A method for detecting triethyl foscarnet in sodium foscarnet
The detection of triethyl phosphonate in sodium phosphonate was solved by liquid chromatography, and the problem of degradation of triethyl phosphonate in gas chromatography was achieved, stable and accurate detection was achieved, cost reduction, and drug safety was ensured.
Patent Information
- Application Number
- CN202510058015.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-14
- Publication Date
- 2025-04-04
AI Technical Summary
When the existing gas chromatography is used to detect the content of triethyl phosphonium in sodium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium phosphonium
The liquid chromatography detection method is used to achieve stable detection of triethyl phosphonate by selecting the appropriate chromatographic column and mobile phase combination. The specific method includes using a Waters C18 chromatography column, the mobile phase is a mixture of anhydrous sodium sulfate, anhydrous sodium acetate, sodium pyrophosphate dehydrate, glacial acetic acid, tetrahexyl ammonium bisulfate and methanol, with a flow rate of 0.8 to 1.2 ml/min, a column temperature of 28 to 32°C, and a detection wavelength of 230 nm.
The stable detection of triethyl phosphonate is achieved, which avoids degradation problems, reduces detection costs, and can accurately control the residual risk of triethyl phosphonate in sodium phosphonate products to ensure the safety of the drug.
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Figure CN119757606A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of drug analysis and detection, and in particular to a method for detecting triethyl foscarnet in sodium foscarnet. Background Art
[0002] In the process of synthesizing sodium foscarnet, triethyl foscarnet is basically used as a raw material. However, according to the ICHM7 (Evaluation and Control of Mutagenic Impurities) guidelines, triethyl foscarnet is a potential mutagenic impurity with potential safety risks, so it is of great significance to control the content of triethyl foscarnet in sodium foscarnet.
[0003] At present, foreign pharmacopoeias USP43 and EP10.0 both use gas chromatography to detect the content of triethyl phosphonoformate in sodium phosphonoformate, and helium is used as the carrier gas, but the detection cost of this method is relatively high. If the carrier gas is replaced with nitrogen for cost considerations, the problem of "triethyl phosphonoformate rapidly degrades during the detection process" will occur, as shown in Table 1, making it impossible to detect.
[0004] Table 1 Stability of triethyl phosphonoformate under nitrogen conditions
[0005]
[0006] Based on this, a new method for detecting triethyl phosphonoformate is urgently needed. Summary of the invention
[0007] The technical problem to be solved by the present invention is to provide a new method for detecting triethyl foscarnet in sodium foscarnet. The method is simple, fast, low in cost, and can achieve the stability of the triethyl foscarnet component. Based on the method, the present invention can effectively control the residual risk of triethyl foscarnet in the sodium foscarnet product and ensure the safety of the drug.
[0008] The structural information of sodium phosphonoformate and triethyl phosphonoformate of the present invention is shown in Table 2.
[0009] Table 2 Structural information of sodium foscarnet and triethyl foscarnet
[0010]
[0011] The present invention adopts the following technical solutions to solve the above technical problems:
[0012] The invention discloses a method for detecting triethyl phosphonoformate in sodium phosphonoformate, which adopts a liquid chromatography detection method, and the chromatographic conditions are as follows: a Waters C18 chromatographic column with a specification of 4.6×100 mm and 3.5 μm is adopted; octadecylsilane bonded silica gel is used as a filler; a mixed liquid of anhydrous sodium sulfate, anhydrous sodium acetate, sodium pyrophosphate decahydrate, glacial acetic acid, tetrahexylammonium hydrogen sulfate and methanol is used as a mobile phase; the flow rate is 0.8-1.2 ml / min; the column temperature is 28-32° C.; the detection wavelength is 230 nm; and the injection volume is 40 μl.
[0013] As one of the preferred embodiments of the present invention, the mobile phase is prepared as follows: 1.42 g of anhydrous sodium sulfate, 1.23 g of anhydrous sodium acetate, 0.268 g of sodium pyrophosphate decahydrate and 3 ml of glacial acetic acid are mixed, water is added to 1000 ml, and then 0.25 g of tetrahexylammonium hydrogen sulfate and 100 ml of methanol are added.
