CN114814005B - Liquid chromatographic analysis method for detecting ziprasidone in blood - Google Patents
Liquid chromatographic analysis method for detecting ziprasidone in blood Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 37
- 239000007788 liquid Substances 0.000 title claims abstract description 24
- 210000004369 blood Anatomy 0.000 title claims abstract description 16
- 239000008280 blood Substances 0.000 title claims abstract description 16
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- 238000004458 analytical method Methods 0.000 claims abstract description 20
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- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 13
- 239000005695 Ammonium acetate Substances 0.000 claims description 13
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- NZORAUGSDRBBBH-UHFFFAOYSA-N 1,3-benzothiazole;piperazine Chemical class C1CNCCN1.C1=CC=C2SC=NC2=C1 NZORAUGSDRBBBH-UHFFFAOYSA-N 0.000 description 1
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- 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
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- 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
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- 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
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- G—PHYSICS
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- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
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- 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
- G01N30/74—Optical detectors
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- G01N30/02—Column chromatography
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- G01N30/8627—Slopes
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- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
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- G01N30/06—Preparation
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Abstract
The invention relates to the technical field of medicine detection, in particular to a liquid chromatography analysis method for detecting ziprasidone in blood. The method comprises the following steps: calibrating the standard solution to obtain a standard curve equation; treating blood to be detected to obtain a sample to be detected, adding the sample to be detected into an online solid phase extraction device, and extracting the sample to be detected to obtain an extract; and (3) adding the extract into a high performance liquid chromatograph for analysis, and calculating through a standard curve equation to obtain the concentration of the ziprasidone in the sample to be detected. The method provided by the invention can simplify the pretreatment step, reduce the sample quantity, shorten the analysis time, be suitable for large-flux sample detection, improve the detection efficiency, and have good sensitivity and accuracy.
Description
Technical Field
The invention relates to the technical field of medicine detection, in particular to a liquid chromatography analysis method for detecting ziprasidone in blood.
Background
Ziprasidone is an atypical antipsychotic, and belongs to benzothiazole piperazine compounds. The chemical structural formula is shown as follows:
In vitro studies have shown that ziprasidone antagonizes dopamine D2, 5HT2A, 5HT1D receptors and agonizes 5HT1A receptors. Has high affinity to receptors such as dopamine D2, D3, 5-hydroxytryptamine 5HT2A, 5HT2C, 5HT1A, 5HT1D, alpha 1-epinephrine and the like, has medium affinity to histamine H1 receptors and has no affinity to other test receptors/binding sites including receptors such as M choline and the like. Ziprasidone inhibits the reuptake of 5-hydroxytryptamine and norepinephrine by synapses. Ziprasidone is mainly used for treating schizophrenia, has good curative effect on acute or chronic, primary or recurrent schizophrenia, and is effective on symptoms related to schizophrenia (including audiovisual hallucinations, delusions, lack of motivation and evasion from society). The effective treatment window of ziprasidone is 50-200 ng/mL, and the laboratory warning value is 400ng/mL, so that the ziprasidone is necessary to be aligned for monitoring therapeutic drugs in clinical application, adverse drug reactions are reduced as much as possible, and medical accidents are avoided.
At present, the method for measuring the ziprasidone content in blood is commonly used as a high performance liquid chromatography and high performance liquid chromatography mass spectrometry combined method. The high performance liquid chromatography-mass spectrometry analysis method has high analysis cost and high site requirement, requires professional personnel to operate, and is not suitable for being widely developed in middle and small hospitals. The high performance liquid chromatography has the advantages of accurate quantification, good reproducibility, low cost and the like, but most reported literature methods have the problems of complex pretreatment operation, large sample amount requirement, long analysis time and the like, and are not suitable for large-flux sample detection.
Disclosure of Invention
In order to solve the technical problems, the invention provides a liquid chromatography method for detecting ziprasidone in blood. At least comprises the following steps:
S1, calibrating a standard solution to obtain a standard curve equation; preferably, an external standard method is adopted;
S2, adding serum or plasma into acetonitrile, uniformly mixing, centrifuging, and taking supernatant as a sample to be detected, wherein the volume ratio of the serum or plasma to the acetonitrile is 1:1 to 10, preferably 1:3, a step of;
s3, detecting the sample to be detected, including:
S31, adding the sample to be detected into an online solid phase extraction device to extract the sample to be detected, so as to obtain an extract;
s32, adding the extract into a high performance liquid chromatograph for analysis, and calculating the concentration of the ziprasidone in the sample to be detected through the standard curve equation.
