CN109490460B - Detection method of L-2-amino-5-guanidino valeric acid related substances - Google Patents
Detection method of L-2-amino-5-guanidino valeric acid related substances Download PDFInfo
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Abstract
The invention discloses a detection method of L-2-amino-5-guanidino valeric acid related substances, which comprises the steps of chromatographic condition and system applicability test and the determination of the L-2-amino-5-guanidino valeric acid related substances; calculating 2-ketoglutaric acid by peak area according to external standard method: (A)S) -2-acetylaminoglutaric acid andE) -content of 2-butenedioic acid; and calculating the content of other single unknown impurities and the sum of other unknown impurities by using the main peak area of the control solution according to a self-comparison method. The detection method has higher sensitivity, accuracy, precision and strong durability, can accurately reflect the content of related substances of the L-2-amino-5-guanidino valeric acid raw material medicine, is suitable for the detection and quality control of the related substances of the L-2-amino-5-guanidino valeric acid in industry, thereby providing a basis for reasonable quality standard establishment, facilitating better control and mastering of product quality and ensuring the safety of eating and medicine.
Description
Technical Field
The invention belongs to the technical field of food and drug analysis, and particularly relates to a detection method of L-2-amino-5-guanidino valeric acid related substances.
Background
L-2-amino-5-guanidinopentanoic acid (CAS registry number 74-79-3) belongs to amino acid compounds, has important physiological functions, is often used as a nutritional supplement, and is also an important component of amino acid capsules and infusion. The L-2-amino-5-guanidino valeric acid can effectively improve immunity, promote the secretion of endogenous substances by an immune system, and is favorable for resisting cancer cells and preventing virus infection; is beneficial to the health care of patients who need a large amount of tissue repair, such as serious trauma, burn and the like. Clinically, the L-2-amino-5-guanidinopentanoic acid is suitable for hepatic coma with high blood ammonia, especially for patients with alkalosis; for aiding in the determination of pituitary function; for male infertility caused by insufficient semen secretion and azoospermia; can be used for infant to supplement deficiency of L-2-amino-5-guanidinopentanoic acid.
At present, L-2-amino-5-guanidino valeric acid is mainly produced by a microbial fermentation method, a large amount of organic acid homolog impurities are generated in the metabolic process (see figure 1), and the L-2-amino-5-guanidino valeric acid is a main source of related substances of raw material medicaments of L-2-amino-5-guanidino valeric acid. The raw materials which are difficult to completely separate and remove mainly comprise 2-ketoglutaric acid, (S) -2-acetamidoglutaric acid, and (E) -2-butenedioic acid, and the like, and are easy to remain in the raw material of L-2-amino-5-guanidinopentanoic acid, thereby affecting the product quality. The L-2-amino-5-guanidino valeric acid recorded in the pharmacopoeia of various countries of the prior edition has no related substance inspection item, so that the existence condition of impurities of the organic acid homologues in the L-2-amino-5-guanidino valeric acid raw material medicine can not be clarified, the quality control of a preparation prepared by using the raw material is not facilitated, and the potential safety hazard is brought to clinical medication. The prior art does not relate to the quantitative detection of substances related to L-2-amino-5-guanidinopentanoic acid raw materials, in particular to the impurities of metabolic homologues such as 2-ketoglutaric acid, (S) -2-acetamidoglutaric acid and (E) -2-butenedioic acid. Therefore, it is necessary to establish a method for detecting related substances, and the method can be used for comprehensively detecting and controlling impurities of L-2-amino-5-guanidinopentanoic acid raw material medicines from microbial fermentation.
The method for detecting the related substances of the L-2-amino-5-guanidinopentanoic acid is established by utilizing a high performance liquid chromatography technology, and has the advantages of good separation degree, high sensitivity, simplicity and convenience in operation and the like.
Disclosure of Invention
The invention aims to provide a high performance liquid chromatography detection method of related substances of L-2-amino-5-guanidino valeric acid, which provides a basis for the establishment of quality standards of the L-2-amino-5-guanidino valeric acid so as to better control the product quality and ensure the edible and medicinal safety.
The invention realizes the purpose by adopting the following technical scheme:
a detection method of related substances of L-2-amino-5-guanidino valeric acid is characterized by adopting a high performance liquid chromatography:
1. chromatographic conditions and system applicability test: a chromatographic column with octadecylsilane chemically bonded silica as a filler is adopted, a buffer salt solution (with the pH value of 2.0-2.8) with the concentration of 1-20 mg/mL is used as a mobile phase A, and acetonitrile is used as a mobile phase B; the flow rate is 0.3-0.8 mL per minute; the elution mode is gradient elution; the detection wavelength is 205-215 nm; the column temperature is 25-35 ℃; the sample injection amount is 10-50 mu L; taking a system applicability test solution to carry out a system applicability test, wherein the separation degree of L-2-amino-5-guanidino valeric acid and adjacent solvent peaks meets the requirement, and the number of theoretical plates is not less than 3000 calculated according to the L-2-amino-5-guanidino valeric acid peak;
wherein the system applicability test solution is a mixed solution of one or more of L-2-amino-5-guanidinopentanoic acid, 2-ketoglutaric acid, (S) -2-acetamidoglutaric acid and (E) -2-butenedioic acid prepared by using a diluent;
2. determination of related substances of L-2-amino-5-guanidino valeric acid: respectively injecting 10-50 mu L of test solution, reference solution and reference solution into a liquid chromatograph, recording a chromatogram, and calculating the content of 2-ketoglutaric acid, (S) -2-acetamido glutaric acid and (E) -2-butenedioic acid by peak area according to an external standard method; calculating the total content of other single unknown impurities and other unknown impurities according to the self-contrast method and the main peak area of the contrast solution;
the test solution is prepared by dissolving L-2-amino-5-guanidino valeric acid raw material in a diluent;
the reference solution is a solution prepared by diluting one or more of 2-ketoglutaric acid, (S) -2-acetamidoglutaric acid and (E) -2-butenedioic acid;
the reference solution is prepared by diluting the test solution with diluent.
