Method for rapidly determining blood concentration of posaconazole and application

Abstract

The invention relates to the technical field of medical examination, and discloses a method for rapidly measuring the blood concentration of posaconazole and application of the method. According to the method for rapidly determining the blood concentration of posaconazole and the application of the method, compared with chromatography in the prior art, the adopted mobile phase is relatively single, the configuration time of the mobile phase is saved, and meanwhile, the fluctuation of a detection result caused by the configuration of the mobile phase is reduced to the greatest extent; the stability of the method is further enhanced; meanwhile, compared with other liquid chromatography methods, the detection method has the advantages that the single-needle sample injection time is short, the reagent preparation is simple, no endogenous and common exogenous substance interference exists around the target peak of posaconazole, and the like, and the detection efficiency and the accuracy of the detection result are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of medical testing, and specifically to a method and application for rapid determination of posaconazole blood drug concentration. Background Technique

[0002] Posaconazole (POS) is a second-generation triazole drug with a broad antibacterial spectrum, strong antibacterial activity, and good tolerance. It has antifungal activity against a variety of yeasts (Candida) and fungi (Aspergillus, Zygomycetes, and Fusarium). Posaconazole is mainly used for the prevention of invasive fungal diseases (IFD): (1) patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) undergoing remission induction chemotherapy, which is expected to cause long-term neutropenia and has a high risk of developing IFD; (2) allogeneic hematopoietic stem cell transplantation (HSCT) recipients who need to receive high-dose immunosuppressive therapy for graft-versus-host disease and have a high risk of onset. In addition, posaconazole is approved for the treatment of oropharyngeal candidiasis, for the treatment of IFD patients intolerant to first-line treatment, and for the rescue of IFD caused by rare pathogens such as hyalohyphomycosis, chromoblastomycosis mycetoma, and coccidioidomycosis.

[0003] The prior art cannot achieve rapid determination of posaconazole blood drug concentration, and there are problems of low efficiency and long time consumption in the actual determination process. In view of this, we have proposed a method and application for rapid determination of posaconazole blood drug concentration. Summary of the Invention

[0004] The purpose of the present invention is to provide a method and application for rapid determination of posaconazole blood drug concentration to solve the problems raised in the above background technique.

[0005] To achieve the above purpose, the present invention provides the following technical solution: A method for rapid determination of posaconazole blood drug concentration, including the following steps:

[0006] S1. Sample pretreatment: Centrifuge whole blood, quantitatively extract plasma samples, add a protein precipitant for protein precipitation, vortex, and centrifuge, and take out the supernatant;

[0007] S2. Sample separation and analysis: Use a C 18 packed chromatographic column as the analytical column, a high-performance liquid chromatograph equipped with an ultraviolet detector or a diode array detector DAD, use a methanol-aqueous solution mixture as the mobile phase, keep the column temperature constant, perform isocratic elution, and the injection volume is 20 μL; Detect the peak area of posaconazole at a UV wavelength of 260 nm and convert it into concentration using the standard curve equation.

[0008] S3. Concentration calculation: Substitute the analysis results into the regression equation to calculate the posaconazole drug concentration. The linear range of posaconazole is 0.28 - 18.00 mg / L, and there is a good linear relationship between the peak area and the concentration.

[0009] Optionally, the protein precipitant is one or more of methanol and acetonitrile, and the ratio of the protein precipitant to plasma can be 3 - 6:1, preferably 4:1.

[0010] Optionally, the whole blood specimen is centrifuged at 4000 rpm for 5 min; the vortex time is 3 min, and the centrifugation condition is: centrifuged at 12000 rpm for 10 min.

[0011] Optionally, the sample pretreatment includes: sucking 100 μL of plasma sample into an EP tube, and then adding 400 μL of protein precipitant.

