WO2020125027A1

WO2020125027A1 - Analysis method for determining substance related to lubiprostone test sample

Info

Publication number: WO2020125027A1
Application number: PCT/CN2019/100185
Authority: WO
Inventors: 李晶晶; 林萍; 吴晶; 王华萍; 兰公剑; 柴雨柱; 徐丹; 朱春霞; 田舟山
Original assignee: 南京正大天晴制药有限公司
Priority date: 2018-12-21
Filing date: 2019-08-12
Publication date: 2020-06-25
Also published as: CN111351867B; CN111351867A

Abstract

Provided is an analysis method for determining a Lubiprostone-related substance, wherein in the method, a Lubiprostone compound, a compound of formula II, a compound of formula III, a compound of formula IV and a compound of formula V are separated and determined using normal-phase high-performance liquid chromatography. The method prevents the problem of traditional analysis methods in which Lubiprostone is bimodal. The method is conducive to the calculation of related substance correction factors and the accurate quantification of related substances, and is an analysis method conducive to the quality control of Lubiprostone.

Description

An analytical method for determination of related substances of lubiprostone test sample

This application requires the priority of the Chinese invention patent application with the application number 201811572959.7 and the application date of 2018.12.21.

Technical field

The invention belongs to the field of pharmaceutical analysis, and particularly relates to a high-performance liquid chromatography analysis method of related substances of lubiprostone.

technical background

Lubiprostone (Compound L) is a localized chloride channel activator that can selectively activate type 2 chloride channels located on the cell membrane of the epithelial tip of the gastrointestinal tract, increasing intestinal fluid secretion and intestinal movement Sex, thereby increasing bowel movements, FDA has approved lubiprostone for the treatment of chronic idiopathic constipation in adults, constipation type irritable bowel syndrome in adult women and constipation caused by taking opioids.

In the quality control of lubiprostone, the existing literature, such as the patent with publication number CN102020625B, discloses a reversed-phase high performance liquid chromatography analysis method of lubiprostone, which Test the related substances in the test product. However, using the currently disclosed analytical method, lubiprostone is a double peak, which is not conducive to the calculation of the correction factor of related substances and the accurate quantification of related substances. Therefore, in order to effectively control the quality, it is necessary to find a new analytical method for the related substances of lubiprostone.

Summary of the invention

On the one hand, the present invention provides a method for analyzing related substances of lubiprostone. The method is high performance liquid chromatography, which uses a normal phase chromatography column, and the mobile phase is composed of n-hexane, ethanol, and glacial acetic acid. The volume ratio of n-hexane, ethanol and glacial acetic acid in the mobile phase is 400-600:30-80:0.5-5.

In some embodiments, the normal phase chromatography column uses a polar stationary phase as a filler; in some typical embodiments, the normal phase chromatography column uses 1,2-dihydroxypropane-functional organosilane-bonded silica gel Is a packing; in some more typical embodiments, the normal phase chromatography column is selected from Li Chrospher 100 Diol/Merck, Waters ACQUITY BEH200SEC, Kromasil Diol, or LiChrosorb Diol; in some of the most typical embodiments, the positive The phase chromatography column uses Li Chrospher 100 Diol/Merck, and its specification is 4.0mm×250mm, 5μm.

In some embodiments, the volume ratio of n-hexane, ethanol and glacial acetic acid in the mobile phase is 450-550:35-55:0.5-3; in some typical embodiments, the n-hexane in the mobile phase The volume ratio of alkanes, ethanol and glacial acetic acid is 480:50:2.

In some embodiments, the flow rate of the mobile phase is 0.7-1.5 ml/min; in some more typical embodiments, the flow rate of the mobile phase is 0.9-1.1 ml/min; in some Typical ranges are 0.9 ml/min, 1.0 ml/min and 1.1 ml/min or any two of them; in some of the most typical embodiments, the flow rate of the mobile phase is 1.0 ml/min.

In some embodiments, the analysis method is performed on a high-performance liquid chromatograph, using a diode array detector, an ultraviolet absorption detector, a refractive index detector, or an evaporative light scattering detector; in some typical embodiments, the The analysis method is performed on a high-performance liquid chromatograph, using an ultraviolet absorption detector; in some more typical embodiments, the analysis method is performed on a high-performance liquid chromatograph, using an ultraviolet absorption detector, and its detection wavelength 292-296nm; in some more typical embodiments, the analysis method is performed on a high-performance liquid chromatograph, using an ultraviolet absorption detector, and its detection wavelength is 292nm, 293nm, 294nm, 295nm or 296nm; In some of the most typical embodiments, the analysis method is performed on a high-performance liquid chromatograph, using an ultraviolet absorption detector, and its detection wavelength is 294 nm.

