Reference marker for everolimus impurity detection and preparation method thereofTechnical Field
The invention relates to an everolimus impurity detection technology, in particular to a reference marker for everolimus impurity detection and a preparation method thereof.
Background
Everolimus is a rapamycin derivative with certain water solubility developed by noval corporation, switzerland, is mainly used clinically to prevent rejection after kidney transplantation and heart transplantation operations, and can be orally administered. The action mechanism mainly comprises an immunosuppressive action, an antitumor action, an antiviral action and a blood vessel protection action, and is often used in combination with other immunosuppressive agents such as cyclosporine and the like to reduce toxicity. In addition, everolimus is also used for the treatment of advanced renal cancer.
It is well known that for the production of active pharmaceutical ingredients for human administration, the requirements for the content of impurities are very high. Generally, a weight ratio of each impurity content of less than 0.15% is required, and a weight ratio of less than 0.1% is required for an unidentified impurity content with undetermined toxicity. However, impurities in the active pharmaceutical ingredient are widely available, may result from degradation of the product itself (which is related to the stability of the product during storage), and may also result from manufacturing processes (including chemical synthesis). The impurities derived from the preparation method include unreacted starting materials, impurities contained in the starting materials and chemical derivatives thereof, synthesis by-products, degradation products, and the like.
In the technical field of medical quality analysis, chemical derivatives, synthesis byproducts and degradation products in active pharmaceutical ingredient impurities can be identified by adopting a spectrum or other physical methods, and the impurities have a correlation with peak positions in a chromatogram, so that the impurities can be identified according to the relative positions of the impurities in the chromatogram. Before analyzing impurities in a compound, a substance with higher purity and the same or similar structure as the impurities is used as a reference marker, a pure compound to be detected is used as a reference standard, the reference marker and the reference standard are detected together, the relative position of the reference marker in a chromatogram is taken as the relative position of the impurities in the chromatogram, and the impurity detection of the compound to be detected is guided. Obviously, the selection and preparation of the reference marker has a direct impact on the scientificity and accuracy of the detection of the impurity content in the active pharmaceutical ingredient. For the detection of the content of the everolimus impurities, a substance reference marker which has the same or similar structure as the chemical derivatives, the synthesis byproducts and the degradation products in the everolimus impurities and has higher purity needs to be selected and prepared, so that the scientificity and the accuracy of the detection of the everolimus impurities are improved.
Disclosure of Invention
In order to improve the scientificity and accuracy of everolimus impurity detection, the invention provides a reference marker for everolimus impurity detection and a preparation method thereof.
The reference marker for detecting the everolimus impurities is a degradation product of everolimus, and the structure of the reference marker is as follows:
according to the preparation method of the reference marker for detecting the everolimus impurities, provided by the invention, the solid everolimus is dissolved by using an alkali solution, excessive alkali is neutralized by using an organic acid after heating reaction, and a neutralized mixture is concentrated into a mixture solid; dissolving the mixture solid by using an aqueous organic solvent, desorbing the chromatographic column by using the aqueous organic solvent as a desorbent, collecting the desorbent containing the target product in sections, and concentrating to obtain a reference marker for detecting the everolimus impurities; wherein,
the alkali solution is one of sodium hydroxide, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium hydroxide, dipotassium hydrogen phosphate and potassium dihydrogen phosphate, and the concentration of the solution is 0.2 to 0.7 mol/L;
the heating reaction temperature is 20 to 90 ℃;
the organic acid is one of formic acid, acetic acid, oxalic acid, benzoic acid and succinic acid;
the concentration temperature is 30-60 ℃, and a rotary evaporator is adopted for concentration;
the water-containing organic solvent is one of water solutions of methanol, ethanol, isopropanol, acetone, acetonitrile and tetrahydrofuran, and the water content of the water-containing organic solvent is 20 to 80 weight percent;
the silica gel column is one of C1, C4, C8 and C18 reverse phase silica gel;
the desorbent is one of water-containing organic solvents of methanol, ethanol, isopropanol, acetone, acetonitrile and tetrahydrofuran, and the water content is 30-70 wt%.
