CN111812218B - Method for simultaneously detecting concentration of multiple antipsychotic drugs in serum - Google Patents

Abstract

The invention provides a method for simultaneously detecting the concentration of multiple antipsychotic drugs in serum, which can detect 16 antipsychotic drugs at one time, simultaneously monitor target drugs and metabolites, has high sensitivity, strong specificity, accuracy and simple pretreatment process, completes the separation and detection of the antipsychotic drugs in the serum within 5.0 minutes, basically meets the requirements on accuracy and precision, can be used for the quantitative analysis of the antipsychotic drugs in the serum in clinic and provides a simple and rapid detection method for the concentration monitoring of the antipsychotic drugs in clinic.

Description

Method for simultaneously detecting concentration of multiple antipsychotic drugs in serum

Technical Field

The invention belongs to the technical field of blood detection, and particularly relates to a method for simultaneously detecting the concentration of multiple antipsychotic drugs in serum, wherein the antipsychotic drugs are respectively as follows: N-Desmethylclozapine (NCZP), olanzapine (OZP), chlorpromazine (CPZ), clozapine (czapine, CZP), sulpiride (SPR), amisulpride (ASP), haloperidol (HLP), quetiapine (QTP), norquetiapine (NQTP), perphenazine (PPZ), risperidone (Risperidone, RPD), ziprasidone (ZPD), 9-hydroxyrisperidone (paliperiperidone, PPD), fluphenazine (Fluphenazine, FPN), dehydroaripiprazole (dedropazoprazole, DHAPZ), aripiprazole (aridazole, DHAPZ)

Background

Antipsychotics are drugs suitable for the treatment of various psychotic disorders such as schizophrenia, hallucinations, mania, sleep disorders and dementia. There are over 9 and over 40 clinically used antipsychotics, among which the classical antipsychotics are the phenothiazines, thioxanthenes, butyrophenones, benzamides and dibenzepins. However, all antipsychotics may cause unpleasant side effects and severe intoxication after excessive use. These problems may be compounded when antipsychotics are used in combination. Unpleasant side effects often increase the non-compliance of psychotic patients. Thus, suitable analytical methods are essential means for Therapeutic Drug Monitoring (TDM) screening, identification and quantification.

At present, methods for detecting the concentration of the antipsychotic drug in vivo mainly comprise high performance liquid chromatography and ultra-high performance liquid-tandem mass spectrometry, and a method for detecting 15 psychotropic drugs and partial metabolites in plasma is reported in the literature of Screening, library-assisted identification and validated quantification of fine neurologic and same of the human beings, but the sample dosage is large, 0.5mL is needed, SPE is needed in the front, the cost is high, the acquisition needs 10 minutes, the linear range is narrow, and the like; the literature, "Therapeutic drug monitoring of seven psychotropic drugs and four methods in human plasma by HPLC-MS" reports a method for detecting various psychotropic drugs and metabolites in plasma, and has the disadvantages of complex pretreatment, long sampling time, narrow linear range and the like. Patent CN109668979A discloses a: "a method for simultaneously detecting 17 antipsychotic drugs in a blood sample", the method has large sample volume, and 20 mu L of sample is injected by one injection; the pretreatment is complex, and nitrogen blowing, concentration and redissolution are needed; the cost is high because filtration is needed; the collection time is long, and needs 9min. Patent CN110455945A discloses a method and a kit for detecting 5 psychotropic drugs and main metabolites thereof in blood, the method needs large sample amount, needs 100 mu L, only can detect 5 drugs at one time, and cannot meet the requirement of high-throughput screening of the drugs. In addition, there are patent reports that the efficiency is relatively low when one or more medicines are detected. The method has the defects of long analysis time of a single sample, low sensitivity, unsuitable linear range, complex sample pretreatment, matrix effect interference and the like.

Disclosure of Invention

It is an object of the present invention to provide a method for simultaneously detecting the concentration of a plurality of antipsychotic agents in serum on the basis of the prior art.

The technical scheme of the invention is as follows:

a method for simultaneously detecting the concentration of antipsychotic drugs in serum,

the antipsychotic drugs are respectively: N-Norclozapine (NCZP), olanzapine (OZP), chlorpromazine (CPZ), clozapine (CZP), sulpiride (SPR), amisulpride (ASP), haloperidol (HLP), quetiapine (QTP), norquetiapine (NQTP), perphenazine (PPZ), risperidone (RPD), ziprasidone (ZPD), 9-hydroxyrisperidone (PPD), fluphenazine (FPN), dehydroaripiprazole (DHAPZ) and Aripiprazole (APZ);

the internal standard substances corresponding to the antipsychotic drugs are respectively: n-norclozapine-d 8 (NCZP-d 8), olanzapine-d 3 (OZP-d 3), chlorpromazine-d 6 (CPZ-d 6), clozapine-d 8 (CZP-d 8), sulpiride-d 3 (SPR-d 3), amisulpride-d 5 (ASP-d 5), haloperidol-d 4 (HLP-d 4), quetiapine-d 8 (QTP-d 8), norquetiapine-13C 4 (NQTP-13C 4), perphenazine-d 8 (PPZ-d 8), risperidone-d 4 (RPD-d 4), ziprasidone-d 8 (ZPD-d 8), 9-hydroxyrisperidone-d 4 (PPD-d 4), fluphenazine-d 8 (FPN-d 8), dehydroaripiprazole-d 8 (DHAPZ-d 8) and aripiprazole-d 8 (APZ-d 8);

after a serum sample is subjected to protein precipitation, oscillating and centrifuging to obtain a supernatant for sample injection, detecting the antipsychotic drug in the pretreated serum by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, separating a substance to be detected from a serum matrix by using the ultra-high performance liquid chromatography, establishing a calibration curve by using a mass spectrum isotope internal standard quantitative method and taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the content of the antipsychotic drug, wherein the specific chromatographic conditions are as follows:

(1) Ultra-high performance liquid chromatography conditions:

a mobile phase A:0.005% -0.1% formic acid water solution; and (3) mobile phase B: acetonitrile;

the type of the chromatographic column: waters BEH C18 (2.1X 100mm,1.7 μm);

adopting a mode of gradient elution by taking a mobile phase A and a mobile phase B as a mixed mobile phase, wherein the initial ratio of the mobile phase A to the mobile phase B is 80-100-20; the gradient elution procedure was as follows: in 0-1.0 min, the volume ratio of the mobile phase A to the mobile phase B is uniformly and gradually changed to 30 from the initial ratio; in 1.0-3.0 minutes, the volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 30; changing the volume ratio of the mobile phase A to the mobile phase B from 2; the collection time of each sample was 5.0 minutes;

(2) Mass spectrum conditions:

in an electrospray ionization positive ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, and the capillary voltage is 2.5kV (ESI +); the source temperature is 120 ℃; the temperature of atomizing gas is 500 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper hole is 150L/h; each target and its corresponding isotope internal standard were monitored simultaneously.