In some embodiments, the column temperature of the normal phase chromatography column is 25-40°C; in some typical embodiments, the column temperature of the normal phase chromatography column is 28-38°C; in some more typical In an embodiment, the column temperature of the normal phase chromatography column is 33-37°C; in some of the most typical embodiments, the column temperature of the normal phase chromatography column is 35°C.

In some embodiments, the analysis method uses isocratic elution or gradient elution; in some typical embodiments, the analysis method uses isocratic elution.

On the one hand, the present invention provides an analysis method of related substances of lubiprostone:

The analysis method is carried out on a high-performance liquid chromatograph;

The detector is an ultraviolet absorption detector with a detection wavelength of 294nm;

The column is a normal phase column with a column temperature of 35°C;

The mobile phase is a mixed solution composed of n-hexane, ethanol and glacial acetic acid, in which the volume ratio of n-hexane, ethanol and glacial acetic acid is 480:50:2, and the flow rate of the mobile phase is: 1.0 ml/min;

Inject Lubiprostone test solution, control solution and system suitability solution separately;

The system suitability solution comprises compound L and one or more mixtures selected from the group consisting of compounds of formula II, compounds of formula III, compounds of formula IV, and compounds of formula V;

The content of the compound of formula II, compound of formula III, compound of formula IV and/or compound of formula V in the sample is calculated by the principal component self-control method.

In some embodiments, the self-control method adds a correction factor for calculation; in some embodiments, the self-control method does not add a correction factor for calculation; in some typical embodiments, the compound of formula III and formula In the calculation of the content of the compound V, a correction factor is added for calculation, and in the calculation of the content of the compound of formula II and the compound of formula IV, the calculation is performed without adding a correction factor. In some more typical embodiments, the compound of formula III has a correction factor of 0.44 and the compound of formula V has a correction factor of 0.5.

In some specific embodiments, the present invention provides an analysis method of lubiprostone related substances:

(1) Mobile phase solution: configure a mixed solution with a volume ratio of 480:50:2 of n-hexane, ethanol and glacial acetic acid;

(2) Formula II compound positioning solution preparation: take compound II compound reference substance, weigh accurately, add mobile phase to dissolve and dilute, dilute to volume, shake well, then get;

Wherein, the compound of formula II has the structural formula:

(3) Formula III compound positioning solution preparation: take compound III compound reference substance, accurately weigh, add mobile phase to dissolve and dilute, dilute to volume, shake well, that is,

Wherein, the compound of formula III has the structural formula:

(4) Formula IV compound positioning solution preparation: take the compound IV compound reference substance, weigh accurately, add mobile phase to dissolve and dilute, dilute to volume, shake well, that is,

Wherein, the compound of formula IV has the structural formula:

(5) Formula V compound positioning solution preparation: take compound V compound reference substance, accurately weigh it, add mobile phase to dissolve and dilute, dilute to volume, shake well, that is,

Wherein, the structural formula of the compound of formula V is:

(6) System suitability solution preparation: take appropriate amount of compound of formula II, compound of formula III, compound of formula IV, compound of formula V and compound L, weigh accurately, add mobile phase to dissolve and dilute, dilute volume, shake well, then ;

(7) Preparation of test solution: take the appropriate amount of lubiprostone for the test product, accurately weigh it, dissolve and dilute with mobile phase, dilute to volume, shake well, then get;

(8) Preparation of control solution: accurately measure the appropriate amount of the test solution, and quantitatively dilute 100 times with mobile phase as the control solution;

(9) Determination of test product: A normal phase chromatographic column of Li Chrospher 100 Diol/Merck, size 4.0mm×250mm, 5μm, and mobile phase n-hexane-ethanol-glacial acetic acid (V:V:V=480:50 :2) Mixed solution, adjust the flow rate to 1.0ml/min, collect 30min, column temperature is 35℃; detection wavelength is 294nm, injection volume is 40μl, respectively measure the mobile phase solution, formula II compound positioning solution, formula III The compound positioning solution, the compound positioning solution of formula IV, the compound positioning solution of formula V, the compound L positioning solution, the system suitability solution, the test solution and the control solution were injected into the liquid chromatograph, and the chromatograms were recorded;

(10) Content calculation: According to each chromatogram, the content of each related substance in the sample is calculated by the main component self-control method with or without correction factor:

Wherein, A _t is the compound of formula II for the chromatogram of the test solution, the compound of Formula III, a compound of Formula IV or Formula V compound respective peak areas; F is a correction factor; A is the total peak area.

