Analysis method for measuring content of analgin in analgin injection

Abstract

The invention relates to the technical field of raw material medicine detection, discloses an analysis method for determining the content of analgin in an analgin injection, and solves the problems that special reagents such as 4-dimethylaminocinnamaldehyde ethanol are needed by adopting an ultraviolet-visible spectrophotometric method, the reagents are not easy to purchase, sample preparation is troublesome, the cost is low, and the like. And subsequent detection operation is not facilitated. According to the method for detecting the content of the analgin injection by adopting the high performance liquid chromatography, octadecyl silane bonded silica gel is used as a filling agent, and a mobile phase is phosphate buffer solution-methanol with the pH value of 7.2; the method is simple to operate, high in sensitivity, strong in specificity and short in detection time.

Description

Technical Field

[0001] The invention relates to the technical field of raw material drug detection, in particular to an analytical method for determining the content of analgin in analgin injection. Background Art

[0002] Analgin is mainly used to reduce fever, and is also used to treat acute arthritis, headache, rheumatic pain, toothache and muscle pain, etc. The synthesis process of Analgin is generally based on pyrazolone as the basic raw material, and antipyrine is obtained through methylation reaction; then it is nitrosated and reduced to 4-aminoantipyrine, and then formylated to obtain 4-formylaminoantipyrine, and then methylated to obtain 4-methylaminoantipyrine; finally, Analgin is obtained through condensation reaction.

[0003] Analgin injection is indicated for antipyretic effect in case of high fever, and can also be used for headache, migraine, muscle pain, joint pain and dysmenorrhea, etc. This product also has a strong anti-rheumatic effect and can be used for acute rheumatic arthritis, but because this product may cause serious adverse reactions, it is rarely used in rheumatic diseases.

[0004] In the prior art, when analyzing the analgin content of analgin injection, the content detection method is mostly based on ultraviolet-visible spectrophotometry, which requires special reagents, such as 4-dimethylaminocinnamaldehyde ethanol. The reagents are not easy to purchase, and the sample preparation is relatively cumbersome, which is not conducive to subsequent detection operations.

[0005] In view of the above problems, an analytical method for determining the content of analgin in analgin injection is proposed. Summary of the invention

[0006] The object of the present invention is to provide an analytical method for determining the content of analgin in analgin injection, the method having the advantages of simple operation, high sensitivity, strong specificity, short detection time and isocratic elution, so that the detection process is more convenient.

[0007] To achieve the above object, the present invention provides the following technical solution: an analytical method for determining the content of analgin in analgin injection, comprising the following steps:

[0008] Step 1: Prepare the sample, prepare the test solution, pipette the injection solution, dilute the sample with methanol, and then make up the volume with the mobile phase to prepare a solution of 0.1 mg per ml;

[0009] Step 2: Prepare a reference solution. Accurately weigh the analgin reference substance, dissolve it in methanol, and then make up the volume with the mobile phase to obtain the reference solution.

[0010] Step 3: respectively measure the test solution and the reference solution, and inject them into a liquid chromatograph for detection to obtain a chromatogram of the test solution and a chromatogram of the reference solution;

[0011] Step 4: Conduct an experiment to optimize the ratio of mobile phases, using phase A: phosphate buffer and phase B: methanol, respectively, to examine the ratios of A: B = 75:25 and A: B = 70:30, and subject the reference solution to liquid chromatography for examination;

[0012] Step 5: Conducting solvent optimization experiments, using pure methanol, methanol and mobile phase as solvents to prepare reference solutions, and subjecting them to liquid chromatography;

[0013] Step 6: Take an appropriate amount of the reference substance solution stock solution, add methanol and mobile phase to dilute, and inject into the above-mentioned liquid chromatograph for detection according to the above-mentioned chromatographic conditions, and record the signal-to-noise ratio S / N of the analgin chromatographic peak;

[0014] Step 7. According to the preparation method of the test solution, take the same batch of analgin injection, prepare six test solutions in parallel, and inject them into the above-mentioned liquid chromatograph for detection according to the above-mentioned chromatographic conditions, and record the chromatogram.

[0015] Preferably, the weight of the analgin reference substance is 10 mg, and it is added into a 100 ml volumetric flask.

