CN111521702A - Liquid quality detection method for antipsychotic drugs in serum or plasma - Google Patents
Liquid quality detection method for antipsychotic drugs in serum or plasma Download PDFInfo
- Publication number
- CN111521702A CN111521702A CN202010369692.2A CN202010369692A CN111521702A CN 111521702 A CN111521702 A CN 111521702A CN 202010369692 A CN202010369692 A CN 202010369692A CN 111521702 A CN111521702 A CN 111521702A
- Authority
- CN
- China
- Prior art keywords
- serum
- solid phase
- phase extraction
- plasma
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/89—Inverse chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to a detection method for anti-psychotropic liquid quality in serum or plasma and the pretreatment of a specific purification material thereof, which comprises the following steps: a serum or plasma sample is subjected to protein precipitation by an organic reagent according to a certain proportion, supernatant obtained by centrifugal precipitation is subjected to pretreatment by a specific purification material to obtain a purified sample solution, liquid chromatography-tandem mass spectrometry detection and analysis are carried out, and simultaneously 8 anti-mental drugs such as olanzapine, clozapine, quetiapine, aripiprazole, haloperidol, risperidone, paliperidone and ziprasidone in the serum or plasma are detected. The pretreatment of the specific purification material is a mixed solid phase extraction column using silica gel as a matrix, and blood is purified by the specific purification material, so that the interference of the matrix in the blood can be removed, the matrix effect of 8 anti-mental drugs can be improved, and the recovery rate of the anti-mental drugs can be better ensured.
Description
Technical Field
The invention belongs to the technical field of medical inspection and analysis, and particularly relates to a liquid chromatography-tandem mass spectrometry detection method for detecting antipsychotic drugs olanzapine, haloperidol, paliperidone, aripiprazole, risperidone, clozapine, quetiapine fumarate and ziprasidone mesylate in a serum or plasma sample.
Technical Field
Therapeutic drug concentration monitoring refers to measuring the concentration of a particular drug in a patient's blood at specified time intervals during a clinically conducted drug therapy. Antipsychotics, also known as nerve blockers, are indicated for schizophreniform and other psychotic disorders with symptoms such as psychomotor delusions and hallucinations, in addition to schizophrenia and paranoid psychotic disorders. Currently, there are a wide variety of antipsychotics, including typical medications that are used for a longer period of time and atypical medications, with typical medications being primarily preferred for positive symptoms and less preferred for negative symptoms. The corresponding symptom spectrum of the non-typical medicine has wide curative effect, small adverse reaction and convenient long-term administration. [ Von Lizhi, et al, journal of clinical and experimental medicine, 2007; 6(5):114-115].
Typical and atypical antipsychotics are monitored for therapeutic drug concentration for the following reasons: the compliance of patients can be effectively improved and the toxic and side effects of the patients can be reduced by keeping the anti-mental drugs to continuously and slowly maintain the minimum effective blood concentration. The steady state blood concentration of the antipsychotic agent shows large inter-patient variation, but the antipsychotic agent is relatively stable in the patient on a daily basis, so it is valuable to monitor the concentration of the antipsychotic agent in the blood.
At present, the domestic clinical monitoring method for the blood concentration of the anti-mental drugs mainly comprises High Performance Liquid Chromatography (HPLC), Radioimmunoassay (RIA), Gas Chromatography (GC), liquid tandem mass spectrometry (LC-MS) and the like. HPLC is a method for measuring blood concentration developed in the seventies and eighties of the last century, and adopts a proper chromatographic column to separate and identify the antipsychotic drugs and metabolites thereof. The method has high specificity and accurate and reliable result, but the method needs more time and energy for removing the interfering substances of the pretreatment sample, the operation is complex, and the HPLC can not achieve accurate detection and quantification. Radioimmunoassay (RIA) is a ultramicroanalytical technique invented in the sixties, which incorporates the high sensitivity of the radioisotope monitoring technique and the high specificity of the immunological antigen-antibody reaction. Its advantages are high sensitivity, less consumption of sample, high precision, simple operation and quick detection. The method has the disadvantages of more operation steps, special monitoring equipment, possibility of radioactive hazard to operators and the like. [ Zhoujing, et al, Anhui preventive medicine journal, 2018; (1):71-73]. The gas chromatography has the characteristics of high separation efficiency, high analysis speed, small sample amount and the like, but the application range is only used for qualitative and quantitative analysis of gas and volatile substances.
The high performance liquid chromatography tandem mass spectrometry is a powerful analysis and detection tool which takes a mass spectrometer as a detection means and integrates the high separation capability of the high performance liquid chromatograph, the high sensitivity and the high selectivity of the mass spectrometer. In the field of drug analysis research, most drugs are compounds with relatively high polarity, and in many analytical instruments, the liquid chromatography analysis range is wide, including nonvolatile compounds, polar compounds, heat-labile compounds and biomacromolecule compounds (including proteins, polypeptides, polysaccharides, polymers, and the like). The mass spectrum specificity is strong, more structural qualitative information can be improved, and the detection sensitivity is very high. The LC-MS technology can align molecular ions for multi-stage fragmentation, thereby providing relative molecular weights of compounds and abundant fragment information. In the impurity research and pharmacokinetic research stages in the drug development, the contents of impurities and the plasma concentration of a pharmacokinetic sample are usually very low, the analysis difficulty is high, the interference is more, and the LC-MS technology can rapidly and accurately determine trace substances in the drug analysis due to strong selectivity and high sensitivity.
