Glapirenn content detection method based on dog plasma

Abstract

The method comprises the following steps: S1, preparing blank dog plasma, selecting beagles of 2-4 years old, collecting blood in a non-anesthetic state, filling the blood into a heparin sodium anticoagulation blood collection tube, centrifuging for 10 minutes at the temperature of 4 DEG C and the speed of 4000 rpm, separating the plasma, and performing low-temperature cryopreservation; s2, preparing an internal standard working solution, a double-blank sample, a matrix effect reference pure solution sample, a matrix effect sample and a recovery rate sample, and preparing an analyte standard substance (glasspirenn) and an internal standard substance (trazodone); the invention relates to the technical field of detection of the content of glasspiren. According to the method, a trazodone internal standard method is adopted for determination, and the established content determination method for analyzing and determining the analyte glasspirenn in the dog plasma by using the ultra-high performance liquid chromatography-tandem mass spectrometer is good in accuracy and high in precision, and can be applied to plasma sample content determination in pharmacokinetic research of the analyte glasspirenn in a dog body.

Description

Technical Field

[0001] The present invention relates to the technical field of the detection of grapiprant content, and in particular to a method for detecting grapiprant content based on canine plasma. Background Art

[0002] Grapiprant is a non-steroidal anti-inflammatory drug mainly used for treating arthritis and osteoarthritis pain in dogs. It is a selective EP4 receptor antagonist that reduces pain and inflammatory responses by inhibiting the action of the inflammatory mediator PGE2. Grapiprant is generally approved for veterinary use after clinical trials and by the FDA. According to research results, it is relatively safe for dogs at the recommended dose.

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

[0004] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a method for detecting grapiprant content based on canine plasma to solve the above problems.

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

[0006] A method for detecting grapiprant content based on canine plasma, comprising the following steps:

[0007] S1. Prepare canine blank plasma. Select beagle dogs aged 2 - 4 years old, collect blood in a non-anesthetized state, 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 at a low temperature;

[0008] S2. Prepare internal standard standard working solution, double blank samples, matrix effect reference pure solution samples, matrix effect samples, and recovery samples, and prepare analyte standard (grapiprant) and internal standard (trazodone);

[0009] S3. Pipette 100 μL of canine blank plasma into a 1.5 mL pipette, accurately add 10 μL of 1000 ng / mL internal standard standard working solution, vortex and mix evenly, add 900 μL of acetonitrile, shake for 5 min, centrifuge at 14000 rpm for 10 min at 4°C, filter through a membrane, take 300 μL of the supernatant and add 300 μL of water, vortex and mix evenly, and analyze and determine by ultra-high performance liquid chromatography-tandem mass spectrometry;

[0010] S4. Take 100 μL of the double blank sample with a 1.5 mL pipette, accurately add 10 μL of the diluent, vortex and mix well, add 900 μL of acetonitrile, shake for 5 min, centrifuge at 14000 rpm for 10 min at 4 °C,

[0011] Take the supernatant through the membrane, vortex and mix well, and analyze and determine by ultra-high performance liquid chromatography-tandem mass spectrometry;

[0012] S5. Take 500 μL of the matrix effect recovery reference pure solution sample, matrix effect sample, and recovery sample, accurately add 5 μL of the 1000 ng / mL internal standard standard working solution, vortex and mix well, pass through the membrane, take 300 μL of the supernatant and add 300 μL of water, vortex and mix well, and analyze and determine by ultra-high performance liquid chromatography-tandem mass spectrometry;

[0013] S6. Data acquisition and integration, use the Aglient Mass Hunter Workstation Data Acquisition system software configured by the mass spectrometer for data acquisition and automatic integration;

[0014] S7. Calculation of the standard curve and sample concentration. Take the labeled concentration of the analyte grapiprant 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 the unknown sample using the standard curve.

[0015] In a preferred example of the present invention, it can be further configured that: the preparation method of the internal standard standard working solution in step S2 is:

[0016] S21. Prepare the diluent. Take a certain amount of acetonitrile and water, prepare it in a ratio of acetonitrile: water of 1:1, and mix well;

[0017] S22. Prepare the internal standard stock solution. Weigh a certain amount of the internal standard (trazodone), dissolve it with methanol, and prepare a stock solution with a concentration of 1.0 mg / mL, store it in the refrigerator at -20 °C in the dark for later use;

[0018] S23. Dilute the internal standard stock solution with the diluent to obtain an internal standard standard working solution with a concentration of 1000.0 ng / mL;

[0019] 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.

