CN113866316A - Method for detecting content of polyethylene glycol 8000 in calcium acetate tablets - Google Patents
Method for detecting content of polyethylene glycol 8000 in calcium acetate tablets Download PDFInfo
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- CN113866316A CN113866316A CN202111211958.1A CN202111211958A CN113866316A CN 113866316 A CN113866316 A CN 113866316A CN 202111211958 A CN202111211958 A CN 202111211958A CN 113866316 A CN113866316 A CN 113866316A
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- 239000001639 calcium acetate Substances 0.000 title claims abstract description 104
- 229960005147 calcium acetate Drugs 0.000 title claims abstract description 104
- 235000011092 calcium acetate Nutrition 0.000 title claims abstract description 104
- VSGNNIFQASZAOI-UHFFFAOYSA-L calcium acetate Chemical compound [Ca+2].CC([O-])=O.CC([O-])=O VSGNNIFQASZAOI-UHFFFAOYSA-L 0.000 title claims abstract description 103
- 239000004353 Polyethylene glycol 8000 Substances 0.000 title claims abstract description 73
- 229940085678 polyethylene glycol 8000 Drugs 0.000 title claims abstract description 73
- 235000019446 polyethylene glycol 8000 Nutrition 0.000 title claims abstract description 73
- 238000000034 method Methods 0.000 title claims abstract description 31
- 238000001514 detection method Methods 0.000 claims abstract description 46
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims abstract description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000000105 evaporative light scattering detection Methods 0.000 claims abstract description 5
- 239000000741 silica gel Substances 0.000 claims abstract description 5
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 81
- 239000013558 reference substance Substances 0.000 claims description 17
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- 239000012085 test solution Substances 0.000 claims description 11
- 239000002904 solvent Substances 0.000 claims description 9
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 5
- 238000002013 hydrophilic interaction chromatography Methods 0.000 claims description 5
- 238000002347 injection Methods 0.000 claims description 5
- 239000007924 injection Substances 0.000 claims description 5
- 239000012088 reference solution Substances 0.000 claims description 5
- 238000012856 packing Methods 0.000 claims description 3
- 239000012071 phase Substances 0.000 abstract description 12
- 229940079593 drug Drugs 0.000 abstract description 4
- 239000003814 drug Substances 0.000 abstract description 4
- 230000007547 defect Effects 0.000 abstract description 2
- 238000005220 pharmaceutical analysis Methods 0.000 abstract description 2
- 239000007791 liquid phase Substances 0.000 abstract 1
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 42
- 239000011550 stock solution Substances 0.000 description 40
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 38
- 238000002360 preparation method Methods 0.000 description 24
- 235000019359 magnesium stearate Nutrition 0.000 description 21
- 238000005303 weighing Methods 0.000 description 16
- 239000002202 Polyethylene glycol Substances 0.000 description 15
- 238000007865 diluting Methods 0.000 description 15
- 229920001223 polyethylene glycol Polymers 0.000 description 15
- 239000002994 raw material Substances 0.000 description 14
- 238000012360 testing method Methods 0.000 description 11
- 238000001914 filtration Methods 0.000 description 10
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 9
- 239000011574 phosphorus Substances 0.000 description 9
- 229910052698 phosphorus Inorganic materials 0.000 description 9
- 238000011084 recovery Methods 0.000 description 9
- 239000012490 blank solution Substances 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 239000000843 powder Substances 0.000 description 7
- 238000000926 separation method Methods 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 6
- 238000009472 formulation Methods 0.000 description 6
- 230000014759 maintenance of location Effects 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 239000000463 material Substances 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000012467 final product Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000011230 binding agent Substances 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 230000002308 calcification Effects 0.000 description 2
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- WGYKZJWCGVVSQN-UHFFFAOYSA-N propylamine Chemical group CCCN WGYKZJWCGVVSQN-UHFFFAOYSA-N 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 208000037147 Hypercalcaemia Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000005770 Secondary Hyperparathyroidism Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- YYRMJZQKEFZXMX-UHFFFAOYSA-L calcium bis(dihydrogenphosphate) Chemical compound [Ca+2].OP(O)([O-])=O.OP(O)([O-])=O YYRMJZQKEFZXMX-UHFFFAOYSA-L 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 208000020832 chronic kidney disease Diseases 0.000 description 1
- 208000022831 chronic renal failure syndrome Diseases 0.000 description 1
- 229940109239 creatinine Drugs 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000000148 hypercalcaemia Effects 0.000 description 1
- 208000030915 hypercalcemia disease Diseases 0.000 description 1
- 201000005991 hyperphosphatemia Diseases 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
