CN109387587B - Detection method of L-2-amino-5-guanidino valeric acid enantiomer - Google Patents
Detection method of L-2-amino-5-guanidino valeric acid enantiomer Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses a detection method of an L-2-amino-5-guanidino valeric acid enantiomer, which belongs to the technical field of food and drug analysis and mainly comprises the steps of preparation of a system applicability solution, preparation of an enantiomer reference solution, preparation of a test sample solution, preparation of a derivatization test solution, pre-column derivatization, test of chromatographic conditions and system applicability, determination of an L-2-amino-5-guanidino valeric acid enantiomer and the like. The detection method has higher sensitivity, accuracy, precision and strong durability, can accurately reflect the content of the L-2-amino-5-guanidino valeric acid enantiomer, is suitable for the detection and quality control of the L-2-amino-5-guanidino valeric acid enantiomer in industry, thereby providing a basis for reasonable quality standard establishment, facilitating better control and mastering of product quality and ensuring the safety of food 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 an L-2-amino-5-guanidino valeric acid enantiomer.
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 supplementing L-2-amino-5-guanidino valeric acid deficiency for infants, and has wide application.
At present, L-2-amino-5-guanidinopentanoic acid is mainly produced by a microbial fermentation method, and a small amount of L-2-amino-5-guanidinopentanoic acid enantiomer (figure 1), namely D-2-amino-5-guanidinopentanoic acid, is produced in a microbial metabolism process. Due to the nature of L-2-amino-5-guanidinopentanoic acid and its enantiomer and its proximity, in the course of separation and purification of L-2-amino-5-guanidinopentanoic acid its enantiomer is difficult to remove (figure 2), and is easy to remain in L-2-amino-5-guanidinopentanoic acid raw material, and can affect product quality. The prior pharmacopoeias of various countries do not contain the enantiomeric search term of L-2-amino-5-guanidino valeric acid. Therefore, how to analyze and prepare the L-2-amino-5-guanidino valeric acid and enantiomers thereof, and controlling the content of D-2-amino-5-guanidino valeric acid impurities so as to ensure the quality of the L-2-amino-5-guanidino valeric acid are very important.
However, there is no report on the analytical separation method of two enantiomers of 2-amino-5-guanidinopentanoic acid. Among them, there may be mainly involved various problems such as the degree of separation of L-2-amino-5-guanidinopentanoic acid from its enantiomer derivative, the detection sensitivity of enantiomer as an impurity, the interference of enantiomer detection by L-2-amino-5-guanidinopentanoic acid, and the like. Therefore, how to establish a method for simply, quickly and efficiently analyzing and separating two enantiomers of 2-amino-5-guanidinopentanoic acid so as to comprehensively detect and control the quality of the L-2-amino-5-guanidinopentanoic acid raw material medicine is a technical problem to be solved at present.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a high performance liquid chromatography detection method of L-2-amino-5-guanidino valeric acid enantiomer, which provides a basis for the establishment of the quality standard of L-2-amino-5-guanidino valeric acid so as to better control the product quality and ensure the edible and medicinal safety.
The invention adopts the following technical scheme: a detection method of L-2-amino-5-guanidino valeric acid enantiomer adopts a pre-column derivatization high performance liquid chromatography:
1) pre-column derivatization: respectively taking a proper amount of a system applicability solution, an enantiomer reference substance solution and a test substance solution, respectively adding equal volume of derivatization test solution to obtain a pre-column derivatization system, uniformly mixing, and standing at room temperature for derivatization for 40-180 min to obtain derivatization solutions of the solutions; the system applicability solution is a mixed solution prepared by dissolving L-2-amino-5-guanidinopentanoic acid and an enantiomer reference substance in a diluent, wherein the concentration range of the L-2-amino-5-guanidinopentanoic acid is 0.2-1.0 mg/mL, and the concentration range of the enantiomer is 10-30 mu g/mL; the enantiomer reference substance solution is prepared by dissolving an L-2-amino-5-guanidinopentanoic acid enantiomer reference substance in a diluent, and the concentration range of the enantiomer reference substance solution is 0.5-5 mu g/mL; the test solution is prepared by dissolving a raw material of L-2-amino-5-guanidino valeric acid in a diluent, and the concentration range of the test solution is 0.1-3.0 mg/mL; the derivatization test solution is an acetonitrile solution dissolved with 2,3,4, 6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate, and the concentration range of the derivatization test solution is 1-5 mg/mL;
2) and (3) testing the applicability of the system: performing high performance liquid chromatography analysis on the derivatization solution of the system applicability solution prepared in the step 1), wherein the separation degree of an L-2-amino-5-guanidino valeric acid enantiomer derivative peak and an adjacent peak 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 enantiomer peak;
3) determination of the enantiomers of L-2-amino-5-guanidinopentanoic acid: and (2) respectively taking the derivatization solution of the test solution prepared in the step 1) and the derivatization solution of the enantiomer reference solution to perform high performance liquid chromatography analysis, recording a chromatogram, and calculating the content of the enantiomer by peak area according to an external standard method.
