CN113075307A

CN113075307A - Detection method of propane fumarate tenofovir isomer

Info

Publication number: CN113075307A
Application number: CN202110249334.2A
Authority: CN
Inventors: 辛秀民; 李广; 高峰; 孔霁虹; 李具伟; 孙永丽; 王辉; 甘兴杰
Original assignee: Reyoung Pharmaceutical Co Ltd
Current assignee: Reyoung Pharmaceutical Co Ltd
Priority date: 2021-03-08
Filing date: 2021-03-08
Publication date: 2021-07-06

Abstract

The invention relates to the technical field of drug analysis and detection, and particularly relates to a detection method of a propane fumarate tenofovir isomer. The detection method of the isomer of the propane fumarate tenofovir comprises the following steps: a chromatographic column: a chiral chromatographic column; mobile phase: n-hexane, absolute ethyl alcohol, methanol and diethylamine; detection wavelength: 260 nm; column temperature: 30 ℃; flow rate: 1.0 ml/min; preparing a solution; detecting by high performance liquid chromatography; the chiral chromatographic column is a normal phase chromatographic column with a stationary phase of amylose derivatives; the volume ratio of the mobile phase n-hexane, the absolute ethyl alcohol and the methanol is 65:25:10, and the volume of the diethylamine is 0.1% of the total sum of the first three. The invention provides a detection method of a propane fumarate tenofovir isomer, which can effectively separate isomer impurities, has high sensitivity and good specificity, accurately evaluates the isomer content of a raw material medicament, and has very important significance on clinical medication safety.

Description

Detection method of propane fumarate tenofovir isomer

Technical Field

The invention relates to the technical field of drug analysis and detection, and particularly relates to a detection method of a propane fumarate tenofovir isomer.

Background

Isomers refer to compounds of the same chemical formula but different structures. Although the chemical compositions of isomers are the same, the atoms are arranged in different spatial arrangements, which make them very different in physical, chemical or toxicity properties. All the chiral compounds involved in drug development require investigation of their isomeric impurities. Since the isomers are only different in spatial structure and have basically no difference in polarity, the separation degree on a conventional reversed-phase high performance liquid chromatography (RP-HPLC) and a common chromatographic column is difficult to meet the requirement, the retention time of the isomers on the RP-HPLC is generally coincident or the separation degree is less than 1.5, and the isomers are usually researched by adopting a normal-phase chromatography and simultaneously selecting a chiral chromatographic column, so that the separation degree is more than 1.5, and the system applicability requirement is met.

The common normal phase chromatography is used for detecting isomers, and the mobile phase is organic solvents such as n-hexane, absolute ethyl alcohol, isopropanol and the like, and is used for separating one or two isomers. The structure of the propane fumarate tenofovir disoproxil fumarate (TAF) contains 3 chiral centers, the configuration is reversed in the synthesis process, theoretically, 8 isomers (including a main component) exist, and according to the research on the propane fumarate tenofovir disoproxil fumarate, only a target configuration (RRS configuration) is found to be an active component. In the field of medicine, the impurity control of chemicals is related to the quality and the use safety of the chemicals, so that the method is very significant for controlling the content of related substances of the chemicals, particularly for accurately analyzing and detecting isomers.

The patent CN01813161.1 nucleotide phosphate analogue prodrug and the screening and preparation method thereof disclose an analysis method for separating and detecting diastereoisomers in the fumaric acid Propofovir, which adopts reversed phase HPLC and has a single detection structure.

Patent CN111189947A discloses an analytical method for separating and detecting propionofovir fumarate isomers, which adopts xylonite bonding type chiral chromatographic column CHIRALPAK IC, the mobile phase is one or a mixture of several of n-hexane, isopropanol, methanol, ethanol, methyl tert-butyl ether, tetrahydrofuran, dichloromethane, chloroform, ethyl acetate and acetonitrile, the additive is diethylamine or ethylenediamine, the separated isomer impurities are diastereoisomer SSS, diastereoisomer RSR, enantiomer SRR, phosphorus isomer RRS, diastereoisomer RRR, diastereoisomer SRS and diastereoisomer SSR, example 2 and fig. 5 show that the combination and proportion of the mobile phase and the selection of the column cannot meet the requirement of baseline separation in the method detection.

