CN114646698A - Method for detecting sodium valproate and related substances thereof in sodium valproate injection - Google Patents
Method for detecting sodium valproate and related substances thereof in sodium valproate injection Download PDFInfo
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Abstract
The invention provides a method for detecting sodium valproate and related substances in sodium valproate injection, which comprises the following steps: and carrying out gas chromatography detection on the sodium valproate injection, wherein the filler in a chromatographic column adopted by the gas chromatography detection is nitroterephthalic acid modified polyethylene glycol, the length of the chromatographic column is 20-40 mm, the inner diameter of the chromatographic column is 0.2-0.4 mm, and the film thickness of the chromatographic column is 0.4-0.6 mu m. By adopting the detection method, the linear relation between the sodium valproate injection and related substances is good, the accuracy and the precision are good, the specificity is strong, the stability is high, the separation degree is good, the reproducibility is good, the detection requirements of the related substances of the sodium valproate injection can be met, and the method can be used for quality control of the sodium valproate injection.
Description
Technical Field
The present invention relates to the field of medicine. Specifically, the invention relates to a detection method of sodium valproate and related substances thereof in a sodium valproate injection.
Background
The valproic acid sodium injection is a nitrogen-free broad-spectrum antiepileptic drug. The product has different degrees of antagonism to convulsion caused by various methods. It is effective on various epilepsy such as small seizures, myoclonic epilepsy, local seizures, grand mal epilepsy and mixed epilepsy. The mechanism of action of sodium valproate is associated with the inhibition of voltage sensitive sodium ion channels. It can inhibit metabolism of gamma-aminobutyric acid, increase accumulation of gamma-aminobutyric acid in brain, and inhibit excessive discharge and abnormal discharge diffusion of focal neuron.
The related substances are initial materials, intermediates, side reaction products, degradation impurities and the like brought in the process of synthesizing the medicament, and the quality and the safety of the medicament can be controlled by detecting the related substances. The existing domestic and foreign pharmacopoeia does not contain a detection method for related substances of the sodium valproate injection, and the detection method for related substances in the raw material medicines is not completely expressed.
Disclosure of Invention
The present invention aims to solve at least to some extent at least one of the technical problems of the prior art. The detection method has the advantages of good linear relation between the sodium valproate injection and the related substances, good accuracy and precision, strong specificity, high stability, good separation degree and good reproducibility, can meet the detection requirements of the related substances of the sodium valproate injection, and can be used for quality control of the sodium valproate injection.
In one aspect of the invention, the invention provides a method for detecting related substances in a sodium valproate injection. According to an embodiment of the invention, the method comprises: performing gas chromatography detection on the sodium valproate injection, wherein the related substances comprise at least one of the following substances: n-pentanoic acid (impurity A for short), (2RS) -2-ethylpentanoic acid (impurity B for short), (2RS) -2- (1-methylethyl) pentanoic acid (impurity C for short), 2-dipropylpentanoic acid (impurity D for short), 2-propylpentanoic acid amine (impurity F for short), (2RS) -2-ethyl-2-methylpentanoic acid (impurity K for short) and (2RS) -2-methylpentanoic acid (impurity L for short), wherein the filler in the chromatographic column adopted for gas chromatography detection is nitroterephthalic acid modified polyethylene glycol; the length of the chromatographic column is 20 to 40mm, the inner diameter is 0.2 to 0.4mm, and the thickness of the membrane is 0.4 to 0.6 μm.
When the inventor adopts the gas chromatography to detect the sodium valproate injection, the inventor finds that the sizes of the filler and the chromatographic column in the chromatographic column can obviously influence the separation effect of various related substances in the sodium valproate injection, and if the selection is not proper, the related substances can not be separated, so that the purpose of accurate separation and detection can not be realized, and the specific content is further difficult to determine. Furthermore, the inventor discovers through intensive research analysis and optimization screening that sodium valproate and related substances in the sodium valproate injection can be effectively separated and detected by using the nitroterephthalic acid modified polyethylene glycol as a filler and controlling the size of a chromatographic column. Further, the content of sodium valproate and related substances thereof can be calculated according to the obtained gas chromatogram. Therefore, the sodium valproate injection and related substances obtained by the detection method disclosed by the embodiment of the invention are good in linear relation, good in accuracy and precision, strong in specificity, high in stability and good in separation degree. The detection method has good reproducibility, can meet the detection requirements of related substances of the sodium valproate injection, and can be used for quality control of the sodium valproate injection.
