Method for detecting concentration of atavtrombopag in human plasma by high performance liquid chromatography-tandem mass spectrometry

Abstract

The invention discloses a method for detecting the concentration of atavtrombopag in human plasma through high performance liquid chromatography-tandem mass spectrometry. The method comprises the following steps: S1, adding an internal standard and a precipitator into a plasma sample, uniformly mixing, centrifuging, and taking a supernatant; s2, taking the supernate, carrying out high performance liquid chromatography-tandem mass spectrometry detection, and analyzing and calculating by combining a standard curvilinear equation to obtain the concentration of the atavalopag in the human plasma, according to methodology verification of specificity, accuracy, precision, matrix effect, dilution reliability, residue, stability and the like, the detection method can be directly applied to detection analysis and pharmacokinetic research of an atavictropag plasma sample of a clinical patient, so that the use dosage of a medicine is reasonably adjusted; and a safer and more effective individualized medication scheme is provided for patients.

Description

Technical Field

[0001] The present invention relates to the technical field of blood drug detection, and in particular to a method for detecting the concentration of avatrombopag in human plasma by high performance liquid chromatography-tandem mass spectrometry. Background Art

[0002] Immune thrombocytopenia (ITP) is a common autoimmune disease characterized by thrombocytopenia, characterized by increased platelet destruction and decreased platelet production. Tumor chemotherapy-induced thrombocytopenia (CIT) refers to the inhibitory effect of anti-tumor chemotherapy drugs on bone marrow megakaryocytes, resulting in insufficient platelet production and excessive destruction, causing the platelet count in the peripheral blood to be lower than normal. The above are all causes of thrombocytopenia in clinical patients. The thrombopoietin receptor (TPO) is the main substance that regulates platelet production in the human body. Its function is achieved by promoting megakaryocyte platelet production. Its receptor (TPO-R) is expressed on the surface of stem cells, platelet-producing megakaryocytes, and mature platelets. The emergence of TPO-R agonists has brought a revolution in the treatment of thrombocytopenia. It is currently the first choice for the second-line clinical treatment of ITP and a commonly used drug for the treatment of CIT.

[0003] Currently, avatrombopag is a TPO-RA drug widely used in clinical practice. Although both preclinical and clinical studies have shown the good efficacy of these drugs, there is still a lack of drug concentration monitoring and specific personalized guidance for treatment plans.

[0004] High performance liquid chromatography (HPLC) is currently the most commonly used and most promising method for monitoring blood drug concentrations in clinical practice. In existing literature, the use of HPLC to monitor the blood drug concentration of anti-epileptic drugs accounts for more than 80% of all methods. This method has high detection sensitivity, precision and specificity. In addition, HPLC has a wide range of applications and can be used in many fields of drug research. It has become increasingly popular. Its principle is that the mobile phase enters the system through a high-pressure pump, the sample solution enters the mobile phase through a syringe, and the mobile phase is loaded into the chromatographic column (stationary phase). Due to the different distribution coefficients of each component in the sample solution in the two phases, after repeated adsorption-desorption distribution in the two phases, the movement speed of each component is very different. It is separated into a single component and then flows out of the column in sequence. When the sample concentration passes through the detector, the sample concentration is converted into an electrical signal and transmitted to the recorder, and the data is printed out in the form of a map.

[0005] The development of liquid chromatography-mass spectrometry technology has provided a broad space for sample analysis. The combination of mass spectrometry has provided more sensitive and accurate technical support for the further development of chromatography, and has greatly expanded the application range of liquid chromatography. For example, when monitoring the concentration of telmisartan in serum, the lower detection limit is 20μg / L when using liquid chromatography alone, but when liquid chromatography-mass spectrometry is used for monitoring, the sensitivity can be increased to 0.5μg / L.

[0006] Therefore, it is necessary to develop a method for detecting the concentration of avatrombopag in plasma using liquid chromatography-mass spectrometry technology, so as to rationally adjust the drug dosage and provide patients with a safer and more effective individualized medication plan. Summary of the invention

[0007] The present invention provides a method for detecting the concentration of avatrombopag in human plasma by high performance liquid chromatography-tandem mass spectrometry, so as to realize drug concentration monitoring, rationally adjust the drug dosage, and provide patients with a safer and more effective individualized medication plan.

