Separation and detection method for related impurities of L-glutamic acid diethyl ester hydrochloride

Abstract

The invention discloses a method for separating and detecting related impurities of L-glutamic acid diethyl ester hydrochloride, which comprises the following steps: carrying out gradient elution under certain conditions by adopting a chromatographic column taking octadecylsilane chemically bonded silica as a filler and taking a mixed solution of a sodium perchlorate buffer salt solution with high concentration and low pH value and acetonitrile as a mobile phase; l-glutamic acid diethyl ester hydrochloride and five related impurities can be effectively separated. The method is good in detection result accuracy and high in sensitivity, so that a good guarantee is provided for quality control of the L-glutamic acid diethyl ester hydrochloride.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis and detection, and particularly relates to a method for separating and detecting L-glutamic acid diethyl ester hydrochloride related impurities by adopting high performance liquid chromatography. Background Art

[0002] L-Glutamic acid diethyl ester hydrochloride is widely used. In the pharmaceutical field, it can be used for the synthesis of multiple drugs, such as raltitrexed, pemetrexed disodium, etc. Its chemical formula is C 9 H 17 NO 4 , the structural formula is shown in Table 1.

[0003] During the production and storage process, L-glutamic acid diethyl ester hydrochloride will introduce related impurities such as L-2-aminoglutaric acid (impurity 1), (S)-4-amino-5-ethoxy-5-oxopentanoic acid (impurity 2), (S)-2-amino-5-ethoxy-5-oxopentanoic acid (impurity 3), (S)-2-pyrrolidone-5-carboxylic acid (impurity 4), and (S)-2-pyrrolidone-5-carboxylic acid ethyl ester (impurity 5). According to research, these impurities are detected in large amounts in L-glutamic acid diethyl ester hydrochloride, and may participate in subsequent reactions and affect the quality control of subsequent products.

[0004] Table 1 Structural formula of L-glutamic acid diethyl ester and its related impurities

[0005] At present, there are few literature reports on the analysis and detection of L-glutamic acid diethyl ester. Patent CN104515815A discloses an analysis and detection method of L-glutamic acid diethyl ester, and patent CN110749692A discloses a separation and detection method of L-glutamic acid diethyl ester hydrochloride and its optical isomers, but the existing method has limited separation effect and cannot separate and quantitatively detect L-glutamic acid diethyl ester and 5 related impurities. Therefore, developing an analysis and detection method that can separate and accurately detect L-glutamic acid diethyl ester related impurities plays a vital role in improving the quality control of L-glutamic acid diethyl ester hydrochloride and subsequent products. Summary of the invention

[0006] The object of the present invention is to provide a separation and detection method for L-glutamate diethyl ester related impurities. The method uses a low pH and high concentration chaotropic agent as a mobile phase and adopts a specific octadecylsilane bonded silica gel chromatographic column to effectively separate L-glutamate diethyl ester and five related impurities and accurately detect them. By controlling the quality of L-glutamate diethyl ester, the quality of subsequent products raltitrexed and pemetrexed disodium is controlled to ensure the effectiveness and safety of the drugs.

[0007] In order to achieve the above-mentioned invention object, the technical solution provided by the present invention is as follows: A method for separating and detecting impurities related to L-glutamic acid diethyl ester hydrochloride, characterized in that high performance liquid chromatography is used, and the chromatographic conditions are: Chromatographic column: Agilent ZORBAX SB-C18, column length 250 mm, particle size 5 μm; Mobile phase: Mobile phase A is a sodium perchlorate buffer-acetonitrile mixed solution, and mobile phase B is acetonitrile; Elution method: gradient elution; Detector: UV detector.

[0008] Furthermore, in the above method of the present invention, the concentration of sodium perchlorate buffer is 100 mmol / L.

[0009] Furthermore, in the above method of the present invention, the pH of the sodium perchlorate buffer is 1.8-2.2, preferably 2.0.

