CN114814005A - Liquid chromatography analysis method for detecting ziprasidone in blood - Google Patents
Liquid chromatography analysis method for detecting ziprasidone in blood Download PDFInfo
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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 a 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 adding the extract into a high performance liquid chromatography analyzer for analysis, and calculating by 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 steps, reduce the sample amount, shorten the analysis time, be suitable for large-flux sample detection, improve the detection efficiency and simultaneously 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, belonging to the benzothiazole piperazine class of compounds. The chemical structural formula is as follows:
in vitro studies indicate that ziprasidone has antagonism to dopamine D2, 5HT2A and 5HT1D receptors and agonism to 5HT1A receptors. The compounds have high affinity for receptors such as dopamine D2, D3, 5-hydroxytryptamine 5HT2A, 5HT2C, 5HT1A, 5HT1D and alpha 1-adrenaline, have moderate affinity for the histamine H1 receptor, and have no affinity for other tested receptors/binding sites including M choline. Ziprasidone inhibits 5-hydroxytryptamine and norepinephrine reuptake by synapses. Ziprasidone is mainly used for treating schizophrenia, has good curative effect on acute or chronic schizophrenia, incipient schizophrenia or recurrent schizophrenia, and is effective on symptoms related to schizophrenia (including audiovisual hallucinations, delusions, lack of motivation and social escape). The effective treatment window of the ziprasidone is 50-200 ng/mL, and the laboratory warning value is 400ng/mL, so that the ziprasidone is required to be monitored for treatment drugs in clinical application, adverse drug reactions are reduced as much as possible, and medical accidents are avoided.
At present, the method commonly adopted for measuring the ziprasidone content in blood is a high performance liquid chromatography and high performance liquid chromatography mass spectrometry combined method. The high performance liquid chromatography-mass spectrometry combined method is high in analysis cost, high in requirement on sites, and not suitable for being widely developed in small and medium-sized hospitals, and needs to be operated by professional personnel. 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 quantity demand, long analysis time and the like, and are not suitable for sample detection with large flux.
Disclosure of Invention
In order to solve the technical problems, the invention provides a liquid chromatography analysis method for detecting ziprasidone in blood. At least comprises the following steps:
s1, calibrating the standard solution to obtain a standard curve equation; preferably adopting an external standard method;
s2, adding acetonitrile into serum or plasma, mixing uniformly, centrifuging, wherein the supernatant is a sample to be detected, and the volume ratio of the serum or plasma to the acetonitrile is 1: 1-10, preferably 1: 3;
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;
and S32, adding the extract into a high performance liquid chromatography analyzer for analysis, and calculating the concentration of ziprasidone in the sample to be detected through the standard curve equation.
Optionally, S1 includes:
s11, preparing at least 3 standard working solutions with different concentrations from ziprasidone standard: preferably, 7 kinds of concentration standard working solutions are prepared, and the concentrations of the standard working solutions are respectively 200ng/mL, 400ng/mL, 600ng/mL, 800ng/mL, 1600ng/mL, 3200ng/mL and 6400 ng/mL;
s12, respectively taking the standard working solution with each concentration, adding blank serum, and uniformly mixing to obtain the standard solution with each concentration;
s13, adding acetonitrile into the standard solution, mixing uniformly, centrifuging, and taking a supernatant;
s14, detecting the supernatant by using a liquid chromatographic analyzer after carrying out online solid-phase extraction to obtain chromatograms of the standard solutions with various concentrations;
and S15, taking the peak area of the standard solution with each concentration as the ordinate y1 of a 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 the standard curve equation of y 1-a x1+ b, and obtaining coefficients a and b of the linear equation.
Optionally, the volume ratio of the standard working solution to the blank serum is 1: 9-19, preferably 1: 9; further preferably, the volume ratio of the standard solution to the acetonitrile is 1: 1-10, more preferably 1: 3.
optionally, S11 includes:
s111, preparing a ziprasidone standard substance by adopting absolute 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 continuously diluting with the methanol aqueous solution to obtain other standard working solutions with various concentrations;
preferably, the volume percentage concentration of the methanol in the methanol water solution is 40-100%, and more preferably 50%.