[0012] Optionally, the chromatographic conditions in S2 are: chromatographic column Shim-pack GIST C 18 , 5 μm, 150 nm × 4.6 nm; mobile phase: methanol: water = 71 - 29 (v / v); flow rate is 1 mL / min; diode array detector or UV detector at 260 nm; column temperature is 40 °C; injection volume is 20 μL; measure the peak area of posaconazole, and substitute it into the standard curve equation to calculate the posaconazole concentration.

[0013] Optionally, S3 includes: linear range, precision, stability, recovery test, and sample stability investigation; among them, the linear range test includes: accurately weigh an appropriate amount of posaconazole reference substance, dissolve it with 50% methanol water and make up to the mark to obtain the mother liquor of the posaconazole standard solution. Then take a certain amount of the mother liquor and dilute it step by step in half. Take 20 μL of each diluted standard solution and transfer it to a 1.5 mL PE tube. Add 80 μL of blank plasma to each tube, vortex and mix well to prepare calibration curve mother liquors with concentrations of 0.28, 0.56, 1.13, 2.25, 4.50, 9.00, 18.00 μg / mL.

[0014] Optionally, the precision, stability, and recovery experiments include: accurately weigh an appropriate amount of posaconazole reference substance, dissolve it with 50% methanol water with one-fifth of the volume, and then dilute and make up to the mark with four-fifths of the volume of blank plasma to prepare a series of concentration standard plasma samples containing posaconazole at 1.13, 2.25, 4.50 μg / mL; take 100 μL of each drug-containing serum and operate according to the sample pretreatment method; calculate the measured concentration according to the linear regression equation. The concentration measured by using the mobile phase instead of blank plasma to prepare the above samples with each concentration is the absolute concentration. Calculate the measured average value and relative standard deviation RSD for each concentration. The recovery formula is as follows:

[0015]

[0016] Optionally, the investigation of sample stability includes: taking standard concentration blood samples and placing them at room temperature for 12 h; placing them at 2 - 8°C for 7 days; storing them frozen at -20°C for 30 days; storing them frozen at -80°C for 6 months; storing them frozen at -80°C for 6 months and subjecting them to 6 cycles of freeze-thaw; taking 100 μL of drug-containing serum each, operating according to the sample pretreatment method, and investigating the stability of posaconazole.

[0017] The present invention also proposes an application of a method for simultaneously determining posaconazole in in vivo drug analysis and determination.

[0018] Compared with the prior art, the present invention provides a method and application for rapidly determining the blood drug concentration of posaconazole, having the following beneficial effects:

[0019] 1. For the method and application for rapidly determining the blood drug concentration of posaconazole, using methanol or acetonitrile as a protein precipitant can minimize the chronic erosion of the chromatographic column and pipeline system by acids or salts as protein precipitants, keeping the method stable in the long term; at the same time, using the protein precipitation method for pretreatment of plasma samples has the characteristics of simple operation, short time consumption, high recovery rate, and low cost compared with the extraction method.

[0020] 2. For the method and application for rapidly determining the blood drug concentration of posaconazole, using the high performance liquid chromatography method preferably provided by the present invention to determine the blood drug concentration of posaconazole can greatly improve the stability and accuracy of the method compared with liquid chromatography-tandem mass spectrometry. The stable period of the calibration curve of this method is at least more than 3 months, while the liquid chromatography-tandem mass spectrometry method usually only has about 3 days, and even a calibration curve needs to be made every day for detection. Therefore, compared with the liquid chromatography-tandem mass spectrometry method, this method greatly improves the detection efficiency, reduces the detection cost, improves the accuracy of the detection results, and is more suitable for clinical tests; at the same time, compared with the liquid chromatography-tandem mass spectrometry method, the coefficient of variation of the method is greatly reduced, making the detection results fluctuate less, the linearity better, and improving the accuracy of the detection results.

