Method for measuring prednisone acetate related substances by HPLC (high performance liquid chromatography) gradient elution

Abstract

The invention discloses a method for determining prednisone acetate related substances by HPLC gradient elution, the method can simultaneously determine a plurality of impurities such as cortisone acetate, cortisone, hydrocortisone and prednisone, and the method has the advantages of strong elution ability, good separation efficiency, short detection time and simple operation, can accurately reflect the impurity content, and is suitable for industrial production. The quality control of the prednisone acetate raw material medicine is facilitated.

Description

Technical Field

[0001] The invention relates to the technical field of drug-related substance detection, in particular to a method for determining prednisolone acetate-related substances by HPLC gradient elution. Background Art

[0002] Prednisolone acetate is a type of adrenal cortex hormone that has anti-inflammatory, anti-allergic, anti-rheumatic and immunosuppressive effects. The anti-inflammatory effect can reduce and prevent the tissue's response to inflammation, thereby reducing the manifestations of inflammation. The oral and injectable formulations of this product are mainly used for allergic and autoimmune inflammatory diseases. It is suitable for connective tissue diseases, systemic lupus erythematosus, severe bronchial asthma, dermatomyositis, vasculitis, etc. Eye drops can be used for steroid-sensitive eye inflammation. Creams are often used for allergic and non-infectious skin diseases.

[0003] Prednisone acetate is mainly obtained through biotransformation. Its main impurities are the substrate cortisone acetate and the degradation impurity prednisone. It may also contain a small amount of cortisone and hydrocortisone. At present, the impurities cortisone acetate and prednisone in prednisone acetate are mainly determined by isocratic elution in pharmacopoeias and literature. Under this condition, the separation degree of prednisone acetate and cortisone acetate is poor. Figure 1 As shown, the separation degree is 1.463, and the baseline separation cannot be achieved. The chromatographic conditions of isocratic elution are not conducive to the elution of strongly retained impurities, resulting in prolonged analysis time or inability of impurities to elute, and inaccurate impurity analysis results.

[0004] Therefore, it is necessary to provide a method for determining related substances in prednisone acetate raw materials by HPLC gradient elution to solve the defect that the existing detection method has poor separation and leads to inaccurate analysis results. Summary of the invention

[0005] The invention provides a method for determining related substances in a prednisone acetate raw material drug by using HPLC gradient elution, which is used to solve the defect that the prior art detection method has poor separation degree and causes inaccurate analysis results.

[0006] In view of this, the scheme of the present invention is:

[0007] The method for determining the related substances of prednisolone acetate by HPLC gradient elution is as follows:

[0008] Use acetonitrile as diluent to prepare reference solutions of cortisone acetate, cortisone, hydrocortisone and prednisone, and test solution;

[0009] The reference solution and the test solution were respectively injected into a high performance liquid chromatograph, and the contents of prednisone and cortisone acetate were detected by HPLC; the HPLC chromatographic conditions were:

[0010] Chromatographic column: octadecyl bonded silica gel chromatographic column;

[0011] Mobile phase: ultrapure water as mobile phase A, acetonitrile as mobile phase B, gradient elution;

[0012] Column temperature: 30°C;

[0013] Flow rate: 1.0 mL / min;

[0014] The injection volume is 1-20 μL;

[0015] Detection wavelength: 240nm;

[0016] The gradient elution procedure is:

[0017]

[0018] Furthermore, during the HPLC detection process, if there are chromatographic peaks of prednisone and cortisone acetate in the chromatogram of the test solution, the reference solution uses a mixed reference solution of prednisone and cortisone acetate, and the impurity content is calculated by the external standard method.

[0019] Furthermore, in the mixed control solution of prednisone and acetic acid, the concentrations of prednisone and acetic acid are both 4.5-5.5 μg / mL.

[0020] Furthermore, the concentration of prednisone acetate in the test solution is 450-550 μg / mL.

[0021] Furthermore, the reference solution and the test solution both use 33% acetonitrile as a diluent.

[0022] Furthermore, during the HPLC detection process, if at least one of hydrocortisone, prednisone and cortisone is present in the chromatogram of the test solution, the impurity content is calculated according to the main component self-reference method.

[0023] Furthermore, the separation degree between any two components in the test solution is greater than 1.5.

