Flurralan content detection method based on cat plasma

Abstract

A method for detecting the content of fluralana based on cat plasma relates to the technical field of detection of the content of fluralana, and comprises the following steps: extracting fluralana in a cat plasma sample by using an acetonitrile precipitation method, adding isotope deuterium-labeled fluralan-D4 as an internal standard, and quantifying the fluralana by using a UPLC-MS / MS method, verification parameters such as the quantitative range, the minimum quantitation limit, the intra-day and inter-day accuracy and precision of the analyte fluralana in the cat plasma and the selectivity and the recovery rate of the method all meet the requirements; after ULOQ is measured every time, an acetonitrile sample is continuously injected, the analyte residual quantity meets the requirement, the established method for measuring the flurala in the plasma through LC / MS / MS meets the requirement of the related guiding principle, the method is applied to the pharmacokinetics method of the flurala drops in the cat body, and the content of the flurala in the cat plasma sample can be accurately quantified.

Description

Technical Field

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

[0002] Cat plasma refers to the liquid containing plasma components extracted from cats. Plasma is a component of blood, equivalent to the liquid part after removing morphological components such as blood cells and platelets. Plasma is mainly composed of water, proteins, inorganic salts, hormones, lipids, etc. It is crucial for maintaining normal physiological functions, including blood circulation, maintaining body temperature, transporting nutrients and drugs, antiviral and antibodies, etc. However, there is no method for detailed analysis and detection of fluralaner content in cat plasma in the prior art.

[0003] Therefore, a method for detecting fluralaner content based on cat plasma is needed. Summary of the Invention

[0004] In order to solve all or part of the above problems, the purpose of the present invention is to provide a method for detecting fluralaner content based on cat plasma to achieve the detection of fluralaner content in cat plasma.

[0005] To achieve the above purpose, the present invention provides the following technical solution: A method for detecting fluralaner content based on cat plasma, which includes the following steps:

[0006] S01: Material preparation;

[0007] S02: Solution preparation;

[0008] S03: Sample treatment, aspirate the sample into an Eppendorf tube, add the standard working solution, vortex, then add acetonitrile, shake and centrifuge, take the supernatant after filtration, add water, mix well and transfer to an injection vial;

[0009] S04: Sampling for LC-MS / MS determination, and perform the determination by controlling chromatographic conditions and mass spectrometry conditions;

[0010] S05: Data processing, perform data acquisition and integration, then calculate the standard curve and sample concentration, calculate the verification parameters again, and finally represent the data.

[0011] Further, the specific steps in S02 are as follows:

[0012] S021: Prepare mobile phase A;

[0013] S022: Prepare diluent;

[0014] S023: Configure reference substance / internal standard stock solution;

[0015] S024: Prepare the standard working solution;

[0016] S025: Prepare the calibration standard samples and quality control samples;

[0017] S026: Prepare the matrix effect samples;

[0018] S027: Prepare the recovery samples;

[0019] S028: Dilute the reliability samples;

[0020] S029: Prepare the stability samples.

[0021] Furthermore, the solution of S023 is prepared by accurately weighing the fluralaner standard, dissolving it in methanol, making up the volume with a volumetric flask to prepare a fluralaner standard stock solution, and storing it in a refrigerator at -20°C.

[0022] Furthermore, in S024, an appropriate amount of the stock solution is taken and serially diluted with the dilution solution to obtain standard working solutions of different concentrations.

[0023] Furthermore, in S025, the calibration standard samples are prepared by accurately measuring appropriate amounts of blank cat mixed plasma and adding standard working solutions of different concentrations to obtain calibration standard samples of different concentrations. A double blank plasma sample is prepared by taking a certain amount of blank mixed plasma and adding the dilution solution. A zero - concentration fluralaner sample is prepared by taking a certain amount of blank mixed plasma and adding the internal standard working solution and the dilution solution.

[0024] Furthermore, in S025, the quality control samples are prepared by measuring an appropriate amount of the quality control working solution and adding it to the blank cat plasma to obtain quality control samples of different concentrations.

[0025] Furthermore, during the data acquisition and integration in S05, data acquisition and automatic integration are performed using the Aglient MassHunter Workstation Data Acquisition system software configured in the mass spectrometer. The same integration method is used for each analysis batch. 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 situation of the analysis batch selectivity samples and distribution, manual integration is used. All manual integration cases should be recorded and reported in the final report.

[0026] Further, in the calculation of the standard curve and the sample concentration in S05, the labeled concentration of the analyte fluralaner in the calibration standard is used as the abscissa, and the ratio of the peak areas of the analyte to the internal standard is used as the ordinate. Linear regression is performed using the least squares method with a weight of 1 / X2 to establish the standard curve and the linear equation. The concentration of the unknown sample is calculated using the standard curve and is calculated by the workstation Aglient MassHunter Quantitative Analysis.

[0027] Further, in the calculation of the standard curve and the sample concentration, the regression equation of the standard curve is

[0028] Y = b + aX, where Y is the ratio of peak areas, X is the labeled concentration, a is the slope, and b is the Y-axis intercept.