Optionally, in S2, the volume of the serum or the plasma is 50-200 μ L, preferably 100 μ L; preferably, the serum or the plasma is added with acetonitrile and then is mixed evenly for 2-6 min in a vortex mode at the rotating speed of 1000-2000 rpm, and then is centrifuged for 3-10 min at 12000-14000 rpm.
Optionally, S14, S31 includes:
the SPE extraction column of the online solid phase extraction device is selected from: a reversed phase SPE column, preferably C18 MG-34.0 mm. times.20 mm;
mobile phase: a: water, B: acetonitrile;
sample introduction amount: 10-100 μ L, preferably 50 μ L;
flow rate: 0.8-1.2 mL/min, preferably 1 mL/min.
Optionally, 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, the following components are used:
0-2 minutes, A: 90%, B: 10 percent;
2-6 minutes, A: 10%, B: 90 percent;
6-9.5 minutes, A: 90%, B: 10 percent;
preferably, the elution product within 2-3 min is collected for high performance liquid chromatography detection.
Optionally, S14, S32 includes:
the analytical chromatographic column is a reversed-phase chromatographic column Thermo Acclaim TM 120C18，4.6×250mm，5μm；
Mobile phase: the volume ratio is 55-65: 45-55 parts of water and acetonitrile; the water contains 0.1-0.3 v/v% of formic acid and 10-60 mmol/L of ammonium acetate; preferably: the volume ratio of water to acetonitrile is 60: 40; the water contained 0.2 v/v% formic acid and 50mmol/L ammonium acetate.
Optionally, the elution mode of the analytical chromatography column: isocratic elution;
flow rate: 0.8-1.2 mL/min, preferably 1 mL/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 steps, reduce the sample amount, shorten the analysis time, is suitable for sample detection with large flux and improves 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 example of the invention;
FIG. 2 is a chromatogram of ziprasidone in a spiked serum sample according to an example of the invention;
FIG. 3 shows that the volume ratio of the serum to the acetonitrile in the example of the present invention is 1: 1 serum sample plus standard (20ng/mL) chromatogram;
FIG. 4 shows that the volume ratio of the serum to the acetonitrile in the example of the present invention is 1: 10 serum sample plus standard (20ng/mL) chromatogram;
FIG. 5 is a chromatogram of a 10mol/L NaOH serum sample spiked with a protein precipitant (20ng/mL) according to comparative example 1 of the present invention;
FIG. 6 is a chromatogram obtained by adding a standard (20ng/mL) to a serum sample containing 6mol/L HCl used in the protein precipitant in comparative example 1 according to the present invention;
FIG. 7 is a chromatogram of a serum sample spiking (640ng/mL) without the use of an online 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 is a mixture of the following mobile phases in a volume ratio of 60: 40 pure water and acetonitrile serum samples plus standard (640ng/mL) chromatograms;
FIG. 9 shows that the mobile phase in comparative example 4 of the present invention is 60: 40 water (50mmol/L ammonium acetate) and acetonitrile serum samples (640ng/mL) chromatograms;
FIG. 10 shows that the mobile phase in comparative example 4 of the present invention is 60: 40 water (0.2 v/v% formic acid) and acetonitrile serum samples plus standard (20ng/mL) chromatograms;
FIG. 11 shows that the mobile phase in comparative example 5 of the present invention is a mixture of the mobile phases in a volume ratio of 50: 50 water (0.2 v/v% formic acid and 50mmol/L ammonium acetate) and acetonitrile blank serum sample chromatograms;
FIG. 12 shows that the mobile phase in comparative example 5 of the present invention is a mixture of the following mobile phases in a volume ratio of 50: water at 50 (0.2 v/v% formic acid and ammonium acetate at 50 mmol/L) and acetonitrile blank serum sample spiked (20ng/mL) chromatograms;
FIG. 13 shows that the mobile phase in comparative example 5 of the present invention is a mixture of the following mobile phases in a volume ratio of 70: 30 water (0.2 v/v% formic acid and 50mmol/L ammonium acetate) and acetonitrile serum samples are spiked (20ng/mL) chromatograms;
FIG. 14 shows the results of the present invention using a column (Thermo Acclaim) in comparative example 6 TM 120C18, 4.6X 150mm, 5 μm) blank serum sample chromatogram;
FIG. 15 shows the results of the present invention obtained in comparative example 6 using a column (Thermo Acclaim) TM 120C18, 4.6X 150mm, 5 μm) serum sample plus standard (20ng/mL) chromatogram;
FIG. 16 is a chromatogram of a blank serum sample obtained in comparative example 7 when the column temperature of a chromatographic column is 30 ℃;
FIG. 17 is a chromatogram obtained by subjecting a serum sample to standard addition (20ng/mL) at 30 ℃ 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 obtained by applying a standard (20ng/mL) to a serum sample at 50 ℃ and a column temperature of a chromatographic column in comparative example 7 of the present invention.