According to the embodiment of the invention, the method for detecting sodium valproate and related substances in the sodium valproate injection can further have the following additional technical characteristics:
according to the embodiment of the invention, the column temperature used for the gas chromatography detection is as follows: initially 95-105 ℃, and maintaining for 1.5-2.5 min; then heating to 140-160 ℃ at the speed of 8-12 ℃/min and maintaining for 0 min; then, the temperature is increased to 180-200 ℃ at a speed of 1-3 ℃/min and maintained for 4-6 min. The inventor obtains the preferable temperature raising program through a large number of experiments, so that sodium valproate and related substances thereof can be effectively separated, and the separation degrees of the main peak and the impurity peak are both greater than 1.5, so that the accuracy of the detection result is ensured.
According to the embodiment of the invention, the temperature of the gasification chamber adopted by the gas chromatography detection is 215-225 ℃, and the temperature of the detector is 215-225 ℃. Therefore, sodium valproate and related substances thereof can be effectively separated, the separation degrees of the main peak and the impurity peak are both greater than 1.5, and the accuracy of the detection result is ensured.
According to the embodiment of the invention, the carrier adopted by the gas chromatography detection is helium, the flow rate is 6-10 mL/min, and the sample injection volume is 0.5-1.5 muL. Therefore, sodium valproate and related substances thereof can be effectively separated, the separation degrees of the main peak and the impurity peak are both greater than 1.5, and the accuracy of the detection result is ensured.
According to the embodiment of the invention, the sodium valproate injection is previously treated as follows: mixing the sodium valproate injection with water and dilute sulfuric acid to obtain a mixed solution; and extracting the mixed solution by using n-heptane, collecting an extracting solution, fixing the volume, and performing gas chromatography detection on the obtained solution to be detected. The n-heptane can be used for effectively separating sodium valproate and related substances in the sodium valproate injection, so that the subsequent gas chromatography detection of the sodium valproate injection is facilitated, and qualitative and quantitative analysis is realized.
According to the embodiment of the invention, the extraction times are 2-4 times, and the addition amount of n-heptane is 3-5 ml each time based on 1ml of the propionic acid injection. Thereby, the extraction efficiency can be effectively improved.
In another aspect of the invention, the invention provides a quality control method of the sodium valproate injection. According to an embodiment of the invention, the method comprises: carrying out gas chromatography detection on the sodium valproate injection to obtain a detection result; comparing the detection result with a threshold value, and determining whether the quality of the sodium valproate injection reaches the standard, wherein the filler in a chromatographic column adopted by the gas chromatography detection is nitroterephthalic acid modified polyethylene glycol; the related substances comprise at least one of the following substances: n-pentanoic acid, (2RS) -2-ethylpentanoic acid, (2RS) -2- (1-methylethyl) pentanoic acid, 2-dipropylpentanoic acid, 2-propylpentanoic acid amine, (2RS) -2-ethyl-2-methylpentanoic acid, and (2RS) -2-methylpentanoic acid; the length of the chromatographic column is 20-40 mm, the inner diameter is 0.2-0.4 mm, and the film thickness is 0.4-0.6 μm. Therefore, whether the sodium valproate injection meets the requirements or not can be accurately judged by adopting the method provided by the embodiment of the invention, so that the quality can be effectively controlled.
According to the embodiment of the invention, the column temperature used for the gas chromatography detection is as follows: initially 95-105 ℃, and maintaining for 1.5-2.5 min; then heating to 140-160 ℃ at the speed of 8-12 ℃/min and maintaining for 0 min; then, the temperature is increased to 180-200 ℃ at a speed of 1-3 ℃/min and maintained for 4-6 min. Therefore, sodium valproate and related substances thereof can be effectively separated, and the accuracy of a detection result is ensured.
According to the embodiment of the invention, the temperature of the gasification chamber adopted by the gas chromatography detection is 215-225 ℃, and the temperature of the detector is 215-225 ℃. Therefore, sodium valproate and related substances thereof can be effectively separated, and the accuracy of a detection result is ensured.
According to the embodiment of the invention, the carrier adopted by the gas chromatography detection is helium, the flow rate is 6-10 mL/min, and the sample injection volume is 0.5-1.5 muL. Therefore, sodium valproate and related substances thereof can be effectively separated, and the accuracy of a detection result is ensured.
