Method for detecting chloroethane in L-glutamic acid diethyl ester hydrochloride by using gas chromatography headspace method

Abstract

The invention relates to the field of analytical chemistry, and provides a method for detecting chloroethane in L-glutamic acid diethyl ester hydrochloride by using a gas chromatography headspace method.The method comprises the following steps that 1, ethyl alcohol is used for diluting a chloroethane-methanol standard solution, and a chloroethane reference substance solution is prepared; 2) dissolving L-glutamic acid diethyl ester hydrochloride by using ethanol to prepare a test solution; and 3) detecting the chloroethane reference substance solution and the test solution by using a gas chromatography headspace method. The method is high in specificity, high in sensitivity and good in repeatability, the content of chloroethane in L-glutamic acid diethyl ester hydrochloride can be accurately and rapidly measured, dangerous reagents are not needed, harm to the human body is smaller, the detection safety is improved, and meanwhile the cost is reduced.

Description

Technical Field

[0001] The present invention relates to the field of analytical chemistry, and more particularly to a method for detecting chloroethane in L-glutamic acid diethyl ester hydrochloride by gas chromatography headspace method. Background Art

[0002] Pemetrexed disodium is an organic compound with the molecular formula C 20 H 19 N5Na2O6. It is a folic acid antagonist that inhibits cell replication by disrupting key folate-dependent metabolic processes essential for cell replication. Pemetrexed disodium can be used to treat non-small cell lung cancer, malignant pleural mesothelioma, etc. L-glutamic acid diethyl ester hydrochloride (LGDE·HCl) is a key intermediate in the synthesis of pemetrexed disodium, and chloroethane is a reaction material of L-glutamic acid diethyl ester hydrochloride (LGDE·HCl). Chloroethane, also known as ethyl chloride, is an organic compound with the chemical formula C2H5Cl. It is a colorless gas at room temperature and atmospheric pressure, has an ether-like odor, is slightly soluble in water, and can be miscible with most organic solvents. Its molecular weight is 64.514, and its boiling point is 12.3 °C. It is a typical small molecule organic compound. On October 27, 2017, the International Agency for Research on Cancer of the World Health Organization released a preliminary list of carcinogens. Chloroethane is in the list of Group 3 carcinogens. It is a definite genotoxic impurity. Therefore, it is necessary to detect the possible residual chloroethane in L-glutamic acid diethyl ester hydrochloride (LGDE·HCl).

[0003] At present, the determination methods of chloroethane have not been included in the pharmacopoeias of various countries. Patent CN201910017911.8 discloses a gas chromatography detection method for chloroethane in L-glutamic acid diethyl ester hydrochloride. It uses water as a diluent to dissolve L-glutamic acid diethyl ester hydrochloride and then conducts detection, and limits the chromatographic column, headspace conditions, temperature programming, etc. However, due to the absence of a pure chloroethane standard product, the commercially available chloroethane standard product uses methanol as a solvent, and the headspace equilibrium temperature set in CN201910017911.8 is 60 ± 5 °C, which is very close to the boiling point of methanol. In the detection, methanol will turn into gas and enter the detector together. The retention times of methanol and chloroethane are very close. Even when methanol is diluted by several times with water, the resolution between methanol and chloroethane is still small. Moreover, methanol will undergo transesterification with the ethyl ester present in L-glutamic acid diethyl ester hydrochloride. Therefore, the presence of methanol will affect the determination of chloroethane. In addition, L-glutamic acid diethyl ester hydrochloride itself has hydrochloric acid and ethyl ester groups. Under certain conditions, the two will react to generate chloroethane, interfering with the detection.

[0004] In addition, for headspace gas chromatography, most of the currently disclosed detection methods use diluents recommended in the Chinese Pharmacopoeia, the United States Pharmacopeia (USP), and the European Pharmacopoeia (EP). That is, usually water is used as the diluent. For water-insoluble substances, organic solvents such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and 1,3-dimethyl-2-imidazolidinone (DMI) are used as diluents. However, the above diluents may interfere with the substances to be detected, and organic diluents are toxic and costly, posing problems such as experimental safety hazards and high costs.

