A method for determining the effective content in tofacitinib citrate tablets by nuclear magnetic resonance hydrogen spectrum

Abstract

The present invention discloses a method for determining the effective content in tofacitinib citrate tablets by nuclear magnetic resonance hydrogen spectrum. Using the chemical shift of tofacitinib δ at 7.13 as the quantitative peak, 3,5-dimethylpyrazole δ at 5.73 as the internal standard peak, deuterated dimethyl sulfoxide as the solvent, under the conditions of pulse sequence zg30, relaxation delay time of 26 s, and number of scans of 16 times at 298 K, the hydrogen spectrum of the mixture is collected to quantitatively determine the active ingredient in tofacitinib citrate tablets. This method has strong specificity and shows a good linear relationship in the range of 0.793-7.925 mg / mL ( r

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for determining the effective content in tofacitinib citrate tablets by nuclear magnetic resonance hydrogen spectrum. Background Art

[0002] Tofacitinib Citrate Tablets, with the chemical name of 3-[(3R,4R)-4-methyl-3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]-1-piperidinyl]-3-oxopropanenitrile-2-hydroxypropane-1,2,3-tricarboxylate, has a chemical structural formula as shown in Formula I.

[0003]

[0004] Chemical structural formula of tofacitinib citrate of Formula I

[0005] Tofacitinib Citrate Tablets was developed by Pfizer Inc. in the United States and was approved by the US Food and Drug Administration (FDA) on November 6, 2012. It is a kinase (JAK) inhibitor mainly used for the treatment of moderate to severe active rheumatoid arthritis (RA) that is insufficiently responsive or intolerant to methotrexate treatment, and it is a first-in-class drug for rheumatoid arthritis. It was approved for marketing by the China Food and Drug Administration (CFDA) on March 10, 2017. Currently, the main detection method for tofacitinib citrate tablets is high performance liquid chromatography, which has a long detection time and requires a reference standard. Nuclear magnetic resonance spectroscopy quantitative technology has the advantages of rapid detection, accuracy, no need for a reference substance, and can achieve qualitative and quantitative analysis simultaneously. Since it was first introduced into the Chinese Pharmacopoeia in 2010, it has been gradually applied to fields such as new drug research and development, pesticides, natural products, drug quality control, food detection, and reference substances.

[0006] Currently, the quality standards for tofacitinib citrate and its preparations have not been included in the pharmacopoeias of various countries, and there are few relevant literature reports. Therefore, the content determination method for tofacitinib citrate tablets needs to be studied. In this study, by using nuclear magnetic resonance hydrogen spectrum method, optimizing nuclear magnetic resonance test parameters, internal standard substances, and deuterated reagents, a content determination method for tofacitinib citrate tablets was developed, and its methodology was verified, and the determination results were verified by high performance liquid chromatography (HPLC). This experiment first used quantitative nuclear magnetic resonance hydrogen spectrum method (qHNMR) to detect the content of tofacitinib in tofacitinib citrate tablets, and achieved qualitative and quantitative analysis simultaneously, providing a rapid, simple, and accurate method for the quality control of tofacitinib citrate tablets. Summary of the Invention

[0007] The object of the present invention is to provide a method for determining the effective content in tofacitinib citrate tablets by nuclear magnetic resonance hydrogen spectrum.

[0008] Based on the above object, the present invention adopts the following technical solutions:

[0009] A method for determining the effective content in tofacitinib citrate tablets by nuclear magnetic resonance hydrogen spectrum, comprising the following steps:

[0010] (1) Determine the quantitative peaks of tofacitinib citrate and the internal standard 3,5-dimethylpyrazole in the solvent dimethyl sulfoxide-d6. The quantitative peak of tofacitinib citrate is the proton peak at δ7.13, and the quantitative peak of the internal standard 3,5-dimethylpyrazole is the proton peak at δ5.73; (2) Set the parameters of the nuclear magnetic resonance spectrometer, pulse sequence zg30; temperature 298K, spectral width (SWH) 20ppm, acquisition time (AQ) 4.09s; pulse width (P1) 14.63μs; dummy scan times (DS) 2 times; relaxation delay time (D1) 26s; scan times (NS) 16 times. Dissolve tofacitinib citrate and the internal standard 3,5-dimethylpyrazole in dimethyl sulfoxide-d6 to obtain a test sample solution, perform on-machine testing. After obtaining the hydrogen spectrum of the mixture, manually adjust the phase and baseline and integrate.

