Separation assay for 16 alpha-hydroxyprednisolone and its impurities by HPLC

Abstract

The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a HPLC method for separating and determining 16alpha-hydroxyprednisolone and impurities thereof. 4h , HYR-Z 4d , HYR-Z 4a , HYR-Z 4g , HYR-Z 4e , HYR-Z 4j , HYR-Z 3c , HYR-Z3, HYR-Z 4b , HYR-Z 3e , HYR-Z2, HYR-Z1. The method is linear gradient elution of high performance liquid chromatography with phosphoric acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, and the chromatographic column uses octylsilane bonded silica gel as the filler, and then enters the detector for detection; then according to the chromatogram, the principal component itself is controlled by multiplying the correction factor to calculate the content of each impurity. The method has the characteristics of good resolution, good durability, high sensitivity and good reproducibility.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to an HPLC method for separating and determining 16α-hydroxyprednisolone and its impurities. Background Technology

[0002] Tinodes are inhaled corticosteroids, characterized by local application, low dosage, strong local anti-inflammatory effects, and minimal systemic side effects. They are widely used to treat refractory asthma and inflammation. 16α-Hydroxyprednisolone is an intermediate in the synthesis of glucocorticoid raw materials and is a key intermediate in the synthesis of approved tinodes, including triamcinolone, budesonide, ciroxonide, and desoxind. Using 16α-Hydroxyprednisolone in the synthesis of tinodes can significantly reduce the economic cost of the raw material, offering advantages such as wide applicability and high cost-effectiveness.

[0003] The molecular formula of 16α-hydroxyprednisolone is C 21 H 28 O6 has a molecular weight of 376.4434 and its structural formula is shown in Formula I.

[0004]

[0005] The study found that 16α-hydroxyprednisolone may contain the following 12 impurities: impurity HYR-Z1, impurity HYR-Z2, impurity HYR-Z3, impurity HYR-Z 3c Impurities HYR-Z 3e Impurities HYR-Z 4a Impurities HYR-Z 4b Impurities HYR-Z 4d Impurities HYR-Z 4e Impurities HYR-Z 4g Impurities HYR-Z 4h Impurities HYR-Z 4j To control the quality of 16α-hydroxyprednisolone and subsequent formulations, appropriate analytical methods are needed to detect and identify these 12 impurities.

[0006] Existing literature and patent information report numerous methods for synthesizing 16α-hydroxyprednisolone, but analytical methods for impurities in 16α-hydroxyprednisolone are rarely reported. The document "Determination of 16α-hydroxyprednisolone in Cosmetics" provides an official detection method for the illegal addition of 16α-hydroxyprednisolone to creams, lotions, and liquid (water) cosmetics; however, this method cannot separate and determine impurities in 16α-hydroxyprednisolone.

[0007] Therefore, there is an urgent need to establish a sensitive, accurate, simple and efficient method to separate and determine 16α-hydroxyprednisolone and its related impurities. Summary of the Invention

[0008] In view of this, one of the objectives of the present invention is to provide a method for separating 16α-hydroxyprednisolone and its impurities based on high performance liquid chromatography, which can complete the separation of multiple substances in a short time.

[0009] To achieve the above objectives, the technical solution of the present invention is as follows:

[0010] A method for separating 16α-hydroxyprednisolone and its impurities using high-performance liquid chromatography, wherein the 16α-hydroxyprednisolone and the impurities together constitute a composition, and the impurities include impurity HYR-Z. 4h Impurities HYR-Z 4d Impurities HYR-Z 4a Impurities HYR-Z 4g Impurities HYR-Z 4e Impurities HYR-Z 4j Impurities HYR-Z 3c Impurity HYR-Z3, Impurity HYR-Z 4b Impurities HYR-Z 3e Any one or more of the following: impurity HYR-Z2, impurity HYR-Z1; the structural formula of each component in the composition is as follows:

[0011]

[0012] In the high-performance liquid chromatography method, the mobile phase is: mobile phase A is an aqueous solution of phosphoric acid, and mobile phase B is acetonitrile; the stationary phase is: the chromatographic column uses octylsilane-bonded silica gel as the packing material; 16α-hydroxyprednisolone and its impurities are separated by linear gradient elution.

[0013] After separation and testing, it is used for the next step of production.

[0014] The aforementioned impurities can be arranged and combined in various ways. For example, combination 1: impurity HYR-Z 4h Impurities HYR-Z 4d For example, combination method 2: impurity HYR-Z 4g Impurities HYR-Z 4e Impurities HYR-Z 4j For example, combination method 3: impurity HYR-Z 4b Impurities HYR-Z 3e Impurity HYR-Z2, Impurity HYR-Z1.

[0015] All possible permutations and combinations will not be listed here. Theoretically, when the upper limit of the substances that this method can separate, identify and / or detect is n (where n is the number of substances), it can naturally detect 1 to n substances.

[0016] As a preferred embodiment, the linear gradient elution procedure is as follows:

[0017] Time - minutes Mobile phase A - (volume parts) Mobile phase B - (volume parts) 0 85±10 15±10 25±1 85±10 15±10 50±1 40±10 60±10 55±1 20±10 80±10 60±1 20±10 80±10 61±1 85±10 15±10 70±1 85±10 15±10 .

