Detection method and verification method for content of cyclosporine in cat whole blood

Abstract

The invention relates to a method for detecting the content of cyclosporine in cat whole blood and a verification method thereof.The method comprises the following steps that S1, a blank biological matrix is collected, the blank biological matrix comprises cat mixed whole blood and cat individual whole blood, the cat whole blood of 1-3 years old is collected in a non-anesthetic state, and the cat whole blood is cryopreserved at the temperature of-80 DEG C after being evenly mixed; and S2, sucking 100 [mu] L of a blank biological matrix by using a transfer pipette, adding 10 [mu] L of an internal standard working solution (W-IS), carrying out vortex for 10 s, adding 900 [mu] L of methanol, carrying out oscillation for 2 min, then carrying out centrifugation for 10 min at a temperature of 4 DEG C under a state of 14000 rpm, taking a supernatant, passing the supernatant through a membrane, transferring the supernatant into a small sample introduction bottle, taking 5 [mu] L, and carrying out sample introduction determination by using a UPLC-MS / MS technology. The invention relates to the technical field of cyclosporine content detection. The ciclosporin-D internal standard method is adopted to measure the content of the ciclosporin, and the measuring method is good in accuracy and high in precision and can be applied to plasma sample content measurement of the ciclosporin in cat in-vivo pharmacokinetic research.

Description

Technical Field

[0001] The present invention relates to the technical field of detecting the content of cyclosporine, and particularly relates to a method for detecting the content of cyclosporine in feline whole blood and a verification method thereof. Background Art

[0002] Cyclosporine is an organic compound, which is applicable to preventing rejection reactions occurring in allogeneic kidney, liver, heart, bone marrow and other organs or tissues transplantation, and preventing and treating graft-versus-host disease occurring during bone marrow transplantation. At the same time, cyclosporine is also widely used in the treatment of immune-mediated diseases in cats, such as chronic allergic dermatitis.

[0003] In the medicinal research of cyclosporine, it is necessary to detect the content of cyclosporine in feline whole blood to study its pharmacokinetic properties. However, there are currently few patent literature reports on the detection methods for the content of cyclosporine in feline plasma. It is difficult for researchers to accurately detect the content of cyclosporine in feline whole blood. Therefore, developing a detection method for the content of cyclosporine in feline whole blood with good accuracy and high precision and its verification method is of great significance for the research of cyclosporine drugs in feline animals. Summary of the Invention

[0004] According to the deficiencies of the existing technology, the purpose of the present invention is to provide a method for detecting the content of cyclosporine in feline whole blood and a verification method thereof to solve the above technical problems.

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

[0006] A method for detecting the content of cyclosporine in feline whole blood, comprising the following steps:

[0007] S1. Collect blank biological matrices, where the blank biological matrices include feline pooled whole blood and feline individual whole blood. Collect feline whole blood from cats aged 1 - 3 years old in a non-anesthetized state, mix well and store at -80 °C;

[0008] S2. Pipette 100 μL of the blank biological matrix, add 10 μL of the internal standard standard working solution (W-IS), vortex for 10 s, add 900 μL of methanol, shake for 2 min, then centrifuge at 4 °C and 14,000 rpm for 10 min. Take the supernatant, filter it through a membrane and transfer it to an injection vial, and inject 5 μL for determination by UPLC-MS / MS technology;

[0009] S3. Pipette 100 μL of the double blank sample (Blank), add 10 μL of methanol, vortex for 10 s, add 900 μL of methanol, shake for 2 min, then centrifuge at 4 °C and 14,000 rpm for 10 min. Take the supernatant, filter it through a membrane and transfer it to an injection vial, and inject 5 μL for determination by UPLC-MS / MS technology;

[0010] S4. Take the zero - concentration cyclosporine sample (Blank IS ), the matrix effect reference pure solution (PS), the matrix effect sample (ME), and the recovery sample (Rs), respectively add 10 μL of the internal standard standard working solution with a concentration of 5000 ng / mL, vortex for 10 s, transfer to a vial after treatment and filtration, and take 5 μL for injection and determination by UPLC - MS / MS technology;

[0011] S5. Process the data of the mass spectrometer and calculate the concentration of cyclosporine in cat whole blood.

[0012] Furthermore: The preparation method of the internal standard standard working solution in step S2 is as follows:

[0013] Accurately weigh 10.53 mg of the internal standard (cyclosporine - D) standard product, dissolve it in methanol, make up the volume to 10 mL with a volumetric flask, and prepare a 1 mg / mL cyclosporine internal standard stock solution (Stock - IS), and store it in a - 20 °C refrigerator;

[0014] Take the cyclosporine internal standard stock solution, dilute it with methanol to obtain an internal standard standard working solution with a concentration of 5000 ng / mL.

