CN115524408A

CN115524408A - Method for detecting related substances in emtricitabine-propofol-tenofovir tablet compound preparation

Info

Publication number: CN115524408A
Application number: CN202110714049.3A
Authority: CN
Inventors: 张晓伟; 黄小燕; 罗鸣; 黄浩喜; 苏忠海
Original assignee: Chengdu Beite Pharmaceutical Co ltd
Current assignee: Chengdu Beite Pharmaceutical Co ltd
Priority date: 2021-06-25
Filing date: 2021-06-25
Publication date: 2022-12-27

Abstract

The invention provides a method for detecting related substances in an emtricitabine propofol tenofovir tablet compound preparation, which adopts a high performance liquid chromatography. The related substance analysis method is excellent in specificity, detection sensitivity, accuracy, durability and the like, can comprehensively detect impurities in the product, is an analysis method with stability indication capability, and can provide important basis for quality control of the product.

Description

Method for detecting related substances in emtricitabine-propofol-tenofovir tablet compound preparation

Technical Field

The invention relates to the field of drug detection.

Background

The research on impurities is an important content of drug research and development, and is directly related to the quality and safety of drugs on the market. Impurities are generally classified into three categories according to their physical and chemical properties: organic impurities, inorganic impurities and residual solvents, wherein the organic impurities comprise impurities, degradation products and the like introduced in the process, and can be known or unknown, volatile or nonvolatile, and the chemical structure of the impurities is generally similar to or has a source relationship with the active ingredients, so the impurities are generally called related substances. The detection methods of related substances comprise a chemical method, a spectroscopic method, a chromatographic method and the like, and the High Performance Liquid Chromatography (HPLC), the Gas Chromatography (GC) and the capillary electrophoresis method are mainly widely applied at present.

HPLC, an important branch of chromatography, is a novel analysis technology developed on the basis of classical liquid chromatography and gas chromatography at the end of the 20 th 60 s, which adopts a high-pressure infusion system to pump a mobile phase into a chromatographic column, elutes each component in the chromatographic column by adopting an isocratic or gradient elution mode, and sequentially enters a detector (ultraviolet detector (UV), diode Array Detector (DAD), evaporative Light Scattering Detector (ELSD) or the like) for detection, so that a sample is analyzed, and the method is widely used in the fields of medicines, foods and the like. The HPLC can be used for analyzing organic compounds with high boiling point, difficult volatilization, unstable heating, large molecular weight and different polarities, bioactive substances, high molecular compounds and the like, is suitable for analyzing 80 percent of organic compounds, makes up the defects of GC, and has the characteristics of high analysis efficiency, high selectivity, high detection sensitivity, high analysis speed and the like.

The emtricitabine-propofol tenofovir tablets are common compound oral solid preparations, the specification is that each 1 tablet contains 200mg of emtricitabine and 25mg of propofol tenofovir (equivalent to 28mg of alanine tenofovir fumarate), the emtricitabine-propofol compound oral solid preparations are successfully researched and developed for the first time by Gillede science company, are sold in the United states as Dacrovy (Descovy) in 04 days in 2016 at the earliest, and are used together with other antiretroviral medicaments for treating adults infected with HIV-1 and pediatric patients (the weight is at least 35 kg); in combination with other antiretroviral drugs (other than those required in combination with CYP3A protease inhibitors) for the treatment of HIV-1 pediatric patients weighing at least 25kg and less than 35 kg. 2016, 21/04, marketed in the european union; 12/19/2016, marketed in japan; listed in china on 11/2018 and 21/month.

The emtricitabine propofol tenofovir tablets are used as compound preparations, the impurity spectrum is complex, and related literature reports of related substance researches are not available at present. Therefore, it is very important to provide a method for analyzing related substances with good specificity, detection sensitivity and accuracy.

Disclosure of Invention

The applicant determines the impurity types and limits to be controlled in the compound preparation through preliminary analysis and experiments as shown in table 1.

TABLE 1 Emtricitabine Propofol Tenofovir tablet related substances impurity List

Note: (1) ICH Q3b indicates that only the content of degradation impurities can be of interest in the formulation, so process impurities do not specify limits;

(2) impurities marked with an "-" are chiral impurities, which may appear as a single peak, a double peak, or a split peak in the chromatogram, if a double peak or a split peak is shown, the peak area and the calculation are not concerned with the degree of separation.

The detection method provided by the invention is used for measuring the related substances by HPLC.

The detection method comprises the following steps:

preparing a solution (a test solution, a control solution, a sensitivity solution and a system applicability solution), setting chromatographic conditions, and calculating and analyzing a detection result.