[0024] Furthermore, the chromatographic column is selected from Ultimate XB-C18.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The HPLC gradient elution determination method provided by the present invention can simultaneously determine multiple impurities such as cortisone acetate, cortisone, hydrocortisone and prednisone. The method has strong elution ability, good separation efficiency, short detection time, simple operation, can accurately reflect the impurity content, and is beneficial to the quality control of prednisone acetate raw material medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1This is a typical spectrum of prednisolone acetate-related substances under the isocratic elution conditions described in the background technology.

[0028] Figures 2 to 7 They respectively correspond to the blank solvent-specific spectrum, the systemic applicability spectrum, the hydrocortisone-specific spectrum, the prednisone-specific spectrum, the cortisone-specific spectrum and the cortisone acetate-specific spectrum in the detection method of the present invention.

[0029] Figure 8 It is the linear spectrum of prednisone in the detection method of the present invention.

[0030] Fig. 9 It is the linear spectrum of cortisone acetate in the detection method of the present invention. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be clearly and completely described below in conjunction with the preferred embodiments. Obviously, the described embodiments are only 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 ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] 1. Solution preparation

[0033] Prednisone reference substance stock solution: Take 9-11 mg of prednisone reference substance (purity 99.7%, sourced from China Food and Drug Inspection Institute, batch number 100199-202104) in a 20 mL volumetric flask, dissolve and dilute with 33% acetonitrile, shake well to make a solution with a concentration of 450-550 μg / mL.

[0034] Cortisone acetate reference substance stock solution: Take 9-11 mg of cortisone acetate reference substance (purity 99.1%, sourced from China Food and Drug Inspection Institute, batch number 100123-201204) in a 20 mL volumetric flask, dissolve and dilute with 33% acetonitrile, shake well, and make a solution with a concentration of 450-550 μg / mL.

[0035] Cortisone reference substance stock solution: Take 9-11 mg of cortisone reference substance (purity 97.2%, sourced from China Food and Drug Inspection Institute, batch number 101128-201001) in a 20 mL volumetric flask, dissolve and dilute with 33% acetonitrile, shake well, and make a solution with a concentration of 450-550 μg / mL.

[0036] Hydrocortisone reference substance stock solution: Take 9-11 mg of hydrocortisone reference substance (purity 99.7%, sourced from China Food and Drug Inspection Institute, batch number 100152-202008) in a 20 mL volumetric flask, dissolve and dilute with 33% acetonitrile, shake well, and make a solution with a concentration of 450-550 μg / mL.

[0037] System suitability solution: Take 45-55 mg of prednisone acetate reference substance (purity 99.9%, sourced from China Food and Drug Inspection Institute, batch number 100012-202108) in a 100-mL volumetric flask, dissolve it with 50 mL of 33% acetonitrile, and then accurately measure 1 mL each of prednisone reference substance stock solution, cortisone acetate reference substance stock solution, cortisone reference substance stock solution, and hydrocortisone reference substance stock solution into the 100-mL volumetric flask, make up to volume with 33% acetonitrile, shake well, and prepare a solution containing 450-550 μg / mL of prednisone acetate, and approximately 4.5-5.5 μg / mL of prednisone, cortisone acetate, cortisone, and hydrocortisone.

[0038] Mixed reference solution: Accurately measure 1 mL of prednisone reference stock solution and 1 mL of cortisone acetate reference stock solution into a 100 mL volumetric flask, dilute to volume with 33% acetonitrile, shake well, and prepare a solution containing approximately 4.5 to 5.5 μg / mL of prednisone and cortisone acetate, respectively.

[0039] Test solution: Take 45-55 mg of prednisone acetate raw material in a 100 mL volumetric flask, dissolve and make up to volume with 33% acetonitrile, shake well to make a solution containing prednisone acetate at a concentration of 450-550 μg / mL.

[0040] Self-control solution: Accurately measure 1 mL of the test solution into a 100 mL volumetric flask, dilute to volume with 33% acetonitrile, and shake well to prepare a solution containing prednisone acetate at a concentration of 4.5 to 5.5 μg / mL.

[0041] Blank solution: 33% acetonitrile.

[0042] 2. Chromatographic conditions: Chromatographic column: octadecylsilane bonded silica column Ultimate XB-C18. Ultrapure water was used as mobile phase A, acetonitrile (chromatographic grade) was used as mobile phase B, the flow rate was 1.0 mL / min, the column temperature was 30°C, the injection volume was 20 μL, the detection wavelength was 240 nm, and gradient elution was performed according to Table 1 below.

