CN110794066A - Method for detecting calcitriol soft capsule related substances by using double-ternary method - Google Patents
Method for detecting calcitriol soft capsule related substances by using double-ternary method Download PDFInfo
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- CN110794066A CN110794066A CN201810877105.3A CN201810877105A CN110794066A CN 110794066 A CN110794066 A CN 110794066A CN 201810877105 A CN201810877105 A CN 201810877105A CN 110794066 A CN110794066 A CN 110794066A
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention relates to the technical field of medicines, in particular to a method for detecting calcitriol soft capsule related substances. The invention relates to a method for detecting calcitriol soft capsule related substances by using a double-ternary high performance liquid chromatography, which is characterized in that a high performance liquid chromatography analyzer is used for measurement, peak areas of a test sample and a reference are respectively measured, and the content of the related substances of the sample is calculated by using a self-reference method. The sensitivity of sample determination is improved, 0.01% of impurities can be detected, both known impurities and unknown impurities can be effectively controlled, the accuracy is obviously improved, the operation is simple, sensitive and good in durability, reliable related substance data are provided for the detection of medicines, and the quality of the medicines is better controlled.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a method for detecting calcitriol soft capsule related substances.
Background
The key role of calcitriol in regulating calcium balance, including the stimulation of osteoblast activity in bones, provides a sufficient pharmacological basis for the treatment of osteoporosis. Is suitable for postmenopausal osteoporosis; chronic renal dysfunction; postoperative hypothyroidism; idiopathic hypoparathyroidism; pseudohypothyroidism; vitamin D dependent rickets; rickets with low blood phosphorus and vitamin D resistance.
The calcitriol soft capsule has specification of 0.5ug and 0.25 ug. At present, the methods for measuring related substances by domestic and foreign documents comprise a high-efficiency liquid phase method and an LC-MS-MS method, wherein the sensitivity of the high-efficiency liquid phase method is too low, only about 1% of impurities can be detected at the lowest, and the content of the related substances cannot be effectively detected; the LC-MSMS method can only measure the content of known impurities, and the required impurity reference substances are not easy to obtain, so that the method cannot effectively measure unknown impurities. At present, no method for simply, efficiently and accurately measuring the related substances of the calcitriol soft capsule exists.
Disclosure of Invention
Aiming at the technical problems, the invention aims to provide a method for detecting calcitriol soft capsule related substances by using a double-ternary high performance liquid chromatography, and the method has the advantages of high sensitivity, simple operation, high efficiency and accuracy.
The purpose of the invention is realized by the following technical scheme:
the invention relates to a method for detecting calcitriol soft capsule related substances by using a double-ternary high performance liquid chromatography, which comprises the following steps:
(a) preparing a test sample: taking a proper amount of calcitriol soft capsules, and adding a diluent to dilute the calcitriol soft capsules to obtain a test solution;
(b) preparation of control solution: diluting the test solution with diluent to obtain a control solution;
(c) performing two-dimensional chromatographic column separation by using a double-ternary high performance liquid chromatography, measuring by using a multi-wavelength ultraviolet detector or a diode array detector, and calculating related substances of calcitriol in the solution to be measured by using a self-control method. Measuring by using a C18 two-dimensional chromatographic column; the first dimension chromatographic column is an enrichment column C18 column with the inner diameter of 4.6mm, the column length of 100mm and the filler particle size of 5 microns, the second dimension chromatographic column is an analysis column C18 column with the inner diameter of 4.6mm, the column length of 250mm and the filler particle size of 5 microns; the high performance liquid chromatography analyzer includes: the device comprises a double ternary pump, an online filter, an enrichment column and an analysis column; the test solution and the test solution enter the online filter, the enrichment column and the analysis column respectively through the double-ternary pump. The invention relates to a method for detecting calcitriol soft capsule medicine related substances on line by using a double-ternary pump, which is characterized by comprising the following steps of: the diluent 1 used in the step (a) is one or more mixed solvents of methanol, ethanol, n-hexane, n-butanol, isopropanol, ethyl acetate, n-propanol, etc., and can be used as a diluent or a sample solution without dilution. The invention relates to a method for detecting calcitriol soft capsule medicine related substances on line by using a double-ternary pump, which is characterized by comprising the following steps of: the dilution of the control solution in step (b) with the test solution is 50 to 1000 times, preferably 100 times. The invention relates to a method for detecting calcitriol soft capsule medicine related substances on line by using a double-ternary pump, which is characterized by comprising the following steps of: in the step (c), the mobile phase is one or more mixed solvents of methanol, ethanol, n-hexane, n-butanol, isopropanol, ethyl acetate and n-propanol. Further optimization can be made to ethyl acetate-isopropanol-ethyl acetate-n-hexane (20: 40: 15: 25).
