Method for determining dissolution curve of calcitriol soft capsule
By using phosphate buffer and liquid mass spectrometry detection of specific surfactants and stabilizers, the mass spectrometry parameters are optimized, and the instability and low sensitivity problems of calcitriol soft capsule dissolution curve determination are solved, and quantitative detection of high-specificity and high sensitivity is achieved.
Patent Information
- Application Number
- CN202510167807.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-14
- Publication Date
- 2025-07-04
AI Technical Summary
In the dissolution curve determination, calcitriol soft capsules are susceptible to temperature and oxygen, poor solubility, common detection methods are cumbersome and have low sensitivity, so they cannot be accurately measured.
Phosphate buffer containing the surfactant lauryl dimethylamine oxide, stabilizer butyl hydroxyanisole and dibutyl hydroxytoluene were used as the dissolution medium. Combined with liquid phase mass spectrometry detection, mass spectrometry parameters were optimized, and calcitriol dissolution curve detection was performed using specific chromatographic columns and gradient elution methods.
The accurate amount of dissolution of calcitriol soft capsules is achieved, which improves the specificity, sensitivity and stability of the detection, and solves the problem of cumbersome operation.
Smart Images
Figure CN120254090A_ABST
Abstract
Description
Technical Field
[0001] The present invention discloses a technology related to pharmaceutical detection, and relates to a method for determining the dissolution curve of calcitriol soft capsules. Background Art
[0002] Calcitriol is a steroid derivative. Calcitriol soft capsules are used for the treatment of postmenopausal osteoporosis, chronic renal failure, especially renal osteodystrophy in patients undergoing hemodialysis, postoperative hypoparathyroidism, pseudohypoparathyroidism, vitamin D-dependent rickets, hypophosphatemic vitamin D-resistant rickets, etc.
[0003] Calcitriol soft capsules are oral preparations. When conducting drug registration and declaration, it is necessary to study their in vitro dissolution curves. At the same time, during production, in order to ensure the quality of drugs, it is also necessary to detect the dissolution rate or dissolution curve. However, due to its chemical properties, calcitriol has very poor stability. When determining the dissolution curve, it is easily affected by conditions such as temperature and oxygen, resulting in degradation and unable to accurately measure; in addition, the specification of calcitriol soft capsules is very small, 0.25 micrograms per capsule. After dissolution determination using the common volume of dissolution medium, the concentration of the dissolution liquid is at the dissolution level of 0.025 ng / ml. Common liquid chromatography detectors, such as ultraviolet detectors, cannot reach its sensitivity; then, calcitriol is a fat-soluble compound and has very poor solubility in common buffer salt media, resulting in the dissolution determination not being able to reach the required solubility, so it is necessary to add a surfactant for solubilization. However, general surfactants will form a matrix effect in mass spectrometry detection, interfering with the ionization of calcitriol, and thus accurate quantification cannot be performed.
[0004] In the prior art, most use an organic phase / water as the dissolution medium, and after the dissolution liquid is rotary evaporated by a rotary evaporator to remove the organic solvent, it is then dissolved with methanol. There are many sample numbers in the dissolution determination itself. This method is cumbersome to operate, and the stability of calcitriol is poor during the rotary evaporation process, and the method applicability is not high. Summary of the Invention
[0005] In view of this, the present application provides a method for determining the dissolution curve of calcitriol soft capsules, which can effectively improve the detection sensitivity, specificity and stability by investigating the types and concentrations of surfactants, different chromatographic columns, the types and concentrations of stabilizers, the selection of mass spectrometry parameters, etc., and can effectively detect the dissolution curve of calcitriol soft capsules.
[0006] According to one aspect of the present application, a method for determining the dissolution curve of calcitriol soft capsules is provided, including the following steps: Step 1: Take calcitriol soft capsules, according to the dissolution test method, use phosphate buffer solution containing surfactant as the dissolution medium, operate according to law, take the dissolution solution, filter, take the subsequent filtrate, add a stabilizer, and mix well to obtain the test solution; Step 2: Take an appropriate amount of calcitriol reference substance, dilute it with the mobile phase to obtain a reference substance stock solution, take an appropriate amount of the stock solution, use phosphate buffer solution containing surfactant and stabilizer as the diluent, and dilute it to obtain a reference substance solution; Step 3: Detect by liquid chromatography-mass spectrometry, obtain the chromatograms of the test solution and the reference substance solution, and quantitatively determine the dissolution amount of calcitriol by the external standard method.