Further, the ratio of the concentration of 2-ketoglutaric acid in the control solution to the concentration of L-2-amino-5-guanidinopentanoic acid in the test solution was 1: 0.4X 103～2×104The ratio of the concentration of (S) -2-acetamidoglutaric acid to the concentration of L-2-amino-5-guanidinopentanoic acid in the test solution is 1: 0.4X 103～2×104The ratio of the concentration of (E) -2-butenedioic acid to the concentration of L-2-amino-5-guanidinopentanoic acid in the sample solution is 1: 0.4X 104～2×105(ii) a The concentration ratio of the control solution to the L-2-amino-5-guanidinopentanoic acid in the test solution is 1:0.4 × 103～2×104。
Preferably, the method for detecting the related substances of the L-2-amino-5-guanidinopentanoic acid comprises the following steps:
1) preparation of buffered salt solution: weighing a certain amount of phosphate or citrate or acetate, adding water to prepare a solution with the concentration of 1-20 mg/mL, and adjusting the pH value of the solution to 2.0-2.8 by using corresponding acid to serve as a buffer salt solution;
2) preparation of a diluent: taking the buffer salt solution obtained in the step 1), adding acetonitrile, and mixing to prepare a buffer salt solution and a solution with the acetonitrile volume ratio of 99: 1-85: 15 as a diluent;
3) preparation of a test solution: accurately weighing an L-2-amino-5-guanidino valeric acid sample to be tested, and preparing a solution with the concentration of 1.0-50.0 mg/mL by using a diluent as a solvent to be used as a test solution;
4) preparation of control solutions: precisely weighing one or more of 2-ketoglutaric acid, (S) -2-acetamidoglutaric acid and (E) -2-butenedioic acid, dissolving with diluent, and diluting to obtain solutions with concentration of 2.5 μ g/mL, 2.5 μ g/mL and 0.25 μ g/mL respectively as control solutions;
5) preparation of control solution: taking the test solution obtained in the step 3), diluting the test solution with a diluent to prepare a solution with the concentration of L-2-amino-5-guanidino valeric acid being one thousandth of that of the test solution, and taking the solution as a reference solution;
6) preparation of system suitability solution: weighing appropriate amount of one or more of L-2-amino-5-guanidinopentanoic acid, 2-ketoglutaric acid, (S) -2-acetamidoglutaric acid and (E) -2-butenedioic acid, dissolving in diluent, and diluting to obtain a mixed solution containing about 2.5mg of L-2-amino-5-guanidinopentanoic acid, 2.5 μ g of 2-ketoglutaric acid, 2.5 μ g of (S) -2-acetamidoglutaric acid and 0.25 μ g of (E) -2-butenedioic acid per 1ml, as a system applicability solution;
7) preparation of a blank solvent: taking the diluent obtained in the step 2) as a blank solvent;
8) chromatographic conditions and system applicability test: a chromatographic column with octadecylsilane chemically bonded silica as a filler is adopted, a buffer salt solution (with the pH value of 2.0-2.8) with the concentration of 1-20 mg/mL is used as a mobile phase A, and acetonitrile is used as a mobile phase B; the flow rate is 0.3-0.8 mL per minute; the elution mode is gradient elution; the detection wavelength is 205-215 nm; the column temperature is 25-35 ℃; the sample injection amount is 10-50 mu L; carrying out a system applicability test on the system applicability test solution prepared in the step 6), wherein the separation degree of L-2-amino-5-guanidinopentanoic acid and an adjacent solvent peak meets the requirement, and the number of theoretical plates is not less than 3000 calculated according to the L-2-amino-5-guanidinopentanoic acid peak;
9) determination of related substances of L-2-amino-5-guanidino valeric acid: respectively injecting 10-50 mu L of the test solution, the reference solution and the reference solution prepared in the steps 3), 4) and 5) into a liquid chromatograph, recording a chromatogram, and calculating the contents of 2-ketoglutaric acid, (S) -2-acetamidoglutaric acid and (E) -2-butenedioic acid by peak areas according to an external standard method; and calculating the content of other single unknown impurities and the sum of other unknown impurities by using the main peak area of the control solution according to a self-comparison method.