[0021] 3. For the method and application for rapidly determining the blood drug concentration of posaconazole, compared with the chromatographic methods in the prior art, the mobile phase used is relatively single, saving the time for preparing the mobile phase, and at the same time minimizing the fluctuations in the detection results caused by the preparation of the mobile phase to further enhance the stability of the method; at the same time, compared with other liquid chromatography methods, the detection method of the present invention has the advantages of short single injection time, simple reagent preparation, and no interference from endogenous and common exogenous substances around the target peak of posaconazole, improving the detection efficiency and the accuracy of the detection results. Description of the Drawings

[0022] Figure 1 It is the ultraviolet absorption spectrum of posaconazole of the present invention;

[0023] Figure 2 This is the liquid chromatography diagram of the blank plasma sample of the present invention;

[0024] Figure 3 This is the liquid chromatography diagram of the blank plasma sample + standard product of the present invention;

[0025] Figure 4 This is the liquid chromatography diagram of the plasma of the patient taking the medicine of the present invention. Detailed implementation manners

[0026] Next, in combination with the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0027] Embodiment 1

[0028] As Figures 1-4 shown, the present invention provides a technical solution: a method for rapidly determining the blood drug concentration of posaconazole, including the following steps:

[0029] S1. Sample pretreatment: Centrifuge whole blood, quantitatively aliquot plasma samples, add a protein precipitant for protein precipitation, vortex, and centrifuge, take out the supernatant. The protein precipitant is one or more of methanol and acetonitrile. The ratio of the protein precipitant to plasma can be 3-6:1, preferably 4:1. Centrifuge the whole blood specimen at 4000 rpm for 5 min; the vortex time is 3 min, and the centrifugation conditions are: centrifuge at 12000 rpm for 10 min. The sample pretreatment includes: pipette 100 μL of plasma sample into an EP tube, and then add 400 μL of protein precipitant;

[0030] S2. Sample separation and analysis: Use a C 18 packed chromatographic column as the analytical column. The high-performance liquid chromatograph is equipped with an ultraviolet detector or a diode array DAD detector. Use a methanol and aqueous solution mixture as the mobile phase, keep the column temperature constant, and perform isocratic elution. The injection volume is 20 μL; Detect the peak area of posaconazole at a UV wavelength of 260 nm, and convert it into concentration using the standard curve equation. The chromatographic conditions are: chromatographic column Shim-pack GIST C 18 , 5 μm, 150 nm×4.6 nm; mobile phase: methanol: water = 71-29 (v / v); flow rate is 1 mL / min; diode array or ultraviolet detector at 260 nm; column temperature 40 °C; injection volume 20 μL; Determine the peak area of posaconazole, and substitute it into the standard curve equation to calculate the posaconazole concentration;

[0031] S3. Concentration calculation: Substitute the analysis results into the regression equation to calculate the posaconazole drug concentration. The linear range of posaconazole is 0.28 - 18.00 mg / L, and there is a good correlation between the peak area and the concentration calculation. Conduct linear range, precision, stability, recovery rate tests, and sample stability investigation; among them, the linear range test includes: Weigh an appropriate amount of posaconazole reference substance precisely, dissolve it with 50% methanol water and dilute to the scale to obtain the mother liquor of the posaconazole standard solution. Then take a certain amount of the mother liquor, dilute it step by step in half, take 20 μL of each diluted standard solution, transfer it to a 1.5 mL PE tube, add 80 μL of blank plasma to each, vortex and mix evenly to prepare calibration curve mother liquors with concentrations of 0.28, 0.56, 1.13, 2.25, 4.50, 9.00, and 18.00 μg / mL.