[0008] To achieve the above object, the scheme of the present invention is as follows:

[0009] The present invention proposes a method for detecting the concentration of avatrombopag in human plasma by high performance liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0010] S1. Add internal standard and precipitant to the plasma sample, mix well, centrifuge and take the supernatant;

[0011] S2. The supernatant was subjected to high performance liquid chromatography-tandem mass spectrometry detection, and the concentration of avatrombopag in human plasma was calculated by combining the standard curve equation;

[0012] The HPLC analysis conditions are as follows: Venusil C18 Plus chromatographic column, specification: 2.1 mm×100 mm, 3.0 μm; 0.1% v / v formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, total flow rate 0.45 mL / min, gradient elution; injector temperature 4-10° C.; column temperature 30-40° C.; injection volume 10 μL;

[0013] The mass spectrometry conditions are: electrospray ionization positive ion mode; scanning mode is multiple reaction monitoring; the monitoring ion pair of avatrombopag is m / z 649.20→267.10, and the monitoring ion pair of the internal standard is m / z 659.40→267.10.

[0014] Furthermore, in step S1, the internal standard is avatrombopag-D8.

[0015] Preferably, the solvent used for the internal standard is DMSO, methanol and water; the concentration of the internal standard is 1×10 -6 g / mL.

[0016] Furthermore, in step S1, the precipitant is methanol or acetonitrile.

[0017] Furthermore, the process of preparing the standard curve equation is to use the concentration of the avatrombopag calibrator as the horizontal coordinate and the peak area ratio of avatrombopag to the internal standard as the vertical coordinate, and to obtain the corresponding standard curve equation by regression; the process of analyzing and calculating the concentration of avatrombopag in plasma is to substitute the ratio of the peak area of ​​avatrombopag to the peak area of ​​the corresponding internal standard into the fitted standard curve equation, and calculate the concentration of avatrombopag in the plasma to be tested.

[0018] Further, in step S2, the gradient elution conditions are as follows:

[0019]

[0020] Further, in step S2, the mass spectrometry conditions include: nebulizer flow rate 3L / min, drying gas flow rate 5L / min, interface voltage 3kV, interface current 9uA, interface temperature 300°C, DL temperature 150°C, heating block temperature 300°C, conversion dynode voltage 10kV, detector voltage 1.75kV, and CID gas pressure 270kPa.

[0021] Furthermore, the plasma sample is fresh plasma or frozen-thawed plasma.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The method for detecting the concentration of avatrombopag in human plasma described in the present invention is verified by methodologies such as specificity, accuracy, precision, matrix effect, dilution reliability, residue, and stability, and can be directly applied to the detection and analysis of avatrombopag plasma samples of clinical patients and the pharmacokinetic study, so as to reasonably adjust the drug dosage and provide patients with a safer and more effective individualized medication regimen.

[0024] Compared with high performance liquid chromatography, the method for detecting the concentration of avatrombopag in human plasma of the present invention has a significantly shorter detection time and is more suitable for clinical testing; the internal standard method is used for quantification, and the accuracy and stability are higher. The detection method is simple to operate for sample pretreatment, does not require expensive reagents, uses a small amount of blood sample, is low in cost, is suitable for routine drug concentration monitoring and related clinical research, and has significant application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a chromatogram of avatrombopag detected with the addition of real matrix in the example of the present invention.

[0026] Figure 2This is a chromatogram of avatrombopag at the detection limit concentration in an embodiment of the present invention. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Example

[0029] 1. Materials and Equipment

[0030] LC-MS equipment: Shimadzu 8050 LC-MS / MS (Shimadzu Corporation, Kyoto, Japan)

[0031] Sample pretreatment equipment:

[0032] Vortex mixer (IKA, Germany)

[0033] KQ-500DE CNC Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd.)

[0034] High-speed refrigerated centrifuge (Shanghai Lu Xiangyi Centrifuge Instrument Co., Ltd.)

[0035] PWN224ZH / E Electronic Analytical Balance (Ohaus Instruments (Changzhou) Co., Ltd.)

[0036] 2. Solution preparation process

[0037] 2.1 Mobile phase and matrix

[0038] The mobile phase A is 0.1% formic acid-water. Add 1000 μL of 98% formic acid to a 1L volumetric flask, dilute to the 1L mark with Watsons drinking water, sonicate for 2 minutes, and store at 4°C (up to 1 week).

[0039] The mobile phase B was acetonitrile.

[0040] The surrogate matrix was human blank plasma.

