CN110596294A - High performance liquid detection method of glycyl-D-glutamine - Google Patents
High performance liquid detection method of glycyl-D-glutamine Download PDFInfo
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- CN110596294A CN110596294A CN201910996192.9A CN201910996192A CN110596294A CN 110596294 A CN110596294 A CN 110596294A CN 201910996192 A CN201910996192 A CN 201910996192A CN 110596294 A CN110596294 A CN 110596294A
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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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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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
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
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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
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
Abstract
The invention discloses a high performance liquid detection method of glycyl-D-glutamine, which comprises the following steps: preparing a test solution; preparing a mixed reference solution; deriving the test solution; derivation of mixed reference substance solution; and (3) chromatography: precisely measuring 2 mu l of each sample derived in the step (3) and the step (4), injecting the samples into a liquid chromatograph after the chiral chromatographic column is well balanced, collecting a chiral separation chromatogram, recording the chromatogram for 60min, and checking the separation effect. The chiral separation method disclosed by the invention realizes the chiral separation of glycyl-L-glutamine and glycyl-D-glutamine, improves the test efficiency, has relatively good chiral column stability, can still obtain a good separation effect after being separated for multiple times, is wide in application range and has good economic value.
Description
Technical Field
The invention belongs to the technical field of high performance liquid chromatography detection, and relates to a high performance liquid detection method of glycyl-D-glutamine.
Background
The existence of amino acids in human body not only provides important raw materials for synthesizing protein, but also provides material basis for promoting growth, carrying out normal metabolism and maintaining life. The amino acid medicine has low side effect, high medicine effect and strong pertinence, and can not accumulate in vivo to cause poisoning.
glycyl-L-glutamine is commonly used to provide glutamine to promote protein synthesis, amino acids constituting proteins are all L-type amino acids, while drugs obtained through chemical synthesis are often enantiomers, and are not single optical isomers, and in many cases, a pair of enantiomers of a compound has significant differences in pharmacological activity, metabolic process, metabolic rate, toxicity and the like in an organism, and there are generally four different cases: both compounds in a pair of enantiomers have equivalent or nearly equivalent qualitative and quantitative pharmacological activity; only one enantiomer has the required pharmacological activity, while the other enantiomer has no significant pharmacological effect; each enantiomer has a quantitatively different activity; the two enantiomers have quantitatively equivalent, but qualitatively different activities. glycyl-L-glutamine has pharmacological activity for a pair of enantiomers of glycyl-glutamine, whereas glycyl-D-glutamine has no significant pharmacological effect. In order to ensure the optical purity of the synthesized product, it is a very important problem to strictly control the production of glycyl-D-glutamine during the synthesis.
When a medicine with an asymmetric center is developed, a plurality of chiral chromatographic columns and chiral mobile phases are required to be used for testing, the testing time is long, and the effect is not ideal. The glycyl glutamine is absorbed at the tail end, the ultraviolet absorption is not strong, and the chiral chromatographic column is adopted for separating and determining the isomers of the glycyl glutamine, so that the cost is high, and the separation difficulty is high. In the prior art, other products with weak ultraviolet absorption are also reported to be separated by a common chromatographic column after being derivatized by a derivatization reagent, but the method needs long reaction time, and also needs the steps of solvent drying, redissolution, filtration and the like, and the operation steps are complicated.
Therefore, the development of a rapid, simple, economical and wide-application method for detecting glycyl-D-glutamine by high performance liquid chromatography is a technical problem that needs to be solved urgently by those skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a rapid, simple, economical and wide-application method for detecting glycyl-D-glutamine by high performance liquid chromatography.
In order to achieve the purpose, the invention adopts the following technical scheme:
a high performance liquid detection method of glycyl-D-glutamine comprises the following steps:
(1) preparing a test solution: accurately weighing glycyl-L-glutamine, and diluting with water to obtain glycyl-L-glutamine solution containing glycyl-L-glutamine 2.5-3.5mg in 1ml water;
(2) preparation of mixed control solutions: accurately weighing glycyl-L-glutamine reference substance and glycyl-D-glutamine reference substance, and diluting with water to obtain 1ml of mixed solution containing glycyl-L-glutamine 2.5-3.5mg and glycyl-D-glutamine 4-5 μ g;
(3) derivation of test solution: precisely measuring 10 μ l of sample solution, placing in a dry derivatization tube, adding 30 μ l of buffer solution, vortex mixing, adding 60 μ l of derivatization reagent, vortex mixing for 15s, sealing the sample tube with parafilm, heating at 55 ℃ for 10min, taking out the sample obtained by derivatization, cooling to 22-28 ℃, and transferring to a microscale sample introduction tube;
(4) mixed control solution derivatization: precisely measuring 10 mu l of mixed reference substance solution, and performing derivatization operation by the same method as the step (3);
(5) and (3) chromatography: precisely measuring 2 mu l of each sample derived in the step (3) and the step (4), injecting the samples into a liquid chromatograph after the chiral chromatographic column is well balanced, collecting a chiral separation chromatogram, recording the chromatogram for 60min, and checking the separation effect.
