A method for the quantitative determination of 21-aldehyde hydrocortisone

Abstract

This invention provides a method for detecting 21-aldehyde hydrocortisone. This method can accurately detect trace amounts of 21-aldehyde hydrocortisone in test samples (such as hydrocortisone or hydrocortisone sodium succinate), thereby achieving better quality control of the drug at the trace level and ensuring patient medication safety.

Description

Technical Field

[0001] This invention relates to the field of analytical methods, and more specifically to a quantitative detection method for 21-aldehyde hydrocortisone. Background Technology

[0002] Hydrocortisone for Injection and Hydrocortisone Sodium Succinate for Injection are both glucocorticoid drugs, widely used clinically due to their various pharmacological effects, including anti-inflammatory, anti-allergic, and immunosuppressive properties. Hydrocortisone Sodium Succinate is a salt compound of hydrocortisone, and both are obtained using hydrocortisone compounds as prodrugs. Therefore, impurity control during the synthesis of hydrocortisone compounds is crucial for impurity control in the final products of hydrocortisone for Injection and Hydrocortisone Sodium Succinate for Injection.

[0003] 21-Dehydrocortisol (CAS NO: 14760-49-7, also known as hydrocortisol EP impurity G) is a Class 3 reproductive toxicity compound with a warning structure. During the synthesis of hydrocortisol and the storage of the injection solution, it will appear as process impurities and degradation impurities due to oxidative degradation.

[0004] Because 21-aldehyde hydrocortisone has reproductive toxicity, to ensure patient safety, the European Pharmacopoeia stipulates that the content of 21-aldehyde hydrocortisone in hydrocortisone raw materials must not exceed 0.4%, and discloses a high-performance liquid chromatography (HPLC) method for detecting hydrocortisone-related substances. However, this method can only be used to detect the content of 21-aldehyde hydrocortisone in hydrocortisone; it is not applicable to other compounds or pharmaceutical preparations using hydrocortisone as a prodrug (such as hydrocortisone sodium succinate for injection). Although the Chinese Pharmacopoeia mentions an HPLC method for the detection of hydrocortisone sodium succinate, this method does not involve the detection of 21-aldehyde hydrocortisone, and the elution method used is isocratic elution, which cannot effectively separate trace amounts of related substances. Therefore, to date, there is no universal detection method suitable for detecting the content of 21-aldehyde hydrocortisone in hydrocortisone and other compounds or pharmaceutical preparations using hydrocortisone as a prodrug. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a universal method for detecting the content of 21-aldehyde hydrocortisone. This method is applicable not only to the detection of the content of 21-aldehyde hydrocortisone in hydrocortisone and hydrocortisone for injection, but also to the detection of the content of 21-aldehyde hydrocortisone in other compounds or pharmaceutical preparations (such as hydrocortisone sodium succinate for injection) using hydrocortisone as a prodrug.

[0006] Specifically, the detection method for 21-aldehyde hydrocortisone provided by this invention includes the following steps:

[0007] S1. Preparation of solvent a:

[0008] Solution a is prepared using 0.2% phosphoric acid solution and acetonitrile solution. The 0.2% phosphoric acid solution and acetonitrile are mixed at a volume ratio of 60:40 (60:40, V / V) to obtain solvent a, which is then set aside.

[0009] S2. Prepare the reference solution (0.5 μg / ml) :

[0010] Take an appropriate amount of reference standard Hydrocortisone Impurity 40 (i.e., 21-aldehyde hydrocortisone, CAS No: 14760-49-7), add an appropriate amount of solvent a to dissolve it, and prepare a reference standard solution with a concentration of 0.5 μg / ml.

[0011] S3. Prepare the sample solution to be tested (10 mg / ml) :

[0012] Take an appropriate amount of the sample to be tested, add an appropriate amount of solvent a, and prepare a sample solution with a concentration of 10 mg / ml (the concentration is based on the actual hydrocortisone content).

[0013] S4, Determination by high performance liquid chromatography :

[0014] High-performance liquid chromatography (HPLC) was performed on the blank solvent (solvent a), the reference solution, and the test sample solution, respectively.

