. However, when the clinical test drug is detected together with other interferents having similar structures, the immunological method has a certain limitation. Because the chemical structures of the medicines are similar, and the medicines have similar antigen surface areas, antigen-antibody reaction can occur, and the medicines are difficult to completely distinguish by adopting an immunization method, so that the detected medicine concentration is deviated from the actual situation, and the curative effect cannot be well predicted and the adverse reaction cannot be well estimated.
In recent years, liquid chromatography tandem mass spectrometry (LC-MS/MS) has been widely used in TDM, which has high specificity and short running time and can simultaneously measure and analyze various chemical structures. In the prior art, for example, the method disclosed in Chinese patent CN111077239A for measuring the drug concentration of aripiprazole, clozapine, chlorpromazine, risperidone and 9-OH risperidone in human serum and the method disclosed in Chinese patent CN106918675A for detecting risperidone and 9-hydroxy risperidone in plasma have the advantages of small majority of flux, simultaneous measurement for one or more drugs, few dozens of drugs, and influence on detection efficiency. The existing partial methods, such as the method and the kit for simultaneously measuring 35 kinds of psychotropic drugs by using high-efficiency liquid chromatography-mass spectrometry disclosed in Chinese patent CN109655568A and the method for simultaneously detecting 17 kinds of antipsychotic drugs in a blood sample disclosed in Chinese patent CN109668979A, adopt an external standard method to measure the concentration of various drugs, have matrix effects, cannot accurately quantify, and also cause excessive cost due to excessive detection of the drug types, so that resource waste is formed, the large-scale clinical detection is not easy, and the sensitivity is low in the process of simultaneously detecting various drugs, so that the problem of relatively low sensitivity of various target substances is caused to influence the accuracy of detection, so that a reasonable method is developed by combining clinical demands. In addition, some of the methods cannot detect drugs and active metabolites simultaneously, and some drugs have pharmacological activities per se, and the metabolites have the same pharmacological actions as the original drugs, for example, clozapine and desmethylclozapine, aripiprazole and dehydroaripiprazole, venlafaxine and O-desmethylvenlafaxine, fluoxetine and norfluoxetine, quetiapine and N-dealkylquetiapine, etc., so that the active metabolites of the drugs are also included in the monitoring range, and the correlation of the blood concentration of patients and the therapeutic effect can be better explained.
In general, the main drawbacks in the clinical test procedure related to psychotropic drug detection in the prior art are:
there is a matrix effect: part of methods still adopt external standard methods for detecting the concentration of the medicine, have matrix effects and cannot be accurately quantified, and most of the current LC-MS/MS methods adopt isotope substitution and other internal standards for correcting the influences of matrix effects and the like, so that the external standard methods are eliminated gradually.
Detection of drug species is not applicable to clinical: the detection drugs are single or too many in types, the detection drugs are single in types and low in flux, the detection efficiency is affected, and more detection projects are not facilitated to develop; the detection of excessive medicine types causes excessive cost and resource waste, and is not easy for clinical detection in large batches.
Linear range is not suitable: the linear range of various medicines in the measuring method in the prior art is unreasonable, and the method is not suitable for clinical examination without verification of clinical samples.
Sensitivity is low: in the process of simultaneously detecting multiple drugs, the problem that the sensitivity of multiple targets is relatively low exists, and the detection accuracy is affected.
Some methods fail to detect both drug and active metabolite: some medicines have pharmacological activity, and the metabolites of the medicines have the same pharmacological action as the original medicines, so that the correlation between the blood concentration of a patient and the treatment effect can be better explained by bringing the active metabolites of the medicines into the monitoring range.