Disclosure of Invention
The invention aims to provide a liquid quality detection method for an antipsychotic drug in serum or plasma and pretreatment of a specific purifying material thereof. The specific purifying material is a mixed solid phase extraction column with silica gel as a matrix, and a solid phase extraction filler is adopted, so that the interference of the matrix in serum or plasma can be removed, the matrix effect of 8 anti-mental drugs can be improved, and the recovery rate of the anti-mental drugs can be better ensured.
In order to solve the technical problems, the technical scheme of the invention is a liquid quality detection method for an antipsychotic drug in serum or plasma, which comprises the following steps:
1) serum or plasma sample extraction: adding a deuterated internal standard into a serum or plasma sample, carrying out vortex mixing, adding a protein precipitator for protein precipitation, carrying out low-temperature high-speed centrifugation, and taking a supernatant for later use;
2) filling a solid phase extraction column;
3) solid phase extraction and separation: the solid phase extraction column is activated by adopting a pure organic solvent; adding pure water for balancing; after the supernatant obtained in the step 1) is sampled; adding pure water for first-step leaching; leaching by using mixed liquor with a volume ratio of (4:6-7:3), leaching by using an organic solvent, eluting by using a pure organic phase, and collecting all elution fractions;
4) analysis of the samples: separating the elution fractions collected in the step 3) by using C18 reversed-phase high performance liquid chromatography, performing tandem mass spectrometry detection analysis, scanning to obtain a sample spectrogram, and detecting 8 anti-psychotropic drugs and deuterated internal standard positive ions thereof.
Preferably, the volume of the serum or plasma sample in step 1) is 5 μ L to 500 μ L; the volume of the deuterated internal standard is 0.5 mu L-500 mu L; the protein precipitator is a mixed solution of an organic solvent and pure water in a volume ratio of 1: 1-5: 1, and the organic solvent is acetonitrile or methanol.
Preferably, the loading specification in step 2): 50-500mg of filler and 1-6mL of column tube volume.
Preferably, the filler in step 2) is a silica gel-based mixed solid phase extraction filler, and the structural formula is as follows:
wherein the bonding phase n is equal to 8-30; the particle size of the silica gel of the solid phase extraction material is 20-100 mu m, and the pore diameter is
Specific surface area 150m2/g-400m2/g。
Preferably, the solid phase extraction column in the step 3) is activated by adopting 0.2mL-2mL of pure organic solvent; then adding 0.2mL-2mL pure water solution for balancing; centrifuging to obtain 0.1-2 mL of supernatant; adding 0.1-2 mL of pure water for first-step leaching; adding 0.1-1 mL of methanol-water (volume ratio is 4:6-7:3) to carry out second-step leaching; adding 0.1-2 mL of methanol for carrying out the third leaching; finally, eluting with 0.1-2 mL of methanol, and collecting all elution fractions; the organic solvent is one or more of methanol, acetonitrile, isopropanol, ethanol and acetone.
Preferably, the high performance liquid chromatography conditions in step 5):
a chromatographic column: c185 μm, 50 x 3 mm; the flow rate is 0.5 mL/min; gradient elution; the column temperature is 30-45 ℃; sample introduction amount: 0.1-20 μ L;
the mass spectrometry conditions are as follows:
ionization mode: ESI+(ii) a Spraying voltage: 5.5 KV; desolventizing gas temperature: 500 ℃; atomizing: 40 psi; assisting atomization gas: 40 psi; air curtain air: 25 psi; the scanning mode is as follows: multiple Reaction Monitoring (MRM).
The method for detecting the anti-psychotic drug liquid quality in serum or plasma and the pretreatment of the specific purifying material thereof determine that the matrix effect of 8 anti-psychotic drugs olanzapine, haloperidol, paliperidone, aripiprazole, risperidone, clozapine, quetiapine fumarate and ziprasidone mesylate is 85.00% -110.00% and the extraction recovery rate is 90.00% -115.00%. The invention can accurately determine the content of 8 anti-mental drugs in serum or plasma.
The invention has the beneficial effects that:
(1) the pretreatment process is novel. The invention extracts and purifies blood serum or blood plasma by a solid phase extraction method for the first time, and provides a mixed solid phase extraction filler taking silica gel as a matrix, wherein the filler has specific enrichment and purification on antipsychotic drugs;
(2) simple operation and high flux. The pretreatment method provided by the invention is simple and reliable to operate, and is beneficial to realizing high flux of clinical serum or plasma samples;
(3) the qualitative and quantitative determination is accurate. The method adopts high performance liquid chromatography-tandem mass spectrometry for detection, the serum or plasma sample is corrected by adding an internal standard, the standard curve is quantitative, the result accuracy is high and stable, and the method can be used for accurate qualitative and quantitative determination of 8 anti-mental drugs in clinical blood samples.
