Analysis method for determining multiple residual solvents in sodium dibutyryladenosine cyclophosphate
Through gas chromatography combined with specific fixing liquid and detector, the problem of determining multiple residual solvents in dibutyryl cyclophosphate adenosine sodium is solved, and high sensitivity and accuracy are achieved, ensuring drug safety and product quality.
Patent Information
- Application Number
- CN202311371246.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-23
- Publication Date
- 2025-04-25
AI Technical Summary
The prior art has failed to effectively determine the various residual solvents in dibutyryl cyclophosphate adenosine sodium, especially the polarity of the five solvents is relatively different, which makes it difficult to develop detection methods and affects drug safety and product quality.
Using gas chromatography, a capillary chromatography column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as the fixed solution was used, combined with a hydrogen flame ionization detector and nitrogen as the carrier gas, N,N-dimethylacetamide as the sample solvent, headspace injection, and the content of residual solvent was calculated using the external standard method based on the peak area, and the test sample was sonicated to improve the sample loading recovery rate.
The high sensitivity, strong specificity and accuracy of various residual solvents in dibutyryl cyclophosphate adenosine sodium is achieved, and it meets the requirements of drug safety and product quality.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of chemical analysis, and in particular to an analytical method for determining multiple residual solvents in sodium dibutyryl adenosine cyclophosphate. Background Art
[0002] Bucladesine Sodium, chemical name: N6, 2′-O-dibutyryladenosine 3′, 5′-cyclic phosphate sodium salt, is a protein kinase activator used for adjuvant treatment of angina pectoris and acute myocardial infarction. It can also be used for myocarditis, cardiogenic shock, postoperative subretinal hemorrhage and psoriasis, and can assist other anticancer drugs in the treatment of leukemia.
[0003] At present, sodium dibutyryladenosine cyclophosphate is obtained through an organic synthesis process, and a large number of organic solvents are used in the entire synthesis process. According to the classification and regulations of residual solvents in the "Pharmacopoeia of the People's Republic of China", the residual amounts of the first, second, and third category solvents must be controlled within a certain range. At present, the solvents used in the synthesis of sodium dibutyryladenosine cyclophosphate raw materials are mainly methyl tert-butyl ether, triethylamine, ethanol, acetone, and 1,4-dioxane. At present, the pharmacopoeias of various countries have not monitored the residual solvents of sodium dibutyryladenosine cyclophosphate raw materials. Considering that the organic solvents used in the synthesis process cannot be completely removed, in order to strictly control the residual solvent limit and ensure the quality of the finished product, a residual solvent determination method needs to be established. At the same time, The existing literature may have corresponding detection methods for these residual solvents alone or for a mixture of two or three solvents, but there are no reports of detection methods that include these five solvents. This is because the polarities of these five solvents vary greatly, making method development more difficult.
[0004] Therefore, it is urgent to develop an analytical method for determining multiple residual solvents in sodium dibutyryladenosine cyclophosphate, which can determine the residual solvents in sodium dibutyryladenosine cyclophosphate and ensure the safety of drugs and product quality. Summary of the invention
[0005] The object of the present invention is to provide a method for determining the residual solvent in sodium dibutyryl adenosine cyclophosphate, which can determine the residual solvent in sodium dibutyryl adenosine cyclophosphate and has the advantages of high sensitivity, strong specificity, high accuracy and the like.
[0006] The present invention provides an analytical method for determining multiple residual solvents in sodium dibutyryladenosine cyclophosphate, which adopts a capillary chromatographic column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as a stationary liquid by gas chromatography, a hydrogen flame ionization detector, nitrogen as a carrier gas, N,N-dimethylacetamide as a sample solvent, headspace injection, and the content of the residual solvent is calculated by peak area according to the external standard method; wherein, the preparation process of the reference solution and the test solution includes the step of ultrasonically treating the headspace bottle, and the power of the ultrasound is 35-45kHz; the gas chromatography conditions are: 6% cyanopropylphenyl-94% dimethylpolysiloxane as a stationary liquid capillary chromatographic column, ... Capillary chromatographic column, 30m×0.53mm, 3μm, FID detector temperature: 240℃~260℃, headspace inlet temperature: 180℃~200℃, N2 gas flow rate: 2.0ml / min~4.0ml / min, split ratio 4~6:1; heating program: start at 45℃ and hold for 8 minutes, increase the temperature to 220℃ at 15℃ / min, and hold for 3min, headspace temperature: 70℃~90℃, equilibrium time: 30min~50min, test sample concentration: 0.05g / ml; the multiple residual solvents are ethanol, acetone, methyl tert-butyl ether, triethylamine and 1,4-dioxane.
