Tofacitinib plasma drug concentration detection method and application thereof

Abstract

The invention discloses a method for detecting the plasma drug concentration of tofacitinib, which is characterized by comprising the following steps of: 1, extracting a sufficient amount of sample to prepare mother liquor, and carrying out liquid chromatography-tandem mass spectrometry detection by adopting an internal standard method preparation method; a mother solution in the internal standard method is prepared from methanol in a gradient manner, and the plasma concentration range of tofacitinib is 100ng / ml-1000ng / ml; an internal standard solution in the internal standard method is prepared from methanol, and the plasma concentration of tofacitinib is 1000ng / ml; and 2, setting detection conditions of liquid chromatography and detection conditions of mass spectrometry, and analyzing the eluent obtained after chromatography gradient elution in combination with mass spectrometry to obtain corresponding tofacitinib plasma drug concentration data. The invention also discloses an application of the composition. The invention provides a tofacitinib plasma drug concentration detection method and application thereof. The market blank of concentration detection and application of the tofacitinib medicine in the prior art is overcome.

Description

Technical Field

[0001] The present invention relates to the field of tofacitinib drug application, and in particular to a method for detecting tofacitinib plasma drug concentration and application thereof. Background Art

[0002] As a Janus kinase (JAK) inhibitor, tofacitinib's chemical structure characteristics enable it to specifically inhibit the JAK signaling pathway, thereby blocking key signal transduction in the immune response and achieving the purpose of treating autoimmune diseases. The chemical components of tofacitinib mainly include the active ingredient tofacitinib citrate and a variety of excipient ingredients. These ingredients together constitute the drug basis of tofacitinib citrate tablets, making it have significant therapeutic effects in the treatment of autoimmune diseases such as rheumatoid arthritis, ankylosing spondylitis and psoriatic arthritis. Despite this, long-term use of tofacitinib citrate tablets may lead to suppression of immune system function and increase the risk of infection, such as respiratory system infections and urinary system infections. In addition, it may also cause side effects such as abnormalities of the blood system, mental and nervous system abnormalities, skin and musculoskeletal abnormalities. Therefore, during the use of tofacitinib, patients need to follow the doctor's advice for regular check-ups in order to promptly detect and deal with possible adverse reactions or complications.

[0003] Dermatomyositis is an autoimmune disease that mainly affects the skin and muscles. Its treatment methods include general treatment, drug therapy, physical therapy, plasma exchange and surgical treatment. Among them, drug therapy is one of the main treatments for dermatomyositis. For patients with dermatomyositis, tofacitinib can be used as an adjuvant therapy to reduce damage to the skin surface and may help improve muscle function. However, patients with dermatomyositis treated with tofacitinib may experience some side effects, such as increased blood pressure, infection, bleeding, etc. In addition, it may also cause anemia, proteinuria, myocardial damage, etc. For tofacitinib, its therapeutic window may be narrow, its half-life is short, and the individual differences in dosage and frequency of medication are large, so drug concentration monitoring is particularly important. Drug concentration monitoring helps to assess the patient's drug exposure level and ensure that the drug is within the effective concentration range, thereby improving the treatment effect and reducing side effects. In addition, drug concentration can closely monitor the patient's physical condition and promptly deal with any discomfort symptoms. According to the drug concentration, patients can receive individualized treatment under the guidance of a doctor. At present, the method for determining drug concentration in plasma is mainly high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). This method has the characteristics of high sensitivity, high specificity and high accuracy, and is suitable for the analysis of large batches of clinical samples.

[0004] At present, there is no commercialized liquid chromatography-tandem mass spectrometry detection kit for tofacitinib drug concentration in the domestic market. Therefore, given the importance of detecting tofacitinib drug concentration, it is particularly important to develop a tofacitinib drug concentration detection kit that can be widely used, is efficient, sensitive and easy to operate, and the corresponding detection method for predicting drug efficacy and adverse reactions and guiding clinical applications. Summary of the invention

[0005] In order to overcome the above-mentioned defects of the prior art, the object of the present invention is to provide a method for detecting the plasma drug concentration of tofacitinib and its application, so as to overcome the market gap of the existing tofacitinib drug concentration detection and application.

[0006] In order to achieve the purpose of the present invention, the technical solution adopted is:

[0007] A method for detecting tofacitinib plasma drug concentration comprises the following steps:

[0008] In the first step, a sufficient amount of sample is extracted to prepare a mother solution, and then an internal standard method is used for liquid chromatography-tandem mass spectrometry detection;

[0009] The mother solution in the internal standard method is prepared using a methanol gradient, wherein the tofacitinib plasma concentration range is 100 ng / ml-1000 ng / ml;

[0010] The internal standard solution in the internal standard method is prepared with methanol, and the plasma concentration of tofacitinib is 1000 ng / ml;

[0011] The second step is to set the detection conditions of liquid chromatography and mass spectrometry.