Drawings
FIG. 1 is an MRM chromatogram of a solution of paliperidone ion channel of the standard curve of example 1;
FIG. 2 is an MRM chromatogram of the standard curve solution aripiprazole ion channel of example 1;
FIG. 3 is an MRM chromatogram of a risperidone ion channel of a standard curve solution of example 1;
FIG. 4 is an MRM chromatogram of clozapine ion channel of a standard curve solution of example 1;
FIG. 5 is an MRM chromatogram of a standard curve solution haloperidol ion channel of example 1;
FIG. 6 is a MRM chromatogram of olanzapine ion channel from a standard curve solution of example 1;
FIG. 7 is an MRM chromatogram of a standard curve solution quetiapine ion channel of example 1;
FIG. 8 is an MRM chromatogram of ziprasidone ion channel of a standard curve solution of example 1;
FIG. 9 is a chromatogram of the total ion current ion channel of the serum sample solution of example 2;
Detailed Description
The invention will now be further described with reference to the following examples, which are intended to be illustrative of the invention and are not to be construed as limiting the invention.
Example 1
Serum containing 8 anti-psychotropic drugs with standard recovery rate experiment:
1. materials and reagents
A chromatographic column: kinetex C185 μm, 50 mm. times.3.0 mm
Risperidone, clozapine, haloperidol, quetiapine, and ziprasidone were purchased from the chinese food & drug testing institute; paliperidone, quetiapine fumarate-d 8, and ziprasidone-d 8 were purchased from SIGMA; risperidone-d 4, aripiprazole-d 8, aripiprazole, olanzapine-d 8, clozapine-d 4, haloperidol-d 4, and-hydroxyrisperidone-d 4 were purchased from Cerilliant; methanol, acetonitrile, isopropanol, formic acid, ammonium formate (chromatographically pure), ammonia (chromatographically pure); ultrapure water: and preparing a Mili-Q ultrapure water machine.
2. Apparatus and device
High performance liquid chromatography-tandem mass spectrometer equipped with Electrospray (ESI) ionization source (TRIPLE QUAD 4500MD, AB SCIEX, USA), liquid chromatography separation mode is reversed phase chromatography separation, detector is TRIPLE quadrupole tandem mass spectrometry; one-ten-thousandth electronic analytical balance; SPE solid phase extraction device.
3. The method for detecting 8 anti-mental drugs by using the high performance liquid chromatography-tandem mass spectrometry comprises the following steps:
1) preparing a standard substance working solution WS and a quality control substance QC: accurately preparing 8 kinds of anti-psychotics mixed standard substance working solution WS _ E (risperidone 5 mu g/mL, clozapine 10 mu g/mL, haloperidol 0.5 mu g/mL, quetiapine 10 mu g/mL, ziprasidone 10 mu g/mL, paliperidone 5 mu g/mL, aripiprazole 10 mu g/mL, olanzapine 5 mu g/mL), diluting with 50% methanol water to obtain the series concentration of the standard substance working solution and quality control substance working solution (standard substance working solution: WS _ A, WS _ B, WS _ C, WS _ D, WS _ E, quality control substance: QC _ L, QC _ M, QC _ H) as follows:
2) preparing an internal standard working solution: accurately preparing isotope internal standard mixed working solution (50 ng/mL each of risperidone-d 4, olanzapine-d 8, 9-hydroxy risperidone-d 4, haloperidol-d 450 ng/mL, clozapine-d 4, aripiprazole-d 8, quetiapine-d 8 and ziprasidone-d 8, 100ng/mL each), and diluting with pure water for later use.
3) Preparation of a standard curve solution: respectively taking 10 mul of WS _ A-E standard solutions with different concentrations in the step 1), adding 90 mul of blank serum solution, adding 20 mul of mixed isotope internal standard working solution, mixing uniformly in a vortex manner, adding 400 mul of precipitator (acetonitrile: water is 4:1), mixing for 5min in a vortex manner, centrifuging for 5min at a low temperature and a high speed of 12500rpm, and taking supernate for later use.
4) Extraction of the spiked serum samples: taking 90 mu L of the same serum sample, respectively adding 50% methanol water and 10 mu L of QC-L, QC-M, QC-H working solution, adding 20 mu L of mixed isotope internal standard working solution, carrying out vortex mixing, adding 400uL of precipitator (acetonitrile: water is 4:1), carrying out vortex mixing, carrying out centrifugation at low temperature and high speed 12500rpm for 5min, and taking the supernatant for later use.
5) Filling a solid phase extraction column: the specific purification material filler 100mg filled in the invention is placed in a 1mL column tube (bonding phase n is equal to 18, the particle size of the filler silica gel is 60 μm, and the pore diameter is
Specific surface area 300m2/g)。
6) Solid phase extraction and separation: the solid phase extraction column is activated by 1mL of acetonitrile; adding 1mL of pure water for balancing; loading 300 mu L of each sample solution in the step 3) and the step 4); adding 500 mu L of pure water for leaching; adding 300 mu L of 60 percent methanol water for leaching; adding 300 mu L of methanol for leaching; finally, 300. mu.L of methanol was used for sample analysis.
7) Analysis of the samples: detecting the final eluent in the step 6) on a machine, separating components to be detected by adopting reverse chromatography, detecting by triple quadrupole tandem mass spectrometry to obtain a sample spectrogram, and detecting positive ions of 8 anti-psychotropic target substances: high performance liquid chromatography conditions and mass spectrometry conditions
i) High performance liquid chromatography conditions
A chromatographic column: kinetex C185 μm, 50 mm. times.3.0 mm; mobile phase: methanol (a), 2mM aqueous ammonium formate solution (B), gradient elution see table 1; column temperature 40 ℃, sample injection volume: 2 μ L.
