Method for detecting content of iron proteinsuccinylate oral solution

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for detecting the content of an iron proteinsuccinylate oral solution. According to the content detection method, the iron proteinsuccinylate is used as a whole to measure ultraviolet absorption, detection can be performed by simply diluting with water after a test sample is obtained, other pretreatment steps are omitted, operation is simpler and more convenient, consumed time is short, the measurement result is accurate, intermediate product control is facilitated, feeding accuracy is ensured, loss in the production process is confirmed, and the method is suitable for industrial production. And the production efficiency is greatly improved.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for detecting the content of an oral ferric protein succinate solution. Background Art

[0002] Anemia is a pathological phenomenon in which the number of red blood cells or hemoglobin in the circulating blood is lower than normal for a long time. Anemia is usually divided into three categories: iron deficiency anemia (IDA), megaloblastic anemia and aplastic anemia. According to the World Health Organization, about 33% of the world's population suffers from anemia, and iron-related cases account for 42-50% of anemia cases.

[0003] Early treatments for iron deficiency anemia were mostly oral inorganic iron salts, with ferrous sulfate being the representative drug. After being ingested by the human body, inorganic iron salts are ionized into metal cations in the acidic gastric juice medium, and then enter the small intestine to be absorbed and utilized by the human body. However, the small intestinal wall has a certain repulsive effect on metal ions with positive charges, which causes some metal ions to not only be difficult to absorb, but also irritate the gastrointestinal tract and cause diarrhea. In order to overcome these shortcomings, the second generation of organic iron preparations including dextran iron, ferrous gluconate, sorbitol iron and ferrous fumarate have been developed one after another. Organic iron preparations solve the problem of low iron absorption rate, but there are still some adverse reactions such as nausea and astringent taste. In the late 1970s, the third generation of new macromolecular iron supplements based on polysaccharide iron complexes and protein iron complexes began to develop, among which iron protein succinylate (ISP) was the main representative.

[0004] Protein succinate iron uses protein as its raw material. After acylation of the amino groups on the protein with succinic anhydride, the number of sites on the protein that can bind to iron increases, and its chemical properties and solubility are improved. Protein succinate iron is in a precipitated state under acidic conditions in the stomach, and is not digested by pepsin, causing no damage to the gastric mucosa. After entering the intestine, it redissolves and is digested by trypsin, and iron begins to be released in the duodenum. Therefore, protein succinate iron does not cause gastrointestinal tolerance problems.

[0005] Iron protein succinate was first launched by Panmark Pharmaceuticals in Italy in 1987 and was first imported into my country in 2001 in the form of an oral solution with the trade name Ferplex. Each bottle is 15 mL and contains 800 mg of iron protein succinate, equivalent to 40 mg of trivalent iron.

[0006] In the production process of iron protein succinate oral solution, the quality of materials, intermediate products and finished products needs to be inspected, which involves measuring the content of the unfiltered liquid after constant volume. Existing methods for detecting the content of iron protein succinate oral solution include: ignition-redox titration, hydrochloric acid digestion-reduction complexation colorimetry, nitric acid digestion-reduction complexation colorimetry, nitric acid digestion-atomic absorption spectrometry. The applicant found the following problems when using the above method to determine the content: ① The ignition-redox titration operation takes a long time (ignition requires 5h), and the crucible is easy to burst during the operation, resulting in inaccurate measurement results; ② Ordinary hydrochloric acid and nitric acid digestion methods cannot completely hydrolyze acylated casein peptide chains and release trivalent iron ions, and the measurement results are inaccurate; ③ Although the nitric acid digestion-atomic absorption method using a digestion instrument can completely digest protein, the digestion instrument operation process takes a long time. Therefore, it is urgent to develop a fast and accurate method for detecting the content of iron protein succinate oral solution to shorten the quality detection time of intermediate products, thereby improving production efficiency. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides a new method for detecting the content of ferric protein succinate oral solution. The method is simple to operate, short in time, high in accuracy, conducive to the control of intermediate products, and can better monitor the quality of ferric protein succinate oral solution.