[0016] Preferably, the chromatogram of the test solution and the chromatogram of the reference solution are used to calculate the content according to the following formula: In the above formula, ms is the sample weight of the reference substance, P is the purity of the reference substance, Ai is the peak area of ​​the test substance, Vi is the dilution multiple of the test solution, Vs is the dilution multiple of the reference solution, As is the peak area of ​​the reference substance, mi is the sample weight of the test substance, and x is the percentage content.

[0017] Preferably, the content of the phosphate buffer is: 6 grams of sodium dihydrogen phosphate, add 1,000 milliliters of water, add 1 milliliter of triethylamine, and adjust the pH to 7.2 with sodium hydroxide solution.

[0018] Preferably, in step six, when the S / N is about 10, the concentration of the solution is the quantitative limit, and when the S / N is about 3, the concentration of the solution is the detection limit.

[0019] Preferably, in the step seven, the contents of analgin in six samples are calculated by external standard method according to the above calculation formula, and the average value and RSD% are calculated, and the RSD% is 0.47%.

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

[0021] 1. The present application proposes an analytical method for determining the content of analgin in analgin injection, which solves the problem that the existing analgin content analysis often requires relatively complicated operations during the detection process. The mobile phase ratio experiment is optimized: phase A: phosphate buffer (6.0 g sodium dihydrogen phosphate, 1000 ml water, 1 ml triethylamine, and sodium hydroxide solution to adjust pH 7.2), phase B: methanol, and the ratios of A:B = 75:25 and A:B = 70:30 are examined respectively. The reference solution is subjected to liquid chromatography for examination. Figure 1 It can be seen that the chromatographic peaks are relatively symmetrical, and the peak times are 9.9min and 7.8min respectively. In order to save analysis time, A:B=70:30 is selected as the mobile phase, and the peak can be emitted within 10min. Figure 2 It can be seen from the peak shape comparison that when pure methanol is used as the solvent, the peak shape fluctuates greatly and the graph is asymmetric, while the chromatogram with methanol and mobile phase as the solvent has a symmetrical and beautiful peak shape, so methanol and mobile phase are selected as the solvent.

[0022] 2. The present application proposes an analytical method for determining the content of analgin in analgin injection, which solves the problem of using ultraviolet-visible spectrophotometry. This method requires special reagents, such as 4-dimethylaminocinnamaldehyde ethanol, which is not easy to purchase and the sample preparation is more troublesome, which is not conducive to the subsequent detection operation. The blank solution, the auxiliary material solution and the reference solution are respectively measured and injected into the above-mentioned liquid chromatograph for detection, and the chromatogram is recorded. The three chromatograms are overlapped to obtain a specific spectrum, as shown in the accompanying drawings. Figure 3 The main peak appeared in 7.8 minutes, and the blank solution chromatographic peak appeared in 1 to 2 minutes. It can be concluded that the solvent and excipient solution had no interference with the detection of analgin and had good specificity. Preparation of reference solution stock solution: Accurately weigh about 40 mg of analgin reference substance into a 20 ml volumetric flask, first dissolve it in methanol, and then make up the volume with mobile phase as the reference solution stock solution.

[0023] Take 1 ml of the reference substance stock solution into a 100 ml volumetric flask, dilute to the mark with solvent, and prepare a solution containing about 0.02 mg of analgin per 1 ml as linear solution 1.

[0024] Take 2.5 ml of the reference substance stock solution into a 100 ml volumetric flask, dilute to the mark with solvent, and prepare a solution containing about 0.05 mg of analgin per 1 ml as linear solution 2.

[0025] Take 1.0 ml of the reference substance stock solution into a 20 ml volumetric flask, dilute to the mark with solvent, and prepare a solution containing about 0.1 mg of analgin per 1 ml as linear solution 3.

[0026] Take 1 ml of the reference substance stock solution into a 10 ml volumetric flask, dilute to the mark with solvent, and prepare a solution containing about 0.2 mg of analgin per 1 ml as linear solution 4.

[0027] Take 2.5 ml of the reference substance stock solution into a 10 ml volumetric flask, dilute to the mark with solvent, and prepare a solution containing about 0.5 mg of analgin per 1 ml as linear solution 5.

[0028] Take 5 ml of the reference substance stock solution into a 10 ml volumetric flask, dilute to the mark with solvent, and prepare a solution containing about 1.0 mg of analgin per 1 ml as linear solution 6.