TABLE 1 ultra high performance liquid chromatography gradient conditions
ii) Mass Spectrometry conditions
Ionization mode: ESI+(ii) a Spraying voltage: 5.5 KV; desolventizing gas temperature: 500 ℃; atomizing GAS (GAS 1): 40 psi; assisting atomising GAS (GAS 2): 40 psi; air curtain air: 25 psi; the scanning mode is as follows: multiple Reaction Monitoring (MRM) qualitative and quantitative ion pairs, residence time collision energy, etc. for 8 antipsychotic drugs and their deuterated internal standards are shown in table 2.
Table 28 mass spectrometric parameters for antipsychotic drugs and deuterated internal standards thereof
4. And (3) analyzing a quantitative calculation result: and (3) according to the ratio of the peak area of the target object ion chromatographic peak to the peak area of the deuterated internal standard ion chromatographic peak in the sample chromatogram as a response, and making a linear regression equation according to the corresponding concentration (shown in table 3).
TABLE 3 Linear regression equation for antipsychotic drugs
Substituting the ratio response of the target object chromatographic peak area and the deuterated internal standard chromatographic peak area of the sample with the standard recovery rate into the random standard curve to calculate the sample concentration, wherein the ratio of the difference value between the concentration of the standard sample and the blank serum sample to the theoretical value of the concentration of the target object is the standard recovery rate, and the standard recovery rates of 8 anti-psychotics are as follows:
the recovery rate of 8 anti-mental drugs in the quantitative analysis of the detected serum sample is between 90 and 110 percent, and the result of the recovery rate of the serum sample is better.
Example 2
1. Materials and reagents
The difference from example 1 is that the protein precipitant used in the pretreatment process is reconstituted in a methanol to pure water (3:1) ratio;
2. apparatus and device
A high performance liquid chromatography-tandem mass spectrometer equipped with an electrospray ionization (ESI) ionization source (API 3200MD, AB SCIEX, USA), wherein the liquid chromatography separation mode is reversed phase chromatography separation, and the detector is triple quadrupole tandem mass spectrometry; one-ten-thousandth electronic analytical balance; SPE solid phase extraction device.
3. Matrix effect of 8 anti-psychotropic drugs in serum (no solid phase extraction pretreatment):
the pretreatment of detecting the matrix effect of 8 anti-psychotropic drugs by high performance liquid chromatography-tandem mass spectrometry comprises the following steps:
1) serum extract + standard: adding 100 μ L of blank serum into a 1.5mL centrifuge tube, adding 300 μ L of protein precipitant, mixing by vortex, centrifuging at high speed of 13500rpm for 5min at low temperature, adding 5 μ L of the supernatant into the working solution (QC-L, QC-M, QC _ H) of the quality control product of example 1, mixing by vortex, and analyzing by sample injection. Respectively measuring peak areas Ax of chromatographic peaks of 8 anti-psychotropic target substances in the sample;
2) pure water extract + standard: 100 μ L of pure water was added to a 1.5mL centrifuge tube, 300 μ L of protein precipitant was added, vortex mixing was performed, centrifugation was performed at 13500rpm for 5min at a low temperature and a high speed, and 5 μ L of the quality control working solution (QC-L, QC-M, QC-H) of example 1 was added to 100 μ L of the supernatant, vortex mixing was performed, and sample analysis was performed. Respectively measuring peak areas As of chromatographic peaks of 8 anti-psychotropic target substances in the sample;
4. matrix effect of 8 anti-psychotropic drugs in serum (pretreatment of solid phase extraction):
the pretreatment of detecting the matrix effect of 8 anti-psychotropic drugs by high performance liquid chromatography-tandem mass spectrometry comprises the following steps:
1) serum extract + standard: adding 100 μ L of blank serum into 1.5mL of centrifugal tube, adding 300 μ L of protein precipitant, vortex mixing, centrifuging at low temperature and high speed of 13500rpm for 5min, and collecting supernatant;
2) pure water extract + standard: adding 100 μ L of pure water into 1.5mL of centrifugal tube, adding 300 μ L of protein precipitant, vortex mixing, centrifuging at low temperature and high speed of 13500rpm for 5min, and collecting supernatant;
3) filling a solid phase extraction column: the specific purification material filler 100mg filled in the invention is placed in a 1mL column tube (bonding phase n is equal to 18, the particle size of the filler silica gel is 60 μm, and the pore diameter is
Specific surface area 300m2/g)。
4) Solid phase extraction process: the solid phase extraction column is activated by 1mL of methanol; adding 1mL of pure water for balancing; loading 300 mu L of each supernatant obtained by centrifuging in the steps 1) and 2); adding 400 mu L of pure water for leaching; adding 300 mu L of 40 percent methanol water for leaching; finally, eluting with 300 mu L of methanol;
5) adding a standard substance into the extracting solution: and (3) adding 250 mu L of eluent obtained in the step 4) into 5 mu L of quality control substance working solution (QC _ L, QC _ M, QC _ H) obtained in the example 1, uniformly mixing by vortex, and waiting for sample loading and analysis.