Further, the chromatographic conditions in the step (5) are as follows:
1) the chromatographic column is a chromatographic column with the surface of silica gel covalently bonded with quinine (8R,9S) - (1R,2R) -cyclohexyl sulfamic acid derivative as a filler, and the chromatographic column is a chromatographic column with the diameter of 3 mu m and the diameter of 150 multiplied by 4 mm;
2) the mobile phase is a mixture of formic acid, diethylamine and methanol, and the volume ratio of the formic acid, diethylamine and methanol is 1.9:2.6: 995.5;
3) the detection wavelength is 248 nm;
4) the flow rate is 0.2-0.22 ml/min;
5) the column temperature is 20-22 ℃.
Further, the glycyl glutamine in the step (1) is a mixture of glycyl-L-glutamine and glycyl-D-glutamine.
Further, the diameter of the derivative pipe in the step (3) is 6mm, and the height of the derivative pipe is 50 mm.
Further, the Buffer in step (3) is Waters AccQ. Fluor boric acid Buffer1 from Waters AccQ. Fluor kit provided by Waters corporation, USA;
the derivatization reagent is prepared from Waters AccQ Fluor derivatization agent powder and Waters AccQ Fluor derivatization agent diluent, wherein the Waters AccQ Fluor derivatization agent powder and the Waters AccQ Fluor derivatization agent diluent are from Waters AccQ Fluor kits provided by the American Waters company.
Further, the preparation method of the derivatization reagent comprises the following steps: sucking 1ml of Waters AccQ. Fluor derivative diluent into a 2B bottle of Waters AccQ. Fluor derivative diluent powder, sealing by a cover, shaking for 10s, heating at 55 ℃ for 30min to completely dissolve the derivative powder, taking out, cooling to 22-28 ℃, and storing in a dryer.
Further, the balance time of the chiral chromatographic column in the step (5) is more than or equal to 100 min.
Further, the mechanism of reaction of the derivatizing reagent with glycyl-L-glutamine is as follows:
the mechanism of reaction of the derivatizing reagent with glycyl-D-glutamine is as follows:
the derivatization reagent undergoes hydrolysis reaction by itself, and the reaction mechanism is as follows:
the invention has the beneficial effects that: the chiral separation of glycyl-L-glutamine and glycyl-D-glutamine is realized, and the testing efficiency is improved. The technical problems of complicated mobile phase system and poor separation effect in a chiral separation system of a high performance liquid chromatography are solved. The chiral separation method only uses a simple mobile phase, has good chiral column stability, can still obtain good separation effect after being separated for many times, has wide application range and has good economic value.
Drawings
FIG. 1 is an HPLC chromatogram for separating glycyl-L-glutamine from glycyl-D-glutamine in example 1.
FIG. 2 is an HPLC chromatogram for separating glycyl-L-glutamine from glycyl-D-glutamine in example 2.
FIG. 3 is an HPLC chromatogram for separating glycyl-L-glutamine from glycyl-D-glutamine in example 3.
FIG. 4 is a chromatogram of a specific test of the high performance liquid detection method of glycyl-D-glutamine of the present invention.
FIG. 5 is a chromatogram of a high performance liquid detection method for glycyl-D-glutamine based on the system applicability test.
FIG. 6 is a linear regression curve of glycyl-D-glutamine by the high performance liquid chromatography method of glycyl-D-glutamine of the present invention.
FIG. 7 is a linear and linear range test chromatogram of the high performance liquid detection method of glycyl-D-glutamine of the present invention.
FIG. 8 is a limited chromatogram for detecting glycyl-D-glutamine by the high performance liquid chromatography method of glycyl-D-glutamine of the present invention.