The invention relates to a method for detecting calcitriol soft capsule medicine related substances on line by using a double-ternary pump, which is characterized by comprising the following steps of: the mobile phase was subjected to gradient elution, the elution procedure was as follows:
the invention relates to a method for detecting calcitriol soft capsule medicine related substances on line by using a double-ternary pump, which is characterized by comprising the following steps of: the switching time of the six-way valve is as follows:
| time (min) | Valve with a valve body |
| 0 | 6-1 |
| 8 | 1-2 |
| 11 | 6-1 |
The invention relates to a method for detecting calcitriol soft capsule medicine related substances on line by using a double-ternary pump, which is characterized by comprising the following steps of: the column temperature is 25-45 deg.C, preferably 35 deg.C.
The invention relates to a method for detecting calcitriol soft capsule medicine related substances on line by using a double-ternary pump, which is characterized by comprising the following steps of: the detection wavelength is 254 nm-270 nm, preferably 265 nm.
The invention relates to a method for detecting calcitriol soft capsule medicine related substances on line by using a double-ternary pump, which is characterized by comprising the following steps of: the sample injection volume is 0.2ml-20ml, preferably 5 ml.
The invention has the beneficial effects that:
the method for detecting the calcitriol soft capsule related substances improves the sensitivity of sample determination, can detect 0.01% of impurities, can effectively control the known impurities and the unknown impurities, obviously improves the accuracy, has simple operation, sensitivity and good durability, provides reliable related substance data for the detection of the medicines, and has better control on the quality of the medicines.
Description of the drawings:
FIG. 1 is a chemical structural formula of calcitriol;
FIG. 2 is a quantitative limit chromatogram of a calcitriol control in example 1;
FIG. 3 chromatogram for measuring calcitriol-related substance in example 1.
Detailed Description
The present invention will be further described by the following examples, however, the present invention is not limited to the following examples, which do not limit the scope of the present invention in any way. Certain changes and modifications within the scope of the claims, which may be made by one skilled in the art, are also considered to be within the scope of the invention.
The method for detecting the calcitriol soft capsule related substances by using the double-ternary high performance liquid chromatography has the advantages of accurate quantification, high sensitivity and good durability.
The purpose of the invention is realized by the following technical scheme:
the invention relates to a method for detecting calcitriol soft capsule related substances by using a double-ternary high performance liquid chromatography, which comprises the following steps:
(a) preparing a test sample: taking a proper amount of calcitriol soft capsules, and adding a diluent to dilute the calcitriol soft capsules to obtain a test solution;
(b) preparation of control solution: taking a proper amount of a test sample, adding a diluent to dilute by 100 times to obtain a reference solution;
(c) performing two-dimensional chromatographic column separation by using a double-ternary high performance liquid chromatography, measuring by using a multi-wavelength ultraviolet detector or a diode array detector, and calculating related substances of calcitriol in a solution to be measured by using an external standard method;
measuring by using a C18 two-dimensional chromatographic column;
the first dimension chromatographic column is a Hypersil GOLD Silica enrichment column, and the second dimension chromatographic column is a Syncronis Silica analysis column;
the high performance liquid chromatography analyzer includes: the device comprises a double ternary pump, an online filter, an enrichment column and an analysis column; the test solution and the test solution enter the online filter, the enrichment column and the analysis column respectively through the double-ternary pump.