[0015] Furthermore: The preparation method of the double - blank sample in step S3 is to add methanol to the cat's mixed whole blood.

[0016] Furthermore: The preparation method of the zero - concentration cyclosporine sample in step S4 is: take the cat's mixed whole blood and add the internal standard standard working solution and methanol to prepare it;

[0017] The preparation method of the matrix effect reference pure solution is: weigh 10.12 mg of the cyclosporine standard product, dissolve it in methanol, make up the volume to 10 mL with a volumetric flask, and prepare a 1 mg / mL cyclosporine quality control stock solution (Stock - QC), and store it in a - 20 °C refrigerator;

[0018] Take the cyclosporine quality control stock solution and serially dilute it with methanol to obtain a series of cyclosporine quality control working solutions (W - QC) with concentrations of 100 ng / mL, 250 ng / mL, 8000 ng / mL, and 16000 ng / mL;

[0019] Dilute the cyclosporine quality control working solution with methanol to obtain the matrix effect reference pure solutions with concentrations of LQC and HQC;

[0020] The preparation method of the matrix effect sample is:

[0021] Take 6 batches of whole blood from cat individuals, add methanol at a ratio of whole blood:methanol of 1:9, vortex and mix for 2 min, centrifuge at 4°C and 14,000 rpm for 10 min, take the supernatant, dry it, and dissolve the dried matter with 990 μL of methanol respectively to obtain blank matrix solution (BM);

[0022] Take 990 μL of blank matrix solution and add 10 μL of low- and high-concentration cyclosporine quality control working solutions to obtain matrix effect samples with concentrations of LQC and HQC;

[0023] The preparation method of the recovery samples is as follows:

[0024] Add 10 μL of low-, medium-, and high-concentration quality control working solutions to 3 batches of 990 μL of blank matrix solution respectively to obtain recovery samples of low, medium, and high concentrations of cyclosporine with concentrations of LQC, MQC, and HQC.

[0025] A verification method for a method for detecting the content of cyclosporine in cat whole blood, including the above detection method, the verification method includes accuracy and precision verification, matrix effect verification, stability verification, re-injection reproducibility verification, and dilution reliability verification;

[0026] Among them, the stability verification includes room temperature stability verification, freeze-thaw stability verification, long-term stability verification, post-treatment stability verification, and stock solution stability verification.

[0027] Furthermore: The method for accuracy and precision verification is as follows:

[0028] Take cyclosporine quality control working solution (W-QC) and add it to four batches of blank biological matrices to obtain quality control samples (QCS) with cyclosporine concentrations of 10.0 ng / mL (LLOQ), 25.0 ng / mL (LQC), 800.0 ng / mL (MQC), and 1600.0 ng / mL (HQC) respectively. The quality control samples are prepared immediately before use;

[0029] Detect the cyclosporine concentration in the quality control samples by the detection method and compare it with the actual concentration of the quality control samples to verify the correctness of the detection method.

[0030] Furthermore: The verification method for the matrix effect verification is as follows:

[0031] Prepare high- and low-concentration matrix effect samples of the analyte cyclosporine from 6 batches of whole blood from cat individuals from different donors, measure the peak areas of the analyte cyclosporine and the internal standard in the presence of the matrix after treatment, compare them with the corresponding peak areas of the matrix-free pure solution samples with the same concentration after treatment, calculate the matrix factors of the analyte cyclosporine and the internal standard, and then divide the matrix factor of the analyte cyclosporine by the matrix factor of the internal standard to calculate the matrix factors normalized by the internal standard respectively.

[0032] Further: The method for verifying room temperature stability is as follows: The cyclosporine quality control working solutions at L, M, and H concentrations are placed at room temperature for 24 hours, then processed and measured, and the accuracy and precision deviations of the cyclosporine quality control working solutions at the three concentrations are analyzed.

[0033] The method for verifying freeze-thaw stability is as follows: The cyclosporine quality control working solutions at L, M, and H concentrations are stored frozen at -80°C. After 3 cycles of repeated freezing and thawing, they are processed and measured, and the accuracy and precision deviations of the cyclosporine quality control working solutions at the three concentrations are analyzed.