Detailed Description
In order that the above objects, features and advantages of the present disclosure may be more clearly understood, aspects of the present disclosure will be further described below. It should be noted that the embodiments and features of the embodiments of the present disclosure may be combined with each other without conflict.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure, but the present disclosure may be practiced in other ways than those described herein; it is to be understood that the embodiments disclosed in the specification are only a few embodiments of the present disclosure, and not all embodiments.
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 the standard solution to obtain a standard curve equation;
s2, processing the blood to be detected to obtain a sample to be detected, wherein the method comprises the following steps:
adding acetonitrile into serum or plasma, mixing, centrifuging, and collecting supernatant as sample to be tested;
s3, detecting the 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;
and S32, adding the extract into a high performance liquid chromatography analyzer for analysis, and calculating the concentration of ziprasidone in the sample to be detected through a standard curve equation.
In the embodiment of the invention, acetonitrile is added into serum or plasma to precipitate the protein, and the volume ratio of the serum or plasma to the acetonitrile is 1: 1-10, preferably 1: 3. if the addition proportion of acetonitrile is too small, the protein precipitation is incomplete, and the service life of the on-line solid-phase extraction cartridge is influenced; if the acetonitrile addition proportion is too large, the dilution is too much, the concentration is reduced, and the signal is influenced.
According to the embodiment of the invention, the online solid-phase extraction device is used together with the high-performance liquid chromatography analyzer, so that the pretreatment steps are simplified, the sample is directly fed to the online solid-phase extraction device after the protein is precipitated through acetonitrile, the detection can be completed without subsequent operation, the personnel operation is reduced, the detection process is simple, convenient and quick, the experiment cost is reduced, and the detection efficiency is improved.
Compared with an internal standard method, the external standard method does not need to search for an internal standard substance, the internal standard substance generally selects other medicines, but if a sample donor to be detected also takes the medicine, the detection result is interfered, and the detection result is inaccurate. The standard curve prepared by the external standard method can be prepared by adopting a method commonly used in the field, and in a specific implementation mode of the embodiment of the invention, the standard curve equation can be obtained by the following method:
s11, preparing at least 3 standard working solutions with different concentrations from ziprasidone standard: preferably, 7 kinds of concentration standard working solutions are prepared, and the concentration of the standard working solution can be 200ng/mL, 400ng/mL, 600ng/mL, 800ng/mL, 1600ng/mL, 3200ng/mL and 6400 ng/mL;
s12, respectively taking the standard working solution with each concentration, adding blank serum, and uniformly mixing to obtain the standard solution with each concentration;
s13, adding acetonitrile into the standard solution, mixing uniformly, centrifuging, and taking the supernatant;
s14, detecting the supernatant by using a liquid chromatography analyzer after online solid-phase extraction, wherein the analysis conditions of the liquid chromatography analyzer are as follows; obtaining a chromatogram of the standard solution with each concentration;
and S15, performing linear regression by taking the peak area of the standard solution with each concentration as the ordinate y1 of the standard curve equation and the concentration of the standard solution as the abscissa x1 of the standard curve equation, fitting to obtain the standard curve equation of y 1-a x1+ b, and obtaining coefficients a and b of the linear equation.
