CN115791999A - Method for detecting impurity C of peramivir injection - Google Patents

Abstract

The invention provides a method for detecting impurity C in a peramivir injection. The method for detecting impurity C in the peramivir injection comprises the following steps: detecting by high performance liquid chromatography under the following conditions: protein bonded silica gel is used as a filling agent for a chromatographic column, and the detection wavelength is as follows: 203-207 nm, column temperature: 18-22 ℃, flow rate: 0.2-0.4 ml/min. The method adopts a reversed-phase chiral chromatography system to establish the detection method of the impurity C in the peramivir injection, performs systematic methodological verification on the method, can be better suitable for detecting the impurity C in the peramivir injection through the investigation of specificity, linearity, range, accuracy test, repeatability test, intermediate precision test, solution stability test, durability test and the like, and is beneficial to improving the quality control level of the peramivir injection.

Description

Method for detecting impurity C of peramivir injection

Technical Field

The invention relates to the field of impurity detection of peramivir injection, in particular to a method for detecting impurity C of the peramivir injection.

Background

Peramivir (Peramivir), a novel anti-influenza virus drug, has been proved to be effective against influenza A and B by current clinical trial data.

The synthesis route of the peramivir bulk drug is as follows:

the specific synthesis method is that the starting material is subjected to ring-opening and reduction reaction under the action of reducing agent sodium borohydride to generate an intermediate I; carrying out esterification reaction on the intermediate I and acetic anhydride to generate an intermediate II; the intermediate II is subjected to hydrolysis reaction under the action of sodium hydroxide to generate an intermediate III; and deprotecting the intermediate III under the action of hydrochloric acid to generate an intermediate IV, directly reacting the unseparated intermediate IV with an initial raw material II to generate a peramivir crude product, and finally carrying out crystal transformation to generate the peramivir. The important dosage form is intravenous injection. Impurity C of the peramivir injection mainly comes from raw material process impurities. The quality standard of the peramivir injection is not recorded in pharmacopoeia of various countries, and a detection method for impurity C of the peramivir injection is not included; the method for detecting the impurity C in the quality standard of the peramivir raw material supplier adopts a normal phase chromatography system, the product is injection, and the solubility in the normal phase chromatography system is poor. Only the State food and drug administration receives the peramivir sodium chloride injection (100 ml: peramivir C) ₁₅ H ₂₈ N ₄ O ₄ 0.3g and 0.9g of sodium chloride) standard (YBH 01412013) also does not describe a method for detecting impurity C in peramivir injection.

The chemical name of the impurity C (corresponding isomer) is (1R, 2R,3S, 4S) -3- [ (1R) -1- (acetylamino) -2-ethylbutyl)]-4-guanidine-2-hydroxycyclopentanecarboxylic acid trihydrate having the molecular formula C ₁₅ H ₂₈ N ₄ O ₄ ·3H ₂ O, molecular weight 382.45, structure as follows:

disclosure of Invention

In view of the above, the invention provides a method for detecting impurity C in peramivir injection, which solves the technical problems.

The technical scheme of the invention is realized as follows:

the method for detecting impurity C in the peramivir injection comprises the following steps: detecting by high performance liquid chromatography under the following conditions: a chromatographic column:

AGP, detection wavelength: 203-207 nm, column temperature: 18-22 ℃, flow rate: 0.2-0.4 ml/min.

Furthermore, each 1L of phosphate buffer solution contains 4.75 to 5.25mmol/L diammonium hydrogen phosphate and 4.75 to 5.25mmol/L ammonium dihydrogen phosphate.

Further, the solution formulation comprises:

impurity C localization solution: precisely weighing impurity C, adding a solvent to dissolve and dilute the impurity C until each 1ml of the impurity C contains 0.015-0.025 mg of solution;

test solution: accurately measuring the peramivir injection, diluting the injection with a solvent until every 1ml of the injection contains 0.4-0.6 mg of peramivir, and uniformly mixing;

control solution: the sample solution is diluted with solvent to a solution containing 0.8-1.2 mug peramivir in each 1ml, and the solution is mixed evenly.