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- 239000012074 organic phase Substances 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
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- 210000002966 serum Anatomy 0.000 description 1
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Images
Classifications
-
- 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
-
- 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
Abstract
The invention relates to the technical field of pharmaceutical analysis, and particularly discloses a method for detecting the content of polyethylene glycol 8000 in a calcium acetate tablet. The invention adopts a high performance liquid phase method and carries out detection according to the following chromatographic conditions: a chromatographic column: propyl amide group bonding silica gel column; mobile phase: acetonitrile water solution with volume ratio of 60-80: 40-20; a detector: an evaporative light scattering detector or a differential detector. The detection method provided by the invention can realize accurate quantitative detection of the polyethylene glycol 8000 in the calcium acetate tablet by a simple and quick method, overcomes the defect that the existing method cannot realize quantitative detection of the polyethylene glycol 8000 in the calcium acetate tablet, provides reliable guarantee for improving and better controlling the quality of the calcium acetate tablet product, and has very important significance for improving the medication safety.
Description
Technical Field
The invention relates to the technical field of pharmaceutical analysis, in particular to a method for detecting the content of polyethylene glycol 8000 in a calcium acetate tablet.
Background
After the renal function of a patient with chronic renal failure is reduced to a certain degree (the endogenous creatinine clearance rate is less than 60mL/min), phosphorus excretion disorder is generated, blood phosphorus begins to rise, but the dialysis rate is limited, the serum phosphorus concentration cannot be effectively and rapidly reduced, and hyperphosphatemia often causes a series of complications, such as secondary hyperparathyroidism, cardiovascular calcification, soft tissue or joint calcification and the like, and seriously affects the functions of a human body, so that the phosphorus binding agent needs to be orally taken to reduce the intake of phosphorus in food.
The calcium acetate tablet is used as a phosphorus binding agent, can be combined with phosphate radicals in food in the digestive tract to form insoluble phosphate-calcium phosphate which is not easy to absorb, and then is discharged out of the body by excrement, so that the absorption of phosphorus is effectively reduced, and the concentration of phosphorus in blood is reduced. In addition, calcium acetate tablets have a strong binding ability to phosphorus and are less likely to cause hypercalcemia than other calcium preparations.
The calcium acetate tablet was approved and marketed in the United states in 1990, the original preparation was produced by FRESENIUS MEDICL, and both the original preparation and the first-copy preparation were released from the market. The current reference pharmaceutical formulation is calcium acetate tablet (PADDOCK LABORATORIES LLC) manufactured by Perrigo corporation. At present, 5 enterprises in China produce and sell calcium acetate tablets, and the specification is 0.667 g.
Accurate detection of the dosage of each auxiliary material in the reference preparation can play a role of getting twice the result with half the effort in research and development of the imitation drugs, so that the product quality and the curative effect of the imitation drugs are closer to those of the reference preparation. However, the calcium acetate in the calcium acetate tablet accounts for more than 95%, the content of the polyethylene glycol 8000 is low, and the calcium acetate and the polyethylene glycol 8000 are difficult to separate, so that accurate quantitative detection of the polyethylene glycol 8000 in the calcium acetate tablet is difficult to realize.
Disclosure of Invention
Aiming at the problem that the accurate quantitative detection of the content of polyethylene glycol 8000 in the calcium acetate tablet is difficult to realize by the existing detection method, the invention provides a detection method of the content of polyethylene glycol 8000 in the calcium acetate tablet. The invention realizes accurate quantitative detection of polyethylene glycol 8000 in calcium acetate tablets by selecting a chromatographic column with specific packing and matching with conditions such as optimized mobile phase, flow rate, column temperature and the like.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a method for detecting the content of polyethylene glycol 8000 in a calcium acetate tablet comprises the following steps:
(1) preparing a test solution and a reference solution:
preparing a test solution from the calcium acetate tablets by using a solvent;
preparing a polyethylene glycol 8000 reference substance into a reference substance solution by using a solvent;
(2) the detection is carried out according to the following high performance liquid chromatography conditions:
a chromatographic column: propyl amide group bonding silica gel column;
mobile phase: acetonitrile water solution with volume ratio of 60-80: 40-20;
a detector: an evaporative light scattering detector or a differential detector.