In a preferred embodiment of the present invention, the volume ratio of the standard working solution to the blank serum is 1: 9-19, the volume of the added 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 further preferably adopts a volume ratio of 1: 9.
in a preferred embodiment of the present invention, the volume ratio of the standard solution to acetonitrile is 1: 1-10, preferably 1: 2-5, more preferably 1: 3. consistent with the pretreatment of the sample.
In a specific implementation manner of the embodiment of the present invention, preparing at least 3 kinds of concentration standard working solutions includes:
s111, preparing a ziprasidone standard substance by adopting absolute methanol to obtain a standard stock solution A;
and S112, diluting the standard stock solution A with a methanol aqueous solution to obtain the standard working solution with the highest concentration, such as: 6400 ng/mL; then, continuously diluting with a methanol water solution to obtain other standard working solutions with various concentrations, such as: 200ng/mL, 400ng/mL, 600ng/mL, 800ng/mL, 1600ng/mL, 3200 ng/mL;
in a preferred embodiment of the present invention, the volume percentage concentration of methanol in the methanol aqueous solution of the diluted standard stock solution a is 40 to 100%, and more preferably 50%. The diluent has the advantages of increased solubility and reduced solvent evaporation.
In the embodiment of the invention, serum or plasma can be obtained under conventional conditions, and the sample size of the serum or plasma required by detection in the embodiment of the invention is 50-200 muL, preferably 100 muL;
in a preferred implementation manner of the embodiment of the present invention, S22 includes: and adding acetonitrile, then, carrying out vortex mixing for 2-6 min at the rotating speed of 1000-2000 rpm, and centrifuging for 3-10 min at 12000-14000 rpm. The condition can ensure the effect of uniform mixing and centrifugation.
In a preferred embodiment of the present invention, the chromatographic conditions in S14 and S31 are specifically:
the extraction column of the on-line solid phase extraction device is selected from: a reversed phase SPE column, preferably C18 MG-34.0 mm. times.20 mm;
mobile phase: a: water, B: acetonitrile;
sample introduction amount: 10-100 μ L, preferably 50 μ L;
flow rate: 0.8-1.2 mL/min, preferably 1 mL/min.
The elution is a gradient elution and 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:
0-2 minutes, A: 90%, B: 10 percent;
2-6 minutes, A: 10%, B: 90 percent;
6-9.5 minutes, A: 90%, B: 10 percent;
and collecting the elution product within 2-3 minutes for high performance liquid chromatography detection.
In 0-2 minutes, 90% of water phase has low elution capacity, the target analyte ziprasidone can be retained on the SPE column, and impurities such as macromolecular protein in the blood sample are eluted into waste liquid; in 3-6 minutes, 90% of the organic phase has strong elution capacity, 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 without impurity interference; and (5) balancing the SPE column for 6-9.5 minutes to prepare for the next injection.
In a preferred embodiment of the present invention, the chromatographic conditions in S14 and S31 are specifically:
the analytical chromatographic column is a reversed-phase chromatographic column Thermo Acclaim TM 120 C18，4.6×250mm，5μm；
Mobile phase: the volume ratio is 55-65: 45-55 parts of water and acetonitrile; the water contains 0.1-0.3 v/v% of formic acid and 10-60 mmol/L of ammonium acetate; preferably: the volume ratio is 60: 40 of water and acetonitrile; the water contained 0.2 v/v% formic acid and 50mmol/L ammonium acetate;
and (3) an elution mode: isocratic elution;
flow rate: 0.8-1.2 mL/min, preferably 1 mL/min;
column temperature: 35-45 ℃, preferably 40 ℃;
sample introduction amount: 10 to 100. mu.L, preferably 50. mu.L.
