CN110632234A - Method for detecting olanzapine in blood - Google Patents
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Abstract
The method for detecting olanzapine in blood comprises the following steps: preparing olanzapine standard stock solution, preparing olanzapine standard intermediate solution, preparing internal standard working solution, preparing standard solution, detecting the standard solution by using a liquid chromatography-mass spectrometer, and fitting to obtain a standard curve equation corresponding to olanzapine as follows: y is1＝a*x1+ b; and (3) processing the blood to be detected, detecting the blood to be detected by using a liquid chromatography-mass spectrometer, and calculating the concentration of olanzapine in the blood to be detected. In the present inventionThe method for processing the blood sample to be detected and the selection of the internal standard substance enable the identification of olanzapine to be more accurate, the analysis time to be short, the interference to be small, the internal standard quantification to be suitable, the specificity to be strong, the sensitivity to be high, meanwhile, all technical indexes such as the recovery rate, the detection limit and the precision to be in line with the requirements, thereby improving the accuracy of the detection result and eliminating the system error.
Description
Technical Field
The invention relates to the technical field of biological detection, and particularly relates to a method for detecting olanzapine in blood.
Background
Olanzapine is a novel atypical neuroleptic, belongs to the second generation antipsychotics, is widely used for the acute phase and maintenance treatment of schizophrenia and other psychoses with serious positive symptoms (such as delusions, hallucinations, thought disorder and suspicion) or negative symptoms (such as apathy and poor speech) clinically, and has important clinical significance for the monitoring of olanzapine.
Currently, high performance liquid chromatography is commonly used to test blood samples from patients taking olanzapine.
However, when the olanzapine content in a blood sample is measured by the above-mentioned method, the blood sample needs to be subjected to a pretreatment such as extraction. The detection time is long due to the long time of the extraction process, which results in low detection efficiency of the blood sample.
Disclosure of Invention
The invention aims to provide a method for detecting olanzapine in blood, which can improve the detection efficiency of a blood sample.
The invention relates to a method for detecting olanzapine in blood, which comprises the following steps: it comprises the following steps:
calibration of a standard solution
(a) Preparation of olanzapine Standard stock solution
Accurately weighing 5.0mg of olanzapine standard substance, placing the olanzapine standard substance into a 2mL freezing storage tube, dissolving the olanzapine standard substance by using a methanol solution with the water content of 0-30%, and determining the olanzapine standard substance to be contained in 1mL to obtain an olanzapine standard stock solution, and storing the olanzapine standard stock solution at the temperature of-80 ℃;
(b) standard intermediate solution for the preparation of olanzapine
Diluting the olanzapine standard stock solution by using a methanol diluent with the water content of 10-80% to prepare a standard intermediate solution containing at least three olanzapine with the concentration of 7.5-2400 ng/mL, and storing at-80 ℃;
(c) preparation of internal standard working solution
Diluting olanzapine-d 8 stock solution by methanol diluent with the water content of 10-80% to obtain an internal standard working solution containing 10-1000 ng/mL olanzapine-d 8, and storing at-80 ℃;
(d) preparation of Standard solutions
Respectively transferring 5 mu L of olanzapine standard intermediate liquid with at least three concentrations, respectively adding 10 mu L of internal standard working liquid and 90 mu L of methanol diluent with the water content of 10-80% into each olanzapine standard intermediate liquid, performing vortex mixing for 30 s-1 min at the rotating speed of 1000-2000 rpm, then adding 300 mu L of precipitated protein reagent acetonitrile, performing vortex mixing for 30 s-1 min at the rotating speed of 1000-2000 rpm, and preparing at least three standard solutions with different concentrations, wherein the internal standard substances contained in each standard solution with different concentrations have the same concentration;
(e) fitting standard curve equation
