Method for detecting D-isomer impurities in L-glutamic acid diethyl ester hydrochloride by HPLC (High Performance Liquid Chromatography) method

Abstract

The invention discloses a method for detecting isomer impurities in L-glutamic acid diethyl ester hydrochloride by HPLC (High Performance Liquid Chromatography), and belongs to the technical field of chemical engineering and pharmacy. D-isomer impurities in L-glutamic acid diethyl ester hydrochloride are detected by adopting high performance liquid chromatography, a CHIRALPAK AY-H column is adopted as a chromatographic column, an ethanol solution of 0.05% diethylamine is adopted as a mobile phase, isocratic elution is carried out, and the content of the D-isomer impurities in L-glutamic acid diethyl ester hydrochloride is detected. According to the present invention, the chromatographic conditions are as follows: the chromatographic conditions are as follows: the chromatographic conditions are as follows: the chromatographic conditions are as follows: the chromatographic conditions are as follows: the chromatographic conditions are as follows: the column temperature is 35 DEG C and the detection wavelength is 220 nm, the L-glutamic acid diethyl ester hydrochloride and the D-isomer thereof can be effectively separated and the content thereof can be quantitatively detected, the method has characteristics of simple operation, strong specificity, high sensitivity, high accuracy and good linearity, and the quality of the p-fluorothiophenol product can be effectively controlled.

Description

Technical Field

[0001] The invention relates to the technical field of chemical industry and pharmaceutical industry, and in particular to a method for detecting D-isomer impurities in L-glutamic acid diethyl ester hydrochloride by HPLC. Background Art

[0002] L-Glutamic acid diethyl ester hydrochloride contains both L and D configurations. Studies have shown that only the L configuration is biologically active, playing a key role in development, learning, memory, adaptability, and cognition. It plays a crucial role in the pathogenesis of neuropathological disorders such as epilepsy, schizophrenia, stroke, ischemia, amyotrophic lateral sclerosis, Huntington's disease, and Parkinson's disease. Glutamate is also responsible for the activation of long-term potentiation (LTP) and long-term depression (LTD), and the induction of glutamate receptors plays a key role in the pathophysiology of migraine. It is also an organic synthesis intermediate and a pharmaceutical intermediate, useful in laboratory research and development and chemical pharmaceutical synthesis, with broad application prospects in pharmaceutical therapy and chemical synthesis.

[0003] Currently, there is no disclosure in the prior art about the detection method of D-isomer impurities in L-glutamic acid diethyl ester hydrochloride. Therefore, it is very necessary to develop an HPLC method that can effectively detect D-isomer impurities in L-glutamic acid diethyl ester hydrochloride. Summary of the Invention

[0004] In order to solve the technical problems mentioned in the above background technology, the present invention provides a method for detecting D-isomer impurities in L-glutamic acid diethyl ester hydrochloride by HPLC, comprising the following steps:

[0005] 1) Instrument selection: High performance liquid chromatography (HPLC); CHIRALPAK AY-H column, 4.6 mm × 250 mm, 5 μm or equivalent; mobile phase: 0.05% diethylamine in ethanol; UV detector, detection wavelength: 200-300 nm; mobile phase flow rate: 0.5-1.5 ml / min; column temperature: 25-45°C; column length: 100-250 mm;

[0006] 2) Prepare the following solutions:

[0007] a. Solvent: The volume ratio of diethylamine to ethanol is 0.5:100;

[0008] b. Blank solution: take an appropriate amount of solvent;

[0009] c. Impurity stock solution: Take 20 mg of D-glutamic acid diethyl ester hydrochloride reference substance, accurately weigh it, place it in a 50 ml volumetric flask, dissolve it with solvent and dilute it to the scale, shake well;

[0010] d. System suitability solution: Take about 80 mg of this product, accurately weigh it, place it in a 10 ml volumetric flask, add an appropriate amount of solvent to dissolve it, add 1.0 ml of impurity stock solution, dilute to the scale with solvent, and shake well;

[0011] e. Reference solution: Accurately measure 1.0 ml of the impurity stock solution and place it in a 10 ml volumetric flask. Dissolve it in solvent and dilute to the mark. Shake well.