[0041] 2.2 Working solution

[0042] Weigh 0.001 g of avatrombopag and dissolve it in a solution of DMSO:methanol = 1:1, with a concentration of 1 mg / mL. Prepare the concentration of standard point 8.

[0043] Internal standard: Weigh 0.001 g of avatrombopag-D8, dissolve in 1 mL of DMSO: methanol = 1:1, and mix to obtain 1×10 -3g / mL internal standard stock solution. The internal standard (avatrombopag-D8, 1×10 -6 g / mL).

[0044] The standard curve (8 points) is prepared as shown in the following table:

[0045]

[0046] The spike recovery preparation is shown in the following table:

[0047]

[0048] 2.3 Pre-processing methods for standard points and punctuation points

[0049] Take 90 μL of blank plasma and add 10 μL of standard point or (90 μL of blank plasma and add 10 μL of standard point), add 10 μL of mixed internal standard working solution (avatrombopag-D8, 1×10 -6 g / mL), add 400 μL of methanol solution, mix well and centrifuge for 5 min, take 100 μL of the supernatant and bottle it.

[0050] 2.4 Pre-processing methods of actual samples

[0051] Take 100 μL of actual sample and add 10 μL of mixed internal standard working solution (avatrombopag-D8, 1×10 -6 g / mL), add 400 μL of methanol solution, mix well and centrifuge for 5 min, take 100 μL of the supernatant and bottle it.

[0052] 3. Liquid phase and mass spectrometry parameters

[0053] 3.1 Liquid phase parameters

[0054] HPLC conditions: analytical column: chromcore aq c18 3um, 2.1×100mm; column oven temperature 40°C; mobile phase A: 0.1% formic acid-water; mobile phase B: acetonitrile; total flow rate 0.4500mL / min;

[0055] Injection volume: 10 μL.

[0056] Time program:

[0057]

[0058] 3.2 Mass spectrometry parameters

[0059] MS conditions: Ionization mode: electrospray ionization positive ion mode (ESI+); Detection method: multiple reaction monitoring (MRM); other conditions are shown in the table below.

[0060]

[0061]

[0062] 3.3 Mass spectrometry methods

[0063]

[0064] 4. Calculation

[0065] Liquid chromatography-tandem mass spectrometry was used to detect the content of avatrombopag in the samples extracted with organic solvents, using avatrombopag-D8 as the internal standard, and the peak area of ​​avatrombopag and the corresponding internal standard peak area in the chromatographic peak diagram of each test sample were recorded.

[0066] By calculating the ratio of the peak area of ​​avatrombopag in each test sample to the peak area of ​​the corresponding internal standard, the ratio of the labeled concentration (x) of the calibrator avatrombopag to the corresponding peak area (y) is fitted into a standard curve, and then the ratio of the peak area of ​​avatrombopag in other test samples to the corresponding internal standard peak area is substituted into the fitted standard curve equation to calculate the concentration of avatrombopag in different test samples.

[0067] The Shimadzu LabSolutions data processing system was used to calculate the peak area ratio of the substance to the internal standard using the standard concentration as the horizontal axis and the weighted (W = 1 / C 2 ) or (W = 1 / C) for linear regression.

[0068] 5. Results

[0069] 5.1 Selectivity and specificity

[0070] Add at least 3 different real matrices to the sample, and only one peak will appear at the retention time, as shown in the following example: Figure 1 shown. Figure 1 The chromatogram results were tested by adding different real matrices to the three samples.

[0071] 5.2 Standard curve and linear range

[0072] The guidelines and clinical applications of mass spectrometry recommend that linearity assessment be performed at least at 6 concentrations (generally 8 concentrations), with the compound meeting the standard curve by at least 75% and at least 6 standards, and R 2 Unless otherwise specified, it must be greater than 0.99.

[0073] The Shimadzu LabSolutions data processing system was used to calculate the peak area ratio of the substance to the internal standard using the standard concentration as the horizontal axis and the weighted (W = 1 / C 2 ) to perform linear regression.

[0074] The linear regression result is, Y = 0.183990X + 0.0470635, R2 =0.9905788, R 2 >0.99. The linear range is 1-2000ng / mL.

[0075] 3.3 Limit of detection and limit of quantification

[0076] The detection limit is the lowest value of the target analyte that can be detected by the established LC-MS / MS method, and the lowest value of the target analyte that can be detected by the quantitative LC-MS / MS method under the premise of meeting the laboratory's requirements for accuracy and precision. The LC-MS / MS method is used for detection, and the concentration when the average signal-to-noise ratio (S / N) = 3 is used as the detection limit of the method. The quantitative limit is the concentration when the average signal-to-noise ratio (S / N) is ≥10, and the (S / N) of STD1 is required to be ≥10.