In some embodiments, the present invention provides a high-purity lubiprostone composition, the content of the compound of formula II is not higher than 0.50%, preferably not higher than 0.10%; the content of the compound of formula III is not higher than 0.50% , Preferably not more than 0.10%; the content of the compound of formula IV is not more than 0.50%, preferably not more than 0.10%; the content of the compound of formula V is not more than 0.50%, preferably not more than 0.10%; other unknown single impurities The content is not higher than 1.0%, preferably not higher than 0.50%.

In yet another aspect, the present invention provides a compound of formula III, the structure of which is:

In some embodiments, the invention provides a compound of formula III with a purity ≥90%; in some typical embodiments, the invention provides a compound of formula III with a purity ≥95%; in some more typical In an embodiment, the present invention provides a compound of formula III with a purity ≥98%.

In still another aspect, the present invention also provides a use of the compound of formula III as a reference marker during the impurity inspection of lubiprostone.

In some embodiments, the present invention provides the use of a compound of formula III with a purity of not less than 90% as a reference marker when checking the impurity of lubiprostone; in some typical embodiments, the present invention provides The use of a compound of formula III with a purity of not less than 95% as a reference marker during the impurity inspection of lubiprostone; in some more typical embodiments, the present invention provides a purity of not less than 98 % Of the compound of formula III is used as a reference marker during the impurity inspection of lubiprostone.

In some embodiments, the content of the compound of formula III in the test product of lubiprostone is not higher than 0.50%; in some more typical embodiments, the content of the compound of formula III in the test product of lubiprostone is not higher than 0.10% .

On the other hand, the present invention provides a method for preparing a compound of formula III, characterized in that compound III-1 is reacted to obtain a compound of formula III:

In some embodiments, the present invention provides a method for preparing a compound of formula III. Compound III-1 is reacted in the presence of acetonitrile, glacial acetic acid, and trifluoroacetic acid to obtain a compound of formula III.

In some embodiments, the present invention provides a method for preparing a compound of formula III, including the steps of: adding compound III-1, acetonitrile, and glacial acetic acid to a reaction vessel, and adding trifluoroacetic acid, after the reaction is completed, to obtain The compound of formula III.

In some embodiments, the present invention provides a method for preparing a compound of formula III, which includes the steps of: adding compound III-1, acetonitrile, and glacial acetic acid to a reaction vessel, heating, stirring, and adding trifluoroacetic acid, to be reacted After the end, extraction, washing and drying to obtain the compound of formula III.

In some embodiments, the present invention provides a method for preparing a compound of formula III, which includes the steps of: adding compound III-1, acetonitrile, and glacial acetic acid to a reaction vessel, heating, stirring, and adding trifluoroacetic acid, the reaction ends After that, water was added and extracted with ethyl acetate. The organic phase was washed with saturated sodium bicarbonate solution, saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered with suction, and the filtrate was concentrated under reduced pressure to obtain the compound of formula III.

In some embodiments, the present invention provides a method for preparing a compound of formula III, including the steps of: adding compound III-1 and acetonitrile to a reaction flask, stirring to dissolve until clear, adding glacial acetic acid to stir at room temperature, heating After warming to 50°C, stirring overnight, adding trifluoroacetic acid to the end of the reaction, adding water to the reaction bottle, and extracting with ethyl acetate, combining organic phases, washing with saturated aqueous sodium bicarbonate solution, and then with saturated sodium chloride solution The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was dried under reduced pressure to obtain an oil, that is, compound III.

Herein, the structural formula of the compound of formula II is

This compound can be obtained by direct purchase or by reference to published literature, including but not limited to WO2007091697.

Herein, the structural formula of the compound of formula III is

The compound can be prepared by the preparation method in the examples.

Herein, the structural formula of the compound of formula IV is

The compound can be obtained by direct purchase or by reference to published documents, including but not limited to US20130184476.

Herein, the structural formula of the compound of formula V is

The compound can be prepared by direct purchase or by reference to published documents, including but not limited to US 20130184476, CN102050808A.

Herein, unless otherwise stated, the lubiprostone includes compound L or a pharmaceutically acceptable salt thereof; the lubiprostone used for "test article configuration" includes but is not limited to newly prepared or stored Lubiprostone API, a pharmaceutical composition containing lubiprostone.