Optionally, S1 includes:
S11, preparing at least 3 concentration standard working solutions from ziprasidone standard products: preferably, 7 concentrations of standard working solutions are prepared, wherein the concentrations of the standard working solutions are respectively 200ng/mL, 400ng/mL, 600ng/mL, 800ng/mL, 1600ng/mL, 3200ng/mL and 6400ng/mL;
s12, respectively taking standard working solutions with various concentrations, adding blank serum, and uniformly mixing to obtain standard solutions with various concentrations;
s13, acetonitrile is added into the standard solution, and after uniform mixing, centrifugation is carried out, and a supernatant is taken;
S14, detecting the supernatant by using a liquid chromatograph after on-line solid-phase extraction to obtain chromatograms of the standard solutions with the concentrations;
S15, respectively taking the peak area of the standard solution with each concentration as the ordinate y1 of the standard curve equation, taking the concentration of the standard solution as the abscissa x1 of the standard curve equation, performing linear regression, fitting to obtain a standard curve equation of y1=a×1+b, and obtaining linear equation coefficients a and b.
Optionally, the volume ratio of the standard working solution to the blank serum is 1:9 to 19, preferably 1:9, a step of performing the process; further preferably, the volume ratio of the standard solution to acetonitrile is 1:1 to 10, more preferably 1:3.
Optionally, S11 includes:
S111, preparing a ziprasidone standard product by adopting anhydrous methanol to obtain a standard stock solution A;
S112, diluting the standard stock solution A with a methanol aqueous solution to obtain a standard working solution with the highest concentration, and then adopting the methanol aqueous solution to continue dilution to obtain standard working solutions with other concentrations;
Preferably, the concentration of methanol in the aqueous methanol solution is 40-100%, more preferably 50% by volume.
Alternatively, in S2, the volume of serum or plasma is 50 to 200. Mu.L, preferably 100. Mu.L; preferably, the serum or the plasma is added with acetonitrile and then is uniformly mixed by vortex for 2 to 6 minutes at the rotating speed of 1000 to 2000rpm, and is centrifuged for 3 to 10 minutes at 12000 to 14000 rpm.
Optionally, S14 and S31 include:
the SPE extraction column of the online solid phase extraction device is selected from the group consisting of: a reverse phase SPE cartridge, preferably C18 MG-34.0mm.times.20mm;
Mobile phase: a: water, B: acetonitrile;
Sample injection amount: 10 to 100. Mu.L, preferably 50. Mu.L;
flow rate: 0.8 to 1.2mL/min, preferably 1mL/min.
Optionally, the elution is gradient elution,
0-2 Minutes, A: 60-100%, B: 0-40%;
2-6 minutes, A: 0-20%, B: 80-100%;
6-9.5 minutes, A: 60-100%, B: 0-40%;
Preferably, it is:
0-2 minutes, A:90%, B:10%;
2-6 minutes, A:10%, B:90%;
6-9.5 minutes, A:90%, B:10%;
Preferably, the eluted product is collected for 2-3 min for high performance liquid chromatography detection.
Optionally, S14, S32 include:
The analytical chromatographic column is a reversed phase chromatographic column Thermo Acclaim TM 120C18,4.6X 250mm,5 μm;
Mobile phase: the volume ratio is 55-65: 45-55 parts of water and acetonitrile; the water contains 0.1-0.3 v/v% formic acid and 10-60 mmol/L ammonium acetate; preferably, it is: the volume ratio of water to acetonitrile is 60:40, a step of performing a; the water contained 0.2v/v% formic acid and 50mmol/L ammonium acetate.
Optionally, the elution mode of the analytical chromatographic column: isocratic elution;
Flow rate: 0.8-1.2 mL/min, preferably 1mL/min;
Column temperature: 35-45 ℃, preferably 40 ℃;
Sample injection amount: 10 to 100. Mu.L, preferably 50. Mu.L.
Compared with the prior art, the technical scheme provided by the embodiment of the invention has the following advantages:
the method provided by the invention can simplify the pretreatment step, reduce the sample quantity, shorten the analysis time, be suitable for large-flux sample detection and improve the detection efficiency.
The method provided by the invention has good sensitivity and accuracy.