[0005] Therefore, it is very necessary to develop a quantitative method for detecting the residual amount of chloroethane in L-glutamic acid diethyl ester hydrochloride (LGDE·HCl) with good accuracy and sensitivity, simple operation, low cost, and greater safety. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a method for detecting chloroethane in L-glutamic acid diethyl ester hydrochloride using headspace gas chromatography. The method includes the following steps: 1) Dilute the chloroethane methanol standard solution with ethanol to prepare a chloroethane reference solution; 2) Dissolve L-glutamic acid diethyl ester hydrochloride with ethanol to prepare a test solution; 3) Detect the chloroethane reference solution and the test solution using headspace gas chromatography; Among them, the chromatographic column is a capillary column with a stationary phase of 6% cyanopropylphenyl 94% dimethylpolysiloxane. The length of the chromatographic column is 60 m, the inner diameter is 0.32 mm, and the film thickness is 1.8 μm; The temperature programming procedure is as follows: the initial temperature is 40°C - 50°C, maintained for 2 - 6 min, heated at a rate of 5 - 20°C / min to 70 - 80°C, and then heated at a rate of 20 - 40°C / min to 200 - 240°C, and held for 2 - 5 min.

[0007] Preferably, the chromatographic conditions in step 3) are as follows: Detector: flame ionization detector, and the detector temperature is 300 ± 5°C; Headspace injector oven temperature: 50°C.

[0008] More preferably, the temperature programming procedure is as follows: the initial temperature is 40°C, maintained for 6 min, heated at a rate of 10°C / min to 80°C, and then heated at a rate of 40°C / min to 240°C, and held for 2 min.

[0009] Preferably, the chromatographic conditions in step 3) further include: Sample flow path temperature: 60°C; Transmission line temperature: 70 ± 5 °C; Headspace sampler constant temperature time: 30 min; GC cycle time of the headspace sampler: 36 min; Sampling is carried out in split mode, and the split ratio is 10:1.

[0010] Preferably, in step 3), a 20 mL headspace vial is used, and the sample loading amount is 1 mL.

[0011] Preferably, the flow rate of the chromatographic column is 3.0 ± 0.2 mL / min.

[0012] Preferably, the chromatographic conditions for step 3) are as follows: Detector: Flame ionization detector, and the detector temperature is 300 °C; Chromatographic column: A capillary column with a stationary phase of 6% cyanopropylphenyl 94% dimethylpolysiloxane, the length of the chromatographic column is 60 m, the inner diameter is 0.32 mm, and the film thickness is 1.8 μm; Headspace sampler oven temperature: 50 °C; Temperature programming: Initial temperature 40 °C, maintained for 6 min, heated to 80 °C at a rate of 10 °C / min, and then heated to 240 °C at a rate of 40 °C / min, held for 2 min; Sample flow path temperature: 60 °C; Transmission line temperature: 70 °C; Headspace sampler constant temperature time: 30 min; GC cycle time of the headspace sampler: 36 min; Sampling is carried out in split mode, and the split ratio is 10:1; The flow rate of the chromatographic column is 3.0 mL / min.

[0013] Preferably, the concentration of chloroethane in the chloroethane reference solution is 0.01 mg / mL, and the concentration of diethyl L - glutamate hydrochloride in the test solution is 10 mg / mL.

[0014] Preferably, the external standard method is used to calculate the content of chloroethane in diethyl L - glutamate hydrochloride.

[0015] The beneficial effects of the present invention are as follows: 1. The sampling method used in the method of the present invention is headspace sampling. Compared with the automatic sampling method, headspace sampling is not easy to contaminate the injection port and the chromatographic system, improves the stability of the method, avoids the influence of baseline fluctuations on the test results, and more avoids frequent maintenance of the injection port, saves the inspection cost, and reduces the operation pressure of the inspection personnel.

[0016] 2. The detector used in the method of the present invention is a common flame ionization detector (FID). Compared with other detectors, FID is more widely used, more mature, and easier to operate. It has lower requirements and training for inspectors and higher sensitivity to organic compounds. In addition, FID is usually a detector built into a gas chromatograph, eliminating the need to purchase other detectors separately and reducing the detection cost. Moreover, compared with the radioactive electron capture detector (ECD), the use of FID in the present invention is safer.

[0017] 3. The diluent used in the method of the present invention is ethanol, eliminating the need to use other hazardous organic reagents, which is less harmful to the human body and cheaper and easier to obtain. Moreover, compared with using water or other organic reagents as diluents, ethanol has no effect on the detection of chloroethane, resulting in better accuracy of the method of the present invention.

[0018] 4. The stationary phase used in the method of the present invention is a capillary column of 6% cyanopropylphenyl 94% dimethylpolysiloxane (specification: 60m × 0.32mm × 1.8μm). By regulating parameters such as the temperature rise degree, it can effectively separate chloroethane from other substances (especially methanol) to reduce the interference of other substances and optimize the recovery rate. In addition, if necessary, the method of the present invention can also simultaneously detect other residual solvents, etc.