Disclosure of Invention

The invention aims to solve the technical problem of providing a detection method of a propane fumarate tenofovir isomer, which is a detection method for effectively separating a plurality of isomer impurities, has high sensitivity and good specificity, can accurately evaluate the isomer content of the raw material medicine, and has very important significance on clinical medication safety.

The target compound of the isomer of the propane fumarate tenofovir disoproxil is RRS configuration, toxicological data of each isomer impurity is not obtained at present, and the quality of a raw material medicament can be determined to be not lower than that of an original developer only according to the detection condition of the original developer, so an isomer method needs to be established to control the content of the isomer. The TAF-6-A reference substance used in the invention is a racemate of TAF-6-A and TAF (free state), the configuration and isomer configuration of the target compound of the valproate tenofovir fumarate, and the limited cases in the raw material medicaments are shown in Table 1:

TABLE 1 Propofol fumarate Tenofovir isomer configuration and limits

The detection method of the isomer of the propane fumarate tenofovir comprises the following steps:

(1) a chromatographic column: a chiral chromatographic column;

(2) mobile phase: n-hexane, absolute ethyl alcohol, methanol and diethylamine;

(3) detection wavelength: 258nm to 262 nm;

(4) column temperature: 25-35 ℃;

(5) flow rate: 0.9ml/min to 1.1 ml/min;

(6) preparing a solution;

(7) detecting by high performance liquid chromatography;

the chiral chromatographic column is a normal phase chromatographic column with a stationary phase of amylose derivatives;

the volume ratio of the mobile phase n-hexane, the absolute ethyl alcohol and the methanol is 65:25:10, and the volume of the diethylamine is 0.1 percent of the total volume of the n-hexane, the absolute ethyl alcohol and the methanol.

The chromatographic column is a xylonite CHIRALPAK AD-H column with particle diameter of 5 μm, inner diameter of 4.6mm, and length of 250 mm.

The detection wavelength is 260nm, the column temperature is 30 ℃, the flow rate is 1.0ml/min, the sample injection amount is 20 mul, and the running time is 60 min.

The detection concentration of the propane fumarate tenofovir isomer is 0.04 mu g/ml to 2.5 mu g/ml.

The preparation of the solution of the invention comprises the following steps:

(1) preparation of isomer control solution: respectively taking each isomer reference substance (all racemates), precisely weighing, respectively placing into measuring bottles, adding a diluent for dilution, and preparing to obtain the required concentration;

(2) preparation of system applicability solution: taking a raw material medicine of the propane fumarate tenofovir, precisely weighing, placing in a measuring flask, adding an isomer reference substance solution, diluting, and preparing to obtain the required concentration;

(3) preparing a test solution: taking the raw material medicine of the propane fumarate tenofovir, precisely weighing, placing in a measuring flask, diluting, and preparing to obtain the required concentration.

The diluent is absolute ethyl alcohol.

The calculation formula of the content of the isomer of the propane fumarate tenofovir is as follows:

wherein:

A_{sample (A)}Is the peak area of the isomer in the test sample;

A_{to pair}Peak area of the isomer in the control;

C_{sample (A)}Is the concentration of the test sample;

C_{to pair}Concentration of control.

Specifically, the detection method of the isomer of the malonic acid Propofovir fumarate comprises the following steps:

(1) preparing a blank solvent: taking the diluent as a blank solvent and taking absolute ethyl alcohol as the blank solvent;

(2) preparation of isomer stock solutions: respectively removing 20mg of each of the rotary body TAF-6-A, TAF-6-A1 and TAF-6-A3 and about 10mg of the TAF-6-A2, precisely weighing, respectively placing into four different 20ml measuring bottles, adding a diluent to dissolve and dilute to a scale, and shaking up;

(3) preparation of system suitability solutions: taking about 10mg of the raw material medicine of the propane fumarate tenofovir, precisely weighing, placing in a 20ml measuring flask, adding 0.2ml of each isomer stock solution in the step (2), adding a diluent to dissolve and dilute to a scale, and shaking up;

(4) preparation of isomer control solutions: precisely measuring 1ml of each isomer stock solution in the step (2), putting the isomer stock solutions into a 100ml measuring flask, diluting the isomer stock solutions to a scale with a diluent, and shaking up; precisely measuring 2ml, placing in a 20ml measuring flask, diluting to scale with diluent, and shaking;

(5) preparing a test solution: taking about 10mg of the raw material medicine of the propane fumarate tenofovir, precisely weighing, placing in a 20ml measuring flask, adding a diluent to dissolve and dilute to a scale, and shaking up;

(6) detecting by high performance liquid chromatography.