[0034] The method for verifying long-term stability is as follows: The cyclosporine quality control working solutions at L, M, and H concentrations are stored frozen at -80°C for a long time, and the accuracy and precision deviations of the cyclosporine quality control working solutions at the three concentrations are analyzed on the 7th day and the 14th day.

[0035] The method for verifying post-treatment stability is as follows: The cyclosporine quality control working solutions at L, M, and H concentrations are stored in the sample tray of the auto-sampler at 8°C for 24 hours and then injected for measurement, and the accuracy and precision deviations of the cyclosporine quality control working solutions at the three concentrations are analyzed.

[0036] The method for verifying the stability of the stock solution is as follows: The freshly prepared cyclosporine quality control stock solution and the cyclosporine internal standard stock solution are stored at -20°C. After that, they are taken out after 21 days respectively and prepared into cyclosporine quality control working solutions with the same concentrations of LQC, MQC, and HQC. The cyclosporine internal standard stock solution is diluted with methanol to the same concentration as the internal standard standard working solution, and the peak areas are compared with the LQC and HQC samples prepared from the standard stock solution and the cyclosporine internal standard stock solution on the same day. 3 samples are prepared in parallel for each concentration to examine the room temperature and long-term stability of the stock solution. The standard stock solution is prepared by dissolving the cyclosporine standard in methanol.

[0037] Further: The method for verifying the reproducibility of re-injection is as follows:

[0038] The cyclosporine quality control working solutions at LQC and HQC concentrations are measured immediately after being processed on the first day, and then stored in the sample tray of the auto-sampler at 8°C for 24 hours, and then injected and measured with freshly prepared calibration standards and the cyclosporine quality control working solutions stored for 24 hours.

[0039] The preparation method of the calibration standard is as follows: Take the standard stock solution and serially dilute it with methanol to obtain standard working solutions with a series of concentrations; measure 90 μL of cat mixed whole blood and add 10 μL of standard working solutions with different concentrations to obtain calibration standards with a series of concentrations.

[0040] Further: The method for verifying dilution reliability is as follows:

[0041] Cat whole blood samples containing the analyte cyclosporine were diluted 10-fold with blank biological matrix, and 6 parallel samples were prepared for each dilution factor, and measured after treatment.

[0042] In summary, the present invention includes at least one of the following beneficial technical effects:

[0043] 1. The method for detecting the content of cyclosporine in cat whole blood of the present invention uses the cyclosporine-D internal standard method to determine the content of cyclosporine. The determination method has good accuracy and high precision, and can be applied to the determination of the content of plasma samples in the pharmacokinetic study of cyclosporine in cats.

[0044] 2. The verification method for the method for detecting the content of cyclosporine in cat whole blood of the present invention is used to verify the effectiveness and correctness of the method for detecting the content of cyclosporine in cat whole blood, so as to ensure that the detection method can accurately determine the content of the analyte cyclosporine in cat whole blood. Description of the Drawings

[0045] Figure 1 is a flowchart of a method for detecting the content of cyclosporine in cat whole blood of the present invention. Detailed Embodiments

[0046] In order to more clearly illustrate the overall concept of the present invention, the following is a detailed description by way of example in conjunction with the drawings of the specification.

[0047] Example 1:

[0048] Refer to Figure 1 , a method for detecting the content of cyclosporine in cat whole blood disclosed by the present invention, includes the following steps:

[0049] S1. Collect blank biological matrix, which includes cat mixed whole blood and cat individual whole blood. Collect the whole blood of cats aged 1 to 3 years under non-anesthetized state, mix well and store at -80 °C;

[0050] S2. Pipette 100 μL of blank biological matrix, add 10 μL of internal standard standard working solution (W-IS), vortex for 10 s, add 900 μL of methanol, shake for 2 min, then centrifuge at 4 °C and 14000 rpm for 10 min, take the supernatant, filter through a membrane and transfer it to an injection vial, and inject 5 μL for determination by UPLC-MS / MS technology.

[0051] The preparation method of the internal standard standard working solution in step S2 is as follows:

[0052] Accurately weigh 10.53 mg of the internal standard (cyclosporine-D) standard product, dissolve it in methanol, make up the volume to 10 mL with a volumetric flask, and prepare a 1 mg / mL cyclosporine internal standard stock solution (Stock-IS), and store it in a -20 °C refrigerator;

[0053] Take the cyclosporine internal standard stock solution and dilute it with methanol to obtain an internal standard standard working solution with a concentration of 5000 ng / mL.