[0020] In a preferred example of the present invention, it can be further configured that: the specific method for preparing the double blank sample in step S2 is:

[0021] A certain amount of blank canine plasma was taken and diluted with a diluent to prepare a double blank sample.

[0022] In a preferred example of the present invention, it can be further configured that: the specific method for preparing the matrix effect reference pure solution sample in step S2 is as follows:

[0023] S41. Prepare a quality control stock solution. Weigh a certain amount of the reference substance of grapiprant accurately, dissolve it with methanol, vortex and mix well, and ultrasonicate for 5 min to prepare a grapiprant quality control stock solution with a concentration of 1.0 mg / mL, and store it in a -20°C refrigerator for later use;

[0024] S42. Prepare a quality control working solution. Dilute the quality control stock solution with a series of diluents to obtain a series of quality control working solutions with concentrations of 8000.0 ng / mL, 4000.0 ng / mL, 60.0 ng / mL, and 20.0 ng / mL respectively;

[0025] S43. Dilute the quality control working solution with a diluent to obtain matrix effect reference pure solution samples of grapiprant with concentrations of 6 ng / mL and 800 ng / mL.

[0026] In a preferred example of the present invention, it can be further configured that: the specific method for preparing the matrix effect sample in step S2 is as follows:

[0027] S51. Take 6 batches of blank canine plasma respectively, mix the blank canine 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;

[0028] S52. Take 495 μL of the blank matrix solution, add 5 μL of low-concentration and high-concentration quality control working solutions respectively to obtain matrix effect samples of grapiprant with concentrations of 6 ng / mL and 800 ng / mL.

[0029] In a preferred example of the present invention, it can be further configured that: the specific method for preparing the recovery sample in step S2 is as follows:

[0030] Take a certain amount of blank canine plasma, prepare a blank matrix solution using step S51, and dilute the quality control working solution with the blank matrix solution to obtain recovery samples of grapiprant with concentrations of 6 ng / mL, 400 ng / mL, and 800 ng / mL.

[0031] In a preferred example of the present invention, it can be further configured that: 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 selectivity samples and distribution are unreasonable, manual integration is adopted.

[0032] In a preferred example, the present invention can be further configured as follows: after step S7, use the workstation software Aglient Mass Hunter Quantitative Analysis or Microsoft Excel 2003 or above to calculate the deviation (%Dev) and precision (%CV) according to the following formula to verify the calculation result of the granisetron concentration. The mean and standard deviation (SD) are calculated using Excel;

[0033] Deviation (%Dev) = (measured concentration - labeled concentration) / labeled concentration × 100%

[0034] Precision (%CV) = measured concentration SD / mean of measured concentration × 100%

[0035] The accuracy of the granisetron content detection in dog plasma is investigated using the deviation.

[0036] In summary, the present invention includes at least one of the following beneficial technical effects:

[0037] A method for detecting the granisetron content based on dog plasma disclosed by the present invention uses the trazodone internal standard method for determination. The established content determination method for analyzing the analyte granisetron in dog plasma by ultra-high performance liquid chromatography-tandem mass spectrometry 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 granisetron in dogs. Description of the Drawings

[0038] Figure 1 is the flowchart of the present invention; Detailed Embodiments

[0039] In order to more clearly explain the overall concept of the present invention, the following will be described in detail by way of examples in combination with the drawings of the specification.

[0040] Example 1:

[0041] Refer to Figure 1 , a method for detecting the granisetron content based on dog plasma disclosed by the present invention includes the following steps:

[0042] S1. Prepare dog blank plasma. Select beagle dogs aged 2 - 4 years old, collect blood in a non-anesthetized state, put 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 at low temperature.

[0043] S2. Prepare the internal standard standard working solution, double blank samples, matrix effect reference pure solution samples, matrix effect samples and recovery samples, and prepare the analyte standard (rapacuronium) and internal standard (trazodone).

[0044] The preparation method of the internal standard standard working solution in step S2 is as follows:

[0045] S21. Prepare the diluent. Take a certain amount of acetonitrile and water, and prepare it in a ratio of acetonitrile: water of 1:1, and mix well;

[0046] S22. Prepare the internal standard stock solution. Weigh a certain amount of the internal standard (trazodone), dissolve it with methanol, and prepare a stock solution with a concentration of 1.0 mg / mL, and store it in the refrigerator at -20 °C in the dark for later use.

[0047] S23. Dilute the internal standard stock solution with the diluent to obtain an internal standard standard working solution with a concentration of 1000.0 ng / mL.

[0048] 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.

[0049] The specific method for preparing the double blank samples in step S2 is as follows:

[0050] Take a certain amount of canine blank plasma and add the diluent to prepare the double blank samples.