Further, the detection method of the related substances of the L-2-amino-5-guanidino valeric acid comprises the following optimization steps:
1) preparation of buffered salt solution: weighing a certain amount of sodium dihydrogen phosphate, adding water to prepare a solution with the concentration of 3-10 mg/mL, and adjusting the pH value of the solution to 2.0-2.5 by using corresponding acid to serve as a buffer salt solution;
2) preparation of a diluent: taking the buffer salt solution obtained in the step 1), adding acetonitrile, and mixing to prepare a buffer salt solution and a solution with the acetonitrile volume ratio of 99: 1-85: 15 as a diluent;
3) preparation of a test solution: accurately weighing an L-2-amino-5-guanidino valeric acid sample to be tested, and preparing a solution with the concentration of 2.5-5.0 mg/mL by using a diluent as a solvent to be used as a test solution;
4) preparation of control solutions: precisely weighing one or more of 2-ketoglutaric acid, (S) -2-acetamidoglutaric acid and (E) -2-butenedioic acid, dissolving with diluent, and diluting to obtain solutions with concentration of 2.5 μ g/mL, 2.5 μ g/mL and 0.25 μ g/mL respectively as control solutions;
5) preparation of control solution: taking the test solution obtained in the step 3), diluting the test solution with a diluent to prepare a solution with the concentration of L-2-amino-5-guanidino valeric acid being one thousandth of that of the test solution, and taking the solution as a reference solution;
6) preparation of system suitability solution: weighing appropriate amount of one or more of L-2-amino-5-guanidinopentanoic acid, 2-ketoglutaric acid, (S) -2-acetamidoglutaric acid and (E) -2-butenedioic acid, dissolving in diluent, and diluting to obtain a mixed solution containing about 2.5mg of L-2-amino-5-guanidinopentanoic acid, 2.5 μ g of 2-ketoglutaric acid, 2.5 μ g of (S) -2-acetamidoglutaric acid and 0.25 μ g of (E) -2-butenedioic acid per 1ml, as a system applicability solution;
7) preparation of a blank solvent: taking the diluent obtained in the step 2) as a blank solvent;
8) chromatographic conditions and system applicability test: a chromatographic column using octadecylsilane chemically bonded silica as a filler, a buffer salt solution (pH value of 2.0-2.5) with the concentration of 3-10 mg/mL as a mobile phase A, and acetonitrile as a mobile phase B; the flow rate is 0.4-0.6 mL per minute; the elution mode is gradient elution; the detection wavelength is 205-215 nm; the column temperature is 28-32 ℃; the sample injection amount is 10-30 mu L; carrying out a system applicability test on the system applicability test solution prepared in the step 6), wherein the separation degree of L-2-amino-5-guanidinopentanoic acid and an adjacent solvent peak meets the requirement, and the number of theoretical plates is not less than 3000 calculated according to the L-2-amino-5-guanidinopentanoic acid peak;
9) determination of related substances of L-2-amino-5-guanidino valeric acid: respectively injecting 10-30 mu L of the test solution, the reference solution and the reference solution prepared in the steps 3), 4) and 5) into a liquid chromatograph, recording a chromatogram, and calculating the contents of 2-ketoglutaric acid, (S) -2-acetamidoglutaric acid and (E) -2-butenedioic acid by peak areas according to an external standard method; and calculating the content of other single unknown impurities and the sum of other unknown impurities by using the main peak area of the control solution according to a self-comparison method.
Further, the gradient elution is the gradient elution shown in the following table.
Compared with the prior art, the invention has the following beneficial effects:
1. the detection method has higher sensitivity, accuracy, precision and strong durability, can accurately reflect the content of related substances of the L-2-amino-5-guanidino valeric acid raw material medicine, is suitable for the detection and quality control of the related substances of the L-2-amino-5-guanidino valeric acid in industry, thereby providing a basis for reasonable quality standard establishment, facilitating better control and mastering of product quality and ensuring the safety of eating and medicine.
2. The method can effectively detect related substances of the L-2-amino-5-guanidino valeric acid, is beneficial to the process control of the production of the L-2-amino-5-guanidino valeric acid, is also beneficial to better control of the product quality, and ensures the safety of eating and medicine. The separation degree of L-2-amino-5-guanidino valeric acid from adjacent solvent peaks in the system applicability test is in accordance with the requirement, and the number of theoretical plates is not less than 3000 calculated according to the L-2-amino-5-guanidino valeric acid peaks; when the high performance liquid chromatography is used for detecting related substances of L-2-amino-5-guanidinopentanoic acid, the sensitivity for detecting 2-ketoglutaric acid and (S) -2-acetamidoglutaric acid is 0.1 percent, the sensitivity for detecting (E) -2-butenedioic acid is 0.01 percent, and the sensitivity for detecting other unknown impurities is 0.1 percent.
