CN116660390A

CN116660390A - HPLC detection method for polyvinylpyrrolidone content in preparation

Info

Publication number: CN116660390A
Application number: CN202210144370.7A
Authority: CN
Inventors: 金方; 周云
Original assignee: Guangzhou Jiankangyuan Respiratory Drug Engineering Technology Co ltd
Current assignee: Guangzhou Jiankangyuan Respiratory Drug Engineering Technology Co ltd
Priority date: 2022-02-17
Filing date: 2022-02-17
Publication date: 2023-08-29

Abstract

The invention provides a detection method of PVP content in a preparation, which is based on high performance liquid chromatography, wherein the chromatographic conditions are as follows: a chromatographic column filled with C18 bonded silica gel resistant to high proportion of water phase is adopted; the mobile phase consists of a mobile phase A and a mobile phase B, wherein the mobile phase A is a mixed solution of NaCl-phosphoric acid-water, the molar concentration of NaCl is 0.05-0.15M, and the mass percentage concentration of phosphoric acid is 0.05-0.2%; the mobile phase B is acetonitrile; performing gradient elution on the mobile phase A and the mobile phase B for 0-35 min; the flow rate of the mobile phase is 0.5-0.8 mL/min; column temperature is 20-30 ℃; the detection wavelength is 200-210 nm; sample injection amount: 20-100 mu L.

Description

HPLC detection method for polyvinylpyrrolidone content in preparation

Technical Field

The invention belongs to the field of analytical chemistry, and particularly relates to a method for detecting polyvinylpyrrolidone content in a preparation by utilizing HPLC.

Background

Polyvinylpyrrolidone (polyvinyl pyrrolidone, PVP), also known as povidone, has the characteristics of adhesion, thickening, suspending, dissolving assisting, dispersing, film forming and the like as a medicinal auxiliary material, and is widely applied to inhalation preparations, ointments, emulsion injection, gel, suppositories and the like. PVP is a water-soluble polymer with various specifications, the main difference being their molecular weight and viscosity, and the specification types of different molecular weights are expressed in terms of K value, for example PVP K25 has an approximate molecular weight of 30000.

There are hundreds of medicines using PVP as an auxiliary material. The development and consistency evaluation of the simulated pharmacy and the exemption of bioequivalence experiments are required to be consistent with the quality and curative effect of the original grinding, and the reverse engineering technology is developed in order to achieve the consistent curative effect with the quality of the original grinding. PVP is used as an auxiliary material commonly used in pharmaceutical preparations, and how to rapidly detect the content of PVP in a preparation prescription is particularly important for imitation drug development, consistency evaluation and the like.

Currently, as for liquid phase detection methods of macromolecular substances such as PVP, permeation chromatography or size exclusion chromatography is commonly adopted, for example, chinese patent application CN112684031A (published year 2021, month 4, and day 20) discloses a method for detecting PVP K30 in a preparation (such as a ferrous succinate tablet) by using size exclusion chromatography. However, these methods have disadvantages such as poor separation, poor peak shape, low sensitivity, and low response value.

The Chinese patent application CN106404952A (2016, 8 and 31) discloses a method for detecting PVP K30 in bromfenac sodium eye drops by taking N-oxidized lycorine M bonded silica gel as a stationary phase. But this method must employ a special stationary phase. The Chinese patent application CN107505424A (publication date 2017, 12, 22) discloses a method for detecting PVP in povidone-iodine oral liquid, which adopts an acetonitrile-water isocratic elution mode. However, the inventor researches and discovers that when the method is transferred to preparations with low PVP concentration (such as inhalation preparations, specific injections, suspensions and the like), particularly when PVP with the concentration as low as 0.01% is detected, the swelling phenomenon at the PVP chromatographic peak is obvious, even the PVP chromatographic peak cannot be detected, and the PVP chromatographic peak is easily interfered by other auxiliary material peaks in a sample. Chinese patent application CN110887901A (17 of the year 2020) discloses a method for detecting residues of N-methylpyrrolidone and PVP K30 in a hemodialyzer based on a high performance liquid phase method. The method adopts acetonitrile and buffer solution for isocratic elution, wherein the buffer solution is aqueous solution of monopotassium phosphate, dipotassium phosphate and phosphoric acid. Similarly, the method is transferred to a preparation with low PVP concentration (such as an inhalation preparation, a specific injection, a suspension and the like), and the problem of interference of peaks of other auxiliary materials in a sample exists.