[0008] The invention provides a method for detecting the content of an oral ferric protein succinate solution, which is determined by ultraviolet-visible spectrophotometry, wherein a reference solution comprises ferric protein succinate, auxiliary materials, a pH regulator and water, and a test solution is obtained by directly diluting the oral ferric protein succinate solution test sample.

[0009] Preferably, the auxiliary material is propylene glycol or sorbitol, or a combination of both.

[0010] Further preferably, the weight ratio of the propylene glycol to the ferric protein succinate is 4.5:4 to 5.5:4; the weight ratio of the sorbitol to the ferric protein succinate is 3.1:2 to 3.9:2.

[0011] Preferably, the pH adjuster is sodium hydroxide.

[0012] Preferably, the concentration of iron protein succinate in the reference solution is 0.29 mg / mL to 0.87 mg / mL.

[0013] Preferably, the pH value of the reference solution is 6.5-8.

[0014] Preferably, the ferric protein succinate oral solution test sample comprises ferric protein succinate, sorbitol, propylene glycol, sodium hydroxide and water.

[0015] Further preferably, in the ferric protein succinate oral solution test sample, the weight ratio of propylene glycol to ferric protein succinate is 4.5:4 to 5.5:4, and the weight ratio of sorbitol to ferric protein succinate is 3.1:2 to 3.9:2.

[0016] Further preferably, the ferric protein succinate oral solution test sample may also contain one or a combination of antibacterial agents, flavoring agents and flavors.

[0017] Furthermore, the antibacterial agent is one or a combination of sorbic acid, potassium sorbate, sodium benzoate, paraben, sodium methylparaben, and sodium propylparaben; the flavoring agent is one or a combination of saccharin sodium, sucralose, sodium cyclamate, sucrose, glucose, maltose, aspartame, and xylitol; the flavor is one or a combination of strawberry flavor, blueberry flavor, banana flavor, apple flavor, cantaloupe flavor, peach flavor, bayberry flavor, sour plum flavor, orange flavor, pineapple flavor, orange flavor, grape flavor, taro flavor, coconut flavor, lemon flavor, lychee flavor, stevia, watermelon flavor, mango flavor, and blackcurrant flavor.

[0018] Furthermore, in the test sample of the oral iron protein succinate solution, each 15 mL of solution contains 0.72 g to 0.88 g of iron protein succinate, 1.26 g to 1.54 g of sorbitol, 0.9 g to 1.1 g of propylene glycol, 46 mg to 57 mg of sodium hydroxide, 36 mg to 54 mg of sodium methylparaben, 12 mg to 18 mg of sodium propylparaben, 13.5 mg to 16.5 mg of sodium saccharin, 13.5 mg to 16.5 mg of flavor and the remainder of water.

[0019] More preferably, in the ferric protein succinate oral solution test sample, each 15 mL of solution contains 0.8 g ferric protein succinate, 1.4 g sorbitol, 1 g propylene glycol, 51 mg sodium hydroxide, 45 mg sodium methylparaben, 15 mg sodium propylparaben, 15 mg sodium saccharin, 15 mg flavor and the remainder water.

[0020] Preferably, the theoretical concentration of iron protein succinate in the test solution is 0.29 mg / mL to 0.87 mg / mL.

[0021] Preferably, the detection wavelength of the UV-visible spectrophotometry is 390 nm to 410 nm, and further, the detection wavelength is preferably 400 nm.

[0022] Furthermore, the specific steps of the method for detecting the protein succinic acid content of the oral solution of iron protein succinate provided by the present invention include: ① preparation of reference solution: heating and mixing the prescribed amount of propylene glycol and / or sorbitol with purified water, adding sodium hydroxide, and then adding iron protein succinate to dissolve it, adding water to make up the volume, stirring until the final solution has a pH value of 6.5 to 8.0, thus obtaining a reference stock solution, taking the reference stock solution, and diluting it with water to obtain a reference solution;

[0023] ② Preparation of test solution: Take the test sample and dilute it with water to obtain the test solution;

[0024] ③ Determination method: Take the reference solution and the test solution according to the UV-visible spectrophotometry (General Rules 0401 of Part IV of the Chinese Pharmacopoeia), use water as the blank, determine the absorbance, and calculate according to the external standard method;

[0025] Among them, steps ① and ② are in no particular order.