[0043] Table 1: Gradient elution conditions for prednisolone acetate related substances

[0044]

[0045] 3. Assay

[0046] According to the chromatographic conditions in "2", take the blank solution, system suitability solution, mixed reference solution, test solution and self-control solution in "1" in turn and inject them into the HPLC and record the chromatogram. The blank should have no interference. In the chromatogram of the system suitability solution, hydrocortisone, prednisone, cortisone, prednisolone acetate and cortisone acetate should flow out in turn, and the separation degree between each component should be not less than 1.5.

[0047] In the chromatogram of the test solution, if there are chromatographic peaks of prednisone and cortisone acetate, calculate the amount of impurities according to the external standard method; if there are other impurities, calculate the amount of impurities according to the main component self-reference method.

[0048] The calculation formula is as follows:

[0049]

[0050] Wherein, ms is the sample weight of the reference substance, P is the purity of the reference substance, Ai is the peak area of ​​prednisone or cortisone acetate in the test substance, Vi is the dilution multiple of the test solution, Vs is the dilution multiple of the reference solution, As is the peak area of ​​impurity F in the reference solution, and mi is the sample weight of the test substance.

[0051]

[0052] Wherein, Ai is the peak area of ​​the unknown impurity in the test sample, Ar is the peak area of ​​prednisone acetate in the self-control solution, and 100 is the dilution volume of the self-control solution.

[0053] Method validation

[0054] 1. Exclusivity

[0055] Blank solution: 33% acetonitrile.

[0056] System suitability solution: the same as the system suitability solution in “1. Preparation of solution” of the invention content.

[0057] Prednisone impurity reference solution: Accurately measure 1 mL of the prednisone reference stock solution in "1. Preparation of solution" of the invention content into a 100 mL volumetric flask, dilute to volume with 33% acetonitrile, and shake well to prepare a solution containing prednisone at a concentration of 4.5 to 5.5 μg / mL.

[0058] Cortisone acetate impurity reference solution: Accurately measure 1 mL of the cortisone acetate reference stock solution in "1. Preparation of solution" of the invention content into a 100 mL volumetric flask, dilute to volume with 33% acetonitrile, shake well, and prepare a solution containing cortisone acetate at a concentration of 4.5 to 5.5 μg / mL.

[0059] Cortisone impurity reference solution: Accurately measure 1 mL of the cortisone reference stock solution in "1. Preparation of solution" of the invention content into a 100 mL volumetric flask, dilute to volume with 33% acetonitrile, shake well, and prepare a solution containing cortisone at a concentration of 4.5 to 5.5 μg / mL.

[0060] Hydrocortisone impurity reference solution: Accurately measure 1 mL of the hydrocortisone reference stock solution in "1. Preparation of solution" of the invention content into a 100 mL volumetric flask, dilute to volume with 33% acetonitrile, shake well, and prepare a solution containing hydrocortisone at a concentration of 4.5 to 5.5 μg / mL.

[0061] Accurately measure the above solution, inject it into the high performance liquid chromatograph according to "2. Chromatographic conditions" in the invention content, and record the chromatogram. Figures 2 to 7 Specificity map.

[0062] like Figures 2 to 7 As shown, the blank had no interference on the peaks of prednisone, prednisone acetate and cortisone acetate, and baseline separation was achieved between the components. The separation degrees are shown in Table 2 below, and the separation degrees are all greater than 1.5.

[0063] Table 2: System suitability solution resolution

[0064]

[0065] 2. Linear

[0066] 2.1 Prednisone linearity

[0067] Prednisone linear solution ①: Accurately measure 2 mL of prednisone impurity reference solution into a 10 mL volumetric flask, dilute to volume with 33% acetonitrile, shake well, and prepare a solution containing prednisone at a concentration of 0.9 to 1.1 μg / mL.

[0068] Prednisone linear solution ②: Accurately measure 5 mL of prednisone impurity reference solution into a 10 mL volumetric flask, dilute to volume with 33% acetonitrile, shake well, and prepare a solution containing prednisone at a concentration of 2.25 to 2.75 μg / mL.

[0069] Prednisone linear solution ③: Same as prednisone impurity reference solution.