[0014] As one of the preferred embodiments of the present invention, the method comprises the following specific steps:
[0015] (1) Solution preparation
[0016] Diluent: glacial acetic acid;
[0017] Test solution: Take the sodium foscarnet raw material to be tested and dissolve it in a diluent for preparation;
[0018] Reference solution: prepare triethyl phosphonoformate by dissolving it in a diluent;
[0019] (2) Liquid chromatography analysis
[0020] The liquid chromatography was used for detection. The chromatographic conditions were as follows: a Waters C18 column with a specification of 4.6×100 mm and 3.5 μm was used; octadecylsilane bonded silica gel was used as a filler; a mixture of anhydrous sodium sulfate, anhydrous sodium acetate, sodium pyrophosphate decahydrate, glacial acetic acid, tetrahexylammonium hydrogen sulfate and methanol was used as a mobile phase; a flow rate of 0.8 to 1.2 ml / min; a column temperature of 28 to 32°C; a detection wavelength of 230 nm; and an injection volume of 40 μl.
[0021] Take the test solution and the reference solution, inject them separately, record the chromatograms, and calculate the content of each component.
[0022] As one of the preferred embodiments of the present invention, in step (1), the diluent is 10% glacial acetic acid.
[0023] As one of the preferred embodiments of the present invention, in step (1), the concentration of the test solution is 2.5 mg / ml.
[0024] As one of the preferred embodiments of the present invention, in step (1), the concentration of the reference solution is 5 μg / ml.
[0025] As one of the preferred embodiments of the present invention, in step (2), a LC-20A high performance liquid chromatograph produced by Shimadzu Corporation of Japan is used.
[0026] As one of the preferred embodiments of the present invention, in step (2), the content of each component is calculated according to the external standard method.
[0027] The advantages of the present invention compared to the prior art are:
[0028] (1) The present invention can achieve good stability of triethyl foscarnet by selecting appropriate liquid chromatography conditions, and is not interfered by sodium foscarnet and other impurities in sodium foscarnet, thereby accurately detecting the content of triethyl foscarnet; based on this method, the present invention can effectively control the residual risk of triethyl foscarnet in sodium foscarnet products and ensure the safety of the drug;
[0029] (2) The detection method provided by the present invention has strong specificity and good durability. Changing the chromatographic conditions such as column flow rate and column temperature within a small range will not affect the detection of triethyl phosphonoformate. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 is the UV scanning image of triethyl phosphonoformate in Experimental Example 1;
[0031] Figure 2 is the system applicability result diagram in Experimental Example 1;
[0032] Figure 3 This is the linear relationship diagram of triethyl phosphonoformate in Experimental Example 1. DETAILED DESCRIPTION
[0033] The embodiments of the present invention are described in detail below. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, the raw materials, reagents, equipment and methods used in the following embodiments are conventional raw materials, reagents, equipment and methods in the art unless otherwise specified, and will not be repeated.
[0034] Example 1
[0035] The method for detecting triethyl phosphonoformate in sodium phosphonoformate of the present embodiment adopts liquid chromatography detection method, and includes the following steps:
[0036] (1) Solution preparation
[0037] Diluent: 10% glacial acetic acid.
[0038] Test solution: Dissolve the sodium foscarnet material to be tested in 10% glacial acetic acid to prepare a solution containing 2.5 mg of sodium foscarnet per 1 ml.
[0039] Reference solution: Take triethyl phosphonoformate and dissolve it in 10% glacial acetic acid to prepare a solution containing 5 μg of triethyl phosphonoformate per 1 ml.