The excitation wavelength of a fluorescence detector of the high performance liquid chromatography is 316nm, and the emission wavelength is 415 nm. The Fluorescence Detector can be selected from an UlltiMate 3000 RS Fluorescence Detector, the response time is 2s, and the acquisition frequency is as follows: 5Hz, and the analysis time is 9.5 min.
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 high-performance liquid chromatography analyzer pump II is used for providing power for the online solid-phase extraction device; a high performance liquid chromatography analyzer pump I; for powering the chromatography column.
Injecting a sample to be detected, enabling the sample to be detected to pass through an extraction column of the online solid-phase extraction device by a pump II, and retaining the analyte ziprasidone on the extraction column;
eluting the analyte ziprasidone from the extraction column to an analysis column of a high performance liquid chromatography analyzer by a pump I, separating the analyte ziprasidone by the pump I through the analysis column, and detecting by a fluorescence detector;
and the pump II is used for cleaning and balancing the extraction column.
According to the embodiment of the invention, after the online solid-phase extraction device is used, serum is directly injected after being subjected to protein precipitation by acetonitrile, so that the operation of personnel is reduced, the detection process is simple, convenient and quick, the experiment cost is reduced, the analysis time is shortened, and the monitoring of the blood concentration of ziprasidone in a patient body in clinical treatment is facilitated.
Examples of the invention the raw materials and equipment used in the following examples are all commercially available products.
Example 1
This example is used to illustrate the specific detection steps of the 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 the ziprasidone standard substance in a 2mL freezing storage 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 50 v/v% methanol water solution to obtain standard working solution with ziprasidone concentration of 6400ng/mL, then continuously diluting with 50 v/v% methanol water solution to prepare ziprasidone solutions with ziprasidone concentrations of 200ng/mL, 400ng/mL, 600ng/mL, 800ng/mL, 1600ng/mL, 3200ng/mL and 6400ng/mL respectively, and storing at-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 a pipettor, and respectively placing the standard working solution and the blank serum into 1.5mL centrifuge tubes to be mixed to prepare seven standard solutions with different concentrations;
and (3) respectively carrying out vortex mixing on the standard solution at the rotating speed of 1000-2000 rpm for 30 s-1 min, then adding 300 mu L of acetonitrile, carrying out vortex mixing at the rotating speed of 1000-2000 rpm for 2-6 min, carrying out high-speed centrifugation at 12000-14000 rpmn for 3-10 min, absorbing 350 mu L of supernatant, and detecting the solution by using a liquid chromatography analyzer 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 solutions of the ziprasidone with different concentrations as an ordinate y1 of a standard curve equation, taking the concentration of the ziprasidone standard solution as an abscissa x1 of the standard curve equation, performing linear regression on the detected data with different concentrations, and fitting to obtain a standard curve equation of y1 to a x1+ b and obtain coefficients a and b of the linear equation; the standard working solution is a solution containing ziprasidone.
2. Treatment of samples to be tested
Transferring 100 mu L of serum or plasma by using a liquid transfer gun, adding 300 mu L of acetonitrile, carrying out vortex mixing for 2-6 min at the rotating speed of 1000-2000 rpm, 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 (3) detecting the standard solution processed in the step (1.2) and the sample to be detected in the step (2) by using a liquid chromatography analyzer and a fluorescence detector to obtain a chromatogram of ziprasidone of the sample to be detected, obtaining a ziprasidone peak area from the ziprasidone chromatogram, substituting the ziprasidone peak area y1 into the standard curve equation y1 ═ a x1+ b in the step (1), and calculating to obtain the concentration x1 of ziprasidone in the sample to be detected.
The high performance liquid chromatograph comprises an online solid phase extraction device.
The extraction column used by the online solid phase extraction device: the Yangtze hall C18 MG-34.0mm is multiplied by 20mm,
extraction column mobile phase: a: water, B: acetonitrile;
gradient elution is 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 was 1 mL/min.