[0032] The precision, stability, and recovery rate experiments include: Weigh an appropriate amount of posaconazole reference substance precisely, dissolve it with 50% methanol water with one-fifth of its volume, and then dilute and fix the volume with four-fifths of the volume of blank plasma to prepare a series of standard plasma samples containing posaconazole with concentrations of 1.13, 2.25, and 4.50 μg / mL; Take 100 μL of each drug-containing serum and operate according to the sample pretreatment method; Calculate its measured concentration according to the linear regression equation. The concentration measured by using the mobile phase to replace the blank plasma to prepare the above-mentioned samples with each concentration is the absolute concentration. Calculate the measured average value and relative standard deviation RSD for each concentration. The recovery rate formula is as follows:

[0033] The sample stability investigation includes: Take standard concentration blood samples and place them at room temperature for 12 h respectively; Place them at 2 - 8 °C for 7 days; Store them frozen at -20 °C for 30 days; Store them frozen at -80 °C for 6 months; Store them frozen at -80 °C for 6 months and thaw and refreeze them 6 times; Take 100 μL of each drug-containing serum and operate according to the sample pretreatment method to investigate the stability of posaconazole.

[0034] The application of the method for simultaneously determining posaconazole in the in vivo drug analysis and determination in this embodiment.

[0035] As an application of this embodiment:

[0036] Compared with the chromatographic method in the prior art in this embodiment, the mobile phase used is relatively single, which saves the mobile phase preparation time. At the same time, it also minimizes the fluctuations in the detection results caused by the mobile phase preparation to the greatest extent, further enhancing the stability of the method; At the same time, compared with other liquid chromatography methods, the detection method of the present invention has the advantages of short single injection time, simple reagent preparation, and no interference from endogenous and common exogenous substances around the posaconazole target peak, improving the detection efficiency and the accuracy of the detection results.

[0037] Example 2

[0038] In view of the drawbacks of the existing posaconazole blood concentration detection technology, the present invention provides a method for rapidly determining the posaconazole blood concentration and its application. The present invention is described in detail below with reference to the accompanying drawings.

[0039] The materials include: posaconazole standard (batch number: content ≥ 98%); acetonitrile and methanol are Aladdin reagent (chromatographic grade); and water is secondary purified water.

[0040] The instruments include: Shimadzu LC-20A high performance liquid chromatograph, equipped with UV detector and autosampler; Shim-pack GIST C 18 Chromatographic column, specification: 4.6×150mm, 5μm.

[0041] Chromatographic conditions included: mobile phase: methanol-water = 71:29 (v / v); UV detection wavelength: 260 nm; column oven temperature: 40°C; mobile phase flow rate: 1.0 mL min -1 ; Injection volume: 20μL.

[0042] 1. Establish a standard curve for determining the concentration of posaconazole in human plasma;

[0043] 1.1. Reagent preparation;

[0044] Prepare standard curve stock solution: accurately weigh an appropriate amount of posaconazole reference substance, add 50% methanol water, dissolve and dilute to 10mL. Prepare a posaconazole reference substance solution with a concentration of 360μg / mL. Then take an appropriate amount of reference substance solution and dilute it in half with 50% methanol water step by step to obtain a series of posaconazole standard solutions with concentrations of 180.00, 90.00, 45.00, 22.50, 11.25, 5.63, 2.82, and 1.41μg / ml, and store them in a -80℃ low-temperature freezer for later use.

[0045] Preparation of quality control stock solution: Accurately weigh an appropriate amount of posaconazole reference substance, add 50% methanol water, dissolve and dilute to 100 mL to prepare a series of posaconazole concentration quality control solutions of 22.5, 11.25, and 5.65 μg / mL.

[0046] Prepare standard curve solution: accurately pipette 20 μL of standard curve stock solution of each concentration, add 80 μL of blank plasma, vortex mix, and obtain a series of standard curve solutions with concentrations of 36.00, 18.00, 9.00, 4.50, 2.25, 1.13, 0.56, and 0.28 μg / mL.

[0047] Preparation of quality control solution: Accurately pipette 20 μL of quality control stock solution of each concentration, add 80 μL of blank plasma, and vortex to mix, thus obtaining a series of standard curve solutions with concentrations of 4.50, 2.25, and 1.13 μg / mL.

