Method for determining impurity aluminum element content in fat emulsion injection by high performance liquid chromatography

Abstract

The present application relates to a method for determining the content of impurity aluminum element in fat emulsion injection by high performance liquid chromatography. 9 mL of test sample and 1 mL of acid reagent are precisely added, heated at 80 DEG C for 30 min in a graphite digestion instrument, then heated at 100 DEG C for 1 h, the obtained product is concentrated at 120 DEG C, cooled, transferred with purified water and diluted to 10 mL, 3 mL of n-heptane is added for extraction, the solution is separated into two layers by standing, the water phase is filtered, and the filtrate is obtained as a test sample solution. 0.1 mL of aluminum element series control solution, blank control solution, test sample solution and blank test sample solution are precisely taken respectively, 0.9 mL of derivative reagent is precisely added, mixed, then injected into a liquid chromatograph respectively, and the chromatogram is recorded. The method of the present application selects a suitable demulsification acid solvent to destroy the structure of fat emulsion oil-in-water, release the wrapped impurity aluminum element, and obtain a clear solution at the same time, which meets the requirements of high performance liquid chromatography injection.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of drug detection, and particularly relates to a method for determining the content of impurity aluminum element in fat emulsion injection based on high performance liquid chromatography. BACKGROUND

[0002] Aluminum element is one of the most abundant metal elements in the earth's crust. Accumulation of aluminum element in the human body increases the risk of disease. The determination of impurity aluminum element in large-volume injection has attracted widespread attention. In the prior art, the methods for detecting aluminum element in drugs mainly include colorimetry, atomic absorption spectrometry, inductively coupled plasma atomic emission spectrometry (ICP-AES), inductively coupled plasma mass spectrometry (ICP-MS), etc. The composition of fat emulsion injection is relatively complex, and the form of aluminum element is diversified. The sensitivity of colorimetry is low; fat emulsion is easily carbonized at high temperature, and thus it is not suitable for atomic absorption spectrometry; ICP-AES and ICP-MS instruments are expensive, which is not conducive to the popularization and use of laboratories. Fat emulsion injection is a kind of large-volume parenteral nutrition injection widely used in clinical practice. The possible impurity aluminum element in fat emulsion injection can directly enter the blood and accumulate in the human body, causing greater harm to the human body, especially to patients with renal insufficiency and infants.

[0003] High performance liquid chromatography has the advantages of high speed, high pressure, high efficiency, high sensitivity, wide application range, reusable column and small sample amount. However, due to the following difficulties of fat emulsion injection, the determination of impurity aluminum element in high performance liquid chromatography is still blank: (1) The composition of fat emulsion injection is complex, and it cannot be directly sampled or simply diluted by purified water or solvent, which will cause the blockage of the chromatographic column and the high performance liquid system. (2) The content of aluminum element impurity in the sample is low (the limit of aluminum element in parenteral nutrition injection is stipulated in the United States Pharmacopoeia as not more than 25 μg / L), and thus the dilution multiple cannot be increased to meet the sampling requirements, otherwise the detection limit and the quantitative limit of the determination method cannot meet the determination requirements. (3) The oil-in-water structure of fat emulsion may wrap the impurity aluminum element from the raw material, resulting in inaccurate determination results. SUMMARY

[0004] In order to solve the above technical problems, the application provides a method for determining the content of impurity aluminum element in fat emulsion injection by high performance liquid chromatography. Under the premise of meeting the quantitative limit and detection limit requirements of high performance liquid chromatography, the oil-in-water structure of fat emulsion is destroyed by selecting a suitable solvent, so that the wrapped impurity aluminum element is released, and a clear solution is obtained, which meets the requirements of high performance liquid chromatography sampling.

[0005] The technical scheme adopted by the application is that the method for determining the content of impurity aluminum element in fat emulsion injection by high performance liquid chromatography comprises the following steps:

[0006] 1) Reagent preparation: separately prepare the derivatization reagent, aluminum element series control solution, blank control solution and mobile phase, and prepare them immediately before use;

[0007] 2) Preparation of sample solution: take a polytetrafluoroethylene digestion tube, accurately add 9 mL of sample and 1 mL of acid reagent, and then place it in a graphite digestion instrument after sealing. First heat it at 80°C for 30 min, and then heat it at 100°C for 1 h. After concentrating the product at 120°C, cool it, transfer it with purified water and dilute it to 10 mL. Add 3 mL of n-heptane for extraction, shake it well, and then separate the layers. Discard the oil phase, filter the water phase, and then take the filtrate to obtain the sample solution. The sample is a fat emulsion injection;

[0008] 3) Preparation of blank sample solution: take a polytetrafluoroethylene digestion tube, accurately add 9 mL of purified water and 1 mL of acid reagent, and then place it in a graphite digestion instrument after sealing. First heat it at 80°C for 30 min, and then heat it at 100°C for 1 h. After concentrating the product at 120°C, cool it, transfer it with purified water and dilute it to 10 mL. Add 3 mL of n-heptane for extraction, shake it well, and then separate the layers. Take the lower water phase, filter it, and then take the filtrate to obtain the blank sample solution;

[0009] 4) Determination: accurately take 0.1 mL of the aluminum element series control solution, blank control solution, sample solution and blank sample solution respectively, and accurately add 0.9 mL of the derivatization reagent respectively. After mixing, inject them into the liquid chromatograph respectively, and record the chromatogram;

[0010] 5) Draw a linear regression equation by corresponding the concentration of the aluminum element series control solution to its peak area and deducting the peak area of the blank control solution. Calculate the content of aluminum element in the sample by deducting the peak area of the blank sample solution from the peak area of the sample solution.

[0011] Preferably, in step 1), the preparation method of the derivatization reagent comprises the following steps: according to the volume ratio, 0.3% 8-hydroxyquinoline acetonitrile solution: 0.2 mol / L ammonium acetate solution = 45:55; take 50 mL of the mixed solution of 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2 mol / L ammonium acetate solution, and add 0.3 mL of 50% NaOH (mass fraction), and mix well.