[0012] f. Test solution: Take about 80 mg of the product, accurately weigh it, place it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well;

[0013] Inject samples according to the following sequence:

[0014] name Number of injection needles blank solution ≥1 injection System suitability solution 1 needle Reference solution 5-pin Test solution 1 needle

[0015] The D-isomer impurity in the L-glutamic acid diethyl ester hydrochloride test solution was calculated by peak area using the external standard method.

[0016] ① The conditions of the high performance liquid chromatography method include: using an ultraviolet detector, the detection wavelength is preferably 220 nm; the optimal flow rate of the mobile phase is 0.6 ml / min; the column temperature is 35°C; the chromatographic column is preferably CHIRALPAK AY-H, 4.6 mm × 250 mm, 5 μm; and the column length is 250 mm.

[0017] ②The impurity is D-glutamic acid diethyl ester hydrochloride.

[0018] The present invention has the following advantages: the present invention can effectively separate L-glutamic acid diethyl ester hydrochloride and its D-isomer impurity; the method is simple to operate, has strong specificity, and is highly sensitive and accurate, thereby ensuring that the quality of L-glutamic acid diethyl ester hydrochloride is controllable; and the present invention can provide a reliable detection method for the quality control of L-glutamic acid diethyl ester hydrochloride. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a linear test result diagram of the present invention;

[0020] Figure 2 is the blank solution spectrum of the present invention;

[0021] Figure 3 is the system suitability solution map of the present invention;

[0022] Figure 4 The reference solution of the present invention is shown in FIG.

[0023] Figure 5 It is a sample solution atlas of the present invention. DETAILED DESCRIPTION

[0024] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0025] Example 1, investigation of system precision and specificity:

[0026] A method for detecting D-isomer impurities in L-glutamic acid diethyl ester hydrochloride comprises the following steps: instrument and chromatographic conditions: instrument: high performance liquid chromatograph; chromatographic column: CHIRALPAK AY-H, 4.6 mm×250 mm, 5 μm; mobile phase: 0.05% diethylamine ethanol solution, wavelength: 220 nm, column temperature: 35° C., flow rate: 0.6 ml / min, injection volume: 20 μL, isocratic elution, and recording the chromatogram to 1.5 times the retention time of the main peak.

[0027] Reference solution: Take 20 mg of D-glutamic acid diethyl ester hydrochloride reference substance, accurately weigh it, place it in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and use it as the D-isomer reference substance stock solution; accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0028] Potential impurity locating solution: Take 20 mg each of L-glutamic acid reference substance, L-glutamic acid-1-ethyl ester, L-glutamic acid-5-ethyl ester, and L-pyroglutamic acid ethyl ester, accurately weigh them, place them in different 50 ml volumetric flasks, add solvent to dissolve and dilute to the scale, shake well, and use them as the stock solution of each potential impurity reference substance; accurately measure 1 ml of each stock solution, place it in different 10 ml volumetric flasks, dilute to the scale with solvent, shake well, and use them as the potential impurity locating solution.

[0029] Mixed solution: Take 80 mg of this product, accurately weigh it, and place it in a 10 ml volumetric flask. First, add solvent to dissolve it, then add 1 ml each of D-isomer reference substance stock solution, L-glutamic acid reference substance stock solution, L-glutamic acid-1-ethyl ester reference substance stock solution, L-glutamic acid-5-ethyl ester reference substance stock solution and L-pyroglutamic acid ethyl ester reference substance stock solution, then dilute to the scale with solvent and shake well.

[0030] Accurately measure 20 μL each of the blank solution, reference solution, each potential impurity locating solution, and the mixed solution, inject them into the liquid chromatograph, and record the liquid chromatogram. System precision results are shown in the table below. The maximum RSD for the peak area of the five reference solutions in the table below is 0.7%.

[0031] The following table is the system precision test results:

[0032]

[0033]

[0034] The specificity test results are shown in the table below. The experimental results of the impurity positioning and separation in the table below show that the blank solution has no interference with the sample detection, other impurities have no interference with the sample determination, and the separation between L-glutamic acid diethyl ester hydrochloride and the D-isomer impurity is 4.0.

[0035] The following table is the specificity test results table:

[0036]

[0037] Example 2, quantification limit investigation:

[0038] Accurately pipette an appropriate amount of the impurity stock solution, dissolve it in solvent, and dilute it to a solution with a D-isomer impurity concentration of approximately 6 μg / mL, which serves as the quantification limit solution. Six consecutive injections of 20 μL of the quantification limit solution were performed, and the chromatograms were recorded. The results are shown in the table below. The quantification limit determination results show that the ratio of the quantification limit concentration of the D-isomer impurity to the test sample concentration is 0.08%. The maximum RSD of the peak areas of the six quantification limit solutions is 6.5%, which is less than 10%.