3. The invention has the most outstanding characteristic of solving the problem of separation degree of impurities of L-2-amino-5-guanidino valeric acid and organic acid homologues. According to the invention, through researches such as flow rate selection, flow phase ratio selection, buffer salt concentration selection, elution mode selection, chromatographic column selection and the like, effective separation of L-2-amino-5-guanidinopentanoic acid from various organic acid homolog impurities is realized, and quantitative detection can be carried out on the contents of homolog impurities such as 2-ketoglutaric acid, (S) -2-acetamino glutaric acid and (E) -2-butenedioic acid. FIG. 1 shows that the related substances of the fermentation broth of L-2-amino-5-guanidinopentanoic acid are very complex, and the removal of impurities in the purification process of the product can be clearly understood by using the present invention.
4. The invention has the second most prominent characteristic of solving the problem of sensitivity of detection of related substances of the L-2-amino-5-guanidino valeric acid. The ultraviolet absorption of impurities of the L-2-amino-5-guanidino valeric acid and the homologous compounds thereof is very weak, and the detection signals of all components are very weak, so that the sensitivity is very low, and the limit requirements of related substances cannot be met. The invention takes the ratio of the impurity concentration of each homologue in the reference solution to the concentration of the L-2-amino-5-guanidino valeric acid in the test solution as a main research object, and improves the ratio by various technical means so as to meet the detection sensitivity of each component.
Drawings
FIG. 1: chromatogram of related substances of L-2-amino-5-guanidino valeric acid fermentation broth
FIG. 2: chromatogram for detecting related substances in sample
FIG. 3: chromatogram for system suitability test
FIG. 4: undisrupted chromatograms
FIG. 5: chromatogram for strong acid destruction
FIG. 6: high temperature disruption of chromatograms
FIG. 7: chromatogram for strong light destruction
Detailed description of the preferred embodiments
The invention is further described below by means of specific preferred embodiments without thereby restricting the invention to the described embodiments.
Example 1: three batches of L-2-amino-5-guanidino valeric acid raw material medicine and related substance detection
Instrument and chromatographic conditions:
adopting Agilent 1260 high performance liquid chromatograph, using octadecylsilane chemically bonded silica as filler (4.6 × 250mm, 5 μm), and column temperature of 30 deg.C; the flow rate is 0.5 mL/min; the detection wavelength is 210 nm; the sample injection amount is 20 mu L; 3.9 g of sodium dihydrogen phosphate is taken, dissolved in water and diluted to 1000mL, the pH value is adjusted to 2.3 by using 20% phosphoric acid as a mobile phase A, acetonitrile is used as a mobile phase B, and elution is carried out according to the following table:
precisely measuring 20 μ L of each of the test solution, the reference solution and the reference solution, respectively injecting into a liquid chromatograph, and recording chromatogram;
calculating the contents of 2-ketoglutaric acid (impurity A), (S) -2-acetamidoglutaric acid (impurity B) and (E) -2-butenedioic acid (impurity C) by peak area according to an external standard method; calculating the content of unknown single impurities and unknown total impurities by a self-contrast method;
the experimental steps are as follows: accurately weighing appropriate amount of L-2-amino-5-guanidino valeric acid, adding diluent [ phosphate buffer solution-acetonitrile (92:8) ] to dissolve and dilute to obtain solution containing about 2.5mg per 1mL as sample solution; precisely weighing an appropriate amount of A, B, C reference substance, adding diluent to dissolve, and quantitatively diluting to obtain a solution containing 2.5 μ g of impurity A, 2.5 μ g of impurity B and 0.25 μ g of impurity C in 1mL as reference substance solution; precisely measuring a proper amount of a test solution, adding a diluent to dilute the test solution into a solution containing about 2.5 mu g of the test solution per 1mL of the test solution, and using the solution as a control solution; taking the diluent as a blank solvent.
Taking appropriate amount of each of the L-2-amino-5-guanidinopentanoic acid raw material drug, the impurity A, the impurity B and the impurity C reference substance, adding a diluent to dissolve and dilute the drug to prepare a solution of 2.5mg of L-2-amino-5-guanidinopentanoic acid, 2.5 mu g of the impurity A, 2.5 mu g of the impurity B and 0.25 mu g of the impurity C in each 1mL of the drug, and taking the solution as a system adaptive solution.
The results of retention time and separation of L-2-amino-5-guanidinopentanoic acid and various impurities in the system applicability test are shown in the following table:
note: unknown impurity 2 is the impurity from the reference sample of impurity B and is calculated as impurity B, as follows.
Under the determination conditions, the separation degree of the main peak and the adjacent impurities of the solution is more than 2.0, the separation degree of each known impurity and the adjacent impurities after the L-2-amino-5-guanidino valeric acid peak is more than 1.5, and the system applicability is good.
The detection results of three batches of L-2-amino-5-guanidino valeric acid raw material medicine related substances are shown in the following table, and the chromatogram is shown in the figure 2:
| sample batch number | Impurity A (%) | Impurity B (%) | Impurity C (%) | Unknown Single hetero (%) | Unknown Total impurities (%) |
| 20180501 | — | — | — | 0.051 | 0.137 |
| 20180502 | — | — | — | 0.057 | 0.123 |
| 20180503 | — | — | — | 0.056 | 0.118 |
Example 2: test for locating related substances
The apparatus and chromatographic conditions were the same as in example 1.