In order to improve the chromatographic separation selectivity, it may be considered to adjust the polarity of the mobile phase. The formic acid is added into the mobile phase A, so that the ionization efficiency of certain target compounds can be effectively improved, and under the coordination of other conditions, compared with the method for detecting the antiepileptic drug in serum by adopting an LC-MS/MS method in the prior art, the method has the advantages of higher sensitivity, simple pretreatment process, low cost, high sensitivity and strong specificity, and can complete the separation and detection of the antiepileptic drug within 5 min. In a preferable embodiment, the mobile phase a is 0.01% to 0.1% formic acid aqueous solution, and in the case of not affecting the effect of the present invention, the mobile phase a is preferably 0.05% formic acid aqueous solution.

In chromatography, the choice of the column is important, and the requirements for the column are: high column efficiency, good selectivity, high analysis speed and the like. The invention adopts acetonitrile and 0.005% -0.1% formic acid aqueous solution as mobile phase, the model of chromatographic column is Waters BEH C18 (2.1X 100mm,1.7 μm), under the coordination of other conditions, the endogenous substance does not interfere the determination of the sample, the sensitivity is high, the specificity is strong, and the accuracy and the precision basically meet the requirements.

When the internal standard method is adopted, the selection of the internal standard substance is very important work. The ideal internal standard should be capable of being added to the sample in an accurate, known amount, and have substantially the same or as consistent as possible physicochemical properties, chromatographic behavior, and response characteristics as the sample being analyzed; under chromatographic conditions, the internal standard must be sufficiently separated from the components of the sample. The invention respectively adopts N-norclozapine-d 8 (NCZP-d 8), olanzapine-d 3 (OZP-d 3), chlorpromazine-d 6 (CPZ-d 6), clozapine-d 8 (CZP-d 8), sulpiride-d 3 (SPR-d 3), amisulpride-d 5 (ASP-d 5), haloperidol-d 4 (HLP-d 4), quetiapine-d 8 (QTP-d 8), norquetiapine-13C 4 (NQTP-13C 4), perphenazine-d 8 (PPZ-d 8), risperidone-d 4 (RPD-d 4), ziprasidone-d 8 (ZPD-d 8), 9-hydroxypiperidone-d 4 (PPD-d 4), fluphenazine-d 8 (FPN-d 8), dehydroaripiprazole-d 8 (DHAPZ-d 8) and aripiprazole-d 8 (DHAPZ-d 8) as internal standard medicaments, and has the same chemical stability and stability, and the internal standard effect can be measured with better determination accuracy and better stability and stability.

In a preferred embodiment, the initial ratio of mobile phase A to mobile phase B is 85 to 95. Further preferably, the initial ratio of mobile phase a and mobile phase B is 90.

In a preferred embodiment, the flow rate is 0.2 to 0.5mL/min, preferably 0.3mL/min.

Further, the column temperature is 30 to 50 ℃, preferably 45 ℃.

In one embodiment, the injection volume is 0.2 to 5. Mu.L, preferably 1. Mu.L.

In a preferred embodiment, when the above antipsychotic drugs in the pretreated serum are detected by the hplc-tandem mass spectrometry, the specific chromatographic conditions are as follows:

(1) Ultra-high performance liquid chromatography conditions:

mobile phase A:0.05% aqueous formic acid; mobile phase B: acetonitrile;

the type of the chromatographic column: waters BEH C18 (2.1X 100mm,1.7 μm);

the initial ratio of mobile phase a to mobile phase B was 90; the gradient elution procedure was as follows: the volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 90 to 10 to 30 in 0-1.0 min; in 1.0-3.0 minutes, the volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 30; the volume ratio of mobile phase a to mobile phase B is changed from 2; the time taken for each sample was 5.0 minutes. The gradient elution mode is specifically shown in table 1; the flow rate is 0.4mL/min, the column temperature is 45 ℃, and the sample injection volume is 1 mu L;

TABLE 1 mobile phase gradient elution parameters

Time (min)	Flow rate (mL/min)	％A	％B	Curve
					0.0	0.4	90	10	-
1.0	0.4	30	70	6
					3.0	0.4	2	98	6
5.0	0.4	90	10	1

(2) Mass spectrum conditions:

in an electrospray ionization positive ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, and the capillary voltage is 2.5kV (ESI +); the source temperature is 120 ℃; the temperature of atomizing gas is 500 ℃, the airflow speed of atomizing gas is 800L/h, and the airflow speed of taper holes is 150L/h; the mass spectrum source parameters are shown in table 2, and the mass spectrum parameters of each target and the corresponding isotope internal standard thereof are monitored at the same time, and are shown in table 3.

TABLE 2 Mass Spectrometry Source parameters

TABLE 3 Mass Spectrometry parameters for antipsychotic drug detection

The serum mentioned in the invention is human or animal serum.

The pretreated serum mentioned in the invention is prepared according to the following method: adding a protein precipitator containing an internal standard into the serum, and then oscillating and centrifuging to obtain a supernatant; the protein precipitator is a mixed solution of isopropanol, methanol and acetonitrile; preferably, the volume ratio of isopropanol, methanol and acetonitrile in the protein precipitant is 1.

In a preferred embodiment, the pretreated serum of the present invention is prepared as follows: putting 50 mu L of serum into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitator containing an internal standard into the centrifuge tube, oscillating for 3-5 min, centrifuging for 4-10 min at 1-5 ℃ at 12000-15000 r/min, transferring 60 mu L of supernatant in the centrifuge tube into a plastic inner lining tube, and feeding a sample; the volume ratio of isopropanol, methanol and acetonitrile in the protein precipitant is 1.

In a more preferred embodiment, the pretreated serum of the present invention is prepared as follows: taking 50 mu L of serum to a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the volume ratio of isopropanol, methanol and acetonitrile is 1; centrifuging at 14000r/min at 4 ℃ for 5min; transfer 60. Mu.L of supernatant from EP tubes to plastic lined tubes for injection.

In one embodiment, the protein precipitant containing the internal standard is prepared according to the following method:

preparing the following isotope internal standard mother liquor by using methanol: n-norclozapine-d 8 (NCZP-d 8) 0.1mg/mL, olanzapine-d 3 (OZP-d 3) 0.1mg/mL, chlorpromazine-d 6 (CPZ-d 6) 0.1mg/mL, clozapine-d 8 (CZP-d 8) 1mg/mL, sulpiride-d 3 (SPR-d 3) 0.25mg/mL, amisulpride-d 5 (ASP-d 5) 0.1mg/mL, haloperidol-d 4 (HLP-d 4) 0.01mg/mL, quetiapine-d 8 (QTP-d 8) 0.1mg/mL, norquetiapine-13C 4 (NQTP-13C 4) 0.1mg/mL, perphenazine-d 8 (PPZ-d 8) 0.01mg/mL, risperidone-d 4 (RPD-d 4) 0.01mg/mL, ziprasidone-d 8 (DHP-d 8) 0.8 mg/mL, and haloperidol-d 8 (PPDp-d 8) 0.1mg/mL.