Disclosure of Invention
Based on the problems in the prior art, the invention provides a detection method for determining 23 psychotropic drugs and main metabolites in a human serum sample, which has the characteristics of simple and convenient sample pretreatment, small required sample volume, high sensitivity, high accuracy and high flux.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a detection method for determining 23 kinds of psychotropic drugs and metabolites in human serum comprises the steps of firstly, carrying out sample pretreatment on a human serum sample to be detected, respectively carrying out liquid chromatography-tandem mass spectrometry detection on each sample subjected to the pretreatment, and accurately and quantitatively analyzing the 23 kinds of psychotropic drugs and metabolites in the human serum; the psychotropic drugs and metabolites include risperidone, 9-hydroxy risperidone, olanzapine, quetiapine, N-dealkylquetiapine, aripiprazole, dehydroaripiprazole, amisulpride, clozapine, N-desmethylclozapine, ziprasidone, venlafaxine, O-desmethylvenlafaxine, levetiracetam, lamotrigine, oxcarbazepine, 10-hydroxy carbamazepine, fluoxetine, norfluoxetine, mirtazapine, escitalopram, sertraline and duloxetine; in the liquid chromatography tandem mass spectrometry detection, mass spectrometry adopts multi-reaction monitoring, and the MRM parameters of the compound are as follows: :
and adopting ESI ion source and positive ion mode to perform sectional scanning analysis.
According to the scheme, the detection method for determining 23 psychotropic drugs and metabolites in human serum adopts an internal standard method, wherein 9 stable isotope internal standards are adopted, namely quetiapine-d8, risperidone-d4, olanzapine-d8, aripiprazole-d8, amisulpride-d5, ziprasidone-d8, fluoxetine-d6, citalopram-d4 and levetiracetam-d 6.
According to the scheme, the detection method for determining 23 psychotropic drugs and metabolites in human serum comprises the following detailed steps:
step S1, preparing a calibrator solution, a quality control solution and an internal standard working solution with serial concentrations;
s2, adding an internal standard working solution into a sample to be tested, and vortex mixing;
and S3, centrifuging the mixed samples in the step S2, adding a diluting solution into the supernatant after centrifuging, diluting, and then carrying out LC-MS/MS analysis.
According to the above scheme, in the step S2, the internal standard working solution is a methanol or acetonitrile solution containing an internal standard, and is added according to an addition amount which is 2-5 times the volume of the sample to be measured; the diluent in the step S3 is 0.1% formic acid aqueous solution and is added according to the volume of 10-30 times.
According to the above scheme, the conditions of the liquid chromatography are as follows:
analytical column: agilent Eclipse Plus C18 RRHD 1.8 μm 2.1X10 mm;
mobile phase: phase A: an aqueous solution containing 0.1% formic acid; and B phase: acetonitrile solution containing 0.1% formic acid;
elution gradient: mobile phase a + mobile phase B = 100%;0 to 0.8min, and the volume of the mobile phase A is kept 95 percent; 0.8-2.0 min, the volume of the mobile phase A is reduced from 95% to 80%;2.0 to 7.0min, the volume of the mobile phase A is reduced from 80 percent to 62 percent; 7.0 to 8.0min, the volume of the mobile phase A is reduced from 62 percent to 5 percent; 8.0 to 9.0min, and the volume of the mobile phase A is kept 5 percent; 9.0 to 9.01min, the volume of the mobile phase A is increased from 5% to 95%; 9.01-10.0 min, the volume of the mobile phase A is kept 95%;
flow rate: 0.35mL/min; column temperature: 40 ℃.
According to the above scheme, the mass spectrometry conditions are as follows:
ion source temperature (Temperature (TEM)): 450 ℃; ion Source atomizing Gas (Ion Source Gas1 (GS 1)): 35psi; the Ion Source heats the assist Gas (Ion Source Gas2 (GS 2)): 35psi; curtain Gas (CUR)): 20psi; spray capillary Voltage (IonSpray Voltage (IS)): 5500V; collision Gas (CAD)): 9.
the beneficial effects of the invention are as follows: by combining with clinical detection requirements, the liquid chromatography-tandem mass spectrometry detection method for simultaneously determining 23 common psychotropic drugs and metabolites thereof is provided, and accurate quantification of up to 23 psychotropic drugs and metabolites thereof is realized by using stable isotope internal standards (9). For each substance to be detected, a pair of quantitative ion pairs is selected respectively, the corresponding retention time is taken as a qualitative basis, and standard curve quantification is manufactured by using standard products; and the accuracy and the effectiveness of the quality control product inspection method with low, medium and high levels are applied; meanwhile, the stable isotope internal standard is adopted for correction, so that the matrix effect can be effectively avoided.