[0012] Preferably, in step 1), the concentration of the aluminum element series control solution is 200 μg / L, 100 μg / L, 50 μg / L, 25 μg / L, 10 μg / L and 5 μg / L respectively.

[0013] Preferably, in step 1), the preparation method of the mobile phase comprises the following steps: taking 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2 mol / L ammonium acetate solution, and mixing them uniformly.

[0014] Preferably, in step 2), the acid reagent is sulfuric acid, nitric acid or hydrochloric acid.

[0015] More preferably, the acid reagent is hydrochloric acid.

[0016] Preferably, in step 3), the liquid chromatography conditions are as follows: a phenyl column with phenethyl-bonded silica gel as the matrix, a column temperature of 30 DEG C, a flow rate of 1.0 mL / min, an injection volume of 100 mu L, and an excitation wavelength of 380 nm and an emission wavelength of 520 nm of the fluorescence detector.

[0017] Preferably, the fat emulsion injection is C6-C 24 medium-chain fat emulsion injection, or fat emulsion amino acid (17) glucose (11%) fat emulsion injection, or medium-chain fat emulsion amino acid (16) glucose (16%) fat emulsion injection.

[0018] The method provided by the application is applied to the determination of the content of impurity aluminum element in a fat emulsion injection.

[0019] The application has the following beneficial effects:

[0020] 1. The method provided by the application selects a suitable demulsification acid solvent to destroy the structure of the oil-in-water emulsion, releases the wrapped impurity aluminum element, and obtains a clear solution, thereby meeting the requirements of high performance liquid chromatography injection.

[0021] 2. The method provided by the application optimizes the sample pretreatment conditions, reduces the introduction of aluminum element, and improves the accuracy of the determination of the impurity aluminum element in the sample.

[0022] 3. The method provided by the application selects appropriate pretreatment methods and chromatography conditions, and realizes the determination of the content of the impurity aluminum element in the fat emulsion injection by high performance liquid chromatography. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a high performance liquid chromatogram of Example 1, wherein the acid solvent is sulfuric acid, and the volume ratio of the sample to sulfuric acid is 9:1.

[0024] Figure 2 is a high performance liquid chromatogram of Example 1, wherein the acid solvent is sulfuric acid, and the volume ratio of the sample to sulfuric acid is 8:2.

[0025] Figure 3is the high performance liquid chromatogram of Example 1, acid solvent is hydrochloric acid, the volume ratio of sample to hydrochloric acid is 9:1.

[0026] Figure 4 is the high performance liquid chromatogram of Example 1, acid solvent is hydrochloric acid, the volume ratio of sample to hydrochloric acid is 8:2.

[0027] Figure 5 is the high performance liquid chromatogram of Example 1, acid solvent is nitric acid, the volume ratio of sample to nitric acid is 9:1.

[0028] Figure 6 is the high performance liquid chromatogram of Example 1, acid solvent is nitric acid, the volume ratio of sample to nitric acid is 8:2.

[0029] Figure 7 is the high performance liquid chromatogram of Example 1, base solvent is NaOH.

[0030] Figure 8 is the high performance liquid chromatogram of Example 1, acid solvent is hydrochloric acid, 60℃ water bath for 5h.

[0031] Figure 9 is the high performance liquid chromatogram of Example 1, acid solvent is hydrochloric acid, 70℃ water bath for 5h.

[0032] Figure 10 is the high performance liquid chromatogram of Example 1, acid solvent is hydrochloric acid, 80℃ water bath for 5h.

[0033] Figure 11 is the high performance liquid chromatogram of Example 1, acid solvent is hydrochloric acid, graphite digestion instrument is heating method, n-heptane extraction.

[0034] Figure 12 is the standard curve of aluminum element series control solution in Example 3. DETAILED DESCRIPTION

[0035] The method for determining the content of impurity aluminum element in fat milk injection by high performance liquid chromatography comprises the following steps:

[0036] 1) Reagent preparation:

[0037] Prepare the derivative reagent, aluminum element series control solution, blank control solution and mobile phase respectively, and prepare them immediately before use.

[0038] 1.1) Preparation of derivative reagent: according to the volume ratio, 0.3% 8-hydroxyquinoline acetonitrile solution: 0.2 mol / L ammonium acetate solution = 45:55; take 50 mL of 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2 mol / L ammonium acetate solution mixture, add 0.3 mL of 50% NaOH (mass percentage), and mix well.

[0039] 1.2) Preparation of aluminum element series control solution: The concentration of the aluminum element series control solution is 200 μg / L, 100 μg / L, 50 μg / L, 25 μg / L, 10 μg / L and 5 μg / L, respectively.

[0040] 1.3) Blank control solution: purified water is used as the blank control solution.

[0041] 1.4) Preparation of mobile phase: 8-hydroxyquinoline acetonitrile solution with a mass percentage concentration of 0.3%: 0.2 mol / L ammonium acetate solution = 45:55 by volume; 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2 mol / L ammonium acetate solution are mixed uniformly.

[0042] 2) Preparation of test sample solution:

[0043] A polytetrafluoroethylene digestion tube is taken, 9 mL of the test sample and 1 mL of the acid reagent are precisely added, and after plugging, it is placed in a graphite digestion instrument, heated at 80°C for 30 min, and then heated at 100°C for 1 h. The obtained product is concentrated at 120°C, cooled, transferred with purified water and diluted to 10 mL, 3 mL of n-heptane is added for extraction, shaken uniformly, and after standing, the oil phase is discarded, the water phase is filtered, and the filtrate is taken to obtain the test sample solution. The test sample is a fat emulsion injection.

[0044] The acid reagent is sulfuric acid, nitric acid or hydrochloric acid. Preferably, the acid reagent is hydrochloric acid.

