A method and kit for detecting enasidenib content in plasma based on HPLC-MS / MS

Abstract

This invention relates to a method and kit for detecting enasidenib content in plasma based on HPLC-MS / MS, using ivosidenib as an internal standard for the detection of enasidenib content in plasma. The HPLC conditions are as follows: Ultimate XB-C18 column; column temperature: 60℃; mobile phase: mobile phase A is 0.1% formic acid methanol solution, mobile phase B is 2 mmol / L ammonium acetate-0.1% formic acid aqueous solution; chromatographic separation is gradient elution, flow rate: 0.8 mL / min; injection volume: 2 μl; mass spectrometry detection conditions are: electrospray ionization source, ion injection voltage: 5500 V, ion source temperature: 550℃, GS1: 55 psi, GS2: 55 psi, Curtain gas: 25 psi, monitoring mode is multiple reaction detection mode (MRM). This invention is accurate, simple, sensitive, highly selective, and allows for rapid determination of enasidenib content in plasma.

Description

Technical Field

[0001] This invention belongs to the field of Enasidenib detection technology, specifically relating to a method and kit for detecting Enasidenib content in plasma based on HPLC-MS / MS. Background Technology

[0002] Enasidenib, a tumor metabolic drug, belongs to the IDH2 inhibitor class. It can inhibit the activity of various enzymes that promote cell proliferation and can be used to treat adult patients with relapsed or refractory acute myeloid leukemia (AML) carrying isocitrate dehydrogenase-2 (IDH2) gene mutations. FDA data shows that enasidenib has an oral bioavailability of approximately 57%, a peak time of about 4 hours, a plasma protein binding rate of 98.5%, and a terminal half-life of 25 hours. It is mainly eliminated through feces, and the area under the concentration-time curve (AUC) increases proportionally with the dose, from 50 to 450 mg daily. Enasidenib is mainly metabolized by cytochrome P450 enzymes (CYP) and glucuronyl transferases (UGTs), and it is prone to drug interactions with other drugs in vivo, leading to significant variability in pharmacokinetic parameters and thus affecting efficacy. Meanwhile, this drug accumulates in the body and is prone to cumulative toxicity; therefore, monitoring the concentration of Enasidenib in the body during medication and making individualized adjustments to the medication is of great significance for patients with excessively low / high concentrations, those who need to prevent serious adverse reactions, and those who do not respond well to long-term medication.

[0003] Therefore, establishing an accurate, simple, sensitive, highly selective, and rapid HPLC-MS / MS method for determining the concentration of Enasidenib in plasma is crucial for evaluating the efficacy and safety of Enasidenib. Summary of the Invention

[0004] The purpose of this invention is to overcome the deficiencies in the prior art and provide a method and kit for detecting the content of Enasidenib in plasma based on HPLC-MS / MS. The method has simple plasma sample pretreatment, requires a small amount of plasma sample, has a short analysis time, and has good sensitivity and specificity.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for detecting the content of enasidenib in plasma based on HPLC-MS / MS, using ivosidenib as an internal standard to detect the content of enasidenib in plasma by HPLC-MS / MS;

[0007] The HPLC-MS / MS conditions for detection were as follows: Ultimate XB-C18 column, 4.6 mm × 50 mm, particle size 5 μm; column temperature: 60℃; mobile phase: mobile phase A was 0.1% formic acid in methanol, and mobile phase B was 2 mmol / L ammonium acetate-0.1% formic acid aqueous solution; chromatographic separation was gradient elution, and the specific gradient elution conditions are shown in the table below.

[0008]

[0009]

[0010] Flow rate: 0.8 mL / min; Injection volume: 2 μl;

[0011] The mass spectrometry detection conditions for HPLC-MS / MS detection were as follows: electrospray ion source, ion injection voltage: 5500V, ion source temperature: 550℃, GS1: 55psi, GS2: 55psi, Curtain gas: 25psi, and the monitoring mode was multiple reaction detection mode (MRM).