The preparation method of the test solution comprises the following steps:

weighing a proper amount of emtricitabine-propofol-tenofovir tablet fine powder, adding a solvent to dissolve emtricitabine and propofol-tenofovir as main components, diluting to a required concentration, standing, taking supernatant for treatment, and preparing a test solution.

Wherein the solvent used is selected from water, acetonitrile-water mixture, most preferably acetonitrile and water mixture (20% acetonitrile-30% acetonitrile), and the amount of the solvent is about 2 ml-3 ml;

wherein the main component is dissolved by adding the solvent in a mode selected from ultrasonic and shaking, preferably shaking for 3-10 min, preferably not less than 5min;

wherein, the adopted diluent is water;

wherein, the concentration of the test solution is that each 1ml of the test solution contains about 4mg of emtricitabine and 0.5mg of Propofol tenofovir;

wherein, the supernatant fluid treatment mode adopts a microporous membrane for filtration, and through a membrane adsorption test, the volume of the primary filtrate to be discarded is preferably 0ml to 10ml, and most preferably not less than 2ml.

The reference substance solution is prepared by the following steps: taking the test solution to prepare a control solution.

The sensitivity solution is prepared by the following steps: taking an emtricitabine reference substance and a tenofovir disoproxil fumarate reference substance to prepare a sensitivity solution.

The system applicability solution of the invention is prepared as follows: preparing system applicability solutions from an emtricitabine reference substance, an acriflavone fumarate reference substance and each known impurity reference substance in table 1.

The chromatographic conditions described in the present invention include: the chromatographic column with octadecylsilane chemically bonded silica as filler is eluted with mobile phase at flow rate of 0.9-1.1 ml/min and column temperature of 33-40 deg.c.

By adopting a diode array detector, the detection wavelength can be screened within the range of 200-400nm to find the wavelength most suitable for the compound preparation, and of course, the wavelength can also be respectively selected according to the pertinence of different related substances. The invention provides a light source with a wavelength of 260nm +/-2 nm.

The amount of sample to be introduced in the present invention may be adjusted depending on the concentration of a sample, and for example, it may be 5 to 100. Mu.l, and usually 5 to 20. Mu.l is preferably selected.

The main components of emtricitabine, part of known impurities (impurity 1, impurity 2, impurity 3 and impurity 5) of emtricitabine and known impurities (impurity 7) of propiolic tenofovir in the emtricitabine and tenofovir are compounds with larger polarity, and the retention on a conventional C18 column is poor, so the chromatographic column is selected from chromatographic columns using octadecylsilane bonded silica gel as a filler, which is compatible with 100% water phase, such as Agilent ZORBAX SB-Aq, waters Xbridge Shield RP18 and Waters XSelectt HST 3.

By adopting the relevant detection method in the embodiment of the invention and examining the three chromatographic columns, the separation efficiency of the Waters Xbridge Shield RP18 or the Waters XSelectt HSS T3 is better, wherein the Waters XSelectt HSS T3 (4.6 mm multiplied by 150mm,3.5 mu m) is optimal.

The term "100% aqueous phase compatibility" as used herein means that the filler of the chromatographic column has good compatibility with the high water content mobile phase, and the filler can maintain a longer service life in the high water content mobile phase.

Wherein the mobile phase is a combination of buffer and acetonitrile, the buffer salt is selected from phosphate, formate and acetate, the preferred buffer salt is volatile acetate (or replaceable ammonium formate), the preferred pH of the buffer salt is between 2.5 and 6.5, and the most preferred pH is 4.6 +/-0.3.

The concentration of the salt in the buffer may be selected from 0.01-0.1mol/L, such as 0.01, 0.02, 0.03, 0.04, 0.05mol/L and the like.

Wherein, the elution mode adopts gradient elution, and preferably adopts a gradient elution mode with double channels and three channels.

Wherein, in the double-channel mode, the mobile phase A is buffer solution-acetonitrile = 95-99.5: 5-0.5, and the mobile phase B is acetonitrile; gradient elution procedures such as gradient one or gradient two:

gradient one:

time/minute	Mobile phase A/%)	Mobile phase B/%)
			0	100	0
2	100	0
			15	95	5
20	95	5
			25	85	15
50	75	25
			60	40	60
70	40	60
			70.1	100	0
75	100	0

