Analysis method of phosphoric acid impurities in acute pancreatitis bulk drug

Abstract

The invention relates to the technical field of medicine analysis, in particular to a method for analyzing phosphoric acid impurities of an acute pancreatitis raw material medicine, and the phosphoric acid impurities are analyzed by high performance liquid chromatography in a gradient elution mode. According to the chromatographic column, positive ions and negative ions are jointly embedded into alkyl bonded porous silica gel to serve as a filling agent; the mobile phase A is selected from an acidic aqueous solution, the mobile phase B is selected from an organic solvent, and the flow velocity is 0.5-0.7 mL / min; the gas flow rate of the evaporative scattered light detector is 1.5-1.7 L / min; experimental results show that the analysis method can be used for trace detection of phosphoric acid in bulk drugs and is high in specificity, and a blank solvent does not interfere with detection of phosphoric acid impurities; the sensitivity is high, the quantitation limit is 5.3650 [mu] g / ml, and the detection limit is 0.26825 [mu] g / ml; the accuracy is good, and the average recovery rate is 80-120%.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to an analysis method for phosphate impurities in a raw material drug for acute pancreatitis. Background Art

[0002] A raw material drug for acute pancreatitis, referred to as WXTJ0262, is an innovative drug for the treatment of acute pancreatitis. It is an inhibitor of calcium ion release-activated channels, mainly through inhibiting calcium release-activated calcium channel protein 1 (Calcium release activates calcium channel 1, Orai1) / matrix acting molecule 1 (Matrixacting molecule 1, STIM1)-mediated Ca 2+ The current and calcium release activate calcium channel protein 2 (Orai2) / STIM1-mediated current, preventing the increase of calcium ions and inappropriate digestive enzyme activation in the acinar cells in the pancreas, preventing acinar cell dysfunction, death or necrosis, inhibiting the Icrac pathway in T cells, and blocking the release of pro-inflammatory cytokines such as IL-2 and IL-17. The molecular structure is shown below: Phosphoric acid is an important fragment of WXTJ0262. It is a process impurity and stability-related degradation impurity in the preparation process, which affects the drug quality of WXTJ0262. Impurity research is the premise and key link to ensure the safety and quality of drugs. Whether such impurities can be accurately detected is related to the quality and safety of WXTJ0262 finished products.

[0003] At present, the main detection methods for phosphoric acid at home and abroad are ion chromatography and HPLC-DRD. The ion chromatograph used in ion chromatography is less popular than HPLC, with high analysis cost and expensive daily maintenance. HPLC-DRD can only be used for isocratic elution and cannot use gradient elution, which makes the elution of the main component more difficult. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the object of the present invention is to provide an analytical method for phosphate impurities in a raw material drug for acute pancreatitis.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: A method for analyzing phosphate impurities in a raw material drug for acute pancreatitis, wherein the structural formula of the raw material drug is as follows: ; The phosphoric acid impurities are analyzed by high performance liquid chromatography in a gradient elution manner; Wherein, liquid chromatography includes the following conditions: Chromatographic column: alkyl-bonded porous silica gel with cations and anions embedded in it as filler; Mobile phase A is selected from an acidic aqueous solution, and mobile phase B is selected from an organic solvent; Flow rate: 0.5~0.7mL / min; The evaporative scattering light detector includes the following conditions: gas flow rate is 1.5 to 1.7 L / min; During the gradient elution process, the volume proportions of mobile phase A and mobile phase B are as follows: after the start of elution, they change uniformly until the 2nd minute, with mobile phase A changing from 0 to 50%, and mobile phase B changing from 100% to 50%; then they change uniformly until the 5th minute, with mobile phase A at 95% and mobile phase B at 5%; from the 5th to the 10th minute, they remain constant, with mobile phase A at 95% and mobile phase B at 5%; then they change uniformly until the 10.5th minute, with mobile phase A at 0 and mobile phase B at 100%; from the 10.5th to the 15th minute, they remain constant, with mobile phase A at 0 and mobile phase B at 100%.

[0006] Furthermore, the chromatographic column is selected from an intelligent liquid chromatography column, preferably a Comixsil ACRP chromatographic column.

[0007] Furthermore, the acidic aqueous solution is selected from an ammonium formate aqueous solution whose pH value is adjusted by formic acid or an ammonium acetate aqueous solution whose pH value is adjusted by acetic acid.

[0008] Furthermore, the pH is 2.5 to 3.5.