[0045] 3) Blank test sample solution:

[0046] A polytetrafluoroethylene digestion tube is taken, 9 mL of purified water and 1 mL of the acid reagent are precisely added, and after plugging, it is placed in a graphite digestion instrument, heated at 80°C for 30 min, and then heated at 100°C for 1 h. The obtained product is concentrated at 120°C, cooled, transferred with purified water and diluted to 10 mL, 3 mL of n-heptane is added for extraction, shaken uniformly, and after standing, the lower water phase is taken and filtered, and the filtrate is taken to obtain the blank test sample solution.

[0047] The acid reagent is sulfuric acid, nitric acid or hydrochloric acid. Preferably, the acid reagent is hydrochloric acid.

[0048] 4) Determination:

[0049] 0.1 mL of the aluminum element series control solution, the blank control solution, the test sample solution and the blank test sample solution, respectively, 0.9 mL of the derivatization reagent is precisely added, mixed uniformly, and then injected into the liquid chromatograph, and the chromatogram is recorded.

[0050] The liquid chromatography conditions are as follows: a phenyl column with phenyl bonded silica gel as the matrix, column temperature is 30°C, flow rate is 1.0 mL / min, injection volume is 100 μL, and the excitation wavelength of the fluorescence detector is 380 nm and the emission wavelength is 520 nm.

[0051] 5) Draw the standard curve and calculate:

[0052] The linear regression equation is obtained by linear regression of the concentration of the aluminum element series control solution corresponding to the peak area of the corresponding peak area minus the peak area of the blank control solution. The content of aluminum element in the test sample is calculated according to the peak area of the test sample solution minus the peak area of the blank test sample solution.

[0053] Example 1

[0054] The following control and test sample are used in the condition screening test of this example.

[0055] Control: single-element standard solution of aluminum metal, National Non-ferrous Metal and Electronic Material Analysis and Testing Center, concentration is 1000 μg / mL.

[0056] Test sample: medium-chain fatty emulsion injection (C6-C 24 ), Liaoning Haishenke Pharmaceutical Co., Ltd.

[0057] (I) Selection of solvent for destroying the oil-in-water structure of the fatty emulsion

[0058] 1. Organic solvent

[0059] As shown in Table 1, ethanol, isopropanol, n-propanol, tetrahydrofuran, n-hexane or a mixture of two reagents in a certain volume ratio are selected as organic solvents and test sample for demulsification test to study the condition of destroying the oil-in-water structure, and the results are shown in Table 1.

[0060] Table 1 Demulsification condition screening (organic solvent)

[0061]

[0062] As shown in Table 1, ethanol, isopropanol, n-propanol and n-hexane cannot destroy the oil-in-water structure. Tetrahydrofuran can destroy the oil-in-water structure, but the dilution multiple is large. Generally, the aluminum content in the test sample is low, and the large dilution multiple of the pretreated sample may result in low final injection concentration, which cannot meet the requirements of the method detection limit and the quantitative limit. Therefore, the organic solvent cannot be used as the demulsification solvent for the fatty emulsion injection in the present application.

[0063] 2. Acid reagent

[0064] Sulfuric acid, hydrochloric acid and nitric acid are selected as acid reagents.

[0065] Derivative reagent: 0.3% 8-hydroxyquinoline acetonitrile solution: 0.2 mol / L ammonium acetate solution = 45:55 by volume ratio; take 50 mL of 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2 mol / L ammonium acetate solution mixed solution, add 0.3 mL of 50% mass percentage NaOH, mix well.

[0066] Preparation of test sample solution: take a polytetrafluoroethylene digestion tube, accurately add 8 mL of test sample and 2 mL of acid reagent or accurately add 9 mL of test sample and 1 mL of acid reagent, heat in 60℃ water bath for 3h, respectively, to obtain test sample solution with different acid reagents and different volume ratios, and observe the demulsification.

[0067] Accurately take 0.1 mL of test sample solution with different acid reagents and different volume ratios, respectively, and accurately add 0.9 mL of derivative reagent, mix well, and then inject into the liquid chromatograph, respectively, to record the chromatogram. The liquid chromatography conditions are: a phenyl column with phenyl bonded silica gel as the substrate, the mobile phase is a mixture of 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2 mol / L ammonium acetate solution with a volume ratio of 45:55; the column temperature is 30℃; the flow rate is 1.0 mL / min; the injection volume is 100 μL; the fluorescence detector has an excitation wavelength of 380 nm and an emission wavelength of 520 nm. Analyze the peak shape and corresponding conditions by high performance liquid chromatography method, and the results are shown in Table 2 and Figures 1-6 .

[0068] 3. Alkali reagent

[0069] NaOH is used as the alkali reagent.

[0070] Derivative reagent: 0.3% 8-hydroxyquinoline acetonitrile solution: 0.2 mol / L ammonium acetate solution = 45:55 by volume ratio; take 50 mL of 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2 mol / L ammonium acetate solution mixed solution, add 0.3 mL of 50% mass percentage NaOH, mix well.

[0071] Preparation of test sample solution: take a polytetrafluoroethylene digestion tube, accurately add 5 mL of test sample and 0.5 mg of sodium hydroxide, heat in 60℃ water bath for 3h, to obtain test sample solution, and observe the demulsification.

[0072] The sample solution of the test sample was precisely taken 0.1 mL, the derivatization reagent was precisely added 0.9 mL, after mixing, it was injected into a liquid chromatograph, and a chromatogram was recorded. The liquid chromatography conditions were as follows: a phenyl column with phenyl bonded silica gel as the matrix, a mobile phase of 8-hydroxyquinoline acetonitrile solution with a mass percentage of 0.3% and ammonium acetate solution with a concentration of 0.2 mol / L at a volume ratio of 45:55; the column temperature was 30°C; the flow rate was 1.0 mL / min; the injection volume was 100 μL; the excitation wavelength of the fluorescence detector was 380 nm, and the emission wavelength was 520 nm. The peak shape and the corresponding conditions were analyzed by using the high performance liquid chromatography method, and the results were shown in Table 2 and Figure 7 .

