An analytical method for phosphoric acid impurities in the active pharmaceutical ingredient for acute pancreatitis

Abstract

The present invention relates to the technical field of pharmaceutical analysis, and particularly relates to an analytical method for phosphoric acid impurities in raw materials for acute pancreatitis. The phosphoric acid impurities are analyzed by gradient elution using high performance liquid chromatography; wherein, the chromatographic column is filled with a cation and an anion co-embedded alkyl-bonded porous silica gel; mobile phase A is selected from acidic aqueous solutions, mobile phase B is selected from organic solvents, and the flow rate is 0.5-0.7 mL / min; the gas flow rate of the evaporative light scattering detector is 1.5-1.7 L / min; the experimental results show that this analytical method can be used for trace detection of phosphoric acid in raw materials, has strong specificity, and the blank solvent does not interfere with the detection of phosphoric acid impurities; it has high sensitivity, the quantitative limit is 5.3650 μg / ml, and the detection limit is 0.26825 μg / ml; it has good accuracy, and the average recovery rate is between 80% and 120%.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis, and particularly to an analytical method for phosphoric acid impurities in the raw material drug for acute pancreatitis. Background Art

[0002] A raw material drug for acute pancreatitis, designated as WXTJ0262, is an innovative drug for the treatment of acute pancreatitis. It belongs to inhibitors of calcium release-activated channels and mainly inhibits the Ca 2+ currents mediated by calcium release-activated calcium channel protein 1 (Orai1) / matrix-acting molecule 1 (STIM1) and the currents mediated by calcium release-activated calcium channel protein 2 (Orai2) / STIM1, preventing the increase in intracellular calcium ions and the inappropriate activation of digestive enzymes in pancreatic acinar cells, 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 structural formula is shown as follows:

[0003]

[0004] Phosphoric acid is an important fragment of WXTJ0262 and is a process impurity and a degradation impurity related to stability during the preparation process, affecting the drug quality of WXTJ0262. Impurity research is a prerequisite and a 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 the finished product of WXTJ0262.

[0005] Currently, the main detection methods for phosphoric acid at home and abroad are ion chromatography and high performance liquid chromatography - refractive index detector method. The ion chromatograph used in ion chromatography is less popular than high performance liquid chromatograph, with high analysis cost and expensive daily maintenance cost. The high performance liquid chromatography - refractive index detector method can only be used for isocratic elution and cannot adopt gradient elution, resulting in difficult elution of the main component. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the related art. For this purpose, the objective of the present invention is to provide an analytical method for phosphoric acid impurities in the raw material drug for acute pancreatitis.

[0007] To achieve the above objective, the technical solutions adopted by the present invention are as follows:

[0008] An analytical method for phosphoric acid impurities in the active pharmaceutical ingredient for acute pancreatitis, the structural formula of the active pharmaceutical ingredient is as follows:

[0009] ;

[0010] The phosphoric acid impurities are analyzed by high performance liquid chromatography in a gradient elution manner;

[0011] Among them, the liquid chromatography includes the following conditions:

[0012] Chromatographic column: filled with porous silica gel with cation and anion co-embedded alkyl bonding;

[0013] Mobile phase A is selected from acidic aqueous solutions, and mobile phase B is selected from organic solvents;

[0014] Flow rate: 0.5 - 0.7 mL / min;

[0015] The evaporative light scattering detector includes the following conditions: gas flow rate is 1.5 - 1.7 L / min;

[0016] During the gradient elution process, the volume ratio of mobile phase A and mobile phase B is as follows: After the start of elution, it changes uniformly to the 2nd minute, mobile phase A changes from 0 to 50%, and mobile phase B changes from 100% to 50%; then it changes uniformly to the 5th minute, mobile phase A is 95%, and mobile phase B is 5%; from the 5th minute to the 10th minute, it is constant, mobile phase A remains at 95%, and mobile phase B remains at 5%, then it changes uniformly to the 10.5th minute, mobile phase A is 0, and mobile phase B is 100%; from the 10.5th minute to the 15th minute, it is constant, mobile phase A is 0, and mobile phase B remains at 100%.