And gradient II:

most preferred is a three-channel elution mode, wherein the mobile phase A is buffer-acetonitrile = 99-99.5: 1-0.5, the mobile phase B is buffer-acetonitrile = 95: 5, and the mobile phase C is acetonitrile, and the gradient procedure is as follows:

the calculation method of the known impurities in the related substances in the invention is selected from an impurity reference substance external standard method and a main component self-comparison method added with a correction factor, and preferably the main component self-comparison method added with the correction factor; the method for attributing the unknown impurities is selected from the unknown sources, directly attributing to small components in the compound preparation, attributing according to the ultraviolet absorption characteristics of the unknown impurities, preferably attributing according to the ultraviolet absorption characteristics of the unknown impurities, and the calculation method selects the corresponding main component self-contrast method according to the preferred attribution mode to calculate the content of the unknown impurities, wherein the calculation formula of the impurities is as follows:

degradation of total impurities (%) = ∑ degradation impurities

In the formula A _X : peak area of impurities in test solution

A _{For is to} : peak area of emtricitabine or propiophenol tenofovir in control solution

f: correction factor for impurities

The related substance analysis method is excellent in specificity, detection sensitivity, accuracy, durability and the like, can comprehensively detect impurities in the product, is an analysis method with stability indication capability, and can provide important basis for quality control of the product.

The method is suitable for quality detection of a preparation formed by compounding emtricitabine and propofol tenofovir, and the compound preparation is selected from a solid preparation or a liquid preparation.

The solid preparation can be selected from tablet, granule, capsule, pill, etc., and the liquid preparation can be selected from oral liquid, soft capsule, injection, etc.

The detection method of the invention can simultaneously detect all the related substances of the impurities 1 to 18, and can also respectively detect partial related substances.

By means of the detection method and the limit of related substances in the compound preparation, the invention also provides an emtricitabine propofol tenofovir tablet compound preparation, wherein the content of the related substances in the preparation is as follows: (ii) less than 0.2% of 5-fluorocytosine, (2R, 3RS, 5S) -5-fluoro-1- [ 2-hydroxymethyl-3-oxo-1, 3-oxathiolan-5-yl ] cytosine less than 0.50%, (2R, 5S) -5-fluoro-1- [ 2-hydroxymethyl-1, 3-oxo-1, 3-oxathiolan-5-yl ] uracil less than 0.50%, (R) -9- (2-phosphonomethoxypropyl) -adenine less than 3%, phenol less than 2%, [ [ (1R) -2- (6-amino-9H-purin-9-yl) -1-methylethoxy ] methyl ] phenyl phosphonate less than 0.75%, tenofovir anhydride triethylamine salt less than 2.5%, 1-methylethylN- [ [ (1R) -2- (6-amino-9H-purin-9-yl) -1-methylethoxy ] methyl ] -phosphono-L ] -L-alanine ester, l-alanine isopropyl ester salt is below 1.0%, and [ (2R, 5S) -5- (4-amino-5-fluoro-2-oxopyrimidin-1 (2H) -yl) -1, 3-oxathiolan-2-yl ] methylhydrogen [ [ (R) -1- (6-amino-9H-purin-9-yl) propan-2-yl ] oxy ] methyl ] phosphonate is below 0.5%.

The related substance analysis method disclosed by the invention is excellent in the aspects of specificity, detection sensitivity, accuracy, durability and the like, can be used for comprehensively detecting impurities in the product, is an analysis method with stability indication capability, can provide an important basis for quality control of the product, and particularly has the following beneficial effects:

(1) the impurities can be detected and separated to the maximum extent by adopting a gradient elution mode;

(2) gradient elution is carried out by adopting a 3-channel mode, so that the condition that the chromatographic peak appearance time is unstable due to the fact that the flow ratio of one flow path in the two-channel elution mode is lower than 5 percent (HPLC of a quaternary low-pressure system is adopted in an experiment, the mixing ratio of the HPLC gradient system is controlled by controlling the opening and closing time of electromagnetic valves of different flow paths, generally speaking, when the flow path ratio is lower than 5 percent, the opening and closing time is too short, and the influence on the accuracy of the mixing ratio is large) can be avoided to a great extent;

(3) in the related substance analysis method, the number of known impurities to be detected is large, the difference of relative retention time of parts is small, and in order to avoid impurity identification errors caused by the small change of the relative retention time when the chromatographic condition is slightly changed, a system applicability solution (prepared by each known impurity reference substance and two main component reference substances) is added in the method so as to facilitate the impurity identification;

(4) the unknown impurities are subjected to attribution according to the light absorption characteristics of the ultraviolet spectrum of the unknown impurities, and then the corresponding main components are calculated according to a self comparison method, so that compared with the calculation of the unknown impurities in most of the existing compound preparations (the specific attribution is not carried out, and the area and the current comparison of the two main components in a comparison solution are calculated), the attribution mode of the unknown impurities is more reasonable, and the level of the unknown impurities can be truly reflected;

(5) the main component self-contrast method added with correction factors is adopted to calculate each known degradation impurity, so that the impurity contrast product can be greatly saved.