[0012] As a further technical solution, the mass spectrometry conditions of the MRM are shown in the table below;

[0013] Enasidenib 474.100 267.100 200 80 42 Ivosidenib 583.200 476.000 150 85 30

[0014] As a further technical solution, plasma sample pretreatment is required before performing HPLC-MS / MS detection on plasma samples;

[0015] The plasma sample pretreatment method includes: taking the plasma to be tested, adding sample processing solution and internal standard solution, shaking to mix, centrifuging to collect the supernatant for later use;

[0016] As a further technical solution, the volume ratio of plasma, sample processing solution and internal standard solution is 1:1:3.

[0017] As a further technical solution, a standard curve needs to be plotted before performing HPLC-MS / MS analysis on plasma samples;

[0018] The process of plotting the standard curve includes: adding Enasidenib standard solutions of different concentration gradients to the sample processing solution and the internal standard solution, respectively, followed by sample injection and detection. A calibration curve is plotted with the concentration of the Enasidenib calibration solution as the abscissa and the ratio of the measured peak area of ​​the Enasidenib standard solution to the peak area of ​​the internal standard as the ordinate.

[0019] As a further technical solution, the sample processing solution is: 2 mmol / L ammonium acetate - 0.1% formic acid aqueous solution;

[0020] The internal standard solution was a 250 ng / mL Ivosidenib methanol standard solution.

[0021] As a further technical solution, the preparation method of Enasidenib standard solutions with different concentration gradients is as follows: prepare standard stock solutions with methanol, and then dilute them with methanol to form 8 concentration gradients, namely 0 μg / mL, 0.05 μg / mL, 1 μg / mL, 1.75 μg / mL, 2.5 μg / mL, 10 μg / mL, 17.5 μg / mL, and 25 μg / mL.

[0022] As a further technical solution, quality control is also included, wherein the quality control involves using quality control samples to verify the testing method;

[0023] The quality control materials consist of single-level low, medium, and high concentration Enasidenib quality control solutions;

[0024] The low-concentration level quality control solution used was a 0.75 μg / mL Enasidenib standard solution;

[0025] The medium-concentration level quality control solution used was a 7.5 μg / mL Enasidenib standard solution;

[0026] The high-concentration level quality control solution used was a 20 μg / mL Enasidenib standard solution.

[0027] A kit for the method of detecting enasidenib content in plasma based on HPLC-MS / MS includes calibrators, quality controls, sample processing solution, internal standard solution and mobile phase, wherein the calibrators include enasidenib standard solutions of 0 μg / mL, 0.05 μg / mL, 1 μg / mL, 1.75 μg / mL, 2.5 μg / mL, 10 μg / mL, 17.5 μg / mL and 25 μg / mL.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] This invention uses ivosidenib as an internal standard and improves the accuracy of quantification of enasidenib by optimizing HPLC-MS / MS detection conditions, avoiding interference from commonly used drugs and concomitant medications. Ultimately, it yields an HPLC-MS / MS method that can accurately, simply, sensitively, selectively, and rapidly determine the concentration of enasidenib in plasma. Furthermore, it features simple plasma sample pretreatment and requires small sample volumes, making it suitable for routine monitoring of clinical blood drug concentrations of enasidenib and pharmacokinetic analysis studies. Attached Figure Description

[0030] Figure 1 This is a secondary scanning fragment ion map of Enasidenib in this invention;

[0031] Figure 2 This is a secondary scanning fragment ion map of Ivosidenib in this invention;

[0032] Figure 3 This is a mass spectrometry chromatogram of Enasidenib and Ivosidenib in this invention; Detailed Implementation

[0033] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1

[0035] The kit used for the HPLC-MS / MS method for detecting enasidenib in plasma includes calibrators, quality controls, sample processing solutions, internal standards, and mobile phases.

[0036] 1) The calibrators include Enasidenib standard solutions at concentrations of 0 μg / mL, 0.05 μg / mL, 1 μg / mL, 1.75 μg / mL, 2.5 μg / mL, 10 μg / mL, 17.5 μg / mL, and 25 μg / mL;

[0037] The calibrator is prepared using a methanol solution.

