Method for separating and detecting ethyl valerate and impurities thereof

Abstract

The invention relates to a method for separating and detecting ethyl valerate and impurities thereof, and belongs to the technical field of quality control of ethyl valerate. The invention provides a method for separating and detecting ethyl valerate and impurities thereof, which adopts a gas chromatographic method, and adopts a capillary column with nitro terephthalic acid esterified polyethylene glycol as a stationary liquid as a chromatographic column; temperature programming is adopted, and the temperature raising program is as follows: the initial temperature is 30-50 DEG C and is maintained for 5-10 minutes, and the temperature is raised to 180-220 DEG C at the speed of 10-30 DEG C per minute and is maintained for 2-10 minutes. Compared with the prior art, the method has the advantages that effective separation and detection of the ethyl valerate and impurities of the ethyl valerate such as valeric acid, ethanol, ethyl n-butyrate, ethyl isovalerate, ethyl n-hexanoate, ethyl n-propionate and ethyl acetate can be realized at the same time, the quality control efficiency is improved, the analysis cost is saved, the separation degree and the sensitivity can meet the requirements of accurate detection, and the method is suitable for large-scale popularization and application. And the possibility is provided for more comprehensive quality control of the ethyl valerate.

Description

Technical Field

[0001] The invention relates to a method for separating and detecting ethyl valerate and its impurities, and belongs to the technical field of quality control of ethyl valerate. Background Art

[0002] Ethyl valerate is also known as ethyl valerate, with the chemical formula C 7 H 14 O 2 It is an important spice in food technology and is widely used in the synthesis of food, cosmetics, medicine and other fields. For example, ethyl valerate is used as raw material to synthesize butylphthalide, a drug clinically used to treat ischemic stroke.

[0003] As an important chemical raw material and pharmaceutical intermediate, ethyl valerate has related substances as its key quality attributes. If the types and contents of related substances are not strictly controlled, when ethyl valerate is used as a starting material to prepare related drugs, the impurities therein may participate in related reactions, thereby further introducing various derivative impurities, which is not only not conducive to improving the yield, but also increases the difficulty of purification, and ultimately directly affects the quality of the product. Therefore, it is of great significance to study the separation and detection methods of ethyl valerate impurities.

[0004] However, at present, most manufacturers of ethyl valerate only conduct physical and chemical tests and content tests on ethyl valerate, and do not strictly control the types and contents of related substances therein, and there are few reports on the development of separation and detection methods for ethyl valerate and its impurities. National standards (GB 1886.287-2016, GB 1886.280-2016) respectively record the content detection methods of ethyl valerate structural analogs (ethyl isovalerate, ethyl 2-methylvalerate), which all use chromatographic columns with polyethylene glycol coating (stationary phase is polyethylene glycol 2000), but the inventors have found that such chromatographic columns cannot solve the problem of separation and detection of ethyl valerate and its impurities. Moreover, in the above detection method, the sample is directly injected without any dilution, and the sample concentration is extremely high. Long-term injection of high-concentration samples will damage the liner. If the sample contains impurities with a high boiling point, it is easy to cause impurity residues and thus affect the continuous detection of the sample (usually there is a risk of residues when the boiling point is greater than 150°C, for example, the boiling point of ethyl hexanoate is 164°C and the boiling point of valeric acid is 186°C). In addition, the above two national standard methods use the area normalization method to determine the content of the test substance, which cannot accurately detect the content of specific impurities.

[0005] Based on the research status described above, in order to better achieve quality control of ethyl valerate, it is urgent to develop a separation and detection method for ethyl valerate and its impurities. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the object of the present invention is to provide a separation and detection method of ethyl valerate and its impurities. Compared with the prior art, the present invention can simultaneously realize the effective separation and detection of ethyl valerate and its impurities valeric acid, ethanol, ethyl n-butyrate, ethyl isovalerate, ethyl n-hexanoate, ethyl n-propionate and ethyl acetate, which not only improves the efficiency of quality control, saves analysis costs, and the separation degree and sensitivity can meet the requirements of accurate detection, and also provides the possibility for more comprehensive quality control of ethyl valerate.