Further, the solvent is water.

Further, assay: precisely measuring the test solution, the reference solution and the blank solvent, respectively injecting into a liquid chromatograph, and recording the chromatogram.

Further, the sample injection volume is as follows: 3-5 mul.

Further mobile phase: 5mmol/L diammonium phosphate and 5mmol/L ammonium dihydrogen phosphate buffer solution.

Further, the specification of the column was 150 mm. Times.4.0 mm,5 μm.

Further, the detection wavelength: 205nm, column temperature: 20 ℃, flow rate: 0.3ml/min, injection volume: 4 μ l.

Further, impurity C localizes the solution: precisely weighing impurity C, adding solvent to dissolve and dilute until each 1ml of the impurity C contains 0.02mg of solution;

test solution: accurately measuring the peramivir injection, diluting the peramivir injection with a solvent until each 1ml of the injection contains 0.5mg of peramivir, and uniformly mixing;

control solution: the test solution is diluted with solvent to a concentration of 1. Mu.g Peramivir per 1ml, and mixed.

Compared with the prior art, the invention has the beneficial effects that:

the method adopts a reversed-phase chiral chromatography system to establish the detection method of the impurity C in the peramivir injection, performs systematic methodological verification on the method, can be better suitable for detecting the impurity C in the peramivir injection through the investigation of specificity, linearity, range, accuracy test, repeatability test, intermediate precision test, solution stability test, durability test and the like, and is beneficial to improving the quality control level of the peramivir injection.

Drawings

FIG. 1 is a solvent HPLC profile in the validation of the method of the invention in a specificity test;

FIG. 2 is a chart of HPLC analysis of blank excipients in the invention for validation of specificity tests;

FIG. 3 is a HPLC chromatogram of a positioning solution for impurity O in a validation specificity test;

FIG. 4 is a HPLC chromatogram of a positioning solution of impurity Q in a validation specificity test;

FIG. 5 is a HPLC chromatogram of an impurity S positioning solution in a validation specificity test;

FIG. 6 is a HPLC chromatogram of an impurity T-positioning solution in a validation specificity test;

FIG. 7 is a HPLC chromatogram of a solution of a test sample to be added in a test for verifying specificity according to the present invention;

FIG. 8 is a HPLC chromatogram of a test solution for a specific test according to the present invention;

FIG. 9 is a HPLC profile of a control solution in the present invention for validation of specificity;

FIG. 10 is a HPLC chromatogram of a spiked mixed solution in a validation specificity test of the present invention;

FIG. 11 is a linear graph of peramivir for the linear test of the present invention;

FIG. 12 is a linear plot of impurity C for the inventive linear test.

Detailed Description

In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.

The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.

The materials, reagents and the like used in the examples of the present invention are commercially available unless otherwise specified.

Examples

Method for detecting impurity C in peramivir injection

By high performance liquid chromatography

And (3) chromatographic column:

AGP，150mm×4.0mm，5μm

detection wavelength: 205nm

Column temperature: 20 deg.C

Flow rate: 0.3ml/min

Sample introduction volume: 4 μ l

Mobile phase: 5mmol/L diammonium hydrogen phosphate and 5mmol/L ammonium dihydrogen phosphate buffer solution

Solvent: water (I)

Solution preparation:

impurity C localization solution: an appropriate amount of impurity C is precisely weighed, and a solvent is added to dissolve and dilute the impurity C until about 0.02mg of the impurity C is contained in each 1ml of the impurity C.

Test solution: 1ml of peramivir injection is precisely measured, placed in a 20ml measuring flask, diluted to scale by adding a solvent and shaken up.

Control solution: precisely measuring 1ml of the test solution, placing the test solution in a 20ml measuring flask, diluting the test solution to a scale with a solvent, shaking up, precisely measuring 1ml of the test solution, placing the test solution in a 25ml measuring flask, diluting the test solution to a scale with a solvent, and shaking up.

The determination method comprises the following steps: precisely measuring the sample solution, the reference solution and the blank solvent by 4 μ l each, injecting into a liquid chromatograph, and recording chromatogram.

