Mirtazapine content detection method based on cat plasma

Abstract

The invention relates to a method for detecting the content of mirtazapine based on cat plasma. The method comprises the following steps: S1, preparing cat blank plasma, collecting blood in a non-anesthetic state, filling the blood into a heparin sodium anticoagulation blood collection tube, centrifuging for 10 minutes at 4 DEG C and 4000 rpm, separating the plasma, freezing and storing in a low-temperature refrigerator, and storing at-20 DEG C; the cat blank plasma comprises blank mixed plasma and blank individual plasma; s2, preparing an internal standard working solution, a double-blank sample, a matrix effect sample and a recovery rate sample, and preparing an analyte standard substance (mirtazapine) and an internal standard (mirtazapine-D4); the invention relates to the technical field of mirtazapine content detection. The method adopts a mirtazapine internal standard method for determination, is suitable for determination of the mirtazapine content in the cat plasma with low mirtazapine content and small sampling amount, and the established LC-MS / MS content determination method of the analyte mirtazapine in the cat plasma is good in accuracy and high in precision; the method can be applied to plasma sample content determination of analyte mirtazapine in cat in-vivo pharmacokinetic research.

Description

Technical Field

[0001] The present invention relates to the technical field of the detection of the content of mirtazapine, and particularly relates to a method for detecting the content of mirtazapine based on cat plasma. Background Art

[0002] Mirtazapine is an oral drug belonging to the piracetam-azepine class of compounds and is also known as a tetracyclic antidepressant. It is usually used to treat human depression and other mood disorders. This drug mainly acts by increasing the levels of neurotransmitters in the brain, and neurotransmitters have been proven to help eliminate depressive feelings. Therefore, mirtazapine can relax and soothe the body. When studying mirtazapine, it is necessary to detect the content level of mirtazapine in plasma to evaluate the preparation situation, determine the drug blood concentration peak value, and the drug administration interval, etc.

[0003] In the clinical practice of cats, mirtazapine is often used to treat the reduced appetite of cats, which can stimulate cats to increase their appetite, reduce problems such as anorexia and weight loss caused by various diseases, and relieve nausea caused by chemotherapy, etc. This method has been widely accepted. Therefore, it is also necessary to detect the content of mirtazapine in cat plasma. Currently, most of the methods for determining the blood drug concentration of mirtazapine are determined after humans take mirtazapine tablets orally. Since the bioavailability of mirtazapine in humans and cats is very different, and some cats are not administered orally, the existing detection methods are difficult to accurately detect the content of mirtazapine in cat plasma. For this reason, the present invention proposes a method for detecting the content of mirtazapine based on cat plasma, which can accurately determine the content of mirtazapine in cat plasma. Summary of the Invention

[0004] Aiming at the deficiencies existing in the prior art, the purpose of the present invention is to provide a method for detecting the content of mirtazapine based on cat plasma, which has the effect of accurately determining the content of mirtazapine in cat plasma.

[0005] The above technical purpose of the present invention is achieved through the following technical solutions:

[0006] A method for detecting the content of mirtazapine based on cat plasma includes the following steps: S1. Prepare cat blank plasma. Collect blood in a non-anesthetized state and fill it into a heparin sodium anticoagulant blood collection tube. Centrifuge at 4°C and 4000 rpm for 10 min to separate the plasma, and store it frozen in a low-temperature refrigerator at -20°C. The cat blank plasma includes blank mixed plasma and blank individual plasma;

[0007] S2. Prepare an internal standard standard working solution, double blank samples, matrix effect samples, and recovery samples, and prepare an analyte standard (mirtazapine) and an internal standard (mirtazapine-D4);

[0008] S3. Pipette 100 μL of cat plasma sample into a 1.5 mL pipette, accurately add 10 μL of the internal standard working solution at a concentration of 100 ng / mL, vortex to mix evenly, add 900 μL of acetonitrile, shake for 5 min, centrifuge at 4 °C and 14,000 rpm for 10 min, take the supernatant, filter through a membrane, vortex to mix evenly, and inject 2 μL for determination by LC-MS / MS;

[0009] S4. Pipette 100 μL of double blank sample into a 1.5 mL pipette, accurately add 10 μL of the diluent at a concentration of 100 ng / mL, vortex to mix evenly, add 900 μL of acetonitrile, shake for 5 min, centrifuge at 4 °C and 14,000 rpm for 10 min, take the supernatant, filter through a membrane, vortex to mix evenly, and inject 2 μL for determination by LC-MS / MS;

[0010] S5. Respectively pipette 990 μL of matrix effect sample and recovery sample, accurately add 10 μL of the internal standard working solution at a concentration of 100 ng / mL, vortex to mix evenly, filter through a membrane, vortex to mix evenly, and inject 2 μL for determination by LC-MS / MS;

[0011] S6. Use the Aglient Mass Hunter Workstation Data Acquisition system software configured in the mass spectrometer for data acquisition and automatic integration;

[0012] S7. Calculation of the standard curve and sample concentration. Take the labeled concentration of the analyte mirtazapine in the calibration standard as the abscissa, and the ratio of the peak area of the analyte to the internal standard as the ordinate, perform linear regression using the least squares method, with a weight of 1 / X2, establish the standard curve and linear equation, and calculate the concentration of mirtazapine using the standard curve.