Preferably, the high performance liquid chromatography conditions are preferably:

a chromatographic column: xylonite CHIRALPAK AD-H chiral chromatographic column with particle diameter of 5 μm, inner diameter of 4.6mm, and length of 250 mm;

column temperature: 30 ℃;

detection wavelength: 260 nm;

flow rate: 1.0 ml/min;

sample introduction amount: 20 μ l.

The invention adopts the diluent to dilute and prepare the reference solution, the system applicability solution and the test solution, and the raw material medicine is easy to dissolve in absolute ethyl alcohol, so the absolute ethyl alcohol is preferably used as the diluent and the blank solvent.

The invention adopts a normal phase chiral chromatographic column taking amylose derivatives as a stationary phase, and adopts normal hexane, absolute ethyl alcohol and methanol as a mobile phase in a specific volume ratio of 65:25:10, so that the mobile phase has strong elution capacity, particularly methanol, improves the separation degree among isomers, and performs isocratic elution; the tail-sweeping agent adopts diethylamine for improving the isomer peak type, and the addition volume of the diethylamine is 0.1 percent of the total sum of the volumes of n-hexane, absolute ethyl alcohol and methanol; in order to achieve a desired degree of separation between the isomers and a suitable running time, the flow rate is preferably 1.0 ml/min.

The selection of the chiral chromatographic column and the mobile phase in the adopted normal-phase chromatography is particularly important, and the mobile phase of n-hexane, absolute ethyl alcohol, diethylamine and methanol is different from the conventional normal-phase system.

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

(1) the method adopts the high performance liquid chromatography to detect the isomer of the propane fumarate tenofovir, has high detection sensitivity, has the minimum detection limit of 0.01 mu g/ml (S/N is 3.9) and the minimum quantification limit of 0.04 mu g/ml (S/N is 10.7), and meets the requirement of detecting the isomer with low concentration;

(2) the method of the invention has wide linear range and is limited in quantificationWithin the concentration range of 500 percent of the limit concentration (0.04 mu g/ml-2.5 mu g/ml), the peak area and the concentration of each isomer impurity present a good linear relationship, and the correlation coefficient R²Are all larger than 0.999;

(3) the method has good accuracy, the recovery rates of all isomer impurities in the three levels of 50%, 100% and 150% of standard sample solution are between 90.0% and 110.0%, and the result is reliable; the solution stability is good, the system applicability solution, the reference solution and the sample solution are continuously fed in 24 hours, the RSD of the peak area% of each isomer impurity in the system applicability solution is between 0.02% and 1.21%, the difference of the detected amount of the isomer impurity in the sample solution is small, and the accurate quantification of each isomer impurity in the product can be realized.

Drawings

FIG. 1 is a typical chromatogram for a column screen of example 1;

FIG. 2 is a typical chromatogram for a column screen of example 1;

FIG. 3 is a typical chromatogram for mobile phase screening of example 1;

FIG. 4 is a typical chromatogram for mobile phase screening of example 1;

FIG. 5 is a specificity overlay for embodiment 2;

FIG. 6 is a graph of the detection limit and quantitative limit overlap in example 3;

FIG. 7 is a linear plot of the peak 1 isomer impurity of TAF-6-A1 of example 4;

FIG. 8 is a linear plot of the peak 2 isomer impurity of example 4 TAF-6-A1;

FIG. 9 is a line graph of the impurities of the TAF-6-A isomer of example 4;

FIG. 10 is a linear graph of the TAF of example 4;

FIG. 11 is a linear plot of the peak 1 isomer impurity of TAF-6-A3 of example 4;

FIG. 12 is a linear plot of the peak 2 isomer impurity of example 4 TAF-6-A3;

FIG. 13 is a line graph of the impurity for the TAF-6-A2 isomer of example 4;

FIG. 14 is a high performance liquid chromatogram of a system suitability solution in example 7;

FIG. 15 is a high performance liquid chromatogram of a control solution of example 7;

FIG. 16 is a high performance liquid chromatogram of the test solution of example 7;

FIG. 17 is a high performance liquid chromatogram of a system suitability solution of comparative example 1;

FIG. 18 is a high performance liquid chromatogram of a system suitability solution of comparative example 2;

FIG. 19 is a high performance liquid chromatogram of a system suitability solution in comparative example 3.