[0073] Table 2 demulsification condition screening (acid reagent and base reagent combined with HPLC analysis)

[0074]

[0075]

[0076] From Table 2, Figure 2 , Figure 4 and Figure 6 it can be seen that when the volume ratio of the test sample sample to the acid reagent is 8:2, the sample can be demulsified to reach the state of oil-water separation. After being filtered through a 0.45 μm filter membrane and injected into a high performance liquid chromatograph, the results show that the sample response is low after being demulsified by sulfuric acid; the peak shape of the sample is poor after being demulsified by hydrochloric acid and nitric acid. The peak shape is not improved by adjusting the amount of the added base in the derivatization reagent.

[0077] From Table 2, Figure 1 , Figure 3 and Figure 5 it can be seen that when the volume ratio of the test sample sample to the acid reagent is 9:1, the sample can be demulsified to reach the state of oil-water separation. After being filtered through a 0.45 μm filter membrane and injected into a high performance liquid chromatograph, the results show that the sample response is low after being demulsified by sulfuric acid, the sample peak bifurcates after being demulsified by nitric acid, and the peak shape is poor, and the peak shape of the sample is good after being demulsified by hydrochloric acid.

[0078] From Table 2 and Figure 7 it can be seen that after the test sample sample is added with sodium hydroxide, the sample is demulsified to reach the state of oil-water separation, and the response value is too high after being injected into a high performance liquid chromatograph for analysis, which is because the sodium hydroxide reagent itself contains a certain amount of aluminum element, and the addition of sodium hydroxide will introduce foreign aluminum element impurities.

[0079] Based on the above investigation, the application preferably uses hydrochloric acid as the acid demulsification reagent, and the volume ratio of the test sample sample to the hydrochloric acid is 9:1.

[0080] (II) Screening of sample pretreatment heating conditions

[0081] The treated sample is determined by HPLC method, and the sample pretreatment process is screened by analyzing the test sample, the spiked test sample and the standard curve.

[0082] 1. Influence of water bath heating on determination results

[0083] 1.1) Reagent preparation

[0084] Aluminum element standard stock solution: Take the single-element aluminum standard solution and dilute it with purified water to prepare an aluminum element standard stock solution with a concentration of 500 μg / L.

[0085] Preparation of aluminum element series control solution: accurately measure an appropriate amount of aluminum element standard stock solution, and quantitatively dilute it with purified water to prepare aluminum element series control solutions with concentrations of 200 μg / L, 100 μg / L, 50 μg / L, 25 μg / L, 10 μg / L and 5 μg / L, respectively.

[0086] Blank control solution: purified water as blank control solution.

[0087] Test sample solution: take 15 mL of polypropylene material calibrated centrifuge tube, add 4.5 mL of test sample and 0.5 mL of hydrochloric acid according to the volume ratio of 9:1, seal, place in water bath, heat at 60℃, 70℃ and 80℃ respectively for 3-5h, take out, filter, and obtain test sample solutions obtained at different temperatures.

[0088] Spiked sample solution: accurately measure 4.5 mL of aluminum element standard stock solution with a concentration of 500 μg / L, add 85.5 mL of test sample solution, mix well to obtain a spiked sample. Take 15 mL of polypropylene material calibrated centrifuge tube, add 4.5 mL of the above spiked sample and 0.5 mL of hydrochloric acid according to the volume ratio of 9:1, seal, place in water bath, heat at 60℃, 70℃ and 80℃ respectively for 3-5h, take out, filter, and obtain spiked sample solutions containing 25 μg / L of standard aluminum element obtained at different temperatures.

[0089] Derivative reagent: according to the volume ratio, 0.3% 8-hydroxyquinoline acetonitrile solution: 0.2 mol / L ammonium acetate solution = 45:55; take 50 mL of 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2 mol / L ammonium acetate solution mixture, add 0.3 mL of 50% mass percentage NaOH, mix well.

[0090] 1.2) Method

[0091] Precisely pipette 0.1 mL of aluminum element series control solution, blank control solution, sample solution obtained at different temperatures and sample solution obtained at different temperatures respectively, precisely add 0.9 mL of derivatization reagent respectively, mix, inject into liquid chromatograph respectively, and record chromatogram.

[0092] The liquid chromatography conditions are as follows: a phenyl column with phenylethyl bonded silica gel as the matrix, a mobile phase of 8-hydroxyquinoline acetonitrile solution with a mass percentage of 0.3% and ammonium acetate solution with a concentration of 0.2 mol / L mixed at a volume ratio of 45:55; column temperature is 30°C; flow rate is 1.0 mL / min; injection volume is 100 μL; excitation wavelength of fluorescence detector is 380 nm, and emission wavelength is 520 nm.

[0093] The concentration of aluminum element series control solution is linearly regressed according to the corresponding peak area, the linear regression equation is obtained, and the content of aluminum element in the solution is calculated according to the peak area of the sample solution obtained at different temperatures and the sample solution with different temperatures. The recovery rate is calculated, and the results are shown in Table 3.

[0094] Table 3: Water bath heating condition screening

[0095]

[0096] From Figures 8-10 It can be seen that hydrochloric acid is selected as the demulsifying solvent for destroying the oil-in-water structure, and the chromatographic peak shape is good. However, as shown in Table 3, the recovery rate is too high, and the deviation between parallel samples is large. This is because the amount of hydrochloric acid added is small, and although the oil-in-water structure is destroyed by heating, the water phase and the oil phase cannot be completely miscible, resulting in inaccurate determination results and the risk of liquid phase system blockage. At the same time, the water bath heating temperature is low, and it takes a long time to destroy the oil-in-water structure. If the temperature is high and the heating time is long, there will be a certain loss of liquid volume, and at the same time, the steam environment of the water bath also has a certain impact on the sample.

