CN113777191A - Method for determining content of calcitriol soft capsule - Google Patents
Method for determining content of calcitriol soft capsule Download PDFInfo
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- CN113777191A CN113777191A CN202111005407.XA CN202111005407A CN113777191A CN 113777191 A CN113777191 A CN 113777191A CN 202111005407 A CN202111005407 A CN 202111005407A CN 113777191 A CN113777191 A CN 113777191A
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- GMRQFYUYWCNGIN-NKMMMXOESA-N calcitriol Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@@H](CCCC(C)(C)O)C)=C\C=C1\C[C@@H](O)C[C@H](O)C1=C GMRQFYUYWCNGIN-NKMMMXOESA-N 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 29
- 239000007901 soft capsule Substances 0.000 title claims abstract description 14
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 claims abstract description 32
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims abstract description 29
- 239000005995 Aluminium silicate Substances 0.000 claims abstract description 28
- 235000012211 aluminium silicate Nutrition 0.000 claims abstract description 28
- 239000003463 adsorbent Substances 0.000 claims abstract description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 9
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 6
- 238000001514 detection method Methods 0.000 claims abstract description 5
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 12
- 238000005086 pumping Methods 0.000 claims description 8
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 7
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- XSTXAVWGXDQKEL-UHFFFAOYSA-N Trichloroethylene Chemical compound ClC=C(Cl)Cl XSTXAVWGXDQKEL-UHFFFAOYSA-N 0.000 abstract 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 abstract 1
- 239000002594 sorbent Substances 0.000 abstract 1
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- 239000000523 sample Substances 0.000 description 5
- 239000012224 working solution Substances 0.000 description 5
- 229930003316 Vitamin D Natural products 0.000 description 4
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 description 4
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- OFHCOWSQAMBJIW-AVJTYSNKSA-N alfacalcidol Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)C[C@H](O)C1=C OFHCOWSQAMBJIW-AVJTYSNKSA-N 0.000 description 2
- 229960002535 alfacalcidol Drugs 0.000 description 2
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 2
- 229910052593 corundum Inorganic materials 0.000 description 2
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- JWUBBDSIWDLEOM-NQZHSCJISA-N 25-hydroxy-3 epi cholecalciferol Chemical compound C1([C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@@H](CCCC(C)(C)O)C)=CC=C1C[C@H](O)CCC1=C JWUBBDSIWDLEOM-NQZHSCJISA-N 0.000 description 1
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- 125000002887 hydroxy group Chemical group [H]O* 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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Abstract
本发明公开了一种测定骨化三醇软胶囊含量的方法，该方法采用自制高岭土‑SPE色谱柱，用高岭土吸附骨化三醇，完成了骨化三醇的纯化和富集，采用骨化三醇软胶囊含量测定的SPE‑HPLC分析方法：固相萃取吸附剂，固相萃取方法；配置分析溶液；使用反相色谱柱，以乙腈和水为流动相，采用梯度洗脱方法；将前处理得到的分析溶液通过HPLC‑DAD测定。本发明能够有效且准确的测定骨化三醇软胶囊的含量，从而确保产品的质量可控。该方法首次尝试将天然高岭土作为固相萃取吸附剂用于脂溶性基质中的样品检测，在详细优化SPE条件后，建立了一种经济高效的SPE净化方法。该方法符合“绿色化学”的思想，并在药物分析领域推广了天然材料。
The invention discloses a method for determining the content of calcitriol soft capsules. The method adopts a self-made kaolin-SPE chromatographic column, uses kaolin to adsorb calcitriol, and completes the purification and enrichment of calcitriol. SPE‑HPLC analytical method for the content determination of triol soft capsules: solid-phase extraction adsorbent, solid-phase extraction method; configuration of analysis solution; use of reversed-phase chromatography column, acetonitrile and water as mobile phases, and gradient elution method; The resulting analytical solution was assayed by HPLC-DAD. The invention can effectively and accurately determine the content of the calcitriol soft capsule, thereby ensuring the quality of the product is controllable. This method is the first attempt to use natural kaolin as a solid-phase extraction sorbent for sample detection in lipid-soluble matrices, and after detailed optimization of the SPE conditions, a cost-effective SPE purification method is established. This method is in line with the idea of "green chemistry" and promotes natural materials in the field of pharmaceutical analysis.
Description
Technical Field
The invention relates to the technical field of pharmaceutical analysis, in particular to a method for determining the content of calcitriol soft capsules by an SPE-HPLC method based on kaolin.