The inventor finds that the polarity of calcium acetate is strong, the peak of calcium acetate is too early if a conventional C18 chromatographic column is adopted, and the proportion of calcium acetate in the product is up to more than 95%, so that the peak of calcium acetate is wide, the chromatographic peak of polyethylene glycol 8000 coincides with the chromatographic peak of calcium acetate, and the separation of polyethylene glycol 8000 and calcium acetate is not easy to realize. Besides, magnesium stearate is added into the calcium acetate tablets as a lubricant in addition to the polyethylene glycol 8000, and if the quantitative detection of the polyethylene glycol 8000 is to be realized, the detection method can also effectively separate the calcium acetate and the magnesium stearate, so that the quantitative detection of the polyethylene glycol 8000 can be ensured. Meanwhile, the conventional dosage of the polyethylene glycol 8000 in the calcium acetate tablet is only about 1-3%, and the content is low, so how to ensure the detection accuracy is one of the difficulties in establishing a detection method.
The method for detecting the content of polyethylene glycol 8000 in the calcium acetate tablet adopts a propyl amide group bonded silica gel chromatographic column, taking acetonitrile water solution with a specific proportion as a mobile phase, adopting an evaporation light scattering detector or a differential refraction detector, the effective detection of the polyethylene glycol 8000 in the calcium acetate tablet is realized through the high performance liquid chromatography, through the research and verification of methodologies such as specificity, sensitivity and the like, the method provided by the invention is found to have better sensitivity, accuracy and reproducibility, can realize accurate quantitative detection on the polyethylene glycol 8000 in the calcium acetate tablet by a simple and quick method, overcomes the defect that the quantitative detection on the polyethylene glycol 8000 in the calcium acetate tablet cannot be realized at present, provides reliable guarantee for improving and better controlling the quality of the calcium acetate tablet product, and has very important significance for improving the medication safety.
Preferably, in the preparation of the test solution and the control solution, the solvent is water.
Preferably, the column has a size of 250mm 4.6mm and a packing diameter of 5 μm.
Further preferably, the column is Venusil HILIC, 4.6mm × 250mm, 5 μm.
The optimal specification and model of the chromatographic column can improve the separation degree between the polyethylene glycol 8000 and magnesium stearate auxiliary materials and the main component of calcium acetate, and the peak shapes of the polyethylene glycol 8000 and calcium acetate are better, so that the quantitative detection of the polyethylene glycol 8000 auxiliary materials with lower content in the calcium acetate tablet is favorably realized, the result is accurate and reliable, and the repeatability is good.
Preferably, the mobile phase is an aqueous acetonitrile solution in a volume ratio of 70: 30.
The preferable mobile phase is more suitable for a propyl amide group bonded silica gel column, can better separate calcium acetate and polyethylene glycol 8000 on the premise of not generating baseline interference, and can effectively improve the peak shape, so that the accuracy and precision of the detection result are higher.
Preferably, the column temperature is 35-45 ℃ and the flow rate is 0.9-1.1 mL/min.
Further preferably, the column temperature is 40 ℃ and the flow rate is 1.0 mL/min.
Preferably, the injection volume is 20. mu.L.
Preferably, the detector temperature is 40 ℃.
The preferable detection condition can ensure that the polyethylene glycol 8000 and the calcium acetate in the calcium acetate tablet reach higher separation degree, and the detection interference of the calcium acetate raw material with higher content to the polyethylene glycol 8000 is avoided, so that the accuracy and the sensitivity of the detection of the content of the polyethylene glycol 8000 in the calcium acetate tablet are improved.
Preferably, the detector is an evaporative light scattering detector.
The preferred detector can obtain higher detection sensitivity, and the chromatographic system is more stable and reliable.
Preferably, the concentration of the test solution is 20.0 mg/mL.
Preferably, the concentration of the polyethylene glycol 8000 in the control solution is 0.1mg/mL-0.5 mg/mL.
The preferable concentration of the test sample is favorable for obtaining better detection sensitivity of the polyethylene glycol 8000 with lower content, and simultaneously effectively avoids the interference of wider peak of calcium acetate on the polyethylene glycol 8000, thereby being favorable for more accurately calculating the content of the polyethylene glycol 8000 in the test sample.