Drawings
FIG. 1 is a standard graph and representative chromatogram of quetiapine determination by the method of the present invention (stabilized isotope internal standard employed: quetiapine-d 8);
FIG. 2 is a standard graph and representative chromatogram of N-dealkylated quetiapine (stable isotope internal standard: ziprasidone-d 8) for the determination of the methods of the present invention;
FIG. 3 is a standard graph and representative chromatogram of risperidone determination by the method of the invention (stable isotope internal standard employed: risperidone-d 4);
FIG. 4 is a standard graph and representative chromatogram of 9-hydroxyrisperidone determination using the method of the invention (stable isotope internal standard: risperidone-d 4);
FIG. 5 is a standard graph and representative chromatogram of olanzapine determination using the method of the present invention (stable isotope internal standard: olanzapine-d 8);
FIG. 6 is a standard graph and representative chromatogram of aripiprazole measured according to the method of the invention (stable isotope internal standard: aripiprazole-d 8);
FIG. 7 is a standard graph and representative chromatogram of the determination of dehydroaripiprazole according to the method of the present invention (stable isotope internal standard: aripiprazole-d 8);
FIG. 8 is a standard graph and representative chromatogram of amisulpride determination by the method of the invention (stable isotope internal standard employed: amisulpride-d 5);
FIG. 9 is a standard graph and representative chromatogram of ziprasidone determination using the method of the present invention (stable isotope internal standard: ziprasidone-d 8);
FIG. 10 is a standard graph and representative chromatogram of fluoxetine determination using the method of the invention (stable isotope internal standard employed: fluoxetine-d 6);
FIG. 11 is a standard graph and representative chromatogram of norfluoxetine determination using the method of the invention (stable isotope internal standard: fluoxetine-d 6);
FIG. 12 is a standard graph and representative chromatogram of sertraline determination using the method of the present invention (stable isotope internal standard: fluoxetine-d 6);
FIG. 13 is a standard graph and representative chromatogram of duloxetine determination according to the method of the present invention (stable isotope internal standard used: fluoxetine-d 6);
FIG. 14 is a standard graph and representative chromatogram of mirtazapine determination using the method of the invention (stable isotope internal standard employed: fluoxetine-d 6);
FIG. 15 is a standard graph and representative chromatogram of the determination of venlafaxine according to the present invention (stable isotope internal standard employed: levetiracetam-d 6);
FIG. 16 is a standard graph and representative chromatogram of an O-desmethylvenlafaxine assay according to the present invention (stable isotope internal standard employed: levetiracetam-d 6);
FIG. 17 is a standard graph and representative chromatogram of oxcarbazepine according to the method of the present invention (stable isotope internal standard employed: levetiracetam-d 6);
FIG. 18 is a standard graph and representative chromatogram of 10-hydroxycarbamoyl level (stable isotope internal standard employed: levetiracetam-d 6) determined according to the methods of the invention;
FIG. 19 is a standard graph and representative chromatogram of levetiracetam measured by the method of the invention (stable isotope internal standard employed: levetiracetam-d 6);
FIG. 20 is a standard graph and representative chromatogram of lamotrigine assay according to the present invention (stable isotope internal standard employed: levetiracetam-d 6);
FIG. 21 is a standard graph and representative chromatogram of clozapine determination using the method of the present invention (stable isotope internal standard employed: levetiracetam-d 6);
FIG. 22 is a standard graph and representative chromatogram of N-desmethylclozapine determination using the method of the present invention (stable isotope internal standard employed: levetiracetam-d 6);
FIG. 23 is a standard graph and representative chromatogram of escitalopram (stable isotope internal standard adopted: citalopram-d 4) for determination according to the method of the invention;
FIG. 24 is a total chromatogram of 23 psychotropic drug cocktails determined by the method of the invention.