[0051] The specific method for preparing the matrix effect reference pure solution samples in step S2 is as follows:

[0052] S41. Prepare the quality control stock solution. Accurately weigh a certain amount of the rapacuronium reference substance, dissolve it with methanol, vortex and mix well, and ultrasonicate for 5 min to prepare a rapacuronium quality control stock solution with a concentration of 1.0 mg / mL, and store it in the refrigerator at -20 °C for later use.

[0053] S42. Prepare the quality control working solution. Serially dilute the quality control stock solution with the diluent to obtain a series of quality control working solutions with concentrations of 8000.0 ng / mL, 4000.0 ng / mL, 60.0 ng / mL and 20.0 ng / mL respectively;

[0054] S43. Dilute the quality control working solution with the diluent to obtain matrix effect reference pure solution samples with a rapacuronium concentration of 6 ng / mL and 800 ng / mL.

[0055] The specific method for preparing the matrix effect samples in step S2 is as follows:

[0056] S51. Take 6 batches of canine blank plasma respectively, mix them in a ratio of canine blank plasma and acetonitrile 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.

[0057] S52. Take 495 μL of blank matrix solution, and add 5 μL of low-concentration and high-concentration quality control working solutions respectively to obtain matrix effect samples with gepirone concentrations of 6 ng / mL and 800 ng / mL.

[0058] The specific method for preparing the recovery samples in step S2 is as follows:

[0059] Take a certain amount of blank dog plasma, prepare blank matrix solution using step S51, and dilute the quality control working solution with the blank matrix solution to obtain recovery samples with gepirone concentrations of 6 ng / mL, 400 ng / mL, and 800 ng / mL.

[0060] S3. Pipette 100 μL of blank dog plasma into a 1.5 mL pipette, accurately add 10 μL of 1000 ng / mL internal standard standard working solution, vortex for mixing, add 900 μL of acetonitrile, shake for 5 min, centrifuge at 14000 rpm for 10 min at 4 °C, filter through a membrane, take 300 μL of the supernatant, add 300 μL of water, vortex for mixing, and analyze and determine by ultra-high performance liquid chromatography-tandem mass spectrometry.

[0061] S4. Pipette 100 μL of double blank sample into a 1.5 mL pipette, accurately add 10 μL of diluent, vortex for mixing, add 900 μL of acetonitrile, shake for 5 min, centrifuge at 14000 rpm for 10 min at 4 °C,

[0062] Take the supernatant through the membrane, vortex for mixing, and analyze and determine by ultra-high performance liquid chromatography-tandem mass spectrometry.

[0063] S5. Take 500 μL of matrix effect recovery reference pure solution sample, matrix effect sample, and recovery sample, accurately add 5 μL of 1000 ng / mL internal standard standard working solution, vortex for mixing, filter through a membrane, take 300 μL of the supernatant, add 300 μL of water, vortex for mixing, and analyze and determine by ultra-high performance liquid chromatography-tandem mass spectrometry.

[0064] The chromatographic conditions of the ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) are as follows:

[0065] a) Ultra-high performance liquid chromatograph: Aglient-1290;

[0066] b) Chromatographic column: Aglient ZORBAX Eclipese Plus C18 (2.1×50 mm, 1.8 μm);

[0067] c) Mobile phase: A: 0.1% formic acid aqueous solution; B: 0.1% formic acid acetonitrile solution;

[0068] d) Flow rate: 0.3 mL / min;

[0069] e) Column temperature: 40 °C;

[0070] f) Injection volume: 2 μL.

[0071] The gradient data of the mobile phase elution table is shown in Table 1:

[0072] Table 1: Gradient of the mobile phase elution table

[0073]

[0074] The mass spectrometry conditions of the ultra-high performance liquid chromatography-tandem mass spectrometer (LC-MS / MS) are as follows:

[0075] a) Mass spectrometer: Aglient-6470;

[0076] b) Ion source: Electrospray ionization source;

[0077] c) Scanning mode: Positive ion scanning;

[0078] d) Detection mode: Multiple reaction monitoring;

[0079] e) Ion source temperature: 250 °C;

[0080] f) Desolvation temperature: 350 °C.

[0081] g) Nebulizing gas pressure: 30 psi;

[0082] h) Capillary voltage: 3500 V;

[0083] i) Dry gas flow rate: 7 L / min;

[0084] j) Sheath gas flow rate: 11 L / min;

[0085] k) Nozzle voltage: 0 V.