[0007] The invention provides a separation and detection method for ethyl valerate and its impurities. The method adopts gas chromatography, wherein a chromatographic column adopts a capillary column with nitroterephthalate-esterified polyethylene glycol as a stationary liquid; and a programmed temperature rise is adopted, wherein the temperature rise program is as follows: the starting temperature is 30-50 DEG C, maintained for 5-10 minutes, and then increased to 180-220 DEG C at a rate of 10-30 DEG C per minute, and maintained for 2-10 minutes.

[0008] Furthermore, the chromatographic column is selected from DB-FFAP, HP-FFAP, and WondaCap FFAP.

[0009] Preferably, the chromatographic column is DB-FFAP.

[0010] Furthermore, the length of DB-FFAP is 10 to 60 m, the inner diameter is 0.1 to 0.53 mm, and the film thickness is 0.1 to 1.5 μm.

[0011] Preferably, the DB-FFAP has a length of 30 m, an inner diameter of 0.32 mm, and a membrane thickness of 0.25 μm.

[0012] Furthermore, a programmed temperature rise was adopted, and the temperature rise program was as follows: the starting temperature was 40° C., maintained for 8 minutes, increased to 200° C. at a rate of 20° C. per minute, and maintained for 4 minutes.

[0013] Furthermore, the diluent for preparing the injection solution is at least one of acetone, n-hexane, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-dimethylacetamide.

[0014] The present invention dilutes the sample with a suitable diluent before injection. On the one hand, the obtained spectrum has a good peak shape and can accurately detect the content of specific impurities. On the other hand, it also reduces the loss rate of the liner and saves detection costs.

[0015] Preferably, the diluent for preparing the injection solution is acetone and / or n-hexane.

[0016] Further preferably, the diluent for preparing the injection solution is acetone.

[0017] According to some specific embodiments of the present invention, ethyl valerate and its impurities can be completely dissolved by using acetone as a diluent, and acetone will not interfere with the detection of the sample; in addition, using acetone as a diluent can also help improve the tailing of each impurity peak and improve the accuracy of the detection result.

[0018] Furthermore, the injection port temperature is 180-250°C.

[0019] Preferably, the injection port temperature is 220°C.

[0020] Furthermore, the detector temperature is 180-250°C.

[0021] Preferably, the detector temperature is 250°C.

[0022] Furthermore, the detector is a FID detector.

[0023] Furthermore, the carrier gas is helium or nitrogen.

[0024] Preferably, the carrier gas is helium.

[0025] According to some specific embodiments of the present invention, using helium as a carrier gas is helpful to improve the separation degree of ethanol and ethyl propionate and enhance the separation effect.

[0026] Furthermore, the carrier gas flow rate is 1.7-1.9 ml / min.

[0027] Preferably, the carrier gas flow rate is 1.8 ml / min.

[0028] Furthermore, the split ratio is 35:1 to 40:1.

[0029] Preferably, the split ratio is 37:1.

[0030] Furthermore, the impurities include ethyl propionate.

[0031] Preferably, the impurities also include ethyl butyrate, ethyl isovalerate, ethyl hexanoate and valeric acid.

[0032] Further preferably, the impurities also include ethyl acetate and ethanol.

[0033] Preferably, the impurity is one or more of ethyl acetate, ethanol, ethyl propionate, ethyl butyrate, ethyl isovalerate, ethyl hexanoate, and valeric acid.

[0034] Furthermore, the concentration of the ethyl valerate test solution is prepared to be 20-40 mg / ml.

[0035] Preferably, the concentration of the ethyl valerate test solution is prepared to be 30 mg / ml.

[0036] Furthermore, the content of the impurity is determined by an external standard method.

[0037] The present invention also provides the use of at least one of ethanol, ethyl acetate, ethyl propionate, ethyl butyrate, ethyl isovalerate, ethyl hexanoate and valeric acid in the preparation of an impurity reference substance or a standard substance for ethyl valerate.

[0038] In some specific embodiments of the present invention, the impurity content % = (A 供试品 *C STD ) / (A STD *C 供试品 )*100%;A 供试品 A is the peak area of ​​impurities in the test solution; STD is the impurity peak area of ​​the reference solution; C STD is the concentration of the reference solution; C 供试品 is the concentration of the test solution.