[0077]

[0078] Figure 2 This is the chromatogram of avatrombopag at the detection limit concentration (0.016 ng / mL), avatrombopag (STD1) (S / N) = 598.1.

[0079] 3.4 Accuracy

[0080] The accuracy is evaluated by the spike recovery method. If the spike recovery rates are within the acceptable range (±15%), it is proved that the accuracy of the established LC-MS / MS method meets the requirements. The specific experimental method is: select a sample of known concentration as the original sample, add three standard solutions of low, medium and high concentrations to the sample, prepare three concentrations of spiked samples, divide each concentration sample into 5 parts, repeat the measurement of each sample 3 times, and calculate the spike recovery rates at each concentration. If the spike recovery rates of each concentration are within the acceptable range, it is proved that the accuracy of the method meets the requirements.

[0081]

[0082]

[0083] 3.5 Precision

[0084] Precision verification should evaluate at least 3 samples distributed in the high, medium and low ranges of analytical measurement. Each sample should be evaluated for both intra-batch precision and total precision. The following method can be used to carry out the verification experiment: select 3 samples with concentrations (low, medium and high) distributed in the analytical measurement range, process each concentration sample in each batch 5 times, and continuously measure 3 batches with a total of 45 samples, 5 times within a day and 5 times between days (1, 3 and 7 days respectively), and evaluate the intra-batch precision and total precision of each concentration sample respectively.

[0085]

[0086] 3.6 Matrix Effects

[0087] The matrix effect mainly evaluates the influence of the matrix of the sample to be tested (rather than the analyte itself) and its characteristics on the specific measurement method and measurement results. The substances that cause the matrix effect can be endogenous (such as salts, lipids) or exogenous (such as polymers, anticoagulants and separation gels in some sample tubes). The matrix effect will affect the accuracy and sensitivity of the measurement method. Three blank matrices from different sources / batches, one 5% hemolysis matrix HE, and the analyte standard can be added after the extract. By adding standard solutions of different concentration levels (at least 3 low, medium, and high concentrations), the signal ratio of the target compound in the matrix and the pure solvent is compared, and the internal standard normalized matrix factor (relative matrix effect) is calculated.

[0088] Matrix effect = response of the blank matrix after extraction when the analyte is added / response of the analyte in the pure solution. Relative matrix effect = concentration of the blank matrix after extraction when the analyte is added / concentration of the analyte in the pure solution = MER / MEP. Extraction recovery rate = response of the analyte added to the blank matrix after extraction at a certain concentration / response of the analyte added to the blank matrix after extraction. Relative extraction recovery rate = concentration of the analyte added to the blank matrix after extraction at a certain concentration / concentration of the analyte added to the blank matrix after extraction = QC / MER.

[0089]

[0090] 3.7 Carryover (residue)

[0091] When the compound meets the upper limit of quantitation (ULOQ), the response of the blank sample does not exceed 20% of the lower limit of quantitation (LLOQ).

[0092]

[0093] 3.8 Dilution Reliability

[0094] Dilution reliability It is recommended to use recovery rate to evaluate dilution reliability. At least 3 parallel samples should be prepared for each dilution factor, including dilutions within and outside the linear range, as well as dilutions of a small amount of samples. Dilutions outside the linear range are more common. High-concentration quality control samples (concentration should be greater than ULOQ) are prepared and tested after dilutions of different proportions (the diluent is an alternative matrix). At least 3 measured values ​​are required for each dilution factor. The mean value of the dilution quality control and the RE of the labeled value should be within ±15%.

[0095] Results of reliability evaluation of avatrombopag dilution:

[0096]

[0097]

[0098] 3.9 Stability

[0099] The evaluation method is the ratio of the sample after storage to the average value of the freshly prepared sample / the sample placed at -80°C after preparation. It can be the concentration or the peak area.

[0100] The evaluation of the stability of the target analyte mainly includes the following aspects:

[0101] (1) Stability of target analytes in solution

[0102] Working solutions ULOQ and LLOQ were prepared from the stock solutions, and the stability of the working solutions at different temperatures and times was investigated, where RT represents room temperature, and the results are shown in the following table.