[0026] Wherein, the sorbitol added in step ① may be sorbitol solid or sorbitol solution, and the sorbitol solution may be a crystalline sorbitol solution or a non-crystalline sorbitol solution. The weight of sorbitol in this application is based on the weight of pure sorbitol.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] Existing methods for detecting the content of oral ferric protein succinate solution are all based on Fe, and complex pretreatment of the protein in the test sample is required. The content detection method of the present invention measures the ultraviolet absorption of ferric protein succinate as a whole, and can be detected after simply diluting the test sample with water after obtaining it. There is no other pretreatment step, the operation is simpler, the time is short, the measurement result is accurate, it is beneficial to the control of intermediate products, ensure the accuracy of feeding and confirm the loss in the production process, and improve production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a standard curve diagram of the protein succinate iron reference substance solution of Example 2 of the present invention.

[0030] Figure 2 This is the standard curve of the ammonium ferric sulfate reference solution of comparative example 2 using the hydrochloric acid digestion-reduction complexation colorimetric method.

[0031] Figure 3 This is the standard curve of the ammonium ferric sulfate reference solution of Comparative Example 3 using the nitric acid digestion-reduction complexation colorimetric method.

[0032] Figure 4 This is the standard curve of the ammonium ferric sulfate reference solution by nitric acid digestion-atomic absorption method of Comparative Example 4. DETAILED DESCRIPTION

[0033] The following examples are used to further illustrate the technical solutions of the present invention, but do not limit the scope of the present invention.

[0034] The API raw materials in this application were purchased from Zhejiang Thermo Pharmaceutical Co., Ltd., and the reference substance ammonium ferric sulfate was purchased from the China Food and Drug Inspection Institutes.

[0035] "API" in this application refers to ferric protein succinate.

[0036] The test instruments used in this application are as follows:

[0037] name model factory UV spectrophotometer TU1901 Beijing Puxi General Instrument Co., Ltd. Magnetic stirring instrument WH260-H WICCENS Electronic balance ME204TE / 02 Mettler pH meter S210 Mettler Muffle furnace SX2 Daoxu Kexi Instrument Factory Electric Ceramic Cooker MC-HW2210 Guangdong Midea Household Electrical Appliances Manufacturing Co., Ltd. Atomic absorption spectrophotometer AA-6880 Shimadzu Graphite Acid Remover GS-16 Zhejiang Tuojie Instrument Equipment Co., Ltd. Microwave Digestion Instrument M6 Shanghai Yiyao Instrument Technology Development Co., Ltd.

[0038] The sources or preparation methods of the test samples used in the examples and comparative examples of this application are as follows:

[0039] Sample A was a self-made product. The preparation method was as follows: 70 mL of purified water was heated, 0.34 g of sodium hydroxide was added, and then 5.33 g of API was added. The pH value was tested until the pH was stable, the volume was adjusted to 100 mL, and stirred for 30 min.

[0040] Test sample B: reference preparation, source: Italfarmaco SA, batch number: 230008.

[0041] Test sample C was a self-made product. The preparation method was as follows: 70 mL of purified water, 13.3 g of 70% sorbitol solution, 6.7 g of propylene glycol, and 0.34 g of sodium hydroxide were heated to dissolve, followed by the addition of 4 g of API, the remaining excipients (0.3 g of sodium methylparaben, 0.1 g of sodium propylparaben, 0.1 g of sodium saccharin) and 1.33 g of API. The dissolution was confirmed and the pH value was tested during the process. After the dissolution was complete, the volume was fixed to 100 mL.

[0042] Sample D was a self-made product, and the preparation method was as follows: sample C was added with black currant essence (concentration percentage 0.1%), and placed in an oven at 60° C. for 9 days.

[0043] Sample E is a self-made product, prepared in the same way as sample D, and is another batch of products prepared at a different time.

[0044] Test product F: reference preparation, source: Italfarmaco SA, batch number: 230035.