The detection method provided by the invention can realize effective separation of the polyethylene glycol 8000 and calcium acetate in the calcium acetate tablet, accurately, qualitatively and quantitatively detect the polyethylene glycol 8000 in the calcium acetate tablet, and has very important significance for improving the quality and curative effect of the calcium acetate tablet imitation pharmaceutical product.
Drawings
FIG. 1 is a chromatogram of a 2.1-specialization standard curve solution 2 of example 2;
FIG. 2 is a chromatogram of a calcium acetate solution of specificity 2.1 from example 2;
FIG. 3 is a chromatogram of a control stock solution of example 2 with specificity of 2.1;
FIG. 4 is a chromatogram of a magnesium stearate solution according to the specificity of 2.1 in example 2;
FIG. 5 is a chromatogram of a solution of the reference formulation under specificity 2.1 in example 2;
FIG. 6 is a chromatogram of a solution of an imitation formulation of specificity 2.1 from example 2;
FIG. 7 is a chromatogram of medium concentration solution 1 at a recovery of 2.3 in example 2;
FIG. 8 is a chromatogram of example 2 with an acetonitrile-water ratio of 60:40 in terms of 2.4 durability;
FIG. 9 is a chromatogram of a reference formulation solution of calcium acetate tablets of comparative example 1;
fig. 10 is a chromatogram of a reference formulation solution of calcium acetate tablets in comparative example 2.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
1.1 preparation of the solution
(1) Blank solvent/diluent: and (3) water.
(2) Test solution: precisely weighing about 500mg of calcium acetate tablet fine powder, placing the calcium acetate tablet fine powder into a 25mL volumetric flask, adding water, ultrasonically dissolving, diluting to a scale, shaking up, and filtering to obtain a test solution.
(3) Control solution: accurately weighing appropriate amount of polyethylene glycol 8000 reference substance, preparing with water to obtain 1mg/mL solution, accurately weighing 1mL, 2mL and 3mL respectively, placing in 10mL measuring flask, diluting with water to scale, shaking up, and using as standard curve solution.
High performance liquid chromatography conditions:
a chromatographic column: venusil HILIC (4.6X 250mm, 5 μm);
mobile phase: acetonitrile: 70 parts of water: 30 (V/V);
flow rate: 1.0 mL/min;
detector temperature: 40 ℃;
column temperature: 40 ℃;
sample introduction amount: 20 μ L.
And (3) respectively injecting the blank solvent, the reference solution and the test solution into a high performance liquid chromatograph, recording a chromatogram, drawing a linear regression equation by taking the natural logarithm of the peak area of each reference solution as a horizontal coordinate and the natural logarithm of the concentration of each reference solution as a vertical coordinate, and calculating the content of the polyethylene glycol 8000 in the test solution according to the linear regression equation.
Example 2
And (3) verification of methodology:
2.1 specificity
Preparing a solution:
blank solution: and (3) water.
Control stock solution: accurately weighing 49.17mg of polyethylene glycol 8000 reference substance, placing in a 50mL volumetric flask, adding water, ultrasonically dissolving, diluting to scale, and shaking up to obtain a reference substance stock solution with the concentration of 1 mg/mL.
Standard curve solution 1: precisely measuring 1mL of the reference substance stock solution, placing the reference substance stock solution in a 10mL volumetric flask, adding water to dilute the reference substance stock solution to a scale, and shaking up to obtain a standard curve solution 1 with the concentration of 0.1 mg/mL.
Standard curve solution 2: precisely measuring 2mL of the reference substance stock solution, placing the reference substance stock solution into a 10mL volumetric flask, adding water to dilute the reference substance stock solution to a scale, and shaking the solution uniformly to obtain a standard curve solution 2 with the concentration of 0.2 mg/mL.
Standard curve solution 3: precisely measuring 3mL of the reference stock solution, putting the reference stock solution into a 10mL volumetric flask, adding water to dilute the reference stock solution to a scale, and shaking the solution uniformly to obtain a standard curve solution 2 with the concentration of 0.3 mg/mL.