[0054] S3. Pipette 100 μL of double blank sample (Blank), add 10 μL of methanol, vortex for 10 s, add 900 μL of methanol, shake for 2 min, then centrifuge at 4 °C and 14000 rpm for 10 min. Take the supernatant, filter it through a membrane and transfer it to an injection vial, and inject 5 μL for determination by UPLC-MS / MS technology.

[0055] The preparation method of the double blank sample in step S3 is to prepare it by adding methanol to the mixed whole blood of cats.

[0056] S4. Take the zero-concentration cyclosporine sample (Blank IS ), matrix effect reference pure solution (PS), matrix effect sample (ME) and recovery sample (Rs), and add 10 μL of the internal standard standard working solution with a concentration of 5000 ng / mL to each of them. Vortex for 10 s, transfer them to an injection vial after treatment and filtration through a membrane, and inject 5 μL for determination by UPLC-MS / MS technology.

[0057] The preparation method of the zero-concentration cyclosporine sample in step S4 is: prepare it by adding the internal standard standard working solution and methanol to the mixed whole blood of cats;

[0058] The preparation method of the matrix effect reference pure solution is: weigh 10.12 mg of cyclosporine standard product, dissolve it in methanol, and make up the volume to 10 mL with a volumetric flask to prepare a cyclosporine quality control stock solution (Stock-QC) with a concentration of 1 mg / mL, and store it in a -20 °C refrigerator;

[0059] Take the cyclosporine quality control stock solution and serially dilute it with methanol to obtain a series of cyclosporine quality control working solutions (W-QC) with concentrations of 100 ng / mL, 250 ng / mL, 8000 ng / mL and 16000 ng / mL;

[0060] Dilute the cyclosporine quality control working solution with methanol to obtain the matrix effect reference pure solution with concentrations of LQC and HQC;

[0061] The preparation method of the matrix effect sample is:

[0062] Take the whole blood of 6 batches of cat individuals, add methanol at a ratio of whole blood:methanol of 1:9, vortex and mix well for 2 min, centrifuge at 4 °C and 14000 rpm for 10 min, take the supernatant, dry it, and then dissolve the dried product with 990 μL of methanol respectively to obtain the blank matrix solution (BM);

[0063] Add 990 μL of blank matrix solution to 10 μL of low- and high-concentration cyclosporine quality control working solution to obtain matrix effect samples with concentrations of LQC and HQC.

[0064] The preparation method of the recovery samples is as follows:

[0065] Add 10 μL of low-, medium-, and high-concentration quality control working solution to 3 batches of 990 μL of blank matrix solution respectively to obtain low-, medium-, and high-concentration cyclosporine recovery samples with concentrations of LQC, MQC, and HQC.

[0066] S5. Data processing of the mass spectrometer to calculate the concentration of cyclosporine in cat whole blood.

[0067] The step S5 includes data acquisition and integration, calculation of the standard curve and sample concentration.

[0068] Data acquisition and integration: Use the Aglient Mass Hunter Workstation Data Acquisition system software configured in the mass spectrometer for data acquisition and automatic integration. 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 analysis batch selectivity samples and distributions, manual integration is used. Record all manual integration cases and report them in the final report.

[0069] Calculation of the standard curve and sample concentration: Use the labeled concentration of the analyte cyclosporine in the calibration standard as the abscissa and the peak area ratio of the analyte to the internal standard as the ordinate, and perform linear regression using the least squares method, with a weight of 1 / X 2 , establish the standard curve and linear equation, and calculate the concentration of cyclosporine using the standard curve. Calculated by the workstation Aglient Mass Hunter Quantitative Analysis.

[0070] Unit of standard sample concentration: ng / mL

[0071] Regression equation of the standard curve: Y = b + aX

[0072] Among them, Y: peak area ratio, X: labeled concentration, a: slope, b: y-axis intercept.

[0073] This example uses the cyclosporine-D internal standard method for the determination of cyclosporine content. The determination method has good accuracy and high precision, and can be applied to the determination of the plasma sample content in the pharmacokinetic study of cyclosporine in cats.

[0074] Example 2:

[0075] The present invention also discloses a verification method for the detection method of cyclosporine content in cat whole blood, which is used to verify the effectiveness and accuracy of the detection method in Example 1.

[0076] The verification method includes accuracy and precision verification, matrix effect verification, stability verification, re-injection reproducibility verification and dilution reliability verification;

[0077] Among them, the stability verification includes room temperature stability verification, freeze-thaw stability verification, long-term stability verification, post-treatment stability verification and stock solution stability verification.