[0019] 5. After methodological verification, the correlation coefficient R of the standard curve constructed by the method of the present invention is 0.9999, indicating good linearity; the detection limit of chloroethane is 0.0001 mg / mL, showing high sensitivity; the relative standard deviation (RSD) of the spiked recovery rate of 6 parallel samples is 3.08% or lower, and the RSD of the spiked recovery rate of 12 samples is 2.55%, indicating excellent repeatability. The method of the present invention has strong specificity, high sensitivity, and good repeatability, and can accurately and rapidly determine the residual amount of chloroethane in L-glutamic acid diethyl ester hydrochloride (LGDE·HCl). Description of the Drawings

[0020] Figure 1 It is the chromatogram of the reference solution, where the lower figure is the corresponding chromatogram with the ordinate scale of the upper figure enlarged; Figure 2 It is the chromatogram of the test solution, where the lower figure is the corresponding chromatogram with the ordinate scale of the upper figure enlarged; Figure 3 It is the chromatogram of the blank solution, where the lower figure is the corresponding chromatogram with the ordinate scale of the upper figure enlarged; Figure 4 It is the chromatogram of the test solution spiked with 100% concentration, where the lower figure is the corresponding chromatogram with the ordinate scale of the upper figure enlarged; Figure 5 It is the chromatogram of the chloroethane single standard localization solution, where the lower figure is the corresponding chromatogram with the ordinate scale of the upper figure enlarged; Figure 6 It is the chromatogram of the tetrahydrofuran single standard positioning solution, and the following figure is the corresponding chromatogram after magnifying the ordinate scale of the above figure; Figure 7 It is the chromatogram of the methanol single standard positioning solution, and the following figure is the corresponding chromatogram after magnifying the ordinate scale of the above figure; Figure 8 It is the standard working curve for detecting the content of chloroethane in diethyl L-glutamate hydrochloride by the method of the present invention; Figure 9 It is the chromatogram of detecting the test solution by using the automatic injection method; Figure 10 It is the chromatogram of detecting by using N,N-dimethylacetamide as the diluent; Figure 11 It is the chromatogram of detecting by using the mixed solution of DMA: water = 9:1 as the diluent; Figure 12 It is the chromatogram of detecting the chloroethane reference substance by using the chromatographic column DB-624 (specification 30m×0.53mm×3.0μm). Specific Embodiments

[0021] The following further describes the present invention in conjunction with the accompanying drawings and embodiments. However, these embodiments are exemplary and are only convenient for those skilled in the art to further understand the characteristics of the present invention, and do not constitute any limitation to the scope of the present invention. The reagents used in the present invention are all conventional commercially available products unless otherwise specified. The percentages appearing in the present invention are all mass percentages unless otherwise specified. The gas chromatograph used in the following embodiments is Shimadzu GC-2030, equipped with Shimadzu's HS-20 headspace sampler; the chromatographic column used is DB-624 (specification 60m×0.32mm×1.8μm); the chloroethane reference substance solution used is a methanol solution of chloroethane, and the concentration of chloroethane is 5 mg / mL. "ND" in the following table means not detected, and "N / A" means not applicable.

[0022] In the specification of the present invention, "chloroethane", "monochloroethane" and "ethyl chloride" all refer to the same organic compound, with the chemical formula C2H5Cl and the structural formula .

[0023] In the specification of the present invention, "diethyl L-glutamate hydrochloride" and "test sample" all refer to the same organic compound, with the chemical formula C9H 17 NO4.HCl and the structural formula .

[0024] The method for detecting chloroethane in diethyl L - glutamate hydrochloride by gas chromatography headspace method of the present invention comprises the following steps: 1) Diluting the chloroethane methanol standard solution with ethanol to prepare a chloroethane reference solution; 2) Dissolving diethyl L - glutamate hydrochloride with ethanol to prepare a test solution; 3) Detecting the chloroethane reference solution and the test solution by gas chromatography headspace method; wherein, the chromatographic column is a capillary column with a stationary phase of 6% cyanopropylphenyl 94% dimethylpolysiloxane, the length of the chromatographic column is 60 m, the inner diameter is 0.32 mm, and the film thickness is 1.8 μm; the temperature programming procedure is that the initial temperature is 40°C - 50°C, maintained for 2 - 6 min, heated at a rate of 5 - 20°C / min to 70 - 80°C, and then heated at a rate of 20 - 40°C / min to 200 - 240°C, and held for 2 - 5 min. As a preferred embodiment of the present invention, the temperature programming procedure is that the initial temperature is 40°C, maintained for 6 min, heated at a rate of 10°C / min to 80°C, and then heated at a rate of 40°C / min to 240°C, and held for 2 min.