Limitation: calculated according to a self-comparison method without adding a correction factor, the impurity C is not more than 0.2 percent.

And (II) complete methodology verification is carried out on the analysis method according to the four guiding principles of the year-round edition of Chinese pharmacopoeia 2020. The method for verifying the content comprises the following steps: specificity, linearity and range, accuracy tests, repeatability tests, intermediate precision tests, solution stability tests, and durability tests. The information on the samples used for the validation is shown in table 1.

Table 1 verification of use sample information

1 specificity test

1.1 brief description

The method has the advantages that peak identification and selectivity are investigated through special research, the separation degree of the impurity C and the main peak is ensured to meet the standard through positioning investigation on each impurity, each impurity can be well separated from the main peak and the impurity C, and the auxiliary material solution and the solvent do not interfere with measurement. 1.2 operating method:

blank adjuvant solution: precisely measuring 1ml of the whole auxiliary material solution, placing the whole auxiliary material solution into a 20ml measuring flask, diluting the whole auxiliary material solution to the scale with a solvent, and shaking up.

Impurity O localization solution: precisely weighing about 2mg of impurity O, placing in a 10ml measuring flask, adding a solvent to dissolve and dilute to a scale, shaking up to obtain a stock solution of the impurity O, precisely weighing 1ml, placing in a 20ml measuring flask, diluting to a scale with the solvent, and shaking up.

Impurity Q localization solution: precisely weighing about 2mg of impurity Q, placing in a 10ml measuring flask, adding a solvent to dissolve and dilute to scale, shaking up to obtain a stock solution of the impurity Q, precisely weighing 1ml, placing in a 20ml measuring flask, diluting to scale with the solvent, and shaking up.

Impurity S localization solution: taking about 2mg of the impurity S, accurately weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and dilute to a scale, shaking up to be used as a stock solution of the impurity S, accurately weighing 1ml, placing in a 20ml measuring flask, diluting to a scale with the solvent, and shaking up.

Impurity T localization solution: precisely weighing about 2mg of the impurity T, placing the impurity T in a 10ml measuring flask, adding a solvent to dissolve and dilute the impurity T to a scale, shaking up the impurity T to serve as a stock solution of the impurity T, precisely weighing 1ml, placing the impurity T in a 20ml measuring flask, diluting the impurity T to the scale with the solvent, and shaking up the impurity T. The structural formulas of the impurity O, the impurity Q, the impurity S and the impurity T are shown in the following table 2:

TABLE 2

Impurity C localization solution: precisely weighing about 5mg of impurity C, placing in a 50ml measuring flask, adding a solvent to dissolve and dilute to scale, shaking up to obtain impurity C stock solution, precisely weighing 5ml, placing in a 25ml measuring flask, adding a solvent to dissolve and dilute to scale, and shaking up.

Impurity mixed solution: precisely measuring 1ml of impurity O, Q, S and T stock solutions respectively, placing in a 10ml measuring flask, adding solvent to dilute to scale, and shaking up.

Test solution: precisely measuring 1ml of peramivir injection, placing the injection into a 20ml measuring flask, adding a solvent to dilute the injection to a scale, and shaking up.

Adding a standard test solution: accurately measuring 1ml of each of the peramivir injection and the impurity C positioning solution, placing the peramivir injection and the impurity C positioning solution into a 20ml measuring flask, adding a solvent to dilute to a scale, and shaking up.

Control solution: precisely measuring 1ml of the sample solution, placing in a 20ml measuring flask, diluting with solvent to scale, shaking, precisely measuring 1ml, placing 25ml, diluting with solvent to scale, and shaking.

Adding a standard mixed solution: precisely measuring 1ml of peramivir injection, 1ml of impurity mixed solution and 1ml of impurity C positioning solution, placing into a 20ml measuring flask, adding a solvent to dilute to a scale, and shaking up to obtain a mixed sample solution with a label.

Precisely measuring the blank solvent, the blank auxiliary material solution, the sample solution, the added standard sample solution, the reference solution, the added standard mixed solution and each impurity positioning solution by 4 mu l respectively, injecting into a liquid chromatograph, and recording the chromatogram. The results are shown in Table 3 and FIGS. 1-10.