Preferably, the high performance liquid chromatography conditions are as follows: a chromatographic column using octadecylsilane chemically bonded silica as a filler, a phosphate solution with the concentration of 5-20 mmol/L and the pH value of 2.5-3.2 is used as a mobile phase A, and methanol is used as a mobile phase B; the flow rate is 0.8-1.2 mL/min; the elution mode is gradient elution; the detection wavelength is 245-255 nm; the column temperature is 20-35 ℃; the sample injection amount is 2-20 mu L.
Preferably, the diluent is a mixed solution of acetonitrile, water and diethylamine in a volume ratio of 50:50: 0.2-0.6, and the diethylamine can also be triethylamine.
Preferably, the mass ratio of the L-2-amino-5-guanidinopentanoic acid to the 2,3,4, 6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate in the pre-column derivatization system of the test solution is 0.05-0.85: 1.
preferably, the content of the enantiomer is calculated by the peak area according to an external standard method, and the peak area of the enantiomer reference product needs to be multiplied by a correction factor of 1.25-1.35.
Preferably, the phosphate is potassium dihydrogen phosphate.
Preferably, the detection method of the L-2-amino-5-guanidinopentanoic acid enantiomer comprises the following specific steps:
1) preparation of system suitability solution: taking the diluent as a solvent, respectively preparing the L-2-amino-5-guanidino valeric acid and enantiomer reference substances thereof into solutions with the concentrations of 0.5mg/mL and 15 mu g/mL, and taking the solutions as system applicability solutions;
2) preparation of enantiomer control solutions: taking the diluent as a solvent, preparing a solution with the concentration of about 1.5 mu g/mL of an L-2-amino-5-guanidinopentanoic acid enantiomer control product as an enantiomer control product solution;
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 0.1-2.0 mg/mL by using a diluent as a solvent to be used as a test solution;
4) preparing a derivatization test solution: weighing a derivatization reagent 2,3,4, 6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate, adding acetonitrile to dissolve the derivatization reagent to prepare a solution with the concentration of 2.4mg/mL, and taking the solution as a derivatization test solution;
5) respectively taking 100 mu L of each of the system applicability solution, the enantiomer reference substance solution and the test substance solution, respectively adding equal volume of derivatization test solution, uniformly mixing, and standing at room temperature for derivatization for 40-180 min to obtain derivatization solutions of the solutions;
6) chromatographic conditions and system applicability test: a chromatographic column using octadecylsilane chemically bonded silica as a filler, 0.01mol/L potassium dihydrogen phosphate solution (pH value is 2.5-3.2) as a mobile phase A, and methanol as a mobile phase B; the flow rate is 0.8-1.2 mL per minute; the elution mode is gradient elution; the detection wavelength is 245-255 nm; the column temperature is 20-35 ℃; the sample injection amount is 2-20 mu L; taking the derivatization solution of the system applicability solution prepared in the step 5) to carry out a system applicability test, wherein the separation degree of the L-2-amino-5-guanidino valeric acid enantiomer derivative peak and the adjacent peak 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 enantiomer peak;
7) determination of the enantiomers of L-2-amino-5-guanidinopentanoic acid: and (3) respectively injecting 2-20 mu L of the derivatization solution of the enantiomer reference substance solution prepared in the step 5) and the derivatization solution of the test substance solution into a liquid chromatograph, recording a chromatogram, calculating the content of the enantiomer according to the peak area of an external standard method, and multiplying the peak area of the enantiomer reference substance by a correction factor of 1.25-1.35.