The experimental steps are as follows: respectively weighing appropriate amount of reference substances of impurity A, impurity B and impurity C, respectively dissolving with diluent, and quantitatively diluting to obtain solution containing impurity A, impurity B2.5 μ g and impurity C0.25 μ g per 1mL as positioning solution of each impurity; the preparation method of the mixed solution was the same as that of the system applicability solution of example 1, and the positioning solution and the mixed solution of each impurity were taken and injected into a liquid chromatograph, and a chromatogram was recorded. The results of the separation of L-2-amino-5-guanidinopentanoic acid from the respective impurities are shown in the following table, and the chromatogram is shown in FIG. 3:
the results show that: the L-2-amino-5-guanidino valeric acid has excellent separation of related matters.
Example 3: specificity failure test
The apparatus and chromatographic conditions were the same as in example 1.
The experimental steps are as follows: weighing appropriate amount of reference substances of the impurity A, the impurity B and the impurity C respectively, dissolving the reference substances by using diluent respectively, and quantitatively diluting the reference substances to prepare a solution containing about 2.5 mu g of the impurity A, 2.5 mu g of the impurity B and 0.25 mu g of the impurity C per 1mL of the reference substances as a positioning solution of each impurity; the preparation method of the mixed solution was the same as that of the system applicability solution of example 1, and the positioning solution and the mixed solution of each impurity were taken and injected into a liquid chromatograph, and a chromatogram was recorded. The results of the separation of L-2-amino-5-guanidinopentanoic acid from the respective impurities are shown in the following table, and the chromatogram is shown in FIG. 3:
accurately weighing a proper amount of L-2-amino-5-guanidino valeric acid, and respectively carrying out forced destruction tests on the L-2-amino-5-guanidino valeric acid under the conditions of 0.5mol/L hydrochloric acid solution, high temperature of 180 ℃ and strong light of 4500Lx, wherein the preparation method of each destruction sample is shown in the following table, and the map is shown in fig. 4-7:
each proprietary failure sample solution was prepared according to the following table:
and (3) taking the solutions and injecting samples respectively and recording chromatograms, wherein the damage results are shown in the following table:
the results show that: impurities A, B and C are not detected in the strong acid and strong light damage samples, which indicates that the strong acid and the strong light can not convert the L-2-amino-5-guanidinopentanoic acid into the impurities A, B and C; impurities B and C can be detected in a high-temperature damage sample; under strong acid and strong light destruction conditions, the separation degrees of the L-2-amino-5-guanidinopentanoic acid, the impurity A, the impurity B and the impurity C peaks and the front and rear peaks are all over 1.3, the quantitative detection of the L-2-amino-5-guanidinopentanoic acid is not influenced, the peak purity of the L-2-amino-5-guanidinopentanoic acid is more than 995, and the peak purity of the L-2-amino-5-guanidinopentanoic acid is reduced to 960-970 under the high-temperature destruction condition; the high temperature destruction sample can detect the impurity B and the impurity C, can not detect the impurity A, and can detect a plurality of other impurities. Therefore, the method has good specificity for detecting the impurity A, the impurity B and the impurity C.
Example 4: quantitative limit and detection limit test
The apparatus and chromatographic conditions were the same as in example 1.
The experimental steps are as follows: and (3) determining the detection limit and the quantification limit of the L-2-amino-5-guanidinopentanoic acid and each related substance thereof by using a signal-to-noise ratio method. Respectively preparing stock solutions of L-2-amino-5-guanidino valeric acid and various related substances, diluting to a certain concentration, injecting a sample, calculating the ratio (signal-to-noise ratio) of peak height to noise, wherein when the sample detection amount of the signal-to-noise ratio (S/N) of about 10 is a quantitative limit, the sample detection amount of the signal-to-noise ratio (S/N) of about 3 is a detection limit, and the specific preparation method comprises the following steps:
accurately weighing appropriate reference substances of L-2-amino-5-guanidino valeric acid, impurity A, impurity B and impurity C, dissolving and diluting with a diluent to prepare a solution containing 0.1 mu g of L-2-amino-5-guanidino valeric acid, 0.1 mu g of impurity A, 0.25 mu g of impurity B and 0.005 mu g of impurity C per 1mL, and taking the solution as a related substance detection limit solution; diluting with diluent to obtain a solution containing about 1 μ g of L-2-amino-5-guanidinopentanoic acid, 0.5 μ g of impurity A, 0.75 μ g of impurity B and 0.025 μ g of impurity C per 1mL, and using the solution as a limit solution; and (3) respectively injecting 20 mu L of the solution, wherein the signal-to-noise ratio (S/N) of each component in the quantitative limiting solution is about 10, and the signal-to-noise ratio (S/N) of the detection limiting solution is about 3.
The specific results of the quantitative limit and the detection limit of each component of the related substances of the L-2-amino-5-guanidino valeric acid are shown in the following table:
the results show that: the quantitative limit concentration of the L-2-amino-5-guanidino valeric acid and the related substances is less than 0.04 percent of the concentration of the test sample, and the content of the related substances can be accurately controlled.