[0011] (3) Calculation formula for the mass content of tofacitinib:

[0012]

[0013] In this formula, A s is the peak area of the tofacitinib quantitative peak; A r is the peak area of the internal standard peak; n r is the number of protons contained in the internal standard quantitative peak; n s is the number of protons contained in the measured sample quantitative peak. In this formula, n r and n s are both 1; M s is the molecular weight of tofacitinib 312.38; M r is the molecular weight of the internal standard 96.13; m r is the weighed mass of the internal standard; m s is the weighed mass of the sample to be measured.

[0014] Furthermore, the specific preparation process of the test sample solution is as follows: Take tofacitinib citrate tablets, place them in an agate mortar and grind them into powder, and transfer them to a sample bottle for standby. Accurately weigh the powdered sample to be measured and 3,5-dimethylpyrazole into a centrifuge tube, add DMSO-d6 for ultrasonic dissolution, then centrifuge, and filter through a 0.45μm microporous filter membrane into a nuclear magnetic resonance tube. That is, the mass ratio of tofacitinib citrate tablets to 3,5-dimethylpyrazole is 40:1.

[0015] Preferably, the ultrasonic power is 250 W, and the concentration of tofacitinib citrate in DMSO-d6 is 200 mg / mL.

[0016] Furthermore, the nuclear magnetic resonance spectrometer is a Bruker Avance III 400 MHz nuclear magnetic resonance spectrometer.

[0017] This method has strong specificity and shows a good linear relationship (r 2 = 0.9984) in the range of 0.793 - 7.925 mg / mL. The relative standard deviations (RSD) of precision, repeatability, and stability are 0.82%, 1.70%, and 0.67% respectively. The mass percentage content of tofacitinib in tofacitinib citrate tablets measured by this method is 2.25%, and the result is basically consistent with that of the high performance liquid chromatography method. Description of the Drawings

[0018] Figure 1 is the 1H NMR spectrum of tofacitinib citrate deuterated DMSO solution; 1 HNMR spectrum;

[0019] Figure 2 is the 1H NMR spectrum of 3,5-dimethylpyrazole deuterated DMSO solution; 1 HNMR spectrum;

[0020] Figure 3 is the 1H NMR spectrum of the mixture of tofacitinib citrate tablets and 3,5-dimethylpyrazole; 1 HNMR spectrum;

[0021] Figure 4 is the linear equation graph of tofacitinib citrate obtained by the method of this application;

[0022] Figure 5 is the HPLC spectrum of tofacitinib citrate tablets. Detailed Embodiments

[0023] Instruments and Reagents

[0024] Bruker Avance III 400 MHz nuclear magnetic resonance spectrometer (Bruker Corporation, Switzerland), one hundred thousandth electronic analytical balance (Mettler Toledo, USA), KQ-250DB digital control ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), Pipet-Lite PL+ pipette (Mettler Toledo Group, USA), centrifuge 5430R (Eppendorf, Germany), Agilent 1260 high performance liquid chromatograph (Agilent Technologies, USA).

[0025] Tofacitinib Citrate Tablets (Qilu Pharmaceutical Co., Ltd., batch number H20193295), tofacitinib citrate reference standard (Wuhan Zhenchun Biotechnology Co., Ltd., content ≥ 99%), 3,5-dimethylpyrazole (Aladdin, content ≥ 99%), maleic acid (Aladdin, content ≥ 99%), dimethyl terephthalate (Aladdin, content ≥ 99%), deuterated dimethyl sulfoxide (DMSO-d6, Shanghai Saen Chemical Technology Co., Ltd., content: 99.8%, TMS 0.03%), trifluoroacetic acid (analytical grade), acetonitrile (chromatographic grade).

[0026] Example 1

[0027] A method for determining the effective content in tofacitinib citrate tablets by nuclear magnetic resonance hydrogen spectrum, comprising the following steps:

[0028] (1) Test conditions

[0029] qHNMR: Pulse sequence zg30; temperature 298K, spectral width (SWH) 20 ppm, acquisition time (AQ) 4.09 s; 90-degree pulse width (P1) 14.63 μs; number of dummy scans (DS) 2 times; relaxation delay time (D1) 26 s; number of scans (NS) 16 times, manually adjust the phase and baseline smoothing and integration.