According to the embodiment of the invention, the sodium valproate injection is previously treated as follows: mixing the sodium valproate injection with water and dilute sulfuric acid to obtain a mixed solution; and extracting the mixed solution by using n-heptane, collecting an extracting solution, fixing the volume, and performing gas chromatography detection on the obtained solution to be detected. The n-heptane can be used for effectively separating sodium valproate and related substances in the sodium valproate injection, so that the subsequent gas chromatography detection of the sodium valproate injection is facilitated, and qualitative and quantitative analysis is realized.
According to the embodiment of the invention, the extraction times are 2-4 times, and the addition amount of n-heptane is 3-5 ml each time based on 1ml of propionic acid injection. Thereby, the extraction efficiency can be effectively improved.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
fig. 1 shows a sample chromatogram of a sodium valproate injection according to an embodiment of the present invention;
FIG. 2 shows a chromatogram peak location diagram of sodium valproate injection and related substances according to an embodiment of the present invention;
FIG. 3 shows a linear plot of impurity A according to one embodiment of the present invention;
FIG. 4 shows a linear plot of impurity L according to one embodiment of the present invention;
FIG. 5 shows a linear plot of impurity B according to one embodiment of the present invention;
FIG. 6 shows a plot of impurity C linearity according to one embodiment of the present invention;
FIG. 7 shows a linear plot of impurity K according to one embodiment of the present invention;
FIG. 8 shows a linear plot of impurity F according to one embodiment of the present invention;
FIG. 9 shows a plot of impurity D linearity according to one embodiment of the present invention;
figure 10 shows a linear plot of sodium valproate according to one embodiment of the present invention.
Detailed Description
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
In this example, the following method was used to perform a gas phase detection method for related substances in sodium valproate injection. The method comprises the following operation steps:
1. medicine and reagent
Valproic acid sodium injection (Saimengda Daddy pharmaceutical industry Co., Ltd.), (2RS) -2-ethyl-2-methylpentanoic acid (impurity K, source: TRC), (2RS) -2- (1-methylethyl) pentanoic acid (impurity C, source: TRC), n-pentanoic acid (impurity A, source: Allantin), (2RS) -2-ethylpentanoic acid (impurity B, source: TRC), 2-dipropylpentanoic acid (impurity D, source: TLC), 2-propylpentanoic acid amine (impurity F, source: TRC), (2RS) -2-methylpentanoic acid (impurity L, source: Allantin), n-heptane (GC (pesticide residue grade), CNW), sulfuric acid (AR, Nanjing chemical reagent Co., Ltd.), hydrochloric acid (AR, Nanjing chemical reagent Co., Ltd.), (Co., Ltd.),), Sodium hydroxide (AR, General-Reagent), 30% hydrogen peroxide solution (AR, national chemical group, Inc.), and ultrapure water (made by Millipore).
2. Step (ii) of
(1) Solution preparation:
precisely transferring 5.0ml of sodium valproate injection, adding 5ml of water, adding 5ml of dilute sulfuric acid (5.7 ml of sulfuric acid is taken, and water is added to dilute the dilute sulfuric acid to 100ml) into a separating funnel, shaking, extracting for 3 times by using n-heptane, 20ml each time, combining n-heptane extracting solutions, transferring into a 100ml measuring flask, fixing the volume to the scale by using the n-heptane, and shaking uniformly to obtain a sample solution.
A test solution 1.0ml to 100ml volumetric flask is taken, and the volume is determined to the scale with n-heptane as a control solution.
Respectively taking the impurity A, the impurity L, the impurity B, the impurity C, the impurity F, the impurity D and the impurity K, adding n-heptane to dilute to obtain a solution containing 5 mu g/ml of each of the impurity A, the impurity B, the impurity C, the impurity L, the impurity D and the impurity F and 7.5 mu g/ml of the impurity K as an impurity reference substance mixed solution.
(2) Respectively injecting the test solution, the reference solution and the impurity reference substance mixed solution into a gas chromatograph, recording a chromatogram, and calculating according to a 1% self-reference method by peak area, wherein the chromatographic conditions are as follows:
a chromatographic column: nitro terephthalic acid modified polyethylene glycol for stationary liquid Agilent DB-FFAP (30m x 0.32mm x 0.5 μm) capillary chromatographic column.
Column temperature: maintaining at 100 deg.C for 2min, heating to 150 deg.C at 10 deg.C/min, and maintaining for 0 min; then raising the temperature to 190 ℃ at the speed of 2 ℃/min, and maintaining the temperature for 5 min.