[0012] The detection conditions of the liquid chromatography are as follows: A C18 column was used as a chromatographic column, and the eluate obtained after gradient elution under the conditions that mobile phase A was 0.1-0.2% by volume formic acid water and mobile phase B was methanol was combined with mass spectrometry to obtain the corresponding tofacitinib plasma drug concentration data;

[0013] The mass spectrometry detection conditions are SCIEX Triple Quad 4500 mass spectrometry;

[0014] The ion source voltage was 5000-5500 V, the ion source temperature was 500 °C, and the electrospray ionization-positive ion mode was used;

[0015] The detection method is multiple reaction monitoring; auxiliary gas 50-60psi, collision gas 8-10psi, nebulizer gas 50psi, and the auxiliary gas, collision gas and nebulizer gas are all nitrogen.

[0016] In a preferred embodiment of the present invention, the standard curve solution configuration curve in the internal standard method is as follows:

[0017] 50ng / ml: mother solution 3*50ul+internal standard solution*20ul+430ul methanol+500ul ultrapure water;

[0018] 20ng / ml: 3*20ul mother solution + 20ul internal standard solution + 460ul methanol + 500ul ultrapure water;

[0019] 10ng / ml: 3*10ul mother solution + 20ul internal standard solution + 470ul methanol + 500ul ultrapure water;

[0020] 5ng / ml: 2*10ul mother solution + 20ul internal standard solution + 470ul methanol + 500ul ultrapure water;

[0021] 2ng / ml: mother solution 1*20ul+internal standard solution*20ul+460ul methanol+500ul ultrapure water;

[0022] 1ng / ml: mother solution 1*10ul+internal standard solution*20ul+470ul methanol+500ul ultrapure water.

[0023] An application of a method for detecting tofacitinib plasma drug concentration, wherein the application is to determine the drug trough concentration by integrating the results obtained by the detection.

[0024] In a preferred embodiment of the present invention, the integral judgment of drug trough concentration includes a total of 6 indicators, namely, aspartate aminotransferase indicator, alanine aminotransferase indicator, glutamyl transpeptidase indicator, blood creatinine indicator, blood uric acid indicator and urea nitrogen indicator. Each indicator is 10 points. The integral algorithm is within a certain drug concentration range, the sum of the number of abnormal cases of the 6 indicators / the total number of cases multiplied by 10 points. A score greater than 1 indicates a risk of drug use.

[0025] The beneficial effects of the present invention are:

[0026] The present invention provides a method for detecting the plasma drug concentration of tofacitinib and its application, so as to overcome the market gap of the existing tofacitinib drug concentration detection and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the standard curve correlation equation and correlation coefficient;

[0028] Figure 2 This is the total ion current of tofacitinib in human plasma samples;

[0029] Figure 3 This is the peak graph of Tofacitinib in human plasma samples;

[0030] Figure 4 Schematic diagram of two different methods for detecting tofacitinib plasma concentration Figure 1 .

[0031] Figure 5 Schematic diagram of two different methods for detecting tofacitinib plasma concentration Figure 2 . DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below through the accompanying drawings and embodiments. However, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention. In addition, in the following structure, the description of the known structure and technology is omitted to avoid unnecessary confusion of the concept of the present invention.

[0033] 1. Establishment of liquid chromatography conditions

[0034] Mobile phase A was 0.1% formic acid in water by volume, mobile phase B was methanol (chromatographic grade), and the chromatographic column was Phenomenex C18 (2.1×100 mm, 2.6 μm), gradient elution: flow rate 0.4 mL / min, column temperature 40°C, injection volume 3 μL. Table 1 shows the ratio of mobile item A to mobile item B at different times.

[0035] Table 1

[0036] Time (min) A(％) B(％) 0 90 10 3 90 10 6 10 90 7.5 10 90 7.51 90 10 10 90 10

[0037] 2. Establishment of mass spectrometry conditions

[0038] SCIEX Triple Quad 4500 mass spectrometer, ion source voltage 5500V, ion source temperature 500℃, electrospray ionization-positive ion mode; detection mode is multiple reaction monitoring; auxiliary gas 55psi, collision gas 9psi, nebulizer gas 50psi, all are nitrogen. As shown in Table 2, the standard (Tofacitinib) and internal standard (Tofacitinib-D3) have specific peaks at 4.96 seconds and 4.93 seconds, respectively.