[0086] Table 2: Characteristic ion pairs, fragmentation voltages and collision energies of grapiprant and internal standard

[0087]

[0088] S6. Data acquisition and integration are carried out using the Aglient Mass Hunter Workstation Data Acquisition system software configured in the mass spectrometer for data acquisition and automatic integration.

[0089] In step S6, for the 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 adopted. All cases of manual integration should be recorded and reported in the final report.

[0090] S7. Calculation of the standard curve and sample concentration: Using the labeled concentration of the analyte grapiprant in the calibration standard as the abscissa and the peak area ratio of the analyte to the internal standard as the ordinate, linear regression is performed using the least squares method with a weight of 1 / X 2 , to establish the standard curve and linear equation, and calculate the concentration of the unknown sample using the standard curve. Calculated by the workstation Aglient MassHunter Quantitative Analysis.

[0091] Unit of the standard sample concentration: ng / mL

[0092] Regression equation of the standard curve: Y = b + aX

[0093] Where, Y: peak area ratio, X: labeled concentration, a: slope, b: y-axis intercept.

[0094] After step S7, calculate the deviation (Deviation,

[0095] %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 grapiprant concentration. The mean (Mean) and standard deviation (SD) are calculated using Excel.

[0096] Deviation (%Dev) = (measured concentration - labeled concentration) / labeled concentration × 100%

[0097] Precision (%CV) = measured concentration SD / mean of measured concentration × 100%

[0098] The accuracy of the detection of grapiprant content in dog plasma is investigated using deviation.

[0099] The peak area data is retained to the integer place, the peak area ratio data is retained to 4 decimal places after the decimal point, and the retention time data is retained to 2 decimal places after the decimal point. The unit of the measured sample concentration is ng / mL, and the data is retained to 2 decimal places after the decimal point. The accuracy, precision, matrix effect, recovery rate, and deviation data are expressed as percentages and retained to 2 decimal places after the decimal point. The correlation coefficient R of the standard curve 2, a and b take the values automatically given by the data system.

[0100] In this example, trazodone internal standard method was used for determination. The established content determination method for analyzing the analyte grapiprant in canine plasma by ultra-high performance liquid chromatography-tandem mass spectrometry has good accuracy and high precision, and can be applied to the determination of the plasma sample content of the analyte grapiprant in the pharmacokinetic study of dogs.

[0101] Example 2:

[0102] In order to prove the accuracy of the grapiprant content detection method based on canine plasma described in Example 1, this example verifies this method.

[0103] The verification contents include the selectivity, carry-over, calibration curve, accuracy and precision, matrix effects, recovery, dilution integrity, stability and re-injection reproducibility of the method.

[0104] 1) Selectivity

[0105] Using at least 6 batches of canine blank plasma samples from different donors as blank samples respectively, after being processed according to the sample processing method, they were determined under the selected UPLC-MS / MS conditions, and the measurement data of interference peaks at the retention times of the analyte grapiprant and the internal standard IS were investigated. The results showed that the peak area of the interference peak of the blank plasma at the retention time of the analyte grapiprant was 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 was less than 5% of the internal standard peak area of the LLOQ.

[0106] 2) Carry-over

[0107] In all batches of experiments, after determining the upper limit of quantification standard sample, double blank plasma samples were injected continuously for three times. The results of 6 batches of experiments showed that the maximum residual amount of gramipran detected in the blank sample after ULOQ determination was 18.30% of the LLOQ residual amount, less than the specification requirement of 20% of the corresponding peak area of the LLOQ, and the peak areas of the residual amounts of the second and third blank samples were smaller; the peak area at the internal standard was less than 1.00%, far lower than the specification requirement of 5% of the corresponding peak area of the LLOQ.

[0108] 3) Calibration Curve

[0109] After the treatment of 7 calibration standard samples, they were injected in sequence according to the concentration from low to high. According to the correlation between the labeled concentration of the calibration standard and the ratio of the peak areas of the analyte grapiprant and the internal standard, the least squares method was used with a weight of 1 / X 2 , and linear regression was performed to obtain the standard curve and the linear equation.

[0110] The results showed that the quantitative range of the analyte grapiprant was 2 - 1000 ng / mL, and the linear relationship of the analyte grapiprant was good within the concentration range of 2 - 1000 ng / mL. The standard curve sample data of the analytical batch for method validation all met the following requirements:

[0111] a) Including the LLOQ, the deviation (%DEV) between the regression value and the theoretical value of all standard samples was between -3.78% and 2.77%, and none exceeded ±15% (the LLOQ exceeded ±20%), meeting the requirements;

[0112] b) After excluding the unqualified samples, the deviation (%Dev) of at least 75% of the calibration standards in the calibration curve obtained by linear regression, including at least 6 valid concentrations, that is, the difference between the concentration regression value (Back - calculated Value) and the labeled value (Nominal Value) should be within ±15% of the labeled value, and the deviation of the LLOQ must be within ±20%;

[0113] c) The correlation coefficient R of all standard curves 2 was greater than 0.99.