Therefore, development of a liquid phase detection method for PVP of trace pharmaceutical excipients in a preparation with strong applicability, wide applicability and high feasibility is urgent and necessary.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides an HPLC detection method for PVP content in a preparation. The method is especially suitable for formulations with PVP content in PPM level, such as inhalant formulations, injection, etc.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the detection method of PVP content in the preparation is based on high performance liquid chromatography, and comprises the following steps of establishing chromatographic conditions:

chromatographic column: a chromatographic column packed with a high proportion of water-resistant C18-bonded silica gel;

mobile phase: the preparation method comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a mixed solution of NaCl-phosphoric acid-water, the molar concentration of NaCl is 0.05-0.15M, and the mass percentage concentration of phosphoric acid is 0.05-0.2%; the mobile phase B is acetonitrile; 0.fwdarw.35 min, mobile phase A and mobile phase B were gradient eluted according to the following procedure:

0 to 15min, 97 to 99 volume percent of mobile phase A and the balance of mobile phase B,

15-25 min, the volume percentage of the mobile phase A is uniformly reduced to 5-20%, the balance is the mobile phase B,

25-30 min, the volume percentage of the mobile phase A and the mobile phase B is kept unchanged,

30-31 min, the volume percentage of the mobile phase A is rapidly increased to 97-99%, the balance is the mobile phase B,

31-35 min, the volume percentage of the mobile phase A and the mobile phase B is kept unchanged;

mobile phase flow rate: 0.5-0.8 mL/min;

column temperature: 20-30 ℃;

detection wavelength: 200-210 nm;

sample injection amount: 20-100 mu L.

Preferably, the chromatographic column is a Waters Atlantis T3 series chromatographic column.

More preferably, the chromatographic column is a Waters Atlantis T3 chromatographic column with a specification of 250mm by 4.6mm,5 μm.

Preferably, in the mobile phase A, the molar concentration of NaCl is 0.05-0.1M.

More preferably, the molar concentration of NaCl in the mobile phase A is 0.1M.

Preferably, in the mobile phase A, the mass percentage concentration of the phosphoric acid is 0.1% -0.2%.

More preferably, the mass percentage concentration of phosphoric acid in the mobile phase a is 0.1%.

Preferably, in the gradient elution procedure, at 15 to 25 minutes, the volume percentage of mobile phase A is uniformly reduced to 10%, the balance being mobile phase B.

As a preferred embodiment, the gradient elution procedure is:

0 to 15min, the volume percentage of the mobile phase A is 98.5 percent, the balance is the mobile phase B,

15-25 min, the volume percentage of the mobile phase A is reduced from 98.5% to 10% at constant speed, the balance is the mobile phase B,

25-30 min, the volume percentage of the mobile phase A is kept to be 10 percent, the balance is the mobile phase B,

30-31 min, the volume percentage of the mobile phase A is rapidly increased from 10% to 98.5%, the balance is the mobile phase B,

31-35 min, the volume percentage of the mobile phase A is kept at 98.5%, and the balance is the mobile phase B.

Preferably, the mobile phase flow rate is 0.8mL/min.

Preferably, the column temperature is 30 ℃.

Preferably, the detection wavelength is 200nm.

Preferably, the sample injection amount is 100 μl.

Preferably, the PVP is selected from PVP K25, PVP K30, PVP K17, PVP K12 or PVP K90; more preferably PVP K25.

Preferably, the detection method further comprises the preparation of a reference substance solution, and the specific operation is as follows:

weighing a proper amount of PVP reference substance, placing the PVP reference substance into a measuring flask, and adding purified water to dissolve and dilute the PVP reference substance into a solution with a specified concentration.

As a preferred embodiment, the present invention provides a method for detecting PVP K25 content in an albuterol sulfate inhalation aerosol, comprising:

I. establishment of chromatographic conditions:

chromatographic column: waters Atlantis T3, 250 mm. Times.4.6 mm,5 μm;

mobile phase: mobile phase A is 0.1M NaCl-0.1% phosphoric acid-water, mobile phase B is acetonitrile; 0.fwdarw.35 min, mobile phase A and mobile phase B were gradient eluted according to the following procedure:

mobile phase flow rate: 0.8mL/min;

column temperature: 30 ℃;

detection wavelength: 200nm;

sample injection amount: 100. Mu.L;

