CN112611821A - High performance liquid chromatography analysis method for calcium gluconate related substances - Google Patents
High performance liquid chromatography analysis method for calcium gluconate related substances Download PDFInfo
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- 229960004494 calcium gluconate Drugs 0.000 title claims abstract description 85
- 235000013927 calcium gluconate Nutrition 0.000 title claims abstract description 85
- 239000004227 calcium gluconate Substances 0.000 title claims abstract description 85
- NEEHYRZPVYRGPP-UHFFFAOYSA-L calcium;2,3,4,5,6-pentahydroxyhexanoate Chemical compound [Ca+2].OCC(O)C(O)C(O)C(O)C([O-])=O.OCC(O)C(O)C(O)C(O)C([O-])=O NEEHYRZPVYRGPP-UHFFFAOYSA-L 0.000 title claims abstract description 85
- 239000000126 substance Substances 0.000 title claims abstract description 40
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 24
- 238000004458 analytical method Methods 0.000 title claims abstract description 16
- 239000000243 solution Substances 0.000 claims abstract description 45
- 239000012535 impurity Substances 0.000 claims abstract description 27
- 239000013558 reference substance Substances 0.000 claims abstract description 26
- 239000012085 test solution Substances 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 21
- 239000012488 sample solution Substances 0.000 claims abstract description 17
- 239000012088 reference solution Substances 0.000 claims abstract description 14
- 239000007788 liquid Substances 0.000 claims abstract description 7
- 238000010812 external standard method Methods 0.000 claims abstract description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 71
- 229960005069 calcium Drugs 0.000 claims description 71
- 239000011575 calcium Substances 0.000 claims description 71
- 229910052791 calcium Inorganic materials 0.000 claims description 71
- AYRXSINWFIIFAE-SCLMCMATSA-N Isomaltose Natural products OC[C@H]1O[C@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)[C@@H](O)[C@@H](O)[C@@H]1O AYRXSINWFIIFAE-SCLMCMATSA-N 0.000 claims description 26
- DLRVVLDZNNYCBX-RTPHMHGBSA-N isomaltose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)C(O)O1 DLRVVLDZNNYCBX-RTPHMHGBSA-N 0.000 claims description 26
- 229940095625 calcium glucarate Drugs 0.000 claims description 24
- UGZVNIRNPPEDHM-SBBOJQDXSA-L calcium;(2s,3s,4s,5r)-2,3,4,5-tetrahydroxyhexanedioate Chemical compound [Ca+2].[O-]C(=O)[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O UGZVNIRNPPEDHM-SBBOJQDXSA-L 0.000 claims description 24
- 239000000523 sample Substances 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 238000005303 weighing Methods 0.000 claims description 13
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 claims description 11
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 10
- 238000007865 diluting Methods 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 6
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 5
- JYTUSYBCFIZPBE-ZNLUKOTNSA-N cellobionic acid Chemical compound OC(=O)[C@H](O)[C@@H](O)[C@@H]([C@H](O)CO)O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O JYTUSYBCFIZPBE-ZNLUKOTNSA-N 0.000 claims description 5
- 235000019253 formic acid Nutrition 0.000 claims description 5
- 230000014759 maintenance of location Effects 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 238000004587 chromatography analysis Methods 0.000 claims description 3
- GPRLSGONYQIRFK-UHFFFAOYSA-N hydron Chemical compound [H+] GPRLSGONYQIRFK-UHFFFAOYSA-N 0.000 claims description 3
- 238000002372 labelling Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 2
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- 229940079593 drug Drugs 0.000 abstract description 4
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- 239000002994 raw material Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 206010006956 Calcium deficiency Diseases 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 208000003217 Tetany Diseases 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 229940069978 calcium supplement Drugs 0.000 description 1
- 230000004856 capillary permeability Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 208000007442 rickets Diseases 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012421 spiking Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
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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
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- General Health & Medical Sciences (AREA)
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Abstract
The invention belongs to the technical field of medicine manufacturing, and particularly relates to a high performance liquid chromatography analysis method for calcium gluconate related substances. The method comprises the following steps: 1) preparing a reference substance solution; 2) preparing a test solution; 3) preparing a standard sample solution; 4) chromatographic conditions; 5) the determination method comprises the following steps: respectively and precisely measuring a reference solution and a test solution, injecting into a liquid chromatograph, recording a chromatogram, and calculating according to the peak area by an external standard method. The invention has the advantages that: a high performance liquid chromatography analysis method for measuring related substances of calcium gluconate is established. The method is convenient and simple, and has high sensitivity; the method can effectively detect known impurities, can accurately detect the content of the impurities according to response factors of the impurities, improves the safety of medicines, controls the quality of calcium gluconate, and provides a basis for formulating the quality standard of calcium gluconate bulk drugs.