Detailed Description

The present invention is further described below with reference to examples.

The starting reagents used in the examples are shown in table 2:

table 2 raw material reagent table

Reagent	Rank of	Manufacturer of the product	Purity of
				N-hexane	AR	Tianjin chemical reagent factory	/
Anhydrous ethanol	AR	TIANJIN GUANGFU TECHNOLOGY DEVELOPMENT Co.,Ltd.	/
				Methanol	HPLC	Fisher	/
Diethylamine	AR	Tianjin chemical reagent factory	/
				TAF-6-A reference substance	Reference (racemate)	Beijing Zhongshuo medicine science and technology development Co Ltd	96.4％
TAF-6-A1 reference	Reference (racemate)	Beijing Zhongshuo medicine science and technology development Co Ltd	96.4％
				TAF-6-A2 reference	Reference (racemate)	Beijing Zhongshuo medicine science and technology development Co Ltd	95.9％
TAF-6-A3 reference	Reference (racemate)	Beijing Zhongshuo medicine science and technology development Co Ltd	96.2％
				Propofol fumarate tenofovir	Sample (I)	BEIJING LEADINGPHARM MEDICINE DEVELOPMENT Co.,Ltd.	100.1％

The instrumentation used in the examples is shown in table 3:

TABLE 3 Instrument and Equipment Table

Example 1

Establishing a detection method and optimizing conditions:

the limits are specified:

isomer impurities are controlled according to common impurities, and the limit is 0.10 percent according to the guidance principle of international harmonization conference (ICH) of registration technology of human drugs, and the isomer impurities are used as a reference basis for development of a detection method.

All isomer impurities have maximum absorption wavelength around 260nm, the detection wavelength can be selected from 258nm to 262nm, and the preferred embodiment is 260 nm; when the sampling amount is 20 mul, the minimum peak height of each isomer impurity is 0.5mV, which can meet the sensitivity requirement of the method, so the concentration of the sample is determined to be 0.5mg/ml, the concentration of the isomer reference substance is determined to be 0.5 mug/ml, and each isomer impurity is prepared into a system applicability solution and an impurity positioning solution according to the concentration of 1 percent of the sample.

Screening of chromatographic column:

preparing a system applicability solution and an impurity positioning solution from the test sample with the concentration of 0.5mg/ml and various isomer impurities according to the concentration of 1% of the test sample, and screening a chromatographic column by using a high performance liquid chromatograph, wherein the screening conditions and the screening results are shown in table 4:

TABLE 4 screening conditions and screening results of chromatography columns

Screening of mobile phase:

the concentration of the test sample is 0.5mg/ml, each isomer impurity is prepared into a system applicability solution according to the concentration of 1% of the test sample, a high performance liquid chromatograph is adopted to carry out the screening of the mobile phase, and the screening type, the screening volume ratio and the screening result are shown in table 5:

TABLE 5 screening types and volume ratios of mobile phases and screening results

The verification analysis method selection conditions and the impurity calculation formula are as follows:

a chromatographic column: CHIRALPAK AD-H (4.6mm X250 mm, 5 μm)

Mobile phase: n-hexane: anhydrous ethanol: methanol: the volume ratio of diethylamine is 65:25:10:0.1

Column temperature: 30 deg.C

Flow rate: 1.0ml/min

Wavelength: 260nm

Sample introduction amount: 20 μ l

Operating time: 60min

The concentration of the test sample: 0.5mg/ml

Diluent agent: anhydrous ethanol

A_{Sample (A)}Is the peak area of the isomer in the test sample;

A_{to pair}Peak area of the isomer in the control;

C_{sample (A)}Is the concentration of the test sample;

C_{to pair}Concentration of control.