The mobile phase is ethyl acetate-isopropanol-ethyl acetate-n-hexane (20: 40: 15: 25), and gradient elution is carried out, wherein the elution procedure is shown in the table; column temperature: 35 ℃, detection wavelength: 265 nm.
The invention relates to a method for detecting calcitriol soft capsule medicine related substances on line by using a double-ternary pump, which is characterized by comprising the following steps of: the switching time of the six-way valve is as follows:
| time (min) | Valve with a valve body |
| 0 | 6-1 |
| 8 | 1-2 |
| 11 | 6-1 |
1. Detection and quantitation limits of the methods of the invention
Taking calcitriol reference substance, adding diluent for dilution, and obtaining the detection limit of 0.3ng and the quantification limit of 0.7ng according to the condition that the chromatographic peak signal-to-noise ratio S/N is larger than 3 and the S/N is larger than 10 as the detection limit
2. In the precision test of the method, 5ml of sample solution to be tested and 5ml of reference solution are respectively injected, 6 parts of sample solution are repeatedly measured, and the RSD value is calculated, and the result is shown in the following table:
| sample (I) | Impurity 1 | Impurity 2 | Impurity 3 | Total miscellaneous |
| 1 part (c) | 0.12% | 0.03% | 1.03% | 1.18% |
| 2 nd part(s) | 0.12% | 0.02% | 1.05% | 1.19% |
| No. 3 part | 0.13% | 0.03% | 1.03% | 1.19% |
| 4 th part by weight | 0.14% | 0.03% | 1.04% | 1.21% |
| 5 th part by weight | 0.13% | 0.03% | 1.03% | 1.19% |
| 6 th part | 0.12% | 0.03% | 1.06% | 1.21% |
| Mean value of | 0.13% | 0.03% | 1.04% | 1.20% |
| RSD | 6.45% | 8.45% | 1.22% | 1.02% |
3. Durability test, changing different chromatographic conditions, determining related substances by the same method, and the durability test result is shown in the following table:
| sample (I) | Impurity 1 | Impurity 2 | Impurity 3 | Total miscellaneous |
| Standard conditions | 0.13% | 0.03% | 1.04% | 1.20% |
| The flow rate was varied: 1.4ml/min | 0.14% | 0.03% | 1.03% | 1.20% |
| The flow rate was varied: 1.6ml/min | 0.15% | 0.03% | 1.05% | 1.23% |
| Changing the column temperature: 30 deg.C | 0.13% | 0.02% | 1.03% | 1.18% |
| Changing the column temperature: 40 deg.C | 0.13% | 0.03% | 1.04% | 1.20% |
| Changing the detection wavelength: 260nm | 0.13% | 0.03% | 1.03% | 1.19% |
| Changing the detection wavelength: 270nm | 0.14% | 0.03% | 1.05% | 1.22% |
| Mean value of | 0.14% | 0.03% | 1.04% | 1.21% |
| RSD | 5.80% | 7.78% | 0.87% | 1.54% |
The calculation result shows that the calcitriol soft capsule related substances have good durability in measurement.