[0010] Furthermore, in the above method of the present invention, the volume ratio of sodium perchlorate buffer to acetonitrile is (96:4) to (98:2), preferably 97:3.

[0011] Furthermore, in the above method of the present invention, the column temperature is 25°C.

[0012] Furthermore, in the above method of the present invention, the flow rate is 1.0 ml / min.

[0013] Furthermore, in the above method of the present invention, the detection wavelength is 210 nm.

[0014] Furthermore, in the above method of the present invention, the elution procedure is:

[0015] The method of the invention realizes the effective separation and detection of L-glutamic acid diethyl ester hydrochloride and five related impurities thereof, has the advantages of high accuracy, good repeatability, high sensitivity, simple operation process, etc., can effectively control the quality of L-glutamic acid diethyl ester hydrochloride, and improves the quality of subsequent final products. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 HPLC chromatogram of the specific solution in Example 1; Figure 2 HPLC chromatogram of the sensitivity solution in Example 1; Figure 3 HPLC chromatogram of the test solution in Example 1; Figure 4 HPLC chromatogram of the mixed solution in Example 2; Figure 5HPLC chromatogram of the mixed solution in Example 3; Figure 6 HPLC chromatogram of the mixed solution in Example 4; Figure 7 HPLC chromatogram of the mixed solution in Example 5; Figure 8 HPLC chromatogram of the mixed solution in Comparative Example 1; Fig. 9 HPLC chromatogram of the mixed solution after replacing the chromatographic column in Comparative Example 1; Fig.10 HPLC chromatogram of the mixed solution in Comparative Example 2; Fig.11 HPLC chromatogram of the mixed solution after replacing the chromatographic column and optimizing the mobile phase in Comparative Example 2. DETAILED DESCRIPTION

[0017] The following examples are used to further illustrate and understand the spirit of the present invention, but are not intended to limit the scope of protection of the present invention in any way.

[0018] Example 1 Chromatographic conditions Chromatographic column: Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm), connected to a ghost peak trapping column Mobile phase A: 100mmol / L sodium perchlorate (pH2.0)-acetonitrile (97:3) Mobile phase B: acetonitrile Detection wavelength: 210nm Column temperature: 25°C Flow rate: 1.0ml / min Injection volume: 15µl Needle washing solution: acetonitrile-water (10:90) Sample plate temperature: 5°C Solvent: Water Elution procedure:

[0019] Experimental steps: Specific solution: Take an appropriate amount of L-glutamic acid diethyl ester hydrochloride test sample, grind it evenly in an agate mortar, take about 60 mg of the test sample, accurately weigh it, and put it in a 5 ml volumetric flask, then take an appropriate amount of 5 impurity reference substances, and use solvent to prepare a mixed solution containing 12 mg of L-glutamic acid diethyl ester, 120 μg of impurity 1 and impurity 4 reference substances, 180 μg of impurity 2 and impurity 3 reference substances, and 48 μg of impurity 5 reference substance per 1 ml.

[0020] Sensitivity solution: Take appropriate amounts of L-glutamic acid diethyl ester hydrochloride reference substance and 5 impurity reference substances, and use solvent to prepare mixed solutions containing 6 μg of L-glutamic acid diethyl ester and 6 μg of 5 impurity reference substances per 1 ml.

[0021] Test solution: Take an appropriate amount of L-glutamic acid diethyl ester hydrochloride test sample, grind it evenly in an agate mortar, take about 60 mg of the test sample, accurately weigh it, put it in a 5 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0022] Determination: Take 15µl of the above-mentioned specific solution, sensitivity solution and test solution respectively and inject them into the high performance liquid chromatograph and record the chromatogram.

[0023] See attached for specific solution results Figure 1 , L-glutamic acid diethyl ester hydrochloride and five related impurities were baseline separated.

[0024] Sensitivity solution results Figure 2 The signal-to-noise ratios of L-glutamic acid diethyl ester hydrochloride and five related impurities were all much greater than 10.