Description
Technical Field
The invention belongs to the technical field of medicine manufacturing, and particularly relates to a high performance liquid chromatography analysis method for calcium gluconate related substances.
Background
Calcium is an inorganic substance necessary for the normal function of human nerve, muscle, skeletal system, cell membrane and capillary permeability in vivo. The calcium gluconate can be used as medicine for preventing and treating calcium deficiency, such as osteoporosis, tetany, bone hypoplasia, rickets, and calcium supplement for children, pregnant women, lactating women, menopausal women, and the elderly.
At present, the quality standards of calcium gluconate are collected in pharmacopoeias of the United states, European pharmacopoeias, Japanese pharmacopoeias, Chinese pharmacopoeias and the like, but the detection method of calcium gluconate-related substances does not exist in each standard, so that the establishment of the detection method for accurately detecting the glucose-related substances has very important significance.
Disclosure of Invention
The invention aims to provide a high performance liquid chromatography analysis method for calcium gluconate-related substances, which is used for measuring the content of the calcium gluconate-related substances by using a high performance liquid differential detector, has the characteristics of simplicity, convenience, exclusiveness, accuracy and high sensitivity, and is suitable for detecting the related substances in a calcium gluconate raw material medicine finished product.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the high performance liquid chromatography analysis method of the calcium gluconate related substances is characterized by comprising the following steps: a method for analyzing the components of a mixture containing related substances of calcium gluconate by using high performance chromatography;
wherein, the related substances of the calcium gluconate comprise: one or more of calcium maltotrionate, calcium isomaltose, calcium maltobionate or calcium glucarate; the method can also detect unknown single impurity in the synthesis of calcium gluconate raw material medicine. However, under the chromatographic condition, the retention time and the corresponding value of the calcium isomaltose and the calcium maltonate are basically consistent when peaks appear, so that only one related substance calcium isomaltose is selected for detection under the same chromatographic condition.
The method comprises the following steps:
1) preparation of a reference solution:
mixing the reference solution: accurately weighing appropriate amount of calcium maltotrigluconate, calcium isomaltose and calcium glucarate reference substances respectively, adding water to dissolve and dilute into solution containing 2.5 μ g of each of calcium maltotrigluconate, calcium isomaltose and calcium glucarate per 1mL as mixed reference substance solution;
calcium maltonate control solution: accurately weighing a proper amount of calcium gluconate reference substances respectively, adding water to dissolve and dilute the calcium gluconate reference substances into a calcium gluconate reference substance solution containing 2.5 mu g of calcium gluconate in each 1 mL;
2) preparing a test solution:
accurately weighing a proper amount of calcium gluconate samples, dissolving with water and diluting into a calcium gluconate solution containing 5mg per 1 mL;
3) preparing a standard sample solution:
mixing the standard sample solution: accurately weighing a proper amount of each of a calcium gluconate sample, calcium maltotrionate, calcium isomaltose and a calcium glucarate reference substance, dissolving the proper amount of each of the calcium gluconate sample, the calcium maltotrionate, the calcium isomaltose and the calcium glucarate reference substance in water, and diluting the solution into a solution containing 5mg of calcium gluconate, 2.5 mu g of each of calcium maltotrionate, calcium isomaltose, calcium maltotrionate and calcium glucarate in each 1mL of solution to be used as a mixed standard sample solution;
calcium maltonate labeling test solution: accurately weighing appropriate amounts of a calcium gluconate sample and a calcium gluconate reference substance, dissolving with water, and diluting into a solution containing 5mg of calcium gluconate and 2.5 μ g of calcium gluconate per 1mL, as a calcium gluconate sample solution;