[0040] (2) Liquid chromatography analysis
[0041] The detection was performed using a liquid chromatograph (LC-20A, Shimadzu, Japan), and the chromatographic conditions were:
[0042] A Waters C18 chromatographic column was used with specifications of 4.6×100mm, 3.5μm; octadecylsilane bonded silica was used as the filler; anhydrous sodium sulfate, anhydrous sodium acetate, sodium pyrophosphate decahydrate, glacial acetic acid, tetrahexylammonium hydrogen sulfate and methanol mixture was used as the mobile phase: first 1.42g of anhydrous sodium sulfate, 1.23g of anhydrous sodium acetate, 0.268g of sodium pyrophosphate decahydrate and 3ml of glacial acetic acid were mixed, water was added to 1000ml, and then 0.25g of tetrahexylammonium hydrogen sulfate and 100ml of methanol were added; the flow rate was 1.0ml / min; the column temperature was 30℃; the detection wavelength was 230nm; the injection volume was 40μl.
[0043] Take the test solution and the reference solution, inject them separately under the above-mentioned chromatographic conditions, and record the chromatograms. If there is a chromatographic peak in the chromatogram of the test solution with the same retention time as the triethyl phosphonoformate peak, calculate the peak area according to the external standard method.
[0044] Example 2
[0045] The method for detecting triethyl foscarnet in sodium foscarnet in this embodiment is basically the same as that in Example 1, except that the flow rate is 0.8 ml / min.
[0046] Example 3
[0047] The method for detecting triethyl foscarnet in sodium foscarnet in this embodiment is basically the same as that in Example 1, except that the flow rate is 1.2 ml / min.
[0048] Example 4
[0049] The method for detecting triethyl phosphonoformate in sodium phosphonoformate in this embodiment is basically the same as that in Example 1, except that the column temperature is 28°C.
[0050] Example 5
[0051] The method for detecting triethyl phosphonoformate in sodium phosphonoformate in this embodiment is basically the same as that in Example 1, except that the column temperature is 32°C.
[0052] Experimental Example 1
[0053] This experimental example is a methodological verification of the method of the present invention.
[0054] 1. Maximum absorption wavelength
[0055] The instrument and chromatographic conditions were the same as those in Example 1.
[0056] Take triethyl phosphonoformate, dissolve it with 10% glacial acetic acid, prepare a solution containing 5 μg triethyl phosphonoformate per 1 ml, scan at a wavelength of 200nm to 400nm, and the result shows that this product has a maximum absorption at 239nm. According to the chromatographic conditions of related substances, 230nm is selected as the detection wavelength. The results are shown in Figure 1 .
[0057] 2. System suitability test
[0058] The instrument and chromatographic conditions were the same as those in Example 1.
[0059] Blank solvent: 10% glacial acetic acid.
[0060] System suitability solution: Accurately weigh the sodium foscarnet raw material and the triethyl foscarnet reference substance, place them in the same volumetric flask, add 10% glacial acetic acid to dissolve and dilute to 2.5 mg sodium foscarnet and 5 μg triethyl foscarnet per 1 ml.
[0061] Take the system suitability solution and inject it into the liquid chromatograph, inject a single sample 6 times in succession (injection volume 40μl), and record the chromatogram ( Figure 2 ). Among them, the separation degree of sodium phosphonoformate and triethyl phosphonoformate meets the requirements, as shown in Table 3.
[0062] Table 3 System suitability data
[0063]
[0064]
[0065] Depend on Figure 2 As shown in the results of Table 3, the separation degrees of sodium foscarnet and triethyl foscarnet were both greater than 2.0, and the tailing factors of triethyl foscarnet were all less than 2.0; the RSDs of the peak areas were all less than 2.0%; and the RSDs of the retention times were all less than 1.0%, indicating that the system applicability of this method was good.
[0066] 3. Durability test
[0067] Referring to the method of Example 1, the durability of the chromatographic conditions was investigated by changing the flow rate and column temperature within a certain range, providing a basis for determining the chromatographic conditions. The tailing factor of each impurity peak shall not be greater than 2.0, the impurity peak and other peaks must be baseline separated, and the area RSD under each condition shall not be greater than 2.0%.