[0007] Furthermore, the cyanopropylphenyl-dimethylpolysiloxane fixing liquid is selected from any one of DB-624, AT-624, Rtx-624, PE-624, 007-624, 007-502, CP-624, ZB-624, VF-624ms, DB-1301 or a combination thereof.
[0008] Further, the FID detector temperature is selected from 240°C, 245°C, 250°C, 255°C or 260°C, the headspace inlet temperature is selected from 180°C, 190°C or 200°C, the N2 gas flow rate is selected from 2.0ml / min, 3.0ml / min or 4.0ml / min, the split ratio is selected from 4:1, 5:1 or 6:1, the headspace temperature is selected from 70, 75°C, 80°C, 85°C or 90°C, and the equilibration time is selected from 30min, 40min or 50min.
[0009] Furthermore, the analytical method for determining multiple residual solvents in sodium dibutyryl adenosine cyclophosphate comprises the following steps: preparation of reference solution, preparation of test solution, data detection, and content calculation.
[0010] Furthermore, in the analytical method for determining multiple residual solvents in sodium dibutyryladenosine cyclophosphate described in item 1, the power of the ultrasound is 35, 40 or 45 kHz.
[0011] Furthermore, the preparation of the reference solution includes the following steps: taking an appropriate amount of residual solvent reference, preparing a mixed solution with a solvent, accurately measuring 2.0 ml into a 20 ml headspace bottle, sealing, and preparing a reference solution. The preparation of the test solution includes the following steps: accurately weighing 0.25 g of sodium dibutyryladenosine cyclophosphate, placing it in a 10 ml volumetric bottle, dissolving and diluting it to the scale with a solvent, shaking well, accurately measuring 2.0 ml into a 20 ml headspace bottle, sealing, and preparing a test solution.
[0012] Furthermore, the residual solvent reference in the reference solution is selected from one or more of ethanol, acetone, methyl tert-butyl ether, triethylamine and 1,4-dioxane, wherein the concentration of ethanol is 0.25 mg / ml, the concentration of acetone is 0.25 mg / ml, the concentration of methyl tert-butyl ether is 0.25 mg / ml, the concentration of triethylamine is 0.016 mg / ml, and the concentration of 1,4-dioxane is 0.019 mg / ml.
[0013] Compared with the prior art, the present invention has the following beneficial effects: the residual solvent determination method of the present invention uses a medium polarity chromatographic column, and can achieve the separation and determination of multiple residual solvents in the presence of polar and weak polar residual solvents. The use of ultrasonic treatment of the test sample can further improve the sample recovery rate of the residual solvent, which is more conducive to the accurate determination of the residual solvent in the test sample. It has been verified that the method of this aspect is highly sensitive, highly specific, and highly accurate. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.
[0015] Figure 1 This is a gas chromatogram of the test solution (batch 01 of crude dibutyryladenosine sodium cyclophosphate API) of Example 1 of the present invention; Figure 2 This is a gas chromatogram of the test solution (batch 02 of crude dibutyryladenosine sodium cyclophosphate bulk drug) of Example 1 of the present invention; Figure 3 This is a gas chromatogram of the test solution (batch 03 of crude dibutyryladenosine sodium cyclophosphate bulk drug) of Example 1 of the present invention; Figure 4 is the gas chromatogram of blank solvent; Figure 5 This is the gas chromatogram of the reference solution (system suitability solution). Implementation
[0016] The present invention will be specifically described below in conjunction with embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and are not intended to limit the essence of the present invention.