[0015] The chromatographic detection conditions are as follows: instrument: Thermo U3000 Chromatographic column: Thermo Hypersil BDS C18, 4.6×250mm, 5μm Detection wavelength: 242nm Flow rate: 1.0 ml / min Column temperature: 25℃ Injection volume: 20μl Mobile phase A: 0.1% phosphoric acid solution - acetonitrile (80:20) Mobile phase B: 0.1% phosphoric acid solution - acetonitrile (30:70) Washing method: Gradient elution

[0016] The elution gradient is as follows: Time (minutes) 0 10 25 28 32 32.1 40 Mobile phase A (%) 100 100 72 0 0 100 100 Mobile phase B (%) 0 0 28 100 100 0 0

[0017] S5, Calculation

[0018] Record the peak area of ​​21-aldehyde hydrocortisone in each solution, and calculate the content of 21-aldehyde hydrocortisone in the sample according to the following formula:

[0019] In the formula:

[0020] A T The peak area of ​​21-aldehyde hydrocortisone in the chromatogram of the sample solution to be tested;

[0021] A S The peak area of ​​21-aldehyde hydrocortisone in the chromatogram of the reference solution;

[0022] W S The sample weight (mg) of the reference standard;

[0023] P represents the purity of the reference standard;

[0024] M is the weight (mg) of hydrocortisone in the sample to be tested.

[0025] Furthermore, S2 includes the following steps: accurately weigh approximately 2 mg of Hydrocortisone Impurity 40, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with solvent a, and shake well to obtain the reference stock solution; take 0.25 ml of the reference stock solution, place it in a 100 ml volumetric flask, dilute to the mark with solvent a, and shake well to obtain the reference solution.

[0026] Furthermore, the test sample in S3 is hydrocortisone and other compounds or pharmaceutical preparations using hydrocortisone as a prodrug.

[0027] In some preferred embodiments, the test sample is selected from hydrocortisone, hydrocortisone sodium succinate, or pharmaceutical preparations thereof.

[0028] In some other preferred embodiments, the test sample is selected from hydrocortisone injection or hydrocortisone sodium succinate for injection.

[0029] Furthermore, S3 includes the following steps: take an appropriate amount of the sample to be tested, dissolve it in solvent a and transfer it to a 10ml volumetric flask, dilute it to the mark with solvent a, shake well, and prepare a sample solution with a concentration of 10mg / ml (concentration is expressed as the labeled amount of hydrocortisone).

[0030] When the sample to be tested is hydrocortisone, the concentration of the sample solution involved in step S3 can be directly calculated using the mass of hydrocortisone as the indicated amount.

[0031] When the sample to be tested is another compound or pharmaceutical preparation with hydrocortisone as a prodrug (e.g., hydrocortisone sodium succinate, hydrocortisone injection, hydrocortisone sodium succinate for injection), the concentration of the sample solution involved in step S3 should be calculated based on the actual weight of hydrocortisone in the sample to be tested. That is, the concentration of the sample solution (10 mg / ml) in step S3 refers to the concentration of hydrocortisone in the sample to be tested, not the concentration of the sample to be tested.

[0032] Similarly, when the sample to be tested is hydrocortisone, M in the above-mentioned formula for calculating the content of 21-aldehyde hydrocortisone is directly the weight of hydrocortisone.

[0033] When the sample to be tested is another compound or pharmaceutical preparation with hydrocortisone as a prodrug (e.g., hydrocortisone sodium succinate, hydrocortisone injection, hydrocortisone sodium succinate for injection), the M in the above-mentioned formula for calculating the content of 21-aldehyde hydrocortisone needs to be calculated according to the actual weight of hydrocortisone in the sample to be tested. That is, the M in the above-mentioned formula for calculating the content of 21-aldehyde hydrocortisone represents the actual weight of hydrocortisone in the sample to be tested, rather than the weight of the sample to be tested.