II preparation of sample solution

Taking salbutamol sulfate to inhale an aerosol 1 tank, inverting the sample tank, adopting a tip cone to prick a small hole at the bottom of the tank, rapidly and positively placing the sample tank in a beaker filled with 30ml of purified water as receiving liquid, immersing the bottom of the tank for more than 25mm, slowly taking out the liquid surface of the sample tank after the escaped liquid medicine is fully absorbed, slightly leaning on the wall of the beaker for 2 seconds, enabling the residual liquid on the sample tank to flow back to the beaker, cutting the sample tank, cleaning the inner wall of the tank by the receiving liquid, transferring the cleaning liquid into a 50ml measuring flask, cleaning the inner wall of the sample tank, the outer wall of the tank contacted with the receiving liquid and the beaker for several times, collecting the cleaning liquid into the measuring flask, adding 2ml of 0.1M NaOH solution, diluting the solution to a fixed volume to a scale by water, ultrasonically processing the solution for 1-3 min, and filtering the solution by 0.45 mu M, thus obtaining a sample solution;

III, preparation of a reference substance solution:

weighing a proper amount of PVP K25 reference substance, placing the reference substance into a measuring flask, and adding purified water for dissolving and diluting to prepare a solution containing 3.6+/-1 mug of PVP K25 per 1 mL;

IV. Determination

Precisely sucking 100 μm of each of the reference solution and the sample solution, respectively injecting into high performance liquid chromatograph, recording chromatogram, and calculating PVP K25 content in the sample solution by external standard method.

As another preferred embodiment, the invention provides a method for detecting PVP K12 content in a propofol emulsion injection, which comprises the following steps:

I. establishment of chromatographic conditions:

chromatographic column: waters Atlantis T3, 250 mm. Times.4.6 mm,5 μm;

mobile phase flow rate: 0.8mL/min;

column temperature: 30 ℃;

detection wavelength: 200nm;

sample injection amount: 100. Mu.L;

II, preparing a test sample solution:

taking a proper amount of the product, precisely weighing, placing into a measuring flask, adding purified water to dilute to a scale, slowly shaking for 2 minutes to prepare a solution containing 9.0+/-3.0 mug of PVP K12 per 1ml, filtering with 0.45 mu m, and taking a subsequent filtrate to obtain the product;

III, preparation of a reference substance solution:

precisely weighing a proper amount of PVP K12 reference substance, placing the reference substance into a measuring flask, and adding purified water for dissolving and diluting to prepare a solution with 9.0+/-3.0 mug of PVP K12 in each 1 mL;

IV. Determination

Precisely sucking 100 μl of each of the control solution and the sample solution, respectively injecting into high performance liquid chromatograph, recording chromatogram, and calculating PVPK12 content in the sample solution by external standard method.

Preferably, in the method for detecting the content of PVP K25 in the salbutamol sulfate inhalation aerosol or the method for detecting the content of PVP K12 in the propofol emulsion injection, the gradient elution procedure is as follows:

The method for measuring the PVP content in the preparation based on the HPLC method provided by the invention can rapidly and accurately measure the PVP content with the concentration as low as 0.01% in the preparation by selecting the C18 chromatographic column with high-proportion water phase and the gradient elution condition, has the advantages of simple operation, fast peak-out time and high accuracy, and good separation degree of PVP chromatographic peaks and other impurity peaks, and can greatly save the cost of manpower, time, solvent and the like, thereby rapidly and efficiently completing the quality control work of PVP in the pharmaceutical preparation.

Drawings

The invention is further described below with reference to the accompanying drawings. In each figure, reference numeral P denotes a chromatographic peak of PVP.

FIG. 1 is a full ultraviolet wavelength scan of PVP control solution from the wavelength selection study of study example 1.

Fig. 2 is an HPLC chromatogram of a PVP control solution obtained from a gradient elution procedure screening study in study example 1, wherein:

the method comprises the steps of 1, gradient elution procedure 1 and reference substance solution chromatogram, 2, gradient elution procedure 2 and reference substance solution chromatogram, and 3 and reference substance solution chromatogram.

FIG. 3 is an HPLC chromatogram obtained from a column screening study in study example 1, wherein:

a: the chromatographic column is ACE Excel 5C 18-PFP,250mm multiplied by 4.6mm,5 μm, and adopts blank auxiliary material solution, PVP reference substance solution and sample solution for sample injection analysis; in the figure: 1 is PVP reference solution chromatogram, 2 is blank adjuvant solution chromatogram.