[0078] The method for accuracy and precision verification is as follows:

[0079] Take the cyclosporine quality control working solution (W-QC) and add it to four batches of blank biological matrices to obtain quality control samples (QCS) with cyclosporine concentrations of 10.0 ng / mL (LLOQ), 25.0 ng / mL (LQC), 800.0 ng / mL (MQC) and 1600.0 ng / mL (HQC) respectively. The quality control samples are prepared immediately before use.

[0080] Detect the cyclosporine concentration in the quality control samples by the detection method and compare it with the actual concentration of the quality control samples to verify the correctness of the detection method.

[0081] The results of accuracy and precision verification are as follows:

[0082] For the analyte cyclosporine samples measured continuously for three days, the within-batch accuracy (%Dev) of each measurement at LLOQ was -19.67~2.20%, -12.85~-0.24% and -15.75~6.16% respectively, and the within-batch precision for the three days was 8.22%, 5.28% and 8.95% respectively; the between-batch accuracy for the three measurements was -5.15%, and the precision was 6.65%.

[0083] For the analyte cyclosporine samples measured continuously for three days, the within-batch accuracy (%Dev) of each measurement at LQC was -14.35~-5.22%, 4.64~9.74% and 4.12~14.44% respectively, and the within-batch precision for the three days was 3.77%, 1.76% and 3.51% respectively; the between-batch accuracy for the three measurements was 2.90%, and the precision was 8.34%.

[0084] For the analyte cyclosporine samples measured continuously for three days, the within-batch accuracy (%Dev) of each measurement at MQC was -11.38~-8.44%, -0.46~5.75% and -2.20~2.61% respectively, and the within-batch precision for the three days was 1.22%, 2.22% and 1.97% respectively; the between-batch accuracy for the three measurements was -2.87%, and the precision was 5.79%.

[0085] The analyte cyclosporine samples were measured continuously for three days. The within-run accuracy (%Dev) of the HQC for each measurement was -14.56 to -7.44%, -3.66 to 2.58%, and -4.27 to -0.02% respectively, and the within-run precision for the three days was 2.96%, 1.89%, and 1.63% respectively; the between-run accuracy for the three measurements was -3.78%, and the precision was 4.90%.

[0086] The above results indicate that "except for the LLOQ, the overall accuracy (%Dev) of the quality control samples at each concentration level should be within ±15% of the labeled value, and the accuracy of the LLOQ should be within ±20% of the labeled value. Except for the LLOQ, the precision (%CV) of the quality control samples at each concentration level should not exceed 15%, and the precision of the LLOQ should not exceed 20%", meeting the requirements of the relevant guiding principles.

[0087] The verification method for the matrix effect verification is as follows:

[0088] High-concentration and low-concentration matrix effect samples of analyte cyclosporine were prepared from the whole blood of 6 batches of cat individuals from different donors. After treatment, the peak areas of analyte cyclosporine 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 at the corresponding concentrations after treatment. The matrix factors of analyte cyclosporine and the internal standard were calculated, and then the matrix factors normalized by the internal standard were calculated by dividing the matrix factor of analyte cyclosporine by the matrix factor of the internal standard respectively.

[0089] The results show that different concentrations of analyte cyclosporine have a greater impact on the matrix factor. The average value of the normalized matrix factors measured for the 6 low-concentration samples was 127.96%, and the precision was 4.46%; the average value of the normalized matrix factors measured for the 6 high-concentration samples was 111.92%, and the precision was 4.19%.

[0090] The precision of the matrix effect determination results of analyte cyclosporine was less than 15%, meeting the requirements of the protocol.

[0091] The method for the room temperature stability verification is as follows: The cyclosporine quality control working solutions at L, M, and H concentrations were placed at room temperature for 24 hours, and after treatment, the accuracy and precision deviations of the cyclosporine quality control working solutions at the three concentrations were analyzed. The accuracy deviations of the three concentration quality control working solutions of analyte cyclosporine were -9.26 to 5.17%, and the precision was 0.21 to 3.77%. The accuracy deviations were all within ±15%, and the precision was less than 15%, indicating that analyte cyclosporine is stable in cat whole blood after being placed at room temperature for 24 hours.