[0007] According to one aspect of the present application, the surfactant is lauryldimethylamine oxide.
[0008] According to one aspect of the present application, the concentration of lauryldimethylamine oxide is 0.2%-1.0%.
[0009] According to one aspect of the present application, the stabilizer is butylated hydroxyanisole and dibutylhydroxytoluene.
[0010] According to one aspect of the present application, the use concentration of butylated hydroxyanisole and dibutylhydroxytoluene is 50-500 μg / ml, and the volume ratio with the dissolution solution is 1:2 to 1:5.
[0011] According to one aspect of the present application, take calcitriol soft capsules, according to the dissolution test method, use phosphate buffer solution containing lauryldimethylamine oxide as the dissolution medium, the rotation speed is 100 revolutions per minute, operate according to law, at 20, 30, 45, and 60 minutes, take the dissolution solution, filter, take the subsequent filtrate, add a mixed solution of butylated hydroxyanisole and dibutylhydroxytoluene, and mix well to obtain the test solution.
[0012] According to one aspect of the present application, the mobile phase includes acetonitrile and 0.1% formic acid solution, and the elution method is gradient elution.
[0013] According to one aspect of the present application, take an appropriate amount of calcitriol reference substance, dilute it with the mobile phase to obtain a reference substance stock solution, take an appropriate amount of the stock solution, use phosphate buffer solution containing surfactant and stabilizer as the diluent, and dilute it to obtain a reference substance solution, and the mobile phase is acetonitrile.
[0014] According to one aspect of the present application, the pH of the phosphate buffer solution is 6.8.
[0015] According to one aspect of the present application, the mass spectrometer detector is a tandem quadrupole mass spectrometer detector, and the chromatographic column uses octadecylsilane-bonded silica gel as the filler.
[0016] Advantages of the present invention: Liquid chromatography - mass spectrometry detection is adopted. Take soft capsules of calcitriol. According to the dissolution determination method, use phosphate buffer solution containing surfactant as the dissolution medium, operate according to the law, take the dissolution solution, filter, take the subsequent filtrate, add a stabilizer, and mix evenly to obtain the test sample solution. Weigh an appropriate amount of calcitriol reference substance, dilute it with the mobile phase to obtain the reference substance stock solution. Take an appropriate amount of the stock solution, use phosphate buffer solution containing surfactant and stabilizer as the diluent, and dilute it to obtain the reference substance solution. Detect by liquid chromatography - mass spectrometry, obtain the chromatograms of the test sample solution and the reference substance solution, and quantitatively determine the dissolution amount of calcitriol by the external standard method, realizing the quantitative detection of the dissolution amount in soft capsules of calcitriol. This determination method has strong specificity, high sensitivity, high accuracy, and good stability, and has important practical significance for accurately quantifying the dissolution curve of soft capsules of calcitriol. Description of the Drawings
[0017] Figure 1 Shows the structural formula of calcitriol;
[0018] Figure 2 Shows the dissolution curves of batches B4287, B4285, and B4281 in Example 1 of the present application;
[0019] Figure 3 Shows the dissolution curves of batch B4281 at different LDAO concentrations in Example 2 of the present application.
[0020] Figure 4 Shows the results of the dissolution curves of batch B4281 at different filter membrane pore sizes in Example 3 of the present application. Detailed Embodiments
[0021] The following embodiments are provided to better further understand the present invention. It is not limited to the best embodiment, and does not limit the content and protection scope of the present invention. Any product that is the same as or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior art features falls within the protection scope of the present invention.
[0022] For those not indicating specific experimental steps or conditions in the embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments not indicating the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.
[0023] According to one aspect of the present application, a method for determining the dissolution curve of calcitriol soft capsules is provided, including the preparation of dissolution curve samples, the selection of surfactants, the selection of stabilizers, and the optimization of mass spectrometry conditions. Specifically, the steps are as follows: Step 1: Take calcitriol soft capsules, according to the dissolution test method, use phosphate buffer solution containing surfactant as the dissolution medium, operate according to law, take the dissolution solution, filter, take the subsequent filtrate, add stabilizer, and mix well to obtain the test solution; Step 2: Take an appropriate amount of calcitriol reference substance, dilute it with the mobile phase to obtain a reference substance stock solution, take an appropriate amount of the stock solution, use phosphate buffer solution containing surfactant and stabilizer as the diluent, and dilute it to obtain a reference substance solution; Step 3: Detect by liquid chromatography-mass spectrometry, obtain the chromatograms of the test solution and the reference substance solution, and quantitatively determine the dissolution amount of calcitriol by the external standard method.