[0039] The following table is the quantitative limit test results:

[0040] name Limit of quantification (μg / ml) Concentration ratio of the test sample % RSD% of 6-needle peak area D-isomer 6.19 0.08％ 6.5％

[0041] Example 3, linear investigation:

[0042] Accurately pipette appropriate amount of impurity stock solution, dissolve it in diluent and dilute it step by step to make solutions with impurity concentration of 8μg / ml, 20μg / ml, 40μg / ml, 60μg / ml and 80μg / ml respectively. Prepare them as linear solutions according to the method of "Preparation of Reference Solution". Take 20μL of each of the above linear solutions, inject them into liquid chromatograph, record the chromatogram, and perform linear regression analysis with concentration as the horizontal axis and peak area as the vertical axis. The linearity is good and the correlation coefficient is 0.9980. The specific results are shown in Figure 1 .

[0043] Example 4, accuracy inspection:

[0044] Accurately weigh 80 mg of L-glutamic acid diethyl ester hydrochloride sample, a total of 9 portions, and place them in 10 ml volumetric flasks. 3 portions form a group. Add 0.5 ml, 1.0 ml, and 1.5 ml of the impurity stock solution at low, medium, and high concentrations, respectively. Prepare solutions according to the "Preparation of System Suitability Solutions" method. Samples are injected and analyzed separately. Record the chromatograms and calculate the recovery. The average recovery of the D-isomer impurity is 101.9%. The results are shown in the following table:

[0045] The following table is the accuracy test results:

[0046]

[0047]

[0048] The present invention is simple to operate, easy to use, and suitable for comprehensive promotion and application. Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting the D-isomer in L-glutamic acid diethyl ester hydrochloride by HPLC, characterized in that: The following steps are involved: 1) Instrument selection: High performance liquid chromatography; Chromatographic column: CHIRALPAK AY-H; mobile phase: 0.05% diethylamine in ethanol; 2) Prepare the following solutions: a. Solvent: The volume ratio of diethylamine to ethanol is 0.5:100; b. Blank solution: take an appropriate amount of solvent; c. Impurity stock solution: Take 20 mg of D-glutamic acid diethyl ester hydrochloride reference substance, accurately weigh it, place it in a 50 ml volumetric flask, dissolve it with solvent and dilute it to the scale, shake well; d. System suitability solution: Take about 80 mg of this product, accurately weigh it, place it in a 10 ml volumetric flask, add an appropriate amount of solvent to dissolve it, add 1.0 ml of impurity stock solution, dilute to the scale with solvent, and shake well; e. Reference solution: Accurately measure 1.0 ml of the impurity stock solution and place it in a 10 ml volumetric flask. Dissolve it in solvent and dilute to the mark. Shake well. f. Test solution: Take about 80 mg of the product, accurately weigh it, place it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well; 3) Measurement record: Accurately measure the blank solution, system suitability solution, reference solution, and test solution and inject them into the liquid chromatograph respectively, record the liquid chromatogram, and calculate the peak area according to the external standard method.

2. The method for detecting D-isomer impurities in L-glutamic acid diethyl ester hydrochloride by HPLC according to claim 1, wherein: An ultraviolet detector is used with a detection wavelength of 200 to 300 nm; the flow rate of the mobile phase is 0.5 to 1.5 ml / min; the column temperature is 25 to 45° C.; and the length of the chromatographic column is 100 to 250 mm.

3. The method for detecting D-isomer impurities in L-glutamic acid diethyl ester hydrochloride by a HPLC method according to claim 1, wherein: The chromatographic column is CHIRALPAK AY-H, 4.6 mm × 250 mm, 5 μm or a chromatographic column with equivalent performance.

4. The method for detecting D-isomer impurities in L-glutamic acid diethyl ester hydrochloride by HPLC according to claim 1, wherein: The mobile phase was 0.05% diethylamine in ethanol.

5. The method for detecting D-isomer impurities in L-glutamic acid diethyl ester hydrochloride by HPLC according to claim 1, characterized in that: The impurity is D-glutamic acid diethyl ester hydrochloride.

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