Detecting the standard solution with each concentration by using a liquid chromatography-mass spectrometer to obtain the spectra of olanzapine and olanzapine-d 8 of at least three standard solutions with different concentrations; respectively obtaining the chromatographic peak area of olanzapine and the chromatographic peak area of olanzapine-d 8 from the spectrum of the standard solution with each concentration, and respectively taking the ratio of the chromatographic peak area of olanzapine in the standard solution with at least three different concentrations to the chromatographic peak area of olanzapine-d 8 as the ordinate y of a standard curve equation1Taking the ratio of the concentration of olanzapine to the concentration of olanzapine-d 8 in the standard solution of at least three different concentrations as the abscissa x of the standard curve1Performing linear regression on the data of at least three different concentrations obtained by the detection, and fitting to obtain a standard curve equation corresponding to olanzapine, wherein the standard curve equation is as follows: y is1＝a*x1+ b and obtaining the weighting coefficients a and b;
(II) treatment of blood samples to be tested
Taking at least 2mL of blood to be detected, centrifuging at a centrifugal speed of 3500rpm for 10min, extracting supernatant of the blood to be detected, and storing at-20 ℃;
transferring 10 mu L of the internal standard working solution of olanzapine-d 8 in the step (c) into a 1.5mL centrifuge tube, adding 100 mu L of the supernatant of the blood to be detected, carrying out vortex oscillation mixing at the rotating speed of 1500rpm for 1min, then adding 300 mu L of the precipitated protein reagent acetonitrile into the centrifuge tube, carrying out vortex oscillation mixing at the rotating speed of 2000rpm for 1min, and then carrying out high-speed centrifugation at the rotating speed of 12000rpm for 10min to obtain the supernatant, namely the blood sample to be detected;
(III) detection of blood sample to be detected
Detecting the blood sample to be detected by using a liquid chromatography-mass spectrometer to obtain a chromatographic peak area of olanzapine and a chromatographic peak area of olanzapine-d 8 in the blood sample to be detected; taking the ratio of the peak area of the chromatographic peak of olanzapine in the blood sample to be detected to the peak area of the chromatographic peak of olanzapine-d 8 as y1Substituting into y obtained in step (e)1＝a*x1In the formula + b, the ratio x of the concentration of olanzapine in the blood sample to be tested to the concentration of olanzapine-d 8 is calculated1Since the concentration of olanzapine-d 8 in the blood sample to be tested is known, the concentration of olanzapine in the blood sample to be tested is calculated.
The invention relates to a method for detecting olanzapine in blood, which comprises the following steps: in step (b), seven standard intermediate solutions containing olanzapine at different concentrations were prepared, which were standard intermediate solutions containing olanzapine concentrations of 7.5, 30, 120, 300, 600, 1200, 2400ng/mL, respectively.
The invention relates to a method for detecting olanzapine in blood, which comprises the following steps: in step (b) and step (d), the methanol dilution is a methanol solution with a water content of 50%, and in step (a), the olanzapine standard is dissolved with pure methanol. In step (c), olanzapine-d 8 standard is dissolved in pure acetonitrile.
The invention relates to a method for detecting olanzapine in blood, which comprises the following steps: in step (c), the olanzapine-d 8 stock solution was diluted with a 50% methanol dilution containing water to give an internal standard working solution containing 500ng/mL olanzapine-d 8.
The invention relates to a method for detecting olanzapine in blood, which comprises the following steps: the chromatographic conditions of the liquid chromatogram-mass spectrum combination instrument comprise: c18 reverse phase chromatography column; the column temperature is 30 ℃; the mobile phase is as follows: water and a methanol solution, wherein the methanol solution is a methanol solution containing 0.1% of formic acid and 10mM of ammonium acetate, and the volume ratio of the water to the methanol solution is 45: 55, the isocratic elution time of the water and the methanol solution is not less than 2.0 min; the flow rate of the mobile phase is 0.2-0.5 mL/min.
The invention relates to a method for detecting olanzapine in blood, which comprises the following steps: the mass spectrum conditions of the liquid chromatogram-mass spectrum combination instrument comprise: the mass spectrum detector of the liquid chromatogram-mass spectrum combination instrument is in an ESI (+) mode; ion source parameters: the air flow is 8-50L/min, the spraying amount is 20-50L/min, the air temperature is 300-400 ℃, and the capillary tube voltage is 3000-5000V.
The invention relates to a method for detecting olanzapine in blood, which comprises the following steps: the C18 reverse phase chromatography column was Waters Xbridge C18.