Drawings
FIG. 1 is a chromatogram of ziprasidone in a standard solution in an embodiment of the invention;
FIG. 2 is a chromatogram of ziprasidone in a labeled serum sample according to an embodiment of the invention;
FIG. 3 shows that the volume ratio of serum to acetonitrile in the examples of the present invention is 1:1 serum sample addition (20 ng/mL) chromatogram;
FIG. 4 shows that the volume ratio of serum to acetonitrile in the examples of the present invention is 1: adding a standard (20 ng/mL) chromatogram to the 10 serum sample;
FIG. 5 is a chromatogram of a protein precipitant of comparative example 1 of the present invention using 10mol/L NaOH serum sample labeled (20 ng/mL);
FIG. 6 is a chromatogram of a protein precipitant of comparative example 1 of the present invention using a 6mol/L HCl serum sample plus standard (20 ng/mL);
FIG. 7 is a chromatogram of a serum sample labeled (640 ng/mL) without the use of an on-line solid phase extraction device in comparative example 2 of the present invention;
Fig. 8 shows that the mobile phase in comparative example 4 of the present invention has a volume ratio of 60:40 pure water and acetonitrile serum samples were subjected to a labeling (640 ng/mL) chromatogram;
Fig. 9 shows that the mobile phase in comparative example 4 of the present invention has a volume ratio of 60:40 (50 mmol/L ammonium acetate) and acetonitrile serum samples were subjected to a standard (640 ng/mL) chromatogram;
fig. 10 shows that the mobile phase in comparative example 4 of the present invention has a volume ratio of 60:40 (0.2 v/v% formic acid) and acetonitrile serum samples were subjected to a standard (20 ng/mL) chromatogram;
Fig. 11 shows that the mobile phase in comparative example 5 of the present invention has a volume ratio of 50:50 (0.2 v/v% formic acid and 50mmol/L ammonium acetate) and acetonitrile blank serum sample chromatograms;
Fig. 12 shows that the mobile phase of comparative example 5 according to the present invention has a volume ratio of 50:50 (0.2 v/v% formic acid and 50mmol/L ammonium acetate) and acetonitrile blank serum samples were subjected to a standard (20 ng/mL) chromatogram;
Fig. 13 shows that the mobile phase in comparative example 5 of the present invention has a volume ratio of 70:30 (0.2 v/v% formic acid and 50mmol/L ammonium acetate) and acetonitrile serum samples were subjected to a standard (20 ng/mL) chromatogram;
FIG. 14 is a chromatogram of a blank serum sample of comparative example 6 of the present invention using a chromatographic column (Thermo Acclaim TM 120C18,4.6X 150mm,5 μm);
FIG. 15 is a chromatogram of a serum sample of comparative example 6 of the present invention using a chromatographic column (Thermo Acclaim TM 120C18,4.6X 150mm,5 μm) labeled (20 ng/mL);
FIG. 16 is a chromatogram of a blank serum sample of comparative example 7 of the present invention using a column temperature of 30 ℃;
FIG. 17 is a chromatogram of a serum sample labeled (20 ng/mL) at 30℃using column temperature in comparative example 7 of the present invention;
FIG. 18 is a chromatogram of a blank serum sample of comparative example 7 of the present invention using a column temperature of 50 ℃;
FIG. 19 is a chromatogram of a serum sample labeled (20 ng/mL) of comparative example 7 according to the present invention using a column temperature of 50 ℃.
Detailed Description
In order that the above objects, features and advantages of the present disclosure may be more clearly understood, a further description of aspects of the present disclosure will be provided below. It should be noted that, without conflict, the embodiments of the present disclosure and features in the embodiments may be combined with each other.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure, but the present disclosure may be practiced otherwise than as described herein; it will be apparent that the embodiments in the specification are only some, but not all, embodiments of the disclosure.
The embodiment of the invention provides a high performance liquid chromatography detection method for ziprasidone content in blood, which at least comprises the following steps:
S1, calibrating a standard solution to obtain a standard curve equation;
S2, treating blood to be tested to obtain a sample to be tested, wherein the method comprises the following steps:
Adding serum or plasma into acetonitrile, uniformly mixing, centrifuging, and taking the supernatant as a sample to be detected;
s3, detecting a sample to be detected, including:
s31, adding a sample to be detected into an online solid-phase extraction device, and extracting the sample to be detected to obtain an extract;
s32, adding the extract into a high performance liquid chromatograph for analysis, and calculating the concentration of the ziprasidone in the sample to be detected through a standard curve equation.
In the embodiment of the invention, the serum or the plasma is added into acetonitrile to precipitate protein, and the volume ratio of the serum or the plasma to the acetonitrile is 1:1 to 10, preferably 1:3. if the acetonitrile adding proportion is too small, the protein precipitation is incomplete, and the service life of the on-line solid phase extraction column is influenced; if the acetonitrile addition ratio is too large, dilution is too large, concentration is lowered, and signals are affected.
The embodiment of the invention is connected with the high performance liquid chromatography analyzer through the online solid phase extraction device, simplifies the pretreatment step, directly feeds the sample to the online solid phase extraction device after the protein is precipitated by acetonitrile, can finish detection without operation in the follow-up process, reduces personnel operation, ensures that the detection process is simple, convenient and quick, reduces the experiment cost and improves the detection efficiency.