[0029] Further, the steps in the data representation in S05 are as follows:

[0030] S051: The peak area data is retained to the integer place, the ratio (Ratio) data of the peak area 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;

[0031] S052: The unit of the measured concentration of the sample is ng / mL, and the data is retained to 2 decimal places after the decimal point;

[0032] S053: The data of accuracy, precision, matrix effect, recovery rate, and deviation are expressed as percentages and retained to 2 decimal places after the decimal point;

[0033] S054: The correlation coefficient R2, a, and b of the standard curve are taken as the values automatically given by the data system;

[0034] S055: Method for retaining significant figures: Round off.

[0035] Compared with the prior art, the beneficial effects of the present invention are:

[0036] A method for detecting the content of fluralaner in cat plasma proposed by the present invention uses the acetonitrile precipitation method to extract fluralaner from cat plasma samples, adds isotope deuterium-labeled fluralaner-D4 as an internal standard, and quantifies fluralaner by UPLC-MS / MS method. The quantitative range of the analyte fluralaner in cat plasma is 10-2500 ng / mL; the lowest quantitative limit is 10 ng / mL; the concentrations of the quality control samples LQC, MQC, and HQC for evaluating the method are 25, 800, and 1800 ng / mL respectively. The accuracy, precision within day and between days, selectivity, and recovery rate of the method, these validation parameters all meet the requirements; after each determination of ULOQ, an acetonitrile sample is continuously injected, and the residual amount of the analyte meets the requirements; a cat plasma biological sample containing the analyte fluralaner is diluted 10-fold with blank cat plasma for determination, and the accuracy and precision of the measurement results meet the requirements; the presence of the matrix has basically no effect on the determination results of the analyte fluralaner in cat plasma; stability: the analyte fluralaner in cat plasma is stable at room temperature for 24 h; stable at -80 °C for 111 days; stable after repeated freezing and thawing 3 times; the processed sample stored in the sample tray of an 8 °C autosampler is stable for 24 h; the stock solutions of the analyte fluralaner and the internal standard fluralaner-D4 are stable when stored frozen at -20 °C for 42 days. The established LC / MS / MS method for determining fluralaner in plasma meets the requirements of relevant guiding principles and is applied to the pharmacokinetic method of fluralaner drops in cats, which can accurately quantify the content of fluralaner in cat plasma samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is the overall flowchart of the method for detecting the content of fluralaner in cat plasma according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0038] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0039] As Figure 1 shown, a method for detecting the content of fluralaner in cat plasma includes the following steps:

[0040] Step 1, sample preparation,

[0041] 1. Preparation of analyte standard (Standard, STD)

[0042] Name or abbreviation: Fluralaner,

[0043] Appearance: White powder,

[0044] Molecular formula: C22H17Cl2F6N3O3,

[0045] Molecular weight: 556.29,

[0046] Purity: 99.9%,

[0047] Storage conditions: Store in a closed container protected from light at 2 - 8°C;

[0048] 2. Preparation of internal standard (Internal Standard, IS)

[0049] Name or abbreviation: Fluralaner - D4,

[0050] Appearance: White powder,

[0051] Molecular formula: C22H13D4Cl2F6N3O3,

[0052] Molecular weight: 560.31,

[0053] Purity: 99.7%,

[0054] Storage conditions: Store in a closed container protected from light at 2 - 8°C;

[0055] 3. Preparation of blank biological matrix

[0056] Both cat mixed blank plasma and cat individual blank plasma were collected and prepared by ourselves. Cats (♂♀) over 1 year old, plasma was collected under non - anesthetized state, filled into "Xinge" heparin sodium anticoagulant blood collection tubes, mixed well and stored at - 80°C;

[0057] 4. Reagent preparation

[0058] 1) Methanol: LC / MS pure,

[0059] 2) Ammonium acetate: Chromatographically pure,

[0060] 3) Ultra - pure water;

[0061] 5. Instrument and consumable preparation

[0062] 1) UPLC - MS / MS: Aglient 6470 - LC - TQ from Aglient, USA, Aglient 1290II / Prime HPLC (the system consists of a quadrupole mass spectrometer, a low - pressure hybrid quaternary ultra - high - pressure pump, an online degasser, a column oven, and a temperature - controlled automatic sampler);

[0063] 2) Analytical balance: Sartorius from Beijing, SQP with a sensitivity of 0.00001 g,

[0064] 3) Vortex mixer: Tongzheng from the north, model: HQ - 60 - 1,

[0065] 4) Centrifuge: Thermo, USA, Model: Micro21R,

[0066] 5) Refrigerator: Gree Jinghong, Model: BCD-430WPSCL,

[0067] 6) Ultra-low temperature freezer: Haier, DW-86L338J,

[0068] 7) Pipette: Eppendorf, Germany, Range: 0.5 - 10 μL, 2 - 20 μL, 10 - 100 μL, 20 - 200 μL, 100 - 1000 μL, 500 - 5000 μL, and tips of various specifications,

[0069] 8) Volumetric flask: 10 mL, 50 mL, 1000 mL,

[0070] 9) Measuring cylinder: 100 mL, 250 mL, 500 mL, 1000 mL.

[0071] Step 2, Solution preparation

[0072] 1. Mobile Phase

[0073] Phase A: 2 mM ammonium acetate aqueous solution; Phase B: Methanol,

[0074] Phase A (2 mM ammonium acetate water): Take 1 mL of 2 M ammonium acetate aqueous solution in a 1 L mobile phase bottle, measure 999 mL of water with a measuring cylinder and add it to the mobile phase bottle, mix well, and you will get mobile phase A.