Drawings

FIG. 1 HPLC chromatogram of mobile phase pH2.5 of example 1

FIG. 2 HPLC chromatogram of mobile phase pH4.6 of example 1

FIG. 3 HPLC chromatogram of mobile phase pH6.5 of example 1

FIG. 4 HPLC profile of sensitive solution in example 3

FIG. 5 HPLC chromatogram of system suitability solution in example 3

FIG. 6 HPLC chromatogram of test solution in example 3

FIG. 7 HPLC chromatogram of control solution in example 3

Detailed Description

The following examples will help demonstrate the beneficial effects of the present invention, but they are only for illustrative purposes and do not limit the present invention in any way.

Example 1: selection of buffer salts and pH in mobile phase

Three systems of phosphate buffer solution (pH2.5) -acetonitrile, acetate buffer salt (pH4.5) -acetonitrile and phosphate buffer solution (pH6.5) -acetonitrile are selected as mobile phases, the influence of different buffer salt types and buffer solution pH on the separation and response of each component is examined, and the specific chromatographic conditions are as follows:

the instrument comprises the following steps: agilent 1260 II liquid chromatograph (DAD detector)

A chromatographic column: waters XSelect HSS T3 (4.6 mm. Times.150mm, 3.5 μm)

Mobile phase: mobile phase A is buffer solution of different buffer salts-acetonitrile (95: 5), and mobile phase B is acetonitrile

Detection wavelength: 260nm/DAD on

Column temperature: 35 deg.C

Flow rate: 1.0ml/min

Sample introduction amount: 10 μ l

And (3) an elution mode: gradient elution, which comprises the following specific steps:

time (minutes)	Mobile phase A (%)	Mobile phase B (%)
			0	100	0
2	100	0
			15	95	5
20	95	5
			25	85	15
50	75	25
			60	40	60
70	40	60
			70.1	100	0
75	100	0

Preparing a mixed solution containing two main components and known impurities, injecting the mixed solution into a high performance liquid chromatograph according to the chromatographic conditions, wherein HPLC (high performance liquid chromatography) spectra are shown in figures 1-3, and the detected number of chromatographic peaks and the response results of the small-component main components are detailed in the following table:

table 2 mobile phase selection results

The above experimental data show that the detected number of the impurities and the response of the main peak of the propofol tenofovir are integrated, the pH value is preferably 4.6-6.5, and the ammonium acetate is volatile salt and is more friendly to an instrument system than potassium dihydrogen phosphate, so that the buffer salt in the mobile phase is determined to be ammonium acetate, and the pH value of the mobile phase is preferably about 4.6.

Example 2: selection of elution mode and elution procedure

The emtricitabine propofol tenofovir tablets have more impurities to be researched, have larger polarity difference, cannot achieve effective separation by isocratic elution and have long time consumption, so a gradient elution mode is preferred.

The gradient elution mode of the double-channel mode and the three-channel mode is selected, the repeatability on different HPLC brands is investigated, and the result shows that the gradient elution mode can effectively separate related impurities under the two-channel mode and the Mulberry island mode. Wherein, the gradient elution mode under the three-channel mode has better reproducibility, and the retention time and the relative retention time of each component in the solution with system applicability are more stable, so the elution is carried out in the most preferable three-channel gradient elution mode. Specific data are shown in the following table. The chromatographic conditions for the two gradient elution modes were as follows:

TABLE 3

TABLE 4 retention time of each component of the system applicability solution, relative retention time in dual channel gradient elution mode

TABLE 5 retention time, relative retention time of each component in system applicability solution in three-channel gradient elution mode

Example 3: detection of related substances of emtricitabine-propofol tenofovir tablets

According to the method for determining related substances of the emtricitabine propiophenol tenofovir tablets, the content of each impurity in the related substances of 181201 batches of emtricitabine propiophenol tenofovir tablets produced by my company in a long-term 24-month sample is determined.

(1) Chromatographic conditions

A chromatographic column: waters XSelect HSS T3 (4.6 mm. Times.150mm, 3.5 μm)

Mobile phase: mobile phase a 0.02mol/L ammonium acetate buffer (pH 4.6) -acetonitrile (99.5: 0.5)

The mobile phase B was 0.02mol/L ammonium acetate buffer (pH 4.6) -acetonitrile (95: 5)

The mobile phase C is acetonitrile

Detection wavelength: 260nm/DAD on

Column temperature: 40 deg.C

Flow rate: 1.0ml/min

Sample injection amount: 10 μ l

And (3) an elution mode: gradient elution, which comprises the following steps:

(2) Solution preparation

25% acetonitrile: respectively weighing 25ml of acetonitrile and 75ml of water, placing the acetonitrile and the water in the same reagent bottle, and uniformly mixing to obtain the reagent.