[0038] The calibrator is prepared as follows: Accurately weigh 5.0 mg of Enasidenib standard, place it in a small beaker, dissolve it in an appropriate amount of methanol, transfer it to a 10.0 mL volumetric flask, dilute to volume with methanol, sonicate to mix, and dispense into portions to obtain a 500 μg / mL Enasidenib stock solution; then dilute the Enasidenib stock solution with methanol to prepare Enasidenib standard solutions of 0 μg / mL, 0.05 μg / mL, 1 μg / mL, 1.75 μg / mL, 2.5 μg / mL, 10 μg / mL, 17.5 μg / mL, and 25 μg / mL, respectively.

[0039] 2) The quality control samples are Enasidenib quality control solutions at three concentration levels: 0.75 μg / mL, 7.5 μg / mL, and 20 μg / mL. The quality control solutions are prepared using methanol as the solvent.

[0040] Low-concentration Enasidenib quality control solution: Take 400 μL of 50 μg / mL Enasidenib stock solution, add 600 μL of methanol, shake to mix, and you will get a 0.75 μg / mL Enasidenib standard solution.

[0041] Medium concentration level Enasidenib quality control solution: Take 150 μL of 50 μg / mL Enasidenib stock solution, add 850 μL of methanol, shake to mix, and you will get a 7.5 μg / mL Enasidenib standard solution.

[0042] High-concentration Enasidenib quality control solution: Take 100 μL of Quality Control M solution, add 900 μL of methanol, shake to mix, and you will have a 20 μg / mL Enasidenib standard solution.

[0043] 3) The sample treatment solution uses a 2 mmol / L ammonium acetate-0.1% (v / v) formic acid aqueous solution, and its preparation method includes:

[0044] Step 1: Weigh 7.7g of ammonium acetate, dissolve it in distilled water from Watsons in Guangzhou, and make up to 100mL in a volumetric flask with distilled water from Watsons in Guangzhou. Mix well by sonication to obtain a 100mmol / L ammonium acetate solution.

[0045] Step 2: Take 1 mL of formic acid solution and 2 mL of 100 mmol / L ammonium acetate solution, and dilute to 1000 mL in a volumetric flask with distilled water from Watsons in Guangzhou. Mix well by sonication to obtain a 2 mmol / L ammonium acetate-0.1% formic acid aqueous solution.

[0046] 4) The internal standard solution used was 250 ng / mL Ivosidenib solution, and its preparation method included:

[0047] Accurately weigh 25.0 mg of Ivosidenib standard, place it in a small beaker, dissolve it with an appropriate amount of methanol, transfer it to a 10.0 mL volumetric flask, dilute to volume with methanol, sonicate to mix, and dispense for use to obtain a 2.5 mg / mL Ivosidenib stock solution.

[0048] Take 25 μL of 2.5 mg / mL Ivosidenib stock solution, dilute to 250 mL with methanol in a volumetric flask, sonicate to mix, and dispense into aliquots to obtain 250 ng / mL Ivosidenib internal standard solution.

[0049] 5) The mobile phase includes mobile phase A and mobile phase B; mobile phase A is a 0.1% formic acid methanol solution; mobile phase B is a 2 mmol / L ammonium acetate-0.1% formic acid aqueous solution;

[0050] Preparation of mobile phase A: Take 1 mL of formic acid solution, dilute to 1000 mL in a volumetric flask with methanol, and sonicate to obtain a formic acid methanol solution with a volume concentration of 0.1%.

[0051] Preparation of mobile phase B: Weigh 7.7 g of ammonium acetate, dissolve it in distilled water (from Watsons, Guangzhou), and bring the volume to 100 mL in a volumetric flask. Mix thoroughly by sonication to obtain 100 mmol / L of mobile phase. -1 Ammonium acetate solution;

[0052] Take 1 mL of formic acid solution, 2 mL of 100 mmol / L... -1 The ammonium acetate solution was diluted to a volumetric flask with distilled water from Watsons (Guangzhou) to a final volume of 1000 mL, and then sonicated to obtain a solution of 2 mmol / L. -1 Ammonium acetate in a 0.1% (v / v) formic acid aqueous solution.