[0075] 2. Diluent Solution

[0076] 50% methanol-aqueous solution: Take a certain amount of methanol and water, prepare it in a ratio of methanol:water of 1:1, and mix well.

[0077] 3. Stock Standard / IS Solution

[0078] 31. Stock Standard Solution (Stock-STD)

[0079] Accurately weigh 10.01 mg of fluralaner standard, dissolve it in methanol, and make up to the mark with a 10 mL volumetric flask,

[0080] Prepare a 1 mg / mL fluralaner stock standard solution Stock-STD, store it in a -20 °C refrigerator for later use,

[0081] The formula for weighing the standard is as follows:

[0082] 10.01 mg (actual weighed amount of reference standard) = 10.00 mg (theoretical required amount) / 99.9% (labeled purity of reference standard),

[0083] 32. Quality control stock solution (Stock-QC)

[0084] Accurately weigh 10.01 mg of fluralaner reference standard, dissolve it in methanol, and dilute to a volume of 10 mL with a volumetric flask to prepare a 1 mg / mL fluralaner quality control stock solution Stock-QC. Store it in a refrigerator at -20 °C for later use.

[0085] The sample weighing is the same as in item 3.1.

[0086] 33. Internal standard stock solution (Stock-IS)

[0087] Accurately weigh 10.03 mg of fluralaner-D4 reference standard, dissolve it in methanol, and dilute to a volume of 10 mL with a volumetric flask to prepare a 1 mg / mL fluralaner internal standard stock solution Stock-IS. Store it in a refrigerator at -20 °C for later use.

[0088] The calculation formula for the weighed amount of the internal standard is as follows:

[0089] 10.03 mg (actual weighed amount of internal standard) = 10 mg (theoretical required amount) / 99.7% (labeled purity of internal standard).

[0090] 4. Reference standard working solution

[0091] 41. Standard fluralaner working solution (W-STD)

[0092] Take an appropriate amount of the stock solution Stock-STD and serially dilute it with the dilution series to obtain a series of standard working solutions W-STD with concentrations of 100, 200, 500, 1000, 5000, 10000, 15000, and 25000 ng / mL.

[0093] 42. Fluralaner quality control working solution (W-QC)

[0094] Take an appropriate amount of the stock solution Stock-QC and serially dilute it with the dilution series to obtain a series of quality control working solutions (W-QCs) with concentrations of 100, 250, 8000, and 18000 ng / mL.

[0095] 43. Internal standard working solution W-IS

[0096] Precisely measure an appropriate amount of the internal standard stock solution Stock-IS, and dilute it with methanol to obtain the internal standard working solution W-IS with a concentration of 10000 ng / mL.

[0097] The above working solutions are freshly prepared for same-day determination, so the stability of the working solutions is not verified in this experiment.

[0098] 5. Preparation of calibration standards, quality control and other samples

[0099] 51. Calibration Standards of fluralaner

[0100] Precisely measure 90 μL of blank cat mixed plasma respectively, add 10 μL of standard working solutions W-STD with different concentrations to obtain calibration standards with concentrations of 10, 20, 50, 100, 500, 1000, 1500 and 2500 ng / mL. Take a certain amount of blank mixed plasma and add diluent to prepare double blank plasma samples (Blank). Take a certain amount of blank mixed plasma and add internal standard working solution and diluent to prepare fluralaner samples with zero concentration (BlankIS).

[0101] 52. Quality Control Samples of fluralaner

[0102] Measure an appropriate amount of the quality control working solution W-QC and add it to blank cat plasma to obtain quality control samples with concentrations of 10 ng / mL (LLOQ), 25 ng / mL (LQC), 800 ng / mL (MQC), and 1800 ng / mL (HQC). Prepare before use.

[0103] 6. Matrix Effects Samples ME

[0104] 61. Matrix Effects Samples ME

[0105] Take 6 batches of blank cat plasma respectively, add acetonitrile at a ratio of plasma 100:acetonitrile 400, vortex and mix well for 2 min, centrifuge at 14000 rpm at 4 °C for 10 min, take the supernatant, and add an equal proportion of water to obtain blank matrix solution (BlankMatrix BM). Take 990 μL of blank matrix solution and add 10 μL of low and high concentration quality control working solutions to obtain fluralaner matrix effect samples of LQC and HQC.

[0106] 62. Pure Solution PS for matrix effect reference

[0107] Dilute the quality control working solution with 40% acetonitrile-water to obtain pure solution samples with concentrations of LQC and HQC,

[0108] which are used for the investigation of matrix effect.

[0109] 7. Recovery samples

[0110] Take a certain amount of blank cat mixed plasma, prepare blank matrix solution according to step 61. Take 990 μL of blank matrix solution and add 10 μL of low, medium, and high concentration quality control working solutions to obtain low, medium, and high concentration recovery samples of fluralaner at LQC, MQC, and HQC.

[0111] 8. Dilution integrity samples

[0112] Take a certain amount of Stock-QC, add it to blank cat mixed plasma to prepare a sample with a concentration of 1000 ng / mL, and then dilute this sample 10-fold with the same blank plasma to obtain a sample with a concentration of 100 ng / mL, which is used for the investigation of 10-fold dilution reliability of fluralaner biological samples.