B: the chromatographic column is Ultimate XB C18, 250mm×4.6mm,5 μm 300A, and adopts blank solution, blank adjuvant solution, PVP reference substance solution and sample solution for sample injection analysis; in the figure: 1 is blank auxiliary material solution chromatogram, 2 is sample solution chromatogram, and 3 is PVP reference solution chromatogram.

C: the chromatographic column is Waters Atlantis T, 250mm×4.6mm,5 μm, and adopts blank solution, blank adjuvant solution, PVP reference substance solution and sample solution for sample injection analysis; in the figure: 1 is a blank solution chromatogram, 2 is a blank auxiliary material solution chromatogram, 3 is a PVP reference substance solution chromatogram, and 4 is a test substance solution chromatogram.

Fig. 4 is an HPLC chromatogram obtained from the mobile phase screening study of study example 1, wherein:

a is a chromatogram obtained by taking aqueous phase 1-acetonitrile as a mobile phase; in the figure, 1 is a blank solution chromatogram, 2 is a blank auxiliary material solution chromatogram, and 3 is a PVP reference solution chromatogram.

B is a chromatogram obtained by taking aqueous phase 2-acetonitrile as a mobile phase; in the figure, 1 is a blank solution chromatogram, 2 is a PVP control solution chromatogram, and 3 is a test solution chromatogram.

FIG. 5 is an HPLC chromatogram of the system applicability experiment of study example 2; in the figure: 1 is blank solution chromatogram, 2 is PVP reference solution chromatogram.

FIG. 6 is an HPLC chromatogram of the specificity experiment of study example 2; in the figure, 1 is a blank solution chromatogram, 2 is a blank auxiliary material solution chromatogram, 3 is a PVP reference substance solution chromatogram, and 4 is a test substance solution chromatogram.

FIG. 7 is a linear regression curve obtained by the linear relationship experiment of study example 2.

FIG. 8 is an HPLC chart recorded in application example 1; in the figure, 1 is a blank solution chromatogram, 2 is a blank auxiliary material solution chromatogram, 3 is a PVP reference solution chromatogram, and 4 and 5 are test solution chromatograms.

FIG. 9 is an HPLC chart recorded in comparative example 1; in the figure, 1 is a blank solution chromatogram, 2 is a PVP control solution chromatogram, and 3 is a test solution chromatogram.

FIG. 10 is an HPLC chart recorded in comparative example 2; in the figure, 1 is a blank solution chromatogram, 2 is a blank auxiliary material solution chromatogram, 3 is a PVP reference substance solution chromatogram, and 4 is a test substance solution chromatogram.

Detailed Description

The invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are for illustration of the invention only and are not intended to limit the scope of the invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagent materials and the like used in the examples described below are commercially available products unless otherwise specified.

Study example 1 establishment of chromatographic conditions

The test solutions, mobile phases used in this study are as follows:

chromatograph: agilent 1260 high performance liquid chromatograph

Test solution: blank solution, blank auxiliary material solution, PVP reference substance solution and test sample solution are prepared by the following methods: .

(1) Blank solution: water was taken as a blank solution.

(2) Blank auxiliary material solution: taking PVP-free salbutamol sulfate to be inhaled into an aerosol 1 tank, inverting the sample tank, adopting a tip cone to prick a small hole at the bottom of the tank, rapidly and positively placing the sample tank into a beaker filled with 30ml of purified water as receiving liquid, ensuring that the bottom of the tank is immersed for more than 25mm, slowly taking out the liquid level of the sample tank after the escaped liquid medicine is fully absorbed, slightly leaning on the wall of the beaker for 2 seconds, enabling the residual liquid on the sample tank to flow back into the beaker, cleaning the inner wall of the tank by the receiving liquid after the sample tank is cut, transferring cleaning liquid into a 50ml measuring flask, cleaning the inner wall of the sample tank, the outer wall of the tank contacted with the receiving liquid and the beaker for several times, collecting the cleaning liquid into the measuring flask, adding 2ml of 0.1M NaOH solution, diluting the solution to a fixed volume to a scale by water, ultrasonically treating for 1-3 min, filtering the liquid by using a 0.45 mu M filter membrane, and obtaining a sample solution;

(3) PVP control solution: precisely weighing a proper amount of PVP K12 reference substance, placing the reference substance into a measuring flask, and adding purified water for dissolving and diluting to prepare 9.0 mug solution containing PVP K12 per 1 mL;

(4) Test solution: the salbutamol sulfate is taken and inhaled into an aerosol 1 tank, and the salbutamol sulfate is prepared according to a blank auxiliary material solution method.