Test solution: precisely weighing an appropriate amount of emtricitabine-propofol-tenofovir tablet fine powder (about 200mg of emtricitabine and 25mg of propofol-tenofovir), adding about 2.5ml of 25% acetonitrile, shaking for no less than 5min, diluting with water to a scale, shaking uniformly, standing, taking an appropriate amount of supernatant, filtering, discarding at least 2ml of primary filtrate, and taking subsequent filtrate as a sample solution.

Control solution: precisely measuring 1ml of the test solution, placing the test solution in a 100ml measuring flask, diluting the test solution to a scale with water, and shaking up to obtain a control solution.

System applicability solution: respectively weighing appropriate amount of each impurity reference substance, emtricitabine reference substance and propane phenol fumarate tenofovir reference substance in the table 1, adding a small amount of water or 25% acetonitrile to dissolve, and diluting with water to prepare a mixed solution containing about 4mg of emtricitabine, 0.5mg of propane phenol tenofovir, 8 mu g of each impurity of emtricitabine and 2.5 mu g of each impurity of propane phenol tenofovir in each 1ml, wherein the mixed solution is used as a system applicability solution.

Sensitivity solution: respectively and precisely weighing a proper amount of emtricitabine reference substance and an appropriate amount of fumaric acid, namely, an appropriate amount of propane phenol tenofovir, adding a proper amount of 25% acetonitrile to dissolve the emtricitabine reference substance and the fumaric acid, and diluting the mixture with water to prepare a mixed solution containing 2 mu g of emtricitabine and 0.25 mu g of propane phenol tenofovir in each 1ml of the mixed solution as a sensitivity solution.

(3) Measurement method

Precisely measuring 10 μ l of each of the sensitivity solution and the system applicability solution, respectively injecting into a liquid chromatograph, and recording chromatogram. The signal-to-noise ratio of the peak heights of two main component peaks (emtricitabine and propiofovir) in a sensitivity solution chromatogram map is not less than 10; system applicability the resolution between the components in the solution chromatogram should not be less than 1.5 (no attention is paid to the resolution between chiral impurities labeled with "") in table 1. Precisely measuring 10 μ l of each of the test solution and the control solution, respectively injecting into a liquid chromatograph, and recording chromatogram. Except a solvent peak and a fumaric acid peak (the retention time is about 1.5 min) in a chromatogram of a test solution, if a known impurity peak (except process impurities) exists, calculating according to a corresponding main component self-comparison method added with a correction factor, and determining that the peaks all accord with the specifications (table 1); if other single impurity peak exists, if the ultraviolet absorption spectrum of the chromatographic peak is consistent with that of the emtricitabine or the tenofovir alafenamide peak in the contrast solution, the ultraviolet absorption spectrum is calculated according to the corresponding main component self contrast method, and the ultraviolet absorption spectrum and the contrast solution both accord with the specification; if the ultraviolet absorption spectrum of the two main component peaks is different from that of the contrast solution, the calculation is carried out according to the self contrast method of the main component of the Propofovir, and the calculation is in accordance with the specification. The total degradation impurity of the emtricitabine is not more than 1.2 percent, and the total degradation impurity of the propiophenol tenofovir is not more than 6.0 percent. The impurity peak in the chromatogram of the test solution, which is smaller than the area of the main peak of the tenofovir disoproxil in the sensitivity solution, is ignored.

(4) Measurement results

The signal-to-noise ratio of two main component peaks in the sensitivity solution is more than 10, and the separation of all components in the system applicability solution is more than 1.5, so that the system applicability test meets the requirements, and the specific data are detailed in table 6. 181201 batches of emtricitabine propiolate tenofovir tablets are placed for 24 months under the long-term condition, the product quality still meets the requirements, and the specific data are shown in table 7.

TABLE 6 detection of related substances of emtricitabine-Propofovir tablets-System suitability test

Note: the emtricitabine isomer is introduced into an emtricitabine reference substance, and attention is not required in a test sample; chiral impurities (labeled with an "+" in table 1, and possibly appearing as single, double or split peaks in the chromatogram) do not require attention to the degree of separation.

TABLE 7 detection results of related substances of emtricitabine-propofol-tenofovir disoproxil fumarate long-term 24-month samples

Note: (1) unknown impurities with retention time of 39.650min, 40.227min and 47.297min in a chromatogram of a test solution are all unknown impurities, wherein ultraviolet spectrum absorption patterns of the unknown impurities with retention time of 39.650min and 47.297min are basically consistent with an ultraviolet absorption spectrum of a main peak of emtricitabine in a contrast solution, so that the unknown impurities belong to emtricitabine, and the ultraviolet spectrum absorption pattern of the unknown impurities with retention time of 40.227min is inconsistent with ultraviolet absorption spectra of two main component peaks in the contrast solution, so that the unknown impurities belong to tenofovir prophenolate; (2) BRL is less than the report limit of 0.05 percent, and ND is detected; (3) "/" means not involved.