[0025] The test solution results are attached. Figure 3 , L-glutamic acid diethyl ester hydrochloride and four related impurities were baseline separated.

[0026] Example 2 Chromatographic conditions Chromatographic column: Agilent ZORBAX SB-C18 (4.6mm×250mm, 5μm), connected with ghost peak column Mobile phase A: 100mmol / L sodium perchlorate (pH1.8)-acetonitrile (97:3) Mobile phase B: acetonitrile Detection wavelength: 210nm Column temperature: 25°C Flow rate: 1.0ml / min Injection volume: 15µl Needle washing solution: acetonitrile-water (10:90) Sample plate temperature: 5°C Solvent: Water Elution procedure:

[0027] Experimental steps: Mixed solution: Take an appropriate amount of L-glutamate diethyl ester hydrochloride test sample (the L-glutamate diethyl ester hydrochloride test sample itself contains impurity 4, which can degrade impurity 5 after being left for a period of time), grind it evenly in an agate mortar, take about 60 mg of the test sample, accurately weigh it, and place it in a 5 ml volumetric flask, then take an appropriate amount of impurity 1, impurity 2, and impurity 3 reference substances respectively, and use solvent to prepare a mixed solution containing 12 mg of L-glutamate diethyl ester and 180 μg of impurity 1, impurity 2, and impurity 3 reference substances per 1 ml.

[0028] Determination: Take 15µl of the above mixed solution and inject it into the high performance liquid chromatograph and record the chromatogram.

[0029] Mixed solution results are attached Figure 4 , L-glutamic acid diethyl ester hydrochloride and five related impurities were baseline separated.

[0030] Example 3 Chromatographic conditions Chromatographic column: Agilent ZORBAX SB-C18 (4.6mm×250mm, 5μm), connected with ghost peak column Mobile phase A: 100mmol / L sodium perchlorate (pH2.2)-acetonitrile (97:3) Mobile phase B: acetonitrile Detection wavelength: 210nm Column temperature: 25°C Flow rate: 1.0ml / min Injection volume: 15µl Needle washing solution: acetonitrile-water (10:90) Sample plate temperature: 5°C Solvent: Water Elution procedure:

[0031] Experimental steps: Mixed solution: Take an appropriate amount of L-glutamate diethyl ester hydrochloride test sample (the L-glutamate diethyl ester hydrochloride test sample itself contains impurity 4, which can degrade impurity 5 after being left for a period of time), grind it evenly in an agate mortar, take about 60 mg of the test sample, accurately weigh it, and place it in a 5 ml volumetric flask, then take an appropriate amount of impurity 1, impurity 2, and impurity 3 reference substances respectively, and use solvent to prepare a mixed solution containing 12 mg of L-glutamate diethyl ester and 180 μg of impurity 1, impurity 2, and impurity 3 reference substances per 1 ml.

[0032] Determination: Take 15µl of the above mixed solution and inject it into the high performance liquid chromatograph and record the chromatogram.

[0033] Mixed solution results are attached Figure 5, L-glutamic acid diethyl ester hydrochloride and five related impurities were baseline separated.

[0034] Example 4 Chromatographic conditions Chromatographic column: Agilent ZORBAX SB-C18 (4.6mm×250mm, 5μm), connected with ghost peak column Mobile phase A: 100mmol / L sodium perchlorate (pH2.0)-acetonitrile (96:4) Mobile phase B: acetonitrile Detection wavelength: 210nm Column temperature: 25°C Flow rate: 1.0ml / min Injection volume: 15µl Needle washing solution: acetonitrile-water (10:90) Sample plate temperature: 5°C Solvent: Water Elution procedure:

[0035] Experimental steps: Mixed solution: Take an appropriate amount of L-glutamate diethyl ester hydrochloride test sample (the L-glutamate diethyl ester hydrochloride test sample itself contains impurity 4, which can degrade impurity 5 after being left for a period of time), grind it evenly in an agate mortar, take about 60 mg of the test sample, accurately weigh it, and place it in a 5 ml volumetric flask, then take an appropriate amount of impurity 1, impurity 2, and impurity 3 reference substances respectively, and use solvent to prepare a mixed solution containing 12 mg of L-glutamate diethyl ester and 180 μg of impurity 1, impurity 2, and impurity 3 reference substances per 1 ml.