[0092] The method for verifying the freeze-thaw stability is as follows: The cyclosporine quality control working solutions at L, M, and H concentrations are stored frozen at -80°C. After 3 cycles of repeated freezing and thawing, they are processed and measured, and the accuracy and precision deviations of the cyclosporine quality control working solutions at the three concentrations are analyzed. The accuracy deviations of the three concentration quality control working solutions of the analyte cyclosporine are -7.33 to 6.30%, and the precision is 3.64 to 5.95%. The accuracy deviations are all within ±15%, and the precision is less than 15%, indicating that after the cyclosporine quality control working solutions at L, M, and H concentrations are stored at -80°C and subjected to 3 cycles of repeated freezing and thawing, the analyte cyclosporine is stable in feline whole blood.

[0093] The method for verifying the long-term stability is as follows: The cyclosporine quality control working solutions at L, M, and H concentrations are stored frozen at -80°C for a long time, and the accuracy and precision deviations of the cyclosporine quality control working solutions at the three concentrations are analyzed on the 7th day and the 14th day. The accuracy deviations of the cyclosporine quality control working solutions at L, M, and H concentrations of the analyte cyclosporine are within ±15% after being stored frozen at -80°C for 7 days or 14 days and then processed and measured, and the precision is less than 15%, indicating that the analyte cyclosporine is stable in feline whole blood for 14 days.

[0094] The method for verifying the stability after pretreatment is as follows: The cyclosporine quality control working solutions at L, M, and H concentrations are stored in the sample tray of the autoinjector at 8°C for 24 h and then injected and measured, and the accuracy and precision deviations of the cyclosporine quality control working solutions at the three concentrations are analyzed. After the cyclosporine quality control working solutions at L, M, and H concentrations of cyclosporine are placed at 8°C for 24 h after pretreatment, the accuracy deviations of the measurement results are within ±15%, and the precision is less than 15%, indicating that the pretreated samples of feline whole blood of the analyte cyclosporine are stable when stored in the sample tray of the autoinjector at 8°C for 24 h.

[0095] The method for verifying the stability of the stock solution is as follows: The freshly prepared cyclosporine quality control stock solution and the cyclosporine internal standard stock solution are stored at -20°C. After that, they are taken out after 21 days respectively and prepared into cyclosporine quality control working solutions with the same concentrations of LQC, MQC, and HQC. The cyclosporine internal standard stock solution is diluted with methanol to the same concentration as the internal standard standard working solution, and the peak areas are compared with the LQC and HQC samples prepared from the standard stock solution and the cyclosporine internal standard stock solution on the same day. 3 samples are prepared in parallel for each concentration to examine the room temperature and long-term stability of the stock solution. The standard stock solution is prepared by dissolving the cyclosporine standard in methanol.

[0096] The accuracy deviation of the internal standard cyclosporine-D is -2.48 to -0.47%, and the precision is 1.37 to 2.63%. After the cyclosporine quality control stock solution is stored at -20°C for 21 days, the mean deviation of the corresponding peak areas for each concentration is less than ±15%. This indicates that the stock solution of the analyte cyclosporine is stable when stored at -20°C for 21 days.

[0097] The method for verifying the reproducibility of re - injection is as follows:

[0098] The cyclosporine quality control working solutions with concentrations of LQC and HQC are measured immediately after the first - day treatment, and then stored at 8 °C in the sample tray of the auto - sampler for 24 hours. Then, the freshly prepared calibration standard samples and the cyclosporine quality control working solutions stored for 24 hours are used for injection and measurement. The accuracy deviations of the measurement results are all within ±15%, and the precisions are all less than 15%. This shows that after storing at 8 °C in the sample tray of the auto - sampler for 24 hours and then re - injecting, the reproducibility meets the experimental requirements.

[0099] The preparation method of the calibration standard samples is as follows: Take the standard stock solution and serially dilute it with methanol to obtain standard working solutions with a series of concentrations; measure 90 μL of cat mixed whole blood and add 10 μL of standard working solutions with different concentrations to obtain calibration standard samples with a series of concentrations.

[0100] The method for verifying the dilution reliability is as follows:

[0101] The cat whole - blood samples containing the analyte cyclosporine are respectively diluted 10 - fold with blank biological matrix. Six parallel samples are prepared for each dilution factor and measured after treatment. The measurement results show that for the analyte cyclosporine, the accuracy deviations of a single 10 - fold dilution are - 0.43% respectively, and the precisions are 6.01% respectively. The accuracy deviations of the measurement results after diluting the samples by a single 10 - fold dilution method are all within ±15%, and the precisions are all less than 15%. This proves that the measurement results after diluting the samples by 10 - fold meet the requirements.

[0102] Therefore, the verification method proposed in this example proves that the UPLC - MS / MS method for determining cyclosporine in cat whole blood established in Example 1 can meet the requirements of relevant guiding principles and can accurately quantify the content of cyclosporine in cat whole - blood samples.