Temperature of the gasification chamber: 220 ℃; detector temperature: 220 ℃; carrier gas: high purity helium gas; no shunt sampling; constant flow rate mode: the flow rate is 8 ml/min; sample introduction volume: 1 μ l.
The sample solution is shown in FIG. 1. It can be seen that the detection method of the invention can effectively realize the separation of sodium valproate and related substances thereof.
Example 2 methodological validation
2.1 specificity
An appropriate amount of an impurity control (impurity A, B, C, D, K, F, L) was measured (see preparation method in example 1), and a test solution was added to prepare a mixed solution containing about 4.3mg/ml of valproic acid, A, B, C, D, F, L5. mu.g/ml of impurity, and 7.5. mu.g/ml of impurity K per 1ml of the test solution. The solution and n-heptane were injected separately, and the gas chromatography conditions were as in example 1, and the chromatogram was as shown in FIG. 2.
The test results show that: under the chromatographic condition, the base line is stable, the solvent has no interference to the measurement of the product, the theoretical plate number of the valproic acid main peak is 148619, the separation degree of each impurity in the mixed control sample is good, each impurity can be detected, and the specificity is good.
2.2 destructive testing
In order to examine whether the degradation products possibly generated by the sodium valproate injection can be detected under the selected chromatographic conditions, the sodium valproate injection is destroyed by respectively using severe conditions such as high temperature, acid, alkali, oxidation, illumination and the like, the destroyed sample is prepared into a test solution according to the method of example 1, 1 μ l of each solution is precisely measured, the test solution is injected into a gas chromatograph, and a chromatogram is recorded, wherein the specific method is shown in table 1.
Table 1 results of the failure test
The sodium valproate injection does not generate new impurities under the conditions of alkali, acid, oxidation and illumination, the product is slightly influenced by the conditions of alkali, acid, oxidation and illumination, the original impurities can be detected after various conditions are damaged, the separation degree of each degradation product and a main peak is good, the determination recovery amount of related substances is good, and the material conservation is good, so the chromatographic condition can be used for determining the related substances of the product.
2.3 determination of limits of quantitation and detection
Samples with certain concentrations are prepared from each impurity control and the sodium valproate injection respectively, and are gradually diluted and injected with 1 mul, and the S/N-3 and S/N-10 signal-to-noise ratio are measured, and the results are shown in table 2.
TABLE 2 detection limit and quantitation limit results
2.4 sample solution stability test
Precisely transferring 5.0ml of sodium valproate injection, adding 5ml of water, adding 5ml of dilute sulfuric acid (5.7 ml of sulfuric acid, and diluting with water to 100ml) into a separating funnel, shaking, extracting with n-heptane for 3 times, 20ml each time, combining n-heptane extracting solutions, transferring into a 100ml measuring flask, adding n-heptane to constant volume to reach the scale, and shaking up to obtain a sample solution.
Precisely transferring 1.0ml to 100ml of the test solution into a volumetric flask, fixing the volume to the scale with n-heptane, and shaking up to obtain the reference solution.
Samples are respectively injected at 0h, 2h, 4h, 6h, 8h, 12h and 24h after preparation to investigate the content of each impurity and main component in the test solution, and the content is calculated by 1 percent of self contrast. The results are shown in Table 3.
TABLE 3 sample solution stability test
The results show that: the sample solution is not generated with new impurities within 14 hours, the RSD value of each impurity is good, the RSD of the main peak area is 1.8%, the RSD of the reference solution peak area is 2.2%, and the stability of the sample solution and the reference solution is good within 24 hours.
2.5 Linear
Linear solution of impurities: weighing appropriate amounts of impurity A, impurity B, impurity C, impurity L, impurity K, impurity D and impurity F respectively, precisely weighing, dissolving with n-heptane, and gradually diluting to obtain a series of mixed solutions with concentrations of 80%, 100%, 120% and 200% relative to the impurity limit (0.15% of impurity K (concentration of about 7.5 μ g/ml) and 0.1% of other impurities (concentration of about 5 μ g/ml)); and (3) taking each impurity solution to dilute the impurity solution to a quantitative limit concentration solution step by step, and taking the solution as a linear regression initial point.
Precisely transferring 5.0ml of sodium valproate injection, adding 5ml of water, adding 5ml of dilute sulfuric acid (5.7 ml of sulfuric acid is taken, and water is added to dilute the dilute sulfuric acid to 100ml) into a separating funnel, shaking, extracting with n-heptane for 3 times, 20ml each time, combining n-heptane extracting solutions, transferring into a 100ml measuring flask, fixing the volume to the scale with n-heptane, and shaking uniformly. As a test solution.