[0070] Prednisone linear solution ④: Accurately measure 1.5 mL of prednisone reference stock solution into a 100 mL volumetric flask, dilute to volume with 33% acetonitrile, shake well, and prepare a solution containing prednisone at a concentration of 6.75-8.25 μg / mL.

[0071] Prednisone linear solution⑤: Accurately measure 2 mL of prednisone reference stock solution into a 100 mL volumetric flask, dilute to volume with 33% acetonitrile, shake well, and prepare a solution containing prednisone at a concentration of 9 to 11 μg / mL.

[0072] Accurately measure the above solution and inject it into the high performance liquid chromatograph according to "2. Chromatographic conditions" in the invention content, record the chromatogram and peak area, use the concentration of the linear solution as the abscissa and the prednisone peak area as the ordinate, perform linear regression, and obtain the linear regression graph and equation, as shown in Figure 8 , r 2 It is 0.9998, indicating that the method has good linearity.

[0073] 2.2 Cortisone acetate linear

[0074] Cortisone acetate linear solution ①: Accurately measure 2 mL of the cortisone acetate impurity reference solution into a 10 mL volumetric flask, dilute to volume with 33% acetonitrile, shake well, and prepare a solution containing cortisone acetate at a concentration of 0.9 to 1.1 μg / mL.

[0075] Cortisone acetate linear solution ②: Accurately measure 5 mL of the cortisone acetate impurity reference solution into a 10 mL volumetric flask, dilute to volume with 33% acetonitrile, shake well, and prepare a solution containing cortisone acetate at a concentration of 2.25 to 2.75 μg / mL.

[0076] Cortisone acetate linear solution ③: Same as cortisone acetate impurity reference solution.

[0077] Cortisone acetate linear solution ④: Accurately measure 1.5 mL of cortisone acetate reference stock solution into a 100 mL volumetric flask, dilute to volume with 33% acetonitrile, shake well, and prepare a solution containing cortisone acetate at a concentration of 6.75-8.25 μg / mL.

[0078] Cortisone acetate linear solution⑤: Accurately measure 2 mL of the cortisone acetate reference stock solution into a 100 mL volumetric flask, dilute to volume with 33% acetonitrile, shake well, and prepare a solution containing cortisone acetate at a concentration of 9 to 11 μg / mL.

[0079] Accurately measure the above solution and inject it into the high performance liquid chromatograph according to "2. Chromatographic conditions" in the invention content, record the chromatogram and peak area, use the concentration of the linear solution as the abscissa and the peak area of ​​cortisone acetate as the ordinate, and perform linear regression to obtain the linear regression graph and equation as follows: Fig. 9 , r 2 It is 0.9998, indicating that the method has good linearity.

[0080] 3. Limit of Quantitation and Limit of Detection

[0081] 3.1 Limit of quantification and detection of prednisone

[0082] Prednisone quantitation limit solution: Accurately measure 1 mL of prednisone impurity reference solution into a 500 mL volumetric flask, dilute to volume with 33% acetonitrile, shake well, and prepare a solution containing prednisone at a concentration of 0.009 to 0.011 μg / mL.

[0083] Prednisone detection limit solution: Accurately measure 3 mL of prednisone quantification limit solution into a 10 mL volumetric flask, dilute to volume with 33% acetonitrile, shake well, and prepare a solution containing prednisone at a concentration of 0.0027 to 0.0033 μg / mL.

[0084] Accurately measure the above solution and inject it into the high performance liquid chromatograph according to "2. Chromatographic conditions" in the invention content, record the chromatogram and prednisone signal-to-noise ratio (S / N). The results are shown in Table 3 below. The S / N of the quantitative limit solution is greater than 10, and the S / N of the detection limit solution is greater than 3.

[0085] Table 3: Results of quantitation limit and detection limit of prednisone

[0086]

[0087] 3.2 Limit of Quantification and Limit of Detection of Cortisone Acetate

[0088] Cortisone acetate quantification limit solution: Accurately measure 1 mL of the cortisone acetate impurity reference solution into a 200 mL volumetric flask, dilute to volume with 33% acetonitrile, shake well, then accurately measure 5 mL of the solution into a 10 mL volumetric flask, dilute to volume with 33% acetonitrile, shake well to make a solution containing cortisone acetate at a concentration of 0.011 to 0.014 μg / mL.

[0089] Cortisone acetate detection limit solution: Accurately measure 3.5 mL of cortisone acetate limit solution into a 10 mL volumetric flask, dilute to volume with 33% acetonitrile, shake well, and prepare a solution containing cortisone acetate at a concentration of 0.0039 to 0.0048 μg / mL.