[0053] Example 2

[0054] A method for detecting enasidenib in plasma based on HPLC-MS / MS includes:

[0055] 1) Preparation of calibrator solutions, quality control solutions, and plasma samples:

[0056] Take 10 μL of each solution from the calibrator and quality control in the kit of Example 1, add 90 μL of blank plasma, 100 μL of sample processing solution, and 300 μL of internal standard solution respectively, vortex to mix, centrifuge and collect the supernatant to obtain calibrator solutions and quality control solutions of different concentrations for later use.

[0057] Take 100 μL of the plasma to be tested, add 100 μL of sample processing solution and 300 μL of internal standard solution, vortex to mix, centrifuge and collect the supernatant to obtain the plasma sample to be tested, for later use.

[0058] 2) The detection conditions for high performance liquid chromatography-tandem mass spectrometry are:

[0059] The HPLC-MS / MS conditions for detection were as follows: Ultimate XB-C18 column, 4.6 mm × 50 mm, particle size 5 μm; column temperature: 60℃; mobile phase: mobile phase A was 0.1% formic acid in methanol, and mobile phase B was 2 mmol / L ammonium acetate-0.1% formic acid aqueous solution; chromatographic separation was gradient elution, and the specific gradient elution conditions are shown in Table 1.

[0060] Table 1: Gradient elution status

[0061] 0.01min 17.5％ 82.5％ 0.5min 82.5％ 17.5％ 1.0min 100％ 0％ 2.0min 100％ 0％ 2.5min 82.5％ 17.5％ 3.0min 17.5min 82.5％

[0062] Flow rate: 0.8 mL / min; Injection volume: 2 μl;

[0063] The mass spectrometry detection conditions for HPLC-MS / MS detection were as follows: electrospray ion source, ion injection voltage: 5500V, ion source temperature: 550℃, GS1: 55psi, GS2: 55psi, Curtain gas: 25psi, and the monitoring mode adopted was multiple reaction detection mode (MRM).

[0064] The mass spectrometry conditions for the MRM are shown in Table 2;

[0065] Table 2: MRM Mass Spectrometry Conditions

[0066] Enasidenib 474.100 267.100 200 80 42 Ivosidenib 583.200 476.000 150 85 30

[0067] 3) Plot the standard curve

[0068] Different concentration gradient calibrator solutions were detected, and the internal standard quantification method of mass spectrometry was used. A standard curve was established with the concentration of Enasidenib standard as the abscissa and the ratio of the measured peak area of ​​Enasidenib standard to the peak area of ​​internal standard as the ordinate. The concentration of Enasidenib in the sample to be tested was calculated based on the standard curve.

[0069] The standard curve was plotted as follows: seven concentrations of Enasidenib standard solutions were used as the abscissa (X), and the ratio of the measured peak area of ​​the seven concentrations of Enasidenib standard solutions to the peak area of ​​their respective internal standards was used as the ordinate (Y). A weighted square regression was performed on Y against X to obtain the standard curve. The linear regression equation was Y = 0.00699x + -0.000231, r = 0.9985.

[0070] 4) Before testing samples each day, the method is validated using a quality control solution. Then, the plasma samples to be tested are tested, and the content of Enasidenib in the plasma samples is obtained using a standard curve.

[0071] Example 3: Validation of the detection method:

[0072] Six aliquots of Enasidenib quality control solutions at low, medium, and high concentrations from the quality control samples in Example 1 were taken, with 10 μL of each sample. 90 μL of blank plasma, 100 μL of sample processing solution, and 300 μL of internal standard solution were added to each aliquot. The mixture was shaken to mix, centrifuged, and the supernatant was collected for later use. The samples were then analyzed under the high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) detection conditions described in Example 2. The measured concentration of the quality control samples was obtained based on the standard curve for that day. Measurements were performed continuously for three days. Intra-day and inter-day RSDs were calculated based on the results, and the ratio of the measured concentration to the standard concentration was used as the relative recovery rate. The results are shown in Table 3.