[0013] In a preferred example of the present invention, it can be further configured as follows: The specific method for preparing the internal standard working solution in step S2 is:

[0014] S21. Prepare the diluent. Take a certain amount of acetonitrile and water, and prepare it in a ratio of acetonitrile: water of 8:2, and mix evenly;

[0015] S22. Prepare the internal standard stock solution. Weigh a certain amount of mirtazapine-D4, dissolve it with methanol, and prepare an internal standard stock solution with a concentration of 1.0 mg / mL, and store it in a -20 °C refrigerator protected from light;

[0016] S23. Dilute the internal standard stock solution with the diluent to obtain an internal standard working solution with a concentration of 100.0 ng / mL;

[0017] The internal standard working solution needs to be freshly prepared and used for determination on the same day to ensure the stability of the internal standard working solution.

[0018] In a preferred embodiment, the present invention can be further configured as follows: The specific method for preparing the double blank sample in step S2 is as follows:

[0019] Take a certain amount of blank mixed plasma and add diluent to prepare a double blank sample.

[0020] In a preferred embodiment, the present invention can be further configured as follows: The specific method for preparing the matrix effect sample in step S2 is as follows:

[0021] S41. Respectively take 6 batches of cat blank individual plasma or mixed plasma cat plasma. Mix the cat blank plasma and acetonitrile in a ratio of 1:9, shake for 5 min, centrifuge at 14000 rpm for 10 min at 4°C, and take the supernatant to prepare a blank matrix solution;

[0022] S42. Prepare a quality control working solution. Accurately weigh a certain amount of mirtazapine, dissolve it in methanol, vortex and mix well, sonicate for 5 min, and prepare a quality control stock solution with a concentration of 1.0 mg / mL, and store it in a -20°C refrigerator;

[0023] Serial dilute the quality control stock solution with diluent to obtain a series of quality control working solutions with concentrations of 750.0 ng / mL, 500.0 ng / mL, 6.0 ng / mL, and 2.0 ng / mL respectively;

[0024] S43. Take 990 μL of the blank matrix solution, and add 10 μL of the low-concentration and high-concentration quality control working solutions respectively to obtain matrix effect samples with mirtazapine concentrations of 0.6 ng / mL and 75 ng / mL.

[0025] In a preferred embodiment, the present invention can be further configured as follows: The specific method for preparing the recovery sample in step S2 is as follows:

[0026] Take a certain amount of blank mixed plasma, prepare a blank matrix solution using step S41, and dilute the quality control working solution with the blank matrix solution to obtain recovery samples with mirtazapine concentrations of 0.6 ng / mL, 50 ng / mL, and 75 ng / mL.

[0027] In a preferred embodiment, the present invention can be further configured as follows: In step S6, for the cases where the retention time of the sample peak drifts resulting in non-integration or unreasonable integration, the integration of the residual determination blank matrix sample is unreasonable, and the analysis batch selective samples and distribution are unreasonable, manual integration is adopted.

[0028] In a preferred embodiment, the present invention can be further configured as follows: after step S7, the deviation (Deviation, %Dev) and precision (Coefficient of Variation, %CV) are calculated according to the following formula using the workstation software Aglient Mass Hunter Quantitative Analysis or Microsoft Excel 2003 or above to verify the calculation result of the mirtazapine concentration. The mean and standard deviation (SD) are calculated using Excel;

[0029]

[0030]

[0031] The accuracy of detecting the mirtazapine content in cat plasma is investigated by deviation.

[0032] In summary, the present invention includes at least one of the following beneficial technical effects:

[0033] A method for detecting the mirtazapine content based on cat plasma disclosed by the present invention uses the mirtazapine internal standard method for determination. It is applicable to the determination of the mirtazapine content in cat plasma with a low mirtazapine content and a small sampling volume. The established method for determining the content of the analyte mirtazapine in cat plasma by LC-MS / MS has good accuracy and high precision, and can be applied to the determination of the plasma sample content in the pharmacokinetic study of the analyte mirtazapine in cats. Description of the Drawings

[0034] Figure 1 is the flow chart of the present invention. Detailed Embodiments

[0035] In order to more clearly illustrate the overall concept of the present invention, the following will be described in detail by way of examples in conjunction with the drawings in the specification.