Example 2

Verifying system applicability and specificity:

solution preparation:

blank solvent: absolute ethanol, a diluent;

isomer stock solution: respectively removing 20mg of each of a rotator TAF-6-A, TAF-6-A1 and TAF-6-A3 and about 10mg of TAF-6-A2, precisely weighing, respectively placing into four different 20ml measuring bottles, adding a diluent to dissolve and dilute to scale to obtain isomer stock solutions, wherein the concentrations of the TAF-6-A, TAF (in a free state), the TAF-6-A1 peak 1, the TAF-6-A1 peak 2, the TAF-6-A3 peak 1, the TAF-6-A3 peak 2 and the TAF-6-A2 peaks are respectively about 0.5 mg/ml;

system applicability solution: taking about 10mg of a raw material drug of the propane fumarate tenofovir, precisely weighing, placing into a 20ml measuring flask, adding 0.2ml of each isomer stock solution, adding a diluent to dissolve and dilute to a scale to obtain a system applicability solution, wherein the concentration of the propane fumarate tenofovir is about 0.5mg/ml, and the concentrations of a TAF-6-A, TAF-6-A1 peak 1 peak, a TAF-6-A1 peak 2 peak, a TAF-6-A3 peak 1 peak, a TAF-6-A3 peak 2 peak and a TAF-6-A2 peak are about 5 mu g/ml respectively;

isomer control solution: precisely measuring 1ml of the isomer stock solutions respectively, placing the isomer stock solutions into a same 100ml measuring flask, diluting the isomer stock solutions to a scale with a diluent, and shaking up; precisely measuring 2ml of the solution, placing the solution into a 20ml measuring flask, diluting the solution to a scale with a diluent, and shaking the solution uniformly to obtain a reference solution, wherein the concentrations of the TAF-6-A, TAF (in a free state), the TAF-6-A1 peak 1, the TAF-6-A1 peak 2, the TAF-6-A3 peak 1, the TAF-6-A3 peak 2 and the TAF-6-A2 peak are respectively about 0.5 mu g/ml;

test solution: taking about 10mg of the raw material medicine of the propane bisphenol tenofovir fumarate, precisely weighing, placing in a 20ml measuring flask, adding a diluent to dissolve and dilute to a scale to obtain a test solution, wherein the concentration of the propane bisphenol tenofovir fumarate is about 0.5 mg/ml.

Sample introduction procedure sample introduction analysis was performed according to table 6

TABLE 6 System applicability and specificity injection procedure

Numbering	Name of solution	Number of samples taken
			1	Blank solvent	≥1
2	System applicability solution	1
			3	Control solution 1	1
4	Control solution 2	1
			5	Test solution	1

The measurement results are shown in Table 7.

TABLE 7 System applicability and specificity results

Obtaining a special overlay according to the table 6, as shown in the special overlay of fig. 5, it can be seen that the blank solvent has no interference peak at the position where each isomer impurity appears; and no impurity interferes with isomer detection in a test solution, so that the method has good specificity; the separation degree among all isomer impurities and between the isomer impurities and a main peak in the system applicability solution is good, the theoretical plate number of all isomer chromatographic peaks is more than 5000, the tailing factor is less than 1.5, and the system applicability is good.

Example 3:

verifying detection limit and quantification limit:

solution preparation:

blank solvent: absolute ethanol, a diluent;

isomer positioning solution: respectively taking 1ml of isomer stock solution, respectively placing the isomer stock solution into four different 50ml measuring flasks, diluting the isomer stock solution to a scale with a diluent, and shaking up to obtain isomer positioning solutions, wherein the concentrations of a TAF-6-A, TAF (free state), a TAF-6-A1 peak 1, a TAF-6-A1 peak 2, a TAF-6-A3 peak 1, a TAF-6-A3 peak 2 and a TAF-6-A2 peak are respectively about 10 mu g/ml;

quantitative limiting solution: respectively taking 0.1ml of TAF-6-A and 0.2ml of TAF-6-A1 isomer positioning solution, putting 0.3ml of TAF-6-A2 isomer positioning solution and 0.2ml of TAF-6-A3 isomer positioning solution into the same 20ml measuring flask, diluting the solution to a scale by using a diluent, and shaking up to obtain a quantitative limiting solution, wherein the concentrations of TAF-6-A, TAF (in a free state), TAF-6-A1 peak 1 and TAF-6-A1 peak 2 are all 0.05 mu g/ml; the concentrations of TAF-6-A3 peak 1 and TAF-6-A3 peak 2 are both 0.1. mu.g/ml; the peak concentration of TAF-6-A2 was 0.15. mu.g/ml;

detection limiting solution: putting 3ml of the quantitative limiting solution into a 10ml measuring flask, diluting the quantitative limiting solution to a scale with a diluent, and shaking up to obtain a detection limiting solution, wherein the concentrations of TAF-6-A, TAF (in a free state), TAF-6-A1 peak 1 and TAF-6-A1 peak 2 are all 0.015 mu g/ml; the concentrations of TAF-6-A3 peak 1 and TAF-6-A3 peak 2 are both 0.03 μ g/ml; the peak concentration of TAF-6-A2 was 0.045. mu.g/ml.