FIG. 9 is a high performance liquid chromatography detection method of glycyl-D-glutamine according to the present invention.
FIG. 10 is a chromatogram of a high performance liquid chromatography for detecting glycyl-D-glutamine according to the present invention.
FIG. 11 is a chromatogram of a high performance liquid chromatography assay for glycyl-D-glutamine for intermediate precision (Analyzer 2, Instrument B).
FIG. 12 is a chromatogram of an accuracy test of a high performance liquid detection method of glycyl-D-glutamine according to the present invention.
FIG. 13 is a chromatogram of a high performance liquid chromatography test for detecting the stability of a glycyl-D-glutamine solution.
FIG. 14 is a chromatogram of a high performance liquid chromatography detection method of glycyl-D-glutamine for testing the stability of a control solution.
FIG. 15 is a chromatogram for examining the range of flow velocity change of a mobile phase in the high performance liquid detection method of glycyl-D-glutamine.
Detailed Description
The present invention will be further illustrated with reference to the accompanying drawings and specific embodiments, which are to be understood as merely illustrative of the invention and not as limiting the scope of the invention.
Example 1
Chromatographic conditions are as follows: a chromatographic column taking silica gel surface covalent bonding-quinine (8R,9S) - (1R,2R) -cyclohexyl sulfamic acid derivative as a filler, a mobile phase of formic acid-diethylamine-methanol (1.9:2.6:995.5), a detection wavelength of 248nm, a flow rate of 0.2ml/min, a column temperature of 20 ℃ and a sample injection amount of 2 mul.
Preparation of a test solution: accurately weighing a proper amount of glycyl-L-glutamine, and diluting with water to obtain a solution containing glycyl-L-glutamine 3mg in 1ml of water as a test solution; an appropriate amount of each of glycyl-L-glutamine and glycyl-D-glutamine was precisely weighed and diluted with water to a mixed solution containing glycyl-L-glutamine (3 mg) and glycyl-D-glutamine (4.5. mu.g) in 1ml of water as a mixed control solution. Precisely measuring 10 μ l of sample solution, placing in a dry derivative tube (diameter 6mm, height 50mm), adding 30 μ l of Buffer solution (Buffer1), vortex mixing, adding 60 μ l of derivative reagent, vortex mixing for 15s, sealing the sample tube with parafilm, heating at 55 deg.C for 10min, taking out, cooling to room temperature, and transferring to a micro sample injection tube.
The determination method comprises the following steps: precisely measuring 2 μ l, injecting into a liquid chromatograph, and recording chromatogram to 60 min; and another 10 mu l of mixed reference substance solution is precisely measured, the same-method derivatization operation is carried out, after the chiral chromatographic column is well balanced, the solution is injected into a chromatograph, a chiral separation chromatogram is collected through a chromatographic workstation, and the separation effect is checked. The result shows that under the chromatographic condition, the separation degree of glycyl-L-glutamine and glycyl-D-glutamine is more than 1.5, the detection requirement is met, and the HPLC (high performance liquid chromatography) spectrum for separating glycyl-L-glutamine and glycyl-D-glutamine is shown in figure 1.
Example 2
Chromatographic conditions are as follows: a chromatographic column taking silica gel surface covalent bonding-quinine (8R,9S) - (1R,2R) -cyclohexyl sulfamic acid derivative as a filler, a mobile phase of formic acid-diethylamine-methanol (1.9:2.6:995.5), a detection wavelength of 248nm, a flow rate of 0.2ml/min, a column temperature of 22 ℃ and a sample injection amount of 2 mul.
Preparation of a test solution: accurately weighing a proper amount of glycyl-L-glutamine, and diluting with water to obtain a solution containing glycyl-L-glutamine 3mg in 1ml of water as a test solution; an appropriate amount of each of glycyl-L-glutamine and glycyl-D-glutamine was precisely weighed and diluted with water to a mixed solution containing glycyl-L-glutamine (3 mg) and glycyl-D-glutamine (4.5. mu.g) in 1ml of water as a mixed control solution. Precisely measuring 10 μ l of sample solution, placing in a dry derivative tube (diameter 6mm, height 50mm), adding 30 μ l of Buffer solution (Buffer1), vortex mixing, adding 60 μ l of derivative reagent, vortex mixing for 15s, sealing the sample tube with parafilm, heating at 55 deg.C for 10min, taking out, cooling to room temperature, and transferring to a micro sample injection tube.