[0034] Understandably, to ensure data accuracy, multiple-needle injection is used when injecting the reference solution and the test sample solution. Therefore, A in the above-mentioned formula for calculating the content of 21-aldehyde hydrocortisone... T The average peak area of ​​21-aldehyde hydrocortisone in the chromatogram of the sample solution can be used; similarly, A in the above-mentioned formula for calculating the content of 21-aldehyde hydrocortisone can be used. s It can be the average peak area of ​​21-aldehyde hydrocortisone in the chromatogram of the reference solution.

[0035] In summary, the 21-aldehyde hydrocortisone content detection method provided by this invention has good system applicability and detection stability. It is not only applicable to the detection of 21-aldehyde hydrocortisone content in hydrocortisone and hydrocortisone for injection, but also applicable to the detection of 21-aldehyde hydrocortisone content in other compounds or pharmaceutical preparations using hydrocortisone as a prodrug (such as hydrocortisone sodium succinate for injection). It has good inter-product versatility and extremely high detection accuracy, achieving a limit of quantitation of 0.00074%, enabling precise detection of trace amounts of 21-aldehyde hydrocortisone in products. Therefore, the 21-aldehyde hydrocortisone content detection method provided by this invention has good versatility and excellent detection accuracy, enabling better control of drug safety and ensuring patient medication safety. Attached Figure Description

[0036] Figure 1 Here is the liquid chromatogram of the blank solvent in Example 1;

[0037] Figure 2 This is the liquid chromatogram of the first injection of the reference solution in Example 1;

[0038] Figure 3 This is the liquid chromatogram of the second injection of the reference solution from Example 1;

[0039] Figure 4 This is the chromatogram of the third injection of the reference solution from Example 1;

[0040] Figure 5 This is the chromatogram of the fourth injection of the reference solution from Example 1;

[0041] Figure 6 This is the chromatogram of the fifth injection of the reference solution from Example 1;

[0042] Figure 7 Here is the liquid chromatogram of the sample solution from Example 1;

[0043] Figure 8 This is a comparison of liquid chromatograms of the limit of quantitation solution in Example 2;

[0044] Figure 9 This is a comparison of liquid chromatography results for Comparative Example 1. Detailed Implementation [definition]

[0045] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein, as they can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0046] The elements of the invention will now be described. These elements are listed together with specific embodiments. However, it should be understood that they can be combined in any manner and in any number to form other embodiments. The various described embodiments and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. This description should be understood to support and cover schemes that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, it should be considered that any permutation and combination of all the elements described in this application is disclosed in the specification of this application unless the context otherwise requires. [Specific Implementation Examples]

[0047] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Example 1: System adaptability verification and 21-aldehyde hydrocortisone in sodium hydrocortisone succinate for injection. Quantitative detection

[0048] S1 prepares solvent a:

[0049] Prepare solvent a with a concentration of 0.2% phosphoric acid solution and acetonitrile (60:40, V / V) for later use.

[0050] S2. Preparation of the reference solution:

[0051] Accurately weigh 2.027 mg of Hydrocortisone Impurity 40 (purchased from China National Standard Pharmaceutical Group Co., Ltd., purity 99.1%), place it in a 10 ml volumetric flask, add solvent a to dissolve and dilute to the mark, shake well to obtain the reference stock solution;

[0052] Take 0.25 ml of the reference standard stock solution, place it in a 100 ml volumetric flask, add solvent a to dilute to the mark, and shake well to obtain the reference standard solution.

[0053] S3. Prepare the sample solution to be tested:

[0054] Take 0.1 g of hydrocortisone sodium succinate for injection, dissolve it in solvent a and transfer it to a 10 ml volumetric flask. Dilute to the mark with solvent a, shake well, and prepare a sample solution with a concentration of 10 mg / ml (concentration is based on the labeled amount of hydrocortisone).

[0055] S4. Determination by high performance liquid chromatography:

[0056] Inject blank solvent a, reference solution, and test sample solution in that order and perform high performance liquid chromatography analysis. Record the peak area of ​​21-aldehyde hydrocortisone in each solution.