1.3 test results

TABLE 3 results of the specificity test

1.4 conclusion

The blank solvent and the blank auxiliary material solution do not interfere with the measurement; in the standard-added mixed solution, the impurities O and Q do not interfere the measurement of the impurities C and main components, the peak-off time of the impurities S and the peak-off time of the impurities C coincide, the impurities are strictly controlled to be combined to calculate the impurity amount, the separation degree of the impurities T and the impurities S/C is 1.41, less than 1.5 but more than 1.2, the baseline separation can be realized, and the specificity of the method is good.

2 Linear test

2.1 brief description

And (4) performing a linear test on the impurity C by combining the impurity analysis condition of the synthesis process of the product. In linear tests, the linear correlation coefficient r should be greater than 0.990 and the ratio of the y-intercept to the 100% theoretical concentration response should be less than 25%.

2.2 method of operation

Linear stock solution: taking 11.5mg of peramivir trihydrate, precisely weighing, placing in a 100ml measuring flask, adding a solvent for dissolving and diluting to a scale, shaking up, precisely weighing 1ml and 1ml of impurity C stock solution under a special test item, placing in a 50ml measuring flask, diluting to the scale with the solvent, shaking up, and taking as a linear stock solution.

Precisely measuring 1ml, 3ml, 5ml, 6ml and 7.5ml of linear stock solutions respectively, placing in different 10ml measuring bottles, diluting to scale with solvent, shaking up to obtain linear solutions of 20%, 60%, 100%, 120% and 150%.

The 20%, 60%, 100%, 120% and 150% linear solutions were measured precisely and measured at 4. Mu.l each, and the solutions were injected into a liquid chromatograph, respectively, and the chromatogram was recorded. And (4) performing linear regression by using the concentration as an abscissa and the peak area as an ordinate by using a least square method, and calculating a correlation coefficient. The results are shown in Table 4.

2.3 test results

TABLE 4 results of the Linear test

2.4 conclusion

The linear correlation coefficient r of the peramivir is 0.99996 which is more than 0.990, and the ratio of the Y-axis intercept to the 100% theoretical concentration response value is 0.46% which is less than 25%; the linear correlation coefficient r of the impurity C is 0.99712 and is more than 0.990, the ratio of the Y-axis intercept to the response value of 100% theoretical concentration is 1.30% and less than 25%, and the correction factor of the impurity C is 1.05 and is in the range of 0.9-1.1, so that the self-control method without adding the correction factor can be adopted for determination.

3 limit of quantitation and limit of detection test

3.1 brief description

The signal-to-noise ratio of each peak in the quantitation limit solution should be greater than 10, the retention time RSD of each peak should be less than 1.0%, the RSD of the peak area should be less than 10%, the quantitation limit should be less than 50% of the limit or the reporting limit (take the minimum). The signal to noise ratio of each peak in the quantitation limit solution should be greater than 3.

3.2 method of operation

Quantitative limiting solution: precisely measuring 5ml of 20% linear solution, placing in a 10ml measuring flask, adding water to dilute to the scale mark, and shaking up.

Detection limiting solution: precisely measuring 5ml of the limiting solution, placing the limiting solution in a 10ml measuring flask, adding water to dilute the limiting solution to a scale, and shaking up the limiting solution.

Precisely measuring 4 μ l of the quantitative limiting solution and the detection limiting solution, respectively injecting into a liquid chromatograph, and recording chromatogram. The results are shown in Table 5.

3.3 test results

TABLE 5 quantitation and detection limits test results

3.4 conclusion

The limit solution concentration of the impurity C is 0.103 mu g/ml, the limit is 0.02 percent and is less than 0.05 percent, the determination is repeated for 6 times, and the signal-to-noise ratio is minimum 12.71 and is more than 10; RSD of retention time 0.11%, less than 1%; the RSD of the peak area is 5.59 percent and less than 10 percent, which meets the requirement. The concentration of the limiting solution for the quantification of the peramivir is 0.115 mu g/ml, the limit is 0.02 percent and is less than 0.05 percent, the repeated determination is carried out for 6 times, and the signal-to-noise ratio is minimum 13.20 and is more than 10; RSD of retention time 0.08%, less than 1%; the RSD of the peak area is 4.75 percent and is less than 10 percent, which meets the requirement. In the detection limit solution, the signal-to-noise ratios of the impurity C and the peramivir are respectively 5.96 and 6.63 and are more than 3, and the requirements are met.

