Progesterone injection content detection method

Abstract

The invention discloses a progesterone injection content detection method, and relates to the technical field of chemical analysis, the detection method adopts instruments and equipment including a high performance liquid chromatograph, an octadecyl silane bonded silica gel chromatographic column needs to be prepared, the theoretical plate number of progesterone peaks is required to be at least 1000, and the degree of separation between peaks meets the standard. In addition, an ultraviolet absorption detector and chromatographic conditions are also needed, a mobile phase is methanol and water in a ratio of 65: 35, and the detection wavelength is 254nm. According to the method, the content of progesterone in the progesterone injection is determined by adopting a high performance liquid chromatography, the blank of progesterone detection by adopting a liquid chromatography method is filled, and the method is suitable for large-scale popularization and application. The detection method can be used for detecting the progesterone content and related substances, and can be used for guiding the investigation of a stable sample, so that an injection person and an injected person can conveniently know the progesterone content in later injection, the amount of the injection is conveniently controlled, and the use safety of the progesterone injection is improved.

Description

Technical Field

[0001] The invention relates to the technical field of chemical analysis, in particular to a method for detecting the content of progesterone injection. Background Art

[0002] Progesterone is an intermediate metabolite in the synthesis of steroid hormones and is the most important natural progestin. In the medical field, progesterone injection is used to treat menstrual disorders, such as amenorrhea and functional uterine bleeding, luteal insufficiency, threatened abortion and habitual abortion, and premenstrual tension syndrome. Progesterone injection is indicated for the treatment of menstrual disorders, such as amenorrhea and functional uterine bleeding, luteal insufficiency, threatened abortion and habitual abortion, and premenstrual tension syndrome. Progesterone injection is a sterile oil solution of progesterone, the main ingredient of which is progesterone, and the excipients are injection oil and the antibacterial agent benzyl alcohol. With the improvement of the level of drug research and development and the increasing attention paid to the adverse reactions of related preparations, the attention of pharmaceutical practitioners to the relevant substances in the preparation products is also increasing.

[0003] A high content of progesterone injection can cause dizziness, headache, hives, breast swelling, and long-term continuous use can cause menstrual reduction or amenorrhea, abnormal liver function, edema, weight gain, etc., so people need to have a necessary understanding of the progesterone content in the injection. For a progesterone injection with a maximum daily dose of 100 mg, the injection form is directly injected into the blood or muscle, and the safety risk level is higher in drugs. It is even more necessary to test the content of the main ingredient progesterone, so that the injector and the injectee can understand the progesterone content, and then it is easier to control the amount of the injection.

[0004] After searching the prior art, we did not find any patent website that describes a method for determining progesterone. We need a new analytical method that is convenient for relevant experimental operators to use for the determination of progesterone content, especially for the determination of progesterone content in progesterone injection.

[0005] In view of the above problems, a method for detecting the content of progesterone injection is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a method for detecting the content of progesterone injection. The device is used to work, thereby solving the problem that the progesterone injection in the above background has a high safety risk level in medicines because the injection dosage form is directly injected into the blood or muscle, and it is even more necessary to detect the content of the main component progesterone. Now, in order to facilitate the injector and the injectee to understand the progesterone content, and then to facilitate the control of the injection amount, we need a new analysis method that is convenient for relevant experimental operators to detect the progesterone content, especially suitable for the detection of the progesterone content of progesterone injection.

[0007] To achieve the above object, the present invention provides the following technical solution: a method for detecting the content of progesterone injection, comprising the following main steps;

[0008] Step 1: Prepare the test sample and the internal standard into a methanol solution, perform chromatographic separation by a high performance liquid chromatograph, use an ultraviolet absorption detector to detect the absorption values ​​of progesterone and the internal standard diethylstilbestrol at a wavelength of 254 nm, and calculate the absorption values ​​to obtain the content of progesterone; the required reagents include methanol and ether;

[0009] Step 2: The sample preparation step includes accurately measuring the test sample, accurately weighing the reference sample to prepare the solution, and preparing the internal standard solution. The test sample is washed and diluted with ether, extracted with methanol and the internal standard solution is added to obtain the test sample solution;

[0010] Step 3: The operation steps are to accurately aspirate 5 μL of the test sample and reference sample solution, inject into the high performance liquid chromatograph, measure the absorbance at a wavelength of 254 nm, and calculate the progesterone content according to the internal standard method;

[0011] Octadecylsilane bonded silica gel is used as the filler, the number of theoretical plates calculated based on the progesterone peak should be no less than 1000, and the separation degree between the progesterone peak and the internal standard substance peak should meet the requirements;

[0012] In the step 2, the sample preparation is as follows: take the test solution, accurately measure an appropriate amount of the product with a volumetric pipette, equivalent to 50 mg of progesterone, and place it in a 50 mL volumetric flask; preparation of the reference solution is as follows: accurately weigh an appropriate amount of progesterone reference, add methanol to dissolve and dilute to a solution.