Respectively transferring the internal standard mother liquor of each isotope: 2.5 μ L of N-norclozapine-d 8 (NCZP-d 8), 5 μ L of olanzapine-d 3 (OZP-d 3), 10 μ L of chlorpromazine-d 6 (CPZ-d 6), 1 μ L of clozapine-d 8 (CZP-d 8), 4 μ L of sulpiride-d 3 (SPR-d 3), 10 μ L of amisulpride-d 5 (ASP-d 5), 10 μ L of haloperidol-d 4 (HLP-d 4), 10 μ L of quetiapine-d 8 (QTP-d 8), 10 μ L of norquetiapine-13C 4 (NQTP-13C 4), 10 μ L of perphenazine-d 8 (PPZ-d 8), 25 μ L of risperidone-d 4 (RPD-d 4), 10 μ L of rasidone-d 8 (ZPD-d 8), 1 μ L of 9-hydroxypiperidone-d 4 (DHP-d 8), 10 μ L of dehydroepiprazole-d 8 (APZ-d 8), and 10 μ L of meperidol-d 8 (PPZ-d 8); additional addition to 860.5. Mu.L of methanol gave 1mL of mixed internal standard solution.

And adding 200uL of the mixed internal standard solution into 19.8mL of protein precipitator to obtain the protein precipitator containing the internal standard.

In one scheme, the protein precipitator is a mixed solution of isopropanol, methanol and acetonitrile; preferably, the volume ratio of isopropanol, methanol and acetonitrile in the protein precipitant is 1; more preferably, the volume ratio of isopropanol, methanol and acetonitrile in the protein precipitant is 1.

In a preferred embodiment, the protein precipitant containing the internal standard is prepared by the following method:

preparing the following isotope internal standard mother liquor by using methanol: n-norclozapine-d 8 (NCZP-d 8) 0.1mg/mL, olanzapine-d 3 (OZP-d 3) 0.1mg/mL, chlorpromazine-d 6 (CPZ-d 6) 0.1mg/mL, clozapine-d 8 (CZP-d 8) 1mg/mL, sulpiride-d 3 (SPR-d 3) 0.25mg/mL, amisulpride-d 5 (ASP-d 5) 0.1mg/mL, haloperidol-d 4 (HLP-d 4) 0.01mg/mL, quetiapine-d 8 (QTP-d 8) 0.1mg/mL, norquetiapine-13C 4 (NQTP-13C 4) 0.1mg/mL, perphenazine-d 8 (PPZ-d 8) 0.01mg/mL, risperidone-d 4 (RPD-d 4) 0.01mg/mL, ziprasidone-d 8 (DHP-d 8) 0.8 mg/mL, and haloperidol-d 8 (PPDp-d 8) 0.1mg/mL.

Respectively transferring the internal standard mother liquor of each isotope: 2.5 μ L of N-norclozapine-d 8 (NCZP-d 8), 5 μ L of olanzapine-d 3 (OZP-d 3), 10 μ L of chlorpromazine-d 6 (CPZ-d 6), 1 μ L of clozapine-d 8 (CZP-d 8), 4 μ L of sulpiride-d 3 (SPR-d 3), 10 μ L of amisulpride-d 5 (ASP-d 5), 10 μ L of haloperidol-d 4 (HLP-d 4), 10 μ L of quetiapine-d 8 (QTP-d 8), 10 μ L of norquetiapine-13C 4 (NQTP-13C 4), 10 μ L of perphenazine-d 8 (PPZ-d 8), 25 μ L of risperidone-d 4 (RPD-d 4), 10 μ L of ziprasidone-d 8 (ZPD-d 8), 1 μ L of 9-hydroxypiperone-d 4 (PPZ-d 8), 5 μ L of haloperidol-d 8 (DHP-d 8), 10 μ L of dehydroepirizole-d 4 (DHP-d 8), 10 μ L of piperazinone-d 8 (DHP-d 8) and 10 μ L of azazole; additional addition to 860.5. Mu.L of methanol gave 1mL of mixed internal standard solution.

Adding 200uL of the mixed internal standard solution into 19.8mL of a protein precipitant (the volume ratio of isopropanol, methanol and acetonitrile is 1. Wherein, the protein precipitator comprises: n-norclozapine-d 8 (NCZP-d 8) 2.5ng/mL, olanzapine-d 3 (OZP-d 3) 5ng/mL, chlorpromazine-d 6 (CPZ-d 6) 10ng/mL, clozapine-d 8 (CZP-d 8) 10ng/mL L, sulpiride-d 3 (SPR-d 3) 10ng/mL, amisulpride-d 5 (ASP-d 5) 10ng/mL, haloperidol-d 4 (HLP-d 4) 1ng/mL, quetiapine-d 8 (QTP-d 8) 10ng/mL, norquetiapine-13C 4 (NQTP-13C 4) 10ng/mL, perphenazine-d 8 (PPZ-d 8) 2.5ng/mL, risperidone-d 4 (RPD-d 4) 1ng/mL, ziprasidone-d 8 (ZPng-d 8) 10ng/mL, perphenazine-d 8 (PPZ-d 8) 2.5ng/mL, risperidone-d 4 (RPD 4-d 4) 1ng/mL, azalazone-d 8 (ZPng-d 8) 10ng/mL, and dehydropiperazinone (DHP-d 8 (DHP-d 8) 2.5 ng/mL).

In one embodiment, the standard mentioned in the present invention is prepared as follows:

the antipsychotic drug was formulated as a master batch at the following concentrations: 2mg/mL of N-Norclozapine (NCZP), 1mg/mL of Olanzapine (OZP), 5mg/mL of Chlorpromazine (CPZ), 5mg/mL of Clozapine (CZP), 5mg/mL of Sulpiride (SPR), 2mg/mL of Amisulpride (ASP), 2mg/mL of Haloperidol (HLP), 5mg/mL of Quetiapine (QTP), 10mg/mL of Norquetiapine (NQTP), 1mg/mL of perphenazine (PPZ), 5mg/mL of Risperidone (RPD), 2mg/mL of Ziprasidone (ZPD), 2mg/mL of 9-hydroxypiperidone (PPD), 2mg/mL of Fluphenazine (FPN), 2mg/mL of Dehydroaripiprazole (DHAPZ) and 1mg/mL of Aripiprazole (APZ).