[0048] 1.2, Sample processing;

[0049] Processing of the standard curve solution and the quality control product solution: Take the standard curve solution and the quality control product solution, add 400 μL of the protein precipitant methanol, vortex for 3 min, and then centrifuge at 13,000 r·min -1 for 10 min to separate the supernatant. Pipette 20 μL of the supernatant for injection and detection analysis, and record the chromatogram and peak area.

[0050] In other embodiments of the present invention, the protein precipitant in the determination method can be selected from methanol and acetonitrile, and its dosage can be arbitrarily selected within the given value range, which basically does not affect the detection result.

[0051] 1.3, Determination of the maximum absorption wavelength;

[0052] Take the processed posaconazole quality control product and detect it under the chromatographic conditions of the present invention. At the peak time point of posaconazole, stop the pump and scan in the range of 200 - 500 nm to obtain the spectrogram (see Figure 1 ). It can be seen from the spectrogram scan that the maximum absorption wavelength of posaconazole is 260 nm. Therefore, the detection wavelength is determined to be 260 nm.

[0053] 1.4, Plotting the standard curve;

[0054] Take the processed posaconazole standard curve solution and detect it under the chromatographic conditions of the present invention. Inject it into the high performance liquid chromatograph for analysis respectively. Using the posaconazole concentration as the abscissa x (μg / mL) and the corresponding chromatographic peak area as the ordinate y, establish a linear regression equation and plot the standard curve. The regression equation of posaconazole is y = 9376.75x - 651.48 (R 2 = 0.9998, n = 8), indicating that within the range of 0.28 - 36.00 μg / mL of the posaconazole content, there is a very good linear relationship between the peak area and the concentration.

[0055] 2, Methodology verification;

[0056] The content of methodology verification includes: specificity, linear range, precision, reproducibility, stability, recovery rate, etc. Among them, the stability study includes: the stability of biological samples during long-term freezing, repeated freezing and thawing, the processing process, and the analysis process after sample preparation. In this example, freshly prepared standard solutions are used to investigate the stability of samples after being stored under certain storage conditions.

[0057] 2.1 Specificity investigation: Take blank plasma from 6 different sources, posaconazole reference solution, and posaconazole standard solution added to blank plasma respectively. Treat the samples according to the method under "Sample Treatment" and determine them according to the conditions under "Chromatographic Conditions". Record the chromatogram and peak area.

[0058] Acceptance criteria: In the obtained chromatogram, judge whether there are interfering peaks according to the retention time. The peak area of the interfering peak should be less than 5.0% of the target peak area. The resolution of the posaconazole target peak is greater than 2.0, and the theoretical plate number is greater than 3000.

[0059] Results No chromatographic peak consistent with the retention time of posaconazole was detected in the chromatogram of blank plasma. The peak areas of interfering peaks in the blank plasma matrix were all less than 2.25%. Moreover, the symmetry of the posaconazole chromatographic peak was good, the resolution between the target peak and other endogenous and exogenous substance peaks was far greater than 2, and the theoretical plate number was greater than 5000, indicating that the method of the present invention has good specificity (see Figure 2 , Figure 3 , Figure 4 ), and is applicable to the quantitative analysis of posaconazole blood drug concentration.

[0060] 2.2 Linear range: Prepare the standard curve solution and draw the standard curve according to the above method. Treat the samples according to the method under "Sample Treatment" and determine them according to the conditions under "Chromatographic Conditions". Record the chromatogram and peak area to obtain the linear range and calculate R 2 .

[0061] y = ax + b (y: peak area, a: slope, b: intercept)

[0062] Acceptance criteria: The correlation coefficient R 2 ≥0.999, and the standard curve should have at least 6 effective concentrations. The low value of the linear range should be less than 0.50 μg / mL, and the high value should be greater than 6.00 μg / mL to cover the common concentrations in clinical treatment and the recommended concentration range of posaconazole.