[0013] The instruments and equipment include a high performance liquid chromatograph equipped with an octadecylsilane bonded silica gel column. The theoretical plate number of the progesterone peak is required to be at least 1000, and the peak separation must meet the standards. In addition, an ultraviolet absorption detector is required.

[0014] The chromatographic conditions required the mobile phase to be a 65:35 ratio of methanol and water, the detection wavelength to be 254 nm, and the column temperature to be maintained at room temperature.

[0015] Preparation of the internal standard solution: Take 25 mg of diethylstilbestrol, place it in a 25 mL volumetric flask, add dilute methanol to dissolve it and dilute it to the scale.

[0016] Preparation of the test solution: Wash the inner wall of the pipette with ether several times, add the washing solution into the volumetric flask, and dilute to the scale with ether;

[0017] Shake well; accurately measure 5 mL into a stoppered centrifuge tube and evaporate the ether in a warm water bath;

[0018] Extract with methanol by shaking for 4 times, shaking for 10 minutes each time, centrifuging for 15 minutes, and transfer the methanol to a 25mL volumetric flask with a dropper, combine the extracts, accurately add 5mL of internal standard solution, dilute to the scale with methanol, shake well, and the test solution is obtained.

[0019] The high performance liquid phase analysis method of progesterone is used in progesterone injection. The method is suitable for progesterone injection. The method adopts high performance liquid chromatography to determine the content of progesterone in progesterone injection.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. In this application, according to the physicochemical properties of progesterone, a high performance liquid chromatography system is developed to measure it, which is used to determine the progesterone content in progesterone injection. This method is specifically suitable for the analysis of progesterone injection. The principle is to prepare the test sample and the internal standard into a methanol solution, perform chromatographic separation by a high performance liquid chromatograph, and use an ultraviolet absorption detector to detect the absorption values ​​of progesterone and the internal standard diethylstilbestrol at a wavelength of 254nm. The content of progesterone is obtained by calculating the absorption value. The required reagents include methanol and ether. The instrument and equipment mainly include a high performance liquid chromatograph equipped with an octadecylsilane bonded silica gel column. The theoretical plate number of the progesterone peak is required to be at least 1000, and the peak separation meets the standard. In addition, an ultraviolet absorption detector is also required; the screening of the detection wavelength, after ultraviolet full scan, the ultraviolet absorption of progesterone is basically close to the terminal absorption, and according to the screening of the mobile phase ratio, it is finally determined to select a detection of progesterone with a moderate retention time and various substances. This method fills the gap in the detection of progesterone by liquid chromatography. The detection method can be used for the detection of progesterone content and related substances, and can be used to guide the inspection of stability samples. In the later injection, since the progesterone injection is directly injected into the blood or muscle, it is convenient for the injector and the injectee to understand the progesterone content, and then it is convenient to control the amount of the injection, thereby improving the safety of the use of progesterone injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The present invention is a flow chart of the main steps of the method for detecting the content of progesterone injection. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] In order to further understand the content of the present invention, the present invention is described in detail in conjunction with the accompanying drawings.

[0025] The present invention provides a technical solution: a method for detecting the content of progesterone injection, comprising the following main steps:

[0026] Use a volumetric pipette to accurately measure an appropriate amount of this product, approximately equivalent to 50 mg of progesterone, and place it in a 50 ml volumetric flask. Wash the inner wall of the pipette with ether several times, add the washing solution into the volumetric flask, dilute with ether to the scale, shake well, accurately measure 5 ml, place in a stoppered centrifuge tube, evaporate the ether in a warm water bath, shake with methanol for 4 times, 5 ml each time for the 1st to 3rd time, and 3 ml for the 4th time. Shake for 10 minutes each time and centrifuge for 15 minutes. Transfer the methanol solution to a 25 ml volumetric flask, combine the extracts, dilute with methanol to the scale, shake well, accurately measure 10 μl, and determine according to the method under the progesterone content determination item.