4) chromatographic conditions are as follows:
detecting with high performance liquid chromatograph
A detector: a differential refractive detector;
a chromatographic column: a hydrogen ion chromatographic column;
column temperature: 25-35 ℃;
mobile phase: 0.45% -0.55% formic acid solution;
flow rate: 0.28-0.32 mL/min;
sample introduction volume: 10-100 μ L;
operating time: 60 min;
5) the determination method comprises the following steps:
precisely measuring the reference solution, the test solution and the added standard test solution, injecting into a liquid chromatograph, and recording the chromatogram; the method separates calcium gluconate and related substances thereof under the same high performance liquid chromatography condition;
determining that a chromatographic peak consistent with the retention time of known impurities exists in the obtained test solution in the chromatogram of the test solution, and calculating according to an external standard method to be not more than 0.05%; among them, known impurities are calcium gluconate, calcium maltotrionate, calcium isomaltose, calcium maltobionate and calcium glucarate,
in the formula: a. thex: peak areas of related substances in the test solution;
AR: peak area of related substance in the reference solution;
CR: the corresponding concentration of the reference solution, mg/mL;
Cx: concentration of test solution, mg/mL.
Preferably, the column is Rezex ROA-Organic Acid H + (8%) Phenomenex 00H-0138-K0.
Preferably, the chromatography column is 300 x 7.8mm in size.
Preferably, the column temperature is 30 ℃.
Preferably, the mobile phase is a 0.5% formic acid solution.
Preferably, the flow rate is 0.3 mL/min.
Preferably, the injection volume is 50 μ L.
Compared with the prior art, the invention has the following beneficial effects:
1. the method of the invention determines the content of the related substances of the calcium gluconate by a high performance liquid differential detector, has the characteristics of simplicity, convenience, exclusiveness, accuracy and high sensitivity, and is suitable for detecting the related substances in the finished product of the calcium gluconate bulk drug.
2. The invention optimizes the optimal analysis condition and the preparation method of the test solution through a large number of experiments. Multiple experiments prove that the HPLC determination method for the calcium gluconate-related substances has good stability and good separation degree, and can accurately, qualitatively and quantitatively detect the main related substances in the calcium gluconate, so that the quality of the calcium gluconate is objectively, accurately and comprehensively evaluated, and the method has important practical significance for controlling the product quality.
3. The invention provides a high performance liquid chromatography analysis method for measuring related substances of calcium gluconate. The method is convenient and simple, has high sensitivity, can effectively detect each known impurity, can accurately detect the content of each impurity according to the response factor of the impurity, improves the safety of the medicine, controls the quality of the calcium gluconate, and provides a basis for formulating the quality standard of the calcium gluconate bulk drug.
Drawings
FIG. 1 is a blank solvent HPLC profile;
FIG. 2 is a HPLC chromatogram of a mixed control solution;
FIG. 3 is an HPLC chromatogram of a calcium maltotrigluconate control solution;
FIG. 4 is an HPLC chromatogram of a test solution;
FIG. 5 is an HPLC chromatogram of a mixed standard sample solution;
FIG. 6 is an HPLC chromatogram of a maltose spiked test sample solution.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail by way of examples, which are only used for explaining the present invention and do not represent the scope of protection of the present invention defined by the claims.
Example 1
The method for detecting the related substances of the calcium gluconate comprises the following steps:
1) preparation of a reference solution:
mixing the reference solution: respectively and precisely weighing appropriate amount of calcium maltotrigluconate, calcium isomaltose and calcium glucarate reference substances, adding water to dissolve and dilute into solutions containing 2.5 mu g of each of the calcium maltotrigluconate, the calcium isomaltose and the calcium glucarate in each 1mL to serve as mixed reference substance solutions.