[0114] 4) Accuracy and precision

[0115] 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 grapiprant were 2, 6, 400, and 800 ng / mL, and 6 samples were prepared in parallel for each concentration. The accuracy and precision of the verification determination were carried out continuously for three days, and the results were as follows:

[0116] a) For the analyte grapiprant samples determined continuously for three days, the within - batch accuracy (%DEV) of the LLOQ for each determination was -0.06 - 5.99%, -2.94 - 1.27%, and 0.80 - 4.07% respectively, and the within - batch precision for the three days was 2.29%, 1.53%, and 1.14% respectively; the between - batch accuracy for the three determinations was 1.27%, and the precision was 2.14%.

[0117] b) For the analyte granisetron samples measured continuously for three days, the within-run accuracy (% DEV) for each measurement of LQC was -3.39 to -1.16%, -5.11 to 1.06%, and -3.46 to 0.25% respectively, and the within-run precision for the three days was 0.85%, 2.21%, and 1.51% respectively; the between-run accuracy for the three measurements was -2.00%, and the precision was 1.56%.

[0118] c) For the analyte granisetron samples measured continuously for three days, the within-run accuracy (% DEV) for each measurement of MQC was -2.67 to 0.39%, -2.34 to 3.36%, and -0.26 to 3.00% respectively, and the within-run precision for the three days was 1.10%, 2.47%, and 1.10% respectively; the between-run accuracy for the three measurements was 0.06%, and the precision was 1.86%.

[0119] d) For the analyte granisetron samples measured continuously for three days, the within-run accuracy (% DEV) for each measurement of HQC was -4.15 to 0.39%, -0.83 to 2.37%, and -0.68 to 1.93% respectively, and the within-run precision for the three days was 1.72%, 1.16%, and 0.99% respectively; the between-run accuracy for the three measurements was 0.37%, and the precision was 1.52%.

[0120] For the accuracy within each single analytical batch (intra-batch) and between different analytical batches (inter-batch) of the analyte granisetron, the deviation (% Dev) of the measured values of the lowest quantitation limit control sample LLOQ was within ±20%, and the measured results of the (intra-batch) precision and between different analytical batches (inter-batch) precision were less than 20%, meeting the requirements of the protocol; for the low, medium, and high concentration control samples of the analyte granisetron, the deviation (% Dev) of the measured values of the accuracy within each single analytical batch (intra-batch) and between different analytical batches (inter-batch) was within ±15%, and the measured results of the (intra-batch) precision and between different analytical batches (inter-batch) precision were less than 15%, meeting the requirements of the protocol. The above data indicate that the established method for the determination of the content of the analyte granisetron in canine plasma in our company has good accuracy and high precision.

[0121] 5) Matrix effect

[0122] Six batches of canine blank plasma from different donors were used to prepare blank matrix solution. W-QC was diluted with the blank matrix solution to obtain high-concentration and low-concentration matrix effect samples (ME) of the analyte grapiprant. The peak areas of the analyte grapiprant 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 grapiprant and the internal standard were calculated. Then, the matrix factor of the internal standard was divided by the matrix factor of the analyte grapiprant to obtain the matrix factor normalized to the internal standard. The measurement data showed that the matrix effect factors of LQC and HQC were 98.97% and 94.05% respectively, and the matrix effect factors normalized to the internal standard were 101.46% and 93.78% respectively, indicating that the presence of the matrix had a certain quenching effect on the ionization of the analyte, and the quenching effect strengthened with the increase of the analyte concentration; through the regression calibration of the standard curve, the slight quenching effect of the matrix on the analyte had little impact on the measurement results. The deviation of accuracy (%DEV) of the LQC and HQC matrix samples was between -3.69% and 4.21% and between -9.07 and -1.69% respectively, and the precision of the corrected concentration of the matrix samples (%CV) was 2.79% and 3.10% 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%.