Respectively transferring mother liquor of each standard product: 20 μ L of N-Norclozapine (NCZP), 20 μ L of Olanzapine (OZP), 8 μ L of Chlorpromazine (CPZ), 10 μ L of Clozapine (CZP), 8 μ L of Sulpiride (SPR), 25 μ L of Amisulpride (ASP), 1 μ L of Haloperidol (HLP), 10 μ L of Quetiapine (QTP), 5 μ L of Norquetiapine (NQTP), 1 μ L of perphenazine (PPZ), 1 μ L of Risperidone (RPD), 25 μ L of Ziprasidone (ZPD), 10 μ L of 9-hydroxypiperidone (PPD), 1 μ L of Fluphenazine (FPN), 20 μ L of Dehydroaripiprazole (DHAPZ) and 50 μ L of Aripiprazole (APZ); additional addition to 785. Mu.L of methanol gave 1mL of mixed standard stock solution. The mixed standard stock solution comprises: N-Norclozapine (NCZP) 40. Mu.g/mL, olanzapine (OZP) 20. Mu.g/mL, chlorpromazine (CPZ) 40. Mu.g/mL, clozapine (CZP) 50. Mu.g/mL, sulpiride (SPR) 40. Mu.g/mL, amisulpride (ASP) 50. Mu.g/mL, haloperidol (HLP) 2. Mu.g/mL, quetiapine (QTP) 50. Mu.g/mL, norquetiapine (NQTP) 50. Mu.g/mL, perphenazine (PPZ) 1. Mu.g/mL, risperidone (RPD) 5. Mu.g/mL, ziprasidone (ZPD) 50. Mu.g/mL, 9-hydroxypiperidone (PPD) 20. Mu.g/mL, fluphenazine (FPN) 2. Mu.g/mL, dehydroaripiprazole (APZ) 40. Mu.g/mL, and Aripiprazole (APZ) 50. Mu.g/mL.

Preparing the mixed standard stock solution into a calibrator solution with seven different concentration points by using a blank serum matrix, wherein the seven concentration points of the calibrator solution are as follows:

the concentrations of Clozapine (CZP), amisulpride (ASP), quetiapine (QTP), norquetiapine (NQTP), ziprasidone (ZPD) and Aripiprazole (APZ) are the same, and the seven concentration points are as follows: 5ng/mL, 12.5ng/mL, 25ng/mL, 125ng/mL, 250ng/mL, 1250ng/mL, 2500ng/mL;

the concentrations of N-Norclozapine (NCZP), chlorpromazine (CPZ), sulpiride (SPR) and Dehydroaripiprazole (DHAPZ) are the same, and the seven concentration points are as follows: 4ng/mL, 10ng/mL, 20ng/mL, 100ng/mL, 200ng/mL, 1000ng/mL, 2000ng/mL;

olanzapine (OZP) and 9-hydroxyrisperidone (PPD) were at the same concentration, with seven concentration points in the order: 2ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL;

the seven concentration points of Risperidone (RPD) are, in order: 0.5ng/mL, 1.25ng/mL, 2.5ng/mL, 12.5ng/mL, 25ng/mL, 125ng/mL, 250ng/mL;

the concentrations of Haloperidol (HLP) and Fluphenazine (FPN) are the same, with seven concentration points being in order: 0.2ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL;

seven concentration Points of Perphenazine (PPZ) are in order: 0.1ng/mL, 0.25ng/mL, 0.5ng/mL, 2.5ng/mL, 5ng/mL, 25ng/mL, 50ng/mL.

In a preferred embodiment, the blank serum base is a blank serum free of antipsychotic agent of interest.

In a more preferred embodiment, the present invention prepares the stock mixed standard solutions into seven different concentration point calibrator solutions in a blank serum base (blank serum without antipsychotic target drug) by the following process:

adding 10 mu L of mixed standard stock solution into 190 mu L of blank serum matrix to serve as a first high-value concentration point; taking the first high-value concentration point, and diluting the first high-value concentration point with an equal volume of blank serum matrix to obtain a second high-value concentration point; diluting the first high-value concentration point with 9 times volume of blank serum substrate to obtain a third high-value concentration point; diluting the second high-value concentration point with 9 times volume of blank serum substrate to obtain a fourth high-value concentration point; diluting the third high-value concentration point with 9 times of volume of blank serum substrate to obtain a fifth high-value concentration point; diluting the fourth high-value concentration point with 9 times of volume of blank serum substrate to obtain a sixth high-value concentration point; and (4) diluting the fifth high-value concentration point with blank serum substrate with 4 times of volume to obtain a seventh high-value concentration point.

Taking 50 μ L of each concentration point of seven different calibrator samples, placing in a 1.5mL centrifuge tube, adding 200 μ L of a protein precipitant containing an internal standard (volume ratio of isopropanol, methanol and acetonitrile is 1; centrifuging at 14000r/min at 4 ℃ for 5min; transfer 60. Mu.L of supernatant from the EP tube to a plastic lined tube for injection.

The invention also comprises preparing a quality control product, wherein the quality control product is blank serum containing the antipsychotic drugs, and the concentration of the blank serum is QC (L), QC (M) and QC (H) respectively;

QC (L) was a 5000-fold dilution of the above mixed standard stock solution in blank serum matrix.

QC (M) was a 500-fold dilution of the above mixed standard stock solution in blank serum matrix.

QC (H) was a 50-fold dilution of the above mixed standard stock solution in blank serum matrix.

Preferably, the blank serum base is a blank serum free of antipsychotic target drugs.

In a preferred embodiment, the quality control product is prepared according to the following method: the mixed standard stock solutions were prepared into three different concentrations of QC (L), QC (M), and QC (H) with blank serum without antipsychotic target drug, as shown in table 4.

TABLE 4 corresponding concentration of quality control article (unit: ng/mL)

QC (L) comprises: N-Norclozapine (NCZP) 8ng/mL, olanzapine (OZP) 4ng/mL, chlorpromazine (CPZ) 8ng/mL, clozapine (CZP) 10ng/mL, sulpiride (SPR) 8ng/mL, amisulpride (ASP) 10ng/mL, haloperidol (HLP) 0.4ng/mL, quetiapine (QTP) 10ng/mL, norquetiapine (NQTP) 10ng/mL, perphenazine (PPZ) 0.2ng/mL, risperidone (RPD) 1ng/mL, ziprasidone (ZPD) 10ng/mL, 9-hydroxypiperidone (PPD) 4ng/mL, fluphenazine (FPN) 0.4ng/mL, dehydroaripiprazole (DHAPZ) 8ng/mL, and Aripiprazole (APZ) 10ng/mL.