[0063] It can be seen from the results of Example 1 that the linear regression equation of posaconazole is y = 9376.75x - 651.48 (R 2 = 0.9998, n = 8), indicating that there is a very good linear relationship between the peak area and the concentration when the content of posaconazole is in the range of 0.28 - 36.00 μg / mL.

[0064] 2.3 Precision: Prepare high, medium, and low concentration sample solutions of posaconazole. Treat the samples according to the method under "Sample Treatment" and determine them according to the conditions under "Chromatographic Conditions". Inject the samples continuously 8 times at each concentration, record the peak area of posaconazole, and calculate the coefficient of variation %CV.

[0065] Acceptance criteria: The %CV for high, medium, and low concentrations should all be less than 5.0%.

[0066] Results: For the high, medium, and low concentration samples, the coefficient of variation %CV was 1.34, 0.72%, and 1.67% respectively, indicating that the precision of this method is very good. The results are shown in Table 1.

[0067] Table 1 Precision results (n = 8)

[0068]

[0069] 2.4 Repeatability: Prepare 6 portions each of posaconazole high, medium, and low concentration sample solutions, and process the samples respectively according to the method under "Sample Handling", and perform the determination according to the conditions under "Chromatographic Conditions". Record the peak area of posaconazole and calculate the coefficient of variation %CV.

[0070] Acceptance criteria: The %CV for high, medium, and low concentrations should all be less than 5.0%.

[0071] Results: For the high, medium, and low concentration samples, the coefficient of variation %CV was 2.22, 1.62%, and 1.79% respectively, indicating that the reproducibility of this method is good. The results are shown in Table 2.

[0072] Table 2 Reproducibility results (n = 6)

[0073]

[0074] 2.5 Stability: Prepare 6 portions each of posaconazole high, medium, and low concentration sample solutions, and take them as 1 portion, for a total of 5 portions. Place 1 portion at room temperature for 12 h to investigate the stability of plasma specimens at room temperature; place 1 portion in a refrigerator at 2 - 8 °C for 7 days to investigate the stability of plasma specimens refrigerated at 2 - 8 °C; place 1 portion in a freezer at -20 °C for 30 days to investigate the stability of freezer storage at -20 °C; place 1 portion in a freezer at -80 °C for 6 months to investigate the stability of freezer storage at -80 °C; subject 1 portion to 6 cycles of freezing and thawing at -80 °C to investigate the stability of repeated freezing and thawing. Process the samples according to the method under "Sample Handling", and perform the determination according to the conditions under "Chromatographic Conditions". Record the peak area of posaconazole and calculate the coefficient of variation %CV.

[0075] Acceptance criteria: Under each storage condition, the %CV for high, medium, and low concentrations should all be less than 5.0%.

[0076] Results: When placed at room temperature for 12 h, refrigerated in a refrigerator at 2 - 8 °C for 7 days, stored in a freezer at -20 °C for 30 days, stored in a freezer at -80 °C for 6 months, and subjected to 6 cycles of freezing and thawing at -80 °C, for the high, medium, and low concentration samples, the maximum value of the coefficient of variation %CV was only 2.52%, far less than the requirement of 1 / 4 Eta for high-standard in-house quality control of blood drug concentration determination (i.e., %CV ≤ 5%), indicating that the stability of posaconazole plasma specimens is very good under the above conditions.

[0077] Table 3 Stability Results (n = 6)

[0078]

[0079]

[0080] 2.6, Recovery Rate; Plasma samples of posaconazole with known low, medium, and high concentrations were selected, and posaconazole standards with a certain concentration were added respectively. Six replicates were prepared in parallel for each concentration. The method recovery rate was calculated. The concentrations of the above samples prepared with mobile phase instead of blank plasma were absolute concentrations (expressed by peak areas). The samples were processed according to the method under "Sample Handling", and determined according to the conditions under "Chromatographic Conditions". The peak areas of posaconazole were recorded, and the relative standard deviation RSD, method recovery rate, and absolute recovery rate were calculated.