[0039] The present invention provides a method for separating and detecting ethyl valerate and its impurities, which has at least the following beneficial technical effects:

[0040] 1. The method provided by the present invention effectively separates ethyl valerate and its related substances on a chromatographic column, thereby finding that impurities valeric acid, ethanol, ethyl butyrate, ethyl isovalerate, ethyl hexanoate, ethyl propionate and ethyl acetate are present in ethyl valerate, which makes it possible to more comprehensively control the quality of ethyl valerate.

[0041] 2. The present invention can simultaneously realize the effective separation and detection of ethyl valerate and its seven impurities, thereby improving the efficiency of quality control and saving analysis costs.

[0042] 3. The method of the present invention is used to separate and detect ethyl valerate and its seven impurities, and the separation degree and sensitivity can meet the requirements of accurate detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is the chromatogram of the reference substance solution in Example 1;

[0044] Figure 2 The chromatogram of the spiked test solution in Example 1;

[0045] Figure 3 is the chromatogram of the test solution in Example 1;

[0046] Figure 4 is the linear relationship diagram of ethyl acetate in Example 4;

[0047] Figure 5 is the linear relationship diagram of ethanol in Example 4;

[0048] Figure 6is the linear relationship diagram of ethyl propionate in Example 4;

[0049] Figure 7 is the linear relationship diagram of ethyl n-butyrate in Example 4;

[0050] Figure 8 is the linear relationship diagram of ethyl isovalerate in Example 4;

[0051] Fig. 9 is the linear relationship diagram of ethyl valerate in Example 4;

[0052] Fig.10 is the linear relationship diagram of ethyl hexanoate in Example 4;

[0053] Fig.11 is the linear relationship diagram of valeric acid in Example 4;

[0054] Fig.12 The chromatogram of the spiked test solution in Comparative Example 1;

[0055] Fig.13 The chromatogram of the spiked test solution in Comparative Example 2;

[0056] Fig.14 The chromatogram of the spiked test solution in Comparative Example 3;

[0057] Fig.15 The chromatogram of the spiked test solution in Comparative Example 4;

[0058] Fig.16 This is the chromatogram of the spiked test solution in Comparative Example 5. DETAILED DESCRIPTION

[0059] The present invention is completed based on the following discovery of the inventors: in order to carry out more stringent quality control on ethyl valerate, the inventors have developed a separation and detection method for ethyl valerate and its impurities. After continuous optimization of detection conditions, the method can effectively separate ethyl valerate and its related substances on a chromatographic column, thereby finding that impurities valeric acid, ethanol, ethyl butyrate, ethyl isovalerate, ethyl hexanoate, ethyl propionate and ethyl acetate exist in ethyl valerate.

[0060] When ethyl valerate is used as a starting material to prepare related drugs such as butylphthalide, some of the above impurities will participate in related reactions, thereby further introducing various types of derivative impurities with similar structures as shown below, and another part of the impurities may also remain in the finished product, which is not only not conducive to improving the yield, but also increases the difficulty of purification, and ultimately directly affects the quality of the product. Specifically, the raw materials used to synthesize butylphthalide are ethyl valerate (SM2) and phthalic anhydride (SM1). The inventors found that the impurities valeric acid, ethyl butyrate, ethyl isovalerate, ethyl hexanoate, and ethyl propionate present in ethyl valerate will react with phthalic anhydride to produce the structural analog impurities K, G, I, H, and F shown below that remain in butylphthalide; at the same time, the impurities ethanol and ethyl acetate are easily left in the finished product as residual solvents, ultimately affecting the quality of the product.

[0061]

[0062] Based on this, the present invention uses related substances valeric acid, ethanol, ethyl butyrate, ethyl isovalerate, ethyl hexanoate, ethyl propionate and ethyl acetate for the quality control of the starting material ethyl valerate. In addition to the physical and chemical items and content detection, adding the detection of related substances can more comprehensively reflect the quality of ethyl valerate.