5. Analysis of the samples: detecting a sample to be detected on a machine, separating components to be detected by adopting reverse chromatography, detecting by triple quadrupole tandem mass spectrometry to obtain a sample spectrogram, and detecting 8 types of anti-psychotropic target positive ions;
high performance liquid chromatography conditions and mass spectrometry conditions
i. High performance liquid chromatography conditions
A chromatographic column: kinetex C185 μm, 50 mm. times.3.0 mm; mobile phase: acetonitrile (a), 2mM aqueous ammonium formate solution (B), gradient elution see table 4; column temperature 40 ℃, sample injection volume: 20 μ L.
TABLE 4 ultra performance liquid chromatography gradient conditions
Mass spectral conditions
Ionization mode: ESI+(ii) a Spraying voltage: 5.5 KV; desolventizing gas temperature: 500 ℃; atomizing GAS (GAS 1): 45 psi; assisting atomising GAS (GAS 2): 45 psi; air curtain air: 27 psi; the scanning mode is as follows: multiple Reaction Monitoring (MRM) qualitative and quantitative ion pairs, residence time collision energies, etc. for 8 antipsychotic drugs and their deuterated internal standards are shown in table 5.
TABLE 58 Mass Spectrometry parameters for anti-psychotic drug targets
6. The experimental results are as follows: the peak areas Ax of the chromatographic peaks of the serum sample extract + the standard substance and the peak areas As of the chromatographic peaks of the pure water sample extract + the standard substance were obtained and substituted into the following formula, and the absolute matrix effects (no solid phase pre-extraction treatment) of the 8 anti-psychotropic target substances in the serum were calculated and shown in table 6, and the absolute matrix effects (solid phase pre-extraction treatment) of the 8 anti-psychotropic target substances in the serum were shown in table 74:
ME％＝Ax/As×100％
TABLE 6 Absolute matrix Effect of 8 anti-psychotic drug targets in serum (pretreatment without solid phase extraction)
TABLE 7 Absolute matrix Effect of 8 anti-psychotic drug targets in serum (pretreatment before solid phase extraction)
The above results show that: the matrix effect of the 8 anti-psychotropic drugs treated by the specific filler is obviously smaller than that of the non-solid phase extraction pretreatment process, and the noise of a detection spectrogram is also obviously different.
Example 3
Comparison of the extraction of 8 anti-psychotropic drugs by different types of commercial solid phase extraction columns C18, C18Phe, and specific purified solid phase extraction columns.
1. Materials and reagents
Commercial solid phase extraction column C18, C18Phe, specific purification packing.
2. Apparatus and device
Same as in example 2.
3. The high performance liquid chromatography-tandem mass spectrometry method for detecting the extraction pretreatment of different types of solid phase extraction columns on 8 anti-psychotropic drugs comprises the following steps:
1)100 μ L of human serum +5 μ L of quality control (M) was added to a 1.5mL centrifuge tube, and a protein precipitant (acetonitrile: water 3:1)300 μ L, vortex mixing, low temperature high speed 13500rpm centrifugation for 5min, taking supernatant for use in triplicate;
2) filling a solid phase extraction column: the C18, C18Phe and the specific purification material of the invention were loaded separately (bonded phase n equals 18, particle size of the filler silica gel was 60 μm, pore size was
Specific surface area 300m2/g) 100mg of packing was placed in a 1mL column tube.
3) Solid phase extraction column C18: activating by using 1mL of methanol; balancing 1mL of pure water; 300uL of supernatant obtained by centrifugation in the step 1) is loaded; respectively adding 0.4mL of pure water and 0.4mL of 5% methanol for leaching in sequence; eluting with 0.3mL of methanol, collecting all eluted fractions, and loading for analysis;
4) solid phase extraction column C18 Phe: activating by using 1mL of methanol; balancing 1mL of pure water; 300uL of supernatant obtained by centrifugation in the step 1) is loaded; respectively adding 0.4mL of pure water and 0.4mL of 10% methanol for leaching in sequence; eluting with 0.3mL of methanol, collecting all eluted fractions, and loading for analysis;
5) a specific solid phase extraction column: activating by adopting 1mL of methanol; balancing 1mL of pure water; loading 300 mu L of supernate obtained by centrifuging in the step 1); adding 400 mu L of pure water for leaching; adding 400 mu L of 40 percent methanol water for leaching; finally, eluting with 300 mu L of methanol;
4. analysis of the samples: detecting a sample to be detected on a computer, separating components to be detected by adopting reverse chromatography, detecting by triple quadrupole tandem mass spectrometry to obtain a sample spectrogram, and detecting peak area responses of positive ions of 8 anti-psychotropic target substances:
high performance liquid chromatography conditions and mass spectrometry conditions
i) High performance liquid chromatography conditions
A chromatographic column: kinetex C185 μm, 50 mm. times.3.0 mm; mobile phase: methanol (a), 2mM aqueous ammonium formate solution (B), gradient elution see table 8; column temperature 40 ℃, sample injection volume: 2 μ L.
TABLE 8 ultra high performance liquid chromatography gradient conditions
ii) Mass Spectrometry conditions
Ionization mode: ESI+(ii) a Spraying voltage: 5.5 KV; desolventizing gas temperature: 500 ℃; atomizing GAS (GAS 1): 40 psi; assisting atomising GAS (GAS 2): 40 psi; air curtain air: 25 psi; the scanning mode is as follows: multiple Reaction Monitoring (MRM) qualitative and quantitative ion pairs, residence time collision energies, etc. for 8 antipsychotic drugs and their deuterated internal standards are shown in table 9.