[0103] The embodiments of this specific implementation manner are all preferred embodiments of the present invention. Without limiting the protection scope of the present invention accordingly, therefore: All equivalent changes made according to the structure, shape, and principle of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for detecting the content of cyclosporine in whole cat blood, characterized in that: It includes the following steps: S1. Collect blank biological matrices, which include cat mixed whole blood and cat individual whole blood. Collect the whole blood of cats aged 1 - 3 years old in a non - anesthetized state, mix well and store at - 80°C; S2. Pipette 100 μL of blank biological matrix, add 10 μL of internal standard standard working solution (W - IS), vortex for 10 s, add 900 μL of methanol, shake for 2 min, then centrifuge at 4°C and 14000 rpm for 10 min. Take the supernatant, filter it and transfer it to an injection vial. Take 5 μL and inject it for determination by UPLC - MS / MS technology; S3. Pipette 100 μL of double - blank sample (Blank), add 10 μL of methanol, vortex for 10 s, add 900 μL of methanol, shake for 2 min, then centrifuge at 4°C and 14000 rpm for 10 min. Take the supernatant, filter it and transfer it to an injection vial. Take 5 μL and inject it for determination by UPLC - MS / MS technology; S4. Take the zero-concentration cyclosporine sample (Blank IS ), matrix effect reference pure solution (PS), matrix effect sample (ME), and recovery sample (Rs), respectively add 10 μL of the internal standard standard working solution with a concentration of 5000 ng / mL, vortex for 10 s, transfer to the injection vial after treatment and filtration, and take 5 μL for injection and determination by UPLC-MS / MS technology; S5. Process the data of the mass spectrometer and calculate the concentration of cyclosporine in cat whole blood.

2. The detection method of the cyclosporine content in the whole blood of cats according to claim 1, characterized in that: The preparation method of the internal standard standard working solution in step S2 is as follows: Accurately weigh 10.53 mg of internal standard (cyclosporine - D) standard product, dissolve it in methanol, make up the volume to 10 mL with a volumetric flask to prepare a 1 mg / mL cyclosporine internal standard stock solution (Stock - IS), and store it in a - 20°C refrigerator; Take the cyclosporine internal standard stock solution and dilute it with methanol to obtain an internal standard standard working solution with a concentration of 5000 ng / mL.

3. The detection method for the content of cyclosporine in the whole blood of cats according to claim 1, wherein: The preparation method of the double - blank sample in step S3 is to add methanol to cat mixed whole blood.

4. The detection method for the content of cyclosporine in the whole blood of cats according to claim 2, wherein: The preparation method of the zero - concentration cyclosporine sample in step S4 is: add internal standard standard working solution and methanol to cat mixed whole blood; The preparation method of the matrix effect reference pure solution is: weigh 10.12 mg of cyclosporine standard product and dissolve it in methanol, make up the volume to 10 mL with a volumetric flask to prepare a 1 mg / mL cyclosporine quality control stock solution (Stock - QC), and store it in a - 20°C refrigerator; Take the cyclosporine quality control stock solution and serially dilute it with methanol to obtain a series of cyclosporine quality control working solutions (W - QC) with concentrations of 100 ng / mL, 250 ng / mL, 8000 ng / mL and 16000 ng / mL; Dilute the cyclosporine quality control working solution with methanol to obtain matrix effect reference pure solutions with concentrations of LQC and HQC; The preparation method of the matrix effect sample is as follows: Take 6 batches of cat individual whole blood, add methanol at a ratio of whole blood:methanol of 1:9, vortex and mix well for 2 min, centrifuge at 4°C and 14000 rpm for 10 min, take the supernatant, dry it, and then dissolve the dried product with 990 μL of methanol respectively to obtain blank matrix solution (BM); Take 990 μL of blank matrix solution and add 10 μL of low - and high - concentration cyclosporine quality control working solutions to obtain matrix effect samples with concentrations of LQC and HQC; The preparation method of the recovery sample is: Add 10 μL of low, medium, and high concentration quality control working solutions to 3 batches of 990 μL blank matrix solutions respectively to obtain recovery samples of low, medium, and high concentrations of cyclosporine with concentrations of LQC, MQC, and HQC.

5. A verification method for a method for detecting the content of cyclosporine in whole cat blood, including the detection method according to any one of claims 1 to 4, characterized in that: The verification method includes accuracy and precision verification, matrix effect verification, stability verification, re-injection reproducibility verification, and dilution reliability verification; Among them, the stability verification includes room temperature stability verification, freeze-thaw stability verification, long-term stability verification, post-treatment stability verification, and stock solution stability verification.