4 accuracy test

4.1 brief description

By combining the impurity analysis of the synthesis process of the product, the accuracy of the impurity C is researched, and the accuracy is inspected by adopting the concentrations of 50%, 100% and 150% so as to ensure the accuracy of the detection result.

4.2 methods of operation

Control solution: precisely measuring 1ml of impurity C positioning solution under a special test item, putting the solution into a 20ml measuring flask, adding a solvent for quantitative dilution to a scale, and shaking up. Two portions were prepared in parallel.

Blank test solution: 1ml of peramivir injection is precisely measured, placed in a 20ml measuring flask, diluted to scale by adding a solvent and shaken up.

50% recovery solution: precisely measuring 1ml of peramivir injection, placing the injection in a 20ml measuring flask, precisely adding 0.5ml of impurity C positioning solution under a special test item, adding a solvent to dilute to a scale, shaking up, adding the solvent to dilute to the scale, and shaking up. (parallel preparation of 3 portions)

100% recovery solution: precisely measuring 1ml of peramivir injection, placing the peramivir injection in a 20ml measuring flask, precisely adding 1ml of impurity C positioning solution under a special test item, adding a solvent to dilute to a scale, shaking up, adding the solvent to dilute to the scale, and shaking up. (parallel preparation of 3 portions)

150% recovery solution: precisely measuring 1ml of peramivir injection, placing the injection in a 20ml measuring flask, precisely adding 1.5ml of impurity C positioning solution under a special test item, adding a solvent to dilute to a scale, shaking up, adding the solvent to dilute to the scale, and shaking up. (parallel preparation 3 parts)

Precisely measuring the above solutions each at 4 μ l, injecting into liquid chromatograph, and recording chromatogram. The results are shown in Table 6.

4.3 test results

TABLE 6 accuracy test results

4.4 conclusion:

the average recovery of 50-150% of the recovered solution was 94.0%, ranging from 80-120%, with an RSD of 2.77%. Less than 10%, the accuracy is good.

5 durability test

5.1 brief description

And (5) inspecting the influence of small changes of chromatographic parameters and changes of instrument chromatographic behavior on the detection result. The bearing degree of the detection result which is not influenced is determined by the variation of flow rate +/-0.1 ml/min, the variation of column temperature +/-2 ℃, and the variation of detection wavelength +/-2 nm.

5.2 methods of operation

Blank adjuvant solution: precisely measuring 1ml of the whole auxiliary material solution, placing the whole auxiliary material solution into a 20ml measuring flask, diluting the whole auxiliary material solution to the scale with a solvent, and shaking up.

Adding a standard mixed solution: precisely measuring 1ml of peramivir injection, 1ml of impurity mixed solution under a special test item and 1ml of impurity C positioning solution under the special test item, placing the mixture in a 20ml measuring flask, adding a solvent to dilute the mixture to a scale, and shaking the mixture uniformly to obtain the labeled mixed solution.

Control solution: precisely measuring 1ml of the added standard mixed solution, placing the mixed solution in a 20ml measuring flask, diluting the mixed solution to the scale by using a solvent, shaking up, precisely measuring 1ml of the mixed solution, placing the mixed solution in a 25ml measuring flask, diluting the mixed solution to the scale by using the solvent, and shaking up.

Precisely measuring the above labeling mixed solution, control solution, blank adjuvant solution and blank solvent by 4 μ l each, respectively injecting into liquid chromatograph under the conditions of flow rate variation of + -0.1 ml/min, column temperature variation of + -2 deg.C, and detection wavelength variation of + -2 nm, and recording chromatogram. The results are shown in Table 7.