Mass spectrum parameters of 98 anti-psychotropic drugs and deuterated internal standard thereof
5. The experimental results are as follows:
TABLE 10 results of solid phase extraction column treatment of 8 anti-psychotic drugs with three different fillers
The above results show that: the response (chromatographic peak area) of the specific solid phase extraction column for treating 8 anti-mental drugs of the same standard human serum is obviously higher than the extraction effect of the commercial C18 and C18Phe solid phase extraction columns.
The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.
Claims (10)
1. A method for testing the quality of an anti-psychotic drug in serum or plasma, comprising the steps of:
1) serum or plasma sample extraction: adding a serum or plasma sample into a target deuterated internal standard, carrying out vortex mixing, adding a protein precipitator for protein precipitation, carrying out low-temperature high-speed centrifugation, and taking supernatant for later use;
2) filling a solid phase extraction column: and (3) filling specification: 50-500mg of solid phase extraction filler, and 1-6mL of column tube volume;
3) solid phase extraction and separation: activating, balancing, loading, leaching and eluting the solid phase extraction column, and finally collecting all elution fractions;
4) and (3) sample analysis: and (3) carrying out high performance liquid chromatography-tandem mass spectrometry detection analysis on the elution fractions collected in the step 3) to detect 8 anti-psychotropic drugs and positive ions of the deuterated internal standard thereof in serum or plasma.
2. The method for detecting liquid quality according to claim 1, wherein the anti-psychotic drug is olanzapine, haloperidol, paliperidone, aripiprazole, risperidone, clozapine, quetiapine fumarate, and ziprasidone mesylate.
3. The method according to claim 1, wherein the volume of the serum or plasma sample sampled is 5 μ L to 500 μ L; adding 1-100 muL of deuterated internal standard of an anti-psychotropic drug into each 5 muL of serum or plasma sample, wherein the concentration of the deuterated internal standard of the anti-psychotropic drugs olanzapine, risperidone and paliperidone is 10-1 mug/mL, the concentration of the deuterated internal standard of haloperidol is 2-200 ng/mL, and the concentration of the deuterated internal standard of aripiprazole, clozapine, quetiapine and ziprasidone is 20-2 mug/mL.
4. The liquid quality detection method according to claim 1, wherein the protein precipitant is a mixture of an organic solvent and pure water at a volume ratio of 1:1 to 5:1, and the volume of the protein precipitant added is 0.1mL to 2 mL; the organic solvent is acetonitrile or methanol.
5. The liquid quality detection method according to claim 1, wherein the solid phase extraction column packing is a mixed solid phase extraction packing with silica gel as a matrix, and the packing has a structural formula as follows:
wherein the bonding phase n is equal to 8 to 30; the particle size of the silica gel of the solid phase extraction material is 20-100 mu m, and the pore diameter is
Specific surface area 150m2/g-400m2/g。
6. The liquid quality detection method according to claim 1, wherein the solid phase extraction column is activated by 0.2mL to 2mL of pure organic solvent; then adding 0.2mL-2mL pure water solution for balancing; centrifuging to obtain 0.1-2 mL of supernatant; adding 0.1-2 mL of pure water for first-step leaching; adding 0.1-1 mL of methanol aqueous solution with the volume ratio of 4:6-7:3 for second-step leaching; adding 0.1-2 mL of methanol for carrying out the third leaching; finally, eluting with 0.1-2 mL of methanol, and collecting all elution fractions;
the organic solvent is one or more of methanol, acetonitrile, isopropanol, ethanol and acetone.
7. The liquid quality detection method of claim 6, wherein the anti-psychotic drug detection adopts C18 reversed-phase high performance liquid chromatography for separation, tandem mass spectrometry detection and analysis, and a sample spectrogram is obtained by scanning, so as to detect 8 anti-psychotic drugs and deuterated internal standard positive ions thereof:
the high performance liquid chromatography conditions are as follows:
a chromatographic column: c185 μm, 50 x 3 mm; the flow rate is 0.5 mL/min; gradient elution; the column temperature is 30-45 ℃; sample introduction amount: 0.1-20 μ L;
the mass spectrometry conditions are as follows:
ionization mode: ESI+(ii) a Spraying voltage: 5.5 KV; desolventizing gas temperature: 500 ℃; atomizing: 40 psi; assisting atomization gas: 40 psi; air curtain air: 25 psi; the scanning mode is as follows: multiple Reaction Monitoring (MRM).
8. The method of claim 2, wherein the ratio of the peak area of the anti-psychotic pharmaceutical compound detected to the peak area of its corresponding deuterated internal standard is used as a response to a regression equation for its concentration.
9. The method for detecting liquid quality according to claim 2, wherein matrix effect of 8 anti-psychotropic drugs olanzapine, haloperidol, paliperidone, aripiprazole, risperidone, clozapine, quetiapine fumarate and ziprasidone mesylate is 85.00% -110.00%.