Aqueous phase 1:5% (v/v) glycerol-0.1% (w/v) phosphoric acid-water solution.

Aqueous phase 2:0.1M NaCl-0.1% (w/v) phosphoric acid-water solution.

Through screening chromatographic parameters such as mobile phase elution gradient, detection wavelength, chromatographic column and the like, a proper method is finally selected, and the specific screening process is as follows:

1. wavelength selection

The PVP K25 control solution was scanned at full wavelength using a Diode Array Detector (DAD), with a pattern shown in fig. 1.

Fig. 1 shows: PVP is only end absorbed. Meanwhile, the cut-off wavelength of the mobile phase system is considered, and finally 200nm is selected as the detection wavelength.

2. Screening of gradient elution programs

Chromatographic column: waters Atlantis T3, 250 mm. Times.4.6 mm,5 μm

The gradient elution procedure is shown in table 1.

100 mu L of the reference substance solution is precisely sucked and respectively injected into a high performance liquid chromatograph, and a chromatogram is recorded, as shown in figure 2.

TABLE 1 gradient elution procedure

^a : volume percent.

Conclusion: figure 2 shows that with procedure 1 and procedure 2 the pvp peak is affected by the system peak, affecting the peak shape or tailing severely, whereas with procedure 3 the pvp peak and system peak separation is satisfactory, the peak shape is symmetrical and the S/N is satisfactory for quantification. Thus, gradient elution procedure 3 is preferred.

3. Screening of chromatographic columns

Chromatographic column 1: ACE Excel 5C 18-PFP,250mm 4.6mm,5 μm;

chromatographic column 2: ultimate XB C18, 250mm 4.6mm,5 μm 300A;

chromatographic column 3: waters Atlantis T3, 250 mm. Times.4.6 mm,5 μm.

And (3) precisely sucking 100 mu L of each of the blank solution, the blank auxiliary material solution, the reference substance solution and/or the test sample solution by adopting the gradient elution procedure 3 screened in the previous section, respectively injecting into a high performance liquid chromatograph, and recording a chromatogram, wherein the chromatogram is shown in figure 3.

Conclusion: the blank adjuvant peak was found to interfere at the PVP peak retention time using a chromatographic column ACE Excel 5C 18-PFP (250 mm. Times.4.6 mm,5 μm) (panel A in FIG. 3); blank adjuvant peak and PVP peak were found to fail to reach baseline separation with chromatographic column Ultimate XB C18 (250 mm. Times.4.6 mm,5 μm 300A) (panel B in FIG. 3); the PVP peak was found to have an optimal separation from the blank peak of greater than 1.5 using column Waters Atlantis T3 (250 mm. Times.4.6 mm,5 μm), baseline separation (panel C in FIG. 3). Therefore, column 3 is preferred: waters Atlantis T3, 250 mm. Times.4.6 mm,5 μm.

4. Screening of mobile phases

The water phase 1 and the water phase 2 respectively form mobile phases with acetonitrile, and chromatograms of a blank solution, a blank auxiliary material, a reference substance solution and a test sample solution are respectively measured and recorded by using a Waters T3, 250mm multiplied by 4.6mm and a 5 mu m chromatographic column according to the gradient elution program 3 screened by the previous section. The map is shown in figure 4.

Fig. 4 shows: when 5% (v/v) glycerol-0.1% (w/v) phosphoric acid-water solution is used as a water phase regulator, the blank auxiliary material solution has interference at PVP peaks (see a diagram A in FIG. 4 in particular), and the method specificity does not meet the requirement; when 0.1M NaCl-0.1% (w/v) phosphoric acid-water solution is adopted, the blank auxiliary material solution has no interference on PVP peaks (see the diagram B of FIG. 4 in particular), and the method specificity meets the requirements. Thus, the aqueous phase is preferably a 0.1M NaCl-0.1% (w/v) phosphoric acid-water solution.

In summary, the preferred chromatographic conditions of the invention are mainly:

chromatographic column: waters Atlantis T3, 250 mm. Times.4.6 mm,5 μm;

15-25 min, the volume percentage of the mobile phase A is uniformly reduced to 10%, the balance is the mobile phase B,

detection wavelength: 200nm.

Study example 2Methodology investigation

The detection method established in study 1 was examined methodically by this study based on albuterol sulfate inhalation aerosol.