Calcium maltonate control solution: respectively and precisely weighing a proper amount of calcium gluconate reference substance, adding water to dissolve and dilute the calcium gluconate reference substance into a calcium gluconate reference substance solution containing 2.5 mu g of calcium gluconate per 1 mL.
2) Preparing a test solution: an appropriate amount of calcium gluconate sample is precisely weighed, dissolved in water and diluted into a calcium gluconate solution containing 5mg per 1 mL.
3) Preparation of a spiked test solution:
mixing the standard sample solution: accurately weighing a proper amount of each of a calcium gluconate sample, calcium maltotrionate, calcium isomaltose and a calcium glucarate reference substance, dissolving the proper amount of each of the calcium gluconate sample, the calcium maltotrionate, the calcium isomaltose and the calcium glucarate reference substance in water, and diluting the solution into a solution containing 5mg of calcium gluconate, 2.5 mu g of each of calcium maltotrionate, calcium isomaltose, calcium maltotrionate and calcium glucarate in each 1mL of solution to be used as a mixed standard sample solution.
Calcium maltonate labeling test solution: an appropriate amount of each of the calcium gluconate sample and the calcium gluconate reference is precisely weighed, and dissolved and diluted by water into a solution containing 5mg of calcium gluconate and 2.5 mug of calcium gluconate per 1mL to be used as a calcium gluconate additive sample solution.
The method for detecting the related substances of the calcium gluconate comprises the following steps of:
a detector: a differential refractive detector;
a chromatographic column: a hydrogen ion chromatography column (Rezex ROA-Organic Acid H + (8%) Phenomenex 00H-0138-K0);
column temperature: 30 ℃;
mobile phase: 0.5% formic acid solution;
flow rate: 0.3 mL/min;
sample introduction volume: 50 mu L of the solution;
blank solvent: and (3) water.
1. System precision: purpose of the experiment: the RSD of peak area and retention time of each component was mainly looked at. The precision test was performed to precisely prepare a control solution with a concentration of 2.5. mu.g/mL, 50. mu.L of the control solution was continuously injected 6 times, and the peak area measurement results of the control are shown in Table 1.
TABLE 1 results of systematic precision test
The results show that: and continuously injecting each impurity and the calcium gluconate reference substance for 6 times, wherein the retention time RSD% is less than 2, and the peak area RSD% is less than 10%.
2. Linear range: purpose of the experiment: each known impurity was studied in the range of the quantitative limit concentration to the 200% limit concentration. The linear relationship is plotted as measured response signal (peak area) against analyte concentration, linear regression is performed by least squares method, and correlation coefficient r is reported to confirm good linear relationship, requiring that the value of the linear correlation coefficient r should be not less than 0.990. The linearity results of the respective substances are shown in Table 2.
TABLE 2 linearity results of the respective substances involved
| Composition (I) | Linear range | Standard curve | Coefficient of linearity r |
| Calcium maltonate | 0.92-4.58μg/mL | y＝1109.5x-87.085 | 0.9993 |
| Calcium maltotrionate | 1.01-5.03μg/mL | y＝1215.3x-47.538 | 0.9999 |
| Calcium isomaltose | 1.00-4.99μg/mL | y＝1200.5x-175.12 | 0.9992 |
| Calcium glucarate | 0.99-4.97μg/mL | y＝727.73x-229.12 | 0.9979 |
| Calcium gluconate | 1.09-5.45μg/mL | y＝1018.2x-70.201 | 0.9999 |
The results show that: the main component and each impurity are linear in the concentration range, and the relationship between the peak area and the linear concentration is good.
3. And (3) quantitative limit determination: purpose of the experiment: the measured signal of each component is compared with the signal (baseline noise) of a blank solution, and the signal-to-noise ratio is required to be more than 10, so that the concentration of the solution to be detected is obtained. The quantitative limit results are determined in table 3.