The invention relates to a method for detecting olanzapine in blood, which comprises the following steps: the on-line filter of the liquid chromatography-mass spectrometer is SSI COL PRE-FILTER WATER 1/160.5M.
The embodiments of the invention have at least the following beneficial effects:
1. in the invention, standard solutions with at least three concentrations are respectively detected by using a liquid chromatography-mass spectrometer, wherein the standard solution is olanzapine standard solution with an internal standard substance, and the internal standard substance concentrations in the standard solutions with at least three concentrations are consistent; respectively fitting a standard curve equation of olanzapine according to the detection results of the standard solutions with at least three concentrations; and (3) carrying out high-speed centrifugation treatment on the blood to be detected, adding internal standard substance working solution with the same amount as that in the standard solution into the centrifuged supernatant, carrying out vortex oscillation mixing at a high rotating speed, adding a precipitated protein reagent, and carrying out high-speed vortex oscillation mixing and high-speed centrifugation treatment again to uniformly mix the solution to obtain the supernatant from which the interferents are removed. And detecting the supernatant by using a liquid chromatography-mass spectrometer, and obtaining the concentration of olanzapine in the blood sample to be detected based on the first detection result of olanzapine and the standard curve equation of olanzapine, so that the detection of olanzapine in the blood is realized, and the detection time is effectively shortened.
2. According to the method, the processing method of the blood sample to be detected and the selection of the internal standard substance enable the identification of olanzapine to be more accurate, the analysis time is short, the interference is small, the internal standard is suitable for quantification and has strong specificity and high sensitivity, and meanwhile, all technical indexes such as the recovery rate, the detection limit and the precision meet the requirements.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a chemical structural formula of olanzapine OLZ provided herein;
FIG. 2 is a chromatogram of olanzapine and olanzapine-d 8 in a standard solution provided by the present invention;
FIG. 3 is a mass spectrum of olanzapine and olanzapine-d 8 in a standard solution provided by the present invention;
FIG. 4 is a chromatogram of olanzapine and olanzapine-d 8 from a test blood sample provided in the present invention;
figure 5 is a mass spectrum of olanzapine and olanzapine-d 8 in a blood sample to be tested provided by the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention.
The method for detecting olanzapine in blood comprises the following steps:
calibration of a standard solution
(a) Preparation of olanzapine Standard stock solution
Accurately weighing 5.0mg of olanzapine standard substance, placing the olanzapine standard substance in a 2mL freezing storage tube, dissolving the olanzapine standard substance by using a pure methanol solution, and determining the volume of the olanzapine standard substance in 1mL to obtain an olanzapine standard stock solution, and storing the olanzapine standard stock solution at the temperature of-80 ℃;
(b) standard intermediate solution for the preparation of olanzapine
Diluting the olanzapine standard stock solution with a methanol diluent with a water content of 50% to prepare a standard intermediate solution containing seven kinds of olanzapine with a concentration of 7.5ng/mL-2400ng/mL, wherein the seven kinds of olanzapine standard intermediate solutions are standard intermediate solutions with a concentration of 7.5, 30, 120, 300, 600, 1200 and 2400ng/mL, and storing at-80 ℃;
(c) preparation of internal standard working solution
Diluting the olanzapine-d 8 stock solution with a methanol diluent with 50% water content to obtain an internal standard working solution containing 500ng/mL olanzapine-d 8, and storing at-80 ℃;
(d) preparation of Standard solutions
Respectively transferring 5 mu L of the seven olanzapine standard intermediate solutions with different concentrations, respectively adding 10 mu L of internal standard working solution and 90 mu L of methanol diluent with 50% of water content into each olanzapine standard intermediate solution, performing vortex mixing for 30 s-1 min at the rotating speed of 1000-2000 rpm, then adding 300 mu L of precipitated protein reagent acetonitrile, performing vortex mixing for 30 s-1 min at the rotating speed of 1000-2000 rpm, and preparing seven standard solutions with different concentrations, wherein the internal standard substances contained in each standard solution with different concentrations have the same concentration;