[0113] 9. Stability samples

[0114] Measure an appropriate amount of quality control working solution W-QC and add it to blank cat mixed plasma to obtain stability samples with concentrations of 25,

[0115] 800, and 1800 ng / mL. Batch prepare and aliquot them for the investigation of stability under various storage and disposal conditions.

[0116] 10. Precautions

[0117] The preparation method of the solution in this method can be adjusted proportionally according to the above method.

[0118] Step 3. Sample treatment method

[0119] Accurately pipette 100 μL of plasma sample into a 1.5 mL Eppendorf tube, accurately add 10 μL of internal standard standard working solution, vortex for 10 s, add 400 μL of acetonitrile, shake for 5 min, centrifuge at 14,000 rpm at 4 °C for 10 min, take 300 μL of the supernatant after passing through the membrane, add 300 μL of water, mix well and transfer to an injection vial, and inject 2 μL for UPLC-MS / MS determination.

[0120] Among them, add 100 μL of Blank double blank sample into a 1.5 mL Eppendorf tube, accurately add 10 μL of 50% methanol water, vortex for 10 s, add 400 μL of acetonitrile, shake for 5 min, centrifuge at 14,000 rpm at 4 °C for 10 min, take 300 μL of the supernatant after passing through the membrane, add 300 μL of water, mix well and transfer to an injection vial, and inject 2 μL for UPLC-MS / MS determination.

[0121] Accurately add 10 μL of 10,000 ng / mL internal standard standard working solution to Blank IS, matrix effect reference pure solution sample, matrix effect sample, and recovery sample respectively, vortex for 10 s, add 400 μL of acetonitrile, shake for 5 min, centrifuge at 14,000 rpm at 4 °C for 10 min, take 300 μL of the supernatant after passing through the membrane, add 300 μL of water, mix well and transfer to an injection vial, and inject 2 μL for UPLC-MS / MS determination.

[0122] The volumes of the sample and internal standard or other solutions can be adjusted proportionally and processed according to the amounts described in the "Sample Treatment Method" as required. When injecting samples in sequence, the standard samples are injected in ascending order of concentration, and after the high-concentration samples, the injection of blank matrix samples and pure acetonitrile samples is carried out multiple or single cycles, and then the next test sample is analyzed to ensure that possible residues do not affect the accuracy and precision.

[0123] Step 4, LC-MS / MS conditions

[0124] 1. Chromatographic conditions

[0125] a) Ultra-high performance liquid chromatograph: Aglient-1290;

[0126] b) Chromatographic column: Phenomenex Kinetex C18 column (50 mm × 2.1 mm, 2.6 μm);

[0127] c) Mobile phase: A: 2 mM ammonium acetate aqueous solution; B: methanol;

[0128] d) Flow rate: 0.4 mL / min;

[0129] e) Column temperature: 40 °C;

[0130] f) Injection volume: 2 μL.

[0131] Mobile phase elution gradient

[0132]

[0133] 2. Mass spectrometry conditions

[0134] a) Mass spectrometer: Aglient-6470;

[0135] b) Ion source: Electrospray ionization source;

[0136] c) Scanning mode: Positive ion scanning;

[0137] d) Detection mode: Multiple reaction monitoring;

[0138] e) Ion source temperature: 250 °C;

[0139] f) Desolvation temperature: 350 °C;

[0140] g) Nebulizing gas pressure: 35 psi;

[0141] h) Capillary voltage: 3000 V;

[0142] i) Desolvation flow rate: 11 L / min;

[0143] j) Nozzle voltage: 0 V.

[0144] Characteristic ion pairs, fragmentation voltages and collision energies of fluralaner and internal standard

[0145]

[0146] Step Five, Data processing

[0147] 1. Data acquisition and integration

[0148] Data acquisition and automatic integration are carried out using the Aglient MassHunter Workstation Data Acquisition system software configured in the mass spectrometer; the same integration method is used for each analysis batch. 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, unreasonable selectivity and distribution of the analysis batch samples, etc., manual integration can be used, and all manual integration cases should be recorded and reported in the final report.

[0149] 2. Calculation of standard curve and sample concentration

[0150] Using the labeled concentration of the analyte fluralaner 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 / X2 to establish the standard curve and linear equation, and the concentration of the unknown sample is calculated using the standard curve, calculated by the workstation Aglient MassHunter Quantitative Analysis, and the unit of the standard sample concentration is ng / mL

[0151] Regression equation of the standard curve: Y = b + aX, Y: Peak area ratio, X: Labeled concentration, a: Slope, b: Y-axis intercept,

[0152] 3. Calculation of verification parameters

[0153] Calculate the deviation (Deviation, %Dev) and precision (Coefficient of Variation, %CV) using the workstation software Aglient MassHunter Quantitative Analysis or Microsoft Excel 2003 or above according to the following formula. The accuracy is examined by the deviation. The mean and standard deviation (SD) are directly calculated using Excel

[0154]

[0155]

[0156] 4. Data representation method

[0157] 1) The peak area data is rounded to the nearest integer, the peak area ratio (Ratio) data is rounded to 4 decimal places after the decimal point, and the retention time data is rounded to 2 decimal places after the decimal point;

[0158] 2) The unit of the measured concentration of the sample is ng / mL, and the data is rounded to 2 decimal places after the decimal point;

[0159] 3) The accuracy, precision, matrix effect, recovery rate, and deviation data are expressed as percentages and rounded to 2 decimal places after the decimal point;

[0160] 4) The correlation coefficient R2, a, and b of the standard curve are taken as the values automatically given by the data system;

[0161] 5) Method for retaining significant figures: Round up or down according to the rule of "rounding off at the fifth digit".