[0017] Furthermore, the chromatographic column is selected from intelligent liquid chromatographic columns, preferably Comixsil ACRP chromatographic column.

[0018] Furthermore, the acidic aqueous solution is selected from ammonium formate aqueous solution adjusted by formic acid or ammonium acetate aqueous solution adjusted by acetic acid.

[0019] Furthermore, the pH is 2.5 - 3.5.

[0020] Furthermore, the organic solvent is selected from acetonitrile.

[0021] Furthermore, the column temperature of the chromatographic column is 33 - 37 °C.

[0022] Furthermore, the conditions of the evaporative light scattering detector also include: the temperature of the nebulization chamber is 40 - 50 °C, and the temperature of the drift tube is 40 - 50 °C.

[0023] Further, the quantitative limit concentration of the phosphoric acid impurity is not higher than 5.40 μg / ml.

[0024] Further, the detection limit concentration of the phosphoric acid impurity is not higher than 0.27 μg / ml.

[0025] Further, when analyzing the phosphoric acid impurity, the bulk drug enters the chromatographic column in the form of its aqueous solution.

[0026] One or more of the above technical solutions in the embodiments of the present invention have at least one of the following technical effects:

[0027] The present invention provides a method for analyzing phosphoric acid impurities in a bulk drug for acute pancreatitis, which can be used for trace detection of phosphoric acid in WXTJ0262. This analysis method has strong specificity, and the blank solvent does not interfere with the detection of phosphoric acid impurities; it has high sensitivity, with a quantitative limit of 5.3650 μg / ml and a detection limit of 0.26825 μg / ml; it has good accuracy, and the average recovery rates are all between 80% and 120%, showing significant advantages in detection effects.

[0028] The additional aspects and advantages of the present invention will be partly given in the following description, partly will become obvious from the following description, or will be understood through the practice of the present invention. Description of the Drawings

[0029] Figure 1 is the HPLC chromatogram of the diluent provided in Example 2 of the present invention.

[0030] Figure 2 is the HPLC chromatogram of the reference substance solution provided in Example 2 of the present invention.

[0031] Figure 3 is the HPLC chromatogram of the test solution provided in Example 2 of the present invention.

[0032] Figure 4 is the HPLC chromatogram of the quantitative limit solution provided in Example 2 of the present invention.

[0033] Figure 5 is the HPLC chromatogram of the detection limit solution provided in Example 2 of the present invention.

[0034] Figure 6 is the HPLC chromatogram of the 50% level standard addition solution provided in Example 2 of the present invention.

[0035] Figure 7 is the HPLC chromatogram of the 100% level standard addition solution provided in Example 2 of the present invention.

[0036] Figure 8 is the HPLC chromatogram of the 105% level standard addition solution provided in Example 2 of the present invention. Detailed implementation manners

[0037] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below with reference to the accompanying drawings in the present invention. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts belong to 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.

[0038] Specificity: It refers to the ability of an analytical method to accurately determine in the presence of other components (such as impurities, degradation products, excipients, etc.).

[0039] Linearity: It refers to the ability of the proportional relationship between the test results and the concentration of the analyte in the sample within the range designed in the experiment.

[0040] Sensitivity: It refers to the degree of change in the response caused by the change of a unit concentration or unit amount of the analyte to be measured in a certain method. It is a basic property of the analytical method, indicating the detection ability of the method, including the limit of detection and the limit of quantitation.

[0041] Limit of Detection (LOD): It refers to the lowest amount of the analyte to be measured in the sample that can be detected.

[0042] Limit of Quantitation (LOQ): It refers to the lowest amount of the analyte to be measured in the sample that can be quantitatively determined. For trace or ultratrace drug analysis and quantitative determination of drug impurities and degradation products, the limit of quantitation of the method should be determined.