[0027] (1) Preparation of test solution for content determination: Use a volumetric pipette to accurately measure an appropriate amount of the product... dilute to the mark with methanol and shake well to obtain the test solution;

[0028] (2) Preparation of reference solution for content determination Take an appropriate amount of progesterone, weigh accurately, dissolve in methanol and dilute to make a solution containing 0.2 mg per 1 ml;

[0029] (3) Mobile phase preparation: the same as the mobile phase preparation method for related substance inspection;

[0030] (4) Chromatographic conditions and system suitability test: octylsilane bonded silica gel was used as filler, methanol-acetonitrile-water (25:35:40) was used as mobile phase, and the detection wavelength was 241 nm. 25 mg of the raw material progesterone of the injection was placed in a 25 ml volumetric flask, 10 ml of 0.1 mol / L sodium hydroxide methanol solution was added to dissolve it, and the mixture was kept warm in a 60°C water bath for 4 hours, cooled, adjusted to neutral with 1 mol / L hydrochloric acid solution, diluted to the scale with methanol, shaken well, and 10 ul was injected into the liquid chromatograph;

[0031] (5) Adjust the flow rate so that the retention time of the progesterone peak is about 12 minutes, adjust the detection sensitivity so that the peak height of the main component chromatographic peak reaches the full scale, and the separation degree between the progesterone peak and the degradation product peak with a relative retention time of about 1.1 in the chromatogram should be greater than 4.0;

[0032] Content determination: Accurately measure 10 μl of the test solution for content determination, inject it into the liquid chromatograph, record the chromatogram, and take another 10 μl of the progesterone reference solution for content determination, determine it in the same way, and calculate it by the external standard method with the peak area.

[0033] Implementation column:

[0034] Example 1

[0035] HPLC Detection of Progesterone Injection

[0036] Sample solution: Dissolve 10 mg of progesterone standard in 1 ml of ether, then dilute to 10 ml with acetonitrile;

[0037] A control solution was prepared by diluting the sample solution 100 times with methanol solution;

[0038] Blank solution: 1:99 tetrahydrofuran:acetonitrile;

[0039] Accurately measure 20 μl of blank solution, sample solution and control solution and inject them into the liquid chromatograph;

[0040] Chromatographic conditions:

[0041] Octylsilane bonded silica gel was used as filler, and methanol-acetonitrile-water (25:35:40) was used as mobile phase;

[0042] The detection wavelength is 241nm. Take 25mg of the raw material progesterone of the injection, put it in a 25ml volumetric bottle, and add 10ml of 0.1mol / L sodium hydroxide methanol solution to dissolve it;

[0043] Keep in a 60℃ water bath for 4 hours, cool, adjust to neutral with 1mol / L hydrochloric acid solution, dilute to scale with methanol, shake well, take 10ul and inject into liquid chromatograph;

[0044] The flow rate was adjusted so that the retention time of the progesterone peak was about 12 minutes, and the detection sensitivity was adjusted so that the peak height of the main component chromatographic peak reached the full scale. The separation degree between the progesterone peak and the degradation product peak with a relative retention time of about 1.1 in the chromatogram should be greater than 4.0.

[0045] Example 2

[0046] Detection of progesterone in progesterone injection

[0047] Preparation of test solution:

[0048] Raw materials (progesterone): dissolve 50 mg of progesterone in 1 ml of acetonitrile, and then make up to 10 ml with acetonitrile;

[0049] Sample (progesterone injection): Dissolve 1 ml of sample in 1 ml of acetonitrile, and then dilute to 10 ml with acetonitrile;

[0050] Soybean oil (auxiliary material): Dissolve 1 ml of soybean oil in 1 ml of acetonitrile, and then dilute to 10 ml with acetonitrile;

[0051] Accurately measure 20 μl of each solution and inject it into the liquid chromatograph;

[0052] Chromatographic conditions:

[0053] Chromatographic column: octylsilane bonded silica gel column (5.6×260mm, 6μm);

[0054] Mobile phase: acetonitrile: water 95:5;

[0055] Column temperature: 40°C;

[0056] Flow rate: 1.0ml / min;

[0057] Detection wavelength: 205nm;

[0058] Test results: The RT of the raw material is 3.856, the RT of the blank solution is 3.733, and the RT of soybean oil is 12.826.

[0059] 14.762, 16.936, each substance has no interference with the detection of progesterone. The peak substances and separation degree are shown in the attached Figure 1 .