High performance liquid chromatograph:
analyzing a chromatographic column: thermo Acclaim TM 120C18(4.6×250mm，5μm)；
Analytical column mobile phase: contains 60 of: isocratic elution with water (50mmol/L ammonium acetate, 0.2V/V% formic acid) and acetonitrile at a ratio of 40 (V/V);
flow rate: 1 mL/min;
column temperature: 40 ℃;
sample introduction amount: 50 mu L of the solution;
the Fluorescence Detector is an UltiMate3000 RS Fluorescence Detector:
the excitation wavelength is 316nm, the emission wavelength is 415nm, the response time is 2s, and the acquisition frequency is as follows: 5Hz, and the analysis time is 9.5 min.
The obtained chromatograms are shown in fig. 1 and fig. 2, wherein the chromatogram of ziprasidone in a serum sample is shown in fig. 2, the chromatogram of ziprasidone in a standard solution is shown in fig. 1, and the retention time of ziprasidone is 8.1 min.
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 an upper pump II of the high-performance liquid chromatography analyzer is used for providing power for the online solid-phase extraction device; the upper pump I of the high performance liquid chromatography is used for providing power for the chromatographic analysis column.
The specific detection process is as follows:
1. and (3) after 0-2 minutes, the sample passes through an automatic sample injector injection system, the pump II carries the sample to pass through the SPE column, the analyte ziprasidone is retained on the SPE column, and part of impurities are eluted and flow into waste liquid.
2. For 2-3 minutes, the chromatographic pump (pump I) elutes the analyte ziprasidone from the SPE column to the analytical column.
3. 3-9.5 minutes, detecting the analyte ziprasidone and impurities after chromatographic separation by a chromatographic analysis pump (pump I); pump II washes the SPE cartridge and equilibrates for the next needle sample analysis.
Example 2
This example serves to illustrate the linear relationship and quantitative limits of the method of the present invention:
respectively adding 90 mul of blank serum into 10 mul of ziprasidone standard working solution with each concentration prepared above, uniformly mixing for 1min, adding 300 mul of acetonitrile, uniformly mixing for 3min in a vortex mode at the rotating speed of 2000rpm, centrifuging at the high speed of 14000r/min for 10min, sucking 350 mul of supernatant, carrying out sample injection analysis, determining the ziprasidone concentration within the range of 20ng/mL to 640ng/mL, determining according to the determination conditions of the embodiment from low to high according to the concentration, and drawing by the peak area-concentration of quantitative chromatography to obtain a standard curve; preparing a series of low-concentration standard solutions, respectively adding 90 mu L of blank serum or plasma into 10 mu L of the prepared ziprasidone standard solutions with various concentrations, uniformly mixing, carrying out pretreatment and sample injection analysis, wherein the detection limit is 3 times of signal-to-noise ratio, the quantitative limit is 10 times of signal-to-noise ratio, and the results show that the linear range and the quantitative limit are as follows:
(1) limit of detection (LOD): ziprasidone is 3 ng/mL.
(2) Limit of quantitation (LOQ): ziprasidone is 9 ng/mL.
(3) Linear range: the ziprasidone has good linearity and a correlation coefficient R within the range of 20 ng/mL-640 ng/mL 2 ＞0.99。
Example 3
This example is provided to illustrate the recovery and precision of the method of the present invention.
The ziprasidone standard working solution is prepared into low, medium and high concentrations of 3 for sample adding recovery rate experiments and precision experiments, the detection is carried out according to the method of the embodiment, 3 batches of analysis and detection are repeated, and the recovery rate and 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%, and the relative standard deviation is 0.83% -1.96%, and the results are shown in table 2.