[0063] The scheme of the present invention will be further described below by specific implementation methods and experiments. It will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area or the product instructions are used. The reagents or instruments used that do not indicate the manufacturer are all conventional products that can be obtained commercially.

[0064] Sources of reference and test products:

[0065]

[0066] Gas chromatograph: Agilent 8890 gas chromatograph system;

[0067] Detector: FID detector (gas chromatography).

[0068] Example 1 Separation and detection of ethyl valerate and its impurities using the method of the present invention

[0069] The chromatographic detection conditions are as follows:

[0070] Chromatographic column: A capillary chromatographic column whose stationary liquid is nitroterephthalate-esterified polyethylene glycol, in this embodiment, Agilent DB-FFAP (0.32 mm×30 m, 0.25 μm) is specifically selected;

[0071] Carrier gas: helium;

[0072] Carrier gas flow rate: 1.8 ml / min, constant flow mode injection;

[0073] Temperature program conditions: column temperature was 40 °C, maintained for 8 min, increased to 200 °C at a rate of 20 °C per minute, and maintained for 4 min;

[0074] Inlet temperature: 220°C;

[0075] The split ratio is 37:1;

[0076] Detector: FID detector;

[0077] Detector temperature: 250°C;

[0078] Test sample concentration: 30 mg / ml;

[0079] The injection volume was 1 μl;

[0080] Thinner: Acetone.

[0081] Preparation of test solution: Take an appropriate amount of the test sample ethyl valerate, accurately weigh it, dissolve it and dilute it to make a solution containing about 30 mg per 1 ml.

[0082] Preparation of reference solution: Take appropriate amount of valeric acid, ethanol, ethyl butyrate, ethyl isovalerate, ethyl hexanoate, ethyl propionate and ethyl acetate, accurately weigh, dissolve and quantitatively dilute to make a mixed solution containing 0.15 mg of each in each ml.

[0083] Preparation of spiked test solution: Take appropriate amounts of ethyl valerate, valeric acid, ethanol, ethyl butyrate, ethyl isovalerate, ethyl hexanoate, ethyl propionate and ethyl acetate, accurately weigh them, dissolve them in diluent and quantitatively dilute them to make a mixed solution containing 30 mg of ethyl valerate and 0.15 mg of each other impurity in each ml.

[0084] Accurately measure the reference solution, spiked test solution, and test solution and inject them into the gas chromatograph respectively. The obtained chromatograms are shown in Figure 1 to Figure 3 .

[0085] from Figure 2 It can be seen that each impurity peak is not interfered by the diluent peak, and the peak shapes of ethyl valerate and each impurity are good, and the separation degree is greater than 1.5, indicating that the method of the present invention can simultaneously realize the effective separation and detection of ethyl valerate and its impurities valeric acid, ethanol, ethyl butyrate, ethyl isovalerate, ethyl hexanoate, ethyl propionate and ethyl acetate; Figure 3It can be seen that the impurities ethyl acetate, ethanol, ethyl propionate, ethyl butyrate, ethyl isovalerate, ethyl hexanoate and valeric acid were detected in the commercially available ethyl valerate test sample, and the contents were 0.012%, 0.061%, 0.002%, 0.046%, 0.061%, 0.057% and 0.200% respectively. The above test results show that the detection method of the present invention has high separation, high sensitivity and good peak shape.

[0086] Example 2 System Applicability and Specificity of the Detection Method of the Present Invention

[0087] The chromatographic detection conditions, diluents, and preparation of the test solution are as in Example 1.

[0088] The preparation of system suitability solution refers to the "Preparation of spiked test solution" in Example 1.

[0089] The preparation of a single impurity localization solution refers to the “Preparation of a reference solution” in Example 1.

[0090] The single impurity localization solution and the system suitability solution were injected into the gas chromatograph respectively, and the separation and detection results are shown in Table 1.