6. The validation method of a method for detecting the content of cyclosporine in feline whole blood according to claim 5, characterized in that: The method for accuracy and precision verification is as follows: Take cyclosporine quality control working solution (W-QC) and add it to four batches of blank biological matrices to obtain quality control samples (QCS) with cyclosporine concentrations of 10.0 ng / mL (LLOQ), 25.0 ng / mL (LQC), 800.0 ng / mL (MQC), and 1600.0 ng / mL (HQC) respectively. The quality control samples are prepared before use. Detect the cyclosporine concentration in the quality control samples by the detection method and compare it with the actual concentration of the quality control samples to verify the correctness of the detection method.

7. A verification method for a method for detecting the content of cyclosporine in whole cat blood according to claim 5, characterized in that: The verification method for matrix effect verification is as follows: Prepare high and low concentration matrix effect samples of analyte cyclosporine from whole blood of 6 batches of cat individuals from different donors. After treatment, measure the peak areas of analyte cyclosporine and internal standard in the presence of matrix, compare them with the corresponding peak areas of the matrix-free pure solution samples at the corresponding concentrations after treatment, calculate the matrix factors of analyte cyclosporine and internal standard, and then divide the matrix factor of analyte cyclosporine by the matrix factor of internal standard to calculate the matrix factors normalized by internal standard respectively.

8. A verification method for a method for detecting the content of cyclosporine in whole cat blood according to claim 5, characterized in that: The method for room temperature stability verification is as follows: The cyclosporine quality control working solutions at L, M, and H concentrations are placed at room temperature for 24 hours, measured after treatment, and analyze the accuracy and precision deviations of the cyclosporine quality control working solutions at the three concentrations. The method for freeze-thaw stability verification is as follows: The cyclosporine quality control working solutions at L, M, and H concentrations are stored frozen at -80 °C. After 3 cycles of repeated freeze-thaw, measure after treatment and analyze the accuracy and precision deviations of the cyclosporine quality control working solutions at the three concentrations. The method for long-term stability verification is as follows: The cyclosporine quality control working solutions at L, M, and H concentrations are stored frozen at -80 °C for a long time, and analyze the accuracy and precision deviations of the cyclosporine quality control working solutions at the three concentrations on the 7th day and the 14th day. The method for post-treatment stability verification is as follows: The cyclosporine quality control working solutions at L, M, and H concentrations are stored in the auto-sampler sample tray at 8 °C for 24 h and then injected for measurement, and analyze the accuracy and precision deviations of the cyclosporine quality control working solutions at the three concentrations. The method for verifying the stability of the stock solution is as follows: The freshly prepared cyclosporine quality control stock solution and the cyclosporine internal standard stock solution are stored at -20°C, and then taken out after 21 days respectively, and prepared into cyclosporine quality control working solutions with the same concentrations of LQC, MQC, and HQC. The cyclosporine internal standard stock solution is diluted with methanol to the same concentration as the internal standard standard working solution, and the peak areas are compared with the LQC and HQC samples prepared from the standard stock solution and the cyclosporine internal standard stock solution on the same day. Three samples are prepared in parallel for each concentration to examine the room temperature and long-term stability of the stock solution. The standard stock solution is prepared by dissolving the cyclosporine standard in methanol.

9. The validation method of a method for detecting the content of cyclosporine in whole cat blood according to claim 8, characterized in that: The method for verifying the reproducibility of re-injection is as follows: The cyclosporine quality control working solutions with concentrations of LQC and HQC are measured immediately after the first-day treatment, and then stored in the sample tray of the autoinjector at 8°C for 24 hours, and then injected and measured with freshly prepared calibration standards and the cyclosporine quality control working solutions stored for 24 hours. The preparation method of the calibration standards is as follows: The standard stock solution is serially diluted with methanol to obtain standard working solutions with a series of concentrations; 90 μL of cat mixed whole blood is taken, and 10 μL of standard working solutions with different concentrations are added to obtain calibration standards with a series of concentrations.

10. The verification method of a method for detecting the content of cyclosporine in feline whole blood according to claim 5, characterized in that: The method for verifying the dilution reliability is as follows: The cat whole blood samples containing the analyte cyclosporine are respectively diluted 10-fold with blank biological matrix, and six parallel samples are prepared for each dilution factor and measured after treatment.

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