TABLE 2 ziprasidone spiking recovery and precision
| Adding quantity of scalar | 40ng/mL | 80ng/mL | 320ng/mL |
| Average recovery rate | 100.5％ | 101.3％ | 99.0％ |
| Precision RSD | 6.76％ | 1.20％ | 1.40％ |
By combining the verification tests, the technical indexes of detection limit, recovery rate, precision and the like of the embodiment of the invention meet the requirements, and the method for detecting the ziprasidone content in blood has good reproducibility and high sample recovery rate, and improves the accuracy of the detection result. The blood sample is directly injected after the protein is precipitated by acetonitrile, so that the operation of personnel is reduced, the detection process is simple, convenient and quick, the experiment cost is reduced, the analysis time is shortened, and the monitoring of the blood concentration of ziprasidone in a patient body in clinical treatment is facilitated.
Example 4:
this example is provided to illustrate the technical effect of the protein precipitant addition ratio of the present invention.
The samples were tested as in example 1, except that:
1. the volume ratio of the serum to the acetonitrile is 1: 1; the experimental results are obtained as shown in fig. 3:
2. the volume ratio of the serum to the acetonitrile is 1: 10; the experimental results are obtained as shown in fig. 4.
The results show that: the expected effect can be obtained by adopting the volume ratio of the serum to the acetonitrile of 1: 1-10.
Comparative example 1:
this example is provided to illustrate the technical effect of acetonitrile as the protein precipitant in the examples of the present invention.
The samples were tested as in 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: the baseline was raised and the signal decreased.
2. The protein precipitant adopts 6mol/L HCl. The obtained experimental results are shown in fig. 6.
The results show that: the base line is raised, the base line is uneven, and impurity interference exists.
Comparative example 2:
this example is provided to illustrate the technical effects of the on-line solid phase extraction apparatus according to the embodiment of the present invention.
The samples were tested as in example 1, except that: extracting and enriching a sample to be detected without using an online solid phase extraction device; the results of the experiment are shown in FIG. 7.
As a result: the peak broadening was severe.
Comparative example 3:
this example is provided to illustrate the technical effect of the gradient elution method of mobile phase in the extraction column of the embodiment of the present invention.
The samples were tested as in example 1, except that:
3.1, using the gradient elution regime shown in 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, using the gradient elution mode shown in 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, using the 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, using the gradient elution regime 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, using the gradient elution regime 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 results obtained are shown in table 8:
TABLE 8
| Initial ratio of gradient elution in extraction column | Peak area of serum standard solution (640ng/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 50/50 initial ratio and the peak area of serum standard solution (640ng/mL) was reduced, indicating that the target analyte ziprasidone was not retained on the SPE extraction column at 50/50 initial ratio and 50/50 initial gradient was not used.
Comparative example 4:
this example serves to illustrate the technical effect of analyzing a mobile phase of a chromatography column according to an embodiment of the present invention.
The samples were tested as in example 1, except that:
1. mobile phase: pure water and acetonitrile in a volume ratio of 60: 40; the experimental results are obtained as shown in fig. 8;
the results show that: no peak appeared within 12 min.
2. Mobile phase: water and acetonitrile in a volume ratio of 60: 40; the water contains 50mmol/L ammonium acetate; the experimental results are obtained as in fig. 9;
the results show that: no peak appeared within 12 min.
3. Mobile phase: water and acetonitrile in a volume ratio of 60: 40; water containing 0.2 v/v% formic acid; the experimental results are obtained as shown in fig. 10;
the results show that: the baseline is not flat.
Comparative example 5:
this example is provided to illustrate the technical effect of analyzing the mobile phase ratio of a chromatographic column according to the embodiment of the present invention.
The samples were tested as in example 1, except that:
1. mobile phase: water and acetonitrile in a volume ratio of 50: 50; the water contained 0.2 v/v% formic acid and 50mmol/L ammonium acetate; the obtained experimental results are shown in fig. 11 and 12.
The results show that: the peak position of the target object is interfered by impurities.
2. Mobile phase: water and acetonitrile in a volume ratio of 70: 30; the water contained 0.2 v/v% formic acid and 50mmol/L ammonium acetate; the obtained experimental results are shown in fig. 13.
The results show that: no peak appeared at 15 min.
Comparative example 6:
this example serves to illustrate the technical effect of using an analytical chromatography column in an embodiment of the present invention.