Further, the detection method of the L-2-amino-5-guanidino valeric acid enantiomer comprises the following optimization steps:
1) preparation of system suitability solution: taking the diluent as a solvent, respectively preparing the L-2-amino-5-guanidino valeric acid and enantiomer reference substances thereof into solutions with the concentrations of 0.5mg/mL and 15 mu g/mL, and taking the solutions as system applicability solutions;
2) preparation of enantiomer control solutions: taking the diluent as a solvent, preparing a solution with the concentration of 1.5 mu g/mL by using an L-2-amino-5-guanidino valeric acid enantiomer reference substance as an enantiomer reference substance solution;
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 1.5mg/mL by using a diluent as a solvent to be used as a test solution;
4) preparing a derivatization test solution: weighing a proper amount of a derivatization reagent 2,3,4, 6-tetra-O-acetyl-beta-D-Glucopyranosyl Isothiocyanate (GITC), adding acetonitrile to dissolve and dilute the mixture to prepare a solution containing 7.2mg of the derivatization reagent solution per 1mL, and taking the solution containing 2.4mg of the derivatization reagent solution per 1mL as a derivatization reagent solution;
5) respectively taking 100 mu L of the system applicability solution, the enantiomer reference substance solution and the test substance solution, respectively adding equal volume of derivatization test solution, uniformly mixing, and standing at room temperature for derivatization for 60min to obtain derivatization solutions of the solutions;
6) chromatographic conditions and system applicability test: a chromatographic column using octadecylsilane chemically bonded silica as a filler, 0.01mol/L potassium dihydrogen phosphate solution (pH value is 2.8) as a mobile phase A, and methanol as a mobile phase B; the flow rate was 1.0mL per minute; the elution mode is gradient elution; the detection wavelength is 250 nm; the column temperature was 25 ℃; the sample injection amount is 5 mu L; taking the derivatization solution of the system applicability solution prepared in the step 5) to carry out a system applicability test, wherein the separation degree of the L-2-amino-5-guanidino valeric acid enantiomer derivative peak and the adjacent peak 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 enantiomer peak;
7) determination of the enantiomers of L-2-amino-5-guanidinopentanoic acid: and (3) respectively taking 5 mu L of the derivatization solution of the enantiomer reference substance solution prepared in the step 5) and the derivatization solution of the test substance solution prepared in the step 5), injecting the solutions into a liquid chromatograph, recording a chromatogram, calculating the content of the enantiomer by using a peak area according to an external standard method, and multiplying the peak area of the enantiomer reference substance by a correction factor of 1.29.
Further, the method for detecting the enantiomer of L-2-amino-5-guanidinopentanoic acid comprises the following steps of gradient elution:
compared with the prior art, the invention has the following beneficial effects:
1. the invention establishes a detection method capable of quantitatively detecting the L-2-amino-5-guanidino valeric acid enantiomer by utilizing the pre-column derivatization high performance liquid chromatography technology, and the method has the advantages of good separation degree, high sensitivity, less interference and capability of effectively controlling the product quality. The method has high sensitivity, accuracy, precision and durability, can accurately reflect the content of the enantiomer in the L-2-amino-5-guanidino valeric acid raw material medicine, is suitable for the detection and quality control of the L-2-amino-5-guanidino valeric acid enantiomer in industry, and thus provides basis for reasonable quality standard formulation, so that the product quality can be better controlled and mastered, and the edible and medicinal safety can be ensured.
2. The method can effectively detect the enantiomer impurities of the L-2-amino-5-guanidino valeric acid raw material, 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 a derivative peak and an adjacent peak of the D-2-amino-5-guanidinopentanoic acid 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 D-2-amino-5-guanidinopentanoic acid peak; the sensitivity of the high performance liquid chromatography for detecting the L-2-amino-5-guanidinopentanoic acid enantiomer is 0.1%.
3. One of the most outstanding characteristics of the invention is to solve the problem of the separation degree of a D-2-amino-5-guanidino valeric acid derivative peak and an adjacent peak. Researches show that the chromatogram derived from GITC has a plurality of interference peaks, the influence on the target peak positioning of the detection method and the test results such as repeatability, system applicability and the like is great, and the requirement of methodology verification is difficult to achieve. Therefore, through researches such as derivation system optimization, buffer salt concentration selection, flow phase ratio selection, elution mode optimization and the like, the problem of the separation degree of a derivation peak and an adjacent peak of D-2-amino-5-guanidinopentanoic acid is solved, and the requirements of indexes such as system applicability test, repeatability, recovery rate, durability and the like verified by methodology are met.