[0009] Furthermore, the organic solvent is selected from acetonitrile.

[0010] Furthermore, the column temperature of the chromatographic column is 33-37°C.

[0011] Furthermore, the conditions of the evaporative scattered light detector also include: the temperature of the atomization chamber is 40-50°C, and the temperature of the drift tube is 40-50°C.

[0012] Furthermore, the quantitative limit concentration of the phosphoric acid impurity is no higher than 5.40 μg / ml.

[0013] Furthermore, the detection limit concentration of the phosphoric acid impurity is no higher than 0.27 μg / ml.

[0014] Furthermore, when analyzing the phosphoric acid impurity, the API enters the chromatographic column in the form of its aqueous solution.

[0015] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: The present invention provides an analytical method for phosphate impurities in a raw material drug for acute pancreatitis, which can be used for trace detection of phosphoric acid in WXTJ0262. The analytical method has strong specificity, and a blank solvent does not interfere with the detection of phosphate impurities; the sensitivity is high, the quantitative limit is 5.3650 μg / ml, and the detection limit is 0.26825 μg / ml; the accuracy is good, and the average recovery rate is between 80% and 120%, showing a significant detection effect advantage.

[0016] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the HPLC chromatogram of the diluent provided in Example 2 of the present invention.

[0018] Figure 2 It is the HPLC chromatogram of the reference substance solution provided in Example 2 of the present invention.

[0019] Figure 3 It is the HPLC chromatogram of the test solution provided in Example 2 of the present invention.

[0020] Figure 4 It is the HPLC chromatogram of the quantitative limit solution provided in Example 2 of the present invention.

[0021] Figure 5 It is the HPLC chromatogram of the detection limit solution provided in Example 2 of the present invention.

[0022] Figure 6 It is the HPLC chromatogram of the 50% level standard solution provided in Example 2 of the present invention.

[0023] Figure 7 It is the HPLC chromatogram of the 100% level standard solution provided in Example 2 of the present invention.

[0024] Figure 8 It is the HPLC chromatogram of the 105% level standard solution provided in Example 2 of the present invention. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention. Some terms used in the present invention are listed below.

[0026] Specificity: refers to the ability of the analytical method to correctly measure the presence of other components (such as impurities, degradation products, excipients, etc.).

[0027] Linearity: refers to the ability to maintain a proportional relationship between the test results and the concentration of the analyte in the sample within the range of the experimental design.

[0028] Sensitivity: refers to the degree of change in the response of a method to a change in unit concentration or unit amount of the substance to be tested. It is the basic attribute of the analytical method, indicating the detection capability of the method, including the detection limit and quantification limit.

[0029] Limit of Detection (LOD): refers to the lowest amount of the analyte that can be detected in the sample.

[0030] Limit of Quantitation (LOQ): refers to the lowest amount of the analyte in the sample that can be quantitatively determined. For the analysis of trace or trace drugs, quantitative determination of drug impurities and degradation products, the quantitative limit of the method should be determined.

[0031] Accuracy: It is the degree of closeness between the measured value and the true value or reference value, which directly affects the effectiveness of drug quality control. Accuracy is a necessary condition for quantitative measurement. The systematic error of the method can be evaluated through recovery experiments (such as adding a standard of known concentration).

[0032] The signal-to-noise ratio (S / N) refers to the ratio of the signal intensity generated by the target analyte to the background noise intensity, which is used to evaluate the sensitivity and reliability of the analytical method.

[0033] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0034] Example 1 1. Selection of mobile phase type in high performance liquid chromatography detection.

[0035] The acidic aqueous solution of mobile phase A was investigated using 0.01mol / L ammonium formate aqueous solution (pH adjusted to 3.0 with formic acid) and 0.01mol / L ammonium acetate aqueous solution (pH adjusted to 3.0 with acetic acid). The results showed that the baseline noise and peaks were basically the same when 0.01mol / L ammonium formate aqueous solution (pH adjusted to 3.0 with formic acid) and 0.01mol / L ammonium acetate aqueous solution (pH adjusted to 3.0 with acetic acid) were used for gradient elution. Therefore, the acidic aqueous solution of mobile phase A was selected to be 0.01mol / L ammonium formate aqueous solution (pH adjusted to 3.0 with formic acid) or 0.01mol / L ammonium acetate aqueous solution (pH adjusted to 3.0 with acetic acid).

[0036] 2. Selection of chromatographic column.