Further, the preparation method of the reference marker for detecting everolimus impurities comprises the following steps:
s1, dissolving everolimus solid by adopting an alkali solution, wherein the alkali solution is one of sodium hydroxide, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium hydroxide, dipotassium hydrogen phosphate and potassium dihydrogen phosphate, and the concentration of the solution is 0.2-0.7 mol/L;
s2, heating for reaction, wherein the heating reaction temperature is 20-90 ℃;
s3, neutralizing the everolimus solution by using organic acid, wherein the organic acid is one of formic acid, acetic acid, oxalic acid, benzoic acid and succinic acid;
s4, concentrating the mixture, and concentrating the mixture at 30-60 ℃ by adopting a rotary evaporator to prepare a mixture solid;
s5, dissolving the solid mixture prepared in the step S4 by using an aqueous organic solvent, wherein the aqueous organic solvent is one of aqueous solutions of methanol, ethanol, isopropanol, acetone, acetonitrile and tetrahydrofuran, and the water content of the solid mixture is 20 to 80 weight percent;
s6, desorbing the chromatographic column, wherein the adopted silica gel column is one of C1, C4, C8 and C18 reverse phase silica gel, the desorbent is one of methanol, ethanol, isopropanol, acetone, acetonitrile and tetrahydrofuran aqueous organic solvent, and the water content is 30 to 70 weight percent;
s7, collecting a desorbent containing a target product;
and S8, concentrating the desorbent containing the target product to obtain the reference marker for detecting the everolimus impurities.
The reference marker for detecting the everolimus impurities and the preparation method thereof provide the reference marker with the structure the same as or similar to that of chemical derivatives, synthesis byproducts and degradation products in the everolimus impurities and the preparation method thereof, and effectively improve the scientificity and accuracy of the everolimus impurity detection.
Drawings
FIG. 1 is a schematic flow chart of a preparation method of a reference marker for everolimus impurity detection in the present invention.
The reference marker for detecting everolimus impurities and the preparation method thereof according to the present invention will be further described with reference to the accompanying drawings and the detailed description.
Detailed Description
Fig. 1 is a schematic flow chart of a preparation method of a reference marker for everolimus impurity detection of the present invention, and it can be seen from the figure that the reference marker for everolimus impurity detection of the present invention is a degradation product of everolimus, and its structure is:
according to the preparation method of the reference marker for detecting the everolimus impurities, provided by the invention, the solid everolimus is dissolved by using an alkali solution, excessive alkali is neutralized by using an organic acid after heating reaction, and a neutralized mixture is concentrated into a mixture solid; dissolving the mixture solid by using an aqueous organic solvent, desorbing the chromatographic column by using the aqueous organic solvent as a desorbent, collecting the desorbent containing the target product in sections, and concentrating to obtain a reference marker for detecting the everolimus impurities; wherein,
the alkali solution is one of sodium hydroxide, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium hydroxide, dipotassium hydrogen phosphate and potassium dihydrogen phosphate, and the concentration of the solution is 0.2 to 0.7 mol/L;
the heating reaction temperature is 20 to 90 ℃;
the organic acid is one of formic acid, acetic acid, oxalic acid, benzoic acid and succinic acid;
the concentration temperature is 30-60 ℃, and a rotary evaporator is adopted for concentration;
the water-containing organic solvent is one of water solutions of methanol, ethanol, isopropanol, acetone, acetonitrile and tetrahydrofuran, and the water content of the water-containing organic solvent is 20 to 80 weight percent;
the silica gel column is one of C1, C4, C8 and C18 reverse phase silica gel;
the desorbent is one of water-containing organic solvents of methanol, ethanol, isopropanol, acetone, acetonitrile and tetrahydrofuran, and the water content is 30-70 wt%.
The preparation method of the reference marker for detecting the everolimus impurities comprises the following steps:
s1, dissolving everolimus solid by adopting an alkali solution, wherein the alkali solution is one of sodium hydroxide, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium hydroxide, dipotassium hydrogen phosphate and potassium dihydrogen phosphate, and the concentration of the solution is 0.2-0.7 mol/L;
s2, heating for reaction, wherein the heating reaction temperature is 20-90 ℃;
s3, neutralizing the everolimus solution by using organic acid, wherein the organic acid is one of formic acid, acetic acid, oxalic acid, benzoic acid and succinic acid;
s4, concentrating the mixture, and concentrating the mixture at 30-60 ℃ by adopting a rotary evaporator to prepare a mixture solid;
s5, dissolving the solid mixture prepared in the step S4 by using an aqueous organic solvent, wherein the aqueous organic solvent is one of aqueous solutions of methanol, ethanol, isopropanol, acetone, acetonitrile and tetrahydrofuran, and the water content of the solid mixture is 20 to 80 weight percent;
s6, desorbing the chromatographic column, wherein the adopted silica gel column is one of C1, C4, C8 and C18 reverse phase silica gel, the desorbent is one of methanol, ethanol, isopropanol, acetone, acetonitrile and tetrahydrofuran aqueous organic solvent, and the water content is 30 to 70 weight percent;
s7, collecting a desorbent containing a target product;
and S8, concentrating the desorbent containing the target product to obtain the reference marker for detecting the everolimus impurities.