[0025] The inventor of the present invention found that the currently commercially available standard product of chloroethane uses methanol as a solvent. However, the retention times of methanol and chloroethane are relatively close, and chloroethane will peak on the methanol tailing. When using the gas chromatography headspace method for detection, in order to completely evaporate the detected chloroethane from the test solution for subsequent detection, a relatively high temperature is required, while the boiling point of methanol is relatively low. In this case, when detecting the reference solution prepared with the chloroethane standard product, methanol will inevitably enter the detector, interfering with the peak of chloroethane. Moreover, transesterification occurs between methanol and the ethyl ester present in diethyl L - glutamate hydrochloride, which will also affect the determination of chloroethane, making the detection sensitivity, accuracy, etc. worse. The present invention effectively solves the above - mentioned technical problems by using ethanol as a diluent, using a capillary column of 6% cyanopropylphenyl 94% dimethylpolysiloxane (length 60 m, inner diameter 0.32 mm, film thickness 1.8 μm), and regulating the temperature programming procedure. Using the method of the present invention, chloroethane can peak within a short time, its retention time is about 4.84, and the resolution from methanol is greater than 3. Through the above settings, not only the detection sensitivity, accuracy are high and the repeatability is good, but also ethanol is less harmful to the human body and is cheap and easily available, improving the safety of the method of the present invention and reducing the experimental cost.

[0026] In a preferred embodiment of the present invention, the chromatographic conditions in step 3) are as follows: Detector: flame ionization detector, and the detector temperature is 300 ± 5 °C; Temperature of the headspace sampler oven: 50 °C. The FID used in the method of the present invention has high sensitivity for the detection of organic substances. And compared with other detectors, FID is more widely used and mature, and the operation is simpler, with lower requirements and training for inspectors. The FID built in the gas chromatograph is used in the present invention, without the need to purchase other detectors additionally, which reduces the detection cost and has higher safety compared with the radioactive ECD. In the method of the present invention, the detector temperature is set to 300 ± 5 °C, which can prevent carbon deposition and condensation of water vapor on the detector. In addition, since L-glutamic acid diethyl ester hydrochloride itself contains hydrochloric acid and ethyl ester groups, if organic solvent diluents such as DMF are used, at higher temperatures, the hydrochloric acid and ethyl ester groups will react to form chloroethane, resulting in poor detection accuracy. However, the diluent used in the present invention is ethanol, and the temperature of the headspace sampler oven can be set relatively low, which can avoid the reaction of the hydrochloric acid and ethyl ester groups in L-glutamic acid diethyl ester hydrochloride to form chloroethane while not affecting the evaporation of chloroethane, thus affecting the accuracy of the determination of the chloroethane content in the test sample.

[0027] In a preferred embodiment of the present invention, the chromatographic conditions in step 3) of the method of the present invention further include: Sample flow path temperature: 60 °C; Transfer line temperature: 70 ± 5 °C; Headspace sampler oven holding time: 30 min; GC cycle time of the headspace sampler: 36 min; Injection is carried out in split mode, and the split ratio is 10:1. By optimizing the chromatographic conditions as above, the sensitivity and accuracy of the method of the present invention can be further improved. And if the split ratio is too low, the peak of methanol will become larger, affecting the resolution between methanol and chloroethane and thus affecting the detection, while if the split ratio is too high, the sensitivity will be too low. In addition, preferably, in order to prevent chloroethane residue, the injection needle cleaning time is set to 15 min. In a preferred embodiment of the present invention, a 20 mL headspace vial is used, and the sample loading amount is 1 mL. In a preferred embodiment of the present invention, high-purity nitrogen is used as the carrier, and the flow rate of the chromatographic column is 3.0 ± 0.2 mL / min.

[0028] In a preferred embodiment of the present invention, the chromatographic conditions in step 3) are as follows: Detector: flame ionization detector, and the detector temperature is 300 °C; Chromatographic column: a capillary column with a stationary phase of 6% cyanopropylphenyl 94% dimethylpolysiloxane, the length of the chromatographic column is 60 m, the inner diameter is 0.32 mm, and the film thickness is 1.8 μm; Headspace injector oven temperature: 50 °C; Temperature programming: initial temperature 40 °C, maintained for 6 min, heated to 80 °C at a rate of 10 °C / min, then heated to 240 °C at a rate of 40 °C / min, and held for 2 min; Sample flow path temperature: 60 °C; Transfer line temperature: 70 °C; Headspace injector constant temperature time: 30 min; GC cycle time of the headspace injector: 36 min; Injection is carried out in split mode, and the split ratio is 10:1; The flow rate of the chromatographic column is 3.0 mL / min.