The determination method comprises the following steps: precisely measuring 2 μ l, injecting into a liquid chromatograph, and recording chromatogram to 60 min; and another 10 mu l of mixed reference substance solution is precisely measured, the same-method derivatization operation is carried out, after the chiral chromatographic column is well balanced, the solution is injected into a chromatograph, a chiral separation chromatogram is collected through a chromatographic workstation, and the separation effect is checked. The result shows that under the chromatographic condition, the separation degree of glycyl-L-glutamine and glycyl-D-glutamine is more than 1.5, the detection requirement is met, and the HPLC (high performance liquid chromatography) spectrum for separating glycyl-L-glutamine and glycyl-D-glutamine is shown in figure 2.
Example 3
Chromatographic conditions are as follows: a chromatographic column taking silica gel surface covalent bonding-quinine (8R,9S) - (1R,2R) -cyclohexyl sulfamic acid derivative as a filler, a mobile phase of formic acid-diethylamine-methanol (1.9:2.6:995.5), a detection wavelength of 248nm, a flow rate of 0.22ml/min, a column temperature of 20 ℃ and a sample injection amount of 2 mul.
Preparation of a test solution: accurately weighing a proper amount of glycyl-L-glutamine, and diluting with water to obtain a solution containing glycyl-L-glutamine 3mg in 1ml of water as a test solution; an appropriate amount of each of glycyl-L-glutamine and glycyl-D-glutamine was precisely weighed and diluted with water to a mixed solution containing glycyl-L-glutamine (3 mg) and glycyl-D-glutamine (4.5. mu.g) in 1ml of water as a mixed control solution. Precisely measuring 10 μ l of sample solution, placing in a dry derivative tube (diameter 6mm, height 50mm), adding 30 μ l of Buffer solution (Buffer1), vortex mixing, adding 60 μ l of derivative reagent, vortex mixing for 15s, sealing the sample tube with parafilm, heating at 55 deg.C for 10min, taking out, cooling to room temperature, and transferring to a micro sample injection tube.
The determination method comprises the following steps: precisely measuring 2 μ l, injecting into a liquid chromatograph, and recording chromatogram to 60 min; and another 10 mu l of mixed reference substance solution is precisely measured, the same-method derivatization operation is carried out, after the chiral chromatographic column is well balanced, the solution is injected into a chromatograph, a chiral separation chromatogram is collected through a chromatographic workstation, and the separation effect is checked. The result shows that under the chromatographic condition, the separation degree of glycyl-L-glutamine and glycyl-D-glutamine is more than 1.5, the detection requirement is met, and the HPLC (high performance liquid chromatography) spectrum for separating glycyl-L-glutamine and glycyl-D-glutamine is shown in figure 3.
Effect test
(1) Apparatus, reagent and chromatographic conditions
The instrument comprises the following steps: waters e2695 high performance liquid chromatograph.
Reagent: formic acid, diethylamine, methanol and ultrapure water.
Reagent testing: glycyl-D-glutamine reference substance, glycyl-L-glutamine
Chromatographic conditions are as follows: a chromatographic column taking silica gel surface covalent bonding-quinine (8R,9S) - (1R,2R) -cyclohexyl sulfamic acid derivative as a filler, a mobile phase of formic acid-diethylamine-methanol (1.9:2.6:995.5), a detection wavelength of 248nm, a flow rate of 0.22ml/min, a column temperature of 20 ℃ and a sample injection amount of 2 mul.
(2) Specificity test
Waters AccQ Fluor boric acid Buffer (Buffer1) was obtained from Waters AccQ Fluor kit supplied by Waters, USA;
waters AccQ Fluor derivative powder (2A), Waters AccQ Fluor derivative diluent (2B) from Waters AccQ Fluor kit available from Waters corporation, USA;
preparation of derivatizing reagent: sucking 1ml of diluent from the bottle 2B into the bottle 2A, sealing with a cover, shaking for 10s, heating at 55 deg.C for 30min to dissolve the derivative powder, taking out, cooling, and storing in a desiccator.