[0017] The analysis method of residual solvent in sodium dibutyryladenosine cyclophosphate of the present invention preferably uses Agilent 7890B gas chromatograph and FID detector. The chromatographic column model is a capillary chromatographic column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as the stationary liquid (column length 30m, inner diameter 0.53mm, liquid film thickness 3μm). Example
[0018] 1. Chromatographic conditions Column temperature: programmed temperature, initial temperature 45°C, maintained for 8 min, increased to 220°C at a rate of 15°C / min, maintained for 3 min; Inlet temperature: 180°C; Detector temperature: 260°C; The carrier gas was nitrogen; the split ratio was 5:1.
[0019] 2. Solution preparation Blank solution: N,N-dimethylacetamide.
[0020] Control stock solution: Take 0.25 g each of anhydrous ethanol, acetone, methyl tert-butyl ether and triethylamine, and 0.19 g of 1,4-dioxane, accurately weigh, place in the same 100 ml volumetric flask, add N,N-dimethylacetamide to dilute to the scale, shake well, and obtain.
[0021] Control solution: Accurately measure 2 ml of the reference stock solution and place it in a 20 ml volumetric flask. Add N,N-dimethylacetamide to dilute to the scale, shake well, accurately measure 2 ml, place it in an empty bottle, and seal.
[0022] Test solution: Take about 0.1 g of the product, weigh it accurately, place it in an empty bottle, add 2 ml of N,N-dimethylacetamide accurately, seal it, and shake it to dissolve.
[0023] 3. Sample testing (1) System suitability test: inject one injection of blank solution, and no solvent peak with the same retention time as the target solvent peak should appear. Inject 5 injections of control solution and record the chromatogram. The separation between the target solvent peak and the adjacent peaks should not be less than 1.5; the theoretical plate number of the target solvent peak should not be less than 5000, and the tailing factor should be less than 2.0; the RSD of the target solvent peak area of 5 injections should be ≤10.0%.
[0024] (2) Sample testing: Inject 2 needles of test solution, record the chromatogram and record the peak area of each solvent.
[0025] 4. Three batches of test samples (using homemade crude dibutyryladenosine sodium cyclophosphate, batch numbers 01, 02, and 03) were tested according to the above determination method.
[0026] .
[0027] The results are shown in Table 1 and the chromatogram is attached. Figure 1 , Attachment Figure 2 With attached Figure 3 The results showed that ethanol and triethylamine were not detected in the three batches of samples; the chromatographic peak separation of the remaining components was greater than 1.5, and they could be completely separated, and the system had good applicability.
[0028] 5. Methodological Validation (1) According to the above solution preparation and chromatographic conditions, inject a blank solution. Take the control solution and inject it into the chromatographic system. Inject 6 injections in succession, record the chromatogram, and record the retention time, separation degree, and theoretical plate number of each impurity.
[0029] The system suitability test data are shown in Table 2. The blank solvent chromatographic peak has no interference with the target impurity peaks. The separation of each impurity peak in the system suitability chromatogram is greater than 1.5. The theoretical plate number and tailing factor meet the requirements. In the 5-injection system suitability solution, the relative standard deviations of the peak areas of ethanol, acetone, methyl tert-butyl ether, triethylamine, and 1,4-dioxane are 1.3%, 1.3%, 1.5%, 1.9%, and 1.1%, respectively, indicating that this method can meet the determination of the above 5 solvents of dibutyryl adenosine cyclophosphate.
[0030] .
[0031] (2) Precision test According to the above solution preparation and chromatographic conditions, inject 1 injection of blank solution, 5 injections of control solution 1, 1 injection of control solution 2, and 1 injection of precision solution respectively, and record the chromatograms, calculate the content of each residual solvent and the corresponding RSD, and calculate the relative standard deviation of each impurity content.
[0032] The precision test data are shown in Table 3. The relative standard deviations of ethanol, acetone, methyl tert-butyl ether, triethylamine and 1,4-dioxane in the 6 precision solutions were 1.7%, 1.8%, 1.9%, 2.0% and 2.1%, respectively, which met the test requirements, indicating that the precision of this method complies with the relevant guidelines.