[0029] In a preferred embodiment of the present invention, the concentration of chloroethane in the chloroethane reference solution is 0.01 mg / mL, and the concentration of diethyl L - glutamate hydrochloride in the test solution is 10 mg / mL. In a preferred embodiment of the present invention, the external standard method is used to calculate the content of chloroethane in the diethyl L - glutamate hydrochloride.

[0030] Example 1: Detection of test sample 1.1 Preparation of solutions Blank solution / diluent: absolute ethanol.

[0031] Chloroethane single - standard positioning solution: Take 1 mL of ethanol and place it in a 20 - mL headspace vial, add 5 μL of a tetrahydrofuran solution containing 15% chloroethane, and shake well; Tetrahydrofuran single - standard positioning solution: Take 1 mL of ethanol and place it in a 20 - mL headspace vial, add 5 μL of tetrahydrofuran, and shake well; Methanol single - standard positioning solution: Take 1 mL of ethanol and place it in a 20 - mL headspace vial, add 5 μL of methanol, and shake well.

[0032] Reference stock solution (0.1 mg / mL): Take 0.5 mL of a 5 - mg / mL chloroethane standard solution and add it to a 20 - mL volumetric flask, and make up the volume with ethanol; Reference solution (0.01 mg / mL): Take 2.0 mL of the reference stock solution (0.1 mg / mL) and add it to a 20 - mL volumetric flask, and make up the volume with ethanol.

[0033] Test solution: Weigh accurately about 100 mg of diethyl L - glutamate hydrochloride and add it to a 10 - mL volumetric flask, and make up the volume with ethanol. The exact concentration of the test sample is calculated based on the actual weight of the diethyl L - glutamate hydrochloride actually weighed; The preparation methods of each linear solution are shown in Table 1: Table 1

[0034] Quantitation limit solution: Use the linear solution with a concentration ratio of 10% as the quantitation limit solution; Detection limit solution: Take 1 mL of the quantitation limit solution and add it to a 10 mL volumetric flask, and make up the volume with ethanol.

[0035] The preparation methods of each spiked solution are shown in Table 2: Table 2

[0036] 1.2 Detection conditions The detection conditions are shown in Table 3: Table 3

[0037] 1.3 Detection Inject the reference solution and the test solution into the headspace sampler respectively, and use a gas chromatograph for detection. The detection conditions are set as described in Section 1.2 above. Record the chromatogram, and calculate the content of chloroethane in the test solution according to the external standard method. The calculation formula is as follows: Chloroethane (ppm) =

[0038] A S is the peak area of chloroethane measured in the test solution; A R is the average peak area of chloroethane measured by injecting the reference solution six times; W R is the concentration of chloroethane in the reference solution (mg / mL); W S is the sample concentration of the test solution (mg / mL).

[0039] Figure 1 and Figure 2 correspond to the chromatograms of the reference solution and the test solution respectively. Repeat the detection three times, and take the average value of the three detection results as the final detection result. Chloroethane was not detected in the test sample.

[0040] Example 2: System suitability According to the detection conditions in Section 1.2 of Example 1, inject the reference solution prepared in Example 1 six times repeatedly and re-inject once, and use a gas chromatograph for detection. The results are shown in Table 4: Table 4

[0041] As shown in Table 4, the RSD of the chloroethane peak in the reference solution for 6 injections was 4.2017%, and the change rate of the chloroethane peak area in the reference solution retest relative to the average value of the chloroethane peak areas in the 6 reference solutions was 3.9235%, indicating good system suitability.

[0042] Example 3: Specificity According to the detection conditions in Part 1.2 of Example 1, the blank solution, chloroethane single standard localization solution, tetrahydrofuran single standard localization solution, methanol single standard localization solution, reference solution, test solution, and test solution spiked with 100% test substance prepared in Example 1 were respectively detected using a gas chromatograph. The chromatogram of the reference solution is as Figure 1 shown, the chromatogram of the test solution is as Figure 2 shown, the chromatogram of the blank solution is as Figure 3 shown, the chromatogram of the test solution spiked with 100% test substance is as Figure 4 shown, the chromatogram of the chloroethane single standard localization solution is as Figure 5 shown, the chromatogram of the tetrahydrofuran single standard localization solution is as Figure 6 shown, and the chromatogram of the methanol single standard localization solution is as Figure 7 shown. The retention time results of each substance are shown in Table 5: Table 5

[0043] As shown in Table 5 and Figures 1 - 7 shown, based on the detection results of the chloroethane single standard localization solution, tetrahydrofuran single standard localization solution, and methanol single standard localization solution, it can be seen that the peak with a retention time of 4.8381 only appears in the chloroethane single standard localization solution. Therefore, it can be determined that the retention time of chloroethane is approximately around 4.84. The retention time of chloroethane in the test solution spiked with the standard substance corresponds to that in the reference solution, and the resolution between chloroethane and methanol is greater than 3. The blank solution does not interfere with the determination of the target analyte.