4. The invention has the second most prominent characteristic of solving the sensitivity problem of detecting the D-2-amino-5-guanidino valeric acid impurity. Research shows that when the concentration of the L-2-amino-5-guanidinopentanoic acid in a test solution exceeds 0.5mg/mL, a very significant solvent effect exists in a D-2-amino-5-guanidinopentanoic acid derivative peak (figure 3), detection of the D-2-amino-5-guanidinopentanoic acid is seriously influenced, and the sensitivity of the method cannot meet the requirement of 0.1%. The invention successfully increases the concentration of a test sample to 2.0mg/mL by various technical means, so that the sensitivity of the method for detecting D-2-amino-5-guanidinopentanoic acid reaches 0.1%.
5. The invention has the most outstanding characteristic of solving the problem of interference in detecting D-2-amino-5-guanidino valeric acid impurities. Researches find that the peak area of a D-2-amino-5-guanidinopentanoic acid derivative in the background of high-concentration L-2-amino-5-guanidinopentanoic acid is greatly different from the peak area of a derivative in the presence of single D-2-amino-5-guanidinopentanoic acid with the same concentration, namely that the peak area of a D-2-amino-5-guanidinopentanoic acid reference substance is used for calculating the content of an enantiomer in a test solution, so that a large error exists, and the content of the enantiomer cannot be accurately detected, therefore, the invention finds a conversion relation among the peak areas, and can accurately detect the content of the enantiomer in a sample by a correction factor 1.29.
Drawings
FIG. 1: a chromatogram of the L-2-amino-5-guanidino valeric acid fermentation broth derivative;
FIG. 2: chromatogram of partial purified liquid derivative of L-2-amino-5-guanidinopentanoic acid;
FIG. 3: a solvent effect chromatogram map for detecting D-2-amino-5-guanidinopentanoic acid;
FIG. 4: chromatogram for detecting enantiomers in three batches of L-2-amino-5-guanidino valeric acid raw materials;
FIG. 5: a separation chromatogram of L-2-amino-5-guanidinopentanoic acid and enantiomers thereof;
FIG. 6: chromatogram of undamaged + D-isomer of L-2-amino-5-guanidinopentanoic acid;
FIG. 7: chromatogram of L-2-amino-5-guanidinopentanoic acid strong acid destruction + D-type isomer;
FIG. 8: chromatogram of L-2-amino-5-guanidinopentanoic acid high temperature destruction + D-type isomer;
FIG. 9: intense light destruction of L-2-amino-5-guanidinopentanoic acid + chromatogram of D-isomer.
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: enantiomer detection of three batches of L-2-amino-5-guanidino valeric acid raw material medicine
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 25 deg.C; the flow rate is 1.0 mL/min; the detection wavelength is 250 nm; the sample injection amount is 5 mu L; taking 1.36 g of monopotassium phosphate, adding water to dissolve and dilute the monopotassium phosphate to 1000mL, adjusting the pH value to 2.8 by using 20% phosphoric acid as a mobile phase A, using methanol as a mobile phase B, and eluting according to the following table, wherein the proportions of the monopotassium phosphate are shown in table 1:
TABLE 1
Precisely measuring 100 μ l of each of the test solution and the enantiomer reference solution, adding equal volume of derivatization test solution 1 (taking appropriate amount of 2,3,4, 6-tetra-O-acetyl-beta-D-Glucopyranose Isothiocyanate (GITC), adding acetonitrile to dissolve and dilute to obtain solution containing 7.2mg per 1 ml), mixing, standing at room temperature for 60min, respectively taking 5 μ l, injecting into a liquid chromatograph, and recording chromatogram;
calculating the content of the enantiomer of the L-2-amino-5-guanidino valeric acid by peak area according to an external standard method;
the experimental steps are as follows: accurately weighing appropriate amount of L-2-amino-5-guanidino valeric acid, adding diluent [ water-triethylamine-acetonitrile (volume ratio 50:0.4:50) ] to dissolve and dilute to obtain solution containing 1.5mg per 1ml as sample solution; precisely weighing a proper amount of D-2-amino-5-guanidinopentanoic acid reference substance, adding a diluent to dissolve and quantitatively diluting to prepare a solution containing about 1.5 mu g of D-2-amino-5-guanidinopentanoic acid in 1ml, and performing pre-column derivatization.
Taking appropriate amount of each raw material of L-2-amino-5-guanidinopentanoic acid and D-2-amino-5-guanidinopentanoic acid as reference substances, adding diluent to dissolve and dilute to obtain a solution containing about 0.5mg of L-2-amino-5-guanidinopentanoic acid and 15 μ g of L-2-amino-5-guanidinopentanoic acid in each 1ml, and using the solution as a system applicability solution.