[0045] Example 1: Detection of protein succinic acid content by the method of the present invention

[0046] Reference substance stock solution: Heat and mix 3.3 g propylene glycol and 6.7 g sorbitol solution with 35 mL purified water, add 0.17 g sodium hydroxide, then add 2.67 g API, stir to dissolve, add water to make up to 50 mL, stir for 30 min, and the final solution pH is 6.5-8.0.

[0047] Reference solution: Take the reference stock solution and dilute it 100 times with water.

[0048] Test sample solution: Take an appropriate amount of the test sample and dilute it 100 times with water.

[0049] Determination method: Take the reference solution and the test solution according to the UV-visible spectrophotometry (Chinese Pharmacopoeia Part IV General Rules 0401), use water as blank, measure the absorbance at wavelengths of 390nm, 400nm, and 410nm, and calculate according to the external standard method. The measurement results of the reference solution and the test solution are shown in Table 1 and Table 2, respectively.

[0050] Table 1 Measurement results of reference solution

[0051]

[0052] Table 2 Test solution measurement results

[0053]

[0054] The results showed that under the condition of 390-410nm, the protein succinic acid content of this product was accurately tested, indicating that this method was durable under the detection wavelength of 390-410nm.

[0055] Example 2: Reference substance linearity experiment

[0056] Preparation method of reference substance solutions of different concentrations: 0.5 mL, 0.8 mL, 1.0 mL, 1.2 mL and 1.5 mL of the reference substance stock solution in Example 1 were respectively measured and placed in a 100 mL volumetric flask, diluted to the mark with water, and shaken well.

[0057] Determination method: Take different concentrations of reference solution according to the UV-visible spectrophotometry (Chinese Pharmacopoeia Part IV General Rules 0401), use water as blank, measure the absorbance at a wavelength of 400nm, and calculate according to the external standard method. The measurement results of the reference solution are shown in Table 3. The standard curve of protein succinate iron reference solution is shown in Figure 1 .

[0058] Table 3 Measurement results of reference solutions of different concentrations

[0059] Concentration of reference solution (mg / mL) Absorbance 0.28950 0.208 0.46321 0.332 0.57901 0.413 0.69481 0.501 0.86851 0.628

[0060] The results showed that the peak area and concentration of protein succinate iron showed a good linear relationship in the concentration range of 0.29 mg / mL to 0.87 mg / mL, proving that the method was accurate in the concentration range of 0.29 mg / mL to 0.87 mg / mL.

[0061] Example 3: Study on auxiliary material components of reference solution

[0062] Reference stock solution (containing only propylene glycol): Heat and mix 3.3 g propylene glycol and 35 mL purified water, add 0.17 g sodium hydroxide, then add 2.67 g API, stir to dissolve, add a small amount of sodium hydroxide to adjust the pH, add water to make up to 50 mL, stir for 30 min, the final solution pH is 6.5-8.0.

[0063] Reference solution (containing only propylene glycol): Take the reference stock solution (containing only propylene glycol) and dilute it 100 times with water.

[0064] Reference stock solution (containing only sorbitol): Heat and mix 6.7 g sorbitol solution with 35 mL purified water, add 0.17 g sodium hydroxide, then add 2.67 g API, stir to dissolve, add a small amount of sodium hydroxide to adjust the pH, add water to make up to 50 mL, stir for 30 min, the final solution pH is 6.5-8.0.

[0065] Reference solution (containing only sorbitol): Take the reference stock solution (containing only sorbitol) and dilute it 100 times with water.

[0066] Reference stock solution (without propylene glycol and sorbitol): Heat 35 mL of purified water, add 0.17 g of sodium hydroxide, then add 2.67 g of API, stir to dissolve, add a small amount of sodium hydroxide to adjust the pH, add water to make up to 50 mL, stir for 30 min, and the final solution pH is 6.5-8.0.

[0067] Reference solution (without propylene glycol and sorbitol): Take the reference stock solution (without propylene glycol and sorbitol) and dilute it 100 times with water.

[0068] The test solution preparation method is the same as in Example 1.