In this article, related substances are also expressed as impurities.

In this article, the appropriate amount means that the amount of each compound is within the detection limit or the limit of quantification of its high-performance liquid chromatograph according to the purpose of the experiment.

The concentration of the positioning solution is 10 ng/ml-100 mg/ml; preferably 0.1 μg/ml-10 mg/ml; more preferably 1 μg/ml-2 mg/ml.

The concentration of compound II, compound III, compound IV, and compound V in the system suitability solution is: 0.1 μg/ml-100 mg/ml; preferably 1 μg/ml-10 mg/ml; more preferably 2 μg/ml-2 mg/ml.

The concentration of the compound L in the system suitability solution is: 1 μg/ml-1000 mg/ml; preferably 10 μg/ml-100 mg/ml; more preferably 50 μg/ml-50 mg/ml.

In the control solution, the concentration of compound L is 0.1 μg/ml-10 mg/ml; preferably 2 μg/ml-2 mg/ml; more preferably 5 μg/ml-1 mg/ml.

The calculation of the correction factor in the present invention can be carried out by accurately weighing the appropriate amount of the main component (compound L) reference substance and the impurity reference substance, respectively prepared into solutions of different concentrations, tested by HPLC, and plotting the concentration of the main component and impurity concentration against their peak areas For the regression curve, the correction factor is calculated as the ratio of the slope of the principal component regression line to the slope of the impurity regression line.

The method of the present invention is not limited to the above four impurities. Any separation and determination of lubiprostone and its impurities using the method of the present invention fall within the scope of protection of the present invention, especially the separation and determination of lubiprostone and the Compound of formula II, compound of formula III, compound of formula IV, compound of formula V.

The technical problem to be solved by the present invention is to provide a high-performance liquid chromatographic analysis method capable of accurately determining the content of related substances in the test sample of lubiprostone. In this detection method, the solvent does not interfere with the detection of impurities, and the method has good specificity. The detection method is simple, fast, accurate, high sensitivity, good repeatability and accuracy. The method provided by the invention can accurately determine the above-mentioned identified impurities in the test product of lubiprostone, and the separation degree between the main peak and the adjacent impurity peak is ≥1.2, and the separation degree between each impurity is ≥1.5.

In the detection method of related substances of the present invention, lubiprostone is a single peak, which is beneficial to the calculation of impurity correction factors and can more accurately quantify impurities; and in this method, each chromatographic peak can be effectively separated and can be quickly Accurately perform qualitative and quantitative analysis of the relevant substances of the test article to ensure the controllability of the quality of lubiprostone.

BRIEF DESCRIPTION

Figure 1 is a chromatogram of system suitability solution.

Figure 2 is a chromatogram of the test product solution.

detailed description

The following embodiments can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention to the scope of the described embodiments.

Example 1 Specificity test-impurity location

Instrument: Shimadzu/LC-20AT high performance liquid chromatograph

Column: Li Chrospher 100 Diol/Merck (4.0mm×250mm, 5μm)

Mobile phase: n-hexane-ethanol-glacial acetic acid (480:50:2) mixed solution, isocratic elution for 30 minutes

Detection wavelength: 294nm

Flow rate: 1ml/min

Column temperature: 35℃

Injection volume: 40μl

Impurity positioning solution preparation: appropriate amount of each standard compound of formula II compound, formula III compound, formula IV compound and formula V compound, accurately weighed, respectively dissolved and diluted with mobile phase to prepare a solution containing about 20 μg per 1 ml.

Preparation of test solution: Take the appropriate amount of lubiprostone for the test product, accurately weigh it, dissolve and dilute with mobile phase to prepare a solution containing approximately 20 mg of lubiprostone compound per 1 ml as the test solution.

Preparation of control solution: Take the appropriate amount of the test solution accurately, dilute it 100 times with the mobile phase quantitatively, and prepare a solution containing about 20 μg of lubiprostone compound per 1 ml as the control solution.

System suitability solution preparation: take appropriate amounts of compound L, compound II, compound III, compound IV, compound V reference substance, accurately weigh them, add mobile phase to dissolve and dilute to make about 1% of Ruby in each 1ml A solution of 20 mg of ketone (Compound L) and each impurity (compound of Formula II, compound of Formula III, compound of Formula IV, compound of Formula V) of about 20 μg was used as a system suitability solution.