[0097] Considering the above factors, water bath heating results in high overall recovery rate of the standard addition, which is not suitable for the present application.

[0098] 2. Graphite digestion instrument heating, selection of organic solvent extractant

[0099] In order to shorten the extraction time and increase the efficiency of solvent destroying the emulsion structure, and considering that the aluminum element should be completely dissolved in the water phase, a graphite digestion instrument in anhydrous environment is used as the heating device. Since the impurity aluminum element exists in the form of aluminum ions in the aqueous solution, an organic solvent extraction step is added to dissolve all the oil phase materials in the extraction solvent and completely separate the water phase, so as to avoid the oil phase materials entering the liquid phase system and affecting the accuracy of the results and increasing the risk of liquid phase system blockage.

[0100] 2.1) Reagent preparation

[0101] Aluminum element standard stock solution: Take the single-element aluminum standard solution, dilute with purified water to prepare an aluminum element standard stock solution with a concentration of 500 μg / L.

[0102] Preparation of aluminum element series control solution: accurately measure an appropriate amount of aluminum element standard stock solution, and quantitatively dilute with purified water to prepare aluminum element series control solutions with concentrations of 200 μg / L, 100 μg / L, 50 μg / L, 25 μg / L, 10 μg / L, and 5 μg / L, respectively.

[0103] Blank control solution: Purified water as blank control solution.

[0104] Test sample solution: Take a polytetrafluoroethylene digestion tube, add 9 mL of test sample and 1 mL of hydrochloric acid according to the volume ratio of 9:1, plug the tube, and place it in a graphite digestion instrument. After heating at 80°C for 30 min, the temperature is increased to 100°C for 1 h. The fat emulsion oil-in-water structure is destroyed, and the solution is turbid with oil and water separation. Concentrate at 120°C, cool, and transfer to 10 mL with purified water. Add the extractant as shown in Table 4, shake well, separate the layers, discard the oil phase, and filter the water phase to obtain the test sample solution obtained with different extractants.

[0105] Spiked sample solution: accurately measure 4.5 mL of aluminum element standard stock solution with a concentration of 500 μg / L, add 85.5 mL of test sample solution, mix well, and obtain the spiked sample. Take a polytetrafluoroethylene digestion tube, add 9 mL of the above spiked sample and 1 mL of hydrochloric acid according to the volume ratio of 9:1, plug the tube, and place it in a graphite digestion instrument. After heating at 80°C for 30 min, the temperature is increased to 100°C for 1 h. The fat emulsion oil-in-water structure is destroyed, and the solution is turbid with oil and water separation. Concentrate at 120°C, cool, and transfer to 10 mL with purified water. Add the extractant as shown in Table 4, shake well, filter the water phase, and prepare the spiked sample containing 25 μg / L of standard aluminum element with different extractants.

[0106] Derivative reagent: according to the volume ratio, 0.3% 8-hydroxyquinoline acetonitrile solution: 0.2 mol / L ammonium acetate solution = 45:55; take 50 mL of 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2 mol / L ammonium acetate solution mixture, add 0.3 mL of 50% mass percentage NaOH, and mix well.

[0107] 2.2) Method

[0108] Precisely pipette 0.1 mL of aluminum element series control solution, blank control solution, sample solution obtained under different extractants and different extractant spiked sample solution respectively, precisely add 0.9 mL of derivatization reagent respectively, mix, inject into liquid chromatograph respectively, and record chromatogram.

[0109] The liquid chromatography conditions are as follows: a phenyl column with phenyl bonded silica gel as the matrix, a mobile phase of 8-hydroxyquinoline acetonitrile solution with a mass percentage of 0.3% and ammonium acetate solution with a concentration of 0.2 mol / L mixed at a volume ratio of 45:55; a column temperature of 30°C; a flow rate of 1.0 mL / min; an injection volume of 100 μL; and an excitation wavelength of 380 nm and an emission wavelength of 520 nm of the fluorescence detector.

[0110] The concentration of the aluminum element series control solution is linearly regressed according to the corresponding peak area, a linear regression equation is obtained, and the content of aluminum element in the solution is calculated according to the peak area of the sample solution obtained at different temperatures and the spiked sample at different temperatures. The recovery rate is calculated, and the results are shown in Table 4.

[0111] Table 4: Extractant screening

[0112]

[0113] As shown in Table 4, the graphite digestion instrument heating method is preferred, and n-heptane is used as the extractant. When 1 mL, 2 mL and 3 mL of n-heptane is used for extraction, the water phase and the oil phase are completely separated. The preferred amount of n-heptane is 2-3 mL, and more preferably 3 mL. The typical chromatogram is shown in Figure 11 .

[0114] Based on the above investigation, the preparation of the sample solution is as follows: a polytetrafluoroethylene digestion tube is taken, 9 mL of sample and 1 mL of hydrochloric acid are added at a volume ratio of 9:1, a plug is added, and the tube is placed in a graphite digestion instrument. After heating at 80°C for 30 min and heating at 100°C for 1 h, the oil-in-water structure of the fat emulsion is destroyed, the solution is turbid and oil-water separation, and the solution is concentrated at 120°C, cooled, transferred with purified water and diluted to 10 mL. 3 mL of n-heptane is added, shaken and shaken, and the oil phase is discarded after separation. The water phase is filtered to obtain the sample solution.

[0115] (Three) selection of mobile phase and derivatization reagent concentration

[0116] 1. Selection of 8-hydroxyquinoline concentration

[0117] 8-hydroxyquinoline combines with aluminum ions to form a fluorescent complex, and the content of the complex is determined by using a fluorescence detector to quantify the aluminum element. 8-hydroxyquinoline is a colored substance, and in the determination system, 8-hydroxyquinoline exists in a dual identity of a derivative reagent and a component of a mobile phase, eliminating the influence of background. The concentration of 8-hydroxyquinoline acetonitrile solution is screened in the application: 8-hydroxyquinoline is selected as 0.1%, 0.3%, 1%, and 3%, respectively.