[0069] Determination method: Take the reference solution and the test solution according to the UV-visible spectrophotometry (General Rules 0401 of Part IV of the Chinese Pharmacopoeia), use water as the blank, measure the absorbance at a wavelength of 400nm, and calculate according to the external standard method. The measurement results of the reference solution and the test solution are shown in Table 4 and Table 5 respectively.

[0070] Table 4 Measurement results of different excipient reference solutions

[0071]

[0072]

[0073] Table 5 Test solution measurement results

[0074]

[0075] The results showed that when sorbitol or propylene glycol or both were added to the reference solution, the content of protein succinate iron in the sample could be accurately determined. When neither of the two was added, the content determination result was inaccurate.

[0076] Comparative Example 1: Determination of content by incineration-redox titration

[0077] Accurately measure 10mL of the sample, place it in a 50mL crucible, evaporate it at low temperature, slowly burn it until it is completely carbonized, let it cool, add 1mL of sulfuric acid to moisten all the carbonized products, and then heat it at low temperature until the sulfuric acid vapor is completely removed. Burn it at 600℃ for 5 hours to completely incense the sample, let it cool, add 10mL of hydrochloric acid and heat it slightly to dissolve it, move it to a 250mL iodine bottle, wash the crucible with 50mL of water several times, combine the washings, add 1g of potassium iodide, shake well, seal it, let it stand in a dark place for 10 minutes, add 20mL of water, and titrate with sodium thiosulfate titrant (0.05mol / L). When it is close to the end point, add 1mL of starch indicator solution and continue to titrate until the blue color disappears. And correct the titration result with a blank test. Each 1mL of sodium thiosulfate titrant (0.05mol / L) is equivalent to 2.7925mg of iron (Fe 3+ ). The measurement results are shown in Table 6.

[0078] Table 6 Ignition-Redox Titration Results

[0079]

[0080] The results show that this method is to detect the content of Fe in protein succinic acid iron, the content of the test sample is close to 100%, and the result is accurate; but the operation process is time-consuming (burning requires 5 hours), and the crucible is easy to crack during the operation, there is a risk of result loss and result difference, resulting in abnormal content results, and retesting is required. This method is suitable for protein succinic acid content detection, but for the control of intermediate products, it undoubtedly increases the production time cost and is not easy to control the production process.

[0081] Comparative Example 2: Determination of content by hydrochloric acid digestion-reduction complexation colorimetric method

[0082] 0.3% 2,2'-bipyridine solution: Accurately weigh 0.3 g of 2,2'-bipyridine, place in a 100 mL volumetric flask, add about 3 mL of water and 3 mL of glacial acetic acid, shake to dissolve, add water to dilute to the scale, and shake well.

[0083] 10% hydroxylamine hydrochloride solution: Accurately weigh 10 g of hydroxylamine hydrochloride, place it in a 100 mL volumetric flask, add water to dilute to the scale, and shake well.

[0084] 10% sulfuric acid solution: Take a 200mL measuring cup, add about 50mL of water, carefully transfer 10mL of concentrated sulfuric acid, place it in a beaker, stir and cool to room temperature, transfer to a 100mL measuring flask, add water to dilute to the scale, and shake well.

[0085] Reference substance stock solution 1: Take 0.2 g of ammonium ferric sulfate reference substance, weigh accurately, place in a 100 mL volumetric flask, add 5 mL of 10% sulfuric acid solution to dissolve it, add water to dilute to the scale, and shake well.

[0086] Reference substance stock solution 2: Accurately pipette 10 mL of reference substance stock solution 1 into a 50 mL volumetric flask, dilute to the mark with water, and shake well.

[0087] Reference solution: Accurately pipette 0mL, 1mL, 2mL, 3mL, 4mL, 5mL, and 6mL of reference stock solution 2, respectively, and place them in 50mL volumetric flasks, add 3mL of 10% hydroxylamine hydrochloride solution, shake well, add 5mL of 0.3% 2,2'-bipyridine solution, shake well, add 3mL of 80% hydrazine hydrate solution, dilute to the scale with water, shake well, and let stand for 30 minutes. Measure the absorbance at a wavelength of 525nm by UV-visible spectrophotometry. The measurement results of the reference solution are shown in Table 7. The standard curve of ammonium ferric sulfate reference solution is shown in Figure 2 .