QC (M) includes: 80ng/mL of N-Norclozapine (NCZP), 40ng/mL of Olanzapine (OZP), 80ng/mL of Chlorpromazine (CPZ), 100ng/mL of Clozapine (CZP), 80ng/mL of Sulpiride (SPR), 100ng/mL of Amisulpride (ASP), 4ng/mL of Haloperidol (HLP), 100ng/mL of Quetiapine (QTP), 100ng/mL of Norquetiapine (NQTP), 2ng/mL of perphenazine (PPZ), 10ng/mL of Risperidone (RPD), 100ng/mL of Ziprasidone (ZPD), 40ng/mL of 9-hydroxyrisperidone (PPD), 4ng/mL of Fluphenazine (FPN), 80ng/mL of Dehydroaripiprazole (DHAPZ) and 100ng/mL of Aripiprazole (APZ).

QC (H) includes: 800ng/mL of N-Norclozapine (NCZP), 400ng/mL of Olanzapine (OZP), 800ng/mL of Chlorpromazine (CPZ), 1000ng/mL of Clozapine (CZP), 800ng/mL of Sulpiride (SPR), 1000ng/mL of Amisulpride (ASP), 40ng/mL of Haloperidol (HLP), 1000ng/mL of Quetiapine (QTP), 1000ng/mL of Norquetiapine (NQTP), 20ng/mL of perphenazine (PPZ), 100ng/mL of Risperidone (RPD), 1000ng/mL of Ziprasidone (ZPD), 400ng/mL of 9-hydroxyrisperidone (PPD), 40ng/mL of Fluphenazine (FPN), 800ng/mL of Dehydroaripiprazole (DHAPZ) and 1000ng/mL of Aripiprazole (APZ).

The invention aims to establish a multi-drug analysis method, which adopts high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC/ESI-MS/MS) and is used for rapidly screening, identifying and quantitatively analyzing 16 antipsychotic drugs from sub-treatment concentration to overdose concentration in serum.

By adopting the technical scheme of the invention, the advantages are as follows:

the invention provides a method for simultaneously detecting concentration of antipsychotic drugs in serum, which can detect 16 antipsychotic drugs at one time, simultaneously monitor target drugs and metabolites, has high sensitivity, strong specificity, accuracy and simple pretreatment process, completes separation and detection of antipsychotic drugs in serum within 5.0 minutes, has accuracy and precision basically meeting requirements, can be used for quantitative analysis of antipsychotic drugs in serum clinically, and provides a simple and rapid detection method for monitoring concentration of antipsychotic drugs clinically.

Drawings

Figure 1 is an extracted ion flow chromatogram of an antipsychotic drug standard;

FIG. 2 is an extracted ion flow chromatogram of antipsychotic agents in serum.

Detailed Description

The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.

Example 1:

1. experimental materials and instruments

1. Material

The samples were obtained from serum samples collected from the clinic of heart disease hospital wuhan asia in 2019, month 8.

(1) The instrument comprises the following steps: xevo TQ-S triple quadrupole mass spectrometer (Waters Corporation); UPLC I-Class ultra high performance liquid chromatography system (with autosampler, waters Corporation); SCILOGEX D2012 high speed bench top centrifuge (usa); ultra pure water meter (ELGA LabWater, uk); multi-tube Vortex mixer (Vortex genie2, usa); an adjustable pipettor (Eppendorf 0.5-10 muL, 10-100 muL, 100-1000 muL); glassware, graduated cylinders, and the like.

(2) Reagent consumables: MS grade methanol (Fisher, usa); HPLC grade methanol (Honeywell, usa); MS grade acetonitrile (Fisher, usa); HPLC grade acetonitrile (Honeywell, usa); HPLC grade isopropanol (Honeywell, usa); MS grade formic acid (Fisher, usa); the type of the chromatographic column: waters BEH C18 (2.1X 100mm,1.7 μm) (Waters Corporation).

(3) And (3) standard substance: the standards and their corresponding internal standards are shown in table 5.

TABLE 5 Standard and internal standards

(4) Quality control product: the blank serum containing antipsychotic drugs has low, medium and high concentrations of QC (L), QC (M) and QC (H), which are shown in Table 4.

2. Liquid condition

(1) Chromatographic conditions are as follows: a mobile phase A:0.05% aqueous formic acid; mobile phase B: and (3) acetonitrile. Type of chromatographic column: waters BEH C18 (2.1X 100mm,1.7 μm), using gradient elution, see Table 1 for details. The flow rate was 0.4mL/min, the column temperature was 45 ℃ and the injection volume was 1. Mu.L.

(2) Mass spectrum conditions: in an electrospray ionization positive ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, and the capillary voltage is 2.5kV (ESI +); the source temperature is 120 ℃; the temperature of atomizing gas is 400 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper holes is 150L/h; the mass spectrum source parameters are shown in table 2, and the mass spectrum parameters of each target and the corresponding isotope internal standard thereof are monitored at the same time and are shown in table 3.

3. Experimental procedures

(1) Preparing a standard substance:

the antipsychotic drug was formulated as a master batch at the following concentrations: 2mg/mL of N-Norclozapine (NCZP), 1mg/mL of Olanzapine (OZP), 5mg/mL of Chlorpromazine (CPZ), 5mg/mL of Clozapine (CZP), 5mg/mL of Sulpiride (SPR), 2mg/mL of Amisulpride (ASP), 2mg/mL of Haloperidol (HLP), 5mg/mL of Quetiapine (QTP), 10mg/mL of Norquetiapine (NQTP), 1mg/mL of perphenazine (PPZ), 5mg/mL of Risperidone (RPD), 2mg/mL of Ziprasidone (ZPD), 2mg/mL of 9-hydroxypiperidone (PPD), 2mg/mL of Fluphenazine (FPN), 2mg/mL of Dehydroaripiprazole (DHAPZ) and 1mg/mL of Aripiprazole (APZ).

Respectively transferring mother liquor of each standard product: 20 μ L of N-Norclozapine (NCZP), 20 μ L of Olanzapine (OZP), 8 μ L of Chlorpromazine (CPZ), 10 μ L of Clozapine (CZP), 8 μ L of Sulpiride (SPR), 25 μ L of Amisulpride (ASP), 1 μ L of Haloperidol (HLP), 10 μ L of Quetiapine (QTP), 5 μ L of Norquetiapine (NQTP), 1 μ L of perphenazine (PPZ), 1 μ L of Risperidone (RPD), 25 μ L of Ziprasidone (ZPD), 10 μ L of 9-hydroxypiperidone (PPD), 1 μ L of Fluphenazine (FPN), 20 μ L of Dehydroaripiprazole (DHAPZ) and 50 μ L of Aripiprazole (APZ); additional addition to 785. Mu.L of methanol gave 1mL of mixed standard stock solution. The mixed standard stock solution comprises: N-Norclozapine (NCZP) 40. Mu.g/mL, olanzapine (OZP) 20. Mu.g/mL, chlorpromazine (CPZ) 40. Mu.g/mL, clozapine (CZP) 50. Mu.g/mL, sulpiride (SPR) 40. Mu.g/mL, amisulpride (ASP) 50. Mu.g/mL, haloperidol (HLP) 2. Mu.g/mL, quetiapine (QTP) 50. Mu.g/mL, norquetiapine (NQTP) 50. Mu.g/mL, perphenazine (PPZ) 1. Mu.g/mL, risperidone (RPD) 5. Mu.g/mL, ziprasidone (ZPD) 50. Mu.g/mL, 9-hydroxypiperone (PPD) 20. Mu.g/mL, fluphenazine (FPN) 2. Mu.g/mL, dehydroaripiprazole (DHAPZ) 40. Mu.g/mL, and Aripiprazole (APZ) 50. Mu.g/mL.