The samples were tested as in example 1, except that:
a chromatographic column: thermo Acclaim TM 120C18, 4.6X 150mm, 5 μm. The obtained experimental results are shown in fig. 14 and 15.
The results show that: the peak position of the target object is interfered by impurities.
Comparative example 7:
this example is provided to illustrate the technical effect of the column temperature used in the embodiments of the present invention.
The samples were tested as in example 1, except that:
1. column temperature of the chromatographic column: 30 ℃; the obtained experimental results are shown in fig. 16 and 17.
The results show that: the peak position of the target object is interfered by impurities.
2. Column temperature of the chromatographic column: 50 ℃; the obtained experimental results are shown in fig. 18 and 19.
The results show that: the peak position of the target object is interfered by impurities.
The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice the present 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 invention 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 (9)
1. A liquid chromatography analysis method for detecting ziprasidone in blood is characterized by at least comprising the following steps:
s1, calibrating the standard solution to obtain a standard curve equation;
s2, adding acetonitrile into serum or plasma, mixing uniformly, centrifuging, wherein the supernatant is a sample to be detected, and the volume ratio of the serum or plasma to the acetonitrile is 1: 1-10;
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;
and S32, adding the extract into a high performance liquid chromatography analyzer for analysis, and calculating the concentration of ziprasidone in the sample to be detected through the standard curve equation.
2. The liquid chromatography analysis method of claim 1, wherein S1 comprises:
s11, preparing at least 3 standard working solutions with different concentrations from ziprasidone standard;
s12, respectively taking the standard working solution with each concentration, adding blank serum, and uniformly mixing to obtain the standard solution with each concentration;
s13, adding acetonitrile into the standard solution, mixing uniformly, centrifuging, and taking a supernatant;
s14, detecting the supernatant by using a liquid chromatographic analyzer after carrying out online solid-phase extraction to obtain chromatograms of the standard solutions with various concentrations;
and S15, taking the peak area of the standard solution with each concentration as the ordinate y1 of a 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 the standard curve equation of y 1-a x1+ b, and obtaining coefficients a and b of the linear equation.
3. The method of claim 2, wherein the volume ratio of the standard working fluid to the blank serum is 1: 9-19; the volume ratio of the standard solution to the acetonitrile is 1: 1 to 10.
4. The liquid chromatography analysis method according to claim 2, wherein S11 includes:
s111, preparing a ziprasidone standard substance by adopting absolute 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 continuously diluting with the methanol aqueous solution to obtain other standard working solutions with various concentrations;
the volume percentage concentration of the methanol in the methanol water solution is 40-100%.
5. The liquid chromatography analysis method according to claim 1,
in S2, the volume of the serum or the plasma is 50-200 mu L;
and adding acetonitrile into the serum or the plasma, and then, uniformly mixing the mixture for 2-6 min in a vortex manner at the rotating speed of 1000-2000 rpm, and centrifuging the mixture for 3-10 min at 12000-14000 rpm.
6. The liquid chromatography analysis method according to claim 1 or 2,
s14, S31 includes: the SPE extraction column of the online solid phase extraction device is selected from: c18 MG-34.0mm × 20 mm;
mobile phase: a: water, B: acetonitrile;
sample introduction amount: 10-100 μ L;
flow rate: 0.8-1.2 mL/min.
7. The liquid chromatography method according to claim 6, wherein 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%;
and collecting the elution product within 2-3 minutes for high performance liquid chromatography detection.
8. The method of liquid chromatography analysis of claim 1 or 2, wherein S14, S32 comprises:
the analytical chromatographic column is a reversed-phase chromatographic column Thermo Acclaim TM 120C18，4.6×250mm，5μm；
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.
9. The liquid chromatography analysis method according to claim 8,
elution mode of the analytical chromatography column: isocratic elution;
flow rate: 0.8-1.2 mL/min;
column temperature: 35-45 ℃;
sample introduction amount: 10 to 100 μ L.
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