Method for detecting concentration content of maropitant based on dog plasma

Abstract

The invention relates to a method for detecting the concentration content of maropitant based on canine plasma, which comprises the following steps: S1, preparing canine blank plasma, selecting 2-4-year-old beagle, 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, and cryopreserving at low temperature; 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 the concentration of maropitant in 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 has generally undergone clinical trials and been approved by the FDA for veterinary use. According to research results, it is relatively safe for dogs at the recommended dose.

[0003] In the pharmaceutical research of grapiprant, it is necessary to detect the content of grapiprant in canine plasma to study its pharmacokinetic properties. However, there are currently few patent literature reports on the detection methods for the content of grapiprant in the plasma of canine animals. It is difficult for researchers to accurately detect the content of grapiprant in canine plasma. Therefore, developing a detection method for the content of grapiprant 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] In view of the deficiencies of the existing technology, the purpose of the present invention is to provide a method for detecting the concentration of maropitant in 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 the concentration of maropitant in 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 4000 rpm for 10 min at 4°C to separate the plasma, and store it frozen at a low temperature;

[0008] S2. Prepare an internal standard working solution, double blank samples, matrix effect reference pure solution samples, matrix effect samples, and recovery samples, and prepare an analyte standard (grapiprant) and an 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 working solution, vortex and mix well, 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 well, and analyze and determine by ultra-high performance liquid chromatography-tandem mass spectrometry;

[0010] S4. Take 100 μL of the double blank sample in 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 under the condition of 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, use 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, perform linear regression 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 the unknown sample using the standard curve.

[0015] In a preferred example of the present invention, it can be further configured as: 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 dark at -20 °C in the refrigerator 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 as: the specific method for preparing the double blank sample in step S2 is:

[0021] A certain amount of canine blank plasma was taken and diluted with 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 ultrasonically treat for 5 min to prepare a grapiprant quality control stock solution with a concentration of 1.0 mg / mL respectively, and store it in a refrigerator at -20 °C for standby;

[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 diluent to obtain matrix effect reference pure solution samples with grapiprant 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 canine blank plasma respectively, mix the canine blank plasma and acetonitrile in a ratio of 1:9, shake for 5 min, centrifuge at 14000 rpm for 10 min under the condition of 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 the low-concentration and high-concentration quality control working solutions respectively to obtain matrix effect samples with grapiprant 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 canine blank plasma, prepare a blank matrix solution by using step S51, and dilute the quality control working solution with the blank matrix solution to obtain recovery samples with grapiprant 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 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 sample 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, the deviation (%Dev) and precision (%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 grapiprant 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 detection of grapiprant content in canine 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 concentration of maropitant in canine plasma disclosed by the present invention uses the trazodone internal standard method for determination. The established method for analyzing and determining 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. Description of the Drawings

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

[0039] In order to more clearly illustrate the overall concept of the present invention, the following is a detailed description by way of examples in combination with the drawings in the specification.

[0040] Example 1:

[0041] Referring to Figure 1 , a method for detecting the concentration of maropitant in canine plasma disclosed by the present invention includes the following steps:

[0042] S1. Prepare canine blank plasma. Select beagle dogs aged 2 to 4 years old, collect blood in a non-anesthetized state, place it in 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 internal standard standard working solution, double blank samples, matrix effect reference pure solution samples, matrix effect samples and recovery samples, and prepare analyte standards (grepiprant) and internal standards (trazodone).

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

[0045] S21. Prepare a 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 an 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 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 double blank samples in step S2 is as follows:

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

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

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

[0053] S42. Prepare quality control working solutions. Serial 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 grepiprant concentrations of 6 ng / mL and 800 ng / mL.

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

[0056] S51. Take 6 batches of canine blank plasma respectively, mix them with canine 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 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 canine blank 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 canine blank 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 to filter through a 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 voltage and collision energy of grapiprant and internal standard

[0087]

[0088] S6. Data acquisition and integration are performed 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 the 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) according to the following formula using the workstation software Aglient Mass Hunter Quantitative Analysis or Microsoft Excel 2003 or above to verify the calculation results of the grapiprant concentration, and calculate the mean (Mean) and standard deviation (SD) 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 the 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, values of a and b are automatically given by the data system.