System applicability test L-2-amino-5-guanidinopentanoic acid and its enantiomeric retention time and resolution results are shown in table 2:
TABLE 2
Under the determination conditions, the separation degrees of the L-2-amino-5-guanidino valeric acid derivative peak and the adjacent peak in the system applicability solution are both larger than 2.0, the separation degrees of the enantiomer derivative peak and the adjacent peak are both larger 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 drug enantiomers are shown in Table 3, and the chromatogram is shown in FIG. 4:
TABLE 3
| Sample batch number | Enantiomeric content (%) |
| 20180501 | Not detected out |
| 20180502 | Not detected out |
| 20180503 | Not detected out |
Example 2: enantiomeric positioning assay
The apparatus and chromatographic conditions were the same as in example 1.
The experimental steps are as follows: respectively weighing appropriate amount of L-2-amino-5-guanidino valeric acid and its enantiomer reference substance, dissolving with diluent, and quantitatively diluting to obtain solution containing 0.5mg per 1mL as positioning solution; the preparation method of the mixed solution is the same as that of the system applicability solution of example 1, then pre-column derivatization is carried out, each derivatization solution is respectively injected into a liquid chromatograph, and a chromatogram is recorded. The results of the degrees of separation of the peaks from the adjacent peaks of the derivatives of the enantiomers of L-2-amino-5-guanidinopentanoic acid are shown in Table 4, and the chromatogram is shown in FIG. 5:
TABLE 4
The results show that: good separation between the peaks and adjacent peaks of the enantiomeric derivative of L-2-amino-5-guanidinopentanoic acid was achieved.
Example 3: specificity failure test
The apparatus and chromatographic conditions were the same as in example 1.
The experimental steps are as follows: an appropriate amount of L-2-amino-5-guanidino valeric acid is precisely weighed, forced destruction tests are respectively carried out 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, the preparation method of each destruction sample is shown in a table 5, and a chromatogram is shown in a graph from 6 to 9:
each proprietary failure sample solution was prepared according to the following table:
TABLE 5
The above derived solutions were sampled and chromatograms recorded, and the destruction results are shown in table 6:
TABLE 6
The results show that: d-2-amino-5-guanidinopentanoic acid is not detected in a strong acid, high temperature and strong light damage sample, which indicates that L-2-amino-5-guanidinopentanoic acid is not converted into D-2-amino-5-guanidinopentanoic acid by the strong acid, the high temperature and the strong light; under various destruction conditions, the separation degree of a D-2-amino-5-guanidinopentanoic acid derivative peak and a front peak and a rear peak is more than 1.5, the quantitative detection of the D-2-amino-5-guanidinopentanoic acid derivative peak is not influenced, and the peak purity of the L-2-amino-5-guanidinopentanoic acid derivative peak is more than 999. As can be seen, the method has good specificity for detecting D-2-amino-5-guanidinopentanoic acid.
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 D-2-amino-5-guanidinopentanoic acid by a signal-to-noise ratio method. Respectively preparing stock solutions of L-2-amino-5-guanidino valeric acid and enantiomer, diluting to a certain concentration, sampling for pre-column derivatization, calculating the ratio (signal-to-noise ratio) of peak height of enantiomer derivative peak and noise, wherein when the sample detection amount of the signal-to-noise ratio (S/N) of about 10 is the quantitative limit, the sample detection amount of the signal-to-noise ratio (S/N) of about 3 is the detection limit, the specific preparation method comprises the following steps:
when the concentration of the D-2-amino-5-guanidinopentanoic acid solution in the test solution was 0.38. mu.g/mL, the amount of the sample was 5. mu.L, the absolute amount of the sample of D-2-amino-5-guanidinopentanoic acid was 0.94ng, and the signal-to-noise ratio of the D-2-amino-5-guanidinopentanoic acid derivative was 5.6, which is the detection limit of D-2-amino-5-guanidinopentanoic acid. When the concentration of the D-2-amino-5-guanidinopentanoic acid solution in the test solution was 0.76. mu.g/mL, the amount of the sample was 5. mu.L, the absolute amount of the sample of D-2-amino-5-guanidinopentanoic acid was 1.89ng, and the signal-to-noise ratio of the D-2-amino-5-guanidinopentanoic acid derivative was 12.5, which is the limit of the quantification of D-2-amino-5-guanidinopentanoic acid.