In the embodiment of the invention, the external standard method is adopted to prepare the standard curve, compared with the internal standard method, the external standard method does not need to search for an internal standard substance, and the internal standard substance generally selects other medicines, but if a sample donor to be detected also takes the medicines, the detection result is interfered, and the detection result is inaccurate. The external standard method may be used to prepare a standard curve, and in one embodiment of the present invention, the standard curve equation may be obtained by:
s11, preparing at least 3 concentration standard working solutions from ziprasidone standard products: preferably, 7 concentration standard working solutions are prepared, and the concentration of the standard working solutions can be specifically 200ng/mL, 400ng/mL, 600ng/mL, 800ng/mL, 1600ng/mL, 3200ng/mL and 6400ng/mL;
s12, respectively taking standard working solutions with various concentrations, adding blank serum, and uniformly mixing to obtain standard solutions with various concentrations;
s13, acetonitrile is added into the standard solution, and after uniform mixing, the mixture is centrifuged, and the supernatant is taken;
S14, detecting the supernatant by using a liquid chromatograph after on-line solid-phase extraction, wherein the analysis conditions of the liquid chromatograph are as follows; obtaining a chromatogram of the standard solution with each concentration;
S15, respectively taking the peak area of the standard solution with each concentration as the ordinate y1 of the standard curve equation, taking the concentration of the standard solution as the abscissa x1 of the standard curve equation, performing linear regression, fitting to obtain a standard curve equation of y1=a x1+b, and obtaining linear equation coefficients a and b.
In a preferred embodiment of the present invention, the volume ratio of the standard working fluid to the blank serum is 1: 9-19, wherein the volume of the standard working solution is as small as possible, the matrix is not changed as much as possible, and the detection accuracy is ensured, and the embodiment of the invention is further preferably 1:9.
In a preferred embodiment of the present invention, the volume ratio of the standard solution to acetonitrile is 1:1 to 10, preferably 1:2 to 5, more preferably 1:3. consistent with sample pretreatment.
In a specific implementation manner of the embodiment of the invention, preparing at least 3 concentration standard working solutions comprises:
S111, preparing a ziprasidone standard product by adopting anhydrous methanol to obtain a standard stock solution A;
S112, diluting the standard stock solution A with a methanol aqueous solution to obtain a standard working solution with the highest concentration, for example: 6400ng/mL; then, the mixture is diluted continuously by adopting methanol aqueous solution to obtain standard working solutions with other concentrations, for example: 200ng/mL, 400ng/mL, 600ng/mL, 800ng/mL, 1600ng/mL, 3200ng/mL;
In a preferred embodiment of the present invention, the concentration of methanol in the aqueous methanol solution of the diluted standard stock solution a is 40 to 100% by volume, more preferably 50%. The benefits of this diluent are increased solubility, reduced solvent evaporation.
In the embodiment of the invention, serum or plasma can be obtained by adopting conventional conditions, and the sample size of the serum or plasma required by the detection of the embodiment of the invention is 50-200 mu L, preferably 100 mu L;
In a preferred implementation manner of the embodiment of the present invention, S22 includes: after acetonitrile is added, vortex mixing is carried out for 2-6 min at the rotating speed of 1000-2000 rpm, and centrifugal is carried out for 3-10 min at the rotating speed of 12000-14000 rpm. The condition can ensure the mixing and centrifuging effect.
In a preferred embodiment of the present embodiment, the chromatographic conditions in S14 and S31 are specifically:
the extraction column of the in-line solid phase extraction device is selected from: a reverse phase SPE cartridge, preferably C18 MG-3.0mmx 20mm;
Mobile phase: a: water, B: acetonitrile;
Sample injection amount: 10 to 100. Mu.L, preferably 50. Mu.L;
flow rate: 0.8 to 1.2mL/min, preferably 1mL/min.
The elution is a gradient elution,
0-2 Minutes, A: 60-100%, B: 0-40%;
2-6 minutes, A: 0-20%, B: 80-100%;
6-9.5 minutes, A: 60-100%, B: 0-40%;
Preferably, it is:
0-2 minutes, A:90%, B:10%;
2-6 minutes, A:10%, B:90%;
6-9.5 minutes, A:90%, B:10%;
And collecting the eluted product in 2-3 min for high performance liquid chromatographic detection.
In 0-2 minutes, 90% of water phase has lower eluting capability, target analyte ziprasidone can be reserved on an SPE column, and macromolecular proteins and other impurities in a blood sample are eluted into waste liquid; in 3-6 minutes, 90% of the organic phase has strong eluting capability, and the SPE column is cleaned to remove impurities remained on the SPE column, so that the service life of the next needle sample and the SPE column is ensured to be free from the interference of the impurities; balance SPE column for 6-9.5 min, and prepare for next needle sample injection.
In a preferred embodiment of the present embodiment, the chromatographic conditions in S14 and S31 are specifically:
The analytical chromatographic column is a reversed phase chromatographic column Thermo Acclaim TM C18, 4.6X105 mm,5 μm;
Mobile phase: the volume ratio is 55-65: 45-55 parts of water and acetonitrile; the water contains 0.1-0.3 v/v% formic acid and 10-60 mmol/L ammonium acetate; preferably, it is: the volume ratio is 60:40 and acetonitrile; the water contains 0.2v/v% formic acid and 50mmol/L ammonium acetate;
Elution mode: isocratic elution;
Flow rate: 0.8-1.2 mL/min, preferably 1mL/min;
Column temperature: 35-45 ℃, preferably 40 ℃;
Sample injection amount: 10 to 100. Mu.L, preferably 50. Mu.L.