[0037] The effects of two types of chromatographic columns on phosphoric acid detection were investigated, namely, octadecyl bonded silica gel as a filler column and cation and anion co-embedded alkyl bonded porous silica gel as a filler column. Through comparative experiments, it was found that when using octadecyl bonded silica gel columns, phosphoric acid impurities could not be retained and the baseline was interfered; when using Comixsil ACRP columns, phosphoric acid was well retained and completely separated from blank and sample interference peaks, achieving an ideal separation effect. Therefore, the chromatographic column should be Comixsil ACRP columns or intelligent liquid chromatography columns with equivalent performance.

[0038] 3. Optimization of evaporative scattered light detector conditions.

[0039] In order to ensure the sensitivity and accuracy of the analytical method for phosphoric acid impurities in WXTJ0262 raw material medicine provided by the present invention, an evaporative scattering light detector was selected to measure phosphoric acid, and the effects of four different parameter settings of the evaporative scattering light detector on the baseline and sensitivity were investigated respectively. The results are as follows: Parameter I: atomization chamber temperature 30°C, drift tube temperature 30°C, gas flow rate 1.5L / min; Parameter II: atomization chamber temperature 40°C, drift tube temperature 40°C, gas flow rate 1.5L / min; Parameter III: atomization chamber temperature 45°C, drift tube temperature 45°C, gas flow rate 1.6L / min; Parameter IV: atomization chamber temperature 50°C, drift tube temperature 50°C, gas flow rate 1.7L / min; The above results show that when the evaporative scattering light detector is set according to parameter Ⅰ, the baseline fluctuation of the detection result is large and the sensitivity is poor; when the evaporative scattering light detector is set according to parameter Ⅱ, parameter Ⅲ and parameter Ⅳ, the baseline and sensitivity of the detection result are good and the difference is not large. Therefore, the optimal parameter settings of the evaporative scattering light detector are: the spray chamber temperature is 40-50℃, the drift tube temperature is 40-50℃, and the gas flow rate is 1.5-1.7L / min.

[0040] Example 2 The chromatographic conditions of High Performance Liquid Chromatography (HPLC) are shown in Table 1.

[0041] 1. Investigation of the specificity of the analytical method for phosphate impurities in the raw material drug for acute pancreatitis provided by the present invention.

[0042] Diluent: Water.

[0043] Reference solution: Take an appropriate amount of phosphoric acid, weigh it accurately, and add diluent to quantitatively dilute it to make a solution containing about 50 μg of phosphoric acid per 1 ml.

[0044] Test solution: Take an appropriate amount of WXTJ0262 raw material, weigh accurately, add an appropriate amount of diluent, dissolve by ultrasonic for 5 minutes, and quantitatively dilute with diluent to make a solution of about 5 mg per 1 ml.

[0045] Take the diluent, reference solution, and test solution and test them according to the set chromatographic conditions. The results are as follows: Figure 1 , Figure 2 and Figure 3 As shown, the results show that the diluent has no interference with the determination of phosphate impurities, and the separation degree between the phosphate impurities in the test solution and the adjacent peaks is greater than 1.5, indicating that the analysis method for phosphate impurities in the raw material drug for acute pancreatitis provided by the present invention has strong specificity.

[0046] 2. Investigation of the sensitivity of the analytical method for phosphate impurities in the raw material drug for acute pancreatitis provided by the present invention.

[0047] Phosphoric acid aqueous solution was prepared with water as solvent, and diluted step by step. The solution with signal-to-noise ratio S / N ≥ 10 was used as the quantitative limit solution; the solution with signal-to-noise ratio S / N ≥ 3 was used as the detection limit solution. The test results were as follows: Figure 4 and Figure 5 As shown, the results show that the analytical method provided by the present invention has high detection sensitivity for the phosphate impurities in the WXTJ0262 raw material drug, wherein the detection limit concentration of the phosphate impurities is 0.26825 μg / ml, and the quantitative limit concentration of the phosphate impurities is 5.3650 μg / ml.

[0048] 3. The linear range of the analytical method for phosphate impurities in the raw material drug for acute pancreatitis provided by the present invention.