(e) fitting standard curve equation
Detecting the standard solution with each concentration by using a liquid chromatography-mass spectrometer to obtain the spectra of olanzapine and olanzapine-d 8 of at least three standard solutions with different concentrations; respectively obtaining the chromatographic peak area of olanzapine and the chromatographic peak area of olanzapine-d 8 from the spectrum of the standard solution with each concentration, and respectively taking the ratio of the chromatographic peak area of olanzapine in the standard solution with at least three different concentrations to the chromatographic peak area of olanzapine-d 8 as the ordinate y of a standard curve equation1Taking the ratio of the concentration of olanzapine to the concentration of olanzapine-d 8 in the standard solution of at least three different concentrations as the abscissa x of the standard curve1Performing linear regression on the data of at least three different concentrations obtained by the detection, and fitting to obtain a standard curve equation corresponding to olanzapine, wherein the standard curve equation is as follows: y is1＝a*x1+ b and obtaining the weighting coefficients a and b;
(II) treatment of blood samples to be tested
Taking at least 2mL of blood to be detected, centrifuging at a centrifugal speed of 3500rpm for 10min, extracting supernatant of the blood to be detected, and storing at-20 ℃;
transferring 10 mu L of the internal standard working solution of olanzapine-d 8 in the step (c) into a 1.5mL centrifuge tube, adding 100 mu L of the supernatant of the blood to be detected, carrying out vortex oscillation mixing at the rotating speed of 1500rpm for 1min, then adding 300 mu L of the precipitated protein reagent acetonitrile into the centrifuge tube, carrying out vortex oscillation mixing at the rotating speed of 2000rpm for 1min, and then carrying out high-speed centrifugation at the rotating speed of 12000rpm for 10min to obtain the supernatant, namely the blood sample to be detected;
(III) detection of blood sample to be detected
Detecting the blood sample to be detected by using a liquid chromatography-mass spectrometer to obtain a chromatographic peak area of olanzapine and a chromatographic peak area of olanzapine-d 8 in the blood sample to be detected; taking the ratio of the peak area of the chromatographic peak of olanzapine in the blood sample to be detected to the peak area of the chromatographic peak of olanzapine-d 8 as y1Substituting into y obtained in step (e)1＝a*x1In the formula + b, the ratio x of the concentration of olanzapine in the blood sample to be tested to the concentration of olanzapine-d 8 is calculated1Since the concentration of olanzapine-d 8 in the blood sample to be tested is known, the concentration of olanzapine in the blood sample to be tested is calculated.
The chromatographic conditions of the liquid chromatogram-mass spectrum combination instrument comprise: c18 is Waters Xbridge C18 reverse phase chromatography column; the column temperature is 30 ℃; the mobile phase is as follows: the volume ratio of the water to the methanol solution is 45: 55, and the isocratic elution time of the water to the methanol solution is not less than 2.0 min; the flow rate of the mobile phase is 0.2-0.5 mL/min.
The mass spectrum conditions of the liquid chromatogram-mass spectrum combination instrument comprise: the mass spectrum detector of the liquid chromatogram-mass spectrum combination instrument is in an ESI (+) mode; ion source parameters: the gas flow is 10L/min, the spray amount is 30L/min, the gas temperature is 350 ℃, and the capillary voltage is 4000V. The on-line filter of the LC-MS is SSI COL PRE-FILTER WATER 1/160.5M.
It is understood that the sample size may be any volume in the range of 0.1. mu.L to 30. mu.L, such as 0.1. mu.L, 0.2. mu.L, 0.5. mu.L, 1. mu.L, 5. mu.L, 10. mu.L, 15. mu.L, 20. mu.L, 25. mu.L and 30. mu.L.
In terms of column temperature, 20 to 40 ℃ means any temperature of 20 ℃ to 40 ℃, for example, 20 ℃, 21 ℃, 22.5 ℃, 23 ℃, 24 ℃, 25 ℃, 27.5 ℃, 29 ℃, 30.5 ℃, 33 ℃, 34 ℃, 35 ℃, 37 ℃, 38.5 ℃, 39 ℃, 40 ℃ and the like, and the detection results are substantially consistent at any temperature within the range.