The excitation wavelength of the fluorescence detector of the high performance liquid chromatograph is 316nm, and the emission wavelength is 415nm. The fluorescence detector can be UltiMate3000 RS Fluorescence Detector detector, the response time is 2s, and the acquisition frequency is as follows: 5Hz, analysis time was 9.5min.
The specific instrument connection mode is as follows: the online solid-phase extraction device is connected with the high-performance liquid chromatograph through a six-way valve, and the high-performance liquid chromatograph pump II is used for providing power for the online solid-phase extraction device; a high performance liquid chromatograph pump I; for powering the chromatography column.
Injecting a sample to be detected, and enabling the sample to be detected to pass through an extraction column of an online solid-phase extraction device by a pump II, wherein an analyte ziprasidone is reserved on the extraction column;
Eluting the analyte ziprasidone from the extraction column to an analysis column of a high performance liquid chromatograph by a pump I, separating the analyte ziprasidone by the pump I, and detecting by a fluorescence detector;
And (3) cleaning and balancing the extraction column by a pump II.
According to the embodiment of the invention, after the online solid-phase extraction device is used, serum is directly injected after the protein is precipitated by acetonitrile, so that the personnel operation is reduced, the detection process is simple, convenient and quick, the experimental cost is reduced, the analysis time is shortened, and the monitoring of the ziprasidone blood concentration in a patient in clinical treatment is facilitated.
The raw materials and equipment used in the following specific examples of the present invention are all commercially available products.
Example 1
The present example is directed to specific detection steps of a liquid chromatography method for detecting ziprasidone in blood:
1. Calibration of standard solutions
1.1 Preparation of standard working solution:
accurately weighing 2mg of ziprasidone standard substance, placing in a 2mL freezing tube, and adding 2mL of anhydrous methanol to obtain a standard stock solution A;
Taking a proper amount of standard stock solution A, diluting with 50v/v% methanol aqueous solution, wherein the concentration of ziprasidone in the obtained standard working solution is 6400ng/mL, then continuously diluting with 50v/v% methanol aqueous solution, preparing ziprasidone solutions with the concentrations of 200ng/mL, 400ng/mL, 600ng/mL, 800ng/mL, 1600ng/mL, 3200ng/mL and 6400ng/mL respectively, and storing at the temperature of minus 80 ℃;
1.2 obtaining a standard curve:
Firstly, respectively transferring 10 mu L of standard working solution and 90 mu L of blank serum by using a pipette, respectively placing the standard working solution and the blank serum into a 1.5mL centrifuge tube, and mixing the standard working solution and the blank serum to prepare seven standard solutions with different concentrations;
After the standard solution is uniformly mixed by vortex for 30s to 1min at the rotating speed of 1000 to 2000rpm, 300 mu L of acetonitrile is added, the standard solution is uniformly mixed by vortex for 2 to 6min at the rotating speed of 1000 to 2000rpm, the standard solution is centrifuged for 3 to 10min at the high speed of 12000 to 14000rpmn, 350 mu L of supernatant is sucked, and the solution is detected by a liquid chromatograph and a fluorescence detector.
Obtaining seven standard solution chromatograms of ziprasidone with different concentrations, respectively obtaining peak areas of the ziprasidone from the standard solution chromatograms of the ziprasidone, respectively taking the peak areas of the seven standard solution of the ziprasidone with different concentrations as an ordinate y1 of a standard curve equation, taking the concentration in the standard solution of the ziprasidone as an abscissa x1 of the standard curve equation, carrying out linear regression on the data with different concentrations obtained by the detection, fitting to obtain a standard curve equation of y1=a x1+b, and obtaining linear equation coefficients a and b; the standard working solution is a solution containing ziprasidone.
2. Sample processing to be tested
Transferring 100 mu L of serum or plasma by a liquid transferring gun, adding 300 mu L of acetonitrile, uniformly mixing by vortex at the rotating speed of 1000-2000 rpm for 2-6 min, centrifuging for 3-10 min at 12000-14000 r/min, and absorbing 350 mu L of supernatant as a sample to be detected;
3. detection of a sample to be tested
And (2) detecting the standard solution processed in the step (1.2) and the sample to be detected in the step (2) by using a liquid chromatograph and a fluorescence detector to obtain a ziprasidone chromatogram of the sample to be detected, obtaining a ziprasidone peak area from the ziprasidone chromatogram, substituting the ziprasidone peak area y1 into a standard curve equation y1=a x1+b of the step (1), and obtaining the concentration x1 of the ziprasidone in the sample to be detected through calculation.
The high performance liquid chromatograph comprises an on-line solid phase extraction device.