The mixed standard stock solutions described above were formulated in a blank serum base (blank serum without antipsychotic target drug) into seven different concentration point calibrator solutions, seven concentration points of which were:

clozapine (CZP), amisulpride (ASP), quetiapine (QTP), norquetiapine (NQTP), ziprasidone (ZPD) and Aripiprazole (APZ) are at the same concentration, and the seven concentration points are as follows: 5ng/mL, 12.5ng/mL, 25ng/mL, 125ng/mL, 250ng/mL, 1250ng/mL, 2500ng/mL;

the concentrations of N-Norclozapine (NCZP), chlorpromazine (CPZ), sulpiride (SPR) and Dehydroaripiprazole (DHAPZ) are the same, and the seven concentration points are as follows: 4ng/mL, 10ng/mL, 20ng/mL, 100ng/mL, 200ng/mL, 1000ng/mL, 2000ng/mL;

olanzapine (OZP) and 9-hydroxyrisperidone (PPD) were at the same concentration, with seven concentration points in the order: 2ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL;

the seven concentration points of Risperidone (RPD) are, in order: 0.5ng/mL, 1.25ng/mL, 2.5ng/mL, 12.5ng/mL, 25ng/mL, 125ng/mL, 250ng/mL;

the concentrations of Haloperidol (HLP) and Fluphenazine (FPN) were the same with seven concentration points in order: 0.2ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL;

seven concentration Points of Perphenazine (PPZ) are in order: 0.1ng/mL, 0.25ng/mL, 0.5ng/mL, 2.5ng/mL, 5ng/mL, 25ng/mL, 50ng/mL.

(2) Preparation of protein precipitant containing internal standard

Preparing the following isotope internal standard mother liquor by using methanol: n-norclozapine-d 8 (NCZP-d 8) 0.1mg/mL, olanzapine-d 3 (OZP-d 3) 0.1mg/mL, chlorpromazine-d 6 (CPZ-d 6) 0.1mg/mL, clozapine-d 8 (CZP-d 8) 1mg/mL, sulpiride-d 3 (SPR-d 3) 0.25mg/mL, amisulpride-d 5 (ASP-d 5) 0.1mg/mL, haloperidol-d 4 (HLP-d 4) 0.01mg/mL, quetiapine-d 8 (QTP-d 8) 0.1mg/mL, norquetiapine-13C 4 (NQTP-13C 4) 0.1mg/mL, perphenazine-d 8 (PPZ-d 8) 0.01mg/mL, risperidone-d 4 (RPD-d 4) 0.01mg/mL, ziprasidone-d 8 (DHP-d 8) 0.1mg/mL, and azapirox-d 8 (PPZP-d 8) 0.1mg/mL;

respectively transferring the internal standard mother liquor of each isotope: 2.5 μ L of N-norclozapine-d 8 (NCZP-d 8), 5 μ L of olanzapine-d 3 (OZP-d 3), 10 μ L of chlorpromazine-d 6 (CPZ-d 6), 1 μ L of clozapine-d 8 (CZP-d 8), 4 μ L of sulpiride-d 3 (SPR-d 3), 10 μ L of amisulpride-d 5 (ASP-d 5), 10 μ L of haloperidol-d 4 (HLP-d 4), 10 μ L of quetiapine-d 8 (QTP-d 8), 10 μ L of norquetiapine-13C 4 (NQTP-13C 4), 10 μ L of perphenazine-d 8 (PPZ-d 8), 25 μ L of risperidone-d 4 (RPD-d 4), 10 μ L of rasidone-d 8 (ZPD-d 8), 1 μ L of 9-hydroxypiperidone-d 4 (DHP-d 8), 10 μ L of dehydroepiprazole-d 8 (APZ-d 8), and 10 μ L of meperidol-d 8 (PPZ-d 8); additional addition to 860.5. Mu.L of methanol gave 1mL of mixed internal standard solution.

And (2) adding 200 mu L of the mixed internal standard solution into 19.8mL of a protein precipitant (the volume ratio of isopropanol, methanol and acetonitrile is 1. Wherein, the protein precipitator comprises: n-norclozapine-d 8 (NCZP-d 8) 2.5ng/mL, olanzapine-d 3 (OZP-d 3) 5ng/mL, chlorpromazine-d 6 (CPZ-d 6) 10ng/mL, clozapine-d 8 (CZP-d 8) 10ng/mL L, sulpiride-d 3 (SPR-d 3) 10ng/mL, amisulpride-d 5 (ASP-d 5) 10ng/mL, haloperidol-d 4 (HLP-d 4) 1ng/mL, quetiapine-d 8 (QTP-d 8) 10ng/mL, norquetiapine-13C 4 (NQTP-13C 4) 10ng/mL, perphenazine-d 8 (PPZ-d 8) 2.5ng/mL, risperidone-d 4 (RPD-d 4) 1ng/mL, ziprasidone-d 8 (ZP-d 8) 10ng/mL, perphenazine-d 8 (PPZ-d 8) 2.5ng/mL, and dehydroperphenazine-d 4 (PPZ-d 8) 10ng/mL, and PPDp-d 8/mL.

(3) Preparing a quality control product:

QC (L) includes: N-Norclozapine (NCZP) 8ng/mL, olanzapine (OZP) 4ng/mL, chlorpromazine (CPZ) 8ng/mL, clozapine (CZP) 10ng/mL, sulpiride (SPR) 8ng/mL, amisulpride (ASP) 10ng/mL, haloperidol (HLP) 0.4ng/mL, quetiapine (QTP) 10ng/mL, norquetiapine (NQTP) 10ng/mL, perphenazine (PPZ) 0.2ng/mL, risperidone (RPD) 1ng/mL, ziprasidone (ZPD) 10ng/mL, 9-hydroxypiperidone (PPD) 4ng/mL, fluphenazine (FPN) 0.4ng/mL, dehydroaripiprazole (DHAPZ) 8ng/mL, and Aripiprazole (APZ) 10ng/mL.