For a liquid chromatography-mass spectrometer used for detecting olanzapine in blood to be detected, the mass spectrum conditions comprise: the mass spectrum detector of the liquid chromatogram-mass spectrum combination instrument is in an ESI (+) mode; the ion source parameters include: the air flow is 8-50L/min, the spraying amount is 20-50L/min, the air temperature is 300-400 ℃, and the capillary tube voltage is 3000-5000V.
For the gas flow, 8-50L/min refers to any flow rate from 8L/min to 50L/min, such as 8L/min, 8.5L/min, 9L/min, 11L/min, 14L/min, 19L/min, 24L/min, 28.5L/min, 35L/min, 38.5L/min, 42L/min, 44L/min, 47.5L/min, 49L/min, 50L/min, and the like.
For the spray amount, 20-50L/min refers to any flow rate of 20L/min to 50L/min, such as 20L/min, 22.5L/min, 26L/min, 30L/min, 32L/min, 35.5L/min, 36L/min, 40L/min, 43L/min, 46.5L/min, 50L/min, and the like.
For the gas temperature, 300-400 ℃ refers to any temperature of 300-400 ℃, such as 300 ℃, 320 ℃, 350 ℃, 380 ℃ and 400 ℃.
For the capillary voltage, 3000 to 5000V means any voltage of 3000V to 5000V, for example, 3000V, 3100V, 3300V, 3500V, 3900V, 4000V, 4200V, 4300V, 4500V, 4700V, 4900V, 5000V, and the like.
Further, the mass spectrum parameters of the lc-ms can be further shown in table 1 below:
TABLE 1
| Name of substance | Parent ion | Daughter ions | Dwell | Fragmentor | CE | CAV |
| Olanzapine | 313.2 | 256.1 | 200 | 120 | 22 | 2 |
| Olanzapine-d 8 | 321.2 | 260.1 | 200 | 120 | 22 | 2 |
In the above table, Dwell represents the scan time, Fragmentor represents the cracking voltage, CE represents the collision voltage, and CAV represents the collision cell acceleration voltage.
The chemical structural formula of olanzapine OLZ detected by the invention is shown in figure 1.
The blood mentioned in the embodiments of the present invention may be blood itself, or may be a sample related to blood, such as whole blood, serum, plasma, etc.
The chromatogram of olanzapine and olanzapine-d 8 for a standard solution of one of the concentrations in the above example is shown in figure 2, and the mass spectrum is shown in figure 3; wherein, numeral 1 in fig. 2 is a chromatogram of olanzapine in a standard solution, and numeral 2 is a chromatogram of olanzapine-d 8 in the standard solution; in figure 3, numeral 1 is the mass spectrum of olanzapine in the standard solution and numeral 2 is the mass spectrum of olanzapine-d 8 in the standard solution. The retention times of olanzapine and olanzapine-d 8 in the standard solution were 1.291min and 1.163min, respectively.
Further, the on-line filter used by the liquid chromatography-mass spectrometer is as follows: SI COL PRE-FILTER WATER 1/160.5M.
In the above examples, the chromatogram of olanzapine and olanzapine-d 8 in the blood sample to be tested is shown in FIG. 4, and the mass spectrum is shown in FIG. 5; wherein, the numeral 1 in fig. 4 is a chromatogram of olanzapine in the blood sample to be tested, and the numeral 2 is a chromatogram of olanzapine-d 8 in the blood sample to be tested; in fig. 5, numeral 1 is a mass spectrum of olanzapine in the blood sample to be tested, and numeral 2 is a mass spectrum of olanzapine-d 8 in the blood sample to be tested. The retention time of olanzapine and olanzapine-d 8 in the blood sample to be tested is 1.191min and 1.263min respectively.
As can be seen by comparing fig. 2, fig. 3, fig. 4 and fig. 5, the retention time of olanzapine in the blood sample to be tested and the retention time of olanzapine in the labeling solution are known, and olanzapine-d 8 is used as an internal standard substance, so that olanzapine identification is more accurate, analysis time is short, interference is small, internal standard quantification is appropriate and has strong specificity, and accuracy and sensitivity are high.