The drawings are all LC-MS/MS detection analysis result graphs, which are the result display in the embodiment, and the characters in the drawings are the result display and can change according to the result of each detection analysis, namely, the characters in the drawings are irrelevant to whether the detection method provided by the invention can be repeatedly implemented or not, and the characters in the drawings are not clear and do not influence the detection method provided by the invention repeatedly implemented by a person skilled in the art.
Detailed Description
The technical scheme of the invention is described below with reference to specific embodiments and drawings.
The method comprises the steps of firstly carrying out sample pretreatment on a human serum sample to be detected, wherein 9 stable isotope internal standard substances are adopted, namely quetiapine-d8, risperidone-d4, olanzapine-d8, aripiprazole-d8, amisulpride-d5, ziprasidone-d8, fluoxetine-d6, citalopram-d4 and levetiracetam-d6, respectively carrying out liquid chromatography tandem mass spectrometry on the pretreated samples, and simultaneously accurately and quantitatively analyzing 23 psychotics and metabolites in the human serum, wherein the psychotics and the metabolites comprise risperidone, 9-hydroxy risperidone, olanzapine, quetiapine, N-desmethylquetiapine, aripiprazole, dehydroaripiprazole, amisulpride, clozapine, N-desmethylclozapine, ziprasidone, venlafaxine, O-norcisapraxine, levocetirizine, oxcarbazone, oxcarbazepine, oxepin, flufaxine, and other than half-moxetine, and the following the steps of detail:
step S1, preparing a calibrator solution, a quality control solution and an internal standard working solution with serial concentrations;
step S11, preparing stable isotope internal standard stock solution: the standard substances of Quetiapine-D8 (Quetiapine-D8 Fumarate), risperidone-D4 (Risperidone-D4), olanzapine-D8 (Olanzapine-D8), aripiprazole-D8 (Aripiprazole-D8), amisulpride-D5 (Amisulpride-D5), ziprasidone-D8 (Ziprasidone-D8), fluoxetine-D6 (fluoxoet), citalopram-D4 (Citalopram-D4), levetiracetam-D6 (Levetiracetam-D6) and the like were respectively weighed to prepare stable isotope internal standard stock solutions with a concentration of 1mg/mL using an organic solvent such as methanol, dimethyl sulfoxide (DMSO) and the like.
Step S12, preparing stable isotope internal standard intermediate working fluid: and (3) accurately transferring the stable isotope internal standard stock solution prepared in the step (S11), and respectively diluting the stable isotope internal standard stock solution into internal standard intermediate working solution with the mass concentration of 200 mug/mL by using methanol as a diluent.
Step S13, preparing stable isotope internal standard working fluid: accurately transferring the stable isotope internal standard intermediate working fluid prepared in the step S12, uniformly mixing, and preparing the stable isotope internal standard working fluid by using acetonitrile as a diluent; the stable isotope internal standard working fluid comprises the following components: quetiapine-d8 at 150ng/mL, risperidone-d4 at 10ng/mL, olanzapine-d8 at 10ng/mL, aripiprazole-d8 at 75ng/mL, amisulpride-d5 at 50ng/mL, ziprasidone-d8 at 50ng/mL, fluoxetine-d6 at 150ng/mL, citalopram-d4 at 18ng/mL, levetiracetam-d6 at 100ng/mL.