TABLE 3 determination of limit of quantitation results
| Name (R) | Sample concentration (μ g/mL) | Blank baseline noise | Sample baseline noise | Signal to noise ratio |
| Calcium maltonate | 0.92 | 1 | 32.0 | 32 |
| Calcium maltotrionate | 0.94 | 2 | 39.0 | 20 |
| Calcium isomaltose | 1.00 | 1 | 37.0 | 37 |
| Calcium glucarate | 1.05 | 1 | 23.0 | 23 |
| Calcium gluconate | 1.09 | 2 | 36.0 | 18 |
The results show that: the signal-to-noise ratio of each related substance is larger than 10 under the quantitative limit concentration, and the verification requirements are met.
4. And (3) recovery rate: purpose of the experiment: a known amount of an impurity is added to a test sample, and the ratio of the measurement result of the known impurity in the sample to the theoretical value (recovery rate), expressed in percent (%), is measured, and the recovery rate is required to be 80.0% to 120.0%, to confirm that the method has good accuracy. The results of the calcium maltotrionate recovery test are summarized in Table 4. The results of the calcium maltonate recovery test are summarized in Table 5. The results of the calcium isomaltose recovery test are summarized in Table 6. The results of the calcium glucarate recovery test are summarized in Table 7.
TABLE 4 summary of calcium maltotrionate recovery test results
TABLE 5 summary of calcium maltonate recovery test results
TABLE 6 summary of calcium isomaltose recovery test results
TABLE 7 summary of calcium glucarate recovery test results
The results show that: under three concentrations, the recovery rates of the calcium maltotrionate, the calcium isomaltose, the calcium maltobionate and the calcium glucarate are all between 85 and 115 percent, and the recovery rates meet the verification requirement (80.0 to 120.0 percent).
5. And (3) investigating the stability of the sample solution: purpose of the experiment: and (3) observing the change rule of the reference substance solution and the standard sample solution along with the standing time, and standing the solutions for 0h, 5h, 10h, 15h, 20h, 25h and 30h at room temperature, wherein the change rate of the content of each impurity in the solution and the initial value is required to be less than or equal to 10.0%, no new impurity larger than the report limit is required to appear, and a stability basis is provided for sample testing. The effect of the control solution standing time on the impurity measurement is shown in Table 8. The effect of the time the test solution was allowed to stand on the impurity determination is shown in Table 9.
TABLE 8 influence of standing time of control solutions on the determination of impurities
TABLE 9 Effect of spiking test solution standing time on impurity determination
The results show that: the peak area change rate of the reference solution and the standard sample solution is within 10 percent within 30 hours, no new impurity is generated, and the stability is good.
6. Durability: the tolerance of the chromatographic conditions with slight changes on the measurement results is evaluated by changing different flow rates, column temperatures and mobile phase concentrations. It is required that the content of each impurity RSD is 10.0% or less under various conditions.
6.1 determination of different column temperatures:
the results of the various column temperature measurements are shown in Table 10.
TABLE 10 results of various column temperature measurements
The result shows that the column temperature is changed, the change rate of the content of each impurity RSD is less than or equal to 10.0 percent, and the measurement result is not influenced.
6.2 measurement of different flow rates
The results of the different flow rate measurements are shown in Table 11.
TABLE 11 results of different flow rate measurements
The results show that: the flow rate is changed, the change rate of the content of each impurity RSD is less than or equal to 10.0 percent, and the measurement result is not influenced.
6.3 determination of the concentration of different mobile phases
The results of the different mobile phase concentration measurements are shown in table 12.
TABLE 12 results of measurement of different mobile phase concentrations
The results show that: the concentration of the mobile phase is changed, the RSD of the content change rate of each impurity is less than or equal to 10.0 percent, and the measurement result is not influenced.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and it will be obvious to those skilled in the art that modifications and improvements can be made on the basis of the present invention, and therefore, any modifications and improvements made within the spirit and principle of the present invention will fall within the protection scope of the present invention.