5.3 test results

Table 7 durability test spiked mixed solution results

5.4 conclusion

In the durability test, under the conditions of the change of the flow rate of +/-0.1 ml/min, the change of the column temperature of +/-2 ℃ and the change of the detection wavelength of +/-2 nm, the separation degree of the impurity C from the main peak is minimum 1.97 and is more than 1.5 in the standard mixed solution; the change rate of the detected amount of the impurity C is at most 14.51% and less than 20%, and the durability is good.

6 repeatability test

6.1 brief description

The repeatability is verified by preparing 6 test solutions with good precision.

6.2 method of operation

And preparing an impurity C positioning solution for the same specificity test.

Adding a standard test solution: precisely measuring 1ml of peramivir injection, placing the injection into a 20ml measuring flask, precisely adding 1ml of impurity C positioning solution, adding a solvent to dilute to a scale, and shaking up to obtain a labeled test sample solution. In parallel, 6 portions of

Control solution: precisely measuring 1ml of the sample solution, placing in a 20ml measuring flask, diluting with solvent to scale, shaking, precisely measuring 1ml, placing 25ml, diluting with solvent to scale, and shaking.

Precisely measuring the blank solvent, the standard sample solution and the reference solution by 4 μ l, respectively, injecting into a liquid chromatograph, and recording chromatogram. The results are shown in Table 8.

6.3 test results

TABLE 8 results of the repeatability tests

6.4 conclusion

In a repeatability test, the RSD of the peak area of the main peak of the continuous 5-point reference solution-1 is 1.43 percent and is less than 2.0 percent, the RSD of the detected amount of the impurity C in 6 parts of the added standard test sample solution is 1.09 percent and is less than 20 percent, and the repeatability is good.

7 intermediate precision test

7.1 brief description

In order to examine the influence of random variation factors on the precision, another analyst independently establishes a system and examines the precision of the determination method.

7.2 method of operation

According to the repeatability test method, the content of the same batch of samples is measured by different operators on different dates and different instruments, and the operation is repeated for 6 times.

Precisely measuring the blank solvent, the standard sample solution and the reference solution by 4 μ l, respectively, injecting into a liquid chromatograph, and recording chromatogram. The results are shown in Table 9.

7.3 test results

TABLE 9 intermediate precision test results

7.4 conclusion

In the intermediate precision test, the RSD of the main peak area of the continuous 5-reference solution-1 is 1.65 percent and is less than 2 percent, the RSD of the impurity C in the 6 parts of the added standard test sample solution is 1.96 percent and is less than 20 percent, and the intermediate precision is good. The RSD of the detected amount of the impurity C in the 12-needle labeled test sample solution is 4.06 percent and less than 30 percent, and the precision is good.

8 solution stability test

8.1 brief description

And (3) observing the change rule of the solution when the solution is placed at room temperature along with the time, and providing a basis for testing the shelf life of the solution in the later detection.

8.2 methods of operation

And preparing an impurity C positioning solution for the same specificity test.

Adding a standard test solution: precisely measuring 1ml of peramivir injection, placing into a 20ml measuring flask, precisely adding 1ml of impurity C positioning solution, adding a solvent to dilute to a scale, and shaking uniformly. Diluting with solvent to scale, and shaking to obtain sample solution.

Control solution: precisely measuring 1ml of the sample solution, placing in a 20ml measuring flask, diluting with solvent to scale, shaking, precisely measuring 1ml, placing 25ml, diluting with solvent to scale, and shaking.

Precisely measuring the blank solvent, the standard sample solution and the control solution at room temperature, respectively in an amount of 4 μ l, injecting into a liquid chromatograph, and recording chromatogram. The results are shown in Table 10.

Calculating the formula:

in the formula A _I Focusing peak-to-peak areas in chromatograms at different times; a. The ₀ The peak-to-peak area of interest in the chromatogram was 0 h.

8.3 test results

TABLE 10 results of the solution stability test

8.4 conclusion

Within 24h, the change rate of the peak area of the main peak in the control solution is 2.49 percent at most and less than 20 percent, and the control solution can be considered to be stable within 24 h. Within 24h, the maximum change rate of the peak area of the impurity C in the standard sample solution is 2.17 percent and is less than 20 percent, and the standard sample solution is considered to be stable within 24 h.