[0118] Through experimental investigation, in combination with the response value of the sample, the durability of the method, and the stability of the mobile phase, and other factors, the application preferably selects that the concentration of 8-hydroxyquinoline in the mobile phase and the derivative reagent is 0.3%, and the solvent is acetonitrile.

[0119] 2. The concentration of sodium hydroxide in the derivative reagent 8-hydroxyquinoline combines with aluminum ions to form a fluorescent complex, and the peak shape of the complex in a solution environment greater than pH 7 is better, so by adding a certain concentration of sodium hydroxide solution to the mobile phase, the pH environment of the sample is changed, thereby improving the chromatographic peak shape. The concentration of NaOH in the derivative reagent is screened in the application: the concentration of NaOH is 0, 0.3%, and 0.5%, respectively.

[0120] Through experimental investigation, in combination with the chromatographic peak shape and the reduction of the introduction of external aluminum elements, 50% NaOH solution is selected to be added to the mobile phase in a small amount, so that the concentration of NaOH in the mobile phase is 0.3%.

[0121] Example 2

[0122] (I) The method for determining the content of impurity aluminum element in fat emulsion injection by high performance liquid chromatography, comprising the following steps:

[0123] 1. Reagent preparation:

[0124] 1.1) Preparation of derivative reagent:

[0125] According to the volume ratio, 0.3% 8-hydroxyquinoline acetonitrile solution: 0.2 mol / L ammonium acetate solution = 45:55; 50 mL of 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2 mol / L ammonium acetate solution mixture solution is added with 0.3 mL of 50% NaOH, and mixed uniformly.

[0126] 1.2) Preparation of aluminum element series control solution:

[0127] Aluminum element standard stock solution preparation: Prepare aluminum element standard stock solution with a concentration of 500 μg / L.

[0128] Preparation of aluminum element series control solution: accurately take appropriate amount of aluminum element standard stock solution, and quantitatively dilute with purified water to prepare aluminum element series control solutions with concentrations of 200 μg / L, 100 μg / L, 50 μg / L, 25 μg / L, 10 μg / L and 5 μg / L, respectively.

[0129] 1.3) Blank control solution:

[0130] The purified water was used as the blank control solution.

[0131] 1.4) Preparation of mobile phase:

[0132] According to the volume ratio, 0.3% 8-hydroxyquinoline acetonitrile solution: 0.2 mol / L ammonium acetate solution = 45:55; take 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2 mol / L ammonium acetate solution, mix well.

[0133] 2. Preparation of test sample solution:

[0134] Take a polytetrafluoroethylene digestion tube, accurately add 9 mL of test sample and 1 mL of hydrochloric acid, plug, and place in a graphite digestion instrument. First heat at 80°C for 30 min, then heat at 100°C for 1 h. Concentrate the obtained product at 120°C, cool, transfer with purified water, and dilute to 10 mL. Then add 3 mL of n-heptane for extraction, shake well, stand for separation, discard the oil phase, filter the water phase, and take the filtrate to obtain the test sample solution. The following test sample is a medium-chain fatty emulsion injection (C6-C 24 ).

[0135] 3. Blank test sample solution:

[0136] Take a polytetrafluoroethylene digestion tube, accurately add 9 mL of purified water and 1 mL of hydrochloric acid, plug, and place in a graphite digestion instrument. First heat at 80°C for 30 min, then heat at 100°C for 1 h. Concentrate the obtained product at 120°C, cool, transfer with purified water, and dilute to 10 mL. Then add 3 mL of n-heptane for extraction, shake well, stand for separation, discard the oil phase, filter the water phase, and take the filtrate to obtain the test sample solution.

[0137] 4. Determination

[0138] Instrument: Shimadzu 20A high performance liquid chromatograph, Shimadzu RF-20A fluorescence detector.

[0139] Accurately take 0.1 mL of aluminum element series control solution, blank control solution, test sample solution, and blank test sample solution, respectively, accurately add 0.9 mL of derivatization reagent, mix well, inject into the high performance liquid chromatograph, and record the chromatogram.

[0140] The liquid chromatography conditions are as follows: a phenyl column (CAPCELL PAK Phenyl 4.6 mm x 250 mm, 5 μm) with phenethyl bonded silica gel as a matrix, a mobile phase of 8-hydroxyquinoline acetonitrile solution with a mass percentage of 0.3% and ammonium acetate solution with a concentration of 0.2 mol / L mixed at a volume ratio of 45:55 (freshly prepared before use), a column temperature of 30°C, a flow rate of 1.0 mL / min, an injection volume of 100 μL, and a fluorescence detector with an excitation wavelength of 380 nm and an emission wavelength of 520 nm.

[0141] 5. Standard curve drawing and calculation

[0142] A linear regression equation is obtained by linear regression of the concentration of the aluminum element series control solution corresponding to the peak area thereof minus the peak area of the blank control solution. The content of the aluminum element in the test sample solution is calculated according to the peak area of the test sample solution minus the peak area of the blank test sample solution.

[0143] (II) Precision test

[0144] The aluminum element control solution with a concentration of 25 μg / L in the aluminum element series control solution is determined by the method determined in (I) 4, and the determination is continuously performed for 6 times. The results are shown in Table 5.

[0145] Table 5: Results of sample injection precision test

[0146]

[0147]

[0148] As shown in Table 5, the sample injection precision RSD of the method of the present application is 0.62%, and the sample injection precision of the method is good.

[0149] (III) Reproducibility test

[0150] The test sample solution is prepared according to (I) 2, and a total of 6 test sample solutions are prepared. Then, the reproducibility experiment is performed according to the method of (I), and the results are shown in Table 6.