[0103]

[0104]

[0105] (2) Stability of target analytes in biological matrices

[0106] Quality control products can be used to evaluate the performance of the LC-MS / MS method and ensure that the accuracy of the test results can meet the expected medical use. Quality control products must be treated identically to samples, and their short-term, long-term, and repeated freeze-thaw stability must be examined to monitor their deviation and precision.

[0107] The results are shown in the table below, such as the stability after being placed at room temperature and 4°C for 3 days, the stability after being placed at -20°C for 7 days, and the stability after being frozen and thawed three times.

[0108]

[0109] (3) Stability of samples after treatment, including stability and reproducibility of autosampler placement after treatment. 3 days at 4°C and 7 days at -20°C.

[0110] Avatrombopag sample stability after extraction:

[0111]

[0112] Avatrombopag sample reproducibility after extraction:

[0113]

[0114] in conclusion:

[0115] (1) Working solution: The working solution corresponding to the LLOQ is tested at room temperature, 4°C, and -20°C for short periods of time (24 hours and

[0116] The working solution corresponding to ULOQ is stable at 4°C, -20°C for short term (24H and 3D) and -20°C for long term (7D), but unstable at room temperature for short term.

[0117] (2) In biological matrices: LQC and HQC were stable at room temperature, 4°C, and -20°C for short term (24 hours and 3 days) and -20°C for long term (7 days), as well as after three freeze-thaw cycles;

[0118] (3) After sample processing: LQC and HQC were placed at 4°C for 3 days and at -20°C for 7 days for good stability and reproducibility.

[0119] 3.10 Non-marking rate

[0120]

[0121] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting the concentration of avatrombopag in human plasma by high performance liquid chromatography-tandem mass spectrometry, characterized in that: The steps include: S1. Add internal standard and precipitant to the plasma sample, mix well, centrifuge and take the supernatant; S2. The supernatant was subjected to high performance liquid chromatography-tandem mass spectrometry detection, and the concentration of avatrombopag in human plasma was calculated by combining the standard curve equation; The HPLC analysis conditions are as follows: Venusil C18 Plus chromatographic column, specification: 2.1 mm×100 mm, 3.0 μm; 0.1% v / v formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, total flow rate 0.45 mL / min, gradient elution; injector temperature 4-10° C.; column temperature 30-40° C.; injection volume 10 μL; The mass spectrometry conditions are: electrospray ionization positive ion mode; scanning mode is multiple reaction monitoring; the monitoring ion pair of avatrombopag is m / z 649.20→267.10, and the monitoring ion pair of the internal standard is m / z 659.40→267.

10.

2. The method for detecting the concentration of avatrombopag in human plasma according to claim 1, characterized in that: In step S1, the internal standard is avatrombopag-D8.

3. The method for detecting the concentration of avatrombopag in human plasma according to claim 2, characterized in that: The solvents used for the internal standard are DMSO, methanol and water; the internal standard concentration is 1×10 -6 g / mL.

4. The method for detecting the concentration of avatrombopag in human plasma according to claim 1, characterized in that: In step S1, the precipitant is methanol or acetonitrile.

5. The method for detecting the concentration of avatrombopag in human plasma according to claim 1, characterized in that: The process of preparing the standard curve equation is to use the concentration of the avatrombopag calibrator as the horizontal coordinate and the peak area ratio of avatrombopag to the internal standard as the vertical coordinate, and to obtain the corresponding standard curve equation through regression; the process of analyzing and calculating the concentration of avatrombopag in plasma is to substitute the ratio of the peak area of ​​avatrombopag to the peak area of ​​the corresponding internal standard into the fitted standard curve equation, and to calculate the concentration of avatrombopag in the plasma to be tested.

6. The method for detecting the concentration of avatrombopag in human plasma according to claim 1, characterized in that: In step S2, the gradient elution conditions are as follows:

7. The method for detecting the concentration of avatrombopag in human plasma according to claim 1, characterized in that: In step S2, the mass spectrometry conditions include: nebulizer flow rate 3 L / min, drying gas flow rate 5 L / min, interface voltage 3 kV, interface current 9 uA, interface temperature 300°C, DL temperature 150°C, heating block temperature 300°C, conversion dynode voltage 10 kV, detector voltage 1.75 kV, and CID gas pressure 270 kPa.

8. The method for detecting the concentration of avatrombopag in human plasma according to claim 1, characterized in that: The plasma sample is fresh plasma or frozen-thawed plasma.

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