[0073] Table 3 Precision and recovery tests of Enasidenib (n=6, )

[0074]

[0075] II. Stability Test

[0076] Six portions of Enasidenib quality control solutions of low, medium and high concentrations were taken from the quality control samples of Example 1. Each sample was 10 μL. 90 μL of blank plasma, 100 μL of sample processing solution and 300 μL of internal standard solution were added to each sample. After being refrigerated at 4℃, -20℃ for 72 h and at room temperature for 72 h, respectively, the samples were injected and analyzed according to the detection conditions of high performance liquid chromatography tandem mass spectrometry in Example 2. The concentration of each sample was determined and its stability was examined. The measured concentrations were compared with the standard concentrations. The results are shown in Table 4.

[0077] Table 4: Stability test results of Enasidenib (n=6, )

[0078]

[0079] In summary, the above experiments show that the kit of the present invention is simple to operate, accurate, precise, with a relative recovery rate of 85% to 115%, good accuracy, RSD of less than 10%, and meets the requirements for precision and stability, and can meet the requirements for clinical batch sample monitoring.

[0080] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible implementations of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting the content of enasidenib in plasma based on HPLC-MS / MS, characterized in that, The content of enasidenib in plasma was detected by HPLC-MS / MS using Ivosidenib as an internal standard; The HPLC-MS / MS detection conditions were as follows: the column was an Ultimate XB-C18, with dimensions of 4.6 mm × 50 mm and a particle size of 5 μm; Column temperature: 60℃; Mobile phase: Mobile phase A was 0.1% formic acid in methanol, and mobile phase B was 2 mmol / L ammonium acetate in 0.1% formic acid aqueous solution; chromatographic separation was performed using gradient elution, and the specific gradient elution conditions are shown in the table below: ； Flow rate: 0.8 mL / min; Injection volume: 2 μL; The mass spectrometry detection conditions for HPLC-MS / MS were as follows: electrospray ionization source, ion injection voltage: 5500V, ion source temperature: 550℃, GS1: 55psi, GS2: 55psi, Curtain gas: 25 psi, monitoring mode uses multiple response detection mode (MRM); The mass spectrometry conditions for the MRM are shown in the table below; ； Before performing HPLC-MS / MS analysis on plasma samples, pretreatment of the plasma samples is required. The plasma sample pretreatment method includes: taking the plasma to be tested, adding sample processing solution and internal standard solution, shaking to mix, centrifuging to collect the supernatant for later use; The sample processing solution was: 2 mmol / L ammonium acetate - 0.1% formic acid aqueous solution; The internal standard solution was: 250 ng / mL Ivosidenib methanol standard solution; Before performing HPLC-MS / MS analysis on plasma samples, a standard curve needs to be plotted. The process of plotting the standard curve includes: adding Enasidenib standard solutions of different concentration gradients to the sample processing solution and the internal standard solution, respectively, then injecting the samples for detection, and plotting the calibration curve with the concentration of the Enasidenib calibration solution as the abscissa and the ratio of the measured peak area of ​​the Enasidenib standard solution to the peak area of ​​the internal standard as the ordinate.

2. The method for detecting the content of enasidenib in plasma based on HPLC-MS / MS according to claim 1, characterized in that, The volume ratio of plasma, sample processing solution, and internal standard solution is 1:1:

3.

3. The method for detecting the content of enasidenib in plasma based on HPLC-MS / MS according to claim 1, characterized in that, The preparation method for Enasidenib standard solutions with different concentration gradients is as follows: prepare standard stock solutions with methanol, and then dilute with methanol to form 8 concentration gradients, namely 0 μg / mL, 0.05 μg / mL, 1 μg / mL, 1.75 μg / mL, 2.5 μg / mL, 10 μg / mL, 17.5 μg / mL, and 25 μg / mL.

4. The method for detecting the content of Enasidenib in plasma based on HPLC-MS / MS according to claim 1, characterized in that, It also includes quality control, which involves using quality control samples to verify the testing methods; The quality control materials consist of single-level low, medium, and high concentration Enasidenib quality control solutions; The low-concentration level quality control solution used was a 0.75 μg / mL Enasidenib standard solution; The medium-concentration level quality control solution used was a 7.5 μg / mL Enasidenib standard solution; The high-concentration quality control solution used was a 20 μg / mL Enasidenib standard solution.

Images