Sample determination: measure 40 μl each of blank solvent, compound II positioning solution, compound III positioning solution, compound IV positioning solution, compound V positioning solution, system suitability solution, control solution and test solution, respectively Liquid chromatograph, record the chromatogram.

Results: The blank solvent did not interfere with the determination; the retention time and resolution of each compound are shown in the table below. Lubiprostone's separation from adjacent impurities and each impurity meets the requirements.

名称name	保留时间(min)Retention time (min)	与相邻杂质峰的分离度Resolution from adjacent impurity peaks
式V化合物Compound of formula V	3.4673.467	//
式II化合物Compound of formula II	5.4845.484	9.59.5
式IV化合物Compound of formula IV	5.9215.921	1.81.8
式III化合物Compound of formula III	7.5067.506	5.25.2
未知化合物Unknown compound	11.60911.609	10.810.8
化合物L(鲁比前列酮)Compound L (Lubiprostone)	12.49512.495	1.61.6

The chromatogram of the test solution is shown in Figure 2. The measurement results indicate that the content of each impurity is calculated according to the self-control method with the addition of the correction factor. The impurities of Formula V, Formula II, and Formula IV compounds are not detected (less than 0.01%). The content of the compound of formula III is 0.01%, the content of the unknown compound is 0.26%, and the purity of lubiprostone (compound L) is 99.72%.

Example 2 durability test

On the basis of the detection chromatographic conditions in Example 1, when the ratio of the mobile phase, the flow rate, the detection wavelength, and the column temperature conditions were changed slightly, the detection of the relevant substances in the test solution was investigated, and the durability of the detection method was investigated.

The solution preparation and determination method refer to Example 1. The chromatographic conditions and detection results are shown in the following table:

Note: "Ethanol-2%, the remaining conditions are unchanged" means that the mobile phase is changed to: n-hexane-ethanol-glacial acetic acid (480:49:2), the rest of the detection conditions remain unchanged; "Ethanol +2%, the remaining conditions "No change" means that the mobile phase is changed to n-hexane-ethanol-glacial acetic acid (480:51:2), and the rest of the detection conditions remain unchanged.

The above results show that the results of the measurement of related substances have not changed significantly when the ratio of ethanol, column temperature, flow rate and wavelength are changed.

Example 3 Limit of detection and limit of quantification

The lubiprostone reference substance and the impurity reference substance solutions were dissolved in a mobile phase and diluted stepwise, and 40 μl were injected respectively. The sample concentration at the signal-to-noise ratio of 3:1 was the detection limit concentration. The results are as follows: the detection limit of the compound of formula II is 5.8ng, the detection limit of the compound of formula III is 3ng, the detection limit of the impurity compound of formula IV is 6.0ng, the detection limit of the compound of formula V is 1.8ng, and the compound L (rubiprostone )12.0ng.

The lubiprostone reference substance and the impurity reference substance solutions were dissolved in a mobile phase and diluted stepwise, and 40 μl were injected respectively. The sample concentration when the signal-to-noise ratio was greater than 10:1 was the limit of quantitation. The results are as follows: the limit of quantitation of the compound of formula II is 40.2 ng, the limit of detection of the compound of formula III is 7 ng, the limit of detection of the impurity compound of formula IV is 19.9 ng, the limit of detection of the compound of formula V is 5.9 ng, and compound L (rubiprostone ) 40.2ng.

Example 4 Specific test-destruction test

Destructive degradation test was performed on the test product of lubiprostone, and the separation between the generated degradation product and the main peak and degradation product was investigated to verify whether the specificity of the method met the requirements.

Undamaged: Take about 20mg of this product, accurately weigh it, place it in a 1.0ml measuring flask, add an appropriate amount of solvent to ultrasonically dissolve and dilute to the mark, shake well, and accurately inject 40μl into the liquid chromatograph and record the chromatogram. Do a blank test at the same time.

Oxidation damage: Take about 20mg of this product, weigh it accurately, put it in a 1.0ml measuring bottle, add 0.1ml of 3% hydrogen peroxide solution, after 5 hours, add solvent to dilute to the mark, shake well, take 40μl of precise amount and inject into the liquid phase Chromatograph, record the chromatogram. Do a blank test at the same time.

Solution photodamage: Take about 20mg of this product, weigh it accurately, place it in a 1.0ml volumetric flask, dilute it to the mark with a solvent, place the illuminance at 6000lx, near ultraviolet 90μW/cm2, irradiate at 25℃ for 7 days, take it out, put it At room temperature, dilute to the mark with a solvent, shake well, accurately inject 40μl into the liquid chromatograph, and record the chromatogram.