[0088] Test sample stock solution: Accurately weigh 3 g of the test sample, place it in a 50 mL volumetric flask, add 3.0 mL of concentrated hydrochloric acid, shake well, and dilute to the scale with water to obtain the test sample stock solution.

[0089] Test solution: Accurately pipette 1 mL of the test stock solution, place it in a 50 mL volumetric flask, add 3 mL of 10% hydroxylamine hydrochloride solution, shake well, add 5 mL of 0.3% 2,2'-bipyridine solution, shake well, add 3 mL of 80% hydrazine hydrate solution, dilute to the mark with water, shake well, leave for 30 minutes, and measure the absorbance at a wavelength of 525 nm by UV-visible spectrophotometry. The test solution measurement results are shown in Table 8.

[0090] Table 7 Measurement results of reference solutions of different concentrations

[0091]

[0092]

[0093] Table 8 Hydrochloric acid digestion-reduction complexation colorimetric determination results

[0094]

[0095] The results showed that the protein succinate iron Fe content determined by the standard curve method after the sample was destroyed by hydrochloric acid and reduced by UV method was inaccurate and the result was too low. It was speculated that hydrochloric acid failed to destroy the protein as a whole and the Fe ions were not completely released. This method is not suitable for the content detection of this product.

[0096] Comparative Example 3: Determination of content by nitric acid digestion-reduction complexation colorimetric method

[0097] 5% nitric acid solution: Accurately measure 20 mL of concentrated nitric acid, dilute to 250 mL with water, and shake well.

[0098] 0.3% 2,2'-bipyridine solution: Accurately weigh 0.3 g of 2,2'-bipyridine, place in a 100 mL volumetric flask, add about 3 mL of water and 3 mL of glacial acetic acid, shake to dissolve, add water to dilute to the scale, and shake well.

[0099] 10% hydroxylamine hydrochloride solution: Accurately weigh 10 g of hydroxylamine hydrochloride, place it in a 100 mL volumetric flask, add water to dilute to the scale, and shake well.

[0100] 10% sulfuric acid solution: Take a 200mL measuring cup, add about 50mL of water, carefully transfer 10mL of concentrated sulfuric acid, place it in a beaker, stir and cool to room temperature, transfer to a 100mL measuring flask, add water to dilute to the scale, and shake well.

[0101] Reference substance stock solution 1: Take 0.2 g of ammonium ferric sulfate reference substance, weigh accurately, place in a 100 mL volumetric flask, add 5 mL of 10% sulfuric acid solution to dissolve, add water to dilute to the scale, and shake well.

[0102] Reference substance stock solution 2: Accurately pipette 10 mL of reference substance stock solution 1 into a 50 mL volumetric flask, dilute to the mark with water, and shake well.

[0103] Reference solution: Accurately pipette 0mL, 1mL, 2mL, 3mL, 4mL, 5mL, and 6mL of reference stock solution 2 respectively, and place them in 50mL volumetric flasks, add 3mL of 10% hydroxylamine hydrochloride solution, shake well, add 5mL of 0.3% 2,2'-bipyridine solution, shake well, add 3mL of 80% hydrazine hydrate solution, dilute to the scale with water, shake well, and let stand for 30 minutes. Measure the absorbance at a wavelength of 525nm by UV-visible spectrophotometry. The measurement results of the reference solution are shown in Table 9. The standard curve of ammonium ferric sulfate reference solution is shown in Figure 3 .

[0104] Test sample stock solution: accurately weigh 3 g of the test sample, place it in a 100 mL beaker, add 10 mL of concentrated nitric acid and shake lightly to allow the nitric acid to fully contact the sample. After the sample is digested from red to white, place it in an electric furnace and heat it at a low setting to drive out the acid until about 5 mL is left. Stop heating, cool to room temperature, wash it with 5% nitric acid several times, transfer the sample to a 50 mL volumetric flask, make up to volume with 5% nitric acid solution and let it stand for a while as the test sample stock solution.