[0030] HPLC: Chromatographic column Potensil C18 (4.6 mm × 250 mm, 5 μm), mobile phase A (0.02 v% trifluoroacetic acid + water), B (0.02 v% trifluoroacetic acid + acetonitrile), flow rate: 1.2 mL / min, gradient elution: 0.0 - 2.0 min, mobile phase B 10%; 2.0 - 10.0 min, mobile phase B 10% - 95%; 10.0 - 12.0 min, mobile phase B 95%; 12.0 - 13.0 min; mobile phase B 95% - 10%; 13.0 - 20.0 min, mobile phase B 10%. Column temperature: 40 °C, detection wavelength: 286 nm, injection volume: 5 μL, external standard method for quantification.

[0031] (2) Solution preparation

[0032] Preparation of nuclear magnetic resonance test solution: Take 30 tofacitinib citrate tablets, place them in an agate mortar and grind them into powder, and transfer them to a sample bottle for standby. Accurately weigh 200 mg of the powdered sample to be tested and 5 mg of 3,5-dimethylpyrazole into a centrifuge tube, add 1.0 mL of DMSO-d6 for dissolution, then ultrasonicate (ultrasonic power 250 W) for 10 min, and centrifuge at a centrifugal force of 16627 g, for 7 min, at a temperature of 25 °C, and filter through a 0.45 μm microporous filter membrane into a 5 mm nuclear magnetic resonance tube.

[0033] High Performance Liquid Chromatography Solution Preparation: Weigh 5 mg of tofacitinib citrate reference standard into a 25 mL volumetric flask, dissolve it with methanol, make up the volume to the mark to obtain the reference standard solution. Then weigh three portions of 30 mg of the powdered sample to be tested into 10 mL volumetric flasks respectively, dissolve and make up the volume with a methanol-water solution with a volume ratio of 50%, sonicate for 10 min under an ultrasonic power of 250 W, and filter through a 0.45 μm microporous filter membrane to obtain the solution to be tested.

[0034] (3) Calculation formula

[0035] Calculation formula for the mass content of tofacitinib:

[0036]

[0037] In this formula, A s is the peak area of the tofacitinib quantification peak; A r is the peak area of the internal standard peak; n r is the number of protons contained in the internal standard quantification peak; n s is the number of protons contained in the quantification peak of the sample to be tested. In this formula, n r and n s are both 1; M s is the molecular weight of tofacitinib, 312.38; M r is the molecular weight of the internal standard, 96.13; m r is the weighed mass of the internal standard; m s is the weighed mass of the sample to be tested.

[0038] Results and Discussion

[0039] 1. Selection of relaxation delay time (D1)

[0040] The relaxation delay time (D1) is an important parameter affecting the accuracy of the proton signal peak area in nuclear magnetic resonance quantitative experiments and is also a key factor in this experiment. D1 should be greater than or equal to 5 times T1 (longitudinal relaxation time) to make the integrated signal intensity be exactly proportional to the number of atomic nuclei and ensure the accuracy of the integrated peak area. If the D1 value is set too large, it will increase the sampling time of this experiment. Therefore, it is very important to select a reasonable D1 value in quantitative nuclear magnetic resonance. In this experiment, the inversion recovery method was used, and the T1 value of the internal standard at δ 5.73 was measured to be 5.17 s, and the T1 value of tofacitinib at δ 7.13 was 0.71 s. Therefore, the D1 value was 26 s.

[0041] 2. Selection of deuterated reagent and quantification peak

[0042] According to the solubility of tofacitinib citrate, DMSO-d6 was selected as the deuterated reagent. The residual solvent peak and water peak of DMSO-d6 have no interference with the tofacitinib quantification peak and the internal standard peak of the internal standard. At the same time, DMSO-d6 can dissolve the internal standard compound well.

[0043] DMSO-d6 was used as a solvent to dissolve tofacitinib citrate, 3,5-dimethylpyrazole, and a mixture of 3,5-dimethylpyrazole and tofacitinib citrate tablets. 1 1H-NMR test (see Figure 1 , Figure 2 and Figure 3 ). Since there are many excipients in tofacitinib citrate tablets and some of them are soluble in deuterated dimethyl sulfoxide, there are many peaks in the high field of the NMR spectrum, but there is no interference in the low field hydrogen. Therefore, the hydrogen in the low field was selected for quantification. The δ8.10 and δ8.09 correspond to the H-1 on the tofacitinib structure in Figure 1 , δ7.13 is H-2, and δ6.56 is H-3. During the experimental calculation process, H-1 and H-3 were used as quantitative peaks for integral quantitative calculation, and the results were almost the same as those of the H-2 quantitative peak detection. Therefore, the three hydrogens in the low field of the 1H-NMR spectrum of tofacitinib can all be used as quantitative peaks. In this application, δ7.13 was selected as the quantitative peak.