Solid light damage: Take about 20mg of this product, weigh it accurately, place it in a 1.0ml measuring bottle, place it at 6000lx, near ultraviolet 90μW/cm2, irradiate at 25℃ for 7 days, take it out, put it to room temperature, and dilute with solvent To the mark, shake well, accurately inject 40μl into the liquid chromatograph, and record the chromatogram.

Solution high temperature destruction: Take about 20mg of this product, weigh it accurately, place it in a 1.0ml volumetric flask, dilute it to the mark with a solvent, put it in a 60°C oven for 17 hours, take it out, put it to room temperature, dilute the solvent to the mark, shake well , Accurately measure 40μl into the liquid chromatograph and record the chromatogram.

Solid high temperature destruction: take about 20mg of this product, accurately weigh it, place it in a 1.0ml measuring bottle, put it in a 60°C oven for 17 hours, take it out, put it to room temperature, dilute it with a solvent to the mark, shake well, take 40μl of precise amount and inject Liquid chromatograph, record the chromatogram.

High humidity destruction: Take about 20mg of this product, weigh it accurately, place it in a 1.0ml measuring bottle, place it in 92.5% saturated KNO ₃ , after 7 days, take it out, put it to room temperature, add solvent to dilute to the mark, shake well, precise amount Take 40μl into the liquid chromatograph and record the chromatogram.

Measurement: According to the chromatographic conditions of Example 1, 40 μl of the above sample solution was injected into a liquid chromatograph, and the chromatogram was recorded. The results are shown in the table below:

The results show that the product has poor stability under high temperature and light conditions. The high temperature destruction of the solution and the solid high temperature destruction, the content of the impurity formula III compound increases significantly, and there are large degradation impurities; the solution light damage, the content of the impurity formula III compound There is a slight increase, and there are larger degradation impurities; under other damage conditions, no obvious degradation impurities are generated. Under each destruction condition, the resolution of the main peak and the adjacent chromatographic peaks meet the requirements, and the degradation products can be completely separated from the main peak and the degradation products, indicating that the method has good specificity.

Example 6 Preparation and Confirmation of Compound III

Add 2.0g of compound L and 15ml of acetonitrile to a 50ml reaction bottle, stir to dissolve to clear, add 24g of glacial acetic acid and stir at room temperature, warm up for 40min, stir at an internal temperature of 50°C overnight, stop heating, add 1.5ml of trifluoroacetic acid to react To the end, add water to the reaction bottle and extract twice with ethyl acetate. Combine the organic phases, wash once with saturated aqueous sodium bicarbonate solution, and once with saturated sodium chloride solution, and dry the organic phase with anhydrous sodium sulfate. After filtration, the filtrate was dried under reduced pressure to obtain 0.6 g of an oil, that is, compound III. (HPLC>97.0%)

1H-NMR: δ7.57(m,1H), δ6.16(m,1H), δ2.77(t,2H), δ2.67(m,1H), δ2.34(t,2H), δ1 .83-2.01(m,5H), δ1.59-1.73(m,3H), δ1.34-1.45(m,11H), δ0.96(t,3H).

13C-NMR: δ211.6, δ200.5(t), δ179.6, δ165.5, δ133.6, δ118.3(t), δ51.2, δ46.6, δ33.9, δ33.6, δ32.1(t), δ31.0, δ29.3, δ28.7, δ26.8, δ26.7, δ24.5, δ23.2(t), δ22.3, δ13.7.

Mass spectrum: [M+H] ⁺ 373.2.