[0024] Among them, Step 1: Take calcitriol soft capsules, according to the dissolution test method, use phosphate buffer solution containing surfactant as the dissolution medium, operate according to law, take the dissolution solution, filter, take the subsequent filtrate, add stabilizer, and mix well to obtain the test solution; Step 2: Take an appropriate amount of calcitriol reference substance, dilute it with the mobile phase to obtain a reference substance stock solution, take an appropriate amount of the stock solution, use phosphate buffer solution containing surfactant and stabilizer as the diluent, and dilute it to obtain a reference substance solution, both of which are the preparation of dissolution curve samples.
[0025] Step 3: Detect by liquid chromatography-mass spectrometry, obtain the chromatograms of the test solution and the reference substance solution, and quantitatively determine the dissolution amount of calcitriol by the external standard method, which is the optimization of mass spectrometry conditions.
[0026] According to one aspect of Step 1 and Step 2 of the present application, the surfactant is lauryldimethylamine oxide, a specific surfactant, which has high solubility and specificity.
[0027] Preferably, the concentration of lauryldimethylamine oxide is 0.2%-1.0%.
[0028] According to one aspect of Step 1 and Step 2 of the present application, the stabilizers are butylated hydroxyanisole and dibutylhydroxytoluene, which are specific stabilizers and have high stability and specificity.
[0029] Preferably, the use concentrations of butylated hydroxyanisole and dibutylhydroxytoluene are 50-500 μg / ml, and the volume ratio to the dissolution solution is 1:2 to 1:5.
[0030] According to one aspect of Step 1 of the present application, take calcitriol soft capsules, according to the dissolution test method, use phosphate buffer solution containing lauryldimethylamine oxide as the dissolution medium, the rotation speed is 100 revolutions per minute, operate according to law, at 20, 30, 45, and 60 minutes, take the dissolution solution, filter, take the subsequent filtrate, add a mixed solution of butylated hydroxyanisole and dibutylhydroxytoluene, and mix well to obtain the test solution.
[0031] According to one aspect of Step 2 of the present application, the mobile phase includes acetonitrile and 0.1% formic acid solution, and the elution method is gradient elution.
[0032] According to one aspect of Step 2 of the present application, an appropriate amount of calcitriol reference substance is diluted with the mobile phase to prepare a reference substance stock solution. An appropriate amount of the stock solution is taken and diluted with a phosphate buffer solution containing a surfactant and a stabilizer as a diluent to prepare a reference substance solution, and the mobile phase is acetonitrile.
[0033] According to one aspect of the present application, the pH of the phosphate buffer solution is 6.8.
[0034] According to one aspect of the present application, the mass spectrometry detector is a tandem quadrupole mass spectrometry detector. The dissolution detection is carried out by mass spectrometry, which has high sensitivity, specificity and accuracy. The chromatographic column uses octadecylsilane-bonded silica gel as the filler, and the specific chromatographic column has high separation efficiency.
[0035] Through the preparation of the dissolution curve samples, the selection of surfactants, the selection of stabilizers and the optimization of mass spectrometry conditions above, liquid chromatography-mass spectrometry detection is used. Octadecylsilane-bonded silica gel is used as the filler, 0.1% formic acid solution and acetonitrile are used as the mobile phase, and gradient elution is used for analysis and detection; take soft capsules of calcitriol, use a phosphate buffer solution with a pH of 6.8 containing lauryldimethylamine oxide as the dissolution medium, the rotation speed is 100 revolutions per minute, and operate according to law. At 20, 30, 45, and 60 minutes, take the dissolution solution, filter it, take the subsequent filtrate, add the mixed solution, mix well, to obtain the test solution; take an appropriate amount of calcitriol reference substance and dilute it with acetonitrile to prepare a reference substance stock solution. Take an appropriate amount of the stock solution and dilute it with a phosphate buffer solution with a pH of 6.8 containing lauryldimethylamine oxide, butylated hydroxyanisole and dibutylhydroxytoluene as a diluent to prepare a reference substance solution; calculate the dissolution amount by the external standard method, and realize the quantitative detection of the dissolution amount in soft capsules of calcitriol. This determination method has strong specificity, high sensitivity, high accuracy and good stability.