[0043] 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 determination. The systematic error of the method can be evaluated through a recovery experiment (such as adding a standard product with a known concentration).

[0044] Signal-to-Noise Ratio (S / N) is the ratio of the signal intensity generated by the target analyte to the background noise intensity, and is used to evaluate the sensitivity and reliability of the analytical method.

[0045] In the following embodiments, unless otherwise specified, the experimental methods used are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0046] Example 1

[0047] I. Selection of mobile phase types in high performance liquid chromatography detection.

[0048] The acidic aqueous solutions of mobile phase A were investigated using 0.01 mol / L ammonium formate aqueous solution (adjusted to pH 3.0 with formic acid) and 0.01 mol / L ammonium acetate aqueous solution (adjusted to pH 3.0 with acetic acid) respectively. The results showed that the baseline noise and peak emergence were basically the same when gradient elution was carried out using 0.01 mol / L ammonium formate aqueous solution (adjusted to pH 3.0 with formic acid) and 0.01 mol / L ammonium acetate aqueous solution (adjusted to pH 3.0 with acetic acid). Therefore, the acidic aqueous solution of mobile phase A was selected as 0.01 mol / L ammonium formate aqueous solution (adjusted to pH 3.0 with formic acid) or 0.01 mol / L ammonium acetate aqueous solution (adjusted to pH 3.0 with acetic acid).

[0049] II. Selection of chromatographic column.

[0050] The effects of two types of chromatographic columns, namely the chromatographic column filled with octadecylsilane bonded silica gel and the chromatographic column filled with porous silica gel with co-embedded cations and anions in the alkyl group, on the detection of phosphoric acid were investigated respectively. Through comparative experiments, it was found that when using the octadecylsilane bonded silica gel chromatographic column, phosphoric acid impurities could not be retained and there was interference in the baseline; when using the Comixsil ACRP chromatographic column, the retention effect of phosphoric acid was good, and it was completely separated from the blank and sample interference peaks, achieving an ideal separation effect. Therefore, the Comixsil ACRP chromatographic column or an intelligent liquid chromatographic column with equivalent performance was selected as the chromatographic column.

[0051] III. Optimization of evaporative light scattering detector conditions.

[0052] To ensure the sensitivity and accuracy of the analytical method for phosphoric acid impurities in the WXTJ0262 bulk drug provided by the present invention, an evaporative light scattering detector was selected to determine phosphoric acid. The effects of four different parameter settings of the evaporative light scattering detector on the baseline and sensitivity were investigated respectively, and the results were as follows:

[0053] Parameter I: Nebulizer temperature 30 °C, drift tube temperature 30 °C, gas flow rate 1.5 L / min;

[0054] Parameter II: Nebulizer temperature 40 °C, drift tube temperature 40 °C, gas flow rate 1.5 L / min;

[0055] Parameter III: Nebulizer temperature 45 °C, drift tube temperature 45 °C, gas flow rate 1.6 L / min;

[0056] Parameter IV: Nebulizer temperature 50 °C, drift tube temperature 50 °C, gas flow rate 1.7 L / min;

[0057] The above results show that when the evaporative light scattering detector is set according to Parameter Ⅰ, the baseline of the detection result fluctuates greatly and the sensitivity is poor; when the evaporative light scattering detector is set according to Parameter Ⅱ, Parameter Ⅲ, and Parameter Ⅳ, both the baseline and sensitivity of the detection result are good and the difference is not significant. Therefore, the preferred parameter settings for the evaporative light scattering detector are: the nebulizer temperature is 40 - 50 °C, the drift tube temperature is 40 - 50 °C, and the gas flow rate is 1.5 - 1.7 L / min.

[0058] Example 2

[0059] The chromatographic conditions of high performance liquid chromatography (HPLC) are shown in Table 1.

[0060]

[0061] I. Specificity investigation of the analytical method for phosphoric acid impurities in the raw material drug for acute pancreatitis provided by the present invention.