[0057] Chromatographic conditions: instrument: Thermo U3000 Chromatographic column: Thermo Hypersil BDS C18, 4.6×250mm, 5μm Detection wavelength: 242nm Flow rate: 1.0 ml / min Column temperature: 25℃ Injection volume: 20μl Mobile phase A: 0.1% phosphoric acid solution - acetonitrile (80:20) Mobile phase B: 0.1% phosphoric acid solution - acetonitrile (30:70) Washing method: Gradient elution

[0058] The elution gradient is as follows: Time (minutes) 0 10 25 28 32 32.1 40 Mobile phase A (%) 100 100 72 0 0 100 100 Mobile phase B (%) 0 0 28 100 100 0 0

[0059] To verify the stability of the testing method provided by this invention, this embodiment performed a 5-needle test on the reference standard, obtaining the peak area of ​​21-aldehyde hydrocortisone in the reference standard solution in 5 separate tests. Figure 1 This is the liquid chromatogram of blank solvent a. Figure 2 This is the liquid chromatogram of the first reference solution. Figure 3This is the liquid chromatogram of the second reference solution. Figure 4 This is the liquid chromatogram of the third reference solution. Figure 5 This is the liquid chromatogram of the fourth reference solution. Figure 6 This is the liquid chromatogram of the fifth reference solution.

[0060] Table 1.5 Peak areas of 21-aldehyde hydrocortisone for reference solutions

[0061] The results are shown in Table 1. As can be seen from Table 1, the peak areas of the reference solution for the five injections were 0.5740, 0.5716, 0.5765, 0.5793, and 0.5758, respectively. The RSD value of the peak area of ​​21-aldehyde hydrocortisone in the reference solution for the five injections was 0.50% (according to the Chinese Pharmacopoeia, the RSD should not exceed 2.0%). This indicates that the detection method provided by the present invention has good system suitability and detection stability.

[0062] This embodiment also used the above method to detect hydrocortisone sodium succinate for injection. The peak area of ​​21-aldehyde hydrocortisone in the obtained hydrocortisone sodium succinate for injection (i.e., the sample to be tested) was 0.2966 (see...). Figure 7 ).

[0063] Calculate the content of 21-aldehyde hydrocortisone in the sample using the external standard method formula:

[0064] In the formula,

[0065] A T In this embodiment, A represents the peak area of ​​21-aldehyde hydrocortisone in the chromatogram of the sample solution to be tested. T =0.2966;

[0066] A S In this example, A represents the peak area of ​​21-aldehyde hydrocortisone in the chromatogram of the reference solution. S The average peak area of ​​21-aldehyde hydrocortisone in the chromatograms of the above 5 reference solutions is A. S =0.57544;

[0067] W S The sample weight (mg) of the reference standard is W in this embodiment. S =2.027mg;

[0068] P represents the purity of the reference standard; in this example, P = 99.1%.

[0069] M represents the weight (mg) of hydrocortisone in the sample to be tested. In this example, the labeled amount of hydrocortisone in the selected sample, hydrocortisone sodium succinate for injection, is 0.1g, i.e., M = 100mg.

[0070] Calculations show that the content of 21-aldehyde hydrocortisone in the injectable hydrocortisone sodium succinate in this embodiment is 0.003%.

[0071] In summary, the detection method provided by this invention has good system applicability and detection stability, and is suitable for detecting the content of 21-aldehyde hydrocortisone in hydrocortisone sodium succinate for injection. Therefore, it can be reasonably inferred that the detection method provided by this application is not only applicable to the detection of the content of 21-aldehyde hydrocortisone in hydrocortisone, but also applicable to the detection of the content of 21-aldehyde hydrocortisone in other compounds or pharmaceutical preparations with hydrocortisone as a prodrug. That is, the detection method provided by this invention has good versatility. Example 2: Limit of Quantitation Validation

[0072] Preparation of limit-of-quantitation solution:

[0073] A 21-aldehyde hydrocortisone solution with a signal-to-noise ratio of approximately 10:1 was prepared for the determination, as follows:

[0074] Preparation of reference stock solution (approximately 200 μg / ml): Accurately weigh 2.000 mg of Hydrocortisone Impurity 40 (purchased from China National Standard Pharmaceutical Group Co., Ltd., purity 99.1%), place it in a 10 ml volumetric flask, add solvent a (see Example 1 for preparation method) to dissolve and dilute to the mark, shake well to obtain the reference stock solution.