[0162] The results achieved by the present invention are as follows:

[0163] 1. Selectivity

[0164] Use at least 6 batches of cat blank plasma samples from different donors as blank samples respectively. After being processed according to the sample processing method, measure them under the selected UPLC-MS / MS conditions, and examine the measurement data of the interfering peaks at the retention time of the analyte fluralaner.

[0165] The results show that the peak area of the interfering peak of the blank plasma at the retention time of the analyte fluralaner is less than 20% of the corresponding peak area of the LLOQ, and the peak area of the interfering peak at the retention time of the internal standard is less than 5% of the internal standard peak area of the LLOQ.

[0166] 2. Carry-over

[0167] During the sample determination process, after each determination of the highest concentration standard sample CAL-1 (Upper Limit of Quantification, ULOQ), the blank sample (Blank) and acetonitrile sample were injected for determination once or multiple times in a cycle.

[0168] The results showed that after the determination of the highest concentration standard sample Cal-1 (ULOQ) in all batches, the peak area of the analyte fluralaner in the blank sample was greater than 20% of the LLOQ three times. However, after injecting acetonitrile to rinse the chromatographic column and then injecting Blank again, the peak area of fluralaner was less than 20% of the LLOQ peak area. Therefore, after the determination of the ULOQ sample, only the Blank and acetonitrile need to be continuously injected once, and then the sample analysis is carried out. The influence of the analyte residue on the analysis result meets the requirements, and the internal standard residue in all results does not exceed 5%.

[0169] 3. Calibration Curve

[0170] After the 8 calibration standard samples were processed, they were injected in ascending order of concentration. According to the correlation between the labeled concentration of the calibration standard sample and the ratio of the peak areas of the analyte fluralaner and the internal standard, the least squares method with a weight of 1 / X2 was used for linear regression to obtain the calibration curve and the linear equation.

[0171] The results showed that the quantification range of the analyte fluralaner was 10 - 2500 ng / mL, and the linear relationship of the analyte fluralaner was good within the concentration range of 10 - 2500 ng / mL. During the method validation process, the calibration curves of all analysis batches met the following requirements:

[0172] a) During linear regression, there was no situation where the standard sample data did not meet the requirements and was excluded.

[0173] b) The deviation (%Dev) between the regression values and the theoretical values of all standard sample data did not exceed ±15%, and the LLOQ did not exceed ±20%.

[0174] The correlation coefficient R2 of all calibration curves was greater than 0.99.

[0175] 4. Accuracy and Precision

[0176] Quality control samples at four concentrations of LLOQ, LQC, MQC, and HQC were prepared using Stock-QC respectively.

[0177] The corresponding concentrations of the analyte fluralaner were 10, 25, 800, and 1800 ng / mL, respectively. Six samples were prepared in parallel for each concentration. The calibration curves for each analytical batch / day were prepared using freshly prepared calibration standards, and the determination was verified for three consecutive batches / three days. The results are as follows:

[0178] 41. Verification results of the lowest quantitation limit

[0179] The analyte fluralaner samples were measured for three consecutive days. The within - batch accuracy (%Dev) of the LLOQ for each measurement was

[0180] -8.14 - 4.22%, -4.51 - 12.68%, and -4.38 - 6.58% respectively. The within - batch precisions for the three days were 4.74%, 5.89%, and 4.03% respectively; the between - batch accuracy for the three measurements was -0.78%, and the precision was 5.01%.

[0181] 42. Verification results of the low - concentration quality control (LQC) samples

[0182] The analyte fluralaner samples were measured for three consecutive days. The within - batch accuracy (%Dev) of the LQC for each measurement was -2.62 - 10.47%, -6.82 - 0.01%, and -3.49 - -0.62% respectively. The within - batch precisions for the three days were 4.78%, 2.34%, and 1.04% respectively; the between - batch accuracy for the three measurements was -1.29%, and the precision was 3.54%.

[0183] 43. Verification results of the medium - concentration quality control (MQC) samples

[0184] The analyte fluralaner samples were measured for three consecutive days. The within - batch accuracy (%Dev) of the MQC for each measurement was -1.89 - 3.53%, -4.10 - -0.53%, and -1.91 - 3.64% respectively. The within - batch precisions for the three days were 1.76%, 1.46%, and 2.03% respectively; the between - batch accuracy for the three measurements was -0.49%, and the precision was 2.38%.

[0185] 44. Verification results of the high - concentration quality control (HQC) samples

[0186] The analyte fluralaner samples were measured for three consecutive days. The within - batch accuracy (%Dev) of the HQC for each measurement was -1.43 - 3.21%, -5.23 - -0.62%, and -1.02 - 5.74% respectively. The within - batch precisions for the three days were 2.12%, 1.64%, and 2.42% respectively; the between - batch accuracy for the three measurements was -0.04%, and the precision was 3.16%.