Preparation of a test solution: taking a proper amount of the product, precisely weighing, adding ultrapure water to dissolve to prepare a solution containing glycyl-L-glutamine 3mg per 1ml, and shaking up to obtain the product.
glycyl-D-glutamine control stock solution: taking glycyl-D-glutamine reference substance about 25mg, precisely weighing, placing in a 50ml measuring flask, adding ultrapure water for dissolving and diluting to scale, shaking uniformly, measuring 5ml, placing in a 50ml measuring flask, adding ultrapure water for dissolving and diluting to scale, and shaking uniformly to obtain the glycyl-D-glutamine contrast substance.
glycyl-D-glutamine control solutions: precisely measuring 2ml of glycyl-D-glutamine reference substance stock solution, placing into a 20ml measuring flask, adding ultrapure water for dissolving, diluting to scale, and shaking up to obtain the glycyl-D-glutamine reference substance stock solution.
Preparation of test solutions: weighing 50mg of the product, accurately weighing, placing in a 20ml measuring flask, accurately adding 2ml of glycyl-D-glutamine reference substance stock solution, adding ultrapure water to dissolve and dilute to scale, and shaking up to obtain the product.
Solution derivatization preparation: precisely measuring 10 mu l of solution to be measured, placing the solution into a dry derivative tube (diameter is 6mm, height is 50mm), adding 30 mu l of Buffer solution (Buffer1), carrying out vortex mixing, adding 60 mu l of derivative reagent, carrying out vortex mixing for 15s, sealing a sample tube by using a paraffin film, heating at 55 ℃ for 10min, taking out, cooling to room temperature, and transferring to a micro sample injection tube to obtain the micro sample injection tube.
Test method and results according to the above chromatographic conditions, 2. mu.l of each of the above solutions was taken, injected into a high performance liquid chromatograph, and the chromatogram was recorded. The results show that the specificity is good, and the specificity test chromatogram is shown in FIG. 4.
(3) System suitability test
Test solutions were formulated and derivatized as per the methods under "specificity". Precisely measuring 2 μ l, injecting into liquid chromatograph, and recording chromatogram. The result shows that the peak area RSD (%, n ═ 6) of glycyl-D-glutamine is 3.06, the theoretical plate number and tailing factor of glycyl-D-glutamine both meet the requirement, and the peak separation degree of glycyl-L-glutamine and glycyl-D-glutamine meets the requirement, which indicates that the system has good applicability. The test result is shown in figure 1, and the chromatogram of the system applicability test is shown in figure 5.
TABLE 1 System suitability results
(4) Linear and range test
Taking a proper amount of glycyl-D-glutamine, precisely weighing, placing into a measuring flask, and adding ultrapure water to prepare a solution with the concentration of about 45 mu g/ml. Measuring the solution in proper amount, and adding ultrapure water to dilute the solution in sequence to prepare a series of linear solutions with concentration. Respectively and precisely measuring 10 mul, and deriving according to the method under the 'specificity' item. Respectively and precisely measuring 2 mu l, injecting into a liquid chromatograph, and recording a chromatogram. Linear regression with concentration (μ g/ml) and peak area gave the regression equation y 13477x +90.537, r 0.9991, indicating that glycyl-D-glutamine has a good linear relationship in the range of 0.4500-6.750 μ g/ml. The results are shown in Table 2, the linear regression curve of glycyl-D-glutamine is shown in FIG. 6, and the chromatogram of the linear and linear range tests is shown in FIG. 7.
TABLE 2 glycyl-D-Glutamine Linearity and Range test results
| — | Concentration (μ g/ml) | 1 | 2 | Average peak area |
| Linear 1 | 0.4500 | 6119 | 6133 | 6126 |
| Linearity 2 | 2.250 | 29839 | 29390 | 29614.5 |
| Line 3 | 3.600 | 49262 | 48366 | 48814 |
| Linearity 4 | 4.500 | 60400 | 61275 | 60837.5 |
| Linear 5 | 5.400 | 74833 | 75213 | 75023 |
| Linear 6 | 6.750 | 88211 | 90647 | 89429 |
(5) Sensitivity test
(5.1) detection Limit test
Precisely measuring 3ml of limiting solution, placing in a 10ml measuring flask, adding ultrapure water for dissolving and diluting to scale, shaking, precisely measuring 10 μ l, deriving to obtain corresponding solution according to the method under the 'special' term, precisely measuring 2 μ l, injecting into a liquid chromatograph, and recording chromatogram. The results showed that the detection limit of glycyl-D-glutamine was 101 ng/ml. The detection limit chromatogram of glycyl-D-glutamine is shown in FIG. 8.