Sample injection procedure sample injection analysis was performed according to table 8:

TABLE 8 detection limit and quantitative sample limit introduction procedures for detection methods

Numbering	Name of solution	Number of samples taken
			1	Blank solvent	≥1
2	Quantitative limiting solution	1
			3	Detection limiting solution	1

The quantitative limit and the detection limit were tested according to the sampling procedure of table 8, the test spectra are shown in fig. 6 and the quantitative limit overlay, and the test results are shown in table 9:

TABLE 9 quantitation limit and detection limit test results

As can be seen from the data in the above Table 9, the signal-to-noise ratios of the quantitative limit solutions of the impurities of the isomers are all about 10, the concentrations are all far smaller than the report limit of 0.05%, the signal-to-noise ratios of the detection limits are all about 3, the concentrations are all one third of the quantitative limit, and the method is indicated to have higher sensitivity.

Example 4

Verification of linearity and range:

solution preparation:

blank solvent: absolute ethanol, a diluent;

linear stock solutions: respectively taking 5ml of the isomer stock solutions, placing the isomer stock solutions into a 50ml measuring flask, diluting the isomer stock solutions to a scale by using a diluent, and shaking the isomer stock solutions uniformly to obtain linear stock solutions, wherein the concentrations of a TAF-6-A, TAF (free state), a TAF-6-A1 peak 1, a TAF-6-A1 peak 2, a TAF-6-A3 peak 1, a TAF-6-A3 peak 2 and a TAF-6-A2 peak are respectively about 0.05 mg/ml;

quantitative limiting solution: respectively taking 0.1ml of each of the TAF-6-A and TAF-6-A1 isomer positioning solutions, 0.3ml of each of the TAF-6-A2 isomer positioning solution and 0.2ml of each of the TAF-6-A3 isomer positioning solutions, putting the solutions in a same 20ml measuring flask, diluting the solutions to a scale by using a diluent, and shaking up to obtain a quantitative limiting solution;

linear 50% (limiting concentration) solution: taking 5ml of the linear stock solution, putting the linear stock solution into a 100ml measuring flask, diluting the linear stock solution to a scale with a diluent, and shaking up; precisely measuring 1ml of the solution, putting the solution into a 10ml measuring flask, diluting the solution to a scale with a diluent, and shaking up;

linear 100% (limiting concentration) solution: taking 1ml of the linear stock solution, putting the linear stock solution into a 100ml measuring flask, diluting the linear stock solution to a scale with a diluent, and shaking up;

linear 200% (limiting concentration) solution: taking 5ml of the linear stock solution, putting the linear stock solution into a 25ml measuring flask, diluting the linear stock solution to a scale with a diluent, and shaking up; precisely measuring 1ml of the solution, putting the solution into a 10ml measuring flask, diluting the solution to a scale with a diluent, and shaking up;

linear 500% (limiting concentration) solution: taking 5ml of the linear stock solution, placing the linear stock solution into a 100ml measuring flask, diluting the linear stock solution to the scale with a diluent, and shaking up the linear stock solution.

The linearity test was performed according to the injection procedure of table 10:

TABLE 10 Linear test sample introduction procedure

Numbering	Name of solution	Number of samples taken
			1	Blank solvent	≥1
2	Quantitative limiting solution	1
			3	Linear 50% (limiting concentration) solution	1
4	Linear 100% (limiting concentration) solution	1
			5	Linear 200% (limiting concentration) solution
6	Linearity 500% (limit)Concentration) solution	1

The results of the linearity test of the high performance liquid analysis performed on the above solutions are shown in Table 11:

TABLE 11 results of the Linear test

Making a linear regression line by taking the concentration value of each isomer as an abscissa and the peak area as an ordinate and taking the concentration value of each isomer as an ordinate within the range of the limit concentration of the quantitative limit to 500 percent, wherein the linear regression coefficient r is²Are all greater than 0.999, the linearity is good, as shown in the linear graphs of fig. 7-13.