Extraction column used in on-line solid phase extraction device: the senior hall C18 MG-34.0mm.times.20mm,
Extraction column mobile phase: a: water, B: acetonitrile;
gradient elution is as shown in table 1:
TABLE 1
| Time(min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 90 | 10 |
| 2 | 90 | 10 |
| 4 | 10 | 90 |
| 6 | 10 | 90 |
| 9.5 | 90 | 10 |
The flow rate of the extraction column is 1mL/min.
High performance liquid chromatograph:
Analytical chromatographic column: thermo Acclaim TM C18 (4.6X250 mm,5 μm);
analytical column mobile phase: the content is 60:40 (V/V) ratio of water (50 mmol/L ammonium acetate, 0.2V/V% formic acid) and acetonitrile;
Flow rate: 1mL/min;
Column temperature: 40 ℃;
Sample injection amount: 50. Mu.L;
The fluorescence detector is UltiMate3000 RS Fluorescence Detector detector:
The excitation wavelength is 316nm, the emission wavelength is 415nm, the response time is 2s, and the acquisition frequency is as follows: 5Hz, analysis time was 9.5min.
The obtained chromatograms are shown in fig. 1 and 2, wherein the chromatogram of ziprasidone in serum sample is shown in fig. 2, the chromatogram of ziprasidone in standard solution is shown in fig. 1, and the retention time of ziprasidone is 8.1min.
The specific instrument connection mode is as follows: the online solid-phase extraction device is connected with a high-performance liquid chromatography analyzer through a six-way valve, and a pump II on the high-performance liquid chromatography analyzer is used for providing power for the online solid-phase extraction device; the pump I on the high performance liquid chromatography analyzer is used for powering the chromatography column.
The specific detection process comprises the following steps:
1. and (3) injecting the sample into the system through an automatic sampler for 0-2 minutes, wherein the pump II carries the sample to pass through the SPE column, the analyte ziprasidone is reserved on the SPE column, and part of impurities are eluted and flow into the waste liquid.
2. The chromatographic pump (pump I) elutes the analyte ziprasidone from the SPE column to the analytical column for 2-3 minutes.
3. 3-9.5 Minutes, and detecting the analyte ziprasidone and impurities after chromatographic separation by a chromatographic pump (pump I); pump II equilibrates the SPE column after washing for next needle sample analysis.
Example 2
This example is presented to illustrate the linear relationship and quantitative limits of the method of the present invention:
Adding 90 mu L of blank serum into 10 mu L of prepared ziprasidone standard working solution respectively, uniformly mixing for 1min, adding 300 mu L of acetonitrile, uniformly mixing by vortex at 2000rpm for 3min, centrifuging at 14000r/min for 10min at high speed, sucking 350 mu L of supernatant, carrying out sample injection analysis, measuring according to the measuring conditions of the embodiment from low concentration to high concentration and plotting quantitative chromatographic peak area-concentration to obtain a standard curve; preparing a series of low-concentration standard solutions, adding 90 mu L of blank serum or plasma into 10 mu L of each concentration of ziprasidone standard solution prepared by the preparation, uniformly mixing, performing pretreatment and sample injection analysis, wherein the 3-time signal-to-noise ratio is a detection limit, the 10-time signal-to-noise ratio is a quantitative limit, and the result shows that the linear range and the quantitative limit are as follows:
(1) Limit of detection (LOD): ziprasidone is 3ng/mL.
(2) Limit of quantitation (LOQ): ziprasidone was 9ng/mL.
(3) Linear range: the ziprasidone has good linearity and a correlation coefficient R 2 of more than 0.99 within the range of 20 ng/mL-640 ng/mL.
Example 3
This example is presented to illustrate the recovery and precision of the method of the present invention.
The standard working solutions of ziprasidone are respectively prepared into low, medium and high concentrations for sample addition recovery rate experiments and precision experiments, the sample addition recovery rate experiments and the precision experiments are carried out, the sample addition recovery rate experiments and the precision experiments are measured according to the method of the embodiment, 3 batches are repeatedly analyzed and measured, and the recovery rate and the precision of the ziprasidone are respectively shown in table 2. The average recovery rate of ziprasidone in the range of 3 addition levels of low, medium and high is 99.0-101.3%, the relative standard deviation is 0.83-1.96%, and the results are shown in table 2.
TABLE 2 ziprasidone labeling recovery and precision
| Scalar addition | 40ng/mL | 80ng/mL | 320ng/mL |
| Average recovery rate | 100.5％ | 101.3％ | 99.0％ |
| Precision RSD | 6.76％ | 1.20％ | 1.40％ |
In the verification test, the detection limit, the recovery rate, the precision and other technical indexes meet the requirements, the method for detecting the ziprasidone content in blood has good repeatability and high sample addition recovery rate, and the accuracy of the detection result is improved. The blood sample is directly injected after acetonitrile is used for precipitating protein, so that the personnel operation is reduced, the detection process is simple, convenient and quick, the experimental cost is reduced, the analysis time is shortened, and the ziprasidone blood concentration in a patient is more conveniently monitored in clinical treatment.