[0090] Accurately measure the above solution and inject it into the high performance liquid chromatograph according to "2. Chromatographic conditions" in the invention content, record the chromatogram and the signal-to-noise ratio (S / N) of cortisone acetate. The results are shown in Table 4 below. The S / N of the quantitative limit solution is greater than 10, and the S / N of the detection limit solution is greater than 3.

[0091] Table 4: Results of quantification limit and detection limit of cortisone acetate

[0092]

[0093] 4. Injection precision

[0094] According to "2. Chromatographic conditions" in the invention content, the mixed impurity reference solution in "1. Preparation of solution" in the invention content was taken and injected into the high performance liquid chromatograph. The injection was repeated 6 times, the chromatogram and the peak areas of the two components were recorded, and the average peak area and RSD% were calculated. The results are shown in Table 5. The peak area RSD% of prednisone and cortisone acetate after 6 repeated injections were all less than 2.0%.

[0095] Table 5: Prednisone and cortisone acetate injection precision results

[0096]

[0097]

[0098] 5. Recovery rate

[0099] Recovery solution: Take 45-55 mg of prednisone acetate API in a 100 mL volumetric flask, dissolve it with 50 mL of 33% acetonitrile, then accurately measure 1 mL of prednisone reference stock solution and 1 mL of cortisone acetate reference stock solution into the 100 mL volumetric flask, dilute to volume with 33% acetonitrile, shake well, and prepare a solution containing 450-550 μg / mL of prednisone acetate API and about 4.5-5.5 μg / mL of prednisone and cortisone acetate reference substances. Prepare 6 copies in parallel.

[0100] Accurately measure the recovery solution and the mixed impurity control solution and the test solution in “1. Preparation of Solution” of the invention content, and inject them into the high performance liquid chromatograph according to “2. Chromatographic Conditions” of the invention content, record the chromatogram, calculate the impurity amount of prednisone and prednisolone acetate in the test solution and the recovery solution by the external standard method, and calculate the recovery rate, mean recovery rate and RSD% of prednisone and prednisolone acetate. The results are shown in Table 6.

[0101]

[0102] Table 6: Prednisone and cortisone acetate recovery results

[0103]

[0104] As can be seen from Table 6, the recoveries of prednisone and cortisone acetate were between 90% and 110%, and the RSD% of the recoveries was within 2.0%.

[0105] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. The method for determining the related substances of prednisolone acetate by HPLC gradient elution is characterized in that: Here are the steps: 1) Prepare the reference solutions of cortisone acetate, cortisone, hydrocortisone and prednisone, and the test solution respectively using acetonitrile as diluent; 2) Inject the reference solution and the test solution into a high performance liquid chromatograph respectively, and detect the contents of prednisone and cortisone acetate by HPLC; the HPLC chromatographic conditions are: Chromatographic column: octadecyl bonded silica gel chromatographic column; Mobile phase: ultrapure water as mobile phase A, acetonitrile as mobile phase B, gradient elution; Column temperature: 30°C; Flow rate: 1.0 mL / min; The injection volume is 1-20 μL; Detection wavelength: 240nm; The gradient elution procedure is:

2. The method according to claim 1, characterized in that During the HPLC detection process, if there are chromatographic peaks of prednisone and cortisone acetate in the chromatogram of the test solution, the reference solution uses a mixed reference solution of prednisone and cortisone acetate, and the impurity content is calculated by the external standard method.

3. The method according to claim 1, characterized in that In the mixed control solution of prednisone and acetic acid, the concentrations of prednisone and acetic acid are both 4.5-5.5 μg / mL.

4. The method according to claim 1, characterized in that: The concentration of prednisone acetate in the test solution is 450-550 μg / mL.

5. The method according to claim 1, characterized in that The reference solution and the test solution both used 33% acetonitrile as a diluent.

6. The method according to claim 1, characterized in that During the HPLC detection process, if at least one of hydrocortisone, prednisone and cortisone is present in the chromatogram of the test solution, the impurity content is calculated according to the main component self-comparison method.

7. The method according to claim 1, characterized in that The separation degree between any two components in the test solution is greater than 1.

5.

8. The method according to claim 1, characterized in that The chromatographic column is selected from Ultimate XB-C18.

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