Background
Calcitriol is a common vitamin D medicament for clinically treating osteoporosis, chronic kidney diseases and abnormal bone metabolism. Vitamin D does not function as a prototype in humans, and requires two-step hydroxylation in the liver and kidney for eventual conversion to 1, 25-dihydroxyvitamin D3I.e. calcitriol. Calcitriol is the most active vitamin D metabolite in human body, and the physiological activity of calcitriol is monohydroxylated 1, 25-hydroxyvitamin D3(alfacalcidol) 1000 times higher. Since 1 α -hydroxylase activity in the kidney of the elderly is almost lost, vitamin D cannot be transformed, and alfacalcidol burdens the liver for patients with liver disease, its application range is limited. On the contrary, calcitriol does not need to be metabolized by liver and kidney, and has high physiological activity, so the calcitriol has more advantages.
Calcitriol is unstable in nature and sensitive to air and light, which can cause isomerization at high temperature. Therefore, the calcitriol is dissolved in a proper fat-soluble matrix to prepare a soft capsule form, so that light and air can be isolated to a certain extent, the stability of the calcitriol is ensured, and the bioavailability is increased. However, the calcitriol soft capsule content was very low (1.55. mu.g.g)-1) Calcitriol accounts for only 0.0015% relative to the very high fat-soluble matrix. Therefore, in order to realize accurate quantification of calcitriol soft capsules, a suitable sample pretreatment method is urgently required to be found to eliminate interference of a fat-soluble matrix.
In recent years, traditional fillers have been widely used to adsorb trace amounts of compounds in complex matrices. However, the traditional adsorbent has complicated preparation procedures, and the preparation process of the material also involves consumption of a plurality of organic reagents and solvents, which has harmful effects on the environment and is not beneficial to environmental protection. Therefore, the search for green adsorbents has become a problem to be solved urgently.
Clay mineral materials have received much attention because of their low cost, ready availability and environmental friendliness. The chemical composition of the kaolin is 39.5 percent of Al2O346.5% SiO2And 14.0% of H2O (in mass%). It is laminated by two different interlayer surfaces. The first side of the layer is composed of silicon dioxide and the second side has six-fold O-coordinated aluminum atoms of hydroxyl groups. The layers of kaolin are held together by hydrogen bonds, dipole-dipole interactions and van der waals forces. The kaolin has abundant mineral resources, and is widely applied to different fields due to the characteristics of abundant reserves, good chemical stability, easy modification and the like. The modified kaolin composites are considered to be effective adsorbents for the removal of dyes and heavy metals. However, the use of kaolin composites for sample detection in a lipid-soluble matrix is lacking.
Based on the research background, the invention firstly tries to use the natural kaolin as the adsorbent for sample detection in the fat-soluble matrix so as to establish an economic and efficient SPE purification method. Calcitriol can be retained by kaolin by polar action. The polarity of the fat-soluble matrix is less compared to calcitriol, so the difference in polarity between the two can be used to achieve selective extraction. At the same time, calcitriol has a pi electron system and exhibits certain lewis base properties. Thus, it may be reacted with Al2O3The Lewis acid sites of the surface interact to achieve a certain retention. Moreover, kaolin is cheap and readily available due to its abundance in nature. In china, the cost of kaolin is only 0.05-0.10% of commercially available SPE fillers. Based on the factors, the invention self-prepares the kaolin-SPE chromatographic column, uses kaolin to adsorb calcitriol, and uses normal hexane as a washing solution to fully remove fat-soluble matrix, thereby completing the purification and enrichment of calcitriol. After optimizing SPE conditions in detail, kaolin-based SPE method was used in combination with HPLC-DAD to quantify calcitriol in soft capsules. The method conforms to the idea of 'green chemistry', and natural materials are popularized in the field of pharmaceutical analysis.
Disclosure of Invention
At present, the existing analysis methods such as Low Temperature Fat Precipitation (LTFP), liquid-liquid extraction (LLE), supercritical fluid chromatography combined with atmospheric pressure chemical ionization tandem mass spectrometry (SFC-APPI-MS/MS) and the like have complex operation and special requirements on instruments, and are not easy to popularize. Based on the difficulties and problems, an analysis method which is easy to operate and accurate in quantification is developed for calcitriol soft capsules by utilizing solid-phase extraction combined with high performance liquid chromatography. A method for measuring the content of calcitriol soft capsules comprises the following steps:
solid phase extraction adsorbent: drying kaolin in an oven at 80 deg.C for 24 hr, weighing 80mg of kaolin, precisely weighing, placing in a solid phase extraction column, and fixing with a sieve plate.