[0075] Solution X (approximately 20 μg / ml): Take 5 ml of the reference stock solution, place it in a 50 ml volumetric flask, add solvent a to dilute to the mark, and shake well to obtain the solution.

[0076] Solution Y (approximately 1 μg / ml): Take 0.5 ml of solution X, place it in a 10 ml volumetric flask, add solvent a to dilute to the mark, and shake well to obtain the solution.

[0077] Limit of Quantification Solution: Take 0.75 ml of solution Y, place it in a 10 ml volumetric flask, add solvent a to dilute to the mark, and shake well to obtain the solution.

[0078] Chromatographic conditions: instrument: Thermo U3000 Chromatographic column: Thermo Hypersil BDS C18, 4.6×250mm, 5μm Detection wavelength: 242nm Flow rate: 1.0 ml / min Column temperature: 25℃ Injection volume: 20μl Mobile phase A: 0.1% phosphoric acid solution - acetonitrile (80:20) Mobile phase B: 0.1% phosphoric acid solution - acetonitrile (30:70) Washing method: Gradient elution

[0079] The elution gradient is as follows: Time (minutes) 0 10 25 28 32 32.1 40 Mobile phase A (%) 100 100 72 0 0 100 100 Mobile phase B (%) 0 0 28 100 100 0 0

[0080] Six consecutive injections were performed, and the retention time and peak area of ​​21-aldehyde hydrocortisone were recorded. The RSD of the retention time and peak area were calculated, as well as the limit of quantitation (μg / ml) and the equivalent content (%) in the test sample.

[0081] Table 2 Results of Limit of Quantitation Test

[0082] From Table 2 and Figure 8 It can be seen that after 6 injections, the RSDs of retention time and peak area were 0.03% and 0.62%, respectively, and the limit of quantitation was 0.00074%. This means that the detection method provided in this application can not only detect the content of 21-aldehyde hydrocortisone in the sample, but also has extremely high detection accuracy, achieving a limit of quantitation of 0.00074%, and can accurately detect trace amounts of 21-aldehyde hydrocortisone in the product. Comparative Example 1

[0083] The method for detecting related substances of hydrocortisone in injectable hydrocortisone sodium succinate was adopted according to the European Pharmacopoeia, as follows:

[0084] 1. Solution preparation:

[0085] 1) Diluent: Acetonitrile-water (40:60, V / V)

[0086] 2) Test solution: Weigh 50 mg of hydrocortisone sodium succinate for injection lyophilized powder, dissolve and dilute with diluent to 10 ml, and shake well.

[0087] 3) Reference stock solution (200 μg / ml): Take 2 mg of Hydrocortisone Impurity 40 (purchased from China National Standard Pharmaceutical Group Co., Ltd., purity 99.1%), place it in a 10 ml volumetric flask, add diluent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0088] 4) Reference solution (0.5 μg / ml): Take 0.25 ml of the reference stock solution, place it in a 100 ml volumetric flask, add mobile phase A to dilute to the mark, and shake well to obtain the solution.

[0089] 2. Chromatographic conditions:

[0090] Column: Thermo Acclaim™ 120C18 4.6mm × 250mm, 5μm;

[0091] Mobile phase A: Water

[0092] Mobile phase B: Acetonitrile

[0093] Perform gradient elution according to the table below; flow rate: 0.8 ml / min; column temperature: 40℃; detection wavelength: 254 nm; injection volume: 10 μl.

[0094] See results Figure 9 ,from Figure 9 As can be seen, the detection method for hydrocortisone published in the European Pharmacopoeia is not applicable to the detection of hydrocortisone sodium succinate for injection. Its solution spectrum is messy and cannot effectively detect 21-aldehyde hydrocortisone in hydrocortisone sodium succinate for injection.