[0036] Determination: Take 15µl of the above mixed solution and inject it into the high performance liquid chromatograph and record the chromatogram.

[0037] Mixed solution results are attached Figure 6 , L-glutamic acid diethyl ester hydrochloride and five related impurities were baseline separated.

[0038] Example 5 Chromatographic conditions Chromatographic column: Agilent ZORBAX SB-C18 (4.6mm×250mm, 5μm), connected with ghost peak column Mobile phase A: 100mmol / L sodium perchlorate (pH2.0)-acetonitrile (98:2) Mobile phase B: acetonitrile Detection wavelength: 210nm Column temperature: 25°C Flow rate: 1.0ml / min Injection volume: 15µl Needle washing solution: acetonitrile-water (10:90) Sample plate temperature: 5°C Solvent: Water Elution procedure:

[0039] Experimental steps: Mixed solution: Take an appropriate amount of L-glutamate diethyl ester hydrochloride test sample (the L-glutamate diethyl ester hydrochloride test sample itself contains impurity 4, which can degrade impurity 5 after being left for a period of time), grind it evenly in an agate mortar, take about 60 mg of the test sample, accurately weigh it, and place it in a 5 ml volumetric flask, then take an appropriate amount of impurity 1, impurity 2, and impurity 3 reference substances respectively, and use solvent to prepare a mixed solution containing 12 mg of L-glutamate diethyl ester and 180 μg of impurity 1, impurity 2, and impurity 3 reference substances per 1 ml.

[0040] Determination: Take 15µl of the above mixed solution and inject it into the high performance liquid chromatograph and record the chromatogram.

[0041] Mixed solution results are attached Figure 7 , L-glutamic acid diethyl ester hydrochloride and five related impurities were baseline separated.

[0042] Comparative Example 1 The liquid chromatography conditions of CN104515815A were used to separate and detect L-glutamic acid diethyl ester hydrochloride and five related impurities.

[0043] Chromatographic column: Yuexu Ultimate XB-C18, (4.6mm×250mm, 5μm) Mobile phase: 50mmol / L sodium dihydrogen phosphate-50mmol / L disodium hydrogen phosphate-acetonitrile (80:5:15) (pH7.0) Detection wavelength: 205nm Column temperature: 27°C Flow rate: 1.0ml / min Injection volume: 15µl Needle washing solution: methanol-water (10:90) Sample plate temperature: 5°C Solvent: Water Elution method: isocratic elution for 30 min

[0044] Experimental steps: Mixed solution: Take an appropriate amount of L-glutamic acid diethyl ester hydrochloride test sample, grind it evenly in an agate mortar, take about 60 mg of the test sample, accurately weigh it, and put it in a 5 ml volumetric flask, then take an appropriate amount of impurity 1, impurity 2, impurity 3, impurity 4, and impurity 5 reference substances, and use solvent to prepare a mixed solution containing 12 mg of L-glutamic acid diethyl ester, 180 μg of impurity 1, impurity 2, and impurity 3 reference substances, and 160 μg of impurity 4 and impurity 5 reference substances per 1 ml.

[0045] Determination: Take 15µl of the above mixed solution and inject it into the high performance liquid chromatograph and record the chromatogram.

[0046] Mixed solution results are attached Figure 8 , the peak shape of L-glutamic acid diethyl ester hydrochloride and impurities is poor, and the separation between L-glutamic acid diethyl ester hydrochloride and 5 related impurities cannot be achieved.