Preparation of test solutions

Taking salbutamol sulfate to inhale an aerosol (self-grinding, batch number 20200616) 1 tank, inverting a sample tank, adopting a tip cone to prick a small hole at the bottom of the tank, rapidly and positively placing the sample tank into a beaker filled with 30ml of purified water as receiving liquid, ensuring that the bottom of the tank is immersed by more than 25mm, slowly lifting the sample tank out of the liquid surface after the escaped liquid medicine is fully absorbed, slightly leaning on the wall of the beaker for 2 seconds, enabling the residual liquid on the sample tank to flow back into the beaker, cutting the sample tank, cleaning the inner wall of the tank by the receiving liquid, transferring the cleaning liquid into a 50ml measuring flask, cleaning the inner wall of the sample tank, the outer wall of the tank contacted with the receiving liquid and the beaker by a small amount of purified water, collecting the cleaning liquid into the measuring flask, adding 2ml of 0.1mol/L NaOH solution, diluting the solution with water to fix the volume to a scale, ultrasonically treating for 1 minute, and taking the continuous filtrate to obtain the sample solution.

Preparation of control solution：

Accurately weighing a proper amount of PVP K25 reference substance, placing into a measuring flask, and adding water to dissolve and dilute to prepare a solution with PVP K25 of 3.6 mug per 1 mL. Two portions are arranged in parallel.

Preparing a blank auxiliary material solution:

respectively weighing salbutamol sulfate, lactose and PEG1000 with proper amounts, adding water for dissolving, and preparing into blank auxiliary material solution.

2.1: system applicability

(1) Solution preparation

The diluent/blank solution is water;

control solution: weighing 18.15mg and 18.14mg of PVP K, respectively placing into 100mL measuring flask, adding water for dissolving and diluting to scale, and mixing to obtain reference stock solution. And precisely transferring 1mL of the reference substance stock solution, placing into a 50mL measuring flask, adding water to dilute to a scale, and uniformly mixing to obtain a reference substance solution.

(2) Measurement

100. Mu.L of each of the blank solution and the control solution was precisely aspirated. Respectively injecting into high performance liquid chromatograph, and recording chromatogram, see figure 5.

(3) Results

Fig. 5 shows: the blank solution had no interference at the main peak retention time.

The control solution 1 (STD 1) and the control solution 2 (STD 2) are continuously injected for 3 needles, and the Relative Standard Deviation (RSD) of response factors is respectively 1.4 percent and 0.8 percent and is less than 5.0 percent; the RSD of the response factor of the 6-needle reference substance solution is 1.0% and less than 5.0%; the ratio of the response factor average of STD2 to the response factor average of STD1 is between 95.0% and 105.0%. The system applicability meets the requirements, and the specific results are shown in Table 2.

Table 2 results of System suitability test

2.2: specialization of

Respectively precisely sucking 100 μl of each of the blank solution, the blank adjuvant solution, the reference solution and the sample solution, performing liquid phase analysis, and recording chromatogram.

Results: the blank solution and the blank auxiliary material solution have no interference at the main peak, and the retention time of PVP peak in the chromatogram of the sample solution is consistent with that of the main peak in the chromatogram of the reference solution; the PVP peak purity of the sample solution is 0.992, the PVP peak purity meets the requirement, and the separation degree of impurities before and after the PVP peak is 3.1 and is more than 1.5; the specificity meets the requirements. The specific results are shown in Table 3 and FIG. 6.

TABLE 3 results of specificity experiments

Sample of	Retention time (min)	Degree of separation	Peak purity
				SPL1-20200616	9.373	3.1	0.992
STD1-1	9.420	2.9	1.000

2.3: quantitative limit

(1) Preparing a solution: weighing 18.38mg of PVP K25, placing into a 100mL measuring flask, diluting with water to a scale, shaking uniformly, taking the PVP K25 stock solution as PVP K25 stock solution, precisely transferring 2.5mL of the stock solution into a 200mL measuring flask, diluting with water to the scale, shaking uniformly, and marking as LOQ.

(2) Results: the quantitative limit measurement results are shown in Table 4, when the PVP K25 reference substance concentration is 2.25 mug/mL and the sample injection amount is 100 mu L, S/N is more than 10, and the quantitative limit is that the RSD of the peak area is 1.9% and less than 10.0%, and the requirements are met.

TABLE 4 quantitative limit measurement results

2.4: linearity of

Preparing a standard solution: precisely weighing PVP K25 mg, placing into a 100ml measuring flask, diluting with water to scale, and shaking. Solutions of the respective concentration levels were prepared in this order according to Table 5.