Example 4: verification method of related substances of emtricitabine-propofol tenofovir tablets

(1) Detection limit

Detection limiting solution: preparing a solution with a certain concentration (the signal-to-noise ratio of each component peak is not less than 3) from each known impurity reference substance, emtricitabine reference substance and the propionic acid propyl phenol fumarate tenofovir reference substance, and taking the solution as a detection limit solution.

The detection limiting solution 10. Mu.l was precisely measured, sample analysis was performed under the chromatographic conditions described in example 3, and the chromatogram was recorded. Test results show that the method has good detection sensitivity (the lowest detection limit is 0.0001%) for each impurity.

TABLE 8 detection limit results of each component in related substances

Note: the unknown impurities are replaced by the reference substance

(2) Linearity and range, correction factor

Linear solution: various known degraded impurity reference substances, emtricitabine reference substances and fumaric acid propyl phenol tenofovir reference substances are prepared into a series of solutions (with quantitative limit concentration-200% limit concentration) as linear solutions.

Precisely measuring 10 μ l of each linear solution, performing sample injection analysis according to the chromatographic conditions in example 3, and recording the chromatogram.

Another person replaces the same instrument to prepare the linear solution for sample injection analysis, and the correction factor f of known degradation impurities is determined, wherein

The calibration factors for known degraded impurities were assigned as the average of the calibration factors for the two-person two-instrument. The results are given in the following table.

TABLE 9 Linear and Range, correction factor results

The test result shows that the linear relation of each component is good under the method, and the ratio of the impurity correction factor f obtained by two persons is between 0.8 and 1.2.

(3) Repeatability of

Impurity control stock solution: preparing a solution with the limit concentration of 10 times of each known degraded impurity reference substance as an impurity reference substance stock solution.

Impurity control solution: and (4) diluting the impurity reference substance stock solution to prepare a solution with a limited concentration as an impurity reference substance solution.

Stock solution of test sample: precisely weighing an appropriate amount of emtricitabine-propofol-tenofovir tablet fine powder (about 2000mg of emtricitabine and 250mg of propofol-tenofovir), putting the emtricitabine-propofol-tenofovir tablet fine powder into a 100ml measuring flask, adding about 12.5ml of 25% acetonitrile solution, shaking for not less than 5min, diluting to scale with water, shaking uniformly, standing, taking an appropriate amount of supernatant, filtering, discarding 2ml of primary filtrate, and taking the subsequent filtrate as a stock solution of a sample. 6 portions of the mixture are prepared by the same method.

Test solution: precisely measuring 2ml of the stock solution of the test sample and 1ml of the stock solution of the impurity reference substance respectively, placing in a 10ml measuring flask, diluting with water to scale, and shaking up.

Control solution: precisely measuring 1ml of the test solution, placing in a 100ml measuring flask, diluting with water to scale, and shaking.

The impurity reference substance solution, the reference solution and the sample solution are precisely measured, sample introduction and analysis are carried out according to the chromatographic conditions in the embodiment 3, and the chromatogram is recorded, so that the results show that the RSD of the results obtained by the external standard method and the main component self-reference method added with the correction factor is less than 10.0 percent, which shows that the difference of the two calculation methods is small, and the method has good repeatability.

Note: (1) all unknown impurities are lower than the report limit (0.05 percent) and are not counted;

(2) in the above table, "1" in the calculation method refers to an external index method, and "2" in the calculation method refers to a principal component self-comparison method with a correction factor.

(3) "RSD1 (%)" refers to RSD values obtained by the same calculation method, and "RSD2 (%)" refers to RSD values obtained by different calculation methods.

(4) Accuracy of

Impurity control stock solution impurity control solution: the same as the corresponding solution in (3) in the present example.

Accuracy test stock solution: the test sample stock solution was prepared as described in (3) of this example. 3 parts of the raw materials are prepared by the same method.

Test solution: and (3) taking 2ml of accurate sample stock solution, putting the sample stock solution into a 10ml measuring flask, diluting the sample stock solution to a scale with water, and shaking up the sample stock solution to obtain the test sample.

Accuracy solution (1) (50% limit concentration): precisely measuring 2ml of accurate stock solution of a test sample and 0.5ml of accurate stock solution of an impurity reference substance respectively, putting the two into a 10ml measuring flask, diluting with water to scale, and shaking up to obtain the test sample. 3 portions of the mixture are prepared by the same method.