[0044] Example 4: Linearity According to the detection conditions in Part 1.2 of Example 1, the linear solution prepared in Example 1 was detected using a gas chromatograph, and the results are shown in Table 6: Table 6

[0045] Taking the concentration of chloroethane as the abscissa and the peak area as the ordinate, a standard working curve was plotted. The linear equation is Y = 7203970X - 234.363, R = 0.9999 ( Figure 8 ), and within the range of 0.001 - 0.020 mg / mL, there is a good linear relationship between the concentration of chloroethane and the peak area.

[0046] Example 5: Quantitation Limit and Detection Limit According to the detection conditions in Section 1.2 of Example 1, the quantitation limit solution and detection limit solution prepared in Example 1 were respectively subjected to gas chromatography detection, and the results are shown in Tables 7 and 8 as follows: Table 7

[0047] Table 8

[0048] As can be seen from Tables 7 and 8, the quantitation limit and detection limit of chloroethane meet the requirements.

[0049] Example 6: Accuracy According to the preparation method of the spiked solution in Example 1, three portions of the spiked solution with each concentration ratio were prepared respectively, and then gas chromatography detection was carried out according to the detection conditions in Section 1.2 of Example 1, and the results are shown in Table 9 as follows: Table 9

[0050] As can be seen from Table 9, the recovery rate is in the range of 85.0% - 115.0%, the recovery is good, the RSD is less than 3, and the accuracy of the method of the present invention is excellent.

[0051] Example 7: Repeatability According to the preparation method of the spiked solution in Example 1, six portions of the spiked solution with a concentration ratio of 100% were prepared respectively, and then gas chromatography detection was carried out according to the detection conditions in Section 1.2 of Example 1, and the results are shown in Table 10: Table 10

[0052] The detection results of the solutions numbered 1 - 3 in Table 10 are taken from the detection results of the solutions numbered 4 - 6 in Table 9. As can be seen from Table 10, the recovery rates of the six parallel detections are in the range of 85.0% - 115.0%, the RSD is less than 4, and the repeatability is good.

[0053] Example 8: Intermediate Precision Experiments in Example 7 were repeated by different experimenters at different times using chromatographs and chromatographic columns of the same manufacturer and model but different batches. The RSD was calculated by combining the obtained results with those of Example 7, and the results are shown in Table 11 as follows: Table 11

[0054] As can be seen from Table 11, the recoveries of 6 parallel detections repeated by different experimenters at different times using chromatographs and chromatographic columns of the same manufacturer and model but different numbers were in the range of 85.0% - 115.0%, with an RSD of less than 3, indicating good repeatability. The RSD of the recovery calculated by comprehensively considering the results of these 6 parallel detections and the results of Example 7 was less than 3, indicating good intermediate precision.

[0055] Example 9: Solution Stability The reference solution prepared in Example 1 was stored under the analysis conditions (room temperature), and samples were injected at different time points. Gas chromatography detection was carried out according to the detection conditions in Part 1.2 of Example 1, and the change rate of the peak area of chloroethane was calculated. The results are shown in Table 12: Table 12

[0056] Among them, the change rate of the peak area of chloroethane at the nth hour relative to the peak area of chloroethane at 0 h is: (peak area at tn - peak area at t0) / peak area at t0, where the peak area at tn is the peak area of chloroethane at the nth hour, and the peak area at t0 is the peak area of chloroethane at 0 h. As can be seen from Table 12, the change rate of the peak area of chloroethane in the reference solution was less than 10% within 32 h, indicating that the reference solution was stable within 32 h.

[0057] Example 10: Robustness The robustness of the method of the present invention was mainly verified from different transfer line temperatures, detector temperatures, flow rate changes, and different chromatographic columns. The conditions were adjusted according to Table 13 below. Except for the conditions listed in Table 13, the other conditions were the same as those in Part 1.2 of Example 1.