Example 5

Verifying the accuracy of the system:

preparing a solution: the preparation of the blank solvent, the isomer stock solution, the isomer reference solution and the test solution is the same as that of the example 2;

accuracy stock solution: respectively taking 1ml of isomer stock solutions, placing the isomer stock solutions into the same 100ml measuring flask, diluting the isomer stock solutions to a scale with a diluent, and shaking up;

spiked test sample solution (50% limit concentration): taking about 10mg of the raw material medicine of the propane fumarate tenofovir, precisely weighing, placing in a 20ml measuring flask, adding 1ml of accuracy stock solution, adding a diluent to dissolve and dilute to a scale, and shaking up; preparing 3 parts by the same method;

standard test sample solution (100% limit concentration): taking about 10mg of the raw material medicine of the propane fumarate tenofovir, precisely weighing, placing in a 20ml measuring flask, adding 2ml of accuracy stock solution, adding a diluent to dissolve and dilute to a scale, and shaking up; preparing 3 parts by the same method;

standard test sample solution (150% limit concentration): taking about 10mg of the raw material medicine of the propane fumarate tenofovir, precisely weighing, placing in a 20ml measuring flask, adding 3ml of accuracy stock solution, adding a diluent to dissolve and dilute to a scale, and shaking up; 3 parts of the composition were prepared in the same manner as above.

The injection procedure for the accuracy experiment is shown in table 12:

TABLE 12 sample introduction procedure for accuracy experiments

Numbering	Name of solution	Number of samples taken
			1	Blank solvent	≥1
2	Control solution 1	1
			3	Control solution 2	1
4	Test solution	1
			5	Standard sample solution (50% limit concentration-1)	1
6	Standard sample solution (50% limit concentration-2)	1
			7	Standard sample solution (50% limit concentration-3)	1
8	Standard sample solution (100% limit concentration-1)	1
			9	Standard sample solution (100% limit concentration-2)	1
10	Standard sample solution (100% limit concentration-3)	1
			11	Standard sample solution (150% limit concentration-1)	1
12	Standard sample solution (150% limit concentration-2)	1
			13	Standard sample solution (150% limit concentration-3)	1

By measuring the recovery rate of the standard sample with different concentrations ((actually measured amount-target content in sample solution)/added amount x 100%), the recovery rate of each isomer of 3 concentration levels is between 90.0% and 110.0%, and the recovery rate of each concentration level and the recovery rate RSD of all levels are less than 10.0%, thus the method has good sample application recovery rate and high accuracy. The test results are shown in table 13.

TABLE 13 spiking recovery test results for each isomer

Example 6

And (3) verifying the stability of the solution:

preparing a solution: the preparation of the blank solvent, isomer stock solution, isomer control solution, test solution and system suitability solution is the same as that in example 2.

The experimental injection procedure for solution stability at room temperature is shown in table 14:

table 14 experimental injection procedure for stability

After the solution is respectively placed for 5h, 8h, 12h and 24h at room temperature, the RSD of the peak area% of each isomer impurity in the system applicability solution is respectively measured to be between 0.02% and 1.21%, and the detected amount of the isomer impurity in the test solution is basically not different, which indicates that the solution is stable after being placed for 24h at room temperature. The results of stability tests on the test solution and the system suitability solution at 5h, 8h, 12h and 24h are shown in Table 15.

TABLE 15 stability test results

Example 7

The isomer in the raw material medicine of the propiofovir fumarate is detected by adopting the detection method provided by the invention.

Diluent agent: anhydrous ethanol.

Isomer stock solution: respectively removing 20mg of each of the rotary body TAF-6-A, TAF-6-A1 and the rotary body TAF-6-A3 and about 10mg of the rotary body TAF-6-A2, precisely weighing, respectively placing into four different 20ml measuring bottles, adding a diluent to dissolve and dilute to a scale, and shaking up.

System applicability solution: taking about 10mg of the raw material medicine of the propane fumarate tenofovir, precisely weighing, placing into a 20ml measuring flask, adding 0.2ml of each isomer stock solution, adding a diluent to dissolve and dilute to a scale, and shaking up.

Isomer control solution: precisely measuring 1ml of the isomer stock solution, placing the isomer stock solution into a 100ml measuring flask, diluting the isomer stock solution to a scale with a diluent, and shaking up; precisely measuring 2ml of the solution, placing the solution into a 20ml measuring flask, diluting the solution to the scale with a diluent, and shaking up.