Reference formulation solution: 499.92mg of fine powder of calcium acetate tablets (batch 7139311) produced by Perrigo company is precisely weighed, placed in a 25mL volumetric flask, added with water for ultrasonic dissolution and diluted to a scale, shaken up and filtered to obtain a reference preparation solution with the concentration of 20 mg/mL.
Imitation preparation solution: 201.20mg of fine powder of calcium acetate tablets (batch No. 20181105) prepared by Kerared company is precisely weighed, placed in a 10mL measuring flask, added with water for ultrasonic dissolution and diluted to scale, shaken up and filtered to obtain an imitation preparation solution with the concentration of 20 mg/mL.
The types and the use amounts of the auxiliary materials of the imitation preparation are completely the same as those of the reference preparation.
Calcium acetate solution: accurately weighing 939.26mg of calcium acetate raw material (batch number C20180420) produced by northeast Hebei Fangfeng pharmaceutical industry, placing in a 50mL measuring flask, adding water, ultrasonically dissolving, diluting to scale, shaking up, and filtering to obtain the final product.
Magnesium stearate solution: precisely weighing 21.31mg of magnesium stearate fine powder (Ron reagent, batch RH119477), placing in a 100mL measuring flask, adding water, ultrasonically dissolving, diluting to scale, shaking, and filtering.
The determination method comprises the following steps: each 20. mu.L of the solutions was measured precisely, and the samples were subjected to sample injection detection under the conditions of high performance liquid chromatography in example 1, and chromatograms were recorded, and the results are shown in tables 1 and 2.
TABLE 1 results of the specificity localization test
| Name (R) | Retention time | Peak area | Calcium acetate peak retention time | Peak area of calcium acetate | Degree of separation |
| Blank solution | 3.944 | 383.114 | — | — | — |
| Standard Curve solution 3 | 3.781 | 3948.401 | — | — | — |
| Calcium acetate solution | 3.946 | 488.800 | 9.005 | 55904.040 | 4.062 |
| Reference formulation solution | 3.854 | 3608.254 | 8.357 | 52270.010 | 3.369 |
| Imitation preparation solution | 3.810 | 2856.078 | 9.598 | 3973.541 | 16.458 |
| Magnesium stearate solution | 3.987 | 609.912 | — | — | — |
TABLE 2 specificity Linear results
Test results show that chromatographic peaks of the magnesium stearate, the calcium acetate and the polyethylene glycol 8000 are not coincident, and the magnesium stearate and the calcium acetate do not interfere with the detection of the polyethylene glycol 8000; the blank solvent is partially overlapped with the chromatographic peak of the polyethylene glycol 8000, the polyethylene glycol presents a linear relation after blank deduction, r is 0.9977, and r is more than 0.99, so the blank solvent has no influence on the content detection of the polyethylene glycol 8000.
2.2 detection and quantitation limits
Solution preparation:
blank solution: and (3) water.
Control stock solution: precisely weighing about 49.17mg of polyethylene glycol 8000 reference substance, placing in a 50mL volumetric flask, adding water, ultrasonically dissolving, diluting to scale, and shaking up to obtain the final product.
Detection limit and quantification limit stock solutions: precisely measuring 2mL of the reference substance stock solution, placing in a 10mL volumetric flask, adding water to dilute to scale, and shaking up to obtain the final product.
Quantitative limiting solution: precisely measuring 1mL of the detection limit and quantification limit stock solution, placing in a 20mL volumetric flask, adding water to dilute to a scale, and shaking up to obtain the product.
Detection limiting solution: precisely measuring 3mL of quantitative limiting solution, placing the quantitative limiting solution in a 10mL volumetric flask, adding water to dilute the quantitative limiting solution to a scale, and shaking up the quantitative limiting solution to obtain the product.
And (3) respectively taking 20 mu L of the blank solution, the quantitative limit solution and the detection limit solution, injecting into a liquid chromatograph, and recording a chromatogram. The quantitative limit solution is continuously injected and detected for 5 times, the repeatability of the peak area of the quantitative limit solution continuously detected for 5 times is calculated, and the test result is shown in tables 3-5.