[0039] Table 2

[0040]

[0041] 3. Plasma sample processing method

[0042] Take 1 mL of tofacitinib plasma in a 2 mL centrifuge tube, add 20 μL of internal standard solution (1 μg / mL), vortex for 1 min to mix, add 1-3 mL of methanol / 1-2 mL of acetonitrile (chromatographic grade), vortex for 3 min, centrifuge at 12000 r / min for 5 min, take 1.5 mL of the supernatant and put it on the machine.

[0043] The tofacitinib plasma is derived from samples of the medication population.

[0044] 4.TOF standard curve

[0045] The concentrations of the standard stock solution and internal standard solution (both prepared in methanol) are:

[0046] Stock solution 1: 100 ng / ml;

[0047] Stock solution 2: 500 ng / ml;

[0048] Mother solution 3: 1000 ng / ml;

[0049] The concentration of the internal standard solution was 1000 ng / ml.

[0050] The standard curve solution was prepared as follows:

[0051] 50ng / ml: 3*50ul mother solution + 20ul internal standard solution + 430ul methanol + 500ul ultrapure water.

[0052] 20ng / ml: 3*20ul mother solution + 20ul internal standard solution + 460ul methanol + 500ul ultrapure water.

[0053] 10ng / ml: 3*10ul mother solution + 20ul internal standard solution + 470ul methanol + 500ul ultrapure water.

[0054] 5ng / ml: 2*10ul mother solution + 20ul internal standard solution + 470ul methanol + 500ul ultrapure water.

[0055] 2ng / ml: mother solution 1*20ul+internal standard solution*20ul+460ul methanol+500ul ultrapure water.

[0056] 1ng / ml: mother solution 1*10ul+internal standard solution*20ul+470ul methanol+500ul ultrapure water.

[0057] like Figure 1 As shown, the equation of the standard curve is y=0.03939x-0.00314, and the coefficient is 0.997.

[0058] 5. Small-scale test of tofacitinib concentration in human plasma samples

[0059] 1 mL of plasma samples from 6 different people taking tofacitinib were taken in 2 mL centrifuge tubes, numbered A, B, C, D, E, and F, and 20 μL of internal standard solution (1 μg / mL) was added respectively, vortexed for 1 min to mix, 1 mL of methanol (chromatographic grade) was added, vortexed for 3 min, centrifuged at 12000 r / min for 5 min, and 1 mL of supernatant was taken and put on the machine. The specific results are shown in Table 3. Tofacitinib showed specific peaks around 4.9 seconds in all 6 patients, and the concentration was measured.

[0060] Table 3

[0061] Sample No. Peak time (min) Concentration (ng / ml) A 4.96 23.64 B 4.93 22.89 C 4.95 23.74 D 4.92 22.13 E 4.96 24.06 F 4.94 23.31

[0062] 6. Description of the mobile phase ratio

[0063] The selection of mobile phase is very important for the detection of target compounds, so it is necessary to screen the mobile phase with good performance on the machine.

[0064] (1) Mobile phase A: 0.1% formic acid in water; Mobile phase B: methanol.

[0065] (2) Mobile phase A: 0.1% formic acid in water; Mobile phase B: acetonitrile.

[0066] It has been verified by experiments, such as Figure 4 , 5 It can be seen that method (1) has better TOF detection effect.

[0067] 7. Screening of plasma sample pretreatment methods

[0068] (1) Take 12 ml of untreated human serum sample and divide it into 4 groups: A, B, C, and D. Each group has 3 portions of 1 mL each. Place them in 2-mL centrifuge tubes and number them as A1, A2, A3, B1, B2, B3, C1, C2, C3, D1, D2, and D3.

[0069] (2) Add 25 ng of tofacitinib standard and 20 μL of internal standard solution (1 μg / mL) respectively and vortex for 1 min to mix.

[0070] (3) Add 1 mL of methanol (chromatographic grade) to groups A and B; add 1 mL of acetonitrile (chromatographic grade) to groups C and D, and vortex mix for 3 min.

[0071] (4) Samples from groups A and C were centrifuged at 6000 r / min for 5 min; samples from groups B and D were centrifuged at 12000 r / min for 5 min.

[0072] (5) Take 1 mL of the supernatant and load it onto the analyzer.

[0073] (6) Take another three portions of 1 mL ultrapure water and place them in 2 mL centrifuge tubes and number them E1, E2, and E3. Add 25 ng of tofacitinib and 20 μL of internal standard solution (1 μg / mL), vortex for 1 min, transfer to a chromatographic bottle, and load onto the machine.

[0074] As shown in Table 4 , the concentration of tofacitinib in the blood sample detected by pretreatment method B was closest to the spiked concentration of the solution, so pretreatment method B was selected.