Test solution: taking 10mg of the raw material medicine of the propane fumarate tenofovir, precisely weighing, placing in a 20ml measuring flask, adding a diluent to dissolve and dilute to a scale, and shaking up.

The detection is carried out by high performance liquid chromatography, and the specific chromatographic conditions are shown in table 16:

TABLE 16 chromatographic conditions

After detection, the high performance liquid chromatograms of the system suitability solution, the reference solution and the test solution are respectively shown in fig. 14, fig. 15 and fig. 16. And (3) analyzing the map, wherein each isomer impurity peak has good pattern and high separation degree.

According to the formula

Calculating the isomer content in the sample;

wherein, the impurity peak area of each isomer in the test solution is 0, so that the isomer in the test solution is not detected.

Comparative example 1

Comparative example 1 and example 7, sample preparation, chromatographic conditions, detection procedure were all the same, mobile phase was changed to: n-hexane: anhydrous ethanol: diethylamine (75:25:0.1), and the detection result is obtained, such as the high performance liquid chromatogram of the system applicability solution in FIG. 17, which can be seen from FIG. 17: after methanol is removed, an isomer peak coincides with a main peak, and the separation degree does not meet the requirement, so that the method cannot be used for detecting the isomer.

Comparative example 2

Comparative example 1 and example 7, the sample preparation, chromatographic conditions, detection procedure were all the same, only the chromatographic column was replaced with: CHIRALPAK AY-H (4.6 mm. times.250 mm, 5 μm), and the detection result is obtained as a high performance liquid chromatogram of the system suitability solution in FIG. 18, as can be seen in FIG. 18: after the chromatographic column is replaced by an CHIRALPAK AY-H chromatographic column, the isomer impurities and the main component peak patterns are poor, do not meet the requirement of system applicability and cannot be used for detecting the isomers.

Comparative example 3

Comparative example 1 and example 7, the sample preparation, chromatographic conditions, detection procedure were all the same, only the chromatographic column was replaced with: CHIRALPAK OD-H (4.6 mm. times.250 mm, 5 μm), and the detection result is obtained as a high performance liquid chromatogram of the system suitability solution in FIG. 19, as can be seen in FIG. 19: after the chromatographic column is replaced by an CHIRALPAK OD-H chromatographic column, the isomer impurities and the main component peak patterns are poor, do not meet the requirement of system applicability and cannot be used for detecting the isomers.

Claims

1. A detection method of a propane fumarate tenofovir isomer is characterized by comprising the following steps: the method comprises the following steps:

(1) a chromatographic column: a chiral chromatographic column;

(2) mobile phase: n-hexane, absolute ethyl alcohol, methanol and diethylamine;

(3) detection wavelength: 258nm to 262 nm;

(4) column temperature: 25-35 ℃;

(5) flow rate: 0.9ml/min to 1.1 ml/min;

(6) preparing a solution;

(7) detecting by high performance liquid chromatography;

2. The method for detecting a tenofovir disoproxil fumarate isomer according to claim 1, wherein: the chromatographic column is a xylonite CHIRALPAK AD-H column with particle diameter of 5 μm, inner diameter of 4.6mm, and length of 250 mm.

3. The method for detecting a tenofovir disoproxil fumarate isomer according to claim 1, wherein: the detection wavelength is 260nm, the column temperature is 30 ℃, the flow rate is 1.0ml/min, the sample injection amount is 20 mul, and the running time is 60 min.

4. The method for detecting a tenofovir disoproxil fumarate isomer according to claim 1, wherein: the detection concentration of the propane fumarate tenofovir isomer is 0.04 mu g/ml to 2.5 mu g/ml.

5. The method for detecting a tenofovir disoproxil fumarate isomer according to claim 1, wherein: preparing a solution, comprising:

6. The method for detecting a tenofovir disoproxil fumarate isomer according to claim 5, wherein: the diluent is absolute ethyl alcohol.

7. The method for detecting a tenofovir disoproxil fumarate isomer according to claim 1, wherein: the calculation formula of the content of the isomer of the propane fumarate tenofovir is as follows:

wherein:

A_{sample (A)}Is the peak area of the isomer in the test sample;

A_{to pair}Peak area of the isomer in the control;

C_{sample (A)}Is the concentration of the test sample;

C_{to pair}Concentration of control.