[0036] The present invention will be further described below in conjunction with the embodiments. The described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.
[0037] Example 1:
[0038] A method for detecting the dissolution curve of soft capsules of calcitriol, including the following content:
[0039] Take the calcitriol soft capsules and, according to the dissolution test method (General Chapter 0931, Method 1, Four Parts of Chinese Pharmacopoeia 2015 Edition), use 500 ml of pH 6.8 phosphate buffer solution (containing 0.4% lauryldimethylamine oxide) as the dissolution medium, rotate at a speed of 100 revolutions per minute, operate according to the law. After 45 minutes, take 10 ml of the solution, filter it through a 0.45 μm polyethersulfone membrane, take 1000 μl of the subsequent filtrate, add 250 μl of the BHA / BHT mixed solution (each 100 μg / ml acetonitrile solution), mix well, and it is the test solution.
[0040] Chromatography - mass spectrometry conditions: Use octadecylsilane chemically bonded silica gel as the filler (chromatographic column: Waters Acquity UPLC BEH C18, 2.1 mm * 50 mm, 1.7 μm); the injection volume is 5 μl. Use 0.1% formic acid as mobile phase A and acetonitrile solution as mobile phase B respectively, with a flow rate of 0.5 ml / min, gradient elution, and the gradient table is as follows:
[0041]
[0042]
[0043] Adopt a tandem quadrupole mass detector, electrospray ionization (ESI), positive ion scanning, selected ion monitoring mode (MRM). The ion pair of calcitriol is 399.2 → 151.1, the collision energy (CE) is 15, the declustering voltage (DP) is 80, the entrance voltage (EP) is 10, the collision cell exit voltage (CXP) is 15, the collision gas (CAD) is 4, the curtain gas (CUR) is 20, the ion source gas 1 (GAS1) is 40, the ion source gas 2 (GAS2) is 60, the ion source spray voltage (LVIS) is 5500, and the ion source temperature (TEM) is 550 °C.
[0044] Precisely weigh an appropriate amount of calcitriol reference substance, dilute it with acetonitrile to a solution containing 100 ng of calcitriol per 1 ml, which is the reference substance stock solution. Take an appropriate amount of the stock solution, use pH 6.8 phosphate buffer solution (containing 0.4% lauryldimethylamine oxide, 0.16 μg / ml BHA / BHT) as the diluent to prepare a solution containing 0.5 ng of calcitriol per 1 ml as the reference substance solution. Precisely measure 5 μl of the test solution and the reference substance solution respectively, inject them into the liquid chromatography - mass spectrometry instrument, and record the chromatogram. Use the external standard method to calculate the dissolution amount per capsule based on the peak area.
[0045] Adopt the above method to determine the dissolution curves of batches B4287, B4285, and B4281 of calcitriol soft capsules, and the results are shown in Table 1 below.
[0046] Table 1 Dissolution curve results of batches B4287, B4285, and B4281
[0047]
[0048] Example 2:
[0049] A method for detecting the dissolution curve of calcitriol soft capsules, in the specific steps, except for the concentration of the surfactant, the rest is the same as in Example 1:
[0050] Under the conditions of Example 2, the dissolution curves of B4281 batch of calcitriol soft capsules were determined at the concentrations of 0.4% LDAO, 0.6% LDAO, and 0.8% LDAO, and the results are shown in Table 2 below.
[0051] Table 2 Results of the dissolution curves of B4281 batch at different LDAO concentrations
[0052]
[0053] Example 3:
[0054] A method for detecting the dissolution curve of calcitriol soft capsules, in the specific steps, except for the pore size of the polyethersulfone filter membrane, the rest is the same as in Example 1:
[0055] Under the conditions of Example 2, the dissolution solutions were filtered through polyethersulfone filter membranes with pore sizes of 0.22 μm and 0.45 μm respectively, and the dissolution curves of B4281 batch of calcitriol soft capsules were determined. The results are shown in Table 3 below.