[0029] The linear solutions 1 to 6 were measured respectively, and injected into the above-mentioned liquid chromatograph for detection according to the above-mentioned chromatographic conditions, and the peak area of ​​analgin was recorded. The concentration of the linear solution was used as the horizontal coordinate, and the peak area of ​​analgin was used as the vertical coordinate. Linear regression was performed, and then the quantitative limit and detection limit test were performed: an appropriate amount of the reference solution stock solution was taken, diluted with methanol and mobile phase, and injected into the above-mentioned liquid chromatograph for detection according to the above-mentioned chromatographic conditions, and the signal-to-noise ratio S / N of the analgin chromatographic peak was recorded. When the S / N was about 10, the concentration of the solution was the quantitative limit, and when the S / N was When the concentration of the solution is about 3, the detection limit is obtained. Finally, a repeatability test is conducted: according to the preparation method of the test solution, the same batch of analgin injection is taken, six test solutions are prepared in parallel, and according to the above chromatographic conditions, they are respectively injected into the above liquid chromatograph for detection, and the chromatogram is recorded to obtain the analgin peak area of ​​six parallel samples. According to the above calculation formula, the analgin content of the six samples is calculated by the external standard method, and the average value and RSD% are calculated. The results are shown in Table 3, and the RSD% is 0.47%, indicating that the method has good repeatability. The specification of the analgin injection sample is 0.5g:2ml, and the content of the analgin injection is detected by high performance liquid chromatography, octadecylsilane bonded silica gel is used as a filler, and the mobile phase is pH7.2 phosphate buffer-methanol; the method is simple to operate, has high sensitivity, strong specificity, and short detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a chromatogram of different mobile phase ratios of the present invention;

[0031] Figure 2 chromatograms of different solvents of the present invention;

[0032] Figure 3 is the specific chromatogram of the present invention;

[0033] Figure 4 It is a linear experiment result diagram of the present invention;

[0034] Figure 5 It is the quantitative limit and detection limit result diagram of the present invention;

[0035] Figure 6 It is a diagram of the repeatability test results of the present invention. DETAILED DESCRIPTION

[0036] 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.

[0037] In order to further understand the content of the present invention, the present invention is described in detail in conjunction with the accompanying drawings.

[0038] Combination Figure 1-Figure 3 , an analytical method for determining the content of analgin in analgin injection, comprising the following steps:

[0039] Step 1: Prepare the sample, prepare the test solution, pipette the injection solution, dilute the sample with methanol, and then make up the volume with the mobile phase to prepare a solution of 0.1 mg per ml;

[0040] Step 2: Prepare a reference solution. Accurately weigh the analgin reference substance, dissolve it in methanol, and then make up the volume with the mobile phase to obtain the reference solution.

[0041] Step 3: respectively measure the test solution and the reference solution, and inject them into a liquid chromatograph for detection to obtain a chromatogram of the test solution and a chromatogram of the reference solution;

[0042] Step 4: Conduct an experiment to optimize the ratio of mobile phases, using phase A: phosphate buffer and phase B: methanol, respectively, to examine the ratios of A: B = 75:25 and A: B = 70:30, and subject the reference solution to liquid chromatography for examination;

[0043] Step 5: Conducting solvent optimization experiments, using pure methanol, methanol and mobile phase as solvents to prepare reference solutions, and subjecting them to liquid chromatography;

[0044] Step 6: Take an appropriate amount of the reference substance solution stock solution, add methanol and mobile phase to dilute, and inject into the above-mentioned liquid chromatograph for detection according to the above-mentioned chromatographic conditions, and record the signal-to-noise ratio S / N of the analgin chromatographic peak;

[0045] Step 7. According to the preparation method of the test solution, take the same batch of analgin injection, prepare six test solutions in parallel, and inject them into the above-mentioned liquid chromatograph for detection according to the above-mentioned chromatographic conditions, and record the chromatogram.

[0046] Weigh ten milligrams of the analgin reference substance and add it into a one hundred milliliter volumetric flask.