To demonstrate the linear relationship that exists for certain concentrations of olanzapine in the solutions of the present application, the following experiments were performed: adding 10 mu L of internal standard working solution and 95 mu L of methanol diluent with 30% water content into the prepared standard working solution of olanzapine with each concentration, adding 300 mu L of acetonitrile, uniformly mixing and injecting, detecting according to the treatment and detection conditions of the application, and drawing a graph by using the peak area and the concentration of a quantitative ion chromatographic peak to obtain a standard curve, wherein the result shows that the linear range and the quantitative limit of olanzapine are as follows:
olanzapine
(1) Detection Line (LOD): 0.003 ng/mL.
(2) Limit of quantitation (LOQ): 0.0375 ng/mL.
(3) Linear range: the linearity is good in the range of 0.375ng/mL to 120ng/mL, and the correlation coefficient R2 is larger than 0.999.
Recovery and precision of a method for detecting olanzapine in blood
And respectively taking olanzapine-containing standard yeast intermediate solution to prepare high, medium and low 3 concentrations to perform sample addition recovery rate experiments and precision experiments, detecting according to the treatment mode and detection conditions of the application, repeatedly analyzing and determining 3 batches, wherein the recovery rate and precision of olanzapine are shown in the following table 2:
TABLE 2
As can be seen from table 2, the average recovery of olanzapine was 99.1% to 109.9% with a relative standard deviation of 1.3% to 5.0% over the range of 3 addition levels, low, medium and high.
By combining the verification tests, the technical indexes such as detection limit, recovery rate and precision and the like of the method meet the requirements, the method for detecting the concentration of olanzapine in blood has good reproducibility and high sample-adding recovery rate, and the accuracy of the detection result is improved.
Finally, it is to be noted that: the above description is only a preferred embodiment of the present invention, and is only used to illustrate the technical solutions of the present invention, and not to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.
Claims (8)
1. A method for detecting olanzapine in blood, comprising: it comprises the following steps:
calibration of a standard solution
(a) Preparation of olanzapine Standard stock solution
Accurately weighing 5.0mg of olanzapine standard substance, placing the olanzapine standard substance into a 2mL freezing storage tube, dissolving the olanzapine standard substance by using a methanol solution with the water content of 0-30%, and determining the olanzapine standard substance to be contained in 1mL to obtain an olanzapine standard stock solution, and storing the olanzapine standard stock solution at the temperature of-80 ℃;
(b) standard intermediate solution for the preparation of olanzapine
Diluting the olanzapine standard stock solution by using a methanol diluent with the water content of 10-80% to prepare a standard intermediate solution containing at least three olanzapine with the concentration of 7.5-2400 ng/mL, and storing at-80 ℃;
(c) preparation of internal standard working solution
Diluting 100mg/L olanzapine-d 8 stock solution by using methanol diluent with the water content of 10-80% to obtain an internal standard working solution containing 10-1000 ng/mL olanzapine-d 8, and storing at-80 ℃;
(d) preparation of Standard solutions
Respectively transferring 5 mu L of olanzapine standard intermediate liquid with at least three concentrations, respectively adding 10 mu L of internal standard working liquid and 90 mu L of methanol diluent with the water content of 10-80% into each olanzapine standard intermediate liquid, performing vortex mixing for 30 s-1 min at the rotating speed of 1000-2000 rpm, then adding 300 mu L of precipitated protein reagent acetonitrile, performing vortex mixing for 30 s-1 min at the rotating speed of 1000-2000 rpm, and preparing at least three standard solutions with different concentrations, wherein the internal standard substances contained in each standard solution with different concentrations have the same concentration;
(e) fitting standard curve equation