② solid phase extraction method
Through a series of condition investigation and optimization, the final solid phase extraction operation steps are determined: taking about 10 products, taking out the contents, taking the amount which is about 1.5 mu g of calcitriol according to the marked amount, diluting the product with 3mL of n-hexane, loading the product to a 1mL n-hexane activated solid phase extraction column, washing a centrifuge tube with 2mL of n-hexane, pumping the product into the solid phase extraction column, leaching the product with 5mL of n-hexane, pumping 1mL of methanol into the solid phase extraction column, and collecting the components. The solution was blown dry with nitrogen gas, and 0.2mL of methanol/ultrapure water (80/20, v/v) was added to the residue to dissolve it, thereby obtaining a sample solution. Taking about 1g of medium-chain triglyceride, diluting with 3mL of n-hexane, loading the diluted medium-chain triglyceride to a 1mL of n-hexane activated solid-phase extraction column, washing a centrifuge tube with 2mL of chromatographically pure hexane, pumping into the solid-phase extraction column, leaching with 5mL of n-hexane, pumping 1mL of methanol into the solid-phase extraction column, and collecting components. The solution was blown dry with nitrogen, and 0.2mL of chromatographic grade methanol/ultrapure water (80/20, v/v) was added to the residue to dissolve it as a blank adjuvant solution.
③ high performance liquid chromatography
Octadecylsilane chemically bonded silica is used as a filler (Agilent XB-C18 column length is 25cm, inner diameter is 4.6mm, and particle size is 5 μm); using purified water as a mobile phase A and acetonitrile as a mobile phase B, and carrying out gradient elution according to the specification in the following table; the flow rate is 1 mL/min-1(ii) a The detection wavelength is 265 nm; the column temperature is 30 ℃; the injection volume was 100. mu.L.
Advantageous effects
A low cost, simple, environmentally friendly and reliable solid phase extraction method has been developed and combined with HPLC-UV analysis to determine calcitriol in pharmaceutical formulations. Untreated kaolin was first used as an SPE adsorbent to scavenge calcitriol from the fat-soluble matrix. It showed high binding capacity (5.495 mg. g-1) and rapid binding kinetics (1 min). Under the optimal condition, the kaolin-SPE can completely eliminate the interference of a fat-soluble matrix and obtain the excellent adsorption performance of calcitriol in the soft capsule. The result shows that the analysis performance of the method provided by the experiment is good. In addition, the kaolin mineral resources are rich, the price is cheap and easy to obtain. Can be directly used as an adsorbent without complex modification and solvent consumption, and the influence on the environment is reduced to the minimum. In addition, its price is 0.05-0.10% of commercial adsorbents. Based on the above factors, the kaolin-SPE-based HPLC-DAD analysis method provided by the invention has great application potential for the economic and effective determination of calcitriol in complex matrixes.
Drawings
FIG. 1(A) is a topographical view of kaolin; (B) fourier transform infrared spectroscopy of kaolin; (C) a temperature contour plot of kaolin; (D) BJH pore volume and pore size curves for kaolin;
FIG. 2 is an SEM image (top) and an EDS image (bottom) of kaolin;
FIG. 3 is a potential diagram (A) of the zero charge point of kaolin; XRD pattern (B);
FIG. 4 is a Langmuir adsorption isotherm model (A); freundlich adsorption isotherm model (B).
Detailed Description
The technical solution of the present invention will be specifically described below with reference to examples.
The analysis method is used for investigating accuracy, repeatability and intermediate precision. The experimental data are as follows.
Accuracy of
Control working solutions (75.0, 150.0, 225.0. mu.g.mL) were prepared with methanol at 3 concentrations-1) Adding 1.00 + -0.01 g of medium-chain triglyceride into 3 working solutions with different concentrations, and pre-treating according to the above solid phase extraction method to obtain calcitriol solutions with the concentration of 50%, 100% and 150%, respectively, of 0.75, 1.50 and 2.25 μ g.g-1And adopting a high performance liquid phase method for sample injection analysis. The chromatographic results are shown in the following table 1, and the recovery rates of 50%, 100% and 150% limit levels meet the requirements and have good accuracy.
TABLE 1 accuracy results
② repeatability
The repeated experiments are carried out by respectively using control solutions (75.0, 150.0, 225.0 mu g. mL) with 3 concentrations-1) Adding 1.00 + -0.01 g of medium-chain triglyceride into 3 working solutions, and pre-treating according to the above solid phase extraction method to obtain final concentrations of 0.75, 1.50 and 2.25 μ g-1The calcitriol solution is prepared into 6 parts in parallel, and is subjected to sample injection analysis by a high performance liquid phase method. The chromatographic results are shown in the following table 2, and the recovery rates of 50%, 100% and 150% limit levels meet the requirements, and the repeatability is good.