Example 5: linear test
The apparatus and chromatographic conditions were the same as in example 1.
The experimental steps are as follows: precisely weighing appropriate amount of L-2-amino-5-guanidino valeric acid and related substances, dissolving with diluent, and preparing solution with different concentrations by the following specific preparation method: an appropriate amount of L-2-amino-5-guanidinopentanoic acid, impurity A, impurity B and impurity C reference substances are precisely weighed, and are respectively dissolved and diluted by a diluent to prepare a solution containing 10 mu g/mL of L-2-amino-5-guanidinopentanoic acid, 10 mu g/mL of impurity A, 5 mu g/mL of impurity B and 10 mu g/mL of impurity C, and the solution is used as a linear stock solution.
Each linearity test solution was prepared for the relevant substance linearity test according to the following table:
respectively sampling the solutions, recording chromatograms, and respectively obtaining linear regression equations of the L-2-amino-5-guanidinopentanoic acid and the impurities A, B and C by taking the concentration as a horizontal coordinate and the peak area as a vertical coordinate;
the linearity results for the relevant substances are given in the following table:
| compound (I) | Linear Range (μ g/mL) | Linear regression equation | Regression coefficient r2 |
| L-2-amino-5-guanidinopentanoic acid | 1～6 | y＝4.6618x+3.6705 | 0.9961 |
| Impurity A | 0.5～6.0 | y＝26.959x-5.3737 | 0.9972 |
| Impurity B | 0.75～3.0 | y＝17.867x+4.5381 | 0.9979 |
| Impurity C | 0.025～6.0 | y＝312.19x+1.6661 | 0.9999 |
The results show that: the L-2-amino-5-guanidino valeric acid has good linear relation with the impurity A, the impurity B and the impurity C within a certain concentration range.
Example 6: accuracy test
The apparatus and chromatographic conditions were the same as in example 1.
The experimental steps are as follows: respectively and precisely weighing L-2-amino-5-guanidino valeric acid and appropriate amount of related substances, and preparing low-concentration solution containing L-2-amino-5-guanidino valeric acid with concentration of 2.5mg/mL and impurity A, impurity B and impurity C with concentrations of 2.0 μ g/mL, 2.0 μ g/mL and 0.20 μ g/mL respectively with diluent; preparing a medium concentration solution containing L-2-amino-5-guanidino valeric acid with the concentration of 2.5mg/mL and the concentrations of impurity A, impurity B and impurity C of 2.5 mug/mL, 2.5 mug/mL and 0.25 mug/mL respectively; preparing a high-concentration solution containing L-2-amino-5-guanidino valeric acid with the concentration of 2.5mg/mL and the concentrations of impurity A, impurity B and impurity C of 3.0 mu g/mL, 3.0 mu g/mL and 0.30 mu g/mL respectively; the above solutions were used for the accuracy tests of the relevant substances, in triplicate for each concentration; and (3) respectively injecting samples and recording chromatograms, and calculating the recovery rate, wherein the results are shown in the following table:
| compound (I) | Low concentration average recovery (%) | Middle concentrationAverage recovery (%) | High concentration average recovery (%) |
| Impurity A | 97.93 | 103.02 | 95.23 |
| Impurity B | 97.84 | 100.26 | 102.24 |
| Impurity C | 99.96 | 99.97 | 101.20 |
The results show that: the average recovery rate of each related substance is between 95 percent and 105 percent in the low, medium and high concentration range, which shows that the method has good accuracy.
Example 7: durability test
The apparatus and chromatographic conditions were the same as in example 1.
The experimental steps are as follows: and (3) taking the system applicability solution, respectively changing chromatographic conditions, such as flow rate variation +/-0.1 mL/min, temperature variation +/-5 ℃, wavelength variation +/-5 nm, flow phase variation +/-5%, chromatographic columns of different types and the like, and carrying out chromatographic analysis according to the method. And evaluating the durability of the method according to the tailing factors of the chromatographic peaks of the impurities A, B and C, the separation degree, the RSD of the content data and the absolute value change of the content of the impurities.
The durability test design for this method is shown in the following table:
the durability test results of this method are shown in the following table:
the results show that: under different chromatographic conditions, the absolute values of the contents of the impurity A, the impurity B and the impurity C respectively change within +/-0.00629%, +/-0.00946% and +/-0.00034%, and the acceptable requirement that the absolute values are not more than +/-0.1% is met; the effective separation (the separation degree is more than 1.17) can be realized with the front peak and the back peak respectively, and the tailing factors are less than 2, which shows that the durability of the method for detecting related substances of the L-2-amino-5-guanidino valeric acid bulk drug is good.