[0047] The chromatographic column was replaced with Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm), and other chromatographic conditions remained unchanged. The results are shown in the attached Fig. 9 , the peak shapes of L-glutamic acid diethyl ester hydrochloride and impurities were improved, but the separation between L-glutamic acid diethyl ester hydrochloride and 5 related impurities could not be achieved.

[0048] Comparative Example 2 The liquid chromatography conditions of CN110749692A were used to separate and detect L-glutamic acid diethyl ester hydrochloride and five related impurities.

[0049] Chromatographic column: DAICEL CROWNPAKCR-I(+), (3.0mm×150mm, 5μm) Mobile phase: perchloric acid aqueous solution (pH 2.0)-acetonitrile (60:40) Detection wavelength: 205nm Column temperature: 25°C Flow rate: 1.0ml / min Injection volume: 15µl Needle washing solution: methanol-water (10:90) Sample plate temperature: 5°C Solvent: Water Elution method: isocratic elution for 30 min

[0050] Experimental steps: Mixed solution: Take an appropriate amount of L-glutamic acid diethyl ester hydrochloride test sample, grind it evenly in an agate mortar, take about 60 mg of the test sample, accurately weigh it, and put it in a 5 ml volumetric flask, then take an appropriate amount of impurity 1, impurity 2, impurity 3, impurity 4, and impurity 5 reference substances, and use solvent to prepare a mixed solution containing 12 mg of L-glutamic acid diethyl ester, 180 μg of impurity 1, impurity 2, and impurity 3 reference substances, and 160 μg of impurity 4 and impurity 5 reference substances per 1 ml.

[0051] Determination: Take 15µl of the above mixed solution and inject it into the high performance liquid chromatograph and record the chromatogram.

[0052] Mixed solution results are attached Fig.10 , L-glutamic acid diethyl ester hydrochloride has poor retention, and it is impossible to separate L-glutamic acid diethyl ester hydrochloride from 5 related impurities. According to the information, acetonitrile is not recommended as the mobile phase for this type of chromatographic column, and organic solvents other than methanol will cause irreversible damage to the chromatographic column.

[0053] The chromatographic column was replaced with Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm), and the proportion of acetonitrile in the mobile phase was reduced. The mobile phase was perchloric acid aqueous solution (pH 2.0)-acetonitrile (60:40). Other chromatographic conditions remained unchanged. The results are shown in the attached Fig.11 , reducing the proportion of acetonitrile in the mobile phase, the retention of L-glutamate diethyl hydrochloride and impurities was not significantly improved, and the L-glutamate diethyl hydrochloride peak was obviously tailing, and the separation of L-glutamate diethyl hydrochloride and the five related impurities could not be achieved.

Claims

1. A method for separating and detecting impurities related to L-glutamic acid diethyl ester hydrochloride, It is characterized in that High performance liquid chromatography was used, and the chromatographic conditions were as follows: Chromatographic column: Agilent ZORBAX SB-C18, column length 250 mm, particle size 5 μm; Mobile phase: Mobile phase A is sodium perchlorate buffer-acetonitrile, and mobile phase B is acetonitrile; Elution mode: gradient elution; Detector: UV detector.

2. method according to claim 1, described sodium perchlorate buffer concentration is 100mmol / L.

3. The method according to claim 1, wherein the pH of the sodium perchlorate buffer is 1.8 to 2.

2.

4. The method according to claim 3, wherein the pH of the sodium perchlorate buffer is 2.

0.

5. The method according to claim 1, wherein the volume ratio of the sodium perchlorate buffer to acetonitrile is (96:4) to (98:2).

6. method according to claim 5, the volume ratio of described sodium perchlorate buffer solution and acetonitrile is 97:

3.

7. The method according to claim 1, wherein the column temperature in the chromatographic conditions is 25°C.

8. The method according to claim 1, wherein the flow rate in the chromatographic conditions is 1.0 ml / min.

9. The method according to claim 1, wherein the detection wavelength in the chromatographic conditions is 210 nm.

10. The method according to claim 1, It is characterized in that The elution procedure is: 。

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