[0095] In summary, the 21-aldehyde hydrocortisone content detection method provided by this invention has good system applicability and detection stability. It is not only applicable to the detection of 21-aldehyde hydrocortisone content in hydrocortisone and hydrocortisone for injection, but also applicable to the detection of 21-aldehyde hydrocortisone content in other compounds or pharmaceutical preparations using hydrocortisone as a prodrug (such as hydrocortisone sodium succinate for injection). It has good inter-product versatility and extremely high detection accuracy, achieving a limit of quantitation of 0.00074%, enabling precise detection of trace amounts of 21-aldehyde hydrocortisone in products. Therefore, the 21-aldehyde hydrocortisone content detection method provided by this invention has good versatility and excellent detection accuracy, enabling better control of drug safety.

[0096] The above description represents only preferred embodiments and is provided as an example only, not as a limitation on the combination of features necessary for carrying out the invention. The provided headings are not intended to limit the various embodiments of the invention. Terms such as “comprising,” “including,” and “including” are not intended to be limiting. Furthermore, unless otherwise stated, the plural form is included when not modified by a numeral, and “or” or “or” means “and / or.” Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0097] All disclosures and patents mentioned in this application are incorporated herein by reference. Various modifications and variations of the methods and compositions described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described through specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, various variations of the described modes of carrying out the invention that will be apparent to those skilled in the art are intended to be included within the scope of the appended claims.

Claims

1. A method for detecting 21-aldehydo hydrocortisone, characterized by, The detection method includes the following steps: S1. Preparation of solvent a: Mix 0.2% phosphoric acid solution and acetonitrile at a volume ratio of 60:40 to obtain solvent a, for later use; S2. Preparation of reference solution: Take Hydrocortisone Impurity 40 and solvent a to prepare a reference solution with a concentration of 0.5 μg / ml; S3. Preparation of the test sample solution: Take the test sample and solvent a, and prepare a test sample solution with a concentration of 10 mg / ml (concentration is based on the content of hydrocortisone); S4. High-performance liquid chromatography (HPLC) determination: Solvent a, reference solution, and test sample solution were analyzed by HPLC, respectively. The chromatographic detection conditions are as follows: The gradient elution procedure is as follows: S5. Calculation: Calculate the content of 21-aldehyde hydrocortisone in the sample according to the following formula. In the formula, A T A21 is the peak area of 21-aldehyde hydrocortisone in the chromatogram of the sample solution to be tested; A S The peak area of 21-hydroxyhydrocortisone in the chromatogram of the control solution; W S for control; (mg) P represents the purity of the reference standard; M is the weight (mg) of hydrocortisone in the sample to be tested.

2. The detection method according to claim 1, characterized in that, The test sample mentioned in step S3 is hydrocortisone and other compounds or pharmaceutical preparations with hydrocortisone as a prodrug.

3. The detection method according to claim 2, characterized in that, The test sample mentioned in step S3 is selected from hydrocortisone, hydrocortisone sodium succinate, and pharmaceutical preparations containing hydrocortisone or hydrocortisone sodium succinate.

4. The detection method according to claim 3, characterized in that, The sample to be tested in step S3 is selected from hydrocortisone, hydrocortisone sodium succinate, hydrocortisone for injection, and hydrocortisone sodium succinate for injection.

5. The application of the detection method according to any one of claims 1-4 in the detection of 21-aldehyde hydrocortisone impurities in hydrocortisone or other compounds or pharmaceutical preparations using hydrocortisone as a prodrug.

6. Use according to claim 5, characterized in that, The other compounds or pharmaceutical preparations using hydrocortisone as a prodrug include hydrocortisone sodium succinate, hydrocortisone for injection, and hydrocortisone sodium succinate for injection.

7. The application of the detection method according to any one of claims 1-4 in detecting the content of 21-aldehyde hydrocortisone in hydrocortisone or other compounds or pharmaceutical preparations using hydrocortisone as a prodrug.

8. Use according to claim 7, characterized in that, The other compounds or pharmaceutical preparations using hydrocortisone as a prodrug include hydrocortisone sodium succinate, hydrocortisone for injection, and hydrocortisone sodium succinate for injection.

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