[0036] Example 1:

[0037] Refer to Figure 1 , a method for detecting the mirtazapine content based on cat plasma disclosed by the present invention includes the following steps:

[0038] S1. Prepare cat blank plasma. Collect blood in a non-anesthetized state and transfer it to a heparin sodium anticoagulant blood collection tube. Centrifuge at 4 °C and 4000 rpm for 10 min to separate the plasma, and store it frozen in a low-temperature refrigerator at -20 °C. The cat blank plasma includes blank mixed plasma and blank individual plasma.

[0039] S2. Prepare the internal standard standard working solution, double blank samples, matrix effect samples and recovery samples, and prepare the analyte standard (mirtazapine) and internal standard (mirtazapine-D4). Both mirtazapine and mirtazapine-D4 should be stored in a sealed and light-proof manner at 2-8°C. The purity of mirtazapine is 97.2%, and the purity of mirtazapine-D4 is 99.9%.

[0040] The specific method for preparing the internal standard standard working solution in step S2 is as follows:

[0041] S21. Prepare the diluent. Take a certain amount of acetonitrile and water, and prepare it according to the ratio of acetonitrile: water = 8:2, and mix well.

[0042] S22. Prepare the internal standard stock solution. Weigh a certain amount of mirtazapine-D4, dissolve it with methanol, and prepare an internal standard stock solution with a concentration of 1.0 mg / mL, and store it in a -20°C refrigerator in the dark.

[0043] S23. Dilute the internal standard stock solution with the diluent to obtain an internal standard standard working solution with a concentration of 100.0 ng / mL.

[0044] The internal standard standard working solution needs to be freshly prepared and used for determination on the same day to ensure the stability of the internal standard standard working solution.

[0045] The specific method for preparing the double blank samples in step S2 is as follows:

[0046] Take a certain amount of blank mixed plasma and add the diluent to prepare the double blank samples.

[0047] The specific method for preparing the matrix effect samples in step S2 is as follows:

[0048] S41. Take 6 batches of cat blank individual plasma or mixed plasma of cats. Mix the cat blank plasma and acetonitrile in a ratio of 1:9, shake for 5 min, centrifuge at 14000 rpm for 10 min at 4°C, and take the supernatant to prepare the blank matrix solution.

[0049] S42. Prepare the quality control working solution. Accurately weigh a certain amount of mirtazapine, dissolve it with methanol, vortex and mix well, sonicate for 5 min, and prepare a quality control stock solution with a concentration of 1.0 mg / mL, and store it in a -20°C refrigerator.

[0050] Serial dilute the quality control stock solution with the diluent to obtain a series of quality control working solutions with concentrations of 750.0 ng / mL, 500.0 ng / mL, 6.0 ng / mL and 2.0 ng / mL respectively.

[0051] S43. Take 990 μL of the blank matrix solution, and add 10 μL of the low-concentration and high-concentration quality control working solutions respectively to obtain matrix effect samples with mirtazapine concentrations of 0.6 ng / mL and 75 ng / mL.

[0052] The specific method for preparing the recovery rate sample in step S2 is as follows:

[0053] Take a certain amount of blank mixed plasma, prepare blank matrix solution using step S41, and dilute the quality control working solution with the blank matrix solution to obtain recovery rate samples with the mirtazapine concentrations of 0.6 ng / mL, 50 ng / mL, and 75 ng / mL.

[0054] S3. Pipette 100 μL of cat plasma sample into a 1.5 mL pipette, accurately add 10 μL of the internal standard working solution at 100 ng / mL, vortex and mix evenly, add 900 μL of acetonitrile, shake for 5 min, centrifuge at 4 °C and 14,000 rpm for 10 min, take the supernatant, filter through a membrane, vortex and mix evenly, and take 2 μL for injection and determination by LC-MS / MS.

[0055] S4. Pipette 100 μL of double blank sample into a 1.5 mL pipette, accurately add 10 μL of the dilution solution at 100 ng / mL, vortex and mix evenly, add 900 μL of acetonitrile, shake for 5 min, centrifuge at 4 °C and 14,000 rpm for 10 min, take the supernatant, filter through a membrane, vortex and mix evenly, and take 2 μL for injection and determination by LC-MS / MS.

[0056] S5. Respectively take 990 μL of matrix effect sample and recovery rate sample, accurately add 10 μL of the internal standard working solution at 100 ng / mL, vortex and mix evenly, filter through a membrane, vortex and mix evenly, and take 2 μL for injection and determination by LC-MS / MS.

[0057] The LC-MS / MS chromatographic conditions are as follows:

[0058] a) Ultra-high performance liquid chromatograph: Aglient-1290;

[0059] b) Chromatographic column: Aglient ZORBAX Eclipese Plus C18 (2.1×50 mm, 1.8 μm);

[0060] c) Mobile phase: A: 0.1% formic acid aqueous solution; B: 0.1% formic acid acetonitrile solution;

[0061] d) Flow rate: 0.3 mL / min;

[0062] e) Column temperature: 40 °C;

[0063] f) Injection volume: 2 μL.