Table 2 repeatability (n ═ 6) results
Third intermediate precision
The intermediate precision was measured with 3 concentrations of working solutions of control (75.0, 150.0, 225.0. mu.g.mL-1), 1.00. + -. 0.01g of medium-chain triglyceride was added to the 3 concentrations of working solutions, and pre-treatment was performed according to the above-mentioned solid phase extraction method to obtain calcitriol solutions with concentrations of 0.75, 1.50 and 2.25. mu.g.g-1, respectively, and they were analyzed by high performance liquid chromatography for three consecutive days. The chromatographic results are shown in the table below, the recovery rates of 50%, 100% and 150% limit levels meet the requirements, and the intermediate precision is good.
TABLE 3 results for intermediate precision (n. 3)
Claims (3)
1. A method for measuring the content of calcitriol soft capsules is characterized by comprising the following specific steps:
solid phase extraction adsorbent: accurately weighing kaolin, putting the kaolin into an oven, putting the kaolin into a small solid-phase extraction column, and fixing the kaolin by using a sieve plate;
② a solid phase extraction method:
taking the kaolin content of the product, taking an amount equivalent to 1.5 mu g of calcitriol according to the marked amount, diluting with 3mL of n-hexane, loading the diluted product to a 1mL n-hexane activated solid-phase extraction column, washing a centrifuge tube with 2mL of n-hexane, pumping the washed centrifuge tube into the solid-phase extraction column, leaching with 5mL of n-hexane, pumping 1mL of methanol into the solid-phase extraction column, and collecting components; blowing the solution to dryness by using nitrogen, and adding 0.2mL of methanol/ultrapure water into the residue for dissolving to obtain a test solution; taking about 1g of medium-chain triglyceride, diluting with 3mL of n-hexane, loading the diluted medium-chain triglyceride to a 1mL of n-hexane activated solid-phase extraction column, washing a centrifugal tube with 2mL of chromatographically pure hexane, pumping into the solid-phase extraction column, leaching with 5mL of n-hexane, pumping 1mL of methanol into the solid-phase extraction column, and collecting components; blowing the solution to dryness by using nitrogen, and adding 0.2mL of chromatographic grade methanol/ultrapure water into the residue for dissolving to obtain a blank auxiliary material solution;
③ high performance liquid chromatography
Octadecylsilane chemically bonded silica is used as a filler Agilent XB-C18, the column length is 25cm, the inner diameter is 4.6mm, and the particle size is 5 mu m; using purified water as a mobile phase A and acetonitrile as a mobile phase B, and carrying out gradient elution according to the specification in the following table; the flow rate is 1 mL/min-1(ii) a The detection wavelength is 265 nm; the column temperature is 30 ℃; injection volume 100 μ L:
2. the method as claimed in claim 1, wherein 80mg of kaolin is weighed in the solid phase extraction adsorbent, and the kaolin is dried in an oven at 80 ℃ for 24 hours.
3. The method as claimed in claim 1, wherein the number of kaolin samples in the solid phase extraction method is 10, and the methanol/ultrapure water is 80/20, v/v.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2140293A1 (en) * | 1992-07-15 | 1994-02-03 | Kowichi Jimbow | Radioimaging and radiochemotherapy phenolic thioether amines and acyl derivatives thereof for use in diagnosing and treating pigmentation disorders |
| CN106890166A (en) * | 2015-12-17 | 2017-06-27 | 北京泰德制药股份有限公司 | Preparation for external application to skin containing Galcium receptor-active compound |
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- 2021-08-30 CN CN202111005407.XA patent/CN113777191A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2140293A1 (en) * | 1992-07-15 | 1994-02-03 | Kowichi Jimbow | Radioimaging and radiochemotherapy phenolic thioether amines and acyl derivatives thereof for use in diagnosing and treating pigmentation disorders |
| CN106890166A (en) * | 2015-12-17 | 2017-06-27 | 北京泰德制药股份有限公司 | Preparation for external application to skin containing Galcium receptor-active compound |
Non-Patent Citations (1)
| Title |
|---|
| XINGCHEN WANG ET AL: "Naturally occurring kaolinite as a sorbent for solid-phase extraction of calcitriol in soft capsules", 《APPLIED CLAY SCIENCE》 * |
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