The specific results of the quantitative limit and the detection limit of the L-2-amino-5-guanidinopentanoic acid enantiomer are shown in Table 7:
TABLE 7
The results show that: the quantitative limit concentration of the D-2-amino-5-guanidinopentanoic acid is less than 0.05 percent of the concentration of a test sample, and the content of the corresponding isomer 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: accurately weighing a proper amount of D-2-amino-5-guanidino valeric acid, adding water to dissolve and dilute to 15 mu g/mL, respectively sucking 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0mL by a pipette, and fixing the volume to 10mL by using a diluent, namely a solution with the concentration of 0.76, 1.51, 2.27, 3.02, 3.78 and 4.53 mu g/mL; various concentrations of D-2-amino-5-guanidinopentanoic acid solution were prepared in the same manner in a background of 1.5mg/mL L-2-amino-5-guanidinopentanoic acid. Taking 200 mu L of various D-2-amino-5-guanidino valeric acid solutions, adding 200 mu L of derivatization test solution, derivatizing for 1 hour, filtering, and injecting 5 mu L of sample for chromatographic analysis. Taking the concentration of the D-2-amino-5-guanidinopentanoic acid as a horizontal coordinate and the peak area as a vertical coordinate, and performing linear regression.
Each linear test solution was prepared for enantiomeric linearity as per table 8:
TABLE 8
And (3) respectively sampling the solutions, recording a chromatogram, and taking the concentration of the D-2-amino-5-guanidinopentanoic acid as a horizontal coordinate and the peak area of the derivative as a vertical coordinate to obtain a linear regression equation of the D-2-amino-5-guanidinopentanoic acid. It can be seen that under the background of single D-2-amino-5-guanidinopentanoic acid or high-concentration L-2-amino-5-guanidinopentanoic acid, the concentration of D-2-amino-5-guanidinopentanoic acid and the peak area of the derivative show good linear relation in a certain concentration range, and the correction factor is 1.29.
Example 6: accuracy test
The apparatus and chromatographic conditions were the same as in example 1.
The experimental steps are as follows: weighing 15mg of L-2-amino-5-guanidinopentanoic acid, respectively adding 0.5, 1.0 and 1.5mL of D-2-amino-5-guanidinopentanoic acid solution with the concentration of 15 mu g/mL, and preparing a mixed solution of low, medium and high L- + D-2-amino-5-guanidinopentanoic acid with the concentration of 1.5mg/mL of L-2-amino-5-guanidinopentanoic acid and the enantiomeric concentrations of 0.76 mu g/mL, 1.51 mu g/mL and 2.27 mu g/mL of L-2-amino-5-guanidinopentanoic acid by using a diluent; taking 200 mu L of each mixed solution, adding 200 mu L of derivative test solution, deriving for 1h, taking each derivative solution for an enantiomer accuracy test, and taking three parts of each concentration; samples were injected and chromatograms were recorded separately, and recovery was calculated, with results shown in table 9:
TABLE 9
| Type of concentration | Low concentration of | Middle concentration | High concentration |
| Recovery (%) | 120.18 | 114.26 | 104.15 |
The results show that: the average recovery rate of each related substance is between 70 and 130 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: taking the system applicability solution, respectively changing chromatographic conditions, such as flow rate variation of +/-0.1 mL/min, temperature variation of +/-5 ℃, wavelength variation of +/-5 nm, mobile phase proportion of +/-2%, chromatographic columns of different models and the like, and carrying out chromatographic analysis according to the method. The durability of the method was evaluated according to the variation of tailing factor, resolution, content, etc. of the enantiomeric derivative peak.
The durability test design of the method is shown in table 10:
watch 10
The durability test results of this method are shown in Table 11:
TABLE 11
The results show that: under different conditions, the absolute value of the content of the D-2-amino-5-guanidinopentanoic acid changes within +/-0.0013 percent, and the acceptable requirement of not more than +/-0.1 percent is met; the separation from the front peak and the back peak is complete (the separation degree is more than 1.5), and the tailing factors are less than 2; however, the method is sensitive to changes of the flow phase ratio, the column temperature, the chromatographic column and the like, and is particularly most sensitive to the flow rate and the chromatographic column, and the RSD is high. The method is used for detecting the D-2-amino-5-guanidinopentanoic acid, the durability is good, and small changes of chromatographic conditions have no obvious influence on system adaptability and D-2-amino-5-guanidinopentanoic acid content determination.