[0047] Preferably, the chromatogram of the test solution and the chromatogram of the reference solution are used to calculate the content according to the following formula: In the above formula, ms is the sample weight of the reference substance, P is the purity of the reference substance, Ai is the peak area of ​​the test substance, Vi is the dilution multiple of the test solution, Vs is the dilution multiple of the reference solution, As is the peak area of ​​the reference substance, mi is the sample weight of the test substance, and x is the percentage content.

[0048] The content of phosphate buffer is: 6 grams of sodium dihydrogen phosphate, add 1,000 milliliters of water, then add 1 milliliter of triethylamine, and adjust the pH to 7.2 with sodium hydroxide solution.

[0049] In step 6, when the S / N is about 10, the concentration of the solution is the quantification limit, and when the S / N is about 3, the concentration of the solution is the detection limit.

[0050] In step 7, according to the above calculation formula, the content of analgin in six samples was calculated by external standard method, and the average value and RSD% were calculated. The RSD% was 0.47%.

[0051] The present invention will be further described below in conjunction with the embodiments.

[0052] Embodiment 1:

[0053] In order to solve the problem that the existing analysis of analgin content often requires relatively complicated operations during the detection process, this embodiment discloses the following technical solutions, specifically Figure 1-Figure 3 As shown in the accompanying drawings Figure 1 It can be seen that the chromatographic peaks are relatively symmetrical, and the peak times are 9.9 minutes and 7.8 minutes respectively. In order to save analysis time, A:B=70:30 is selected as the mobile phase, and the peak can be emitted within 10 minutes. According to the attached figure Figure 2 It can be seen from the peak shape comparison that when pure methanol is used as the solvent, the peak shape fluctuates greatly and the graph is asymmetric, while the chromatogram with methanol and mobile phase as the solvent has a symmetrical and beautiful peak shape, so methanol and mobile phase are selected as the solvent.

[0054] At the same time, in order to solve the problem that the UV-visible spectrophotometry method requires special reagents, such as 4-dimethylaminocinnamaldehyde ethanol, which is not easy to purchase, and the sample preparation is more troublesome, which is not conducive to the subsequent detection operation, the blank solution, the auxiliary material solution and the reference solution are respectively measured and injected into the above-mentioned liquid chromatograph for detection, and the chromatograms are recorded. The three chromatograms are overlapped to obtain a specific spectrum, as shown in the attached figure Figure 3The main peak appeared in 7.8 minutes, and the blank solution chromatographic peak appeared in 1 to 2 minutes. It can be concluded that the solvent and excipient solution had no interference with the detection of analgin and had good specificity. Preparation of reference solution stock solution: Accurately weigh about 40 mg of analgin reference substance into a 20 ml volumetric flask, first dissolve it in methanol, and then make up the volume with mobile phase as the reference solution stock solution.

[0055] Take 1 ml of the reference substance stock solution into a 100 ml volumetric flask, dilute to the mark with solvent, and prepare a solution containing about 0.02 mg of analgin per 1 ml as linear solution 1.

[0056] Take 2.5 ml of the reference substance stock solution into a 100 ml volumetric flask, dilute to the mark with solvent, and prepare a solution containing about 0.05 mg of analgin per 1 ml as linear solution 2.

[0057] Take 1.0 ml of the reference substance stock solution into a 20 ml volumetric flask, dilute to the mark with solvent, and prepare a solution containing about 0.1 mg of analgin per 1 ml as linear solution 3.

[0058] Take 1 ml of the reference substance stock solution into a 10 ml volumetric flask, dilute to the mark with solvent, and prepare a solution containing about 0.2 mg of analgin per 1 ml as linear solution 4.

[0059] Take 2.5 ml of the reference substance stock solution into a 10 ml volumetric flask, dilute to the mark with solvent, and prepare a solution containing about 0.5 mg of analgin per 1 ml as linear solution 5.

[0060] Take 5 ml of the reference substance stock solution into a 10 ml volumetric flask, dilute to the mark with solvent, and prepare a solution containing about 1.0 mg of analgin per 1 ml as linear solution 6.