Detecting the standard solution with each concentration by using a liquid chromatography-mass spectrometer to obtain the spectra of olanzapine and olanzapine-d 8 of at least three standard solutions with different concentrations; respectively obtaining the chromatographic peak area of olanzapine and the chromatographic peak area of olanzapine-d 8 from the spectrum of the standard solution with each concentration, and respectively taking the ratio of the chromatographic peak area of olanzapine in the standard solution with at least three different concentrations to the chromatographic peak area of olanzapine-d 8 as the ordinate y of a standard curve equation1Taking the ratio of the concentration of olanzapine to the concentration of olanzapine-d 8 in the standard solution of at least three different concentrations as the abscissa x of the standard curve1Performing linear regression on the data of at least three different concentrations obtained by the detection, and fitting to obtain a standard curve equation corresponding to olanzapine, wherein the standard curve equation is as follows: y is1＝a*x1+ b and obtaining the weighting coefficients a and b;
(II) treatment of blood samples to be tested
Taking at least 2mL of blood to be detected, centrifuging at a centrifugal speed of 3500rpm for 10min, extracting supernatant of the blood to be detected, and storing at-20 ℃;
transferring 10 mu L of the internal standard working solution of olanzapine-d 8 in the step (c) into a 1.5mL centrifuge tube, adding 100 mu L of the supernatant of the blood to be detected, carrying out vortex oscillation mixing at the rotating speed of 1500rpm for 1min, then adding 300 mu L of the precipitated protein reagent acetonitrile into the centrifuge tube, carrying out vortex oscillation mixing at the rotating speed of 2000rpm for 1min, and then carrying out high-speed centrifugation at the rotating speed of 12000rpm for 10min to obtain the supernatant, namely the blood sample to be detected;
(III) detection of blood sample to be detected
Detecting the blood sample to be detected by using a liquid chromatography-mass spectrometer to obtain a chromatographic peak area of olanzapine and a chromatographic peak area of olanzapine-d 8 in the blood sample to be detected; taking the ratio of the peak area of the chromatographic peak of olanzapine in the blood sample to be detected to the peak area of the chromatographic peak of olanzapine-d 8 as y1Substituting into y obtained in step (e)1＝a*x1In the formula + b, the ratio x of the concentration of olanzapine in the blood sample to be tested to the concentration of olanzapine-d 8 is calculated1Since the concentration of olanzapine-d 8 in the blood sample to be tested is known, the concentration of olanzapine in the blood sample to be tested is calculated.
2. The method for detecting olanzapine in blood as claimed in claim 1 wherein: in step (b), seven standard intermediate solutions containing olanzapine at different concentrations were prepared, which were standard intermediate solutions containing olanzapine concentrations of 7.5, 30, 120, 300, 600, 1200, 2400ng/mL, respectively.
3. The method for detecting olanzapine in blood according to claim 2, wherein: in step (b) and step (d), the methanol dilution is a methanol solution with a water content of 50%, and in step (a), the olanzapine standard is dissolved with pure methanol.
4. The method for detecting olanzapine in blood according to claim 3 wherein: in step (c), the olanzapine-d 8 stock solution was diluted with a 50% methanol dilution containing water to give an internal standard working solution containing 500ng/mL olanzapine-d 8.
5. The method for detecting olanzapine in blood according to claim 4, wherein: the chromatographic conditions of the liquid chromatogram-mass spectrum combination instrument comprise: c18 reverse phase chromatography column; the column temperature is 30 ℃; the mobile phase is as follows: the volume ratio of the water to the methanol solution is 45: 55, and the isocratic elution time of the water to the methanol solution is not less than 2.0 min; the flow rate of the mobile phase is 0.2-0.5 mL/min.
6. The method for detecting olanzapine in blood according to claim 5, wherein: the mass spectrum conditions of the liquid chromatogram-mass spectrum combination instrument comprise: the mass spectrum detector of the liquid chromatogram-mass spectrum combination instrument is in an ESI (+) mode; ion source parameters: the air flow is 8-50L/min, the spraying amount is 20-50L/min, the air temperature is 300-400 ℃, and the capillary tube voltage is 3000-5000V.
7. The method for detecting olanzapine in blood according to claim 6 wherein: the C18 reverse phase chromatography column was Waters Xbridge C18.
8. The method for detecting olanzapine in blood according to claim 7 wherein: the on-line filter of the liquid chromatography-mass spectrometer is SSI COL PRE-FIL TER WATER 1/160.5M.
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