[0058] Table 13

[0059] Experiments were carried out according to the conditions listed in Table 13 above. For each condition, the blank solution was injected into the system until it reached equilibrium, and then 6 injections of the reference solution, 1 injection of the test solution, and 1 injection of the 100% test solution spiked solution were made, and a reference solution was injected again at the end of the sequence. The obtained results are shown in Table 14: Table 14

[0060] As can be seen from Table 14, under different detection conditions, when the reference solution was injected 6 times repeatedly, the RSD of the peak area of chloroethane was ≤ 10.0%. The recovery rate of chloroethane in the test solution spiked solution was 85.0% - 115.0%. Changes in transfer line temperature, detector temperature, flow rate, and replacement of the same model chromatographic column had no obvious effect on the detection results, indicating good robustness.

[0061] Comparative Example 1: Using a Shimadzu GC-2030 gas chromatograph, equipped with an FID, in the automatic injection mode, set the inlet temperature to 200 °C, the injection volume to 1 μL, the split ratio to 40:1, and the detector temperature to 300 °C. Also use a DB-624 (60 m × 0.32 mm × 1.8 μm) chromatographic column, set the column flow rate to 3.0 mL / min, and modify the temperature programming as follows: hold at 50 °C for 2 min, increase to 70 °C at a rate of 5 °C / min and hold for two minutes, then increase to 200 °C at a rate of 20 °C / min and hold for 10.5 min.

[0062] Prepare a 100% spiked solution of the test sample: Pipette 1 mL of the reference stock solution prepared in Example 1 into a 10 mL volumetric flask, add 500 mg of the test sample, and make up to the final volume with ethanol.

[0063] The results of the 100% spiked solution of the test sample are as Figure 9 shown. After testing, the recovery rate of its accuracy is between 88% and 94%. Compared with the headspace injection method used in the present invention, the recovery rate is significantly worse, which is caused by the interference of high-boiling test samples and the inherent hydrochloric acid in the test samples, resulting in severe baseline fluctuations. And from Figure 9 it can also be seen that there is an unknown impurity peak immediately after chloroethane. Considering that high-boiling test samples and the inherent hydrochloric acid in the test samples are likely to contaminate the inlet of the gas chromatograph, frequent maintenance of the inlet is required, which will increase the workload of the inspection personnel and the detection cost, and moreover, the risk of inaccurate detection cannot be completely avoided. The headspace injection method of the present invention is superior to the automatic injection method.

[0064] Comparative Example 2: Using N,N-dimethylacetamide (DMA) as a diluent, prepare each solution according to the ratio in Part 1.1 of Example 1, set the headspace oven temperature to 100 °C, and the other conditions are the same as in Part 1.2 of Example 1. Detect the test sample solution, each spiked solution, and the reference solution prepared with DMA, respectively. The results are as Figure 10As shown in the figure, from top to bottom, the integrated spectra are the 100% spiked test solution, the integrated spectra of the test solution and the reference solution, the spectrum of the 100% spiked test solution (data 1 represented by the black line), the spectrum of the test solution (data 2 represented by the purple line), and the spectrum of the reference solution (data 3 represented by the blue line). The peak with a retention time between 4.25 - 4.50 corresponds to chloroethane. It can be seen that a large amount of chloroethane is detected in the test sample. When detecting each spiked solution, the recovery rate is extremely abnormal. The reason for the abnormal recovery rate is that the inherent hydrochloric acid and ethyl ester in the test sample will react at high temperatures to produce chloroethane, thus interfering with the detection. N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) also belong to high-melting-point diluents. Those skilled in the art can reasonably infer that when using the above two diluents, a large amount of chloroethane will also be produced in the test sample, making it impossible to accurately measure the chloroethane in the test sample.

[0065] Comparative Example 3: Using a mixed solution of DMA:water = 9:1 as the diluent, and the rest is the same as in Comparative Example 2 for detection. The results are as Figure 11 shown. Figure 11 In the figure, the black line is the result of detection using a mixed solution of DMA:water = 9:1 as the diluent, and the purple line is the result of detection using pure DMA as the diluent. It can be seen that compared with the test solution prepared with a mixed solution of DMA:water = 9:1 as the diluent for injection, the chloroethane produced in the case of pure DMA is about 10 times that of the mixed solution of DMA:water = 9:1 as the diluent. This may be because in the presence of water, the process of hydrochloric acid and ethyl ester reacting to produce chloroethane will be inhibited. However, although water inhibits the above process, a large amount of chloroethane is still detected in the test solution, and it is impossible to accurately measure the chloroethane in the test sample using a mixed solution of DMA:water = 9:1 as the diluent.