[0068] ① Different flow rates:
[0069] The flow rate was changed to 0.8ml / min, 1.0ml / min, and 1.2ml / min to examine the tolerance of this method to flow rate changes.
[0070] ② Different column temperatures:
[0071] The column temperature was changed to 28℃, 30℃, and 32℃ to investigate the tolerance of this method to column temperature changes.
[0072] Take the above system suitability sample solution, change the flow rate and column temperature, accurately measure 40 μl and inject it into the liquid chromatograph, record the chromatogram, and the data results are shown in Table 4.
[0073] Table 4 Durability test results
[0074]
[0075]
[0076] From the above results, it can be seen that by changing the flow rate and column temperature in a small range, the separation degree of sodium foscarnet and triethyl foscarnet meets the requirements, indicating that different flow rates and column temperatures have little effect on the chromatographic conditions.
[0077] 4. Column Selection Specificity
[0078] Referring to the method of Example 1, the experiments were carried out using Shiseido (4.6×150 mm) and Waters C18 (4.6×100 mm, 3.5) chromatographic columns respectively. The tailing factor of each impurity peak shall not be greater than 2.0, the impurity peak and other peaks must be baseline separated, and the area RSD under each condition shall not be greater than 2.0%.
[0079] Take the above system suitability sample solution, accurately measure 40 μl and inject it into the liquid chromatograph, record the chromatogram, and the data results are shown in Table 5.
[0080] Table 5 Test results of different chromatographic columns
[0081]
[0082] From the above results, it can be seen that for the detection of triethyl phosphonoformate in sodium phosphonoformate, based on specific chromatographic conditions, the Waters C18 (4.6×100mm, 3.5) chromatographic column has the best effect.
[0083] 5. Solution stability
[0084] Reference solution: Take triethyl phosphonoformate, dissolve it in 10% glacial acetic acid and dilute it into a solution containing 5 μg of triethyl phosphonoformate per 1 ml.
[0085] Test solution: Accurately weigh the sodium foscarnet raw material, dissolve it in 10% glacial acetic acid and dilute it into a solution containing 2.5 mg of sodium foscarnet per 1 ml.
[0086] Referring to the method of Example 1, 40 μl was injected at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h and 12 h, the peak area was measured and the solution stability was investigated. The results are shown in Tables 6 and 7.
[0087] Table 6 Stability of reference solution
[0088]
[0089] As can be seen from Table 6, the control solution was basically stable within 12 hours, and the RSD of the peak area of triethyl phosphonoformate was 3.85%, indicating that the control solution had good stability.
[0090] Table 7 Stability of raw material solution
[0091]
[0092] As can be seen from Table 7, the raw material solution was basically stable within 12 hours, and triethyl phosphonoformate was not detected, indicating that the raw material solution had good stability.
[0093] 6. Linear relationship test
[0094] Accurately measure triethyl phosphonoformate and use 10% glacial acetic acid solution to prepare solutions containing triethyl phosphonoformate at concentrations of ①1.06μg / ml, ②2.66μg / ml, ③5.32μg / ml, ④7.98μg / ml, and ⑤10.64μg / ml, respectively. Accurately pipette 40μl of each solution and inject it into the liquid chromatograph. Record the chromatogram and draw a standard curve with peak area as the ordinate and concentration as the abscissa. The results are shown in Figure 3 , Table 8.
[0095] Table 8 Linear relationship test results
[0096] Concentration (μg / ml) 1.06 2.66 5.32 7.98 10.64 Peak area (A) 1670 5063 10013 15030 20157
[0097] From the above results, it can be seen that triethyl phosphonoformate has a good linear relationship in the range of 1.06 μg / ml to 10.64 μg / ml. The equation is: y = 1915.6x-212.02, R 2 =0.9997.
[0098] 7. Limit of Quantitation and Precision of Limit of Quantitation
[0099] Accurately measure the solution under the linear item, add 10% glacial acetic acid to dilute it step by step. When the S / N is about 10, the corresponding concentration is the quantitative limit of the substance. The sample is injected 6 times continuously, and the chromatogram is recorded to examine the precision of the quantitative limit. The results are shown in Tables 9-10.