TABLE 3 quantitative Limit test results
| 1 | 2 | 3 | 4 | 5 | Mean value of | RSD(％) | |
| Retention time | 3.832 | 3.842 | 3.844 | 3.846 | 3.831 | 3.839 | 0.18 |
| Peak area | 141.132 | 140.656 | 146.552 | 137.645 | 145.116 | 142.2202 | 2.53 |
| Signal to noise ratio | 11.71 | 13.72 | 13.35 | 14.31 | 11.84 | / | / |
TABLE 4 detection Limit test results
| Name (R) | Retention time (min) | Peak area | Signal to noise ratio |
| Polyethylene glycol 8000 | 3.811 | 47.658 | 3.56 |
TABLE 5 detection limit and quantitation limit test results
And (4) conclusion: the detection limit concentration of the polyethylene glycol 8000 is 0.00295mg/mL, which is equivalent to 0.00559% of the sample, and the signal-to-noise ratio is 3.56; the limit concentration of polyethylene glycol 8000 is 0.009834mg/mL, which is equivalent to 0.0197% of the sample, and the signal-to-noise ratio is more than 10. The RSD of the peak area of the limiting solution of polyethylene glycol 8000 after 5 times of continuous sample injection is 2.53 percent, and the RSD of the retention time is 0.18 percent. The results show that the method has high detection sensitivity and is suitable for quantitative detection of the polyethylene glycol 8000 in the calcium acetate tablets.
2.3 recovery rate
Solution preparation:
blank solution: and (3) water.
Control stock solution: precisely weighing 52.34mg of polyethylene glycol 8000 reference substance, placing in a 50mL volumetric flask, adding water, ultrasonically dissolving, diluting to scale, and shaking.
Standard curve solution 1: precisely measuring 1mL of the reference stock solution, placing the reference stock solution in a 10mL volumetric flask, adding water to dilute the reference stock solution to a scale, and shaking up the solution to obtain the reagent.
Standard curve solution 2: precisely measuring 2mL of the reference stock solution, placing the reference stock solution in a 10mL volumetric flask, adding water to dilute the reference stock solution to a scale, and shaking up the reference stock solution to obtain the reagent.
Standard curve solution 3: precisely measuring 3mL of the reference stock solution, placing the reference stock solution in a 10mL volumetric flask, adding water to dilute the reference stock solution to a scale, and shaking up the reference stock solution to obtain the reagent.
Recovery rate solution:
low-concentration solution 1: accurately weighing 1442.64mg of calcium acetate raw material, 800016.32mg of polyethylene glycol and 15.11mg of magnesium stearate, putting the calcium acetate raw material, the polyethylene glycol and the magnesium stearate into a same 100mL volumetric flask, adding water, ultrasonically dissolving, diluting to a scale, shaking up, and filtering to obtain the calcium acetate.
Low-concentration solution 2: accurately weighing 1443.11mg of calcium acetate raw material, 800014.06mg of polyethylene glycol and 14.99mg of magnesium stearate, putting the calcium acetate raw material, the polyethylene glycol and the magnesium stearate into a same 100mL volumetric flask, adding water, ultrasonically dissolving, diluting to a scale, shaking up, and filtering to obtain the calcium acetate.
Medium concentration solution 1: accurately weighing 1449.26mg of calcium acetate raw material, 800019.14mg of polyethylene glycol and 15.67mg of magnesium stearate, putting the calcium acetate raw material, the polyethylene glycol and the magnesium stearate into a same 100mL volumetric flask, adding water, ultrasonically dissolving, diluting to a scale, shaking up, and filtering to obtain the calcium acetate.
Medium concentration solution 2: accurately weighing 1451.23mg of calcium acetate raw material, 800019.28mg of polyethylene glycol and 15.77mg of magnesium stearate, putting the calcium acetate raw material, the polyethylene glycol and the magnesium stearate into a same 100mL volumetric flask, adding water, ultrasonically dissolving, diluting to a scale, shaking up, and filtering to obtain the calcium acetate.
High-concentration solution 1: accurately weighing 1439.46mg of calcium acetate raw material, 800024.46mg of polyethylene glycol and 15.52mg of magnesium stearate, putting the calcium acetate raw material, the polyethylene glycol and the magnesium stearate into a same 100mL volumetric flask, adding water, ultrasonically dissolving, diluting to a scale, shaking up, and filtering to obtain the calcium acetate.
High-concentration solution 2: accurately weighing 1443.62mg of calcium acetate raw material, 800023.41mg of polyethylene glycol and 14.96mg of magnesium stearate, putting the calcium acetate raw material, the polyethylene glycol and the magnesium stearate into a same 100mL volumetric flask, adding water, ultrasonically dissolving, diluting to a scale, shaking up, and filtering to obtain the calcium acetate.