[0075] Table 4

[0076] sample Treatment Upper concentration (ng / ml) A Methanol, 6000r / min 18.56±1.66 B Methanol, 12000r / min 22.96±0.84 C Acetonitrile, 6000r / min 16.43±0.41 D Acetonitrile, 12000r / min 18.97±0.68 E - 24.57±0.33

[0077] 8. Drug concentration integration test

[0078] Table 5 shows the test results of 245 MDA5 antibody-positive dermatomyositis patients treated with tofacitinib. The sampling times are all the trough values ​​of the drug concentration. The results show that as the dosage increases, the mean plasma drug concentration we detected also increases, indicating that the detection method is reliable.

[0079] Table 6 shows the calculation of the safe drug concentration threshold for medication dosage. The clinical criteria are mainly liver and kidney function. A total of 6 indicators are included, namely aspartate aminotransferase index, alanine aminotransferase index, glutamyl transpeptidase index, blood creatinine index, blood uric acid index and urea nitrogen index.

[0080] Each indicator is worth 10 points, and the scoring algorithm is the sum of the number of abnormal cases of the six indicators within a certain drug concentration range / the total number of cases multiplied by 10 points. A score greater than 1 indicates a risk of drug use.

[0081] It can be seen that the drug trough concentration below 10ng / ml is a safe concentration, and patients with drug trough concentrations of 10-30ng / ml need regular monitoring. Drug trough concentrations greater than 30ng / ml are prone to complications of liver and kidney damage, and medication needs to be adjusted in time.

[0082] Table 5

[0083] Frequency of taking medication Number of cases Drug concentration (ng / ml) qod 3 1.95±2.92 qd 21 9.34±15.94 bid 17 10.79±10.41 tid 4 14.81±15.13 total 45 9.88±13.33

[0084] Table 6

[0085]

[0086] The basic principles and main features of the invention and the advantages of the invention are shown and described above.

[0087] Those skilled in the art should understand that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which shall fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for detecting tofacitinib plasma drug concentration, characterized in that: The steps include: In the first step, a sufficient amount of sample is extracted to prepare a mother solution, and then an internal standard method is used for liquid chromatography-tandem mass spectrometry detection; The mother solution in the internal standard method is prepared using a methanol gradient, wherein the tofacitinib plasma concentration range is 100 ng / ml-1000 ng / ml; The internal standard solution in the internal standard method is prepared with methanol, and the plasma concentration of tofacitinib is 1000 ng / ml; The second step is to set the detection conditions of liquid chromatography and mass spectrometry. The detection conditions of the liquid chromatography are as follows: A C18 column was used as a chromatographic column, and the eluate obtained after gradient elution under the conditions that mobile phase A was 0.1-0.2% formic acid water by volume and mobile phase B was methanol was combined with mass spectrometry to obtain the corresponding tofacitinib plasma drug concentration data; The mass spectrometry detection conditions are SCIEX Triple Quad 4500 mass spectrometry; The ion source voltage was 5000-5500 V, the ion source temperature was 500 °C, and the electrospray ionization-positive ion mode was used; The detection method was multiple reaction monitoring; Auxiliary gas 50-60psi, collision gas 8-10psi, nebulizer gas 50psi, auxiliary gas, collision gas and nebulizer gas are all nitrogen.

2. A method for detecting tofacitinib plasma drug concentration according to claim 1, characterized in that: The standard curve solution configuration curve in the internal standard method is as follows: 50ng / ml: 3*50ul mother solution + 20ul internal standard solution + 430ul methanol + 500ul ultrapure water; 20ng / ml: 3*20ul mother solution + 20ul internal standard solution + 460ul methanol + 500ul ultrapure water; 10ng / ml: 3*10ul mother solution + 20ul internal standard solution + 470ul methanol + 500ul ultrapure water; 5ng / ml: 2*10ul mother solution + 20ul internal standard solution + 470ul methanol + 500ul ultrapure water; 2ng / ml: mother solution 1*20ul+internal standard solution*20ul+460ul methanol+500ul ultrapure water; 1ng / ml: mother solution 1*10ul+internal standard solution*20ul+470ul methanol+500ul ultrapure water.

3. An application of the method for detecting the plasma drug concentration of tofacitinib as described in any one of claims 1 or 2, characterized in that: The application is to determine the drug trough concentration by integrating the results obtained by the test.

4. The use of the method for detecting tofacitinib plasma drug concentration as claimed in claim 3, characterized in that: The integral method for judging the trough concentration of drugs includes a total of 6 indicators, namely, aspartate aminotransferase indicator, alanine aminotransferase indicator, glutamyl transpeptidase indicator, blood creatinine indicator, blood uric acid indicator and urea nitrogen indicator. Each indicator is 10 points. The integral algorithm is within a certain drug concentration range, the sum of the number of abnormal cases of the 6 indicators / the total number of cases multiplied by 10 points. A score greater than 1 indicates a risk of drug use.

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