[0123] 6) Recovery

[0124] After the quality control samples at three concentrations were processed, the peak areas of grapiprant and the internal standard were measured by injection and compared with the corresponding peak areas measured by injection of the recovery samples (RE). The recoveries (%RE) of grapiprant and the internal standard were calculated respectively. The measurement data showed that the average recoveries of the low, medium, and high concentration QC samples of the analyte grapiprant were between 102.23% and 104.38%, and the measurement precision at each concentration was between 0.61% and 3.04%; the average recovery of the internal standard IS was between 101.32% and 105.55%, and the precision was between 1.46% and 2.84%.

[0125] The recoveries of the quality control samples of the analyte grapiprant at the low, medium, and high concentrations and the internal standard IS were between 115% and 85%, and the relative standard deviations were all less than 15%, meeting the requirements.

[0126] 7) Dilution reliability

[0127] Canine plasma samples containing the analyte grapiprant were diluted 10-fold and 50-fold with blank canine plasma respectively. Six parallel samples were prepared for each dilution factor. After treatment, the determination results were as follows: the average accuracies of the 10-fold dilution and 50-fold dilution were -11.02% and -9.30% respectively, and the precisions were 1.51% and 1.93% respectively. The accuracy deviations of the determination results after diluting the samples by the two dilution methods were within ±15%, and the precisions were less than 15%, indicating that the determination results after diluting the samples by the two methods met the requirements.

[0128] 8) Stability

[0129] a) Room temperature stability

[0130] QC plasma samples of the analyte grapiprant at low, medium and high concentrations were placed at room temperature for 24 hours, and after treatment, the determination was carried out. The accuracy deviations of the three concentration QC samples of the analyte grapiprant were -4.87% to 0.95%, and the precisions were 0.96% to 2.15%. The accuracy deviations of the determination results of the three concentration QC plasma samples of the analyte grapiprant after being placed at room temperature for 24 hours and treated were within ±15%, and the precisions were less than 15%, indicating that the analyte grapiprant was stable in canine plasma after being placed at room temperature for 24 hours.

[0131] b) Freeze-thaw stability

[0132] QC plasma samples of the analyte grapiprant at low, medium and high concentrations were stored frozen at -80°C and determined after 3 repeated freeze-thaw cycles. The range of accuracy deviations of the three concentration QC samples of the analyte grapiprant was -8.64% to -1.49%, and the precisions were 0.72% to 2.58%.

[0133] The accuracy deviations of the determination results of the three concentration QC plasma samples of the analyte grapiprant after being repeatedly freeze-thawed 3 times at -80°C and treated were within ±15%, and the precisions were less than 15%, indicating that the analyte grapiprant was stable in canine plasma after being stored at -80°C and subjected to 3 repeated freeze-thaw treatments.

[0134] c) Long-term stability

[0135] QC plasma samples of the analyte grapiprant at low, medium and high concentrations were stored frozen at -80°C for 21 days, and the stability under the -80°C storage condition was investigated. The range of accuracy deviations of the three concentration QC samples of the analyte grapiprant was 1.56% to 8.03%, and the precisions were 0.57% to 2.21%. The accuracy deviations of the above determination results were within ±15%, and the precisions were less than 15%, indicating that the analyte grapiprant was stable in canine plasma for 21 days after being stored at -80°C.

[0136] d) Stability after pretreatment

[0137] For the low, medium, and high concentration QC plasma samples of the analyte grapiprant, after treatment and storage at 8 °C in the autosampler sample tray for 24 h, freshly prepared calibration standards were injected for determination to investigate the stability of the samples after treatment when stored at 8 °C for 24 h. The accuracy deviation ranges of the three concentration QC samples of the analyte grapiprant were from -9.07% to -3.76%, and the precision was from 0.13% to 2.31%.

[0138] The accuracy deviations of the above measurement results were all within ±15%, and the precision was less than 15%, indicating that the analyte grapiprant was stable when stored at 8 °C in the autosampler sample tray for 24 h after pretreatment in canine plasma.

[0139] f) Stability of stock solutions

[0140] The freshly prepared stock solutions of the analyte grapiprant and the internal standard were stored at -20 °C. After 30 days of storage, they were taken out respectively and prepared into two grapiprant pure solution samples with low and high concentrations of 0.5 ng / mL and 5 ng / mL. The internal standard stock solution was diluted with the 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 solutions when stored at -20 °C.

[0141] The average accuracy values of the low and high concentration samples of the analyte grapiprant were -3.48% and -3.28% respectively, and the corresponding average accuracy values of the internal standard IS were -4.86% and -4.39%. For the 12 QC samples with low and high concentrations, the precision ranges for the determination of the analyte grapiprant and the internal standard samples were from 0.29% to 0.92%.