The determination method comprises the following steps: and (4) injecting the recovery rate solution into a liquid chromatograph, and recording a chromatogram. The test results are shown in tables 6 to 7.
TABLE 6 recovery Linear results
TABLE 7 recovery results
And (4) conclusion: from the data, the recovery rate of the polyethylene glycol 8000 is 96.67-100.82%, the average value is 98.8%, the recovery rate is within the specified range of 95-105%, the recovery rate meets the specification, the accuracy is good, and the method is suitable for detecting the content of the polyethylene glycol 8000 in the calcium acetate tablets.
2.4 durability
Solution preparation:
blank solution: and (3) water.
Control stock solution: precisely weighing 51.38mg of polyethylene glycol 8000 reference substance, placing in a 50mL volumetric flask, adding water, ultrasonically dissolving, diluting to scale, and shaking.
Standard curve solution 1: precisely measuring 1mL of the reference stock solution, placing the reference stock solution in a 10mL volumetric flask, adding water to dilute the reference stock solution to a scale, and shaking up the solution to obtain the reagent.
Standard curve solution 2: precisely measuring 2mL of the reference stock solution, placing the reference stock solution in a 10mL volumetric flask, adding water to dilute the reference stock solution to a scale, and shaking up the reference stock solution to obtain the reagent.
Standard curve solution 3: precisely measuring 3mL of the reference stock solution, placing the reference stock solution in a 10mL volumetric flask, adding water to dilute the reference stock solution to a scale, and shaking up the reference stock solution to obtain the reagent.
Imitation preparation solution: accurately weighing about 506.24mg of fine powder of calcium acetate tablets (batch No. 20181105) prepared by Kerared corporation, placing the fine powder into a 25mL measuring flask, adding water, ultrasonically dissolving, diluting to scale, shaking up, and filtering to obtain an imitation preparation solution with the concentration of 20 mg/mL.
The determination method comprises the following steps:
the chromatogram was recorded by examining the mobile phase ratio (acetonitrile: water: 60:40 and 80:20), the flow rate variation. + -. 0.1mL/min, and the column temperature variation. + -. 5 ℃ when measured.
Precisely measuring 20 μ L of blank solution, imitation preparation solution and standard curve solution 1-3 respectively, performing sample injection detection under the above chromatographic conditions, and recording chromatogram. The results are shown in Table 8.
Acceptance criteria: the RSD of the polyethylene glycol 8000 content in the imitation preparation solution is less than or equal to 10 percent.
TABLE 8 durability test results
The results show that when the detection conditions are changed within a certain range (flow rate +/-0.1 mL/min, column temperature +/-5 ℃, mobile phase proportion +/-20 ℃), the mean value of the content of polyethylene glycol 8000 is 1.44%, RSD is 2.63%, and is less than 10%, thus the method has good durability.
Example 3
Three batches of home-made simulated calcium acetate tablets (made by Kerared, lots 20190701, 20190702, 20190703) and reference preparation (Perrigo, lot 7139311) were tested according to the chromatography method of example 1, and the results are shown in Table 9.
TABLE 9
Comparative example 1
The calcium acetate reference preparation solution of example 1 was tested under exactly the same chromatographic conditions by replacing the Venusil HILIC (4.6X 250mm, 5 μm) column of example 1 with an Intersil ODS-3 (4.6X 250mm, 5 μm) column, and the results of the tests are shown in Table 10.
Watch 10
| Chromatographic column | Calcium acetate time to peak (min) | Polyethylene glycol time to peak (min) |
| Venusil HILIC | 9.005 | 3.843 |
| Intersil ODS-3 | 3.658 | 3.765 |
And (4) conclusion: as can be seen from the above table, after the Intersil ODS-3 chromatographic column is replaced, calcium acetate and polyethylene glycol 8000 are not separated, and the separation degree of the method cannot meet the requirement.
Comparative example 2
The acetonitrile in the mobile phase in example 1 was replaced with methanol, and other chromatographic conditions were completely the same, and the calcium acetate reference preparation solution in example 1 was examined, and a chromatogram was recorded, and the examination results are shown in table 11.