[0091] Table 1 System suitability and specificity test results

[0092] name Single impurity location retention time / min System suitability solution retention time / min Separation Ethyl acetate 2.651 2.650 N / A Ethanol 3.111 3.111 11.92 Ethyl propionate 3.345 3.345 5.25 Ethyl butyrate 4.723 4.724 25.02 Ethyl isovalerate 5.582 5.584 11.50 Ethyl valerate / 8.438 24.54 Ethyl hexanoate 10.538 10.543 22.04 Valeric acid 14.749 14.746 115.37

[0093] According to the test results in Table 1, the retention time of impurities in the single impurity positioning solution and the system suitability solution is consistent; the diluent does not interfere with the detection of related substances such as valeric acid, ethanol, ethyl butyrate, ethyl isovalerate, ethyl hexanoate, ethyl propionate, and ethyl acetate, and the location of each impurity is clear; the separation degree of each impurity in the system suitability solution is greater than 1.5, indicating that the system applicability and specificity of the detection method of the present invention are good.

[0094] Example 3 Quantitative Limit and Detection Limit of the Detection Method of the Present Invention

[0095] The chromatographic detection conditions and diluents are the same as those in Example 1.

[0096] Take appropriate amounts of valeric acid, ethanol, ethyl butyrate, ethyl isovalerate, ethyl hexanoate, ethyl propionate, ethyl acetate and ethyl valerate reference substances, dissolve and dilute them with acetone to prepare solutions of certain concentrations, dilute them step by step, and perform detection according to the detection method of Example 1, with a signal-to-noise ratio of about 10 to 50 as the quantitative limit, and a signal-to-noise ratio of about 3 to 10 as the detection limit. The results are shown in Tables 2 and 3.

[0097] Table 2 Results of limit of quantitation test

[0098]

[0099]

[0100] Table 3 Detection limit test results

[0101]

[0102]

[0103] From the test results in Table 2 and Table 3, it can be seen that the quantitative limit solution was injected 6 times continuously, the peak area RSD≤10%, and the signal-to-noise ratio was in the range of 10 to 50; the detection limit solution signal-to-noise ratio was in the range of 3 to 10. The detection method of the present invention has high sensitivity.

[0104] Example 4 Linearity and range of the detection method of the present invention

[0105] The chromatographic detection conditions and diluents are the same as those in Example 1.

[0106] Take appropriate amounts of valeric acid, ethanol, ethyl butyrate, ethyl isovalerate, ethyl hexanoate, ethyl propionate, ethyl acetate and ethyl valerate reference substances, dissolve them in acetone and dilute them to prepare stock solutions of a certain concentration, dilute the stock solutions to prepare a series of linear solutions of different concentrations, perform detection according to the detection method of Example 1, record the chromatogram, perform linear regression on the concentration with the peak area of ​​each component, calculate the correlation coefficient r and the y-axis intercept, and evaluate the linearity of each component in this method. The results are shown in Table 4, and the linear regression diagram is shown in Figures 4 to 11 .

[0107] Table 4 Linearity and range test results

[0108]

[0109]

[0110]

[0111] According to the test results, the correlation coefficients r of the impurities were all greater than 0.999, and the percentage of the y-axis intercept to the limit concentration peak area was less than 25%, indicating that the method had a good linear relationship.

[0112] Example 5 Repeatability of the detection method of the present invention

[0113] The chromatographic detection conditions and diluents are the same as those in Example 1.

[0114] Take appropriate amounts of valeric acid, ethanol, ethyl butyrate, ethyl isovalerate, ethyl hexanoate, ethyl propionate and ethyl acetate reference substances to make stock solutions, add them to the ethyl valeric acid test sample, and add diluent to make a spiked test solution containing approximately 30 mg of ethyl valeric acid and 0.15 mg of each impurity per 1 ml. Prepare 6 solutions in the same way to examine the repeatability of the method. The results are shown in Table 5.

[0115] Table 5 Repeatability test results

[0116]

[0117]

[0118] It can be seen from the test results in Table 5 that the RSDs of the contents of valeric acid, ethanol, ethyl butyrate, ethyl isovalerate, ethyl hexanoate, ethyl propionate and ethyl acetate in the 6 repeatability solutions are 1.45%, 1.71%, 1.68%, 1.66%, 1.59%, 1.57% and 1.69%, respectively, all of which are less than 10%. Therefore, the method of the present invention has good repeatability in determining the contents of valeric acid, ethanol, ethyl butyrate, ethyl isovalerate, ethyl hexanoate, ethyl propionate and ethyl acetate in ethyl valerate.