[0105] Test solution: Accurately pipette 1 mL of the test stock solution, place it in a 50 mL volumetric flask, add 3 mL of 10% hydroxylamine hydrochloride solution, shake well, add 5 mL of 0.3% 2,2'-bipyridine solution, shake well, add 3 mL of 80% hydrazine hydrate solution, dilute to the mark with water, shake well, leave for 30 minutes, and measure the absorbance at a wavelength of 525 nm by UV-visible spectrophotometry. The test solution measurement results are shown in Table 10.

[0106] Table 9 Measurement results of reference solutions of different concentrations

[0107] Concentration of reference solution (μg / mL) Absorbance 0.0000 0.000 1.0004 0.141 2.0008 0.298 3.0012 0.457 4.0016 0.613 5.0020 0.770 6.0024 0.922

[0108] Table 10 Nitric acid digestion-reduction complexation colorimetric determination results

[0109]

[0110] The results showed that the protein succinate iron Fe content was inaccurate and low when the sample was destroyed by nitric acid, heated and acid-removed by induction cooker and measured by UV method after reduction complexation using the standard curve method. It was speculated that the nitric acid failed to destroy the protein as a whole and the Fe ions were not completely released. This method is not suitable for the content detection of this product.

[0111] Comparative Example 4: Nitric Acid Digestion-Atomic Absorption Spectrometry Content Determination

[0112] Diluent: Measure 5 mL of concentrated nitric acid into a 500 mL volumetric flask, dilute to the mark with water and shake well.

[0113] Reference substance stock solution 1: Take 215.8 mg of ammonium ferric sulfate reference substance, accurately weigh it, put it in a 100 mL volumetric flask, add 5 mL of 10% sulfuric acid solution to dissolve it, add water to dilute to the scale, and shake well.

[0114] Reference substance stock solution 2: Accurately pipette 5 mL of reference substance stock solution 1 into a 25 mL volumetric flask, add diluent to dilute to the mark, and shake well.

[0115] Reference substance solution: Accurately pipette 0mL, 0.5mL, 1mL, 1.5mL, 2mL, 2.5mL, and 3mL of reference substance stock solution 2 respectively, place them in 25mL volumetric flasks, add diluent to dilute to the scale, and shake well.

[0116] Sample blank stock solution: Measure 5 mL of nitric acid and place it in a digestion tank. Heat it at 100°C for 30 min. Digest it in a microwave digester at 140°C for 2 min, 180°C for 2 min, and 200°C for 30 min. Heat it at 110°C on a graphite acid remover to remove the acid to 1 mL to 2 mL. After cooling, transfer it quantitatively to a 50 mL volumetric flask, dilute it to the scale with a diluent, and shake well.

[0117] Sample blank solution: Accurately pipette 1.5 mL of sample blank stock solution into a 25 mL volumetric flask, add diluent to dilute to the mark, and shake well.

[0118] Test sample stock solution (digestion instrument method): Measure 1 mL of the test sample and place it in a digestion tank, add 5 mL of nitric acid, heat at 100°C for 30 min, digest in a microwave digester at 140°C for 2 min, 180°C for 2 min, and 200°C for 30 min, then heat at 110°C on a graphite acid remover to remove the acid to 1 mL to 2 mL, transfer quantitatively to a 50 mL volumetric flask after cooling, dilute to the scale with diluent, and shake well.

[0119] The preparation method of the test sample stock solution (electric furnace method) is the same as the test sample stock solution of Comparative Example 3.

[0120] Test sample solution: Accurately pipette 1.5 mL of the test sample stock solution into a 25 mL volumetric flask, add diluent to dilute to the mark, and shake well.

[0121] Sample determination: Take the reference solution and the test solution, and determine the absorbance at a wavelength of 248.3nm according to the atomic absorption spectrophotometry (General Rules 0406 of Part IV of the Chinese Pharmacopoeia). The measurement results of the reference solution and the test solution are shown in Table 11 and Table 12 respectively. The standard curve of the ammonium ferric sulfate reference solution is shown in Figure 4 .