[0100] In this example, trazodone internal standard method was used for determination. The established content determination method for 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] 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 processing according to the sample processing method, they were determined under the selected UPLC-MS / MS conditions, and the determination 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 continuously injected three times for determination. The results of 6 batches of experiments showed that the maximum residual amount of grapimuron 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 even 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 gepirone 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 gepirone was 2 - 1000 ng / mL, and the linear relationship of the analyte gepirone was good within the concentration range of 2 - 1000 ng / mL. The standard curve sample data of the analytical batch in the 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% (LLOQ exceeded ±20%), meeting the requirements;

[0112] b) After removing 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 gepirone 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 gepirone 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 grapiprant samples, measurements were carried out continuously for three days. The within - batch accuracy (%DEV) of the LQC for each measurement was -3.39 to -1.16%, -5.11 to 1.06%, and -3.46 to 0.25% respectively, and the within - batch precision for the three days was 0.85%, 2.21%, and 1.51% respectively; the between - batch accuracy for the three measurements was -2.00%, and the precision was 1.56%.

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

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

[0120] For the accuracy of each single analytical batch (within - batch) and different analytical batches (between - batch) of the analyte grapiprant, the deviation (%Dev) of the measured values of the lowest - quantitation - limit control sample LLOQ was within ±20%, and the measured 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 control samples of the analyte grapiprant, 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 measured results of the (within - batch) precision and different analytical batches (between - 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 grapiprant 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. The 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, by dividing the matrix factor of the analyte grapiprant by the matrix factor of the internal standard, the matrix factor normalized to the internal standard was obtained. 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 precision of the measurements 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 low, medium, and high concentration quality control samples of the analyte grapiprant 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 of the samples after dilution by the two dilution methods were both within ±15%, and the precisions were both less than 15%, indicating that the determination results of the samples after dilution by the two methods met the requirements.

[0128] 8) Stability

[0129] a) Room temperature stability

[0130] Low, medium, and high concentration QC plasma samples of the analyte grapiprant 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 24h were all within ±15%, and the precisions were all less than 15%, indicating that after being placed at room temperature for 24h, the analyte grapiprant was stable in canine plasma.

[0131] b) Freeze-thaw stability

[0132] Low, medium, and high concentration QC plasma samples of the analyte grapiprant were stored frozen at -80°C and determined after 3 cycles of freeze-thaw. The accuracy deviation range of the three concentration QC samples of the analyte grapiprant was -8.64% to -1.49%, and the precision was 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 3 cycles of freeze-thaw at -80°C were all within ±15%, and the precisions were all less than 15%, indicating that after storage at -80°C and 3 cycles of freeze-thaw treatment, the analyte grapiprant was stable in canine plasma.

[0134] c) Long-term stability

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

[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 and storage at 8 °C for 24 h. The accuracy deviation ranges of the three concentration QC samples of the analyte grapiprant were -9.07% to -3.76%, and the precision was 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 after pretreatment in dog plasma and storage at 8 °C in the autosampler sample tray for 24 h.

[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 separately 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 peak areas of 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. Six samples were prepared in parallel for each concentration to investigate the long-term stability of the stock solutions 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 average accuracy values of the corresponding internal standard IS were -4.86% and -4.39%. For the 12 QC samples with low and high concentrations, the precision ranges of the determinations of the analyte grapiprant and the internal standard samples were 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 areas 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 was accurate and the operations were 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 in the sample tray of the autoinjector at 8 °C, 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 obtained 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 were all within ±15%, and the precisions were all less than 15%. It shows that after the same batch of QCs was processed and stored in the sample tray of the autoinjector at 8 °C for 24 h and then re-injected, their reproducibilities all met 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 limits of quantification LLOQ and ULOQ are 2 ng / mL and 1000 ng / mL respectively;

[0151] 3. Measure the QCs of low, medium, and high concentrations 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 defined 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; it 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 shall be covered within the protection scope of the present invention.

Claims

1. A method for detecting the concentration of maropitant based on canine plasma, characterized in that: The following steps are involved: S1. Prepare canine blank plasma. Select beagles aged 2 to 4 years old, collect blood under non-anesthesia, put it into sodium heparin anticoagulation blood collection tubes, centrifuge at 4°C and 4000 rpm for 10 minutes, separate the plasma, and freeze it at low temperature; S2, prepare internal standard working solution, double blank samples, matrix effect reference pure solution samples, matrix effect samples and recovery samples, prepare analyte standards (glapiram) and internal standards (trazodone); S3, draw 100 μL of canine blank plasma into a 1.5 mL pipette, accurately add 10 μL of 1000 ng / mL internal standard working solution, vortex mix, add 900 μL of acetonitrile, shake for 5 min, centrifuge at 14000 rpm for 10 min at 4 °C, pass through a membrane, take 300 μL of the supernatant, add 300 μL of water, vortex mix, and analyze and determine by ultra-performance liquid chromatography-tandem mass spectrometry; S4. Take 100 μL of double blank sample in a 1.5 mL pipette, accurately add 10 μL of diluent, vortex mix, add 900 μL of acetonitrile, shake for 5 min, centrifuge at 14000 rpm for 10 min at 4 °C, take the supernatant and pass it through a membrane, vortex mix, and analyze by ultra-performance liquid chromatography-tandem mass spectrometry; S5. Take 500 μL of the matrix effect recovery reference pure solution sample, matrix effect sample, and recovery sample, accurately add 5 μL of 1000 ng / mL internal standard working solution, vortex mix, pass the membrane, take 300 μL of the supernatant, add 300 μL of water, vortex mix, and analyze by ultra performance liquid chromatography-tandem mass spectrometry; S6, data acquisition and integration, using the Aglient Mass Hunter Workstation DataAcquisition system software configured for the mass spectrometer for data acquisition and automatic integration; S7. Calculation of standard curve and sample concentration, with the labeled concentration of analyte glapiram in the calibration standard as the horizontal axis and the peak area ratio of the analyte to the internal standard as the vertical axis, the least squares method was used for linear regression with a weight of 1 / X 2 , establish a standard curve and linear equation, and use the standard curve to calculate the concentration of glapiram.