10. The method for detecting liquid quality according to claim 2, wherein the recovery rate of 8 anti-psychotropic drugs olanzapine, haloperidol, paliperidone, aripiprazole, risperidone, clozapine, quetiapine fumarate, and ziprasidone mesylate is 90.00% -115.00%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010369692.2A CN111521702A (en) | 2020-05-05 | 2020-05-05 | Liquid quality detection method for antipsychotic drugs in serum or plasma |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010369692.2A CN111521702A (en) | 2020-05-05 | 2020-05-05 | Liquid quality detection method for antipsychotic drugs in serum or plasma |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111521702A true CN111521702A (en) | 2020-08-11 |
Family
ID=71906838
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010369692.2A Pending CN111521702A (en) | 2020-05-05 | 2020-05-05 | Liquid quality detection method for antipsychotic drugs in serum or plasma |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111521702A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112162046A (en) * | 2020-09-25 | 2021-01-01 | 宁波大红鹰药业股份有限公司 | Quantitative analysis method for extremely-low-concentration haloperidol in blood plasma |
| CN112730686A (en) * | 2020-12-28 | 2021-04-30 | 林萍 | Method for detecting concentration of psychotropic drugs by liquid-phase mass spectrometry and solid-phase extraction method |
| CN112834679A (en) * | 2020-12-31 | 2021-05-25 | 苏州海科医药技术有限公司 | Analysis method for clinically researching concentration of quetiapine in plasma sample |
| CN113267579A (en) * | 2021-05-18 | 2021-08-17 | 中南民族大学 | Method for monitoring blood concentration of risperidone |
| CN113933422A (en) * | 2021-10-09 | 2022-01-14 | 上海中科新生命生物科技有限公司 | Detection method and kit for quetiapine, risperidone and 9-hydroxy risperidone |
| CN114034805A (en) * | 2021-11-12 | 2022-02-11 | 大连润生康泰医学检验实验室有限公司 | Metabolic kinetic analysis method of pentafluorouracil drugs |
| CN114814005A (en) * | 2022-04-01 | 2022-07-29 | 北京和合医学诊断技术股份有限公司 | Liquid chromatography analysis method for detecting ziprasidone in blood |
| CN115078612A (en) * | 2022-06-01 | 2022-09-20 | 长沙理工大学 | Analysis method for detecting chemicals based on modified Cr-MOF |
| CN115290775A (en) * | 2022-07-25 | 2022-11-04 | 上海市徐汇区中心医院 | Quality control product of mental drugs, kit, preparation method and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080050838A1 (en) * | 2006-08-26 | 2008-02-28 | Yuhui Yang | Methods for matrix cleanup and analysis of drugs and metabolites in biological matrices |
| JP2013184923A (en) * | 2012-03-07 | 2013-09-19 | Iwate Univ | Extract derived from paecilomyces tenuipes, astrocyte growth promoter including the same, and method of producing the astrocyte growth promoter |
| WO2016090228A1 (en) * | 2014-12-05 | 2016-06-09 | Myriad Genetics, Inc. | Biomarkers for distinguishing mood disorders |
| CN110108809A (en) * | 2019-04-29 | 2019-08-09 | 南京大学 | A kind of measuring method of essence crudefiber crop drug |
| CN110455943A (en) * | 2019-07-25 | 2019-11-15 | 苏州艾迪迈医疗科技有限公司 | A kind of on-line mixing filler solid-phase extraction column and preparation method thereof |
| CN111060638A (en) * | 2019-08-31 | 2020-04-24 | 河南省兽药饲料监察所(河南省畜产品质量监测检验中心) | Screening and confirming method for 207 veterinary drugs and additives in animal food |
-
2020
- 2020-05-05 CN CN202010369692.2A patent/CN111521702A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080050838A1 (en) * | 2006-08-26 | 2008-02-28 | Yuhui Yang | Methods for matrix cleanup and analysis of drugs and metabolites in biological matrices |
| JP2013184923A (en) * | 2012-03-07 | 2013-09-19 | Iwate Univ | Extract derived from paecilomyces tenuipes, astrocyte growth promoter including the same, and method of producing the astrocyte growth promoter |
| WO2016090228A1 (en) * | 2014-12-05 | 2016-06-09 | Myriad Genetics, Inc. | Biomarkers for distinguishing mood disorders |
| CN110108809A (en) * | 2019-04-29 | 2019-08-09 | 南京大学 | A kind of measuring method of essence crudefiber crop drug |
| CN110455943A (en) * | 2019-07-25 | 2019-11-15 | 苏州艾迪迈医疗科技有限公司 | A kind of on-line mixing filler solid-phase extraction column and preparation method thereof |
| CN111060638A (en) * | 2019-08-31 | 2020-04-24 | 河南省兽药饲料监察所(河南省畜产品质量监测检验中心) | Screening and confirming method for 207 veterinary drugs and additives in animal food |
Non-Patent Citations (4)
| Title |
|---|
| C. A. MUELLER 等: "Development of a multi‐target screening analysis for 301 drugs using a QTrap liquid chromatography/tandem mass spectrometry system and automated library searching", 《RAPID COMMUN. MASS SPECTROM.》 * |
| MATTEO MORETTI 等: "Determination of Antidepressants and Antipsychotics in Dried Blood Spots (DBSs) Collected from Post-Mortem Samples and Evaluation of the Stability over a Three-Month Period", 《MOLECULES》 * |
| YUNFENG CAO等: "A simple and rapid LC-MS/MS method for the simultaneous determination of eight antipsychotics in human serum, and its application to therapeutic drug monitoring", 《JOURNAL OF CHROMATOGRAPHY B》 * |