(5.2) quantitative Limit test
Precisely weighing a proper amount of glycyl-D-glutamine reference substance, adding ultrapure water for dissolving, gradually diluting, precisely measuring 10 mu l, deriving according to a method under the 'specificity' item to obtain a corresponding solution, precisely measuring 2 mu l, injecting into a liquid chromatograph, recording a chromatogram, and displaying the result that the quantitative limit of glycyl-D-glutamine is 435ng/ml and the quantitative limit chromatogram of glycyl-D-glutamine is shown in figure 9.
(6) Precision test
(6.1) repeatability test
glycyl-D-glutamine control solutions and test solutions were prepared and derivatized as described under "specificity". Respectively and precisely measuring 2 mu l, injecting into a liquid chromatograph, and recording a chromatogram. The results show that the RSD (%, n ═ 6) value of the measured content of glycyl-D-glutamine in the test solution was 1.85, indicating that the method was highly reproducible. The test results are shown in Table 3, and the chromatogram of the repeatability test is shown in FIG. 10.
TABLE 3 glycyl-D-Glutamine repeatability test results
(6.2) intermediate precision test
According to the method under the item of 'precision test', different testers use different test instruments to test on different test dates, the content of glycyl-D-glutamine is compared with the result measured in the precision test, and the intermediate precision of the method is inspected. The results showed that the RSD (%, n-12) value of the content of glycyl-D-glutamine was 2.51, indicating that the method was good in intermediate precision. The results are shown in Table 4, and the chromatogram of the intermediate precision (Analyzer 2, instrument B) is shown in FIG. 11.
TABLE 4 measurement results of intermediate precision test of glycyl-D-glutamine
(7) Accuracy test
Taking a glycyl-D-glutamine reference substance of about 25mg, precisely weighing, placing in a 50ml measuring flask, adding ultrapure water for dissolving and diluting to scale, shaking up, precisely weighing 2ml, placing in a 20ml measuring flask, adding ultrapure water for dissolving and diluting to scale, shaking up, and taking as a reference substance storage solution.
Taking about 50mg of the product, precisely weighing, respectively placing into 20ml measuring flasks, adding glycyl-D-glutamine reference substance stock solutions 1.6ml, 2.0ml and 2.4ml respectively, adding ultrapure water for dissolving, diluting to scale, and shaking uniformly to obtain corresponding 80%, 100% and 120% sample loading recovery rate solutions.
Respectively and precisely measuring 10 μ l of the above solutions, deriving to obtain corresponding solutions according to the method under the term of "specificity", precisely measuring 2 μ l, injecting into high performance liquid chromatograph, recording chromatogram, and calculating recovery rate of glycyl-D-glutamine. The test results show that the average recovery rate (%, n ═ 9) is 94.9, and the RSD (%, n ═ 9) is 3.14, which indicates that the method has good accuracy and reliable test results. The test results are shown in Table 5, and the accuracy test chromatogram is shown in FIG. 12.
TABLE 5 glycyl-D-Glutamine sample recovery test results
(8) Stability test of solution
(8.1) stability of test solutions
glycyl-D-glutamine control solutions and test solutions were prepared and derivatized as described under "specificity". After 0h, 4h, 8h, 12h and 19h of derivatization, 2 mul is precisely measured and injected into a liquid chromatograph. The results show that the measured glycyl-D-glutamine content RSD (%, n ═ 5) in the test solution is 2.91, indicating that the test solution is stable for at least 19h when stored at room temperature. The test results are shown in Table 6, and the chromatogram for the stability test of the test solution is shown in FIG. 13.
TABLE 6 test solution stability test results
(8.2) stability of control solutions
glycyl-D-glutamine reference solutions were prepared and derivatized according to the methods described under "specificity". After 0h, 4h, 8h, 12h and 19h of derivatization, 2 mul is precisely measured and injected into a liquid chromatograph. The results showed that the RSD (%, n-5) of the glycyl-D-glutamine peak area in the control solution was 2.76, indicating that the control solution was stable for at least 19h when stored at room temperature. The test results are shown in Table 7, and the chromatogram for the solution stability test of the control is shown in FIG. 14.
TABLE 7 stability test results for glycyl-D-Glutamine control solutions
(9) Durability test
TABLE 8 durability test results
(9.1) range of variation of flow velocity of mobile phase
And under the condition that other detection conditions are not changed, changing the flow velocity of the mobile phase, and inspecting the influence of the change of the flow velocity on the detection result. The test result shows that the flow rate of 0.18-0.22ml/min has no significant influence on the glycyl-D-glutamine determination. The test results are shown in Table 9, and the chromatogram for examining the flow velocity variation range of the mobile phase is shown in FIG. 15.