Accuracy solution (2) (100% limit concentration): precisely measuring 2ml of accurate stock solution of a test sample and 1ml of accurate stock solution of an impurity reference substance respectively, putting the two into a 10ml measuring flask, diluting with water to scale, and shaking up to obtain the test sample. 3 portions of the mixture are prepared by the same method.

Accuracy solution (3) (150% limit concentration): precisely measuring the accurate stock solution of the sample and the accurate stock solution of the impurity reference substance respectively by 2ml and 1.5ml, placing the two solutions in the same 10ml measuring flask, diluting with water to scale, and shaking up to obtain the final product. 3 parts of the raw materials are prepared by the same method.

Precisely measuring 10 μ l of each of the impurity reference solution, the sample solution and each of the accuracy solutions, performing sample injection analysis under the chromatographic conditions of example 7, and recording the chromatogram. It can be known that the recovery rates of all impurities are between 90.0% and 110.0% under the limit concentrations of 50%, 100% and 150%, which indicates that the method has good accuracy.

(4) Durability of chromatographic conditions

On the basis of the chromatographic conditions in example 3, the influence of the changes in the flow rate (0.9 ml/min and 1.1 ml/min), the column temperature (33 ℃), the mobile phase pH (buffer salts pH 4.3 and pH 4.9 in mobile phases A and B) on the analytical method was examined, respectively.

Control solution, test solution, sensitivity solution and system applicability solution: the corresponding solution was prepared as in example 3.

Under various chromatographic conditions, 10. Mu.l of each solution was measured, injected into a liquid chromatograph, and the chromatogram was recorded.

Under various chromatographic conditions, the peak-to-peak signal-to-noise ratio of two main components in a sensitive solution is greater than 10, the separation condition among the components in a system applicability solution chromatogram is good (the separation degree is greater than 1.5), and the detection result difference of related substances in a test solution is small (the range of detection values of various impurities and degraded total impurities is less than 0.03 percent, and the maximum is 0.02 percent). In summary, the method is robust when there is a slight change in chromatographic conditions

TABLE 10 durability results for chromatographic conditions for related materials methods-test solutions

Note: (1) the impurity of the process is not limited (only the investigation of specificity and detection limit is carried out during verification), so the statistical quantitative detection result is not needed;

(2) BRL means less than the reported limit (0.05%), ND means not detected.

Claims

1. The method for detecting related substances in the emtricitabine-propofol-tenofovir tablet compound preparation is characterized by comprising the following steps of: adopting high performance liquid chromatography, the chromatographic conditions are as follows:

a chromatographic column: octadecylsilane chemically bonded silica is used as a filler, and further, the chromatographic column can be compatible with 100% water;

mobile phase: a combination of buffer and acetonitrile;

detection wavelength: 200-400 nm.

2. The detection method according to claim 1, characterized in that: in the mobile phase, the mobile phase A is buffer solution-acetonitrile = 95-99.5: 5-0.5, and the mobile phase B is acetonitrile; gradient elution procedures such as gradient one or gradient two:

gradient one:

time per minute Mobile phase A/%) Mobile phase B/%) 0 100 0 2 100 0 15 95 5 20 95 5 25 85 15 50 75 25 60 40 60 70 40 60 70.1 100 0 75 100 0

And gradient II:

or, in the mobile phase, the mobile phase a is buffer-acetonitrile = 99-99.5: 1-0.5, the mobile phase B is buffer-acetonitrile = 95: 5, and the mobile phase C is acetonitrile, and the gradient procedure is as follows:

time/min Mobile phase A/%) Mobile phase B/%) Mobile phase C/%) 0 95 5 0 2 95 5 0 10 78 22 0 20 78 22 0 25 0 100 0 32 0 100 0 32.1 95 0 5 35 88 0 12 40 88 0 12 45 78 0 22 65 70 0 30 70 40 0 60 80 40 0 60 80.1 95 5 0 90 95 5 0

。

3. The detection method according to claim 1, characterized in that: in the mobile phase, the pH value of the buffer solution is 2.5-6.5; further, the buffer pH 4.6. + -. 0.3.

4. The detection method according to claim 1 or 2, characterized in that: in the mobile phase, the buffer solution is selected from phosphate, formate and acetate buffer solution; further selected from ammonium formate or ammonium acetate buffer.

5. The detection method according to claim 1, characterized in that: in the mobile phase, the concentration of the salt in the buffer solution is selected from 0.01-0.1mol/L.

6. The detection method according to claim 1, characterized in that: the chromatographic column is selected from Agilent ZORBAX SB-Aq, waters Xbridge Shield RP18 or Waters XSelection HSST 3, further selected from Waters Xbridge Shield RP18 or Waters XSelection HSST 3, more preferably Waters XSelection HSST 3.6 mm x 150mm,3.5 μm or a chromatographic column with equivalent efficiency.