[0064] The gradient data of the mobile phase elution table is shown in Table 1:

[0065] Table 1: Gradient of the mobile phase elution table

[0066]

[0067] The LC-MS / MS chromatographic conditions are as follows:

[0068] a) Mass spectrometer: Aglient-6470;

[0069] b) Ion source: electrospray ionization source;

[0070] c) Scanning mode: positive ion scanning;

[0071] d) Detection mode: multiple reaction monitoring;

[0072] e) Ion source temperature: 250 °C;

[0073] f) Desolvation temperature: 350 °C.

[0074] g) Nebulizing gas pressure: 30 psi;

[0075] h) Capillary voltage: 3500 V;

[0076] i) Dry gas flow rate: 7 L / min;

[0077] j) Sheath gas flow rate: 11 L / min;

[0078] k) Nozzle voltage: 0 V.

[0079] Table 2: Characteristic ion pairs, fragmentation voltages and collision energies of mirtazapine and internal standard

[0080]

[0081] S6. Data acquisition and automatic integration are performed using the Aglient Mass Hunter Workstation Data Acquisition system software configured in the mass spectrometer. The same integration method is used for each analysis batch.

[0082] In step S6, for cases where the retention time of the sample peak drifts resulting in non-integration or unreasonable integration, unreasonable integration of the residual determination blank matrix sample, and unreasonable selectivity and distribution of the analysis batch samples, manual integration is used. All cases of manual integration should be recorded and reported in the final report.

[0083] S7. Calculation of the standard curve and sample concentration. Using the labeled concentration of the analyte mirtazapine in the calibration standard as the abscissa and the ratio of the peak areas of the analyte to the internal standard as the ordinate, linear regression is performed using the least squares method with a weight of 1 / X2 to establish the standard curve and linear equation, and the concentration of mirtazapine is calculated using the standard curve. Calculated by the workstation Aglient Mass Hunter Quantitative Analysis.

[0084] Standard sample concentration unit: ng / mL

[0085] Regression equation of the standard curve: Y = b + aX

[0086] Among them, Y: peak area ratio, X: labeled concentration, a: slope, b: y-axis intercept.

[0087] After the said step S7, use the workstation software Aglient Mass Hunter Quantitative Analysis or Microsoft Excel 2003 or above to calculate the deviation (Deviation, %Dev) and precision (Coefficient of Variation, %CV) according to the following formula to verify the calculation result of the mirtazapine concentration. The mean (Mean) and standard deviation (SD) are calculated using Excel;

[0088] Deviation (%Dev) = (measured concentration - labeled concentration) / labeled concentration × 100%

[0089] Precision (%CV) = measured concentration SD / mean of measured concentration × 100%

[0090] The accuracy of the detection of mirtazapine content in cat plasma is investigated by deviation.

[0091] Example 2:

[0092] In order to prove the accuracy of the mirtazapine content detection method based on cat plasma described in Example 1, this example verifies this method.

[0093] 1) Use at least 6 batches of cat blank individual plasma samples from different donors as blank samples respectively. After processing according to the sample processing method, measure them under the selected UPLC-MS / MS conditions, and investigate the measurement data of interference peaks at the retention times of the analyte mirtazapine and the internal standard IS.

[0094] The results show that the peak area of the interference peak of the blank plasma at the retention time of the analyte mirtazapine is less than 20% of the corresponding peak area of the LLOQ, and the peak area of the interference peak at the retention time of the internal standard is less than 5% of the internal standard peak area of the LLOQ.

[0095] 2) In all batches of experiments, after measuring the upper limit of quantification standard sample, inject and measure the double blank plasma samples. The results of 6 batches of experiments show that the peak area of the analyte mirtazapine in the blank sample after ULOQ measurement is less than 20% of the corresponding peak area of the LLOQ, and the peak area of the internal standard does not exceed 5% of the corresponding peak area of the LLOQ.

[0096] 3) After the 8 calibration standard samples were processed, they were injected in ascending order of concentration. According to the correlation between the labeled concentration of the calibration standard and the ratio of the peak areas of the analyte mirtazapine and the internal standard, the least squares method with a weight of 1 / X2 was used for linear regression to obtain the standard curve and the linear equation. The results showed that the quantitative range of the analyte mirtazapine was 0.2 - 100 ng / mL, and the linear relationship of the analyte mirtazapine was good within the concentration range of 0.2 - 100 ng / mL. The standard curves of the analytical batch for method validation all met the following requirements:

[0097] a) Including the LLOQ, the deviation (%DEV) between the regression value and the theoretical value of all standard samples was between -9.55% and 10.74%, and none exceeded ±15% (LLOQ exceeded ±20%), meeting the requirements;

[0098] b) After excluding unqualified samples, the calibration line obtained by linear regression had at least 75% of the calibration standards, including at least

[0099] 6 valid concentrations with a deviation (%Dev), that is, the difference between the back-calculated value and the nominal value of the concentration should be within ±15% of the nominal value, and the deviation of the LLOQ must be within ±20%;

[0100] c) The correlation coefficient R2 of all standard curves was greater than 0.99.