2. The method for detecting the concentration of maropitant based on dog plasma according to claim 1, characterized in that: The preparation method of the internal standard working solution in step S2 is: S21, prepare a diluent, take a certain amount of acetonitrile and water, prepare acetonitrile: water in a ratio of 1:1, and mix well; S22, prepare an internal standard stock solution, weigh a certain amount of internal standard (trazodone), dissolve it in methanol, prepare a stock solution with a concentration of 1.0 mg / mL, and store it in a -20°C refrigerator away from light for later use; S23, diluting the internal standard stock solution with a diluent to obtain an internal standard working solution with a concentration of 1000.0 ng / mL; The internal standard working solution needs to be freshly prepared and used in the measurement on the same day to ensure the stability of the internal standard working solution.

3. The method for detecting the concentration of maropitant based on dog plasma according to claim 2, characterized in that: The specific method for preparing the double blank sample in step S2 is: A certain amount of canine blank plasma was taken and added with diluent to prepare double blank samples.

4. The method for detecting the concentration of maropitant based on dog plasma according to claim 2, characterized in that: The specific method for preparing the matrix effect reference pure solution sample in step S2 is: S41, prepare quality control stock solution, accurately weigh a certain amount of glapiram reference substance, dissolve it in methanol, vortex mix, sonicate for 5 minutes, prepare glapiram quality control stock solution with a concentration of 1.0 mg / mL, and store it in a -20°C refrigerator for future use; S42, prepare quality control working solution, and serially dilute the quality control stock solution with 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; S43. Dilute the quality control working solution with diluent to obtain matrix effect reference pure solution samples with glapiren concentrations of 6 ng / mL and 800 ng / mL.

5. The method for detecting the concentration of maropitant based on dog plasma according to claim 4, characterized in that: The specific method for preparing the matrix effect sample in step S2 is: S51, respectively taking 6 batches of canine blank plasma, mixing the canine blank plasma with acetonitrile in a ratio of 1:9, shaking for 5 minutes, centrifuging at 14000 rpm for 10 minutes at 4°C, and taking the supernatant to prepare a blank matrix solution; 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 glapiren concentrations of 6 ng / mL and 800 ng / mL.

6. The method for detecting the concentration of maropitant based on dog plasma according to claim 5, characterized in that: The specific method for preparing the recovery sample in step S2 is: A certain amount of canine blank plasma was taken, and a blank matrix solution was prepared using step S51. The blank matrix solution was used to dilute the quality control working solution to obtain recovery samples with glapiran concentrations of 6 ng / mL, 400 ng / mL, and 800 ng / mL.

7. The method for detecting the concentration of maropitant based on dog plasma according to claim 1, characterized in that: In step S6, manual integration is used for situations where sample peak retention time drifts, resulting in no integration or unreasonable integration, unreasonable integration of blank matrix samples for residual determination, and unreasonable selective samples and distribution of analytical batches.

8. The method for detecting the concentration of maropitant based on dog plasma according to claim 1, characterized in that: After step S7, the deviation (%Dev) and precision (%CV) are calculated using the workstation software Aglient Mass Hunter Quantitative Analysis or Microsoft Excel 2003 or above according to the following formula to verify the calculated results of glapiram concentration, and the mean (Mean) and standard deviation (SD) are calculated using Excel; Deviation (% Dev) = (measured concentration - labeled concentration) / labeled concentration × 100% Precision (%CV) = SD of measured concentration / mean of measured concentration × 100% The accuracy of the detection of glapiram content in dog plasma was investigated by bias.

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