[0119] Example 6 Accuracy of the detection method of the present invention

[0120] The chromatographic detection conditions and diluents are the same as those in Example 1.

[0121] Valeric acid, ethanol, ethyl butyrate, ethyl isovalerate, ethyl hexanoate, ethyl propionate and ethyl acetate reference substances were taken in appropriate amounts to prepare stock solutions, which were added to the ethyl valerate sample at limit concentrations of 50%, 100% and 150%, respectively, to prepare solutions containing about 30 mg of ethyl valerate and 0.08 mg, 0.15 mg and 0.225 mg of each impurity per 1 ml of solution, respectively. The solutions were tested according to the detection method of Example 1, and the results are shown in Tables 6 to 12.

[0122] Table 6 Ethyl acetate accuracy test results

[0123]

[0124] Table 7 Ethanol accuracy test results

[0125]

[0126] Table 8 Ethyl propionate accuracy test results

[0127]

[0128]

[0129] Table 9 Ethyl butyrate accuracy test results

[0130]

[0131] Table 10 Ethyl isovalerate accuracy test results

[0132]

[0133] Table 11 Ethyl hexanoate accuracy test results

[0134]

[0135]

[0136] Table 12 Valeric acid accuracy test results

[0137]

[0138] The results showed that the average recoveries of 9 solutions of each concentration of valeric acid, ethanol, ethyl butyrate, ethyl isovalerate, ethyl hexanoate, ethyl propionate and ethyl acetate were all between 90% and 110%, and the RSDs of the recoveries were 2.51%, 1.34%, 1.20%, 1.19%, 3.44%, 1.32% and 1.49%, respectively, all less than 10%, indicating that the method of the present invention has high accuracy in detecting the contents of valeric acid, ethanol, ethyl butyrate, ethyl isovalerate, ethyl hexanoate, ethyl propionate and ethyl acetate in ethyl valerate.

[0139] Example 7 Stability of the detection method of the present invention

[0140] The chromatographic detection conditions and diluents are the same as those in Example 1.

[0141] The preparation of reference solution, test solution and spiked test solution was as described in Example 1.

[0142] The reference solution and the test solution spiked with the sample were placed at 25°C for different periods of time to examine the changes in the peak area of ​​each component in the reference solution and the test solution spiked with the sample within 24 hours. The results of the impurity peak areas detected at each time point in the reference solution and the test solution spiked with the sample are shown in Tables 13 and 14, respectively.

[0143] Table 13 Stability test results - reference solution

[0144]

[0145]

[0146] Table 14 Stability test results - spiked test solution

[0147]

[0148] According to the test results in Table 13 and Table 14, the reference solution and the spiked test solution were placed at 25°C for different periods of time, and the peak area RSD of each impurity within 24 hours was less than 5%, indicating that the solution had good stability.

[0149] Example 8 Durability of the detection method of the present invention

[0150] The chromatographic detection conditions and diluents are the same as those in Example 1.

[0151] The preparation of the spiked test solution was carried out according to Example 1.

[0152] The durability test conditions are shown in Table 15.

[0153] Table 15 Durability inspection conditions

[0154]

[0155] The spiked test solution was sampled and tested under the above conditions to investigate the separation and detection of impurities under each condition. The results are shown in Table 16.

[0156] Table 16 Durability test results

[0157]

[0158] The results show that under each durability test condition, each impurity is well separated, with a minimum separation of 5.20 (greater than 1.5); the diluent does not interfere with the separation and detection of valeric acid, ethanol, ethyl butyrate, ethyl isovalerate, ethyl hexanoate, ethyl propionate, and ethyl acetate in ethyl valerate; the ratio of the impurity content under each durability test condition to the original condition is within 90-110%, indicating that the detection method of the present invention has good durability.