[0101] 4) Quality control working solutions were used to prepare quality control samples at four concentrations of LLOQ, LQC, MQC, and HQC. The corresponding concentrations of the analyte mirtazapine were 0.2, 0.6, 50, and 75 ng / mL, respectively. Six samples were prepared in parallel for each concentration. The accuracy and precision of the verification measurements were carried out continuously for three days, and the results were as follows:

[0102] 41) For the analyte mirtazapine samples measured continuously for three days, the within-run accuracy (%Dev) of the LLOQ for each measurement was -0.55 - 12.95%, -7.30 - -1.05%, and -1.70 - 7.25% respectively, and the within-run precision for the three days was 4.63%, 2.37%, and 5.82% respectively; the between-run accuracy for the three measurements was -0.05%, and the precision was 6.09%.

[0103] 42) For the analyte mirtazapine samples measured continuously for three days, the within-run accuracy (%Dev) of the LQC for each measurement was -5.97 - 4.40%, -8.97 - 2.67%, and -3.37 - -2.83% respectively, and the within-run precision for the three days was -2.36%, -4.60%, and -1.58% respectively; the between-run accuracy for the three measurements was -2.85%, and the precision was 3.09%.

[0104] 43) The analyte mirtazapine samples were assayed continuously for three days. The within - batch accuracy (%Dev) of MQC for each assay was - 3.39 to - 1.84%, - 4.89 to - 1.95%, and - 6.07 to - 2.92% respectively, and the within - batch precision for the three days was - 2.21%, - 4.04%, and - 4.14% respectively; the between - batch accuracy for the three assays was - 3.50%, and the precision was 1.42%.

[0105] 44) The analyte mirtazapine samples were assayed continuously for three days. The within - batch accuracy (%Dev) of HQC for each assay was - 4.34 to - 2.10%, - 10.39 to - 6.88%, and - 4.32 to 8.02% respectively, and the within - batch precision for the three days was - 3.58%, - 8.98%, and - 1.31% respectively; the between - batch accuracy for the three assays was - 4.62%, and the precision was 4.64%.

[0106] For the accuracy of each single analytical batch (within - batch) and different analytical batches (between - batch) of the analyte mirtazapine, the deviation (%Dev) of the measured value of the lowest - quantitation - limit quality - control sample LLOQ was within ±20%, and the results of the (within - batch) precision and different analytical batches (between - batch) precision were less than 20%, meeting the requirements of the protocol; for the low, medium, and high - concentration quality - control samples of the analyte mirtazapine, the deviation (%Dev) of the measured values of the single - analytical - batch (within - batch) accuracy and different analytical batches (between - batch) accuracy was within ±15%, and the results of the (within - batch) precision and different analytical batches (between - batch) precision were less than 15%, meeting the requirements of the protocol.

[0107] The above data indicate that the method for determining the content of the analyte mirtazapine in cat plasma established in Example 1 has good accuracy and high precision.

[0108] Further verify the determination method in Example 1.

[0109] 5) Six batches of cat blank plasma from different donors were used to prepare blank matrix solutions. W - QC was diluted with the blank matrix solution to obtain high - concentration and low - concentration matrix - effect samples (ME) of the analyte mirtazapine. The peak areas of the analyte mirtazapine and the internal standard in the presence of the matrix were measured and compared with the corresponding peak areas of the matrix - free pure solution samples (PS) at the corresponding concentrations after treatment. The matrix factors (Matrix Factor, MF) of the analyte mirtazapine and the internal standard were calculated. Then, by dividing the matrix factor of the analyte mirtazapine by the matrix factor of the internal standard, the matrix factor normalized to the internal standard was obtained.

[0110] The measured data showed that the matrix effect factors of LOQ and HOQ were 94.11% and 95.40% respectively, indicating that the presence of the matrix had a weak quenching effect on the ionization of the analyte, and this effect had basically no difference for low concentration (LOQ) and high concentration (HOQ) of the analyte. The normalized matrix effect factors of LOQ and HOQ were 100.87% and 103.55% respectively, indicating that after correction by the internal standard matrix factor, the weak matrix effect had been almost completely eliminated. The deviations (%DEV) of the accuracy of the LOQ and HOQ matrix samples were between -5.38% and 4.27% and between -3.86 and 2.26% respectively, and the precision (%CV) of the corrected concentration of the matrix samples were 3.35% and 2.57% respectively, meeting the requirements of the relevant guiding principles that the accuracy of the matrix effect samples should be within ±15% of the labeled concentration and the precision (%CV) should not be greater than 15%.