[0044] 3. Selection of internal standard and internal standard peak

[0045] The standard for selecting an internal standard in quantitative NMR is that it does not react with the analyte, has good solubility in the deuterated reagent, there should be no interference between the internal standard peak and the sample peak, and the resolution is good. Common internal standards such as maleic acid, dimethyl terephthalate, and 3,5-dimethylpyrazole were used for determination. The results showed that the proton peak of 3,5-dimethylpyrazole at δ5.73 did not overlap with the signal peak of tofacitinib citrate and there was no interference. Therefore, 3,5-dimethylpyrazole was selected as the internal standard, and δ5.73 was the internal standard peak.

[0046] 4. Methodological verification

[0047] (1) Investigation of linear relationship

[0048] Using an electronic balance, accurately weigh 51.49 mg of the internal standard 3,5-dimethylpyrazole and add it to 10 mL of DMSO-d6 to dissolve to obtain an internal standard solution with a concentration of 5.149 mg / mL. Accurately weigh 51.20 mg of tofacitinib citrate standard (the content of tofacitinib is 31.70 mg) into a volumetric flask and add 4.0 mL of the above internal standard solution to obtain a test solution with a tofacitinib concentration of 7.925 mg / mL. A series of solutions with tofacitinib concentrations of 7.925, 6.3468, 4.7601, 3.1734, 1.5867, and 0.79335 mg / mL were prepared by diluting the internal standard solution. NMR measurement, phase correction, and integration were performed (see Table 1). Using the ratio of the sample and internal standard concentrations as the abscissa (x) and the ratio of the quantitative peak and internal standard area as the ordinate (y) for linear regression, the linear regression equation of tofacitinib citrate was obtained as y = 0.303x - 0.010 (R2 = 0.998), (see Figure 4 ). From Figure 4 it can be seen that it is good in the concentration range of 0.80 - 7.93 mg / mL.

[0049] C s represents the concentration of tofacitinib reference standard, and C r represents the concentration of the internal standard.

[0050] Table 1 Linear Results

[0051]

[0052]

[0053] (2) Repeatability Test

[0054] Prepare 6 samples in magnetic tubes in parallel according to the method of preparing the test solution in "Step (2) of Example 1", and measure them according to the nuclear magnetic resonance conditions in "Step (1) of Example 1" to collect the hydrogen spectrum. Calculate the content of tofacitinib in tofacitinib citrate tablets respectively, and calculate the RSD value to be 1.70%. The results are shown in Table 2 in detail. From the results in Table 2, it can be seen that this method has good repeatability.

[0055] Table 2 Repeatability Results

[0056]

[0057] (3) Precision Test

[0058] Take the solution No. 6 in the repeatability test item, and continuously test it 6 times according to the nuclear magnetic resonance conditions in "Step (1) of Example 1", perform phase correction, baseline smoothing and manual integration. Calculate the relative standard deviation (RSD) as 0.82% based on the ratio of the quantitative peak area of the reference standard to the quantitative peak area of the internal standard (A s / A r ). The results are shown in Table 3 in detail. From Table 3, it can be seen that this method has good precision.

[0059] Table 3 Precision Results

[0060]

[0061] (4) Stability Test

[0062] Take the solution No. 6 in the repeatability test item and place it at room temperature. According to the nuclear magnetic resonance conditions in "Step (1) of Example 1", collect the hydrogen spectrum at 0, 6, 18, 24, 36, and 48 h respectively. Calculate the ratio of the quantitative peak area of the reference standard to the quantitative peak area of the internal standard (A s / A r) The relative standard deviation (RSD) was obtained as 0.67%, and the results are shown in Table 4. As can be seen from Table 4, the sample is stable within 48 hours at room temperature.

[0063] Table 4 Stability Results

[0064]

[0065] (5) HPLC and qHNMR Determination Results

[0066] qHNMR determination: According to the method in step (2) of Example 1, accurately weigh 3 portions of powdered tofacitinib citrate tablets, and use nuclear magnetic resonance hydrogen spectroscopy (qHNMR) respectively according to the test conditions in "step (1) of Example 1".