7. The detection method according to claim 1, characterized in that: the method also comprises one of the following conditions: (1) the flow rate of the mobile phase is 0.9 ml/min-1.1 ml/min; (2) the column temperature is 33-40 ℃.

8. The detection method according to claim 1, characterized in that: the compound preparation is selected from a solid preparation or a liquid preparation; further, the solid preparation is selected from tablets, granules, capsules and pills.

9. The detection method according to claim 1, characterized in that: the related substances are selected from 5-fluorocytosine, (2RS, 5S) -5- (4-amino-5-fluoro-2-oxo-1 (2H) -pyrimidin-1-yl) -1, 3-oxathiane-2-carboxylic acid, (2R, 3RS, 5S) -5-fluoro-1- [ 2-hydroxymethyl-3-oxo-1, 3-oxathiolan-5-yl ] cytosine, (2R, 5S) -5-fluoro-1- [ 2-hydroxymethyl-1, 3-oxo-1, 3-oxathiolan-5-yl ] uracil, (-) -1- [ (2R, 5S) -2- (hydroxymethyl) -1, 3-oxathiolan-5-yl ] cytosine, (2R, 5S) -5- (5-fluorocytosin-1-yl) -1, 3-oxathiolan-2-carboxylic acid menthyl ester, (R) -9- (2-phosphonic acid [ methoxypropyl) -phenyl ester, phenol, [ < 1R) -2- (6-amino-2-oxo-1-methyl ] -1, 3-oxathiolan-5-yl ] phosphate, [ (2R, 5-hydroxy-methyl-1, 3-oxathiolan-5-yl ] uracil, tenofovir anhydride triethylamine salt, 1-methylethyl N- [ [ [ (1R) -2- (6-amino-9H-purin-9-yl) -1-methylethoxy ] methyl ] phenoxyphosphono ] -D-alanine ester fumarate, [ [ (1R) -2- (6-amino-9H-purin-9-yl) -1-methylethoxy ] methyl ] phenoxyphosphono-isopropyl ester, 1-methylethyl N- [ [ (1R) -2- (6-amino-9H-purin-9-yl) -1-methylethoxy ] methylphosphono ] -L-alanine ester, L-alanine isopropyl ester salt, 1-methylethyl N- [ [ [ (1R) -2- (6-amino-9H-purin-9-yl) -1-methylethoxy ] methyl ] phenoxyphosphono ] -L-alanine ester, 1-ethylN- [ [ [ (1R) -2- (6-amino-9H-purin-9-yl) -1-methylethoxy ] methyl ] phenoxyl-alanine ester, [ [ (1R) -2- (6-amino-9H-purin-9-yl) -1-methylethoxy ] methyl ] phenoxyl ] -alanine ester, and ethyl N- [ [ [ (1R) -2- (6-amino-9H-purin-9-yl ] phenoxy ] methyl ] phenoxy ] ethyl ester, at least one of [ (2r, 5s) -5- (4-amino-5-fluoro-2-oxopyrimidin-1 (2H) -yl) -1, 3-oxathiolan-2-yl ] methylhydrogen [ [ [ (R) -1- (6-amino-9H-purin-9-yl) propan-2-yl ] oxy ] methyl ] phosphonate.

10. The emtricitabine-propofol-tenofovir tablet compound preparation is characterized in that the limit of each related substance is as follows: (ii) less than 0.2% of 5-fluorocytosine, (2R, 3RS, 5S) -5-fluoro-1- [ 2-hydroxymethyl-3-oxo-1, 3-oxathiolan-5-yl ] cytosine less than 0.50%, (2R, 5S) -5-fluoro-1- [ 2-hydroxymethyl-1, 3-oxo-1, 3-oxathiolan-5-yl ] uracil less than 0.50%, (R) -9- (2-phosphonomethoxypropyl) -adenine less than 3%, phenol less than 2%, [ [ (1R) -2- (6-amino-9H-purin-9-yl) -1-methylethoxy ] methyl ] phenyl phosphonate less than 0.75%, tenofovir anhydride triethylamine salt less than 2.5%, 1-methylethyl N- [ [ (1R) -2- (6-amino-9H-purin-9-yl) -1-methylethoxy ] methyl ] -L-alanine ester, l-alanine isopropyl ester salt is 1.0% or less, and [ (2R, 5S) -5- (4-amino-5-fluoro-2-oxopyrimidin-1 (2H) -yl) -1, 3-oxathiolan-2-yl ] methylhydrogen [ [ (R) -1- (6-amino-9H-purin-9-yl) propan-2-yl ] oxy ] methyl ] phosphonate is 0.5% or less.