[0111] 6) After processing the quality control samples at 3 concentrations, inject them for determination of the peak areas of mirtazapine and the internal standard, and compare with the corresponding peak areas determined by injecting the recovery samples (RE), and calculate the recoveries (%Recovery) of mirtazapine and the internal standard respectively.

[0112] The measured data showed that the average recoveries of the analyte mirtazapine in low, medium and high concentration QC samples were 100.55% - 104.60%, and the precision of the determination at each concentration was between 87.78% and 105.35%; the average recovery of the internal standard IS was 90.45% - 104.16%, and the precision was between 2.14% and 5.12%. The relative standard deviations of the recoveries of the analyte mirtazapine in low, medium and high concentration QC samples and the internal standard IS were all less than 15%, meeting the requirements.

[0113] 7) Dilute the cat plasma samples containing the analyte mirtazapine by 10-fold and 50-fold with blank cat plasma respectively, prepare 6 parallel samples for each dilution factor, process and then determine. The average accuracies of the 10-fold dilution and 50-fold dilution were -3.14% and -6.34% respectively, and the precisions were 3.71% and 3.98% respectively. The accuracy deviations of the results determined after diluting the samples by both dilution methods were within ±15%, and the precisions were less than 15%, proving that the results determined after diluting the samples by the two methods met the requirements.

[0114] 81) The low, medium and high concentration QC plasma samples of the analyte mirtazapine were placed at room temperature for 24 hours, processed and then determined. The accuracy deviations of the three concentration QC samples of the analyte mirtazapine were -2.02% - 2.83%, and the precisions were 1.02% - 2.00%. The accuracy deviations of the determination results of the 3 concentration QC plasma samples of the analyte mirtazapine were within ±15% after being placed at room temperature for 24h and processed, and the precisions were all less than 15%, indicating that the analyte mirtazapine was stable in cat plasma after being placed at room temperature for 24h.

[0115] 82) The low, medium, and high concentration QC plasma samples of the analyte mirtazapine were stored frozen at -80 °C. After three cycles of freeze-thaw treatment, they were processed and measured. The accuracy deviations of the three concentration QC samples of the analyte mirtazapine were -9.79% to -0.67%, and the precision was 0.33% to 3.57%. The accuracy deviations of the measured results of the QC plasma samples of the analyte mirtazapine at three concentrations after three cycles of freeze-thaw treatment at -80 °C were all within ±15%, and the precision was less than 15%. This indicates that after storage at -80 °C and three cycles of freeze-thaw treatment, the analyte mirtazapine is stable in cat plasma.

[0116] 83) The low, medium, and high concentration QC plasma samples of the analyte mirtazapine were stored frozen at -80 °C for a long term to investigate the stability under the condition of -80 °C storage for 22 days. The accuracy deviations of the three concentration QC samples of the analyte mirtazapine were -14.00% to -0.29%, and the precision was 1.28% to 4.87%. The accuracy deviations of the above measured results were all within ±15%, and the precision was less than 15%. This indicates that after storage at -80 °C, the analyte mirtazapine is stable in cat plasma for 22 days.

[0117] 84) The low, medium, and high concentration QC plasma samples of the analyte mirtazapine were stored in the sample tray of the auto-sampler at 8 °C for 24 h after being processed, and then injected and measured with freshly prepared calibration standards to investigate the stability of the samples after being placed at 8 °C for 24 h after sample treatment. The accuracy deviations of the three concentration QC samples of the analyte mirtazapine were -0.41% to 7.33%, and the precision was 1.54% to 2.52%. The accuracy deviations of the above measured results were all within ±15%, and the precision was less than 15%. This indicates that after pretreatment of the analyte mirtazapine in cat plasma, it is stable when stored in the sample tray of the auto-sampler at 8 °C for 24 h.

[0118] 85) The freshly prepared stock solution of the analyte mirtazapine and the internal standard stock solution were stored at -20 °C. After 31 days of storage, they were taken out separately and prepared into mirtazapine pure solution samples at two concentrations, low and high. The internal standard stock solution was diluted with a diluent to the same concentration as the internal standard working solution. The above low and high concentration samples were compared with the low and high concentration pure solution samples prepared from the standard stock solution and the internal standard stock solution on the same day in terms of peak area. Six samples were prepared in parallel for each concentration to investigate the long-term stability of the stock solution stored at -20 °C.

[0119] The average accuracy values of the low and high concentration samples of the analyte mirtazapine were -3.50% and 8.31% respectively, and the average accuracy values of the corresponding internal standard IS were -9.45% and -7.03%. For the 12 QC samples at the low and high two concentrations, the precision ranges of the measurements of the analyte mirtazapine and the internal standard samples were 0.94% to 6.11%.