[0056] Table 3 Results of the dissolution curves of B4281 batch at different filter membrane pore sizes
[0057]
[0058] Through the above steps of sample preparation for the curve, selection of surfactant, selection of stabilizer, and optimization of mass spectrometry conditions, this application uses liquid chromatography - mass spectrometry detection. Octadecylsilane - bonded silica gel is used as the filler, 0.1% formic acid solution and acetonitrile are used as the mobile phase, and gradient elution is used for analysis and detection; for the calcitriol soft capsule, a phosphate buffer solution with a pH of 6.8 containing lauryldimethylamine oxide is used as the dissolution medium, the rotation speed is 100 revolutions per minute, and the operation is carried out according to the law. At 20, 30, 45, and 60 minutes, the dissolution solution is taken, filtered, and the continued filtrate is taken, and a mixed solution is added and mixed evenly to obtain the test solution; an appropriate amount of calcitriol reference substance is taken and diluted with acetonitrile to obtain the reference substance stock solution. An appropriate amount of the stock solution is taken and diluted with a phosphate buffer solution with a pH of 6.8 containing lauryldimethylamine oxide, butylated hydroxyanisole, and dibutylhydroxytoluene to obtain the reference substance solution; the dissolution amount is calculated by the external standard method, realizing the quantitative detection of the dissolution amount in the calcitriol soft capsule. This determination method has strong specificity, high sensitivity, high accuracy, and good stability, solving the technical problems of cumbersome operation, poor stability of calcitriol during the rotary evaporation process, and low applicability of the method.
[0059] As described above, it is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the scope disclosed by the present invention, according to the technical solution and its concept of the present invention, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.
Claims
1. A method for determining the dissolution curve of calcitriol soft capsules, characterized in that, The steps are as follows: Step 1: Take the calcitriol soft capsules. According to the dissolution test method, use the phosphate buffer solution containing surfactant as the dissolution medium, operate according to the law, take the dissolution solution, filter it, take the subsequent filtrate, add the stabilizer, and mix well to obtain the test solution; Step 2: Weigh an appropriate amount of calcitriol reference substance, dilute it with the mobile phase to obtain the reference substance stock solution. Take an appropriate amount of the stock solution, and use the phosphate buffer solution containing surfactant and stabilizer as the diluent to dilute it to obtain the reference solution; Step 3: Detect by liquid chromatography-mass spectrometry, obtain the chromatograms of the test solution and the reference solution, and quantitatively determine the dissolution amount of calcitriol by the external standard method.
2. The method for determining the dissolution curve of calcitriol soft capsules according to claim 1, characterized in that The surfactant is lauryldimethylamine oxide.
3. The method for determining the dissolution curve of calcitriol soft capsules according to claim 2, characterized in that, The concentration of lauryldimethylamine oxide is 0.2%-1.0%.
4. The method for determining the dissolution curve of calcitriol soft capsules according to claim 3, characterized in that, The stabilizer is butylated hydroxyanisole and dibutylhydroxytoluene.
5. The method for determining the dissolution curve of calcitriol soft capsules according to claim 4, characterized in that The use concentration of butylated hydroxyanisole and dibutylhydroxytoluene is 50-500 μg / ml, and the volume ratio to the dissolution solution is 1:2 to 1:
5.
6. The method for determining the dissolution curve of calcitriol soft capsules according to claim 5, characterized in that, Take the calcitriol soft capsules. According to the dissolution test method, use the phosphate buffer solution containing lauryldimethylamine oxide as the dissolution medium, with a rotation speed of 100 revolutions per minute, operate according to the law. At 20, 30, 45, and 60 minutes, take the dissolution solution, filter it, take the subsequent filtrate, add the mixed solution of butylated hydroxyanisole and dibutylhydroxytoluene, and mix well to obtain the test solution.
7. The method for determining the dissolution curve of calcitriol soft capsules according to claim 6, characterized in that, The mobile phase includes acetonitrile and 0.1% formic acid solution, and the elution method is gradient elution.
8. The method for determining the dissolution curve of calcitriol soft capsules according to claim 7, characterized in that, Weigh an appropriate amount of calcitriol reference substance, dilute it with the mobile phase to obtain the reference substance stock solution. Take an appropriate amount of the stock solution, and use the phosphate buffer solution containing surfactant and stabilizer as the diluent to dilute it to obtain the reference solution, and the mobile phase is acetonitrile.
9. The method for determining the dissolution curve of calcitriol soft capsules according to any one of claims 1-8, characterized in that, The pH of the phosphate buffer solution is 6.
8.
10. The method for determining the dissolution curve of calcitriol soft capsules according to claim 9, characterized in that, The mass spectrometer detector is a tandem quadrupole mass spectrometer detector, and the chromatographic column uses octadecylsilane-bonded silica gel as the filler.