[0142] After the grapiprant and internal standard stock solutions were stored at -20 °C for 30 days, the mean deviation of the corresponding peak area for each concentration was less than ±15%. This indicates that the stock solutions of the analyte grapiprant and the internal standard were stable when stored at -20 °C for 30 days.

[0143] 9) Inspection of the accuracy of weighing and preparation of stock solutions

[0144] 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 manner to prepare pure solution samples of grapiprant with concentrations of 0.5 ng / mL and 5 ng / mL. Prepare 6 samples for each concentration, and compare the measured peak areas of the low- and high-concentration samples diluted from the two prepared stock solutions. The DEV(%) of the low-concentration samples prepared from the two stock solutions in the two measurements was -1.40% and -6.02%; the DEV(%) of the high-concentration samples was -2.86% and -7.02%. Among them, the accuracy deviation of the second time was relatively large, which might be caused by the operation. For verification, the working solution was re-prepared with the stock solution of the second time, and the results showed that the (%DEV) of the low- and high-concentration samples prepared from the two stock solutions was 1.03% and -2.26%. It shows that the sample weighing for preparing the stock solution is accurate and the operations are basically consistent, meeting the requirements.

[0145] 10) Re-injection reproducibility

[0146] Extract the LQC, MQC, and HQC plasma samples of the analyte grapiprant, and measure them immediately after the first-day treatment; then continue to store them at 8 °C in the sample tray of the autosampler, and re-inject and measure them after 24 h. For the 18 QC samples of the low, medium, and high concentrations of the analyte grapiprant, the 36 measurement results from 2 measurements, the accuracy deviation range of the back-calculated concentration was -3.46% to 4.47%, and the precision range was 1.40% to 1.44%.

[0147] The accuracy deviations of the above measurement results are all within ±15%, and the precisions are all less than 15%. It shows that after the same batch of QCs is processed and stored at 8 °C in the sample tray of the autosampler for 24 h and then re-injected, their reproducibilities all meet the experimental requirements.

[0148] Based on the above verification results, the quantitative method for grapiprant in beagle dog plasma samples established is summarized as follows:

[0149] 1. The quantitative range of the analyte grapiprant in dog plasma is 2 - 1000 ng / mL;

[0150] 2. The lower limit of quantitation LLOQ and upper limit of quantitation ULOQ are 2 ng / mL and 1000 ng / mL respectively;

[0151] 3. Measure the low, medium, and high concentrations of QCs for 3 consecutive days, and the accuracy and precision within the day and between days all meet the requirements of the protocol;

[0152] 4. The selectivity, residue, and recovery rate of the method all meet the requirements of the protocol;

[0153] 5. Dilute the plasma samples containing the analyte grapiprant by a factor of 10 and 50 times in a single time, and the measurement results meet the requirements of the protocol;

[0154] 6. There is no obvious matrix effect for the analyte grapiprant in plasma;

[0155] 7. The analyte grapiprant in canine plasma is stable when stored at room temperature for 24 h, repeatedly frozen and thawed 3 times after freezing at -80 °C, and stable when stored at -80 °C for 21 days;

[0156] 8. After the plasma sample of the analyte grapiprant is processed, it is stored in the sample tray of the autosampler at 8 °C and is stable for 24 h;

[0157] 9. The stock solutions of the analyte grapiprant and the internal standard are stable when stored at -20 °C for 30 days;

[0158] 10. After the plasma sample is processed, it is immediately assayed together with the calibration standards processed in the same batch, and then continues to be stored under the condition of 8 °C in the autosampler sample tray. After being stored for 24 h, it is re-assayed, and the reproducibility of the re-injection meets the requirements.

[0159] In summary, under the limited conditions of this validation, the established LC-MS / MS method for the determination of the content of the analyte grapiprant in beagle dog plasma has good accuracy and high precision, and can be applied to the determination of the plasma sample content in the bioequivalence study of the analyte grapiprant in beagle dogs.