Step S14, preparation of standard stock solution: the appropriate amounts of lamotrigine, oxcarbazepine, 10-hydroxycarbazepine, levetiracetam, quetiapine, N-dealkylquetiapine, risperidone, 9-hydroxyrisperidone, olanzapine, aripiprazole, dehydroaripiprazole, clozapine, N-desmethylclozapine, amisulpride, ziprasidone, fluoxetine, norfluoxetine, sertraline, escitalopram, duloxetine, mirtazapine, venlafaxine and O-desmethylvenlafaxine were each weighed, dissolved with methanol and formulated into standard stock solutions, the concentrations shown in the following table:
step S15, preparing a mixed standard intermediate working solution: sequentially and accurately transferring the stock solution solutions of the standard substances prepared in the step S14, and preparing intermediate working solution of the mixed standard substances by using methanol as a diluent, wherein the concentrations are shown in the table below;
| names of Compounds | Concentration (μg/mL) | Names of Compounds | Concentration (μg/mL) |
| Lamotrigine | 0 | Clozapine | 108.0 |
| Oxcarbazepine | 0 | N-desmethylclozapine | 108.0 |
| 10-hydroxy carbamazepine | 0 | Amisulpride | 64.8 |
| Levetiracetam | 0 | Ziprasidone | 43.2 |
| Quetiapine | 81 | Fluoxetine | 108.0 |
| N-dealkylquetiapine | 54 | Norfluoxetine | 80.0 |
| Risperidone | 16.2 | Sertraline | 32.4 |
| 9-hydroxy risperidone | 16.2 | Escitalopram (escitalopram) | 21.6 |
| Olanzapine | 16.2 | Duloxetine | 27.0 |
| Aripiprazole | 108.0 | Mirtazapine | 10.8 |
| Dehydroaripiprazole | 108.0 | Venlafaxine | 81.0 |
| O-desmethylvenlafaxine | 81.0 |
Step S16, preparing mixed standard working solution: sequentially and accurately transferring the lamotrigine standard substance stock solution, oxcarbazepine standard substance stock solution, 10-hydroxy carbamazepine standard substance stock solution, levetiracetam standard substance stock solution and the mixed standard substance intermediate working solution prepared in the step S14, respectively preparing 7 gradient mixed standard substance working solutions by using human serum as a diluent, wherein the concentrations are shown in the following table;
| sequence number | Names of Compounds | C1 | C2 | C3 | C4 | C5 | C6 | C7 |
| 1 | Quetiapine | 18 | 37.5 | 75 | 225 | 360 | 450 | 900 |
| 2 | N-dealkylquetiapine | 12 | 25 | 50 | 150 | 240 | 300 | 600 |
| 3 | Risperidone | 3.6 | 7.5 | 15 | 45 | 72 | 90 | 180 |
| 4 | 9-hydroxy risperidone | 3.6 | 7.5 | 15 | 45 | 72 | 90 | 180 |
| 5 | Olanzapine | 3.6 | 7.5 | 15 | 45 | 72 | 90 | 180 |
| 6 | Aripiprazole | 24 | 50 | 100 | 300 | 480 | 600 | 1200 |
| 7 | Dehydroaripiprazole | 24 | 50 | 100 | 300 | 480 | 600 | 1200 |
| 8 | Clozapine | 24 | 50 | 100 | 300 | 480 | 600 | 1200 |
| 9 | N-desmethylclozapine | 24 | 50 | 100 | 300 | 480 | 600 | 1200 |
| 10 | Amisulpride | 14.4 | 30 | 60 | 180 | 288 | 360 | 720 |
| 11 | Ziprasidone | 9.6 | 20 | 40 | 120 | 192 | 240 | 480 |
| 12 | Fluoxetine | 24 | 50 | 100 | 300 | 480 | 600 | 1200 |