9 durability

9.1 test methods

Under the conditions of flow rate variation of +/-0.1 ml/min, column temperature variation of +/-2 ℃ and detection wavelength variation of +/-2 nm, the separation degree between the main peak and the impurity C peak is in accordance with the requirement in the standard mixed solution. In the standard-added mixed solution, the change rate of the detected amount of the impurity C is less than 20%.

9.2 test results

Under the conditions that the flow rate changes +/-0.1 ml/min, the column temperature changes +/-2 ℃ and the detection wavelength changes +/-2 nm, the separation degree of the impurity C and the main peak in the standard mixed solution is minimum 1.97 and is more than 1.5; the change rate of the detected amount of the impurity C is at most 14.51% and less than 20%, and the durability is good.

The results of the (III) verification are summarized in Table 11

TABLE 11 summary of validation results

In conclusion, the impurity C detection method for the peramivir injection disclosed by the invention is better suitable for detecting the impurity C of the peramivir injection through the investigation of specificity, linearity, range, accuracy test, repeatability test, intermediate precision test, solution stability test, durability test and the like, and is beneficial to improving the quality control level of the peramivir injection.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (10)

1. The method for detecting impurity C in the peramivir injection is characterized by comprising the following steps: detecting by high performance liquid chromatography under the following conditions: protein bonded silica gel for chromatographic column is used as a filling agent, and the detection wavelength is as follows: 203-207 nm, column temperature: 18-22 ℃, flow rate: 0.2-0.4 ml/min.

2. The method for detecting impurity C in peramivir injection according to claim 1, wherein the type of the chromatographic column is

AGP。

3. The method for detecting impurity C in peramivir injection according to claim 1, wherein the mobile phase: phosphate buffer solution, wherein each 1L of the phosphate buffer solution contains 4.75 to 5.25mmol/L diammonium hydrogen phosphate and 4.75 to 5.25mmol/L ammonium dihydrogen phosphate.

4. The method for detecting impurity C in peramivir injection according to claim 1, wherein the solution preparation comprises:

impurity C localization solution: taking impurity C, precisely weighing, adding a solvent to dissolve and dilute the impurity C until each 1ml of the impurity C contains 0.015-0.025 mg of solution;

test solution: accurately measuring the peramivir injection, diluting the peramivir injection with a solvent until each 1ml of the injection contains 0.4-0.6 mg of peramivir, and uniformly mixing;

control solution: the sample solution is diluted with solvent to a solution containing 0.8-1.2 mug peramivir in each 1ml, and the solution is mixed evenly.

5. The method for detecting impurity C in peramivir injection according to claim 4, wherein the solvent is water.

6. The method for detecting impurity C in peramivir injection according to claim 1, wherein the determination method comprises: precisely measuring a test solution, a reference solution and a blank solvent, respectively injecting into a liquid chromatograph, and recording a chromatogram; the sample injection volume is as follows: 3-5 mul.

7. The method for detecting impurity C in peramivir injection according to claim 1, wherein the mobile phase: 5mmol/L diammonium hydrogen phosphate and 5mmol/L ammonium dihydrogen phosphate buffer.

8. The detection method according to claim 1, characterized in that: the specification of the chromatographic column is 150mm × 4.0mm,5 μm.

9. The method for detecting impurity C in peramivir injection according to claim 1,

detection wavelength: 205nm, column temperature: 20 ℃, flow rate: 0.3ml/min, injection volume: 4 μ l.

10. The method for detecting impurity C in peramivir injection according to claim 1,

impurity C localization solution: precisely weighing impurity C, adding solvent to dissolve and dilute until each 1ml of the impurity C contains 0.02mg of solution;

test solution: accurately measuring the peramivir injection, diluting the peramivir injection with a solvent until each 1ml of the injection contains 0.5mg of peramivir, and uniformly mixing;

control solution: the test solution is diluted with solvent to a concentration of 1. Mu.g Peramivir per 1ml, and mixed.

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