TABLE 9 examination of flow Rate variation Range of glycyl-D-Glutamine Mobile phase
From the foregoing description, it is clear that while the method of the present invention has been described in terms of preferred embodiments, it is obvious to those skilled in the art that the technology can be implemented and applied by modifying or appropriately combining the methods and applications described herein within the spirit, scope and spirit of the present invention. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included within the invention.
Claims (7)
1. A high performance liquid detection method of glycyl-D-glutamine is characterized by comprising the following steps:
(1) preparing a test solution: accurately weighing glycyl-L-glutamine, and diluting with water to obtain glycyl-L-glutamine solution containing glycyl-L-glutamine 2.5-3.5mg in 1ml water;
(2) preparation of mixed control solutions: accurately weighing glycyl-L-glutamine reference substance and glycyl-D-glutamine reference substance, and diluting with water to obtain a mixed solution containing glycyl-L-glutamine 2.5-3.5mg and glycyl-D-glutamine 4-5 μ g in 1ml of water;
(3) derivation of test solution: precisely measuring a sample solution, placing the sample solution in a dry derivatization tube, adding a buffer solution, carrying out vortex mixing, adding a derivatization reagent, carrying out vortex mixing, sealing the sample tube by using a paraffin film, heating for 10min, taking out a derivatization sample, cooling to 22-28 ℃, and transferring the derivatization sample into a micro sample injection tube;
(4) mixed control solution derivatization: precisely measuring a mixed reference substance solution, and performing derivatization operation in the same method as the step (3);
(5) and (3) chromatography: precisely measuring 2 mu l of each sample derived in the step (3) and the step (4), injecting the samples into a liquid chromatograph after the chiral chromatographic column is well balanced, collecting a chiral separation chromatogram, recording the chromatogram, and checking the separation effect.
2. The method for detecting glycyl-D-glutamine according to claim 1, wherein the chromatographic conditions in the step (5) are as follows:
1) the chromatographic column is a chromatographic column with the surface of silica gel covalently bonded with quinine (8R,9S) - (1R,2R) -cyclohexyl sulfamic acid derivative as a filler, and the chromatographic column is a chromatographic column with the diameter of 3 mu m and the diameter of 150 multiplied by 4 mm;
2) the mobile phase is a mixture of formic acid, diethylamine and methanol, and the volume ratio of the formic acid, diethylamine and methanol is 1.9:2.6: 995.5;
3) the detection wavelength is 248 nm;
4) the flow rate is 0.2-0.22 ml/min;
5) the column temperature is 20-22 ℃.
3. The method for detecting glycyl-D-glutamine according to claim 1, wherein said glycyl-glutamine in step (1) is a mixture of glycyl-L-glutamine and glycyl-D-glutamine.
4. The method for detecting glycyl-D-glutamine according to claim 1, wherein the diameter of the derivative tube of step (3) is 6mm and the height thereof is 50 mm.
5. The method for the high performance liquid chromatography detection of glycyl-D-glutamine according to claim 1, wherein the Buffer in step (3) is Waters AccQ Fluor boric acid Buffer1 from Waters CCQ Fluor kit provided by Waters corporation, USA;
the derivatization reagent is prepared from Waters AccQ Fluor derivatization agent powder and Waters AccQ Fluor derivatization agent diluent, wherein the Waters AccQ Fluor derivatization agent powder and the Waters AccQ Fluor derivatization agent diluent are from Waters AccQ Fluor kits provided by the American Waters company.
6. The method for detecting glycyl-D-glutamine with high performance liquid according to claim 5, wherein the method for preparing the derivatization reagent comprises the following steps: sucking 1ml of Waters AccQ. Fluor derivative diluent into a 2B bottle of Waters AccQ. Fluor derivative diluent, sealing the bottle with a cover, shaking for 10s, heating at 55 ℃ for 30min to completely dissolve the derivative powder, taking out and cooling to 22-28 ℃, and storing in a dryer.
7. The high performance liquid chromatography detection method of glycyl-D-glutamine according to claim 1, wherein the equilibrium time of the chiral chromatographic column in the step (5) is not less than 100 min.
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