TABLE 11
| Organic phase | Calcium acetate time to peak | Time to peak of polyethylene glycol |
| Acetonitrile-water (70:30) | 9.005 | 3.843 |
| Methanol-water (70:30) | 3.867 | 4.325 |
And (4) conclusion: as can be seen from the above table, after the mobile phase is replaced, the retention of calcium acetate and polyethylene glycol 8000 is weakened, the peak time is too fast, and the separation of calcium acetate and polyethylene glycol 8000 cannot be realized.
As can be seen from the comparative example 1 and the comparative example 2, the detection effect in the embodiment of the application cannot be achieved by adopting other C18 chromatographic columns and other mobile phases, which indicates that the detection method provided by the invention can realize the qualitative and quantitative determination of the polyethylene glycol 8000 in the calcium acetate tablet, thereby realizing the more effective control of the detection of the polyethylene glycol 8000 in the calcium acetate tablet and improving the product effectiveness.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method for detecting the content of polyethylene glycol 8000 in a calcium acetate tablet is characterized by comprising the following steps:
(1) preparing a test solution and a reference solution:
preparing a test solution from the calcium acetate tablets by using a solvent;
preparing a polyethylene glycol 8000 reference substance into a reference substance solution by using a solvent;
(2) the detection is carried out according to the following high performance liquid chromatography conditions:
a chromatographic column: propyl amide group bonding silica gel column;
mobile phase: acetonitrile water solution with volume ratio of 60-80: 40-20;
a detector: an evaporative light scattering detector or a differential detector.
2. The method for detecting the content of polyethylene glycol 8000 in the calcium acetate tablet according to claim 1, wherein the chromatographic column has a specification of 250mm x 4.6mm and a packing diameter of 5 μm.
3. The method for detecting the content of polyethylene glycol 8000 in the calcium acetate tablet according to claim 1 or 2, wherein the chromatographic column is Venusil HILIC, 4.6mm x 250mm, 5 μm.
4. The method for detecting the content of polyethylene glycol 8000 in the calcium acetate tablet as claimed in claim 1, wherein the mobile phase is an aqueous acetonitrile solution with a volume ratio of 70: 30.
5. The method for detecting the content of polyethylene glycol 8000 in the calcium acetate tablet as claimed in claim 1, wherein the column temperature is 35-45 ℃ and the flow rate is 0.9-1.1 mL/min.
6. The method for detecting the content of polyethylene glycol 8000 in the calcium acetate tablet as claimed in claim 5, wherein the column temperature is 40 ℃ and the flow rate is 1.0 mL/min.
7. The method for detecting the content of polyethylene glycol 8000 in the calcium acetate tablet of claim 1, wherein the injection volume is 20 μ L.
8. The method for detecting the content of polyethylene glycol 8000 in the calcium acetate tablet of claim 1, wherein the detector is an evaporative light scattering detector.
9. The method for detecting the content of polyethylene glycol 8000 in calcium acetate tablets of claim 1, wherein the concentration of the test solution is 20.0 mg/mL.
10. The method for detecting the content of polyethylene glycol 8000 in the calcium acetate tablet of claim 1, wherein the concentration of polyethylene glycol 8000 in the control solution is 0.1mg/mL-0.5 mg/mL.
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| CN101366692A (en) * | 2007-08-15 | 2009-02-18 | 江苏豪森药业股份有限公司 | Stable Exenatide formulation |
| WO2009029231A2 (en) * | 2007-08-24 | 2009-03-05 | Millipore Corporation | Method of detecting filter extractables in biopharmaceutical products by liquid chromatography-mass spectrometry |
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| CN101366692A (en) * | 2007-08-15 | 2009-02-18 | 江苏豪森药业股份有限公司 | Stable Exenatide formulation |
| WO2009029231A2 (en) * | 2007-08-24 | 2009-03-05 | Millipore Corporation | Method of detecting filter extractables in biopharmaceutical products by liquid chromatography-mass spectrometry |
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| CN116223677A (en) * | 2023-03-08 | 2023-06-06 | 浙江萃泽医药科技有限公司 | Method for detecting content of polyethylene glycol 400 in meloxicam powder preparation |
| CN116223677B (en) * | 2023-03-08 | 2023-07-28 | 浙江萃泽医药科技有限公司 | Method for detecting content of polyethylene glycol 400 in meloxicam powder preparation |
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