TABLE 5 Linear solution formulation

Name of the name	Concentration level	Concentration (μg/mL)	Stock solution volume (mL)	Final volume (mL)
					L1	25％	2.25	2.5	200
L2	50％	4.50	2.5	100
					L3	80％	7.20	4.0	100
L4	100％	9.00	1.0	20
					L5	200％	18.00	1.0	10
L6	300％	27.00	3.0	20

100 mu L of each of the prepared gradient standard solutions was precisely sucked, and the solution was injected into a high performance liquid chromatograph, the chromatogram was recorded, and peak area integration was performed, and the results are shown in Table 6.

TABLE 6 Linear test results

The PVP K25 linear regression results are shown in FIG. 7, and PVP K25 has good linear relationship in the concentration range of 2.298-27.570 mug/mL. The linear equation is:

y=836, 425.5653x-76,976.7167, r=1.0000, RSD not less than 0.995, RSD of response factor of 1.4% and less than 10%, all meet the requirements.

2.5: accuracy of

The blank auxiliary material solution is used as a matrix, three levels of solutions with the concentration of 65%, 100% and 150% of the solution of the test sample are respectively prepared, and 3 parts of the solutions are respectively prepared in parallel. The above solutions were each sampled once, and the average recovery and Relative Standard Deviation (RSD) are shown in table 7. The result shows that the average recovery rate of PVP K25 is 99.4%, the relative standard deviation is 1.2%, and the accuracy of the method meets the analysis requirement.

TABLE 7 accuracy test results

2.6: repeatability of

(1) Preparing a solution: according to the method for preparing the sample solution in study example 2, 6 parts of sample solution were prepared in parallel.

(2) Results: RSD of PVP K25 content in 6 test sample solutions is 5.3%, and meets the requirements, which shows that the repeatability of the method is good, and specific results are shown in Table 8.

TABLE 8 repeatability test results

Application example 1 PVP K12 content determination in Propofol emulsion injection

Preparation of test solution:

taking a proper amount of the product, precisely weighing, placing into a measuring flask, adding water for dilution to a scale, slowly shaking for 2 minutes to prepare a solution with PVP K12 of 9.0 mug per 1ml, filtering with 0.45 mu m, and taking the subsequent filtrate.

Preparation of a control solution:

accurately weighing a proper amount of PVP K12 reference substance, placing the reference substance into a measuring flask, and adding water to dissolve and dilute the reference substance to prepare 9.0 mug solution containing PVP K12 per 1 mL.

And (3) measuring:

precisely sucking 100 μl of each of the above sample solution and control solution, respectively injecting into high performance chromatograph, and recording chromatogram, as shown in figure 8. And calculating the concentration of the sample solution by adopting an external standard method.

Fig. 8 shows: the PVP K12 main peak and other impurity peaks reach good separation degree, and the peak shape is good. The content determination method established by the invention can be applied to the content detection of PVP K12 in the injection.

Calculated, PVP K12 content in the 1 branch of the propofol emulsion injection is 9.02 mug/ml.

Comparative example 1: transfer of CN105353052A detection method

The content of PVP K25 in the salbutamol sulphate inhalation aerosol was measured as described in CN105353052A, example 1.

Preparation of a blank solution: same as in study example 2.

Preparing a blank auxiliary material solution: same as in study example 2.

Preparation of test solution: same as in study example 2.

Preparation of a control solution: same as in study example 2.

Chromatographic conditions: octadecylsilane chemically bonded silica is used as a packed column, and the volume ratio is 95:5, 0.01mol/L dipotassium hydrogen phosphate solution-acetonitrile is taken as a mobile phase, the detection wavelength is 210nm, the flow rate is 0.8ml/min, and the column temperature is as follows: 25 ℃.

And (3) measuring: 100 mu L of each of the sample solution and the control solution is precisely sucked and injected into a high performance liquid chromatograph, and a chromatogram is recorded, which is shown in figure 9.

Fig. 9 shows: in the test solution, PVP K25 has serious main peak swelling phenomenon. Therefore, the method for detecting the content of PVP monomer disclosed in CN105353052a cannot be applied to the detection of the content of PVP K25 in inhalation aerosol.

Comparative example 2 transfer of the method for determining the content of CN110887901A

The content of PVP K25 in the salbutamol sulphate inhalation aerosol was measured as described in CN110887901A, example 1.

Preparation of a blank solution: same as in study example 2.

Preparing a blank auxiliary material solution: same as in study example 2.