[0066] Comparative Example 4: Using water as the diluent, and setting the headspace oven temperature to 80°C, the rest is the same as in Example 1. Each spiked solution prepared with water is detected respectively, and the recovery rate is between 73 - 95%, with a poor recovery rate.

[0067] Comparative Example 5: Using a chromatographic column of DB-624 (specification 30m × 0.53mm × 3.0μm), setting the initial temperature of the chromatographic column to 50°C, and the rest is the same as in Comparative Example 1. The chloroethane standard solution is detected. The results are as Figure 12 shown. It can be seen that chloroethane peaks on the tailing of methanol, and the presence of methanol interferes with the detection of chloroethane, thereby affecting the recovery rate.

[0068] As can be seen from the above examples and comparative examples, the detection method of the present invention has strong specificity, high sensitivity, good precision, good stability, and strong durability, and can accurately detect chloroethane in L-glutamic acid diethyl ester hydrochloride (LGDE·HCl). Moreover, the method of the present invention has high safety and low experimental cost.

[0069] The above are only the preferred embodiments of the present invention and do not impose any limitation on the present invention. Any person skilled in the art, within the scope of the technical solution of the present invention, makes any form of equivalent substitution or modification and other changes to the technical solution and technical content disclosed by the present invention, which are all within the content of the technical solution of the present invention and still fall within the protection scope of the present invention.

Claims

1. A method for detecting chloroethane in diethyl L - glutamate hydrochloride by headspace gas chromatography, characterized in that, The method includes the following steps: 1) Dilute the chloroethane methanol standard solution with ethanol to prepare a chloroethane reference solution; 2) Dissolve diethyl L-glutamate hydrochloride with ethanol to prepare a test solution; 3) Detect the chloroethane reference solution and the test solution by gas chromatography headspace method; Among them, the chromatographic column is a capillary column with a stationary phase of 6% cyanopropylphenyl 94% dimethylpolysiloxane. The length of the chromatographic column is 60 m, the inner diameter is 0.32 mm, and the film thickness is 1.8 μm; The temperature programming procedure is as follows: the initial starting temperature is 40°C - 50°C, maintained for 2 - 6 min, heated to 70 - 80°C at a rate of 5 - 20°C / min, and then heated to 200 - 240°C at a rate of 20 - 40°C / min, and held for 2 - 5 min.

2. The method according to claim 1, characterized in that, The chromatographic conditions in step 3) are as follows: Detector: hydrogen flame ionization detector, and the detector temperature is 300 ± 5°C; Headspace injector oven temperature: 50°C.

3. The method according to claim 1, wherein The temperature programming procedure is: the initial starting temperature is 40°C, maintained for 6 min, heated to 80°C at a rate of 10°C / min, and then heated to 240°C at a rate of 40°C / min, and held for 2 min.

4. The method according to claim 1, wherein The chromatographic conditions in step 3) also include: Sample flow path temperature: 60°C; Transfer line temperature: 70 ± 5°C; Headspace injector constant temperature time: 30 min; GC cycle time of the headspace injector: 36 min; Inject in split mode, and the split ratio is 10:

1.

5. The method according to claim 1, characterized in that, In step 3), a 20 mL headspace vial is used, and the sample loading volume is 1 mL.

6. The method according to claim 2, wherein The flow rate of the chromatographic column is 3.0 ± 0.2 mL / min.

7. The method according to claim 1, wherein The chromatographic conditions in step 3) are as follows: Detector: hydrogen flame ionization detector, and the detector temperature is 300°C; Chromatographic column: a capillary column with a stationary phase of 6% cyanopropylphenyl 94% dimethylpolysiloxane. The length of the chromatographic column is 60 m, the inner diameter is 0.32 mm, and the film thickness is 1.8 μm; Headspace injector oven temperature: 50°C; Temperature programming procedure: the initial starting temperature is 40°C, maintained for 6 min, heated to 80°C at a rate of 10°C / min, and then heated to 240°C at a rate of 40°C / min, and held for 2 min; Sample flow path temperature: 60°C; Transfer line temperature: 70°C; Headspace injector constant temperature time: 30 min; GC cycle time of the headspace injector: 36 min; Inject in split mode, and the split ratio is 10:1; The flow rate of the chromatographic column is 3.0 mL / min.

8. The method according to claim 1, wherein The concentration of chloroethane in the chloroethane reference solution is 0.01 mg / mL, and the concentration of diethyl L-glutamate hydrochloride in the test solution is 10 mg / mL.

9. The method according to any one of claims 1-8, characterized in that Use the external standard method to calculate the content of chloroethane in the diethyl L-glutamate hydrochloride.

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