[0151] Table 6: Results of reproducibility test

[0152]

[0153] As shown in Table 6, the RSD of the method of the present application is 5.5%, and the reproducibility of the method is good.

[0154] (IV) Accuracy test (spiked recovery rate):

[0155] Aluminum element control solution: precisely take 25 mL of aluminum element standard stock solution with a concentration of 500 μg / L into a 100 mL volumetric flask, and dilute with purified water to prepare an aluminum element control solution with a concentration of 125 μg / L.

[0156] Test sample solution: take a polytetrafluoroethylene digestion tube, precisely add 1 mL of hydrochloric acid and 9 mL of test sample, cap it, and place it in a graphite digestion instrument. First heat at 80°C for 30 min, then heat at 100°C for 1 h. After the product is concentrated at 120°C, cool it, transfer it with purified water, and dilute to 10 mL. Add 3 mL of n-heptane for extraction, shake well, and let it stand to separate the layers. Discard the oil phase, filter the water phase, and take the filtrate to obtain the test sample solution.

[0157] Blank test sample solution: take a polytetrafluoroethylene digestion tube, precisely add 1 mL of hydrochloric acid and 9 mL of purified water, cap it, and place it in a graphite digestion instrument. First heat at 80°C for 30 min, then heat at 100°C for 1 h. After the product is concentrated at 120°C, cool it, transfer it with purified water, and dilute to 10 mL. Add 3 mL of n-heptane for extraction, shake well, and let it stand to separate the layers. Take the lower water phase, filter it, and take the filtrate to obtain the blank test sample solution.

[0158] Spiked recovery test sample solution: precisely take 0.5, 1, and 1.5 mL of aluminum element control solution with a concentration of 125 μg / L into polytetrafluoroethylene digestion tubes, respectively. Precisely add 4, 3.5, and 3 mL of purified water, respectively. Then precisely add 4.5 mL of test sample, mix well, and precisely add 1 mL of hydrochloric acid (3 preparations for each concentration). Cap it, heat at 80°C for 30 min in a graphite digestion instrument, and heat at 100°C for 1 h. The fatty oil-in-water structure is destroyed, and the solution is turbid with oil and water separation. Concentrate at 120°C. Cool it, transfer it with purified water, and dilute to 10 mL. Add 3 mL of n-heptane for extraction, shake well, and let it stand to separate the layers. Discard the oil phase, filter the water phase, and take the filtrate as the spiked recovery test sample solution. Replace the test sample with 9 mL of purified water to prepare a blank spiked recovery test sample solution.

[0159] Precisely take 0.1 mL of aluminum element series control solution, blank control solution, test sample solution, blank test sample solution, spiked recovery test sample solution, and blank spiked recovery test sample solution, respectively. Precisely add 0.9 mL of derivatization reagent, mix well, and determine as in method (I) 4. The results are shown in Table 7.

[0160] Table 7 Recovery determination results

[0161]

[0162] From table 7, the method of the application, the recovery rate of sample addition is 109.4%, and the RSD is 1.4% (n=9), and the method accuracy is good.

[0163] Practical application of example 3

[0164] (I) The method for determining the content of impurity aluminum element in fat emulsion injection by high performance liquid chromatography comprises the following steps:

[0165] 1. Reagent preparation:

[0166] 1.1) Preparation of derivatization reagent:

[0167] According to the volume ratio, 0.3% 8-hydroxyquinoline acetonitrile solution: 0.2 mol / L ammonium acetate solution = 45:55; take 50 mL of 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2 mol / L ammonium acetate solution mixture, add 0.3 mL of 50% mass percentage NaOH, mix well.

[0168] 1.2) Preparation of aluminum element series control solution:

[0169] Aluminum element standard stock solution preparation: Take the metal aluminum single element standard solution (National Non-ferrous Metals and Electronic Materials Analysis and Testing Center, concentration is 1000 μg / ml), dilute with purified water to prepare aluminum element standard stock solution with a concentration of 500 μg / L.

[0170] Preparation of aluminum element series control solution: accurately take the aluminum element standard stock solution, add purified water to dilute quantitatively, and prepare aluminum element series control solution with a concentration of 200 μg / L, 100 μg / L, 50 μg / L, 25 μg / L, 10 μg / L and 5 μg / L, respectively.

[0171] 1.3) Blank control solution: purified water as blank control solution.

[0172] 1.4) Preparation of mobile phase: according to the volume ratio, 0.3% 8-hydroxyquinoline acetonitrile solution: 0.2 mol / L ammonium acetate solution = 45:55; take 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2 mol / L ammonium acetate solution, mix well.

[0173] 2. Preparation of sample solution:

[0174] Take polytetrafluoroethylene material digestion tube, accurately add 9 mL of test sample and 1 mL of hydrochloric acid, plug, place in graphite digestion instrument, first heat at 80℃ for 30 min, then heat at 100℃ for 1 h, the product is concentrated at 120℃, cooled, transferred with purified water and diluted to 10 mL, then 3 mL of n-heptane is added for extraction, shaken and shaken, separated, the oil phase is discarded, the water phase is filtered, the filtrate is taken, and the test sample solution is obtained.

[0175] As shown in Table 8, different varieties and different batches of fat emulsion injection were taken as test sample.

[0176] 3. Blank test solution:

[0177] Take polytetrafluoroethylene material digestion tube, accurately add 9 mL of test sample and 1 mL of hydrochloric acid, plug, place in graphite digestion instrument, first heat at 80℃ for 30 min, then heat at 100℃ for 1 h, the product is concentrated at 120℃, cooled, transferred with purified water and diluted to 10 mL, then 3 mL of n-heptane is added for extraction, shaken and shaken, separated, the oil phase is discarded, the water phase is filtered, the filtrate is taken, and the test sample solution is obtained.

[0178] 4. Determination

[0179] Instrument: Shimadzu 20A high performance liquid chromatograph.