[0159] Comparative Example 1

[0160] The chromatographic detection conditions are as follows:

[0161] Chromatographic column: A capillary column with polyethylene glycol as the stationary liquid, specifically Agilent DB-WAX, 30m×0.25mm, 0.25μm;

[0162] Carrier gas: nitrogen;

[0163] Column front pressure: 0.06MPa, injection in constant pressure mode;

[0164] Temperature program conditions: the initial column temperature was 60 °C, maintained for 5 min, increased to 200 °C at a rate of 8 °C per minute, and maintained for 3 min;

[0165] Inlet temperature: 220°C;

[0166] Split ratio: 10:1;

[0167] Detector: FID detector;

[0168] Detector temperature: 250°C;

[0169] Diluent: dimethyl sulfoxide;

[0170] Test sample concentration: 30 mg / ml;

[0171] Injection volume: 1μl.

[0172] The preparation of the reference solution and the spiked test solution was carried out according to Example 1.

[0173] Accurately measure the reference solution and the spiked test solution and inject them into the gas chromatograph respectively. The chromatogram of the spiked test solution is shown in Fig.12 .

[0174] Depend on Fig.12 It can be seen that under the detection conditions of this experiment, the separation degree of ethyl propionate is less than 1.5 and cannot be accurately detected; in addition, the tailing factors of multiple impurity peaks are too large, the impurity peaks have obvious tailing, and the peak shape is poor, which will lead to a decrease in detection sensitivity and seriously affect the accuracy and reliability of the chromatographic analysis results.

[0175] Comparative Example 2

[0176] On the basis of Comparative Example 1, only the diluent was changed to N,N-dimethylformamide, and the other chromatographic detection conditions were the same as those of Comparative Example 1.

[0177] The preparation of the reference solution and the spiked test solution was carried out according to Example 1.

[0178] Accurately measure the reference solution and the spiked test solution and inject them into the gas chromatograph respectively. The chromatogram of the spiked test solution is shown in Fig.13 .

[0179] Depend on Fig.13 The test results shown in the figure show that, compared with Comparative Example 1, the impurity peaks of ethanol, ethyl butyrate, ethyl isovalerate, ethyl hexanoate and valeric acid have more severe tailing and worse peak shape when detected by the method of Comparative Example 2; the column efficiency of ethyl isovalerate, ethyl valerate and ethyl hexanoate is lower.

[0180] Comparative Example 3

[0181] On the basis of Comparative Example 1, only the diluent was changed to acetone, and the other chromatographic detection conditions were the same as those of Comparative Example 1.

[0182] The preparation of the spiked test solution and reference solution was carried out according to Example 1.

[0183] Accurately measure the reference solution and the spiked test solution and inject them into the gas chromatograph respectively. The chromatogram of the spiked test solution is shown in Fig.14 .

[0184] From the detection spectrum, it can be seen that the tailing phenomenon of each impurity peak has been significantly improved, but the tailing factor of valeric acid is still high, and the peaks of ethanol and ethyl propionate overlap and cannot be separated.

[0185] Comparative Example 4

[0186] On the basis of Comparative Example 3, only the carrier gas was changed to helium (flow rate of 1.0 ml / min, constant flow mode injection), and the other chromatographic detection conditions were all referred to Comparative Example 3.

[0187] The preparation of the spiked test solution and reference solution was carried out according to Example 1.

[0188] Accurately measure the reference solution and the spiked test solution and inject them into the gas chromatograph respectively. The chromatogram of the spiked test solution is shown in Fig.15 .

[0189] From the detection spectrum, it can be seen that the peak shapes of each impurity are good, but the tailing factor of valeric acid is still high; the peaks of ethyl propionate and ethanol no longer completely overlap, but under the detection conditions of this experiment, the separation degree is still far less than 1.5 and cannot be accurately detected; ethyl acetate and the blank interference peak cannot be completely separated (the separation degree is only 1.2).

[0190] It can be seen from comparative examples 1 to 4 that using acetone as a diluent is beneficial to improving the tailing phenomenon of the impurity peak. However, when nitroterephthalate-esterified polyethylene glycol is not used as the stationary phase, the problem of separation and detection of all impurities cannot be solved.