The above description is only exemplary of the present invention and should not be taken as limiting, and any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. A detection method of related substances of L-2-amino-5-guanidino valeric acid is characterized by adopting a high performance liquid chromatography:
1) chromatographic conditions and system applicability test: a chromatographic column with octadecylsilane chemically bonded silica as a filler is adopted, a buffer salt solution with the concentration of 1-20 mg/mL is used as a mobile phase A, and acetonitrile is used as a mobile phase B; the flow rate is 0.3-0.8 mL per minute; the elution mode is gradient elution; the detection wavelength is 205-215 nm; the column temperature is 25-35 ℃; the sample injection amount is 10-50µL; taking a system applicability test solution to carry out a system applicability test, wherein the separation degree of L-2-amino-5-guanidino valeric acid and adjacent solvent peaks meets the requirement, and the number of theoretical plates is not less than 3000 calculated according to the L-2-amino-5-guanidino valeric acid peak;
wherein the system suitability test solution is L-2-amino-5-guanidinopentanoic acid and 2-ketoglutaric acid: (S) -2-acetylaminoglutaric acid andE) 4 mixed solutions of 2-butenedioic acid prepared by using the diluent;
determination of related substances of L-2-amino-5-guanidino valeric acid: getThe sample solution, the reference solution and the reference solution are 10-50 of eachµL, respectively injecting into a liquid chromatograph, recording chromatogram, and calculating 2-ketoglutaric acid and (A) according to peak area by an external standard methodS) -2-acetylaminoglutaric acid andE) -content of 2-butenedioic acid; calculating the total content of other single unknown impurities and other unknown impurities according to the self-contrast method and the main peak area of the contrast solution;
the test solution is prepared by dissolving L-2-amino-5-guanidino valeric acid raw material in a diluent;
the control solution is prepared from 2-ketoglutaric acid (II)S) -2-acetylaminoglutaric acid andE) -one or more solutions of 2-butenedioic acid in a diluent;
the reference solution is prepared by diluting the test solution with diluent;
wherein the buffered salt solution is selected from a phosphate buffer, a citrate buffer, and an acetate buffer; the preparation method comprises the following steps: weighing a certain amount of phosphate or citrate or acetate, adding water to prepare a solution with the concentration of 1-20 mg/mL, and adjusting the pH value of the solution to 2.0-2.8 by using corresponding acid;
the gradient elution is the gradient elution shown in the following table:
。
2. The method for detecting a substance related to L-2-amino-5-guanidinopentanoic acid as claimed in claim 1, wherein the preparation of the dilution solution comprises: and adding acetonitrile into the buffer salt solution, and mixing to prepare a solution with the volume ratio of the buffer salt solution to the acetonitrile of 99: 1-85: 15 as a diluent.
3. The method for detecting a substance related to L-2-amino-5-guanidinopentanoic acid as claimed in claim 1, wherein the ratio of the concentration of 2-ketoglutaric acid in the control solution to the concentration of L-2-amino-5-guanidinopentanoic acid in the test solution is 1: 0.4X 103～2×104，（S) The ratio of the concentration of the (E) -2-acetylglutamic acid to the concentration of the L-2-amino-5-guanidinopentanoic acid in the test solution is 1: 0.4X 103～2×104，（E) Concentration of (E) -2-butenedioic acid and L-2-amino-5-guanidinopentanoic acid in test solutionThe concentration ratio of (A) to (B) is 1: 0.4X 104～2×105(ii) a The concentration ratio of the control solution to the L-2-amino-5-guanidinopentanoic acid in the test solution is 1:0.4 × 103～2×104。
4. The method for detecting a substance related to L-2-amino-5-guanidinopentanoic acid as claimed in any one of claims 1 to 3, comprising the steps of:
1) preparation of buffered salt solution: weighing a certain amount of phosphate or citrate or acetate, adding water to prepare a solution with the concentration of 1-20 mg/mL, and adjusting the pH value of the solution to 2.0-2.8 by using corresponding acid to serve as a buffer salt solution;
2) preparation of a diluent: taking the buffer salt solution obtained in the step 1), adding acetonitrile, and mixing to prepare a buffer salt solution and a solution with the acetonitrile volume ratio of 99: 1-85: 15 as a diluent;
3) preparation of a test solution: accurately weighing an L-2-amino-5-guanidino valeric acid sample to be tested, and preparing a solution with the concentration of 1.0-50.0 mg/mL by using a diluent as a solvent to be used as a test solution;
4) preparation of control solutions: precisely weighing 2-ketoglutaric acid (a), (b), (c), (d), (c) and (d)S) -2-acetylaminoglutaric acid andE) One or more of-2-butenedioic acid, dissolved in a diluent and diluted to a prepared concentration of 2.5 eachμg/mL、2.5μg/mL and 0.25μg/mL solution as reference solution;
5) preparation of control solution: taking the test solution obtained in the step 3), diluting the test solution with a diluent to prepare a solution with the concentration of L-2-amino-5-guanidino valeric acid being one thousandth of that of the test solution, and taking the solution as a reference solution;