Example 4:
this example is used to illustrate the technical effect of the protein precipitant addition ratio of the present invention.
The samples were tested using the method of example 1, except that:
1. the volume ratio of the serum to the acetonitrile is 1:1; experimental results were obtained as in fig. 3:
2. The volume ratio of the serum to the acetonitrile is 1:10; the experimental results are shown in FIG. 4.
The results illustrate: the expected effect can be obtained by adopting the volume ratio of serum to acetonitrile of 1:1-10.
Comparative example 1:
The embodiment of the invention is used for explaining the technical effect of acetonitrile as the protein precipitant.
The samples were tested using the method of example 1, except that:
1. the protein precipitant adopts 10mol/L NaOH. The experimental results obtained are shown in fig. 5.
The results show that: baseline raised and signal lowered.
2. The protein precipitant adopts 6mol/L HCl. The experimental results obtained are shown in fig. 6.
The results show that: baseline elevation, uneven baseline, and impurity interference.
Comparative example 2:
the embodiment is used for explaining the technical effect of the online solid phase extraction device.
The samples were tested using the method of example 1, except that: extracting and enriching a sample to be detected without using an online solid-phase extraction device; the experimental results obtained are shown in fig. 7.
Results: peak broadening is severe.
Comparative example 3:
the embodiment is used for explaining the technical effect of the gradient elution mode of the mobile phase of the extraction column.
The samples were tested using the method of example 1, except that:
3.1, gradient elution mode shown in Table 3:
TABLE 3 Table 3
| Time(min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 2 | 100 | 0 |
| 4 | 10 | 90 |
| 6 | 10 | 90 |
| 7 | 100 | 0 |
3.2, Gradient elution mode shown in Table 4:
TABLE 4 Table 4
| Time(min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 80 | 20 |
| 2 | 80 | 80 |
| 4 | 10 | 90 |
| 6 | 10 | 90 |
| 7 | 80 | 20 |
3.3, Gradient elution mode shown in Table 5:
TABLE 5
| Time(min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 70 | 30 |
| 2 | 70 | 30 |
| 4 | 10 | 90 |
| 6 | 10 | 90 |
| 7 | 70 | 30 |
3.4, Gradient elution mode shown in Table 6:
TABLE 6
| Time(min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 60 | 40 |
| 2 | 60 | 40 |
| 4 | 10 | 90 |
| 6 | 10 | 90 |
| 7 | 60 | 40 |
3.5, Gradient elution mode shown in Table 7:
TABLE 7
| Time(min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 50 | 50 |
| 2 | 50 | 50 |
| 4 | 10 | 90 |
| 6 | 10 | 90 |
| 7 | 50 | 50 |
The experimental results obtained are shown in table 8:
TABLE 8
| Gradient elution initial proportion of extraction column | Peak area of serum standard solution (640 ng/mL) | |
| Comparative example 3.1 | 100/0 | 260492.9083 |
| Example 1 | 90/10 | 278306.1800 |
| Comparative example 3.2 | 80/20 | 288577.8767 |
| Comparative example 3.3 | 70/30 | 291881.8617 |
| Comparative example 3.4 | 60/40 | 273039.3817 |
| Comparative example 3.5 | 50/50 | 53824.8550 |
The results show that: the extraction column was eluted with a 50/50 initial ratio and the peak area of the serum standard solution (640 ng/mL) was reduced, indicating that the target analyte ziprasidone was not retained on the SPE extraction column at an initial ratio of 50/50, and a 50/50 initial gradient was not available.
Comparative example 4:
This example is presented to illustrate the technical effect of the present invention in analyzing the mobile phase of a chromatographic column.
The samples were tested using the method of example 1, except that:
1. Mobile phase: pure water and acetonitrile in a volume ratio of 60:40; experimental results were obtained as shown in fig. 8;
the results show that: no peak was seen within 12 min.
2. Mobile phase: water and acetonitrile in a volume ratio of 60:40; the water contains 50mmol/L ammonium acetate; experimental results were obtained as shown in fig. 9;
the results show that: no peak was seen within 12 min.
3. Mobile phase: water and acetonitrile in a volume ratio of 60:40; the water contains 0.2v/v% formic acid; experimental results were obtained as shown in fig. 10;
the results show that: the baseline is uneven.
Comparative example 5:
this example is presented to illustrate the technical effect of the present invention in analyzing the mobile phase ratio of a chromatographic column.
The samples were tested using the method of example 1, except that:
1. Mobile phase: water and acetonitrile in a volume ratio of 50:50; the water contains 0.2v/v% formic acid and 50mmol/L ammonium acetate; the experimental results obtained are shown in fig. 11 and 12.
The results show that: the peak position of the target is interfered by impurities.
2. Mobile phase: water and acetonitrile in a volume ratio of 70:30; the water contains 0.2v/v% formic acid and 50mmol/L ammonium acetate; the experimental results obtained are shown in fig. 13.