Linear valproic acid solution: the test solution is diluted step by step to prepare a series of mixed solutions which are 50%, 80%, 100%, 120% and 150% of the self control.
Precisely measuring 1 μ l of each solution with the above concentration, analyzing, recording chromatogram, performing linear regression with each solution concentration (C: μ g/ml) as abscissa and each peak area (A) as ordinate, and determining the results as shown in Table 4 and FIGS. 3-10.
TABLE 4 results of Linear examination
2.6 sample introduction precision test
Taking 100% linear solution under the linear term, continuously injecting and measuring for 6 times, and inspecting the change conditions of peak area and retention time. The results are shown in Table 5.
TABLE 5 sample introduction precision test results
The result shows that the injection precision is good.
2.7 stability of Mixed control solution
The mixed control solution was taken and measured at 0, 2, 4, 6, 8, 12, and 24 hours after preparation. The results are shown in Table 6.
TABLE 6 stability test results for impurity control solutions
The results show that the impurity control solution is relatively stable when placed at room temperature for 24 hours after being prepared.
2.8 repeatability test
Test solution: taking 5.0ml of sodium valproate injection, adding 5ml of water, adding 5ml of dilute sulfuric acid (taking 5.7ml of sulfuric acid, and adding water to dilute to 100ml to obtain the sodium valproate injection) into a separating funnel, shaking, extracting with n-heptane for 3 times, 20ml each time, combining n-heptane extracting solutions, transferring into a 100ml measuring flask, using n-heptane to fix the volume to the scale, and shaking up to obtain the sodium valproate injection.
Control solution: precisely measuring the sample solution in a volumetric flask of 1ml to 100ml, and diluting the solution to the scale to obtain the product.
Six test samples and reference solutions are prepared in parallel, and the content of each impurity in the product is calculated by peak area according to a 1% self-reference method. The results are shown in Table 7.
TABLE 7 results of the repeatability tests
The method has good repeatability.
2.9 recovery test
Precisely transferring 5.0ml of sodium valproate injection, adding 5ml of water and 5ml of dilute sulfuric acid into a separating funnel, shaking, measuring a proper amount of impurity reference substance stock solution into the separating funnel to prepare 80%, 100% and 120% solutions containing impurities relative to the limit (the limit of the impurity K is 0.15%, and the limits of other impurities are 0.1%), extracting with n-heptane for 3 times, each time 20ml, combining n-heptane extracting solutions, transferring into a 100ml measuring flask, diluting with n-heptane to scale, and shaking up; triplicate samples were prepared for each limit. Separately, 1. mu.l of the solution was measured precisely and injected into a gas chromatograph to measure the recovery rate. The results are shown in tables 8 to 14.
TABLE 8 test results on recovery of impurity A
TABLE 9 test results on the recovery of impurity L
TABLE 10 test results for impurity B recovery
TABLE 11 test results of recovery of impurity C
TABLE 12 test results for recovery of impurity K
TABLE 13 test results for recovery of impurity F
TABLE 14 test results for recovery of impurity D
2.10 intermediate precision test
Test solution: taking 5.0ml of sodium valproate injection, adding 5ml of water, adding 5ml of dilute sulfuric acid (taking 5.7ml of sulfuric acid, and adding water to dilute to 100ml to obtain the sodium valproate injection) into a separating funnel, shaking, extracting with n-heptane for 3 times, 20ml each time, combining n-heptane extracting solutions, transferring into a 100ml measuring flask, using n-heptane to fix the volume to the scale, and shaking up to obtain the sodium valproate injection.
Control solution: precisely measuring the sample solution in a volumetric flask of 1ml to 100ml, and diluting the solution to the scale to obtain the product.
Six test samples and reference solutions are prepared in parallel, the content of each impurity in the product is calculated by peak area according to a 1% self-reference method, and the result is shown in table 15.
TABLE 15 results of intermediate precision test
The test result shows that: the method has good intermediate precision.
2.11 durability examination
The invention further discusses the tolerance degree of the detection method when the chromatographic condition is slightly changed, and carries out durability test, the test solution is taken as a mixed control solution and a sample solution, the investigation items comprise initial column temperature, injection port temperature, detector temperature, chromatographic column replacement, and the durability of the method is investigated by taking the number of peaks of the mixed control solution, the number of theoretical plates of main components in the sample solution of the separation degree of the main peak and adjacent peaks and the detection content of impurities as investigation indexes.