| 13 | Norfluoxetine | 17.8 | 37.0 | 74.0 | 222.0 | 355.6 | 444.0 | 888.0 |
| 14 | Sertraline | 7.2 | 15 | 30 | 90 | 144 | 180 | 360 |
| 15 | Escitalopram (escitalopram) | 4.8 | 10 | 20 | 60 | 96 | 120 | 240 |
| 16 | Duloxetine | 6 | 12.5 | 25 | 75 | 120 | 150 | 300 |
| 17 | Venlafaxine | 18 | 37.5 | 75 | 225 | 360 | 450 | 900 |
| 18 | O-desmethylvenlafaxine | 18 | 37.5 | 75 | 225 | 360 | 450 | 900 |
| 19 | Mirtazapine | 2.4 | 5 | 10 | 30 | 48 | 60 | 120 |
| 20 | Oxcarbazepine | 1.2 | 2.5 | 5 | 15 | 24 | 30 | 60 |
| 21 | 10-hydroxy carbamazepine | 1.2 | 2.5 | 5 | 15 | 24 | 30 | 60 |
| 22 | Levetiracetam | 1.2 | 2.5 | 5 | 15 | 24 | 30 | 60 |
| 23 | Lamotrigine | 0.3 | 0.625 | 1.25 | 3.75 | 6 | 7.5 | 15 |
In the above table, the concentration units of oxcarbazepine, 10-hydroxy carbamazepine, levetiracetam and lamotrigine are μg/mL, and the rest are ng/mL;
step S17, preparing mixed quality control working solution: sequentially and accurately transferring the lamotrigine standard substance stock solution, oxcarbazepine standard substance stock solution, 10-hydroxy carbamazepine standard substance stock solution, levetiracetam standard substance stock solution and mixed standard substance intermediate working solution prepared in the step S14, and respectively preparing mixed quality control working solutions with low, medium and high concentration levels by using human serum as a diluent, wherein the concentrations are shown in the following table;
| sequence number | Names of Compounds | LQC | MQC | HQC |
| 1 | Quetiapine | 54 | 360 | 450 |
| 2 | N-dealkylquetiapine | 36 | 240 | 480 |
| 3 | Risperidone | 10.8 | 72 | 144 |
| 4 | 9-hydroxy risperidone | 10.8 | 72 | 144 |
| 5 | Olanzapine | 10.8 | 72 | 144 |
| 6 | Aripiprazole | 72 | 480 | 960 |
| 7 | Dehydroaripiprazole | 72 | 480 | 960 |
| 8 | Clozapine | 72 | 480 | 960 |
| 9 | N-desmethylclozapine | 72 | 480 | 960 |
| 10 | Amisulpride | 43.2 | 288 | 576 |
| 11 | Ziprasidone | 28.8 | 192 | 384 |
| 12 | Fluoxetine | 72 | 480 | 960 |
| 13 | Norfluoxetine | 53.3 | 355.6 | 711.1 |
| 14 | Sertraline | 21.6 | 144 | 288 |
| 15 | Escitalopram (escitalopram) | 14.4 | 96 | 192 |
| 16 | Duloxetine | 18 | 120 | 240 |
| 17 | Venlafaxine | 54 | 360 | 720 |
| 18 | O-desmethylvenlafaxine | 54 | 360 | 720 |
| 19 | Mirtazapine | 7.2 | 48 | 96 |
| 20 | Oxcarbazepine | 3.6 | 24 | 48 |
| 21 | 10-hydroxy carbamazepine | 3.6 | 24 | 48 |
| 22 | Levetiracetam | 3.6 | 24 | 48 |
| 23 | Lamotrigine | 0.9 | 6 | 12 |
In the above table, the concentration units of oxcarbazepine, 10-hydroxy carbamazepine, levetiracetam and lamotrigine are μg/mL, and the rest are ng/mL;
step S2, a sample to be tested is subjected to a volume ratio of 1:4, adding an internal standard working solution with the volume of 4 times, and vortex mixing;
step S3, centrifuging the samples mixed in the step S2, and taking supernatant after centrifuging according to the volume ratio of 1:20 was diluted with 20 volumes of 0.1% formic acid in water and then analyzed by LC-MS/MS.