[0081]

[0082] Acceptance Criteria: The method recovery rate is 85% - 115%, the absolute recovery rate ≥ 70%, and the relative standard deviation RSD is less than 10%.

[0083] Results: For low, medium, and high concentration specimens, the average method recovery rates were 100.45%, 99.49%, and 98.34% respectively, and the relative standard deviations were 2.53%, 1.95%, and 2.13% respectively; the average absolute recovery rates were 99.35%, 98.10%, and 97.35% respectively, and the relative standard deviations were 1.66%, 1.49%, and 1.22% respectively, indicating that both the method recovery rate and absolute recovery rate of the present invention are relatively high, and the differences between groups are small, fully meeting the requirements of analytical testing. The results are shown in Table 4 and Table 5.

[0084] Table 4 Method Recovery Rate Results (n = 6)

[0085]

[0086] Table 5 Absolute Recovery Rate Results (n = 6)

[0087]

[0088]

[0089] 3. Sample Detection and Study on Storage Stability of Clinical Plasma Specimens

[0090] 3.1. Sample Detection; Sample Pretreatment: Take clinical specimens to be tested, centrifuge at 4000 r·min -1 for 5 min, precisely pipette 100 μL of plasma into a 1.5 mL EP tube, add 400 μL of protein precipitant methanol, vortex for 3 min, and then centrifuge at 13000 r·min-1 Centrifuge for 10 min to separate the supernatant, and determine according to the conditions under "Chromatographic Conditions". Record the peak area of posaconazole, record the chromatogram and peak area, substitute the analysis results into the standard curve regression equation, and calculate the concentration of posaconazole in plasma.

[0091] In other embodiments of the present invention, the addition amounts of plasma and protein precipitant are halved, that is, 50 μL of plasma is taken and 200 μL of protein precipitant is added, which does not affect the detection results.

[0092] 3.2 Storage stability of clinical specimens: Take clinical specimens submitted for inspection, with concentrations covering low, medium, and high concentrations as much as possible. Centrifuge at 4000 r·min -1 for 5 min to separate plasma, accurately determine the initial posaconazole concentration, then divide it into 8 aliquots, 100 μL for each aliquot, and store them in a refrigerator at 2 - 8 °C, at room temperature of 10 - 20 °C, and at 30 °C respectively. Measure at 1, 4, 8, 24, 48, 72, 96, and 120 h. Calculate the ratio of the concentration at each time point to the initial concentration and the coefficient of variation %CV to evaluate the storage stability of clinical specimens.

[0093] Acceptance criteria: The absolute value of the bias from the initial concentration at each time point is less than or equal to 10%, that is, |bias| ≤ 10%, and the coefficient of variation %CV ≤ 10%

[0094] Results: When stored in a refrigerator at 2 - 8 °C, at room temperature of 10 - 20 °C, and at 30 °C, the bias from the initial concentration is less than 10%, and the coefficient of variation is less than 10%, indicating that the clinical posaconazole plasma specimens have good stability when stored under the above conditions. The results are shown in Table 6.

[0095] Table 6 Storage stability of clinical specimens

[0096]

[0097]

[0098] The above has generally described the present invention in detail, but based on the present invention, some modifications or improvements can be made, which are obvious to those of ordinary skill in the art. Therefore, modifications or improvements made without departing from the spirit of the present invention are within the protection scope of the present invention.