[0187] The above results indicate that "except for the LLOQ, the overall accuracy (%Dev) of the quality control samples at each concentration level should be within ±15% of the labeled value, and the accuracy of the LLOQ should be within ±20% of the labeled value.

[0188] Except for the LLOQ

[0189] the precision (%CV) of the quality control samples at each concentration level should not exceed 15%, and the precision of the LLOQ

[0190] should not exceed 20%", meeting the requirements of the relevant guiding principles.

[0191] The maximum quantitation limit (ULOQ) of the established method is 2500 ng / mL, which is consistent with the concentration of the highest concentration standard sample Cal-1 in the linear standard curve. The corresponding quality control samples for ULOQ were not evaluated for intra-day / inter-day determination during the method validation. However, from the results of the standard curve regression parameters in multiple experiments, it can be seen that the back-calculated concentrations at the maximum quantitation limit of 2500 ng / mL each time did not exceed the range of ±15%, meeting the requirements.

[0192] The above data indicate that the established method for determining the content of the analyte fluralaner in cat plasma has good accuracy and high precision.

[0193] 5. Matrix Effects

[0194] High-concentration and low-concentration matrix effect samples of the analyte fluralaner were prepared from 6 batches of cat blank plasma from different donors. After treatment, the peak areas of the analyte fluralaner and the internal standard in the presence of the matrix were measured and compared with the corresponding peak areas of the matrix-free pure solution samples at the corresponding concentrations. The matrix factors (Matrix Factor, MF) of the analyte fluralaner and the internal standard were calculated, and then the matrix effect factors normalized by the internal standard were calculated by dividing the matrix factor of the analyte fluralaner by the matrix factor of the internal standard.

[0195] The results show that the matrix effect factors for the LOQ and HOQ are 99.14% and 99.04% respectively, and the normalized matrix effect factors are 101.58% and 101.42% respectively. The deviation in accuracy (%DEV) of the matrix sample determination is between -1.44% to -6.01% and 0.34 to -1.76 respectively, and the precision (%CV) is 2.06% and 1.02% respectively, meeting the requirements 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% of the relevant guiding principles.

[0196] The above results indicate that the presence of the matrix has basically no effect on the determination results of the analyte fluralaner.

[0197] 6. Recovery

[0198] After processing 3 concentrations of quality control samples (QCs), inject them for determination of the peak areas of fluralaner and the internal standard, compare with the corresponding peak areas determined by injecting the recovery samples, and calculate the recoveries (%Recovery) of fluralaner and the internal standard respectively.

[0199] The recoveries of 18 QCs samples of the analyte fluralaner at L, M, and H concentrations are between 92.95% and 107.55%, and the precisions are between 3.35% and 5.71%; the recovery of the internal standard IS is between 92.01% and 107.45%, and the precision is between 3.37% and 5.18%.

[0200] The relative standard deviations of the recoveries of the quality control samples of the analyte fluralaner at L, M, and H concentrations and the internal standard IS are all within 85% - 115%, and the precisions are all less than 15%, meeting the requirements.

[0201] 7. Dilution Integrity

[0202] Take cat plasma samples containing the analyte fluralaner, dilute them 10 - fold with blank cat plasma respectively to prepare 6 parallel samples, process and then determine. The results show that for a single 10 - fold dilution of cat plasma of the analyte fluralaner, the average accuracy is - 4.72% and the average precision is 1.72%. The accuracy deviation of the determined results is within ±15%, and the precision is less than 15%, proving that the results of the samples after 10 - fold dilution meet the requirements.

[0203] 8. Stability

[0204] 81. Bench - Top Stability (BTS)

[0205] QCs plasma samples of the analyte fluralaner at L, M, and H concentrations are placed at room temperature for 24 hours, processed and then determined. The accuracy deviations of the three - concentration QCs samples of the analyte fluralaner are - 5.99% - 3.10%, and the precisions are 1.56% - 3.73%.

[0206] The accuracy deviations are all within ±15%, and the precisions are all less than 15%, indicating that the analyte fluralaner is stable in cat plasma after being placed at room temperature for 24 h.

[0207] 82. Freeze and Thaw Stability (FTS)

[0208] QCs plasma samples of the analyte fluralaner at L, M, and H concentrations were stored frozen at -80°C. After three cycles of freeze-thaw, they were processed for determination. The accuracy deviations of the three concentration QCs samples of the analyte fluralaner were -4.55 to 0.62%, and the precision was 0.50 to 1.50%.

[0209] The accuracy deviations were all within ±15%, and the precision was less than 15%, indicating that after storage at -80°C and three cycles of freeze-thaw, the analyte fluralaner was stable in cat plasma.

[0210] 83. Long-Term Stability (LTS)

[0211] QCs plasma samples of the analyte fluralaner at L, M, and H concentrations were stored frozen at -80°C for a long time. The stability of the samples after storage at -80°C for 35 days, 60 days, and 111 days was investigated. The accuracy deviations of the three concentration QCs samples of the analyte fluralaner were -10.31 to 3.73%, and the precision was 0.61 to 5.47%.