[0100] Table 9 Limit of Quantification of Triethyl Foscarnet
[0101] sample Triethyl phosphonoformate Limit of quantification concentration (μg / ml) 0.665 Limit of quantification (ng) 26.6 S / N 10.65 Equivalent to test sample concentration (%) 0.03
[0102] Table 10 Precision limit of quantification of triethyl phosphonoformate
[0103] serial number 1 2 3 4 5 6 RSD(%) Peak area 1316 1637 1449 1699 1377 1524 9.91
[0104] From the above results, it can be seen that the precision of the quantitative limit of triethyl phosphonoformate is good, with an RSD of 9.91%.
[0105] 8. Detection Limit
[0106] Accurately measure an appropriate amount of the solution below the quantitative limit and dilute it gradually with 10% glacial acetic acid. Inject the sample according to the above method and record the chromatogram. When the S / N is about 3, the corresponding concentration is the detection limit of the substance. The results are shown in Table 11.
[0107] Table 11 Detection limit of triethyl phosphonoformate
[0108]
[0109]
[0110] From the above results, we can see that the detection limit of this method is 8.08ng, and the sensitivity is relatively high.
[0111] IX. Intermediate Precision
[0112] Reference solution: Accurately weigh the triethyl phosphonoformate reference substance, dissolve it in 10% glacial acetic acid and dilute it into a solution containing approximately 5 μg of triethyl phosphonoformate per 1 ml.
[0113] Test solution: Accurately weigh the sodium foscarnet raw material, dissolve it with 10% glacial acetic acid and dilute it into a solution containing 2.5 mg of sodium foscarnet per 1 ml; prepare 6 portions of test solution according to the method.
[0114] According to the above chromatographic conditions and detection methods, the chromatogram was recorded, and the content of triethyl phosphonoformate in 6 samples was calculated by external standard method, and the RSD of the content was statistically analyzed. Different personnel tested the samples and the reference substance, and the rest were different. The content was determined by external standard method, and combined with the results of 6 replicates, the intermediate precision was investigated. The results are shown in Tables 12 to 13.
[0115] Table 12 Intermediate precision results 1
[0116] NO. Triethyl phosphonoformate (%) 1 Not detected 2 Not detected 3 Not detected 4 Not detected 5 Not detected 6 Not detected Average content (%) - RSD(%) -
[0117] Table 13 Intermediate precision results 2
[0118]
[0119] As can be seen from the table above, triethyl phosphonoformate was not detected in 6 bulk drugs, indicating that the repeatability of this method is good. Triethyl phosphonoformate was not detected in 12 samples, indicating that the intermediate precision of this method is good.
[0120] 10. Recovery rate
[0121] Reference solution: Take triethyl phosphonoformate and dissolve it in 10% glacial acetic acid to prepare a solution containing 5 μg of triethyl phosphonoformate per 1 ml.
[0122] 50% recovery solution: Take appropriate amount of sodium foscarnet raw material and triethyl foscarnet reference stock solution, dissolve and dilute with 10% glacial acetic acid to a mixed solution containing 2.5 μg triethyl foscarnet and 2.5 mg sodium foscarnet per 1 ml of solution. Prepare 3 portions in the same way.
[0123] 100% recovery solution: Take appropriate amount of sodium foscarnet raw material and triethyl foscarnet reference stock solution, dissolve and dilute with 10% glacial acetic acid to a mixed solution containing 5 μg triethyl foscarnet and 2.5 mg sodium foscarnet per 1 ml of solution. Prepare 3 portions in the same way.
[0124] 150% recovery solution: Take appropriate amount of sodium foscarnet raw material and triethyl foscarnet reference stock solution, dissolve and dilute with 10% glacial acetic acid to a mixed solution containing 7.5 μg triethyl foscarnet and 2.5 mg sodium foscarnet per 1 ml of solution. Prepare 3 portions in the same way.