[0180] Precisely take 0.1 mL of aluminum element series control solution, blank control solution, test sample solution and blank test sample solution respectively, accurately add 0.9 mL of derivatization reagent respectively, mix, inject into high performance liquid chromatograph respectively, and record chromatogram;

[0181] The liquid chromatography conditions are: using phenyl column with phenyl bonded silica gel as matrix (CAPCELL PAK Phenyl 4.6mmx250mm, 5μm), the mobile phase is 8-hydroxyquinoline acetonitrile solution with mass percentage of 0.3% and ammonium acetate solution with concentration of 0.2mol / L mixed in volume ratio of 45:55 (freshly prepared); column temperature is 30℃; flow rate is 1.0mL / min; injection volume is 100μL; fluorescence detector excitation wavelength is 380nm, and emission wavelength is 520nm.

[0182] 5. Results

[0183] As Figure 12 The concentration of aluminum element series control solution corresponds to the peak area of the corresponding standard curve, and the blank control peak area is deducted, and a linear regression equation is obtained: y=1480.66x+14529.46, R 2 =0.9994. In the range of 5-200ug / L, the linear relationship is good.

[0184] The content of aluminum element in the sample solution of the test product (C0) is obtained by deducting the peak area of the blank test product from the peak area of the sample solution of the test product, and then the content of aluminum element in the sample of the test product is obtained according to formula (1). The results are shown in Table 8.

[0185]

[0186] C: the content of aluminum element in the sample of the test product, g / mL

[0187] C0: the content of aluminum element in the sample solution of the test product, μg / L

[0188] C 油 : the oil phase concentration of the sample of the test product, g / mL

[0189] ρ 油 : the oil phase density of the sample of the test product, g / mL

[0190] Note: The oil phase concentration and the oil phase density of the sample of the test product are provided by the pharmaceutical production enterprise.

[0191] Table 8 sample determination

[0192]

[0193] As can be seen from Table 8, the method of the present application has accurate measurement results, simple method and instrument equipment, low price, and is more easy to promote the use in the laboratory.

Claims

1. A method for determining the content of impurity aluminum element in fat emulsion injection by high performance liquid chromatography, characterized in that, It comprises the following steps: 1) reagent preparation: respectively prepare the derivative reagent, aluminum element series control solution, blank control solution and mobile phase, use it as soon as possible; the preparation method of the derivative reagent comprises the following steps: according to the volume ratio, the mass percentage concentration of 0.3% 8-hydroxyquinoline acetonitrile solution: 0.2mol / L ammonium acetate solution = 45:55; take 50mL of 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2mol / L ammonium acetate solution mixed solution, add 0.3mL mass percentage concentration of 50% NaOH, mix evenly; 2) preparation of sample solution: take a polytetrafluoroethylene digestion tube, accurately add 9mL of sample and 1mL of hydrochloric acid, after plugging, place it in a graphite digestion instrument, first heat at 80℃ for 30min, then heat at 100℃ for 1h, the obtained product is concentrated at 120℃, cooled, transferred with purified water and constant volume to 10mL, add 3mL of n-heptane extraction, shake evenly, stand for separation, discard the oil phase, filter the water phase, take the filtrate, and obtain the sample solution; the sample is a fat emulsion injection; 3) preparation of blank sample solution: take a polytetrafluoroethylene digestion tube, accurately add 9mL of purified water and 1mL of hydrochloric acid, after plugging, place it in a graphite digestion instrument, first heat at 80℃ for 30min, then heat at 100℃ for 1h, the obtained product is concentrated at 120℃, cooled, transferred with purified water and constant volume to 10mL, add 3mL of n-heptane extraction, shake evenly, stand for separation, take the lower water phase and filter, take the filtrate, and obtain the blank sample solution; 4) determination: accurately take 0.1mL of aluminum element series control solution, blank control solution, sample solution and blank sample solution respectively, accurately add 0.9mL of derivative reagent respectively, mix evenly, then inject into the liquid chromatograph respectively, and record the chromatogram; 5) linear regression is made by corresponding the concentration of aluminum element series control solution to the peak area of the corresponding blank control solution, the linear regression equation is obtained, the peak area of the sample solution is deducted from the peak area of the blank sample solution, and the content of aluminum element in the sample is calculated.

2. The method for determining the content of impurity aluminum element in fat emulsion injection by high performance liquid chromatography according to claim 1, characterized in that, In step 1), the concentration of the aluminum element series control solution is 200μg / L, 100μg / L, 50μg / L, 25μg / L, 10μg / L and 5μg / L respectively.

3. The method for determining the content of impurity aluminum element in fat emulsion injection by high performance liquid chromatography according to claim 1, characterized in that, In step 1), the preparation method of the mobile phase comprises the following steps: according to the volume ratio, the mass percentage concentration of 0.3% 8-hydroxyquinoline acetonitrile solution: 0.2mol / L ammonium acetate solution = 45:55; take 0.3% 8-hydroxyquinoline acetonitrile solution and 0.2mol / L ammonium acetate solution, mix evenly.

4. The method for determining the content of impurity aluminum element in fat emulsion injection by high performance liquid chromatography according to claim 1, characterized in that, In step 3), the liquid chromatography conditions are: a phenyl column with phenylethyl bonded silica gel as the matrix, the column temperature is 30℃; the flow rate is 1.0mL / min; the injection volume is 100μL; the excitation wavelength of the fluorescence detector is 380nm, and the emission wavelength is 520nm.

5. The method for determining the content of impurity aluminum element in fat emulsion injection by high performance liquid chromatography according to any one of claims 1-4, characterized in that, The fat emulsion injection is fat emulsion amino acid (17) glucose (11%) fat emulsion injection or medium-long chain fat emulsion amino acid (16) glucose (16%) fat emulsion injection.

6. Use of the method according to any one of claims 1 to 4 for determining the content of the impurity aluminum in a fat emulsion injection.

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