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 (8)
1. A detection method of L-2-amino-5-guanidino valeric acid enantiomer is characterized by adopting a pre-column derivatization high performance liquid chromatography:
1) pre-column derivatization: respectively taking a proper amount of a system applicability solution, an enantiomer reference substance solution and a test substance solution, respectively adding equal volume of derivatization test solution to obtain a pre-column derivatization system, uniformly mixing, and standing at room temperature for derivatization for 40-180 min to obtain derivatization solutions of the solutions;
the system applicability solution is a mixed solution prepared by dissolving L-2-amino-5-guanidinopentanoic acid and an enantiomer reference substance in a diluent, wherein the concentration range of the L-2-amino-5-guanidinopentanoic acid is 0.2-1.0 mg/mL, and the concentration range of the enantiomer is 10-30 mu g/mL; the enantiomer reference substance solution is prepared by dissolving an L-2-amino-5-guanidinopentanoic acid enantiomer reference substance in a diluent, and the concentration range of the enantiomer reference substance solution is 0.5-5 mu g/mL; the test solution is prepared by dissolving a raw material of L-2-amino-5-guanidino valeric acid in a diluent, and the concentration range of the test solution is 0.1-3.0 mg/mL; the derivatization test solution is dissolved with 2,3,4, 6-tetra-OAcetyl-βAn acetonitrile solution of D-glucopyranosyl isothiocyanate with the concentration range of 1-5 mg/mL;
2) and (3) testing the applicability of the system: performing high performance liquid chromatography analysis on the derivatization solution of the system applicability solution prepared in the step 1), wherein the separation degree of an L-2-amino-5-guanidino valeric acid enantiomer derivative peak and an adjacent peak 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 enantiomer peak;
3) determination of the enantiomers of L-2-amino-5-guanidinopentanoic acid: respectively taking the derivatization solution of the test solution prepared in the step 1) and the derivatization solution of the enantiomer reference solution for high performance liquid chromatography analysis, recording a chromatogram, and calculating the content of the enantiomer by peak area according to an external standard method;
the high performance liquid chromatography analysis conditions are as follows: a chromatographic column using octadecylsilane chemically bonded silica as a filler, a phosphate solution with the concentration of 5-20 mmol/L and the pH value of 2.5-3.2 is used as a mobile phase A, and methanol is used as a mobile phase B; the flow rate is 0.8-1.2 mL/min; the elution mode is gradient elution; the detection wavelength is 245-255 nm; the column temperature is 20-35 ℃; the sample injection amount is 2-20 mu L;
the gradient elution was performed with a linear gradient as follows:
。
2. The method for detecting an enantiomer of L-2-amino-5-guanidinopentanoic acid as claimed in claim 1, wherein the diluent is a mixed solution of acetonitrile, water and diethylamine in a volume ratio of 50:50: 0.2-0.6.
3. The method for detecting an enantiomer of L-2-amino-5-guanidinopentanoic acid as claimed in claim 1, wherein the diluent is a mixed solution of acetonitrile, water and triethylamine in a volume ratio of 50:50:0.2 to 0.6.
4. The method for detecting enantiomers of L-2-amino-5-guanidinopentanoic acid according to claim 1, wherein the mass ratio of L-2-amino-5-guanidinopentanoic acid to 2,3,4, 6-tetra-O-acetyl- β -D-glucopyranosyl isothiocyanate in the pre-column derivative system of the test solution is 0.05 to 0.85: 1.
5. the method for detecting an enantiomer of L-2-amino-5-guanidinopentanoic acid as claimed in claim 1, wherein the phosphate is potassium dihydrogen phosphate.
6. The method for detecting an enantiomer of L-2-amino-5-guanidinopentanoic acid as claimed in claim 1, wherein the content of the enantiomer is calculated by peak area according to external standard method, and the peak area of the enantiomer control is multiplied by a correction factor of 1.25-1.35.