[0062] Diluent: water.

[0063] Reference solution: Take an appropriate amount of phosphoric acid, weigh it accurately, and quantitatively dilute it with the diluent to prepare a solution containing about 50 μg of phosphoric acid per 1 ml.

[0064] Test solution: Take an appropriate amount of the raw material drug WXTJ0262, weigh it accurately, add an appropriate amount of the diluent, dissolve it by ultrasonic treatment for 5 minutes, and quantitatively dilute it with the diluent to prepare a solution containing about 5 mg per 1 ml.

[0065] Take the diluent, reference solution, and test solution and perform detection according to the set chromatographic conditions. The results are as Figure 1 , Figure 2 and Figure 3 shown. The results show that the diluent has no interference on the determination of phosphoric acid impurities, and the resolution between the phosphoric acid impurities in the test solution and the adjacent peaks nearby is greater than 1.5, indicating that the analytical method for phosphoric acid impurities in the raw material drug for acute pancreatitis provided by the present invention has strong specificity.

[0066] II. Sensitivity investigation of the analytical method for phosphoric acid impurities in the raw material drug for acute pancreatitis provided by the present invention.

[0067] Prepare an aqueous phosphoric acid solution with water as the solvent and dilute it step by step. The solution with a signal-to-noise ratio S / N ≥ 10 of the aqueous phosphoric acid solution is used as the quantitative limit solution; the solution with a signal-to-noise ratio S / N ≥ 3 is used as the detection limit solution. The detection results are as Figure 4 and Figure 5As shown, the results indicate that the analytical method provided by the present invention has high detection sensitivity for the determination of phosphoric acid impurities in the WXTJ0262 bulk drug. Among them, the detection limit concentration of phosphoric acid impurities is 0.26825 μg / ml, and the quantitation limit concentration of phosphoric acid impurities is 5.3650 μg / ml.

[0068] III. Linear range of the analytical method for phosphoric acid impurities in the bulk drug for acute pancreatitis provided by the present invention.

[0069] Using water as the solvent, linear solutions of phosphoric acid with different concentrations (concentrations are 5 μg / ml, 25 μg / ml, 75 μg / ml, 100 μg / ml, 125 μg / ml respectively) were prepared and detected according to the set chromatographic conditions. Taking the lg(peak area) of phosphoric acid as the ordinate y and the lg(concentration) of phosphoric acid as the abscissa x, a standard curve was plotted. The results show that in the concentration range of 5 μg / ml to 125 μg / ml for phosphoric acid, the linear regression equation is y = 1.6139x - 0.1150, and the correlation coefficient is 0.9990, indicating that the analytical method for determining phosphoric acid impurities in the WXTJ0262 bulk drug by the high performance liquid chromatography method provided by the present invention has good linearity.

[0070] IV. Investigation on the accuracy of the analytical method for phosphoric acid impurities in the bulk drug for acute pancreatitis provided by the present invention.

[0071] Using the WXTJ0262 bulk drug whose phosphoric acid content has been determined as the sample, phosphoric acid solutions with concentration levels of 26.8250 μg / ml, 53.6500 μg / ml, and 80.4750 μg / ml (each addition level was determined in parallel 3 times) were added for accuracy detection. The detection chromatograms are as Figure 6 、 Figure 7 and Figure 8 shown. The detection results are shown in Table 2. From the data provided in this 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 for the analysis of phosphoric acid impurities in the WXTJ0262 bulk drug.

[0072]

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate 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; Wherein, the acidic aqueous solution is selected from an aqueous solution of ammonium formate whose pH value is adjusted to 2.5 to 3.5 by formic acid or an aqueous solution of ammonium acetate whose pH value is adjusted to 2.5 to 3.5 by acetic acid, and the organic solvent is selected from acetonitrile; 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 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.

4. The method for analyzing phosphoric acid 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.

5. 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.

6. 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.

7. 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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