Preparation of test solution: same as in study example 2.

Preparation of a control solution: same as in study example 2.

Chromatographic conditions: octadecylsilane chemically bonded silica is used as a packed column, acetonitrile-buffer solution (preparation of buffer solution: 2.085g of monopotassium phosphate and 0.2175g of anhydrous dipotassium phosphate are precisely weighed in a 1L volumetric flask, dissolved and diluted by water, 1.0mL of phosphoric acid is added, the volume is fixed to a scale with water) with the volume ratio of 3:97 is used as a mobile phase, the detection wavelength is 200nm, the flow rate is 0.5mL/min, and the column temperature is as follows: the sample injection amount is 100 mu L at 25 ℃.

And (3) measuring: 100 mu L of each of the sample solution and the control solution is precisely sucked and injected into a high performance liquid chromatograph, and a chromatogram is recorded, which is shown in figure 10.

Fig. 10 shows: the interference of the blank auxiliary material solution at the retention time of the PVP K25 main peak is serious, and no peak is generated at the retention time of the PVP K25 main peak in the chromatogram of the test solution. Therefore, the method for detecting the content of N-methylpyrrolidone and PVP K30 disclosed in CN110887901A cannot be applied to the detection of the content of PVP K25 in inhalation aerosol.

The invention is not limited in its application to the examples described above, but may be modified or varied by a person skilled in the art from the description given above, all of which are intended to be within the scope of the appended claims.

Claims

1. The detection method of PVP content in the preparation is based on high performance liquid chromatography, and comprises the following steps of establishing chromatographic conditions:

mobile phase flow rate: 0.5-0.8 mL/min;

column temperature: 20-30 ℃;

detection wavelength: 200-210 nm;

sample injection amount: 20-100 mu L.

2. The method of claim 1, wherein the chromatographic column is a Waters Atlantis T3 series chromatographic column;

preferably, the chromatographic column is a Waters Atlantis T3 chromatographic column with a specification of 250mm by 4.6mm,5 μm.

3. The method according to claim 1, wherein the molar concentration of NaCl in the mobile phase A is 0.05 to 0.1M;

preferably, in the mobile phase A, the molar concentration of NaCl is 0.1M;

still preferably, in the mobile phase a, the mass percentage concentration of phosphoric acid is 0.1% -0.2%;

4. A detection method according to claim 1 or 3, wherein in the gradient elution procedure, the volume percentage of mobile phase a is reduced to 10% at a constant rate at 15 to 25min, the balance being mobile phase B.

5. The method of claim 1, 3 or 4, wherein the gradient elution procedure is:

6. The method of claim 1, 3, 4, or 5, wherein the mobile phase flow rate is 0.8mL/min;

preferably, the column temperature is 30 ℃;

preferably, the detection wavelength is 200nm;

preferably, the sample injection amount is 100 μl.

7. The method of claim 1, wherein the PVP is selected from PVP K25, PVP K30, PVP K17, PVP K12, or PVP K90; PVP K25 is preferred.

8. The method according to any one of claims 1 to 7, further comprising the preparation of a control solution, specifically comprising the steps of:

9. A method for detecting PVP K25 content in salbutamol sulfate inhalation aerosol, comprising:

I. establishment of chromatographic conditions:

chromatographic column: waters Atlantis T3, 250 mm. Times.4.6 mm,5 μm;

preferably, 0.fwdarw.35 min, mobile phase A and mobile phase B were gradient eluted according to the following procedure:

31-35 min, the volume percentage of the mobile phase A is kept to be 98.5 percent, and the balance is the mobile phase B;

mobile phase flow rate: 0.8mL/min;

column temperature: 30 ℃;

detection wavelength: 200nm;

sample injection amount: 100. Mu.L;

II preparation of sample solution

III, preparation of a reference substance solution:

IV. Determination

Precisely sucking 100 μl of each of the control solution and the sample solution, respectively injecting into high performance liquid chromatograph, recording chromatogram, and calculating PVP K25 content in the sample solution by external standard method.

10. A method for detecting PVP K12 content in a propofol emulsion injection comprises the following steps:

I. establishment of chromatographic conditions:

chromatographic column: waters Atlantis T3, 250 mm. Times.4.6 mm,5 μm;

mobile phase flow rate: 0.8mL/min;

column temperature: 30 ℃;

detection wavelength: 200nm;

sample injection amount: 100. Mu.L;

II, preparing a test sample solution:

III, preparation of a reference substance solution:

IV. Determination