[0125] According to the above chromatographic conditions and detection method, 40 μl of each of the above solutions was taken, and the content of triethyl phosphonoformate in each test sample was calculated by peak area according to the external standard method, and the recovery rate was calculated. The results are shown in Table 14.
[0126] Table 14 Recovery
[0127]
[0128] From the above results, it can be seen that the recovery rates of triethyl phosphonoformate are between 98.15% and 104.31%, the average recovery rate is 100.34%, and the RSD is 1.86%, indicating that the recovery rate is good.
[0129] In summary, the detection method provided by the present invention has strong specificity and good durability. Changing chromatographic conditions such as column flow rate and column temperature within a small range will not affect the detection of triethyl phosphonoformate; the method provided by the present invention can accurately detect the triethyl phosphonoformate impurity in sodium phosphonoformate, the separation degree between sodium phosphonoformate and triethyl phosphonoformate is good, the detection result is accurate and reliable, and the quality of sodium phosphonoformate can be controlled more scientifically.
[0130] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for detecting triethyl foscarnet in sodium foscarnet, characterized in that, Liquid chromatography detection method was adopted, and the chromatographic conditions were as follows: Waters C18 chromatographic column with specifications of 4.6×100mm, 3.5μm; octadecylsilane bonded silica gel was used as filler; a mixture of anhydrous sodium sulfate, anhydrous sodium acetate, sodium pyrophosphate decahydrate, glacial acetic acid, tetrahexylammonium hydrogen sulfate and methanol was used as mobile phase; flow rate was 0.8-1.2ml / min; column temperature was 28-32℃; detection wavelength was 230nm; injection volume was 40μl.
2. The method for detecting triethyl foscarnet in sodium foscarnet according to claim 1, characterized in that, The mobile phase was prepared by first mixing 1.42 g of anhydrous sodium sulfate, 1.23 g of anhydrous sodium acetate, 0.268 g of sodium pyrophosphate decahydrate and 3 ml of glacial acetic acid, adding water to 1000 ml, and then adding 0.25 g of tetrahexylammonium hydrogen sulfate and 100 ml of methanol.
3. The method for detecting triethyl foscarnet in sodium foscarnet according to claim 1 or 2, characterized in that, The specific steps include: (1) Solution preparation Diluent: glacial acetic acid; Test solution: Take the sodium foscarnet raw material to be tested and dissolve it in a diluent for preparation; Reference solution: prepare triethyl phosphonoformate by dissolving it in a diluent; (2) Liquid chromatography analysis The liquid chromatography was used for detection. The chromatographic conditions were as follows: a Waters C18 column with a specification of 4.6×100 mm and 3.5 μm was used; octadecylsilane bonded silica gel was used as a filler; a mixture of anhydrous sodium sulfate, anhydrous sodium acetate, sodium pyrophosphate decahydrate, glacial acetic acid, tetrahexylammonium hydrogen sulfate and methanol was used as a mobile phase; a flow rate of 0.8 to 1.2 ml / min; a column temperature of 28 to 32°C; a detection wavelength of 230 nm; and an injection volume of 40 μl. Take the test solution and the reference solution, inject them separately, record the chromatograms, and calculate the content of each component.
4. The method for detecting triethyl foscarnet in sodium foscarnet according to claim 3, characterized in that, In the step (1), the diluent is 10% glacial acetic acid.
5. The method for detecting triethyl foscarnet in sodium foscarnet according to claim 3, characterized in that, In the step (1), the concentration of the test solution is 2.5 mg / ml.
6. The method for detecting triethyl foscarnet in sodium foscarnet according to claim 3, characterized in that, In the step (1), the concentration of the reference solution is 5 μg / ml.
7. The method for detecting triethyl foscarnet in sodium foscarnet according to claim 3, characterized in that, In the step (2), a LC-20A high performance liquid chromatograph produced by Shimadzu Corporation of Japan is used.
8. The method for detecting triethyl foscarnet in sodium foscarnet according to claim 3, characterized in that, In the step (2), the content of each component is calculated according to the external standard method.