[0191] Comparative Example 5

[0192] On the basis of Comparative Example 4, only the chromatographic column was changed to a capillary chromatographic column whose stationary liquid was polyethylene glycol esterified with nitroterephthalate. Agilent DB-FFAP, 0.32 mm×30 m, 0.25 μm, was specifically selected for this comparative example. The other chromatographic detection conditions were all referred to Comparative Example 4.

[0193] The preparation of the spiked test solution and reference solution was carried out according to Example 1.

[0194] Accurately measure the spiked test solution and reference solution and inject them into the gas chromatograph respectively. The chromatogram of the spiked test solution is shown in Fig.16 .

[0195] From the detection spectrum, it can be seen that ethyl acetate and blank interference peaks are not completely separated (separation degree is only 1.15); and the tailing factors of ethyl acetate and valeric acid peaks are too large, and the tailing is obvious.

[0196] It can be seen from Comparative Example 5 that, in addition to the stationary phase, the temperature program also has a significant impact on the separation and detection of ethyl valerate and its impurities. In this experiment, the initial column temperature was too high and the heating rate was slow, and the separation and detection problem of all impurities could not be solved.

Claims

1. A method for separating and detecting ethyl valerate and its impurities, characterized in that: Gas chromatography was used, and the chromatographic column used a capillary column with nitroterephthalate-esterified polyethylene glycol as the stationary liquid; programmed temperature was used, and the temperature rise program was as follows: the initial temperature was 30-50°C, maintained for 5-10 minutes, and then increased to 180-220°C at a rate of 10-30°C per minute, and maintained for 2-10 minutes.

2. The separation and detection method according to claim 1, characterized in that: The chromatographic column is selected from DB-FFAP, HP-FFAP, and WondaCap FFAP; preferably, the chromatographic column is DB-FFAP.

3. The separation and detection method according to claim 2, characterized in that: The length of DB-FFAP is 10 to 60 m, the inner diameter is 0.1 to 0.53 mm, and the film thickness is 0.1 to 1.5 μm; preferably, the length of DB-FFAP is 30 m, the inner diameter is 0.32 mm, and the film thickness is 0.25 μm.

4. The separation and detection method according to claim 1, characterized in that: The temperature was programmed as follows: the initial temperature was 40°C, maintained for 8 minutes, increased to 200°C at a rate of 20°C per minute, and maintained for 4 minutes.

5. The separation and detection method according to claim 1, characterized in that: The diluent for preparing the injection solution is at least one of acetone, n-hexane, dimethyl sulfoxide, N,N-dimethylformamide, and N,N-dimethylacetamide; preferably, the diluent for preparing the injection solution is acetone and / or n-hexane; further preferably, the diluent for preparing the injection solution is acetone.

6. The separation and detection method according to claim 1, characterized in that: At least one of the following is met: The injection port temperature is 180-250°C; Preferably, the injection port temperature is 220°C; The detector temperature is 180-250°C; Preferably, the detector temperature is 250°C; The detector is a FID detector; The carrier gas is helium or nitrogen; Preferably, the carrier gas is helium; The carrier gas flow rate is 1.7-1.9 ml / min; Preferably, the carrier gas flow rate is 1.8 ml / min; The split ratio is 35:1 to 40:1; Preferably, the split ratio is 37:

1.

7. The separation and detection method according to claim 1, characterized in that: The impurities include ethyl propionate; preferably, the impurities also include ethyl butyrate, ethyl isovalerate, ethyl hexanoate, and valeric acid; further preferably, the impurities also include ethyl acetate and ethanol; preferably, the impurities are one or more of ethyl acetate, ethanol, ethyl propionate, ethyl butyrate, ethyl isovalerate, ethyl hexanoate, and valeric acid.

8. The separation and detection method according to claim 1, characterized in that: The concentration of the ethyl valerate test solution is 20-40 mg / ml; preferably, the concentration of the ethyl valerate test solution is 30 mg / ml.

9. The separation and detection method according to claim 1, characterized in that: The content of the impurities was determined by external standard method.

10. Use of at least one of ethanol, ethyl acetate, ethyl propionate, ethyl butyrate, ethyl isovalerate, ethyl hexanoate and valeric acid in the preparation of an impurity reference substance or standard substance for ethyl valerate.

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