[0067] High performance liquid chromatography (HPLC) determination: The mass content of tofacitinib was determined by the external standard method. The high performance liquid detection solution was prepared according to "step (2) of Example 1". The standard product solution was detected by HPLC according to the HPLC detection method in "step (1) of Example 1", and the injection volumes were 1 μL, 2 μL, 4 μL, 6 μL, 8 μL, 10 μL, and 12 μL respectively for HPLC detection. With the concentration of tofacitinib as the abscissa x and the HPLC peak area as the ordinate y, the standard curve equation y = 262.4x + 4.766 (r 2 = 0.9999) was obtained. Then, the content of tofacitinib was obtained by substituting the peak area of the sample to be tested into the HPLC detection method in "step (1) of Example 1". The HPLC chromatogram of tofacitinib citrate tablets is as Figure 5 shown.

[0068] The same tofacitinib citrate tablet sample was quantitatively detected by HPLC and nuclear magnetic resonance hydrogen spectroscopy respectively, and the quantitative results of the content of tofacitinib citrate tablets were calculated. The results are shown in Table 5. As can be seen from the determination results, the average HPLC determination result (mass percentage) is 2.24%, and the average qHNMR determination result is 2.25%. The determination results of the two methods are relatively consistent.

[0069] Table 5 Quantitative Results of the Content of Tofacitinib Citrate Tablets Determined by HPLC and qHNMR Methods

[0070]

[0071] 5. Conclusion

[0072] In this paper, nuclear magnetic resonance hydrogen spectroscopy was used to determine the content of tofacitinib in tofacitinib citrate tablets, and the methodology of this method was verified. This analytical method has good repeatability and stability, high specificity, convenient operation, short time consumption, and does not require standard samples, and can achieve qualitative and quantitative analysis at the same time. The detection results are basically consistent with those detected by HPLC method. Quantitative nuclear magnetic resonance hydrogen spectroscopy provides an accurate and rapid new method for the quality control of tofacitinib citrate tablets.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the specific implementation technical solutions of the present invention rather than to limit it. Those of ordinary skill in the art should understand that any equivalent substitution or obvious modification of the implementation manner of the present invention without changing its performance or use should be covered within the scope of the present invention claimed.

Claims

1. A method for determining the effective content in tofacitinib citrate tablets by nuclear magnetic resonance hydrogen spectrum, characterized in that, It includes the following steps: (1) Determine the quantitative peaks of tofacitinib citrate and the internal standard 3,5-dimethylpyrazole in the solvent deuterated dimethyl sulfoxide. The quantitative peak of tofacitinib citrate is the proton peak at δ 7.13, and the quantitative peak of the internal standard 3,5-dimethylpyrazole is the proton peak at δ 5.73; (2) Set the parameters of the nuclear magnetic resonance spectrometer, pulse sequence zg30; temperature 298K, spectral width SWH 20 ppm, acquisition time AQ 4.09 s; Pulse width P1 14.63 μs; number of dummy scans DS 2 times; relaxation delay time D1 26 s; number of scans NS 16 times. Dissolve tofacitinib citrate and the internal standard 3,5-dimethylpyrazole in deuterated dimethyl sulfoxide to obtain a test sample solution, and perform on-machine testing. After obtaining the hydrogen spectrum of the mixture, manually adjust the phase and baseline and integrate; (3) Calculation formula for the mass content of tofacitinib: In this formula, is the peak area of the tofacitinib quantification peak; is the peak area of the internal standard peak; is the number of protons contained in the internal standard quantification peak; is the number of protons contained in the quantification peak of the sample to be measured. In this formula, and are both 1; is the molecular weight of tofacitinib, 312.38; is the molecular weight of the internal standard, 96.13; is the weighed mass of the internal standard; is the weighed mass of the sample to be tested.

2. The method for determining the effective content in tofacitinib citrate tablets by nuclear magnetic resonance hydrogen spectrum according to claim 1, characterized in that, The specific preparation process of the test sample solution is as follows: Take tofacitinib citrate tablets, place them in an agate mortar and grind them into powder, and transfer them to a sample bottle for standby. Accurately weigh the powdered sample to be tested and 3,5-dimethylpyrazole into a centrifuge tube, add DMSO-d6 for ultrasonic dissolution, then centrifuge, and filter through a 0.45 μm microporous filter membrane into a nuclear magnetic tube. That is, the mass ratio of tofacitinib citrate tablets to 3,5-dimethylpyrazole is 40:

1.

3. The method for determining the effective content in tofacitinib citrate tablets by nuclear magnetic resonance hydrogen spectrum according to claim 1, characterized in that, The nuclear magnetic resonance spectrometer is a Bruker Avance III 400 MHz nuclear magnetic resonance spectrometer.

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