Example 1
10.00g of everolimus was dissolved in 50ml of 0.5mol/L sodium dihydrogen phosphate solution, heated to 35 ℃ for 30min, then adjusted to neutral with acetic acid, and concentrated at 40 ℃ by a rotary evaporator to obtain 10.23g of a mixture solid.
Example 2
Dissolving 12.00g everolimus in 60ml 0.6mol/L disodium hydrogen phosphate solution, heating to 40 deg.C, heating for 45min, adjusting pH to neutral with acetic acid, and concentrating at 45 deg.C with rotary evaporator to obtain 12.13g mixture solid.
Example 3
10.00g of everolimus was dissolved in 50ml of 0.5mol/L disodium hydrogenphosphate solution, heated to 40 ℃ for 40min, adjusted to neutral with formic acid, and concentrated at 45 ℃ using a rotary evaporator to obtain 10.17g of a mixture solid.
Example 4
Dissolving 5g of the concentrated mixture solid (the content of the reference marker for detecting the everolimus impurities is 35.77 percent, and the detection is carried out by adopting a high performance liquid chromatography area normalization method), using 100ml of acetone with the concentration of 60 percent by weight to dissolve the mixture solid, feeding the mixture solid into a 1000ml of reverse phase C4 silica gel chromatographic column, using acetone with the concentration of 73 percent by weight as a desorbent, desorbing the chromatographic column, collecting the mixture in sections, monitoring the liquid phase, combining and concentrating the mixture solid to obtain 1.6g of the reference marker for detecting the everolimus impurities, wherein the content of the reference marker for detecting the everolimus impurities is 95.33 percent, the content of the everolimus is 2.31 percent, and the content of other impurities is 2.36 percent.
Example 5
Dissolving 3g of the concentrated mixture solid (the content of the reference marker for detecting everolimus impurities is 44.28 percent and detecting the everolimus impurities by adopting a high performance liquid chromatography area normalization method) by using 100ml of 65 weight percent isopropanol, feeding the solution into a 1000ml of reverse phase C8 silica gel chromatographic column, using 76 weight percent isopropanol as a desorbent, desorbing the chromatographic column, collecting the solution in sections, monitoring the liquid phase, merging and concentrating the solution to obtain 0.83g of the reference marker for detecting everolimus impurities, wherein the content of the reference marker for detecting everolimus impurities is 96.18 percent, the content of everolimus is 2.17 percent and the content of other impurities is 1.65 percent.
Example 6
Dissolving 5g of the concentrated mixture solid (the content of the reference marker for detecting the everolimus impurities is 48.97 percent and the detection is carried out by adopting a high performance liquid chromatography area normalization method) by using 100ml of acetonitrile with the concentration of 45 percent by weight, feeding the mixture solid into a 1000ml of reverse phase C8 silica gel chromatographic column, using acetonitrile with the concentration of 58 percent by weight as a desorbent, desorbing the chromatographic column, collecting the mixture in sections, monitoring the liquid phase, combining and concentrating the mixture to obtain 1.34g of the reference marker for detecting the everolimus impurities, wherein the content of the reference marker for detecting the everolimus impurities is 97.02 percent, the content of the everolimus is 1.31 percent and the content of other impurities is 1.67 percent.
From the above examples, it can be seen that the reference marker for everolimus impurity detection of the present invention is a degradation product of everolimus, and the structure of the reference marker is the same as or similar to that of a chemical derivative, a synthesis byproduct and a degradation product in everolimus impurities, so that the scientificity and accuracy of everolimus impurity detection can be effectively improved.
Obviously, the reference marker for detecting the everolimus impurities and the preparation method thereof provide the reference marker with the structure the same as or similar to that of chemical derivatives, synthesis byproducts and degradation products in the everolimus impurities and the preparation method thereof, and effectively improve the scientificity and accuracy of the everolimus impurity detection.