[0060] In summary, the invention proposes a method for detecting the content of progesterone injection: in this application, according to the physicochemical properties of progesterone, a set of high-performance liquid chromatography systems is developed to measure it, which is used to determine the progesterone content in progesterone injection. This method is specifically suitable for the analysis of progesterone injection. The principle is to prepare the test sample and the internal standard into a methanol solution, perform chromatographic separation by a high-performance liquid chromatograph, and use an ultraviolet absorption detector to detect the absorption values ​​of progesterone and the internal standard diethylstilbestrol at a wavelength of 254nm. The content of progesterone is obtained by calculating the absorption values. The required reagents include methanol and ether. The instrument and equipment mainly include a high-performance liquid chromatograph equipped with an octadecylsilane bonded silica gel column. The theoretical plate number of the progesterone peak is required to be at least 1000, and the peak separation meets the standard. In addition, an ultraviolet absorption detector is also required; the screening of the detection wavelength, after ultraviolet full scan, the ultraviolet absorption of progesterone is basically close to the terminal absorption, and according to the screening of the mobile phase ratio, it is finally determined to select a detection of progesterone with a moderate retention time and various substances. This method fills the gap in the detection of progesterone by liquid chromatography. The detection method can be used for the detection of progesterone content and related substances, and can be used to guide the inspection of stability samples. In the later injection, since the progesterone injection is directly injected into the blood or muscle, it is convenient for the injector and the injectee to understand the progesterone content, and then it is convenient to control the amount of the injection, thereby improving the safety of the use of progesterone injection.

[0061] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0062] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting the content of progesterone injection, characterized in that: The main steps include: Step 1: Prepare the test sample and the internal standard into a methanol solution, perform chromatographic separation by a high performance liquid chromatograph, use an ultraviolet absorption detector to detect the absorption values ​​of progesterone and the internal standard diethylstilbestrol at a wavelength of 254 nm, and calculate the absorption values ​​to obtain the content of progesterone; the required reagents include methanol and ether; Step 2: The sample preparation step includes accurately measuring the test sample, accurately weighing the reference sample to prepare the solution, and preparing the internal standard solution. The test sample is washed and diluted with ether, extracted with methanol and the internal standard solution is added to obtain the test sample solution; Step 3: The operation procedure is to accurately aspirate 5 μL of the test sample and reference sample solutions, inject them into the high performance liquid chromatograph, measure the absorbance at a wavelength of 254 nm, and calculate the progesterone content according to the internal standard method.

2. A method for detecting the content of progesterone injection according to claim 1, characterized in that: Octadecylsilane bonded silica gel is used as the filler, the number of theoretical plates calculated based on the progesterone peak should be no less than 1000, and the separation degree between the progesterone peak and the internal standard substance peak should meet the requirements; In the step 2, the sample preparation is as follows: take the test solution, accurately measure an appropriate amount of the product with a volumetric pipette, equivalent to 50 mg of progesterone, and place it in a 50 mL volumetric flask; preparation of the reference solution is as follows: accurately weigh an appropriate amount of progesterone reference, add methanol to dissolve and dilute to a solution.

3. A method for detecting the content of progesterone injection according to claim 2, characterized in that: The instruments and equipment include a high performance liquid chromatograph equipped with an octadecylsilane bonded silica gel column. The theoretical plate number of the progesterone peak is required to be at least 1000, and the peak separation must meet the standards. In addition, an ultraviolet absorption detector is required.

4. A method for detecting the content of progesterone injection according to claim 3, characterized in that: The chromatographic conditions required a mobile phase of methanol and water in a ratio of 65:35, a detection wavelength of 254 nm, and an environment maintained at room temperature.

5. A method for detecting the content of progesterone injection according to claim 4, characterized in that: Preparation of the internal standard solution: Take 25 mg of diethylstilbestrol, place it in a 25 mL volumetric flask, add dilute methanol to dissolve it and dilute it to the scale.

6. A method for detecting the content of progesterone injection according to claim 5, characterized in that: Preparation of the test solution: Wash the inner wall of the pipette with ether several times, add the washing solution into the volumetric flask, and dilute to the scale with ether; Shake well; accurately measure 5 mL into a stoppered centrifuge tube and evaporate the ether in a warm water bath; Extract with methanol by shaking for 4 times, shaking for 10 minutes each time, centrifuging for 15 minutes, and transfer the methanol to a 25mL volumetric flask with a dropper, combine the extracts, accurately add 5mL of internal standard solution, dilute to the scale with methanol, shake well, and the test solution is obtained.

7. A method for detecting the content of progesterone injection according to claim 6, characterized in that: The high performance liquid phase analysis method of progesterone is used in progesterone injection. The method is suitable for progesterone injection. The method adopts high performance liquid chromatography to determine the content of progesterone in progesterone injection.

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