[0212] The accuracy deviations were all within ±15%, and the precision was less than 15%, indicating that after storage at -80°C, the analyte fluralaner was stable in cat plasma for 111 days.

[0213] 84. Processed Sample Stability (PSS)

[0214] The extracts of QCs plasma samples of the analyte fluralaner at L, M, and H concentrations were stored in the autosampler sample tray at 8°C for 24 h and then injected for determination. The accuracy deviations of the three concentration QC samples of the analyte fluralaner were -4.00 to 4.55%, and the precision was 0.64 to 2.82%.

[0215] The accuracy deviations were all within ±15%, and the precision was less than 15%, indicating that the pre-treated cat plasma samples of the analyte fluralaner were stable when stored in the autosampler sample tray at 8°C for 24 h.

[0216] 85. Stock Solution Stability (SSS)

[0217] The freshly prepared analyte stock solution and internal standard stock solution were stored at -20°C. After 42 days, they were taken out respectively and prepared into fluralaner samples with the same concentrations of LQC, MQC, and HQC. Stock-IS was diluted with the diluent to the same concentration as the internal standard working solution, and the peak areas were compared with the LQC, MQC, and HQC samples prepared from the standard stock solution and internal standard stock solution on the same day. Three samples were prepared in parallel for each concentration to investigate the long-term stability of the stock solution. The accuracy deviations of the three concentration QCs samples of the analyte fluralaner were -5.43 to -1.47%, and the precision was 1.01 to 1.90%. The accuracy deviations of the internal standard were -9.19 to -3.79%, and the precision was 1.64 to 4.12%.

[0218] The mean deviation of the corresponding peak areas for each concentration was within ±15%, and the precision was less than 15%. This indicates that the stock solutions of the analyte fluralaner and the internal standard were stable when stored at -20°C for 42 days.

[0219] 9. Stock Solution Accuracy Check

[0220] Take a certain amount of freshly prepared Stock-STD and Stock-QC, dilute them by a certain multiple in the same way so that the concentration of fluralaner is in the range of 10 - 2500 ng / mL, prepare two concentrations, and prepare 6 samples for each concentration. After adding the internal standard, measure them. Compare the measured peak areas of the low and high concentration samples diluted from the two prepared stock solutions, and calculate the deviation value according to the formula listed below:

[0221] %DEV = (Average peak area of Stock-STD - Average peak area of Stock-QC) * 100 / ((Average peak area of Stock-STD + Average peak area of Stock-QC) / 2)

[0222] The results showed that the %Dev of the low and high concentration samples were -1.48% and -5.81% respectively, indicating that the accuracy of the prepared standard sample stock solution and quality control sample stock solution met the requirements.

[0223] 10. Reinjection Reproducibility

[0224] The extraction solutions of the LQC and HQC plasma samples of the analyte fluralaner were measured immediately after being processed on the first day.

[0225] Then, after storing them in the autosampler sample tray at 8°C for 24 h, they were injected again for measurement. The accuracy deviations of the measurement results were all within ±15%, and the precision was less than 15%. This indicates that after storing them in the autosampler sample tray at 8°C for 24 h and injecting them again, their reproducibility met the experimental requirements.

[0226] 11. Acceptance Criteria for Analytical Batches

[0227] An analytical batch includes double blank samples (Blank), zero - concentration samples with internal standard (BlankIS), calibration standards at at least 6 concentration levels, at least 3 concentration levels of QCs samples (low, medium, and high concentration duplicate samples or at least 5% of the total number of samples to be measured, whichever is greater), and the validation samples to be analyzed.

[0228] All samples within an analytical batch are processed and extracted in the same batch in the order in which they will be analyzed. The same batch includes processing at the same time, by the same analyst, using the same batch of reagents, and successive processing under consistent conditions without time intervals.

[0229] Calibration standard samples are all injected singly during the method validation phase.

[0230] QCs are dispersed throughout the analytical batch to ensure balanced monitoring of the accuracy and precision of the entire analytical batch.

[0231] The accuracy values of LQC, MQC, and HQC are within ±15% of the labeled concentration. At least 67% of the QCs,

[0232] and at least 50% of the samples at each concentration level meet this standard.

[0233] Based on the above verification results, the established quantitative method for fluralaner in cat plasma is summarized as follows:

[0234] The quantitative range of the analyte fluralaner in cat plasma is 10 - 2500 ng / mL;

[0235] The lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) are 10 ng / mL and 2500 ng / mL respectively;

[0236] The QCs at low, medium, and high concentrations are measured continuously for 3 days, and the accuracy and precision within - day and between - day are both in line with the requirements;

[0237] The selectivity and recovery rate of the method meet the requirements of the protocol;

[0238] After each ULOQ (Cal - 1) measurement, Blank and acetonitrile samples are injected continuously, and the residue amount of the analyte meets the requirements

[0239] The plasma samples are diluted by a factor of 10 singly, and the measurement results meet the requirements of the protocol;

[0240] The presence of the matrix has basically no effect on the measurement results of the analyte fluralaner;

[0241] The analyte fluralaner in cat plasma was stored at room temperature for 24 h; after freezing and thawing three times repeatedly after storage at -80 °C; stable after storage at -80 °C for 111 days; after the plasma sample was processed, it was stored in the sample tray of the autosampler at 8 °C and was stable for 24 h.