Claims

An analysis method of related substances of lubiprostone, characterized in that the analysis method is high-performance liquid chromatography, which adopts a normal-phase chromatography column, and the mobile phase is composed of n-hexane, ethanol and glacial acetic acid. The volume ratio of n-hexane, ethanol and glacial acetic acid in the phase is 400～600:30～80:0.5～5.
The analysis method according to claim 1, characterized in that the normal phase chromatography column uses a polar stationary phase as a filler, preferably using 1,2-dihydroxypropane functional group organosilane-bonded silica gel as a filler.
The analysis method according to claim 2, wherein the normal phase chromatography column is selected from Li Chrospher 100 Diol/Merck, Waters ACQUITY BEH200SEC, Kromasil Diol or Li Chrosorb Diol.
The analysis method according to claim 3, wherein the normal phase chromatography column uses Li Chrospher 100 Diol/Merck, and its specification is 4.0 mm×250 mm, 5 μm.
The analysis method according to claim 1, wherein the volume ratio of n-hexane, ethanol and glacial acetic acid in the mobile phase is 450-550:35-55:0.5-3.
The analysis method according to claim 5, wherein the volume ratio of n-hexane, ethanol and glacial acetic acid in the mobile phase is 480:50:2.
The analysis method according to claim 1, wherein the flow rate of the mobile phase is 0.7-1.5 ml/min.
The analysis method according to claim 7, wherein the flow rate of the mobile phase is 0.9-1.1 ml/min.
The analysis method according to claim 7, wherein the flow rate of the mobile phase is 0.9ml/min, 1.0ml/min and 1.1ml/min or a range consisting of any two values.
The analysis method according to claim 7, wherein the flow rate of the mobile phase is 1.0 ml/min.
The analysis method according to claim 1, characterized in that it is performed on a high-performance liquid chromatograph, using a diode array detector, an ultraviolet absorption detector, a refractive index detector or an evaporative light scattering detector.
The analysis method according to claim 11, wherein an ultraviolet absorption detector is used.
The analysis method according to claim 11, wherein an ultraviolet absorption detector is used, and the detection wavelength is 292-296 nm.
The analysis method according to claim 11, wherein an ultraviolet absorption detector is used, and the detection wavelength is 292nm, 293nm, 294nm, 295nm or 296nm.
The analysis method according to claim 11, wherein an ultraviolet absorption detector is used, and the detection wavelength is 294 nm.
The analysis method according to claim 1, wherein the column temperature of the chromatography column is 25-40°C.
The analysis method according to claim 16, wherein the column temperature of the chromatography column is 28-38°C.
The analysis method according to claim 16, wherein the column temperature of the chromatography column is 33-37°C.
The analysis method according to claim 16, wherein the column temperature of the chromatography column is 35°C.
The analysis method according to claim 1, characterized in that isocratic elution or gradient elution is used.
The analysis method according to claim 20, wherein isocratic elution is used.
An analysis method of related substances of lubiprostone, characterized in that the analysis method is performed on a high-performance liquid chromatograph; the detector is an ultraviolet absorption detector, and the detection wavelength is 294 nm; the chromatography column is a normal phase chromatography column , The column temperature is 35 ℃; the mobile phase is a mixed solution composed of n-hexane, ethanol and glacial acetic acid, wherein the volume ratio of n-hexane, ethanol and glacial acetic acid is 480:50:2, the flow rate of the mobile phase is: 1.0ml /min; inject lubiprostone test solution, control solution and system suitability solution separately; the system suitability solution contains lubiprostone compound and is selected from compound of formula II, compound of formula III, compound of formula IV and formula One or a mixture of two or more compounds of V; the content of the compound of formula II, compound of formula III, compound of formula IV and/or compound of formula V in the sample is calculated by the main component self-control method,

Wherein the compound of formula II has the structural formula:

The structural formula of the compound of formula III is:

The structural formula of the compound of formula IV is:

The compound of formula V has the structural formula:
The analysis method according to claim 22, wherein the self-control method is calculated by adding a correction factor or without adding a correction factor.
The analysis method according to claim 23, characterized in that: in the calculation of the content of the compound of formula III and the compound of formula V, a correction factor is added for calculation, and in the calculation of the content of the compound of formula II and compound of formula IV, the calculation is not added .
The analytical method according to claim 24, wherein the correction factor of the compound of formula III is 0.44, and the correction factor of the compound of formula V is 0.5.
An analysis method of related substances of lubiprostone, characterized by:

(1) Mobile phase solution: configure a mixed solution with a volume ratio of 480:50:2 of n-hexane, ethanol and glacial acetic acid;

(2) Formula II compound positioning solution preparation: take compound II compound reference substance, weigh accurately, add mobile phase to dissolve and dilute, dilute to volume, shake well, then get;

Wherein, the compound of formula II has the structural formula:

(3) Formula III compound positioning solution preparation: take compound III compound reference substance, accurately weigh, add mobile phase to dissolve and dilute, dilute to volume, shake well, that is,

Wherein, the compound of formula III has the structural formula:

(4) Formula IV compound positioning solution preparation: take the compound IV compound reference substance, weigh accurately, add mobile phase to dissolve and dilute, dilute to volume, shake well, that is,

Wherein, the compound of formula IV has the structural formula:

(5) Formula V compound positioning solution preparation: take compound V compound reference substance, accurately weigh it, add mobile phase to dissolve and dilute, dilute to volume, shake well, that is,