The conditions of the liquid chromatography are as follows:
analytical column: agilent Eclipse Plus C18 RRHD 1.8 μm 2.1X10 mm;
mobile phase: phase A: an aqueous solution containing 0.1% formic acid; and B phase: acetonitrile solution containing 0.1% formic acid;
elution gradient: mobile phase a + mobile phase B = 100%;0 to 0.8min, and the volume of the mobile phase A is kept 95 percent; 0.8-2.0 min, the volume of the mobile phase A is reduced from 95% to 80%;2.0 to 7.0min, the volume of the mobile phase A is reduced from 80 percent to 62 percent; 7.0 to 8.0min, the volume of the mobile phase A is reduced from 62 percent to 5 percent; 8.0 to 9.0min, and the volume of the mobile phase A is kept 5 percent; 9.0 to 9.01min, the volume of the mobile phase A is increased from 5% to 95%; 9.01-10.0 min, the volume of the mobile phase A is kept 95%;
flow rate: 0.35mL/min; column temperature: 40 ℃.
The mass spectrum conditions are as follows:
ion source temperature (Temperature (TEM)): 450 ℃; ion Source atomizing Gas (Ion Source Gas1 (GS 1)): 35psi; the Ion Source heats the assist Gas (Ion Source Gas2 (GS 2)): 35psi; curtain Gas (CUR)): 20psi; spray capillary Voltage (IonSpray Voltage (IS)): 5500V; collision Gas (CAD)): 9, a step of performing the process; mass spectra were monitored using multiple reactions, compound MRM parameters were as follows:
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and adopting ESI ion source and positive ion mode to perform sectional scanning analysis.
Detecting the 7 gradient mixed standard working solutions by using a high performance liquid chromatography-mass spectrometer to obtain chromatographic peak areas of 7 standard solutions with different concentrations, respectively taking the ratio of the chromatographic peak areas of the 7 standard solutions with different concentrations to the chromatographic peak areas of corresponding isotope internal standards as an ordinate y of a standard curve equation, taking the concentrations of the 7 standard solutions with different concentrations as an abscissa x of the standard curve, carrying out linear regression on the 7 data with different concentrations obtained by the detection, and fitting to obtain standard curve equations corresponding to all the standard solutions, wherein the standard curve equations are as follows: y=a×x+b, and the result is shown in the figure.
The detection method provided by the invention is further subjected to feasibility detection, and the process and the result are as follows:
firstly, the method provided by the invention is further analyzed in-batch and inter-batch precision, and the process and the result are as follows:
precision describes the proximity of repeated analyte determinations, defined as the relative standard deviation of the measured values (coefficient of variation, CV%) that should be used to obtain the precision of low, medium, and high concentration quality control samples within the same batch (intra-batch precision) and between different batches (inter-batch precision) using the results of analyzing the batch samples with the same demonstrated accuracy.
Precision in batch: the invention adopts blank human serum to prepare low, medium and high concentration mixed quality control working solution; each concentration was measured in 6 replicates for 1 day.
Precision between batches: the invention adopts blank human serum to prepare low, medium and high concentration mixed quality control working solution; each concentration was measured in 6 replicates and measured continuously for 3 days.
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The results of the batch-to-batch precision detection are: the variation coefficient (CV%) of 23 analytes to be detected among batches is less than or equal to 15%; the results of the in-batch precision detection are: the coefficient of variation (CV%) in the batch of all analytes to be tested is less than or equal to 15%.
Residual test:
the residue should be estimated by injecting a blank sample after injecting a high concentration sample or a calibration standard sample, and the residue in the blank sample after injecting the blank sample should not exceed 20% of the lower limit of quantification.
The invention adopts blank human serum to prepare a correction standard sample ULOQ, and is evaluated by continuously injecting 5 needles of blank samples after injecting the correction standard sample ULOQ; continuous measurement for 3 days, the detection results show that: the residues in the blank samples after ULOQ of 23 analytes to be detected are all less than 20% LLOQ;
matrix effect test:
the invention adopts 6 batches of blank human serum matrixes from different donors to examine the matrix effect of the analyte to be detected under the low concentration and the high concentration.