Claims

1. A method for rapidly determining the blood drug concentration of posaconazole, characterized in that: It includes the following steps: S1. Sample pretreatment: Centrifuge whole blood, quantitatively aliquot plasma samples, add a protein precipitant for protein precipitation, vortex, and centrifuge, then take out the supernatant; S2. Sample separation and analysis: Using a C 18 packed chromatographic column as the analytical column, a high-performance liquid chromatograph equipped with an ultraviolet detector or a diode array detector (DAD), using a methanol and aqueous solution mixture as the mobile phase, maintaining a constant column temperature, isocratic elution, with an injection volume of 20 μL; detecting the peak area of posaconazole at a UV wavelength of 260 nm and converting it into concentration using the standard curve equation; S3. Concentration calculation: Substitute the analysis results into the regression equation to calculate the posaconazole drug concentration. The linear range of posaconazole is 0.28 - 18.00 mg / L, and there is a good correlation between the peak area and the concentration calculation.

2. The method for rapidly determining the blood drug concentration of posaconazole according to claim 1, wherein: The protein precipitant is one or more of methanol and acetonitrile, and the ratio of the protein precipitant to plasma can be 3 - 6:1, preferably 4:

1.

3. A method for rapid determination of posaconazole blood drug concentration according to claim 1, characterized in that: The whole blood specimen is centrifuged at 4000 rpm for 5 min; the vortex time is 3 min, and the centrifugation condition is: centrifuged at 12000 rpm for 10 min.

4. The method for rapid determination of posaconazole blood drug concentration according to claim 1, characterized in that: The sample pretreatment includes: Pipette 100 μL of plasma sample into an EP tube, and then add 400 μL of protein precipitant.

5. A method for rapid determination of posaconazole blood drug concentration according to claim 1, characterized in that: The chromatographic conditions in S2 are as follows: chromatographic column Shim-pack GIST C 18 , 5 μm, 150 nm × 4.6 nm; mobile phase: methanol: water = 71 - 29 (v / v); flow rate is 1 mL / min; secondary array tube or ultraviolet detector at 260 nm; column temperature 40 °C; injection volume 20 μL; measure the peak area of posaconazole, and substitute it into the standard curve equation to calculate the concentration of posaconazole.

6. The method for rapidly determining the blood drug concentration of posaconazole according to claim 1, wherein: S3 includes: linear range, precision, stability, recovery test, and sample stability investigation; among them, the linear range test includes: Weigh an appropriate amount of posaconazole reference substance accurately, dissolve it with 50% methanol water and make up to the mark to obtain the mother liquor of the posaconazole standard solution. Then take a certain amount of the mother liquor and dilute it step by step in half. Take 20 μL of each diluted standard solution and transfer it to a 1.5 mL PE tube. Add 80 μL of blank plasma to each tube, vortex and mix evenly to prepare calibration curve mother liquors with concentrations of 0.28, 0.56, 1.13, 2.25, 4.50, 9.00, and 18.00 μg / mL.

7. The method for rapid determination of posaconazole blood drug concentration according to claim 6, wherein: The precision, stability, and recovery experiments include: Weigh an appropriate amount of posaconazole reference substance accurately, dissolve it with 50% methanol water with one-fifth of its volume, and then dilute and make up to the mark with four-fifths of the volume of blank plasma to prepare a series of standard plasma samples containing posaconazole at concentrations of 1.13, 2.25, and 4.50 μg / mL; Take 100 μL of each drug-containing serum and operate according to the sample pretreatment method; Calculate its measured concentration according to the linear regression equation. The concentration measured by using the mobile phase instead of blank plasma to prepare the above samples with each concentration is the absolute concentration. Calculate the measured average value and relative standard deviation RSD for each concentration. The recovery formula is as follows:

8. The method for rapidly determining the blood drug concentration of posaconazole according to claim 7, characterized in that: The sample stability investigation includes: Take standard concentration blood samples and place them at room temperature for 12 h; at 2 - 8°C for 7 days; stored frozen at -20°C for 30 days; stored frozen at -80°C for 6 months; stored frozen at -80°C for 6 months and thawed and refrozen 6 times; Operate according to the sample pretreatment method to investigate the stability of posaconazole.

9. Use of a method for simultaneously determining posaconazole according to any one of claims 1 - 8 in in vivo drug analysis and determination.

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