The results show that: no peak was seen for 15 min.
Comparative example 6:
this example is presented to illustrate the technical effect of the present invention using analytical chromatographic columns.
The samples were tested using the method of example 1, except that:
Chromatographic column: thermo Acclaim TM 120C18,4.6 X150 mm,5 μm. The experimental results obtained are shown in fig. 14 and 15.
The results show that: the peak position of the target is interfered by impurities.
Comparative example 7:
The embodiment is used for explaining the technical effect of the column temperature in the embodiment of the invention.
The samples were tested using the method of example 1, except that:
1. column temperature of chromatographic column: 30 ℃; the experimental results obtained are shown in fig. 16 and 17.
The results show that: the peak position of the target is interfered by impurities.
2. Column temperature of chromatographic column: 50 ℃; the experimental results obtained are shown in fig. 18 and 19.
The results show that: the peak position of the target is interfered by impurities.
The foregoing is only a specific embodiment of the invention to enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present disclosure is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (7)
1. A liquid chromatography method for detecting ziprasidone in blood, comprising at least the steps of:
S1, calibrating a standard solution to obtain a standard curve equation;
S2, adding serum or plasma into acetonitrile, uniformly mixing, centrifuging, and taking supernatant as a sample to be detected, wherein the volume ratio of the serum or plasma to the acetonitrile is 1:1 to 10;
s3, detecting the sample to be detected, including:
S31, adding the sample to be detected into an online solid phase extraction device to extract the sample to be detected, so as to obtain an extract;
the SPE extraction column of the online solid phase extraction device is selected from the group consisting of: c18 MG-3.0mmx20mm;
Mobile phase: a: water, B: acetonitrile;
The elution is a gradient elution,
0-2 Minutes, A: 60-100%, B: 0-40%;
2-6 minutes, A: 0-20%, B: 80-100%;
6-9.5 minutes, A: 60-100%, B: 0-40%;
Collecting the eluted product in 2-3 min for high performance liquid chromatographic detection;
S32, adding the extract into a high performance liquid chromatograph for analysis, and calculating to obtain the concentration of ziprasidone in the sample to be detected through the standard curve equation;
The analytical chromatographic column is a reversed phase chromatographic column Thermo Acclaim TM 120C18,4.6X 250mm,5 μm;
mobile phase: the volume ratio is 55-65: 35-45 parts of water and acetonitrile; the water contains 0.1-0.3 v/v% formic acid and 10-60 mmol/L ammonium acetate; the elution mode of the analytical chromatographic column is as follows: isocratic elution.
2. The liquid chromatography method according to claim 1, wherein S1 comprises:
s11, preparing at least 3 concentration standard working solutions from ziprasidone standard;
s12, respectively taking standard working solutions with various concentrations, adding blank serum, and uniformly mixing to obtain standard solutions with various concentrations;
s13, acetonitrile is added into the standard solution, and after uniform mixing, centrifugation is carried out, and a supernatant is taken;
S14, detecting the supernatant by using a liquid chromatograph after on-line solid-phase extraction to obtain chromatograms of the standard solutions with the concentrations;
S15, respectively taking the peak area of the standard solution with each concentration as the ordinate y1 of the standard curve equation, taking the concentration of the standard solution as the abscissa x1 of the standard curve equation, performing linear regression, fitting to obtain a standard curve equation of y1=a×1+b, and obtaining linear equation coefficients a and b.
3. The liquid chromatography method according to claim 2, wherein the volume ratio of the standard working fluid to the blank serum is 1:9 to 19; the volume ratio of the standard solution to acetonitrile is 1:1 to 10.
4. The liquid chromatography method according to claim 2, wherein S11 comprises:
S111, preparing a ziprasidone standard product by adopting anhydrous methanol to obtain a standard stock solution A;
S112, diluting the standard stock solution A with a methanol aqueous solution to obtain a standard working solution with the highest concentration, and then adopting the methanol aqueous solution to continue dilution to obtain standard working solutions with other concentrations;
the volume percentage concentration of the methanol in the methanol aqueous solution is 40-100%.
5. The method of liquid chromatography according to claim 1, wherein,
In S2, the volume of the serum or the plasma is 50-200 mu L;
After acetonitrile is added into the serum or the plasma, the mixture is uniformly mixed by vortex for 2 to 6 minutes at the rotating speed of 1000 to 2000rpm, and the mixture is centrifuged for 3 to 10 minutes at 12000 to 14000 rpm.
6. The method for liquid chromatography according to claim 1 or 2, wherein,
S14 and S31 include: sample injection amount of the online solid phase extraction device: 10-100 mu L;
Flow rate: 0.8-1.2 mL/min.
7. The method of liquid chromatography according to claim 1, wherein,
Flow rate of the analytical chromatographic column: 0.8-1.2 mL/min;
Column temperature: 35-45 ℃;
sample injection amount: 10-100 mu L.
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