[0061] The linear solutions 1 to 6 were measured respectively, and injected into the above-mentioned liquid chromatograph for detection according to the above-mentioned chromatographic conditions, and the peak area of ​​analgin was recorded. The concentration of the linear solution was used as the horizontal coordinate, and the peak area of ​​analgin was used as the vertical coordinate. Linear regression was performed, and then the quantitative limit and detection limit test were performed: an appropriate amount of the reference solution stock solution was taken, diluted with methanol and mobile phase, and injected into the above-mentioned liquid chromatograph for detection according to the above-mentioned chromatographic conditions, and the signal-to-noise ratio S / N of the analgin chromatographic peak was recorded. When the S / N was about 10, the concentration of the solution was the quantitative limit, and when the S / N was When the concentration of the solution is about 3, the detection limit is obtained. Finally, a repeatability test is conducted: according to the preparation method of the test solution, the same batch of analgin injection is taken, six test solutions are prepared in parallel, and according to the above chromatographic conditions, they are respectively injected into the above liquid chromatograph for detection, and the chromatogram is recorded to obtain the analgin peak area of ​​six parallel samples. According to the above calculation formula, the analgin content of the six samples is calculated by the external standard method, and the average value and RSD% are calculated. The results are shown in Table 3, and the RSD% is 0.47%, indicating that the method has good repeatability. The specification of the analgin injection sample is 0.5g:2ml, and the content of the analgin injection is detected by high performance liquid chromatography, octadecylsilane bonded silica gel is used as a filler, and the mobile phase is pH7.2 phosphate buffer-methanol; the method is simple to operate, has high sensitivity, strong specificity, and short detection time.

[0062] 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.

[0063] 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. An analytical method for measuring the content of Analgin in Analgin injection, characterized in that: The steps include: Step 1: Prepare the sample, prepare the test solution, pipette the injection solution, dilute the sample with methanol, and then make up the volume with the mobile phase to prepare a solution of 0.1 mg per ml; Step 2: Prepare a reference solution. Accurately weigh the analgin reference substance, dissolve it in methanol, and then make up the volume with the mobile phase to obtain the reference solution. Step 3: respectively measure the test solution and the reference solution, and inject them into a liquid chromatograph for detection to obtain a chromatogram of the test solution and a chromatogram of the reference solution; Step 4: Conduct an experiment to optimize the ratio of mobile phases, using phase A: phosphate buffer and phase B: methanol, respectively, to examine the ratios of A: B = 75:25 and A: B = 70:30, and subject the reference solution to liquid chromatography for examination; Step 5: Conducting solvent optimization experiments, using pure methanol, methanol and mobile phase as solvents to prepare reference solutions, and subjecting them to liquid chromatography; Step 6: Take an appropriate amount of the reference substance solution stock solution, add methanol and mobile phase to dilute, and inject into the above-mentioned liquid chromatograph for detection according to the above-mentioned chromatographic conditions, and record the signal-to-noise ratio S / N of the analgin chromatographic peak; Step 7. According to the preparation method of the test solution, take the same batch of analgin injection, prepare six test solutions in parallel, and inject them into the above-mentioned liquid chromatograph for detection according to the chromatographic conditions, and record the chromatogram.

2. The analytical method for measuring the content of Analgin in Analgin injection according to claim 1, characterized in that: The weight of the analgin reference substance is 10 mg, and it is added into a 100 ml volumetric flask.

3. A method for analyzing the content of Analgin in Analgin injection according to claim 1, characterized in that: The chromatograms of the test solution and the reference solution were used to calculate the content according to the following formula: In the above formula, ms is the sample weight of the reference substance, P is the purity of the reference substance, Ai is the peak area of ​​the test substance, Vi is the dilution multiple of the test solution, Vs is the dilution multiple of the reference solution, As is the peak area of ​​the reference substance, mi is the sample weight of the test substance, and x is the percentage content.

4. A method for analyzing the content of Analgin in Analgin injection according to claim 1, characterized in that: The content of the phosphate buffer is: 6 grams of sodium dihydrogen phosphate, add 1,000 milliliters of water, add 1 milliliter of triethylamine, and adjust the pH to 7.2 with sodium hydroxide solution.

5. The analytical method for measuring the content of Analgin in Analgin injection according to claim 1, characterized in that: In step 6, when the S / N is about 10, the concentration of the solution is the quantitative limit, and when the S / N is about 3, the concentration of the solution is the detection limit.

6. A method for analyzing the content of Analgin in Analgin injection according to claim 1, characterized in that: In the step 7, the contents of analgin in six samples were calculated by external standard method according to the calculation formula, and the average value and RSD% were calculated. The RSD% was 0.47%.

Images