2.11.1 Change in the number of extractions
According to a solution preparation mode: taking 5.0ml of sodium valproate injection, adding 5ml of water, adding 5ml of dilute sulfuric acid (taking 5.7ml of sulfuric acid, and adding water to dilute to 100ml to obtain the sodium valproate injection) into a separating funnel, shaking, extracting with n-heptane for 3 times, 20ml each time, combining n-heptane extracting solutions, transferring into a 100ml measuring flask, using n-heptane to fix the volume to the scale, and shaking up to obtain the sodium valproate injection. The detection of impurities was examined 2 times, 3 times, and 4 times.
The experimental results show that: the detection results of the related substances of the sodium valproate injection are not obviously changed after 2, 3 and 4 times of extraction. The results are shown in Table 16.
TABLE 16 durability test (variation of the number of extractions test)
2.11.2 extraction Using changes in volume of n-heptane
According to a solution preparation mode: taking 5.0ml of sodium valproate injection, adding 5ml of water, adding 5ml of dilute sulfuric acid (taking 5.7ml of sulfuric acid, and adding water to dilute to 100ml to obtain the sodium valproate injection) into a separating funnel, shaking, extracting with n-heptane for 3 times, 20ml each time, combining n-heptane extracting solutions, transferring into a 100ml measuring flask, using n-heptane to fix the volume to the scale, and shaking up to obtain the sodium valproate injection. The detection of impurities was examined when n-heptane was used in the extraction in a volume of 15ml, 20ml or 25 ml.
The experimental results show that: when the volume of the n-heptane used for extraction is 15ml, 20ml and 25ml, the detection result of related substances in the sodium valproate injection does not change obviously. The results are shown in Table 17.
TABLE 17 durability test (extraction using n-heptane volume)
2.11.3 Change in column temperature
Other gas chromatography conditions were the same as those described above, and the degrees of separation of the main peak from adjacent peaks, the number of theoretical plates of the main component in the sample solution, the retention time of the main component, and the change in impurity detection were examined at column temperatures of 95 ℃, 100 ℃, and 105 ℃. The experimental results show that: when the column temperature is changed within the range of 95-105 ℃, the influence on each parameter is not great. The results are shown in Table 18.
TABLE 18 durability examination (column temperature)
2.11.4 variation of injection port temperature
Other gas chromatography conditions were the same as those described above, and the degrees of separation of the main peak from adjacent peaks, the number of theoretical plates of the main component in the sample solution, the retention time of the main component, and the change in impurity detection were examined at the inlet temperatures of 215 ℃, 220 ℃, and 225 ℃. The experimental results show that: when the sample inlet is changed within the range of 215-225 ℃, the influence on each parameter is not great. The results are shown in Table 19.
TABLE 19 durability examination (sample inlet temperature)
2.11.5 change in detector temperature
Other gas chromatography conditions were the same as those described above, and the degrees of separation of the main peak from adjacent peaks, the number of theoretical plates of the main component in the sample solution, the retention time of the main component, and the change in impurity detection were examined at detector temperatures of 215 ℃, 220 ℃, and 225 ℃. The experimental results show that: when the detector is changed within the range of 215-225 ℃, the influence on each parameter is not great. The results are shown in Table 20.
TABLE 20 durability examination (detector temperature)
2.11.6 variations of the column
Other gas chromatography conditions were the same as those described above, and the degrees of separation of the main peak from adjacent peaks, the number of theoretical plates of the main component in the sample solution, the retention time of the main component, and the change in impurity detection were examined. The experimental results show that: different chromatographic columns are used, and the influence on each parameter is small. The results are shown in Table 21.
TABLE 21 durability examination (different columns)
2.12 determination of the measurement method of the relevant substance
The methodology verification shows that the preliminarily formulated method is suitable for the inspection of related substances of the product.