[0122] Table 11 Measurement results of reference solutions of different concentrations

[0123] Concentration of reference solution (μg / mL) Absorbance 0.0000 -0.0004 1.0004 0.0918 2.0008 0.1829 3.0012 0.2657 4.0016 0.3467 5.0020 0.4203 6.0024 0.5075

[0124] Table 12 Nitric acid digestion-atomic absorption spectrometry results

[0125]

[0126]

[0127] The results showed that the standard curve method, the sample was destroyed by microwave nitric acid using a digestion instrument, and then the acid was driven away. The protein succinate iron Fe content was accurately determined by atomic absorption spectrometry after reduction and complexation. However, the operation process of the digestion instrument was time-consuming and the detection cost was high. Therefore, this method was suitable for the detection of protein succinic acid content. However, for the control of intermediate products, the same as comparative example 1, it undoubtedly increased the production time cost. The digestion instrument, digestion tank, and acid-dripping instrument used all increased the detection cost, which was not easy to control the production process.

[0128] In summary, compared with the existing method, the content detection method of the oral solution of iron protein succinate provided by the present invention is simpler to operate, takes less time, and has accurate measurement results, which is beneficial to the control of intermediate products and can improve the quality inspection and production efficiency of drugs.

[0129] Although the present invention has been fully described through its embodiments, it is noteworthy that various changes and modifications will be apparent to those skilled in the art, and such changes and modifications should be included within the scope of the appended claims of the present invention.

Claims

1. A method for detecting the content of ferric protein succinate oral solution, using ultraviolet-visible spectrophotometry, characterized in that: The reference solution contains ferric protein succinate, excipients, a pH adjuster and water, and the test solution is obtained by directly diluting the ferric protein succinate oral solution test sample.

2. The detection method according to claim 1, characterized in that: The auxiliary material is one or a combination of propylene glycol or sorbitol.

3. The detection method according to claim 2, characterized in that: The weight ratio of the propylene glycol to the ferric protein succinate is 4.5:4-5.5:4; the weight ratio of the sorbitol to the ferric protein succinate is 3.1:2-3.9:

2.

4. The detection method according to claim 1, characterized in that: The pH regulator is sodium hydroxide.

5. The detection method according to claim 1, characterized in that: The concentration of iron protein succinate in the reference solution is 0.29 mg / mL to 0.87 mg / mL.

6. The detection method according to claim 1, characterized in that: The pH value of the reference solution is 6.5-8.

7. The detection method according to claim 1, characterized in that: The ferric protein succinate oral solution test sample comprises ferric protein succinate, sorbitol, propylene glycol, sodium hydroxide and water.

8. The detection method according to claim 7, characterized in that: In the test sample of the ferric protein succinate oral solution, the weight ratio of propylene glycol to ferric protein succinate is 4.5:4-5.5:4, and the weight ratio of sorbitol to ferric protein succinate is 3.1:2-3.9:

2.

9. The detection method according to claim 1, characterized in that: The theoretical concentration of iron protein succinate in the test solution is 0.29 mg / mL~0.87 mg / mL.

10. The detection method according to claim 1, characterized in that: The detection wavelength of the UV-Vis spectrophotometry is 390 nm to 410 nm, preferably 400 nm.

11. The detection method according to any one of claims 1 to 10, characterized in that: The specific steps of the detection method include: ① Preparation of reference substance solution: Heat and mix the prescribed amount of propylene glycol and / or sorbitol with purified water, add sodium hydroxide, then add ferric protein succinate to dissolve it, add water to make up to volume, and stir until the final solution has a pH value of 6.5-8.0 to obtain the reference substance stock solution. Take the reference substance stock solution and dilute it with water to obtain the reference substance solution; ② Preparation of test solution: Take the test sample and dilute it with water to obtain the test solution; ③ Determination method: Take the reference solution and the test solution according to the UV-visible spectrophotometry (General Rules 0401 of Part IV of the Chinese Pharmacopoeia), use water as the blank, determine the absorbance, and calculate according to the external standard method; Among them, steps ① and ② are in no particular order.

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