[0049] Phosphoric acid linear solutions of different concentrations (5 μg / ml, 25 μg / ml, 75 μg / ml, 100 μg / ml, 125 μg / ml, respectively) were prepared with water as solvent, and the detection was carried out according to the set chromatographic conditions. The standard curve was drawn with lg (peak area) of phosphoric acid as the ordinate y and lg (concentration) of phosphoric acid as the abscissa x. The results showed that the linear regression equation of phosphoric acid in the concentration range of 5 μg / ml to 125 μg / ml was y=1.6139x-0.1150, and the correlation coefficient was 0.9990, indicating that the analytical method for determining phosphoric acid impurities in WXTJ0262 raw materials by high performance liquid chromatography provided by the present invention had good linearity.

[0050] 4. Investigation of the accuracy of the analytical method for phosphate impurities in the raw material drug for acute pancreatitis provided by the present invention.

[0051] The WXTJ0262 API with determined phosphoric acid content was used as a sample, and phosphoric acid solutions with concentrations of 26.8250μg / ml, 53.6500μg / ml, and 80.4750μg / ml were added (each addition level was measured in parallel 3 times) for accuracy testing. The test spectrum is shown in the figure below. Figure 6 , Figure 7 and Figure 8 The test results are shown in Table 2. From the data provided in the table, it can be seen that the recovery rate of phosphoric acid is in the range of 97% to 119%, and the relative standard deviation is 7.6%, indicating that the high performance liquid chromatography method provided by the present invention has good accuracy in analyzing phosphoric acid impurities in WXTJ0262 raw materials.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for analyzing phosphoric acid impurities in a raw material drug for acute pancreatitis, characterized in that: The structural formula of the raw material is as follows: ； The phosphoric acid impurities are analyzed by high performance liquid chromatography in a gradient elution manner; Wherein, liquid chromatography includes the following conditions: Chromatographic column: alkyl-bonded porous silica gel with cations and anions embedded in it as filler; Mobile phase A is selected from an acidic aqueous solution, and mobile phase B is selected from an organic solvent; Flow rate: 0.5~0.7mL / min; The evaporative scattering light detector includes the following conditions: gas flow rate is 1.5 to 1.7 L / min; During the gradient elution process, the volume proportions of mobile phase A and mobile phase B are as follows: after the start of elution, they change uniformly until the 2nd minute, with mobile phase A changing from 0 to 50%, and mobile phase B changing from 100% to 50%; then they change uniformly until the 5th minute, with mobile phase A at 95% and mobile phase B at 5%; from the 5th to the 10th minute, they remain constant, with mobile phase A at 95% and mobile phase B at 5%; then they change uniformly until the 10.5th minute, with mobile phase A at 0 and mobile phase B at 100%; from the 10.5th to the 15th minute, they remain constant, with mobile phase A at 0 and mobile phase B at 100%.

2. The method for analyzing phosphoric acid impurities in a bulk drug for acute pancreatitis according to claim 1, wherein: The chromatographic column is selected from an intelligent liquid chromatography column.

3. The method for analyzing phosphate impurities in a bulk drug for acute pancreatitis according to claim 1, wherein: The acidic aqueous solution is selected from an aqueous ammonium formate solution whose pH value is adjusted by formic acid or an aqueous ammonium acetate solution whose pH value is adjusted by acetic acid.

4. The method for analyzing phosphoric acid impurities in a bulk drug for acute pancreatitis according to claim 3, wherein: The pH is 2.5-3.

5.

5. The method for analyzing phosphoric acid impurities in a bulk drug for acute pancreatitis according to claim 1, wherein: The organic solvent is selected from acetonitrile.

6. The method for analyzing phosphoric acid impurities in a bulk drug for acute pancreatitis according to claim 1, wherein: The column temperature of the chromatographic column is in the range of 33-37°C.

7. The method for analyzing phosphate impurities in a bulk drug for acute pancreatitis according to claim 1, wherein: The conditions of the evaporative scattered light detector also include: the temperature of the atomization chamber is 40-50°C, and the temperature of the drift tube is 40-50°C.

8. The method for analyzing phosphoric acid impurities in a bulk drug for acute pancreatitis according to claim 1, wherein: The quantitative limit concentration of phosphoric acid impurity is not higher than 5.40μg / ml.

9. The method for analyzing phosphoric acid impurities in a bulk drug for acute pancreatitis according to claim 1, wherein: The detection limit concentration of phosphate impurities is no higher than 0.27μg / ml.

10. The method for analyzing phosphoric acid impurities in a bulk drug for acute pancreatitis according to claim 1, wherein: When analyzing for phosphate impurities, the API enters the column as an aqueous solution.

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