[0120] After the mirtazapine and internal standard stock solutions were stored at -20°C for 31 days, the mean deviation of the peak area corresponding to each concentration was within ±15%. This indicates that the stock solutions of the analyte mirtazapine and the internal standard are stable when stored at -20°C for 31 days.

[0121] 9) Take a certain amount of freshly prepared standard stock solution and quality control stock solution, add an internal standard with the same concentration as the internal standard in the sample, and dilute it by a certain multiple in the same way so that the concentration of mirtazapine is in the range of 0.2 - 100 ng / mL. Prepare two concentrations, and prepare 6 samples for each concentration. Compare the measured peak areas of the low and high concentration samples diluted from the two prepared stock solutions. The DEV(%) range of the low and high concentration samples prepared from the two stock solutions is -0.09% - 1.70%. The concentrations of the two stock solutions are almost the same, indicating that the sample weighing for preparing the stock solution is accurate and the operation of preparing the solution is completely consistent, meeting the requirements.

[0122] 10) The extraction solutions of the LQC, MOQ, and HQC plasma samples of the analyte mirtazapine were measured immediately after being processed on the first day; then they were continuously stored in the sample tray of the autoinjector at 8°C and injected again for measurement after 24 h and 48 h. For the 18 QC samples of the analyte mirtazapine at low, medium, and high concentrations, the 54 measurement results obtained from 3 measurements, the accuracy deviation range of the back-calculated concentration is -6.17% - 4.33%, and the precision range is 0.77% - 3.37%. The accuracy deviations of the above measurement results are all within ±15%, and the precision is less than 15%. This indicates that after the same batch of QCs is processed and stored in the sample tray of the autoinjector at 8°C for 24 h and 48 h and then reinjected, their reproducibility meets the experimental requirements.

[0123] Based on the verification methods described in Example 2 above, the method for detecting the content of mirtazapine in cat plasma samples established in Example 1 is summarized as follows:

[0124] a) The quantitative range of the analyte mirtazapine in cat plasma is 0.2 - 100 ng / mL;

[0125] b) The lower limit of quantitation LLOQ and ULOQ are 0.2 ng / mL and 100 ng / mL respectively;

[0126] c) The low, medium, and high concentration QCs were measured continuously for 3 days, and the accuracy and precision within and between days both meet the requirements of the protocol;

[0127] d) The selectivity, residue, and recovery rate of the method all meet the requirements of the protocol;

[0128] e) The plasma samples containing the analyte mirtazapine were diluted by a factor of 10 and 50 times singly, and the measurement results meet the requirements of the protocol;

[0129] f) The presence of the matrix has a weak quenching effect on the ionization of the analyte mirtazapine. However, after normalization by the matrix effect factor, it has basically no impact on the determination results of the analyte mirtazapine in plasma.

[0130] g) The analyte mirtazapine in cat plasma was stored at room temperature for 24 h; repeatedly frozen and thawed 3 times after storage at -80 °C; stable after storage at -80 °C for 22 days.

[0131] h) After the plasma sample of the analyte mirtazapine was processed, it was stored in the sample tray of an 8 °C autosampler and was stable for 24 h.

[0132] i) The stock solutions of the analyte mirtazapine and the internal standard were stored at -20 °C and were stable for 31 days.

[0133] j) After the plasma sample was processed, it was immediately determined together with the calibration standards processed in the same batch. Then, it was continuously stored under the condition of 8 °C in the autosampler sample tray. After storage for 24 h and 48 h respectively, re-determination was carried out, and the reproducibility of the re-injection met the requirements.

[0134] Therefore, under the limited conditions of this validation, the established LC-MS / MS method for the determination of the analyte mirtazapine in cat plasma has good accuracy and high precision; it can be applied to the determination of the plasma sample content in the pharmacokinetic study of the analyte mirtazapine in cats.

[0135] The embodiments of this specific implementation manner are all preferred embodiments of the present invention. The protection scope of the present invention is not limited thereby. Therefore, all equivalent changes made according to the structure, shape, and principle of the present invention shall be covered within the protection scope of the present invention.