For each batch of matrix, calculating matrix factors of each analyte and stable isotope internal standard by calculating the ratio of the peak area (measured by adding mixed standard working solution and stable isotope internal standard after extraction from blank human serum matrix) in the presence of the matrix to the corresponding peak area (pure solution of analyte and stable isotope internal standard) of the matrix without human serum; and further dividing the matrix factor of the analyte by the matrix factor of the corresponding stable isotope internal standard, calculating the matrix factor normalized by the internal standard, wherein the detection results are shown in the following table, and the variation coefficient of the matrix factor normalized by the stable isotope internal standard calculated by 23 analytes from 6 batches of matrixes is less than 15%, which indicates that the matrix effect is not obvious in the invention.
The above examples are provided for illustration only and not for limitation of the technical solution of the present invention, although the above examples illustrate the present invention in detail, it should be understood by those skilled in the art: modifications and equivalents may be made thereto without departing from the spirit and scope of the invention, and any modifications and equivalents are intended to be included within the scope of the claims.
Claims (4)
1. The detection method for determining 23 kinds of psychotropic drugs and metabolites in human serum is characterized by comprising the following steps of:
step S1, preparing a calibrator solution, a quality control solution and an internal standard working solution with serial concentrations;
s2, adding an internal standard working solution into a sample to be tested, and vortex mixing;
step S3, centrifuging the mixed samples in the step S2, adding a diluting solution into supernatant after centrifuging, diluting, and then carrying out LC-MS/MS analysis;
the psychotropic drugs and metabolites include risperidone, 9-hydroxy risperidone, olanzapine, quetiapine, N-dealkylquetiapine, aripiprazole, dehydroaripiprazole, amisulpride, clozapine, N-desmethylclozapine, ziprasidone, venlafaxine, O-desmethylvenlafaxine, levetiracetam, lamotrigine, oxcarbazepine, 10-hydroxy carbamazepine, fluoxetine, norfluoxetine, mirtazapine, escitalopram, sertraline and duloxetine;
the conditions of the liquid chromatography are as follows:
analytical column: agilent Eclipse Plus C18 RRHD 1.8 μm 2.1X10 mm;
mobile phase: phase A: an aqueous solution containing 0.1% formic acid; and B phase: acetonitrile solution containing 0.1% formic acid;
elution gradient: mobile phase a + mobile phase B = 100%;0 to 0.8min, and the volume of the mobile phase A is kept 95 percent; 0.8-2.0 min, the volume of the mobile phase A is reduced from 95% to 80%;2.0 to 7.0min, the volume of the mobile phase A is reduced from 80 percent to 62 percent; 7.0 to 8.0min, the volume of the mobile phase A is reduced from 62 percent to 5 percent; 8.0 to 9.0min, and the volume of the mobile phase A is kept 5 percent; 9.0 to 9.01min, the volume of the mobile phase A is increased from 5% to 95%; 9.01-10.0 min, the volume of the mobile phase A is kept 95%;
flow rate: 0.35mL/min; column temperature: 40 ℃;
in the liquid chromatography tandem mass spectrometry detection, mass spectrometry adopts multi-reaction monitoring, and the MRM parameters of the compound are as follows:
and adopting ESI ion source and positive ion mode to perform sectional scanning analysis.
2. The method according to claim 1, wherein 9 stable isotope internal standard substances are used, namely quetiapine-d8, risperidone-d4, olanzapine-d8, aripiprazole-d8, amisulpride-d5, ziprasidone-d8, fluoxetine-d6, citalopram-d4 and levetiracetam-d 6.
3. The method for detecting 23 kinds of psychotropic drugs and metabolites in human serum according to claim 1, wherein in the step S2, the internal standard working solution is a methanol or acetonitrile solution containing an internal standard, and the internal standard working solution is added according to an addition amount which is 2-5 times the volume of the sample to be detected; the diluent in the step S3 is 0.1% formic acid aqueous solution and is added according to the volume of 10-30 times.
4. A method for detecting 23 psychotropic drugs and metabolites in human serum according to claim 3, wherein said mass spectrometry conditions are as follows:
ion source temperature: 450 ℃; ion source atomizing gas: 35psi; the ion source heats the auxiliary gas: 35psi; air curtain gas: 20psi; spray capillary voltage: 5500V; collision gas: 9.
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