Claims (10)
1. A method for on-line detection of calcitriol soft capsule medicine related substances by using a double-ternary pump is characterized by comprising the following steps: it comprises the following steps:
(a) preparing a test sample: taking a proper amount of calcitriol soft capsules, and adding a diluent to dilute the calcitriol soft capsules to obtain a test solution;
(b) preparation of control solution: diluting the test solution with diluent to obtain a control solution;
(c) separating with two-dimensional chromatographic column by double-ternary high performance liquid chromatography, and passing through multi-wavelength ultraviolet detector or diode
Measuring by a tube array detector, and calculating related substances of calcitriol in the solution to be measured by using a self-contrast method;
measuring by using a C18 two-dimensional chromatographic column;
the first dimension chromatographic column is enrichment column C18 with inner diameter of 4.6mm, column length of 100mm, filler particle size of 5 μm, and the second dimension
The chromatographic column is an analytical column C18 column, the inner diameter is 4.6mm, the column length is 250mm, and the particle size of the filler is 5 μm;
the high performance liquid chromatography analyzer includes: the device comprises a double ternary pump, an online filter, an enrichment column and an analysis column; the test solution and the test solution enter the online filter, the enrichment column and the analysis column respectively through the double-ternary pump.
2. The method for on-line detection of calcitriol soft capsule drug related substances by using a double triple pump as claimed in claim 1, wherein the method comprises the following steps: the diluent 1 used in the step (a) is one or more mixed solvents of methanol, ethanol, n-hexane, n-butanol, isopropanol, ethyl acetate, n-propanol and the like as a diluent.
3. The method for on-line detection of calcitriol soft capsule drug related substances by using a double triple pump as claimed in claim 1, wherein the method comprises the following steps: the dilution of the control solution in step (b) with the test solution is 50 to 1000 times, preferably 100 times.
4. The method for on-line detection of calcitriol soft capsule drug related substances by using a double triple pump as claimed in claim 1, wherein the method comprises the following steps: in the step (c), the mobile phase is one or more mixed solvents of methanol, ethanol, n-hexane, n-butanol, isopropanol, ethyl acetate and n-propanol.
5. The mobile phase of claim 4 may be further optimized as ethyl acetate-isopropanol-ethyl acetate-n-hexane (20: 40: 15: 25).
6. The method for on-line detection of calcitriol soft capsule pharmaceutical related substances by using a double triple pump as claimed in claim 1, wherein the mobile phase is subjected to gradient elution, and the elution procedure is as follows:
7. the method for on-line detection of calcitriol soft capsule drug related substances by using a double triple pump as claimed in claim 1, wherein the method comprises the following steps: the switching time of the six-way valve is as follows:
。
8. The method for on-line detection of calcitriol soft capsule drug related substances by using a double triple pump as claimed in claim 1, wherein the method comprises the following steps: the column temperature is 25-45 deg.C, preferably 35 deg.C.
9. The method for on-line detection of calcitriol soft capsule drug related substances by using a double triple pump as claimed in claim 1, wherein the method comprises the following steps: the detection wavelength is 254 nm-270 nm, preferably 265 nm.
10. The method for on-line detection of calcitriol soft capsule drug related substances by using a double triple pump as claimed in claim 1, wherein the method comprises the following steps: the sample injection volume is 0.2ml-20ml, preferably 5 ml.
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| CN201810877105.3A CN110794066A (en) | 2018-08-03 | 2018-08-03 | Method for detecting calcitriol soft capsule related substances by using double-ternary method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112552265A (en) * | 2020-09-09 | 2021-03-26 | 四川国为制药有限公司 | Calcitriol pharmaceutical composition |
| CN116466016A (en) * | 2023-04-21 | 2023-07-21 | 正大制药（青岛）有限公司 | Method for measuring content of related substances in calcitriol oral liquid |
-
2018
- 2018-08-03 CN CN201810877105.3A patent/CN110794066A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112552265A (en) * | 2020-09-09 | 2021-03-26 | 四川国为制药有限公司 | Calcitriol pharmaceutical composition |
| CN116466016A (en) * | 2023-04-21 | 2023-07-21 | 正大制药（青岛）有限公司 | Method for measuring content of related substances in calcitriol oral liquid |
| CN116466016B (en) * | 2023-04-21 | 2023-09-15 | 正大制药（青岛）有限公司 | Method for measuring content of related substances in calcitriol oral liquid |
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Application publication date: 20200214 |