Taking 5ml of the product, placing the product in a separating funnel, adding 5ml of water, adding 5ml of dilute sulfuric acid, shaking, extracting with n-heptane for 3 times, 20ml each time, combining n-heptane liquids, placing in a 100ml measuring flask, diluting with n-heptane to scale, shaking up, precisely measuring 1ml as a sample solution, placing in a 100ml measuring flask, diluting with n-heptane to scale, shaking up to serve as a control solution. Taking a proper amount of valproic acid system applicability solution containing the impurity I, and quantitatively diluting with n-heptane to prepare a solution containing 5mg per 1ml as the system applicability solution. Performing gas chromatography (0521 of the four ministry of general regulations of the national pharmacopoeia 2015 edition) test on a capillary chromatographic column (Agilent DB-FFAP 30m multiplied by 0.32mm multiplied by 0.5 mu m) taking nitroterephthalic acid modified polyethylene glycol as a stationary liquid, wherein the initial temperature is 100 ℃, the capillary chromatographic column is maintained for 2 minutes, the temperature is increased to 150 ℃ at the rate of 10 ℃ per minute, and then is increased to 190 ℃ at the rate of 2 ℃ per minute, and the capillary chromatographic column is maintained for 5 minutes; the temperature of a sample inlet is 220 ℃; the temperature of the detector is 220 ℃; the flow rate was 1ml per minute; the carrier gas was helium. Injecting 0.5 mu l of system applicability solution into a gas chromatograph, and recording a chromatogram, wherein the relative retention time of the impurity I is about 0.95, and the separation degree of the impurity I and valproic acid is not lower than 2.0; precisely measuring 0.5 μ l of the reference solution and the sample solution, injecting into a gas chromatograph, and recording chromatogram. If an impurity peak exists in a chromatogram of a test solution, the peak area of the impurity I is not more than 0.15 times (0.15) of the main peak area of the control solution, the peak area of other single impurities is not more than 0.1 times (0.1) of the main peak area of the control solution, the sum of the peak areas of the impurities is not more than 0.3 times (0.3) of the main peak area of the control solution, and chromatographic peaks which are less than 0.03 times of the main peak area of the control solution are ignored.
The impurity content was calculated according to the following formula:
(1) content (%) of single impurity ═ aSimple impurity/AControl
ASimple impurity: peak area of impurities in sample solution
AControl: area of valproic acid peak in self control
(2)General assemblyContent (%) of hetero ═ aTotal miscellaneous/AControl
ATotal miscellaneous: sum of impurity peak areas in sample solution
The results of the tests on the test samples by this method are shown in Table 22.
Table 22 examination results of substances relevant to the test
Comparative example 1
In this comparative example, sodium valproate injection and related substances thereof were tested as follows:
1. solution preparation
The same as in example 1.
2. Respectively injecting the test solution, the reference solution and the impurity reference substance mixed solution into a gas chromatograph, recording a chromatogram, and calculating according to a 1% self-reference method by peak area, wherein the chromatographic conditions are as follows:
a chromatographic column: nitro terephthalic acid modified polyethylene glycol as a stationary liquid Agilent DB-FFAP (30m x 0.32mm x 0.5 μm) capillary chromatographic column.
Column temperature: the temperature program is as follows in Table 23.
Temperature of the gasification chamber: 220 ℃; temperature of the detector: 220 ℃; carrier gas: high purity helium gas; no shunt sampling; constant flow rate mode: the flow rate is 8 ml/min; sample injection volume: 1 μ l.
See table 24 for the results of the measurements. It can be seen that, by adopting the column temperature rise program, the separation degree R of the impurity C and the impurity K is less than 1.5, and the baseline separation is not achieved, so that the accurate determination of the impurity K cannot be realized.
TABLE 23 Effect of different warming procedures
| Method | Rate (. degree.C./min) | Temperature (. degree.C.) | Retention time (min) |
| Initial value | / | 80 | 5 |
| Gradient 1 | 7 | 150 | 0 |
| Gradient 2 | 3 | 190 | 30 |
TABLE 24 results of comparative example 1
Comparative example 2
In this comparative example, sodium valproate injection and related substances thereof were tested as follows:
1. solution preparation
The same as in example 1.
2. Respectively injecting the test solution, the reference solution and the impurity reference substance mixed solution into a gas chromatograph, recording a chromatogram, and calculating according to a 1% self-reference method by peak area, wherein the chromatographic conditions are as follows:
a chromatographic column: nitro terephthalic acid modified polyethylene glycol as a stationary liquid Agilent DB-FFAP (30m x 0.53mm x 1.0 μm) capillary chromatographic column.
Column temperature: maintaining at 100 deg.C for 2min, heating to 150 deg.C at 10 deg.C/min, and maintaining for 0 min; then raising the temperature to 190 ℃ at the speed of 2 ℃/min, and maintaining the temperature for 5 min.