6) preparation of system suitability solution: separately weighing L-2-amino-5-guanidinopentanoic acid and 2-ketoglutaric acid (a) ((b))S) -2-acetylaminoglutaric acid andE) One or more appropriate amount of-2-butenedioic acid is dissolved in a diluent and diluted to obtain a solution containing about 2.5mg of L-2-amino-5-guanidinopentanoic acid and 2.5mg of 2-ketoglutaric acid per 1mlμg、（S) 2.5-Acetaminoglutaric acid 2μg and (a)E) -2-butene-diAcid 0.25μg of a mixed solution as a system suitability solution;
7) preparation of a blank solvent: taking the diluent obtained in the step 2) as a blank solvent;
8) chromatographic conditions and system applicability test: a chromatographic column using octadecylsilane chemically bonded silica as a filler, a buffer salt solution with the pH value of 2.0-2.8 and the concentration of 1-20 mg/mL is used as a mobile phase A, acetonitrile is used as a mobile phase B, and the flow rate is 0.3-0.8 mL per minute; the elution mode is gradient elution; the detection wavelength is 205-215 nm; the column temperature is 25-35 ℃; the sample injection amount is 10-50µL; carrying out a system applicability test on the system applicability test solution prepared in the step 6), wherein the separation degree of L-2-amino-5-guanidinopentanoic acid and an adjacent solvent peak meets the requirement, and the number of theoretical plates is not less than 3000 calculated according to the L-2-amino-5-guanidinopentanoic acid peak;
9) determination of related substances of L-2-amino-5-guanidino valeric acid: taking 10-50 parts of the test solution, the reference solution and the reference solution prepared in the steps 3), 4) and 5) respectivelyµL, respectively injecting into a liquid chromatograph, recording chromatogram, and calculating 2-ketoglutaric acid and (A) according to peak area by an external standard methodS) -2-acetylaminoglutaric acid andE) -content of 2-butenedioic acid; and calculating the content of other single unknown impurities and the sum of other unknown impurities by using the main peak area of the control solution according to a self-comparison method.
5. The method for detecting a substance related to L-2-amino-5-guanidinopentanoic acid as claimed in claim 4, wherein the optimization step comprises:
1) preparation of buffered salt solution: weighing a certain amount of sodium dihydrogen phosphate, adding water to prepare a solution with the concentration of 3-10 mg/mL, and adjusting the pH value of the solution to 2.0-2.5 by using corresponding acid to serve as a buffer salt solution;
2) preparation of a diluent: taking the buffer salt solution obtained in the step 1), adding acetonitrile, and mixing to prepare a buffer salt solution and a solution with the acetonitrile volume ratio of 99: 1-85: 15 as a diluent;
3) preparation of a test solution: accurately weighing an L-2-amino-5-guanidino valeric acid sample to be tested, and preparing a solution with the concentration of 2.5-5.0 mg/mL by using a diluent as a solvent to be used as a test solution;
4) preparation of control solutions: precisely weighing 2-ketoglutaric acid (a), (b), (c), (d), (c) and (d)S) -2-acetylaminoglutaric acid andE) One or more of-2-butenedioic acid, dissolved in a diluent and diluted to a prepared concentration of 2.5 eachμg/mL、2.5μg/mL and 0.25μg/mL solution as reference solution;
5) preparation of control solution: taking the test solution obtained in the step 3), diluting the test solution with a diluent to prepare a solution with the concentration of L-2-amino-5-guanidino valeric acid being one thousandth of that of the test solution, and taking the solution as a reference solution;
6) preparation of system suitability solution: separately weighing L-2-amino-5-guanidinopentanoic acid and 2-ketoglutaric acid (a) ((b))S) -2-acetylaminoglutaric acid andE) One or more appropriate amount of-2-butenedioic acid is dissolved in a diluent and diluted to obtain a solution containing about 2.5mg of L-2-amino-5-guanidinopentanoic acid and 2.5mg of 2-ketoglutaric acid per 1mlμg、（S) 2.5-Acetaminoglutaric acid 2μg and (a)E) 0.25 of (E) -2-butenedioic acidμg of a mixed solution as a system suitability solution;
7) preparation of a blank solvent: taking the diluent obtained in the step 2) as a blank solvent;
8) chromatographic conditions and system applicability test: a chromatographic column using octadecylsilane chemically bonded silica as a filler, a buffer salt solution with the pH value of 2.0-2.5 and the concentration of 3-10 mg/mL is used as a mobile phase A, and acetonitrile is used as a mobile phase B; the flow rate is 0.4-0.6 mL per minute; the elution mode is gradient elution; the detection wavelength is 205-215 nm; the column temperature is 28-32 ℃; the sample injection amount is 10-30µL; carrying out a system applicability test on the system applicability test solution prepared in the step 6), wherein the separation degree of L-2-amino-5-guanidinopentanoic acid and an adjacent solvent peak meets the requirement, and the number of theoretical plates is not less than 3000 calculated according to the L-2-amino-5-guanidinopentanoic acid peak;
9) determination of related substances of L-2-amino-5-guanidino valeric acid: taking 10-30 parts of the test solution, the reference solution and the reference solution prepared in the steps 3), 4) and 5) respectivelyµL, respectively injecting liquid phase colorA spectrometer for recording chromatogram and calculating 2-ketoglutaric acid and (C) according to peak area by an external standard methodS) -2-acetylaminoglutaric acid andE) -content of 2-butenedioic acid; and calculating the content of other single unknown impurities and the sum of other unknown impurities by using the main peak area of the control solution according to a self-comparison method.
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