7. The method for detecting an enantiomer of L-2-amino-5-guanidinopentanoic acid as claimed in any of claims 1 to 6, comprising the steps of:
1) preparation of system suitability solution: taking the diluent as a solvent, respectively preparing the L-2-amino-5-guanidino valeric acid and enantiomer reference substances thereof into solutions with the concentrations of 0.5mg/mL and 15 mu g/mL, and taking the solutions as system applicability solutions;
2) preparation of enantiomer control solutions: using the diluent as solvent, preparing the reference substance of L-2-amino-5-guanidino valeric acid enantiomer with the concentration of 1.5μg/mL solution as an enantiomer control solution;
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 0.1-2.0 mg/mL by using a diluent as a solvent to be used as a test solution;
4) preparing a derivatization test solution: weighing the derivatization reagent 2,3,4, 6-tetra-OAcetyl-βDissolving D-glucopyranosyl isothiocyanate in acetonitrile to prepare a solution with the concentration of 2.4mg/mL as a derivatization test solution;
5) respectively taking 100 parts of system applicability solution, enantiomer reference substance solution and test solutionµL, adding equal volume of derivatization test solution respectively, mixing uniformly, and standing at room temperature for derivatization for 40-180 min to obtain derivatization solutions of the solutions;
6) chromatographic conditions and system applicability test: a chromatographic column using octadecylsilane chemically bonded silica as a filler, a potassium dihydrogen phosphate solution with the pH value of 2.5-3.2 of 0.01mol/L as a mobile phase A, and methanol as a mobile phase B; the flow rate is 0.8-1.2 mL/mL; the elution mode is gradient elution; the detection wavelength is 245-255 nm; the column temperature is 20-35 ℃; the sample injection amount is 2-20µL; taking the derivatization solution of the system applicability solution prepared in the step 5) to carry out a system applicability test, wherein the separation degree of the L-2-amino-5-guanidino valeric acid enantiomer derivative peak and the adjacent peak 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 enantiomer peak;
7) process for preparing enantiomers of L-2-amino-5-guanidinopentanoic acidAnd (3) determination: taking the derivatization solution of the enantiomer reference substance solution prepared in the step 5) and the derivatization solution of the test substance solution for 2-20 respectivelyµAnd L, respectively injecting the L into a liquid chromatograph, recording a chromatogram, calculating the content of the enantiomer according to an external standard method by using a peak area, and multiplying the peak area of an enantiomer reference product by a correction factor of 1.25-1.35.
8. The method for detecting an enantiomer of L-2-amino-5-guanidinopentanoic acid as claimed in any of claims 1 to 6, comprising the steps of:
1) preparation of system suitability solution: taking the diluent as solvent, respectively preparing the L-2-amino-5-guanidino valeric acid and enantiomer thereof into the reference substances with concentrations of 0.5mg/mL and 15 mg/mLμg/mL of solution as a system suitability solution;
2) preparation of enantiomer control solutions: using the diluent as solvent, preparing the reference substance of L-2-amino-5-guanidino valeric acid enantiomer with the concentration of 1.5μg/mL solution as an enantiomer control solution;
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 1.5mg/mL by using a diluent as a solvent to be used as a test solution;
4) preparing a derivatization test solution: weighing the derivatization reagent 2,3,4, 6-tetra-OAcetyl-βDissolving a proper amount of D-glucopyranosyl isothiocyanate in acetonitrile to prepare a solution with the concentration of 2.4mg/mL as a derivatization test solution;
5) respectively taking a system applicability solution, an enantiomer reference substance solution and a test substance solution 100µL, adding equal volume of derivatization test solution respectively, mixing uniformly, and standing at room temperature for derivatization for 60min to obtain derivatization solutions of the solutions;
6) chromatographic conditions and system applicability test: a chromatographic column using octadecylsilane chemically bonded silica as a filler, a potassium dihydrogen phosphate solution with the pH value of 2.8 of 0.01mol/L as a mobile phase A, and methanol as a mobile phase B; the flow rate is 1.0 mL/min; the elution mode is gradient elution; the detection wavelength is 250 nm; the column temperature was 25 ℃; the sample injection amount is 5µL; taking the derivatization solution of the system applicability solution prepared in the step 5) to carry out a system applicability test, wherein the separation degree of the L-2-amino-5-guanidino valeric acid enantiomer derivative peak and the adjacent peak 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 enantiomer peak;
7) determination of the enantiomers of L-2-amino-5-guanidinopentanoic acid: taking the derivatization solution of the enantiomer reference substance solution prepared in the step 5) and the derivatization solution of the test substance solution 5 respectivelyµAnd L, respectively injecting the mixture into a liquid chromatograph, recording a chromatogram, calculating the content of the enantiomer according to the peak area of an external standard method, and multiplying the peak area of an enantiomer reference product by a correction factor of 1.29.
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