Temperature of the gasification chamber: 220 ℃; temperature of the detector: 220 ℃; carrier gas: high purity helium gas; no shunt sampling; constant flow rate mode: the flow rate is 8 ml/min; sample introduction volume: 1 μ l.
The results are shown in Table 25. It can be seen that with the above size column, the degree of separation R of impurity C and impurity K is less than 1.5, and baseline separation is not achieved.
TABLE 25 results of comparative example 2
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (10)
1. A method for detecting related substances in a sodium valproate injection is characterized by comprising the following steps:
performing gas chromatography detection on the sodium valproate injection, wherein the related substances comprise at least one of the following substances: n-pentanoic acid, (2RS) -2-ethylpentanoic acid, (2RS) -2- (1-methylethyl) pentanoic acid, 2-dipropylpentanoic acid, 2-propylpentanoic acid amine, (2RS) -2-ethyl-2-methylpentanoic acid and (2RS) -2-methylpentanoic acid,
wherein, the filler in the chromatographic column adopted by the gas chromatography detection is nitroterephthalic acid modified polyethylene glycol;
the length of the chromatographic column is 20-40 mm, the inner diameter is 0.2-0.4 mm, and the film thickness is 0.4-0.6 μm.
2. The method of claim 1, wherein the gas chromatography assay uses column temperatures as follows: initially 95-105 ℃, and maintaining for 1.5-2.5 min; then heating to 140-160 ℃ at the speed of 8-12 ℃/min and maintaining for 0 min; then, the temperature is increased to 180-200 ℃ at a speed of 1-3 ℃/min and maintained for 4-6 min.
3. The method of claim 1, wherein the gas chromatography detection uses a vaporizer temperature of 215-225 ℃ and a detector temperature of 215-225 ℃.
4. The method according to claim 1, wherein the carrier used for gas chromatography detection is helium, the flow rate is 6-10 mL/min, and the sample injection volume is 0.5-1.5 μ L.
5. The method of claim 1, wherein the sodium valproate injection is pre-treated as follows:
mixing the sodium valproate injection with water and dilute sulfuric acid to obtain a mixed solution;
and extracting the mixed solution by using n-heptane, collecting an extracting solution, fixing the volume, and performing gas chromatography detection on the obtained solution to be detected.
6. The method according to claim 5, wherein the number of the extractions is 2-4, and the amount of n-heptane added per time is 3-5 ml based on 1ml of the propionic acid injection.
7. A quality control method of a sodium valproate injection is characterized by comprising the following steps:
carrying out gas chromatography detection on the sodium valproate injection to obtain a detection result;
comparing the detection result with a threshold value to determine whether the quality of the sodium valproate injection reaches the standard or not,
wherein, the filler in the chromatographic column adopted by the gas chromatography detection is nitroterephthalic acid modified polyethylene glycol;
the related substances comprise at least one of the following substances: n-pentanoic acid, (2RS) -2-ethylpentanoic acid, (2RS) -2- (1-methylethyl) pentanoic acid, 2-dipropylpentanoic acid, 2-propylpentanoic acid amine, (2RS) -2-ethyl-2-methylpentanoic acid, and (2RS) -2-methylpentanoic acid;
the length of the chromatographic column is 20-40 mm, the inner diameter is 0.2-0.4 mm, and the film thickness is 0.4-0.6 μm.
8. The method of claim 7, wherein the gas chromatography detection uses column temperatures as follows: initially 95-105 ℃, and maintaining for 1.5-2.5 min; then heating to 140-160 ℃ at the speed of 8-12 ℃/min and maintaining for 0 min; then, the temperature is increased to 180-200 ℃ at a speed of 1-3 ℃/min and maintained for 4-6 min.
9. The method according to claim 7, wherein the gas chromatography detection adopts a gasification chamber with a temperature of 215-225 ℃ and a detector with a temperature of 215-225 ℃;
optionally, the carrier used for gas chromatography detection is helium, the flow rate is 6-10 mL/min, and the sample injection volume is 0.5-1.5 muL.
10. The method of claim 7, wherein the sodium valproate injection is pre-treated as follows:
mixing the sodium valproate injection with water and dilute sulfuric acid to obtain a mixed solution;
extracting the mixed solution with n-heptane, collecting the extract, fixing the volume, and performing gas chromatography detection on the obtained solution to be detected;
optionally, the number of times of extraction is 2-4;
optionally, the addition amount of the n-heptane is 3-5 ml based on 1ml of the propionic acid injection.
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