[0242] The stock solutions of the analyte fluralaner and the internal standard fluralaner-D4 were stored at -20 °C and were stable for 42 days.

[0243] For the processed sample, after storing in the autosampler sample tray at 8 °C for 24 h and then injecting again, the reproducibility results met the requirements.

[0244] Conclusion: Under the defined conditions of this validation, the established LC / MS / MS method for the determination of fluralaner in plasma meets the requirements of relevant guiding principles and can be applied to the pharmacokinetic method of fluralaner drops in cats to accurately quantify the content of fluralaner in cat plasma samples.

[0245] It should be noted that in the description of this application, it should be understood that the orientation or positional relationships indicated by terms such as "length", "thickness", "inner", "outer", "axial", "radial", etc. are based on the orientation or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be construed as a limitation to the present invention.

[0246] In addition, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprising", "including" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device.

[0247] The above is only the preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution of the present invention and its inventive concept, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

Claims

1. A method for detecting the content of fluralaner in cat plasma, characterized in that, It includes the following steps: S01: Material preparation; S02: Solution preparation; S03: Sample treatment. Pipette the sample into an Eppendorf tube, add the standard working solution, vortex, then add acetonitrile, shake and centrifuge. Take the supernatant after filtration, add water, mix well and transfer to an injection vial; S04: Sampling for LC-MS / MS determination, and perform the determination by controlling the chromatographic conditions and mass spectrometry conditions; S05: Data processing. After data acquisition and integration, calculate the standard curve and sample concentration, calculate the verification parameters again, and finally represent the data.

2. The method for detecting the content of fluralaner based on cat plasma according to claim 1, wherein, The specific steps in S02 are as follows: S021: Prepare mobile phase A phase; S022: Prepare diluent; S023: Prepare reference substance / internal standard stock solution; S024: Prepare standard working solution; S025: Prepare calibration standard samples and quality control samples; S026: Prepare matrix effect samples; S027: Prepare recovery samples; S028: Dilute reliability samples; S029: Prepare stability samples.

3. The method for detecting the content of fluralaner based on cat plasma according to claim 2, wherein The solution preparation in S023 is to accurately weigh the fluralaner reference substance, dissolve it in methanol, make up the volume with a volumetric flask to prepare a fluralaner standard stock solution, and store it in a -20°C refrigerator.

4. The method for detecting the content of fluralaner based on cat plasma according to claim 2, wherein, In S024, take an appropriate amount of the stock solution and serially dilute it with the diluent series to obtain standard working solutions with different concentrations.

5. The method for detecting the content of fluralaner based on cat plasma according to claim 2, characterized in that, The preparation of calibration standard samples in S025 is to accurately pipette appropriate amounts of blank cat mixed plasma, add standard working solutions with different concentrations to obtain calibration standard samples with different concentrations. Take a certain amount of blank mixed plasma and add diluent to prepare double blank plasma samples. Take a certain amount of blank mixed plasma and add internal standard working solution and diluent to prepare zero-concentration fluralaner samples.

6. The method for detecting the content of fluralaner based on cat plasma according to claim 2, wherein, The preparation of quality control samples in S025 is to pipette an appropriate amount of quality control working solution and add it to blank cat plasma to obtain quality control samples with different concentrations.

7. The method for detecting the content of fluralaner based on cat plasma according to claim 1, wherein In the data acquisition and integration process in S05, use the Aglient MassHunter Workstation Data Acquisition system software configured in the mass spectrometer for data acquisition and automatic integration. The same integration method is used for each analysis batch. For cases where the retention time of the sample peak drifts resulting in non-integration or unreasonable integration, unreasonable integration of the residual determination blank matrix sample, and unreasonable selectivity and distribution of the analysis batch selective samples, manual integration is used. All manual integration cases should be recorded and reported in the final report.

8. The method for detecting the fluralaner content based on cat plasma according to claim 1, wherein In the calculation of the standard curve and sample concentration in S05, use the labeled concentration of the analyte fluralaner in the calibration standard sample as the abscissa, and the peak area ratio of the analyte to the internal standard as the ordinate. Perform linear regression using the least squares method with a weight of 1 / X2 to establish the standard curve and linear equation, and calculate the concentration of the unknown sample using the standard curve, which is calculated by the workstation Aglient MassHunter Quantitative Analysis.

9. The method for detecting the content of fluralaner based on cat plasma according to claim 8, characterized in that, In the calculation of the standard curve and the sample concentration, the regression equation of the standard curve is Y: Peak area ratio, X: Labeled concentration, a: Slope, b: Y-axis intercept.

10. A method for detecting the content of fluralaner based on cat plasma according to claim 1, characterized in that, The steps in the data representation in S05 are as follows: S051: Round the peak area data to the nearest integer, retain 4 digits after the decimal point for the peak area ratio (Ratio) data, and retain 2 digits after the decimal point for the retention time data; S052: The unit of the measured concentration of the sample is ng / mL, and retain 2 digits after the decimal point for the data; S053: The accuracy, precision, matrix effect, recovery rate, and deviation data are expressed as percentages and retain 2 digits after the decimal point; S054: Take the values automatically given by the data system for the correlation coefficient R2, a, and b of the standard curve; S055: Method for retaining significant figures: Round off.

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