| 华萌萌等: "HPLC-MS/MS法检测鸡肉中8种镇静剂残留 ", 《食品研究与开发》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112162046A (en) * | 2020-09-25 | 2021-01-01 | 宁波大红鹰药业股份有限公司 | Quantitative analysis method for extremely-low-concentration haloperidol in blood plasma |
| CN112730686A (en) * | 2020-12-28 | 2021-04-30 | 林萍 | Method for detecting concentration of psychotropic drugs by liquid-phase mass spectrometry and solid-phase extraction method |
| CN112834679A (en) * | 2020-12-31 | 2021-05-25 | 苏州海科医药技术有限公司 | Analysis method for clinically researching concentration of quetiapine in plasma sample |
| CN113267579A (en) * | 2021-05-18 | 2021-08-17 | 中南民族大学 | Method for monitoring blood concentration of risperidone |
| CN113267579B (en) * | 2021-05-18 | 2022-08-30 | 中南民族大学 | Method for monitoring blood concentration of risperidone |
| CN113933422A (en) * | 2021-10-09 | 2022-01-14 | 上海中科新生命生物科技有限公司 | Detection method and kit for quetiapine, risperidone and 9-hydroxy risperidone |
| CN114034805A (en) * | 2021-11-12 | 2022-02-11 | 大连润生康泰医学检验实验室有限公司 | Metabolic kinetic analysis method of pentafluorouracil drugs |
| CN114814005A (en) * | 2022-04-01 | 2022-07-29 | 北京和合医学诊断技术股份有限公司 | Liquid chromatography analysis method for detecting ziprasidone in blood |
| CN114814005B (en) * | 2022-04-01 | 2024-05-03 | 北京和合医学诊断技术股份有限公司 | Liquid chromatographic analysis method for detecting ziprasidone in blood |
| CN115078612A (en) * | 2022-06-01 | 2022-09-20 | 长沙理工大学 | Analysis method for detecting chemicals based on modified Cr-MOF |
| CN115290775A (en) * | 2022-07-25 | 2022-11-04 | 上海市徐汇区中心医院 | Quality control product of mental drugs, kit, preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111521702A (en) | Liquid quality detection method for antipsychotic drugs in serum or plasma | |
| EP2405273B1 (en) | Methods for detecting vitamin D metabolites by mass spectrometry | |
| EP2633325B1 (en) | Lc-ms separation and detection of vitamin d metabolites | |
| CN107247105B (en) | A kind of method that Solid Phase Extraction-high performance liquid chromatography-tandem mass method detects perchlorate in tealeaves | |
| CN111537634B (en) | Method for detecting NDMA content in tini-class medicines | |
| CN109738565B (en) | Method for determining illegally added compounds in health food | |
| CN106018602B (en) | Combine detection reagent and its detection method for detecting sulfonamides compound | |
| Lihl et al. | High-performance liquid chromatographic determination of penicillins by means of automated solid-phase extraction and photochemical degradation with electrochemical detection | |
| US20220074900A1 (en) | Monitoring mycotoxins and its metabolites in the blood of pigs or broiler chickens | |
| EP4310505A2 (en) | Detection and quantitation of guanidinoacetate, creatine, and creatinine by mass spectrometry | |
| KR20190087900A (en) | Simultaneous determination method of donepezil and memantine | |
| Zhao et al. | An LC‐MS/MS method for determination of curculigoside with anti‐osteoporotic activity in rat plasma and application to a pharmacokinetic study | |
| RU2683032C1 (en) | Method for determining dabigatran in human blood serum | |
| Pichini et al. | High-performance liquid chromatographic—mass spectrometric assay of busulfan in serum and cerebrospinal fluid | |
| CN112834680B (en) | Method for determining concentrations of tegafur, gimeracil and 5-fluorouracil in blood plasma of tumor patient | |
| Cao et al. | Study of the determination and pharmacokinetics of bufadienolides in dog's plasma after administration of Liu-Shen-Wan by high performance liquid chromatography time-of-flight mass spectrometry | |
| Zhou et al. | An LC-MS/MS method for the simultaneous determination of lycorine and galanthamine in rat plasma and its application to pharmacokinetic study of Lycoris radiata extract in rats | |
| CN108802237B (en) | Detection and analysis method for trace triptolide in biological sample | |
| Patel et al. | Development of highly-sensitive method and its validation for the determination of fluticasone in human plasma by UPLC-MS/MS | |
| KR20060123270A (en) | Method for the determination of 25-hydroxycholecalciferol in feed | |
| CN112748203B (en) | Biological analysis method for Jactinib and ZG0244 concentrations in plasma sample in clinical research of Jettitinib cream serving as innovative medicine | |
| Tartarotti et al. | Simultaneous measurement of 1.25-dihydroxy-vitamin D, 24.25-dihydroxy-vitamin D and 25-hydroxy-vitamin D from a single two milliliters serum specimen. Preliminary clinical application | |
| CN110824039B (en) | Method for detecting concentration of cycloserine in bone tuberculosis specimen | |
| CN111337597A (en) | Method for rapidly detecting concentration of tadalafil in blood plasma | |
| RU2665164C1 (en) | Method of quantification of levodopa in blood plasma |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200811 |
|
| RJ01 | Rejection of invention patent application after publication |