[0160] The embodiments of this specific implementation manner are all preferred embodiments of the present invention, and do not limit 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 grapiprant in canine plasma, characterized in that: It includes the following steps: S1. Prepare canine blank plasma. Select beagle dogs aged 2 - 4 years old. Collect blood in a non - anesthetized state and transfer it into a heparin - sodium - anticoagulated blood collection tube. Centrifuge at 4℃ and 4000 rpm for 10 min to separate the plasma, and store it frozen at low temperature. S2. Prepare internal standard standard working solution, double - blank samples, matrix effect reference pure solution samples, matrix effect samples and recovery samples. Prepare analyte standard (rapacuronium) and internal standard (trazodone). S3. Pipette 100 μL of canine blank plasma into a 1.5 mL pipette, accurately add 10 μL of 1000 ng / mL internal standard standard working solution, vortex and mix well. Add 900 μL of acetonitrile, shake for 5 min, centrifuge at 14000 rpm for 10 min at 4℃, filter through a membrane. Take 300 μL of the supernatant and add 300 μL of water, vortex and mix well, and analyze and determine by ultra - performance liquid chromatography - tandem mass spectrometry. S4. Pipette 100 μL of double - blank samples into a 1.5 mL pipette, accurately add 10 μL of diluent, vortex and mix well. Add 900 μL of acetonitrile, shake for 5 min, centrifuge at 14000 rpm for 10 min at 4℃, take the supernatant and filter through a membrane, vortex and mix well, and analyze and determine by ultra - performance liquid chromatography - tandem mass spectrometry. S5. Pipette 500 μL of matrix effect recovery reference pure solution samples, matrix effect samples and recovery samples, accurately add 5 μL of 1000 ng / mL internal standard standard working solution, vortex and mix well, filter through a membrane. Take 300 μL of the supernatant and add 300 μL of water, vortex and mix well, and analyze and determine by ultra - performance liquid chromatography - tandem mass spectrometry. S6. Data collection and integration. Use the Aglient Mass Hunter Workstation Data Acquisition system software configured in the mass spectrometer for data collection and automatic integration. S7. Calculation of standard curve and sample concentration: Taking the labeled concentration of the analyte gepirone 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 / X 2 , establish a standard curve and a linear equation, and calculate the concentration of gepirone using the standard curve.

2. The method for detecting the content of galiprant in canine plasma according to claim 1, wherein: The preparation method of 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 = 1:1, and mix well. S22. Prepare the internal standard stock solution. Weigh a certain amount of internal standard (trazodone), dissolve it with methanol, and prepare a stock solution with a concentration of 1.0 mg / mL. Store it in a - 20℃ refrigerator protected from light for later use. S23. Dilute the internal standard stock solution with the diluent to obtain an internal standard standard working solution with a concentration of 1000.0 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 method for detecting the content of galiprant in canine plasma according to claim 2, wherein: The specific method for preparing double - blank samples in step S2 is as follows: Take a certain amount of canine blank plasma and add diluent to obtain double - blank samples.

4. The method for detecting the content of grapiprant in canine plasma according to claim 2, wherein: The specific method for preparing matrix effect reference pure solution samples in step S2 is as follows: S41. Prepare the quality control stock solution. Accurately weigh a certain amount of rapacuronium reference substance, dissolve it with methanol, vortex and mix well, and ultrasonicate for 5 min to prepare rapacuronium quality control stock solutions with concentrations of 1.0 mg / mL respectively. Store them in a - 20℃ refrigerator for later use. S42. Prepare the quality control working solution by serially diluting the quality control stock solution with a series of diluents to obtain a series of quality control working solutions with concentrations of 8000.0 ng / mL, 4000.0 ng / mL, 60.0 ng / mL, and 20.0 ng / mL, respectively. S43. Dilute the quality control working solution with a diluent to obtain matrix effect reference pure solution samples with concentrations of 6 ng / mL and 800 ng / mL of gepirone.

5. The method for detecting the content of galiprant in canine plasma according to claim 4, characterized in that: The specific method for preparing the matrix effect samples in step S2 is as follows: S51. Take 6 batches of canine blank plasma, mix it with acetonitrile at 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. S52. Take 495 μL of the blank matrix solution, add 5 μL of low-concentration and high-concentration quality control working solutions respectively to obtain matrix effect samples with concentrations of 6 ng / mL and 800 ng / mL of gepirone.

6. The method for detecting the content of grapiprant based on canine plasma according to claim 5, characterized in that: The specific method for preparing the recovery samples in step S2 is as follows: Take a certain amount of canine blank plasma, prepare the blank matrix solution using step S51, and dilute the quality control working solution with the blank matrix solution to obtain recovery samples with concentrations of 6 ng / mL, 400 ng / mL, and 800 ng / mL of gepirone.

7. A method for detecting the content of grapiprant based on canine plasma according to claim 1, characterized in that: 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 analysis batch selectivity samples and distributions, manual integration is used.

8. A method for detecting the content of galiprant in canine 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 gepirone concentration. The mean (Mean) and standard deviation (SD) are calculated using Excel. Deviation (%Dev) = (measured concentration - labeled concentration) / labeled concentration × 100% Precision (%CV) = measured concentration SD / measured concentration mean × 100% The accuracy of the gepirone content detection in canine plasma is investigated using the deviation.

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