QC (M) includes: 80ng/mL of N-Norclozapine (NCZP), 40ng/mL of Olanzapine (OZP), 80ng/mL of Chlorpromazine (CPZ), 100ng/mL of Clozapine (CZP), 80ng/mL of Sulpiride (SPR), 100ng/mL of Amisulpride (ASP), 4ng/mL of Haloperidol (HLP), 100ng/mL of Quetiapine (QTP), 100ng/mL of Norquetiapine (NQTP), 2ng/mL of perphenazine (PPZ), 10ng/mL of Risperidone (RPD), 100ng/mL of Ziprasidone (ZPD), 40ng/mL of 9-hydroxyrisperidone (PPD), 4ng/mL of Fluphenazine (FPN), 80ng/mL of Dehydroaripiprazole (DHAPZ) and 100ng/mL of Aripiprazole (APZ).

QC (H) includes: 800ng/mL of N-Norclozapine (NCZP), 400ng/mL of Olanzapine (OZP), 800ng/mL of Chlorpromazine (CPZ), 1000ng/mL of Clozapine (CZP), 800ng/mL of Sulpiride (SPR), 1000ng/mL of Amisulpride (ASP), 40ng/mL of Haloperidol (HLP), 1000ng/mL of Quetiapine (QTP), 1000ng/mL of Norquetiapine (NQTP), 20ng/mL of perphenazine (PPZ), 100ng/mL of Risperidone (RPD), 1000ng/mL of Ziprasidone (ZPD), 400ng/mL of 9-hydroxyrisperidone (PPD), 40ng/mL of Fluphenazine (FPN), 800ng/mL of Dehydroaripiprazole (DHAPZ) and 1000ng/mL of Aripiprazole (APZ).

(4) Sample processing

1) And (3) standard product treatment: taking 50 μ L of each concentration point of seven different calibrator samples, placing in a 1.5mL centrifuge tube, adding 200 μ L of protein precipitant containing internal standard (volume ratio of isopropanol, methanol and acetonitrile is 1; centrifuging at 14000r/min at 4 ℃ for 5min; transfer 60. Mu.L of supernatant from the EP tube to a plastic lined tube in a 1. Mu.L sample volume.

2) Pretreatment of a serum sample: taking 50 mu L of serum to a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing an internal standard (the volume ratio of isopropanol, methanol and acetonitrile is 1; centrifuging at 14000r/min at 4 ℃ for 5min; transfer 60. Mu.L of supernatant from the EP tube to a plastic lined tube in a 1. Mu.L sample volume.

3) Pretreatment of quality control products: taking 50 μ L of quality control solution QC (L), QC (M), QC (H) respectively in a 1.5mL centrifuge tube, and then consistent with the pretreatment of the serum sample, which is not described herein again.

4. Method verification

1. Extracting an ion current chromatogram: the peak shapes of the antipsychotic drug standard substance and the serum sample are symmetrical, and no peak interference exists, which indicates that the antipsychotic drug standard substance and the serum sample can be well detected under the condition, and fig. 1 is an extracted ion current chromatogram of the antipsychotic drug standard substance, and fig. 2 is an extracted ion current chromatogram of the antipsychotic drug in the serum.

2. Calibration curve: and establishing a calibration curve by adopting an isotope internal standard quantitative method and utilizing TargetLynx software to calculate the concentration of the substance to be detected in the serum by taking the concentration ratio of the standard substance to the internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis. The linear fit equation of antipsychotic drugs in the respective concentration ranges is good in linearity, the correlation coefficient is above 0.99, and the quantitative requirements are met, see table 6.

TABLE 6 Linear regression equation and Linear correlation coefficient for antipsychotic drugs

3. Accuracy survey: and evaluating the accuracy of the method by adopting a standard recovery rate test. A mixed blank serum sample was prepared, 3 concentrations of mixed standard, low, medium and high, were added, and the same procedure was repeated 5 times to determine the recovery rate of antipsychotic drug at 93.84% -108.65%, RSD of 5 repeated experiments ranged from 0.51% -10.35%, and the statistical results are shown in Table 7.

Table 7 recovery results for antipsychotic drug addition

4. And (3) precision test: taking an interference-free blank serum sample, adding antipsychotic drug standard substances with different concentrations to obtain serum samples with low, medium and high concentrations, repeatedly processing 6 batches in one day for three days continuously, quantitatively determining the concentration of the antipsychotic drug by an isotope internal standard method, wherein the internal precision of the batch is 0.96-14.33%, processing 3 batches in three days, and calculating the inter-batch precision to be 3.29-11.89%, and the results are shown in Table 8.

TABLE 8 results of in-batch and inter-batch precision measurements

5. Discussion of the preferred embodiments

The concentration of the antipsychotic drug in human serum is measured by an ID-UPLC-MS/MS method. Meanwhile, the method detects the peak time and the ion pair of the target object, has high sensitivity, can greatly eliminate matrix interference by adopting an isotope internal standard method for quantification, is not influenced by the conditions of pretreatment process, sample loading volume and flow and the like, and can achieve accurate quantification.

The accuracy of the method is evaluated by a standard recovery test, and the result shows that the standard recovery of the antipsychotic drug is between 93.84% and 108.65%, and the RSD of 5 times of repeated tests is between 0.51% and 10.35%, so that the accuracy is good.

The reproducibility of the method results show that the antipsychotic drug has an intra-batch precision of 0.96-14.33% and an inter-batch precision of 3.29-11.89%. The established serum sample pretreatment process is very simple, the protein precipitation is completed in one step, and the serum dosage is only 50 mu L.

In a word, the detection method disclosed by the invention is high in sensitivity, strong in specificity, accurate and simple in pretreatment process, can be used for completing the separation and detection of the compound within 5.0 minutes, meets the requirements on accuracy and precision, can be used for quantitative analysis of clinical serum antipsychotic drug concentration, and provides a reliable detection method for clinical treatment and monitoring of the antipsychotic drug concentration.

The above examples are only intended to illustrate the technical solution of the present invention, and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (1)

1. A method for simultaneously detecting the concentration of a plurality of antipsychotic drugs in serum,

the antipsychotic drugs are respectively: n-norclozapine, olanzapine, chlorpromazine, clozapine, sulpiride, amisulpride, haloperidol, quetiapine, norquetiapine, perphenazine, risperidone, ziprasidone, 9-hydroxyrisperidone, fluphenazine, dehydroaripiprazole, and aripiprazole;

the internal standards corresponding to the antipsychotic drugs are respectively: n-norclozapine-d 8, olanzapine-d 3, chlorpromazine-d 6, clozapine-d 8, sulpiride-d 3, amisulpride-d 5, haloperidol-d 4, quetiapine-d 8, norquetiapine-13C 4, perphenazine-d 8, risperidone-d 4, ziprasidone-d 8, 9-hydroxyrisperidone-d 4, fluphenazine-d 8, dehydroaripiprazole-d 8, and aripiprazole-d 8;

detecting the antipsychotic drug in the preprocessed serum by adopting an ultra-high performance liquid chromatography tandem mass spectrometry technology, firstly separating a substance to be detected from a serum matrix by utilizing the ultra-high performance liquid chromatography, then establishing a calibration curve by utilizing a mass spectrometry isotope internal standard quantitative method and taking the concentration ratio of a standard substance to an internal standard substance as an X axis and the peak area ratio of the standard substance to the internal standard substance as a Y axis, and calculating the content of the antipsychotic drug, wherein the specific chromatographic conditions are as follows:

(1) Ultra-high performance liquid chromatography conditions:

a mobile phase A:0.05% aqueous formic acid; mobile phase B: acetonitrile;

type of chromatographic column: waters BEH C18;

adopting a mode of gradient elution with a mixed mobile phase of a mobile phase A and a mobile phase B, wherein the initial ratio of the mobile phase A to the mobile phase B is 90; the gradient elution procedure was as follows: in 0-1.0 min, the volume ratio of the mobile phase A to the mobile phase B is uniformly and gradually changed to 30 from the initial ratio; in 1.0-3.0 minutes, the volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 30; changing the volume ratio of the mobile phase A to the mobile phase B from 2; each sample was collected for 5.0 minutes; the flow rate is 0.4mL/min; the column temperature was 45 ℃; the sample introduction volume is 1 mu L;

mass spectrum conditions:

in an electrospray ionization positive ion detection mode, a mass spectrum scanning mode of multi-reaction monitoring is adopted, and the capillary voltage is 2.5kV (ESI +); the source temperature is 120 ℃; the temperature of atomizing gas is 500 ℃, the airflow speed of atomizing is 800L/h, and the airflow speed of taper hole is 150L/h; simultaneously monitoring each target object and the corresponding isotope internal standard thereof;

the pretreated serum was prepared as follows: taking 50 mu L of serum into a 1.5mL centrifuge tube, adding 200 mu L of protein precipitant containing internal standard into the centrifuge tube, oscillating for 3 to 5min, centrifuging for 4 to 10min at 12000 to 15000r/min at 1 to 5 ℃, and transferring 60 mu L of supernatant in the centrifuge tube into a plastic inner lining tube for sample injection;

the protein precipitant containing the internal standard is prepared according to the following method:

preparing the following isotope internal standard mother liquor by using methanol: n-norclozapine-d 8.1 mg/mL, olanzapine-d 3.1 mg/mL, chlorpromazine-d 6.1 mg/mL, clozapine-d 8 mg/mL, sulpiride-d 3.25 mg/mL, amisulpride-d 5.1 mg/mL, haloperidol-d 4.01 mg/mL, quetiapine-d 8.1 mg/mL, norquetiapine-1 c4.1mg/mL, perphenazine-d 8.01 mg/mL, risperidone-d 4.01 mg/mL, ziprasidone-d 8 mg/mL, 9-hydroxypiperidone-d 4.0.1 mg/mL, fluphenazine-d 8.01 mg/mL, dehydroaripiprazole-d 8 mg/mL and aripiprazole-d 8.1 mg/mL;

respectively transferring the internal standard mother liquor of each isotope: n-norclozapine-d 8.5. Mu.L, olanzapine-d 3. Mu.L, chlorpromazine-d 6. Mu.L, clozapine-d 8. Mu.L, sulpiride-d 3. Mu.L, amisulpride-d 5. Mu.L, haloperidol-d 4. Mu.L, quetiapine-d 8. Mu.L, norquetiapine-13C4. Mu.L, perphenazine-d 8. Mu.L, risperidone-d 4. Mu.L, ziprasidone-d 8. Mu.L, 9-hydroxypiperidone-d 4. Mu.L, fluphenazine-d 8. Mu.L, dehydroaripiprazole-d 8. Mu.L and aripiprazole-d 8. Mu.L; then adding the mixed solution into 860.5 mu L of methanol to obtain 1mL of mixed internal standard solution; adding 200 mul of the mixed internal standard solution into 19.8mL of mixed solution of isopropanol, methanol and acetonitrile with the volume ratio of 1;

the standard is prepared according to the following method:

the above antipsychotic drugs were formulated as standard stock solutions at the following concentrations: 2mg/mL of N-norclozapine, 1mg/mL of olanzapine, 5mg/mL of chlorpromazine, 5mg/mL of clozapine, 5mg/mL of sulpiride, 2mg/mL of amisulpride, 2mg/mL of haloperidol, 5mg/mL of quetiapine, 10mg/mL of norquetiapine, 1mg/mL of perphenazine, 5mg/mL of risperidone, 2mg/mL of ziprasidone, 2mg/mL of 9-hydroxypiperone, 2mg/mL of fluphenazine, 2mg/mL of dehydroaripiprazole, and 1mg/mL of aripiprazole;

respectively transferring and taking mother liquor of each standard product: 20 μ L of N-norclozapine, 20 μ L of olanzapine, 8 μ L of chlorpromazine, 10 μ L of clozapine, 8 μ L of sulpiride, 25 μ L of amisulpride, 1 μ L of haloperidol, 10 μ L of quetiapine, 5 μ L of norquetiapine, 1 μ L of perphenazine, 1 μ L of risperidone, 25 μ L of ziprasidone, 10 μ L of 9-hydroxypiperidone, 1 μ L of fluphenazine, 20 μ L of dehydroaripiprazole, and 50 μ L of aripiprazole; then adding into 785. Mu.L of methanol to obtain 1mL of mixed standard stock solution;

the mixed standard stock solution is prepared into a calibrator solution with seven different concentration points by using a blank serum matrix, wherein the blank serum matrix is blank serum without antipsychotic target drugs, and the seven concentration points of the calibrator solution are as follows:

the concentration of clozapine, amisulpride, quetiapine, norquetiapine, ziprasidone and aripiprazole is the same, and seven concentration points are as follows: 5ng/mL, 12.5ng/mL, 25ng/mL, 125ng/mL, 250ng/mL, 1250ng/mL, 2500ng/mL;

the concentrations of N-norclozapine, chlorpromazine, sulpiride and dehydroaripiprazole are the same, and seven concentration points are as follows: 4ng/mL, 10ng/mL, 20ng/mL, 100ng/mL, 200ng/mL, 1000ng/mL, 2000ng/mL;

the concentration of olanzapine and 9-oxycodone is the same, and seven concentration points are as follows: 2ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL;

the seven concentration points of risperidone are, in order: 0.5ng/mL, 1.25ng/mL, 2.5ng/mL, 12.5ng/mL, 25ng/mL, 125ng/mL, 250ng/mL;

the concentrations of haloperidol and fluphenazine are the same, and seven concentration points are as follows: 0.2ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL, 100ng/mL;

seven concentration points of perphenazine are as follows: 0.1ng/mL, 0.25ng/mL, 0.5ng/mL, 2.5ng/mL, 5ng/mL, 25ng/mL, 50ng/mL.

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