Claims

1. A method for detecting the content of mirtazapine based on cat plasma, characterized in that: It includes the following steps: S1. Prepare cat blank plasma. Collect blood in a non - anesthetized state and transfer it into a heparin - sodium anticoagulant blood collection tube. Centrifuge at 4°C and 4000 rpm for 10 min to separate the plasma, and store it frozen in a low - temperature refrigerator at - 20°C. The cat blank plasma includes blank mixed plasma and blank individual plasma; S2. Prepare internal standard standard working solution, double - blank samples, matrix effect samples and recovery samples, and prepare analyte standard (mirtazapine) and internal standard (mirtazapine - D4); S3. Pipette 100 μL of cat plasma sample into a 1.5 - mL pipette, accurately add 10 μL of 100 ng / mL internal standard standard working solution, vortex - mix, add 900 μL of acetonitrile, shake for 5 min, centrifuge at 4°C and 14000 rpm for 10 min, take the supernatant, filter through a membrane, vortex - mix, and inject 2 μL for determination by LC - MS / MS; S4. Pipette 100 μL of double - blank sample into a 1.5 - mL pipette, accurately add 10 μL of 100 ng / mL diluent, vortex - mix, add 900 μL of acetonitrile, shake for 5 min, centrifuge at 4°C and 14000 rpm for 10 min, take the supernatant, filter through a membrane, vortex - mix, and inject 2 μL for determination by LC - MS / MS; S5. Respectively pipette 990 μL of matrix effect samples and recovery samples, accurately add 10 μL of 100 ng / mL internal standard standard working solution, vortex - mix, filter through a membrane, vortex - mix, and inject 2 μL for determination by LC - MS / MS; S6. Use the Aglient Mass Hunter Workstation Data Acquisition system software configured in the mass spectrometer for data acquisition and automatic integration; S7. Calculate the standard curve and sample concentration. Take the labeled concentration of mirtazapine to be measured in the calibration standard as the abscissa, and the peak - area ratio of the analyte to the internal standard as the ordinate, perform linear regression using the least - squares method, with a weight of 1 / X2, establish the standard curve and linear equation, and calculate the concentration of mirtazapine using the standard curve.

2. The mirtazapine content detection method based on cat plasma according to claim 1, wherein: The specific method for preparing the internal standard standard working solution in step S2 is as follows: S21. Prepare the diluent. Take a certain amount of acetonitrile and water, and prepare it according to the ratio of acetonitrile: water = 8:2, and mix well; S22. Prepare the internal standard stock solution. Weigh a certain amount of mirtazapine - D4, dissolve it in methanol, and prepare an internal standard stock solution with a concentration of 1.0 mg / mL, and store it in the - 20°C refrigerator protected from light; S23. Dilute the internal standard stock solution with the diluent to obtain an internal standard standard working solution with a concentration of 100.0 ng / mL; The internal standard standard working solution needs to be freshly prepared and used for determination on the same day to ensure the stability of the internal standard standard working solution.

3. The mirtazapine content detection method based on cat plasma according to claim 2, characterized in that: The specific method for preparing the double - blank samples in step S2 is as follows: Take a certain amount of blank mixed plasma and add diluent to prepare double - blank samples.

4. The mirtazapine content detection method based on cat plasma according to claim 2, wherein: The specific method for preparing the matrix effect samples in step S2 is as follows: S41. Take 6 batches of blank cat plasma or pooled cat plasma respectively. Mix the blank cat plasma and acetonitrile in a ratio of 1:9, shake for 5 min, centrifuge at 14,000 rpm for 10 min under the condition of 4 °C, and take the supernatant to prepare the blank matrix solution; S42. Prepare the quality control working solution. Weigh a certain amount of mirtazapine accurately, dissolve it with methanol, vortex and mix evenly, ultrasonicate for 5 min, and prepare a quality control stock solution with a concentration of 1.0 mg / mL, and store it in a -20 °C refrigerator; Dilute the quality control stock solution with a series of diluents to obtain a series of quality control working solutions with concentrations of 750.0 ng / mL, 500.0 ng / mL, 6.0 ng / mL, and 2.0 ng / mL respectively; S43. Take 990 μL of the blank matrix solution, and add 10 μL of the low-concentration and high-concentration quality control working solutions respectively to obtain matrix effect samples with mirtazapine concentrations of 0.6 ng / mL and 75 ng / mL.

5. The mirtazapine content detection method based on cat plasma according to claim 4, wherein: The specific method for preparing the recovery samples in step S2 is as follows: Take a certain amount of blank pooled plasma, prepare the blank matrix solution using step S41, and dilute the quality control working solution with the blank matrix solution to obtain recovery samples with mirtazapine concentrations of 0.6 ng / mL, 50 ng / mL, and 75 ng / mL.

6. The mirtazapine content detection method based on cat plasma according to claim 1, wherein: In step S6, for the situations where the retention time of the sample peak drifts resulting in non-integration or unreasonable integration, the integration of the residual determination blank matrix sample is unreasonable, and the analysis batch selectivity samples and distributions are unreasonable, manual integration is adopted.

7. A method for detecting the content of mirtazapine based on cat plasma according to claim 1, characterized in that: After step S7, calculate the deviation (Deviation, %Dev) and precision (Coefficient of Variation, %CV) using the workstation software Aglient Mass Hunter Quantitative Analysis or Microsoft Excel 2003 or above according to the following formula to verify the calculation results of the mirtazapine concentration, and calculate the mean (Mean) and standard deviation (SD) using Excel; The accuracy of the mirtazapine content detection in cat plasma is investigated by deviation.

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