Wherein, the structural formula of the compound of formula V is:

(6) System suitability solution preparation: take appropriate amount of compound of formula II, compound of formula III, compound of formula IV, compound of formula V and compound L, weigh accurately, add mobile phase to dissolve and dilute, dilute volume, shake well, then ;

(7) Preparation of test solution: take the appropriate amount of lubiprostone for the test product, accurately weigh it, dissolve and dilute with mobile phase, dilute to volume, shake well, then get;

(8) Preparation of control solution: accurately measure the appropriate amount of the test solution, and quantitatively dilute 100 times with mobile phase as the control solution;

(9) Determination of the test product: A normal phase chromatography column of Li Di Chrospher 100 Diol/Merck, size 4.0 mm × 250 mm, 5 μm, and mobile phase n-hexane-ethanol-glacial acetic acid (V:V:V=480:50: 2) For the mixed solution, adjust the flow rate to 1.0 ml/min, collect for 30 min, the column temperature is 35 °C, the detection wavelength is 294 nm, the injection volume is 40 μl, and measure the mobile phase solution, the formula II compound positioning solution, and the formula III compound, respectively The positioning solution, the positioning solution of the compound of formula IV, the positioning solution of the compound of formula V, the positioning solution of compound L, the system suitability solution, the test solution and the control solution were injected into the liquid chromatograph, and the chromatograms were recorded;

(10) Calculation of content: According to each chromatogram, the content of each relevant substance in the sample is calculated by the main component self-control method with or without correction factor:

Wherein, A t is the compound of formula II for the chromatogram of the test solution, the compound of Formula III, a compound of Formula IV or Formula V compound respective peak areas; F is a correction factor; A is the total peak area.
A high-purity lubiprostone composition, characterized in that the content of the compound of formula II is not more than 0.50%, the content of the compound of formula III is not more than 0.50%, the content of the compound of formula IV is not more than 0.50%, the content of the compound of formula V It is not higher than 0.50%, and the contents of other unknown single impurities are not higher than 1.0%.
The composition of claim 27, wherein the content of the compound of formula II is not more than 0.10%; the content of the compound of formula III is not more than 0.10%; the content of the compound of formula IV is not more than 0.10%; the content of the compound of formula V is not It is higher than 0.10%; the content of other unknown single impurities is not higher than 0.50%.
A compound whose structure is:
The compound according to claim 29, characterized in that its purity is ≥ 90%.
The compound according to claim 29, characterized in that its purity is ≥ 95%.
The compound according to claim 29, characterized in that its purity is ≥98%.
Use of the compound according to claims 29-33 as a reference marker in the examination of impurities of lubiprostone.
The method for preparing a compound according to claim 29, wherein compound III-1 is reacted to obtain a compound of formula III:
The preparation method according to claim 34, wherein the compound III-1 is reacted in the presence of acetonitrile, glacial acetic acid, and trifluoroacetic acid to obtain the compound of formula III.
The preparation method according to claim 35, wherein compound III-1, acetonitrile and glacial acetic acid are added to the reaction vessel, trifluoroacetic acid is added, and the compound of formula III is obtained after the reaction is completed.
The preparation method according to claim 36, characterized in that: compound III-1, acetonitrile and glacial acetic acid are added to the reaction vessel, the temperature is raised, stirred, trifluoroacetic acid is added, after the reaction is completed, extraction, washing and drying, The compound of formula III is obtained.
The preparation method according to claim 37, characterized in that: compound III-1, acetonitrile and glacial acetic acid are added to the reaction vessel, the temperature is raised, stirred, trifluoroacetic acid is added, after the reaction is completed, water is added, and ethyl acetate is extracted The organic phase was washed with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered with suction, and the filtrate was concentrated under reduced pressure to obtain the compound of formula III.
The preparation method according to claim 38, characterized in that: compound III-1 and acetonitrile are added to the reaction bottle, stirred and dissolved until clear, glacial acetic acid is added to stir at room temperature, the temperature is raised, and the internal temperature is raised to 50°C and stirred overnight, Add trifluoroacetic acid to the end of the reaction, add water to the reaction bottle and extract with ethyl acetate, combine the organic phases, wash with saturated aqueous sodium bicarbonate solution, and then wash with saturated sodium chloride solution, and dry the organic phase with anhydrous sodium sulfate , Filtered, and the filtrate was dried under reduced pressure to give an oil, that is, compound III.