Claims (7)
1. A high performance liquid chromatography analysis method of calcium gluconate related substances is characterized by comprising the following steps: a method for analyzing the components of a mixture containing related substances of calcium gluconate by using high performance chromatography;
wherein, the related substances of the calcium gluconate comprise: one or more of calcium maltotrionate, calcium isomaltose, calcium maltobionate or calcium glucarate;
the method comprises the following steps:
1) preparation of a reference solution:
mixing the reference solution: accurately weighing appropriate amount of calcium maltotrigluconate, calcium isomaltose and calcium glucarate reference substances respectively, adding water to dissolve and dilute into solution containing 2.5 μ g of each of calcium maltotrigluconate, calcium isomaltose and calcium glucarate per 1mL as mixed reference substance solution;
calcium maltonate control solution: accurately weighing a proper amount of calcium gluconate reference substances respectively, adding water to dissolve and dilute the calcium gluconate reference substances into a calcium gluconate reference substance solution containing 2.5 mu g of calcium gluconate in each 1 mL;
2) preparing a test solution:
accurately weighing a proper amount of calcium gluconate samples, dissolving with water and diluting into a calcium gluconate solution containing 5mg per 1 mL;
3) preparing a standard sample solution:
mixing the standard sample solution: accurately weighing a proper amount of each of a calcium gluconate sample, calcium maltotrionate, calcium isomaltose and a calcium glucarate reference substance, dissolving the proper amount of each of the calcium gluconate sample, the calcium maltotrionate, the calcium isomaltose and the calcium glucarate reference substance in water, and diluting the solution into a solution containing 5mg of calcium gluconate, 2.5 mu g of each of calcium maltotrionate, calcium isomaltose, calcium maltotrionate and calcium glucarate in each 1mL of solution to be used as a mixed standard sample solution;
calcium maltonate labeling test solution: accurately weighing appropriate amounts of a calcium gluconate sample and a calcium gluconate reference substance, dissolving with water, and diluting into a solution containing 5mg of calcium gluconate and 2.5 μ g of calcium gluconate per 1mL, as a calcium gluconate sample solution;
4) chromatographic conditions are as follows:
detecting with high performance liquid chromatograph
A detector: a differential refractive detector;
a chromatographic column: a hydrogen ion chromatographic column;
column temperature: 25-35 ℃;
mobile phase: 0.45% -0.55% formic acid solution;
flow rate: 0.28-0.32 mL/min;
sample introduction volume: 10-100 μ L;
operating time: 60 min;
5) the determination method comprises the following steps:
precisely measuring the reference solution, the test solution and the added standard test solution, injecting into a liquid chromatograph, and recording the chromatogram; the method separates calcium gluconate and related substances thereof under the same high performance liquid chromatography condition;
determining that a chromatographic peak consistent with the retention time of known impurities exists in the obtained test solution in the chromatogram of the test solution, and calculating according to an external standard method to be not more than 0.05%; among them, known impurities are calcium gluconate, calcium maltotrionate, calcium isomaltose, calcium maltobionate and calcium glucarate,
in the formula: a. thex: peak areas of related substances in the test solution;
AR: peak area of related substance in the reference solution;
CR: the corresponding concentration of the reference solution, mg/mL;
Cx: concentration of test solution, mg/mL.
2. The high performance liquid chromatography analysis method for calcium gluconate-related substances according to claim 1, wherein: the chromatographic column is Rezex ROA-Organic Acid H + (8%) Phenomenex 00H-0138-K0.
3. The high performance liquid chromatography analysis method for calcium gluconate-related substances according to claim 1, wherein: the size of the chromatographic column is 300X 7.8 mm.
4. The high performance liquid chromatography analysis method for calcium gluconate-related substances according to claim 1, wherein: the column temperature was 30 ℃.
5. The high performance liquid chromatography analysis method for calcium gluconate-related substances according to claim 1, wherein: the mobile phase is 0.5% formic acid solution.
6. The high performance liquid chromatography analysis method for calcium gluconate-related substances according to claim 1, wherein: the flow rate was 0.3 mL/min.
7. The high performance liquid chromatography analysis method for calcium gluconate-related substances according to claim 1, wherein: the injection volume is 50 muL.
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