[0033] .
[0034] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the scope of protection of the claims of the present invention.
Claims
1. An analytical method for determining various residual solvents in sodium dibutyryladenosine cyclophosphate, using a capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as the stationary phase, a hydrogen flame ionization detector, nitrogen as the carrier gas, N,N-dimethylacetamide as the sample solvent, headspace injection, and calculating the residual solvent content by peak area according to the external standard method; wherein, The preparation process of the reference solution and the test solution includes the step of ultrasonically treating the headspace bottle, wherein the power of the ultrasound is 35-45kHz; the gas chromatography conditions are: a capillary chromatographic column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as the stationary liquid, 30m×0.53mm, 3μm, FID detector temperature: 240℃~260℃, headspace injection port temperature: 180℃~200℃, N2 gas flow rate: 2.0ml / min~4.0ml / min, split ratio 4~6:1; heating program: starting at 45℃ and holding for 8 minutes, heating to 220℃ at 15℃ / min, holding for 3min, headspace temperature: 70℃~90℃, equilibrium time: 30min~50min, test sample concentration: 0.05g / ml; the multiple residual solvents are ethanol, acetone, methyl tert-butyl ether, triethylamine and 1,4-dioxane.
2. The analytical method for determining multiple residual solvents in sodium dibutyryladenosine cyclophosphate according to claim 1, characterized in that: The cyanopropylphenyl-dimethylpolysiloxane fixing liquid is selected from any one of DB-624, AT-624, Rtx-624, PE-624, 007-624, 007-502, CP-624, ZB-624, VF-624ms, DB-1301 or a combination thereof.
3. The analytical method for determining multiple residual solvents in sodium dibutyryladenosine cyclophosphate according to claim 1, characterized in that: The FID detector temperature is selected from 240°C, 245°C, 250°C, 255°C or 260°C, the headspace inlet temperature is selected from 180°C, 190°C or 200°C, the N2 gas flow rate is selected from 2.0ml / min, 3.0ml / min or 4.0ml / min, the split ratio is selected from 4:1, 5:1 or 6:1, the headspace temperature is selected from 70, 75°C, 80°C, 85°C or 90°C, and the equilibration time is selected from 30min, 40min or 50min.
4. The analytical method for determining multiple residual solvents in sodium dibutyryladenosine cyclophosphate according to claims 1 to 3, characterized in that: The following steps are involved: Preparation of reference solution, preparation of test solution, data detection, and content calculation.
5. The analytical method for determining multiple residual solvents in sodium dibutyryladenosine cyclophosphate according to any one of claims 1 to 4, characterized in that: The power of the ultrasound is 35, 40 or 45 kHz.
6. The analytical method for determining multiple residual solvents in sodium dibutyryladenosine cyclophosphate according to claim 5, characterized in that: The preparation of the reference solution comprises the following steps: Take an appropriate amount of residual solvent reference substance, prepare a mixed solution with the solvent, accurately measure 2.0 ml into a 20 ml headspace bottle, seal it, and prepare the reference solution. The preparation of the test solution includes the following steps: accurately weigh 0.25 g of dibutyryladenosine sodium cyclophosphate, place it in a 10 ml volumetric bottle, dissolve and dilute it to the scale with the solvent, shake well, accurately measure 2.0 ml into a 20 ml headspace bottle, seal it, and prepare the test solution.
7. The analytical method for determining multiple residual solvents in sodium dibutyryladenosine cyclophosphate according to claim 6, characterized in that: The residual solvent reference in the reference solution is selected from one or more of ethanol, acetone, methyl tert-butyl ether, triethylamine and 1,4-dioxane, wherein the concentration of ethanol is 0.25 mg / ml, the concentration of acetone is 0.25 mg / ml, the concentration of methyl tert-butyl ether is 0.25 mg / ml, the concentration of triethylamine is 0.016 mg / ml, and the concentration of 1,4-dioxane is 0.019 mg / ml.