[0018] Preferably, the linear gradient elution procedure is as follows:

[0019] Time - minutes Mobile phase A - (volume parts) Mobile phase B - (volume parts) 0 84-86 14-16 25 84-86 14-16 50 38-42 58-62 55 20 80 60 20 80 61 84-86 14-16 70 84-86 14-16 .

[0020] For example, at 0, 25, 61, and 70 minutes, mobile phase A accounted for 86 parts, and mobile phase B accounted for 14 parts. Figure 11 ); or at 0, 25, 61, and 70 minutes, mobile phase A accounts for 85 parts and mobile phase B accounts for 15 parts. Figure 3 ); or at 0, 25, 61, and 70 minutes, mobile phase A accounts for 84 parts and mobile phase B accounts for 16 parts. Figure 12 For example, at 50 minutes, mobile phase A accounted for 42 parts and mobile phase B accounted for 58 parts. Figure 13 ); or at 50 minutes, mobile phase A accounts for 41 parts and mobile phase B accounts for 59 parts; or at 50 minutes, mobile phase A accounts for 40 parts and mobile phase B accounts for 60 parts. Figure 3 ); or at 50 minutes, mobile phase A accounts for 39 parts and mobile phase B accounts for 61 parts; or at 50 minutes, mobile phase A accounts for 38 parts and mobile phase B accounts for 62 parts. Figure 14 ).

[0021] As the preferred option, the linear gradient elution procedure is as follows:

[0022] Time - minutes Mobile phase A - (volume parts) Mobile phase B - (volume parts) 0 85 15 25 85 15 50 40 60 55 20 80 60 20 80 61 85 15 70 85 15 .

[0023] Furthermore, the flow rate was 0.7-1.3 mL / min; the column temperature was 15-30℃.

[0024] Preferably, the flow rate is 0.9-1.1 mL / min; the column temperature is 18-22 °C. For example, a flow rate of 0.9 mL / min ( Figure 7 ); or a flow rate of 1.0 mL / min ( Figure 3 ); or a flow rate of 1.1 mL / min ( Figure 8 For example, the column temperature is 18℃. Figure 9 ); or column temperature of 19℃; or column temperature of 20℃. Figure 3 ); or column temperature of 21℃; or column temperature of 22℃ Figure 10 ).

[0025] The optimal flow rate is 1.0 mL / min, and the column temperature is 20 °C.

[0026] Preferably, the concentration of the phosphoric acid aqueous solution in the mobile phase A is 0.1%.

[0027] Preferably, the chromatographic column has dimensions of 4.6 mm × 150 mm and a diameter of 2.6 μm.

[0028] As the preferred option, the chromatographic column is a phenomenex Kinetex C8, 4.6 mm × 150 mm, 2.6 μm.

[0029] Preferably, the injection volume is 10 μl.

[0030] Preferably, the method uses ghost peaks.

[0031] As a preferred option, the running time is 70 minutes.

[0032] Furthermore, the sample was prepared using a mixed solution of mobile phase A and acetonitrile, wherein the volume ratio of mobile phase A to acetonitrile was 70:30.

[0033] Preferably, before separation, the test solution is prepared using a sample preparation solvent; the test solution includes a sample solution, a control solution, and / or a system suitability solution.

[0034] As a preferred method, the sample solution is prepared by accurately weighing an appropriate amount of 16α-hydroxyprednisolone, adding solvent, sonicating to dissolve and diluting it to prepare a solution containing approximately 1 mg per 1 ml.

[0035] As a preferred method, the preparation of the control solution is as follows: accurately measure 2 ml of the sample solution, place it in a 100 ml volumetric flask, dilute it to the mark with solvent, and shake well; accurately measure 1 ml of the sample solution, place it in a 10 ml volumetric flask, dilute it to the mark with solvent, and shake well.

[0036] As a preferred method, the preparation method of the system suitability solution is as follows:

[0037] Preparation of system suitability solution: Take 16α-hydroxyprednisolone system suitability reference standard (containing 16α-hydroxyprednisolone, impurity HYR-Z3, and impurity HYR-Z) 4d Impurities HYR-Z 4h Impurities HYR-Z 4j Add an appropriate amount of solvent, sonicate to dissolve and dilute to prepare a solution containing approximately 1 mg per 1 ml.

[0038] Preparation of System Suitability Solution 1: Take 16α-hydroxyprednisolone system suitability reference standard 1 (containing 16α-hydroxyprednisolone and impurity HYR-Z) 4a Impurities HYR-Z 4g Impurities HYR-Z 4eImpurities HYR-Z 4b Impurities HYR-Z 3c Impurities HYR-Z 3e Add appropriate amounts of impurities HYR-Z2 and HYR-Z1, dissolve and dilute with solvent using ultrasound to prepare a solution containing approximately 1 mg per 1 ml.

[0039] The second objective of this invention is to provide a method for identifying 16α-hydroxyprednisolone and its impurities, which can complete the identification of multiple substances in a short time.

[0040] To achieve the above objectives, the technical solution of the present invention is as follows:

[0041] A method for identifying 16α-hydroxyprednisolone and its impurities involves separating the composition using the aforementioned method and detecting it in a detector to obtain a chromatogram; by comparing the chromatographic characteristics of the test sample and the reference sample, it is determined whether the test sample contains 16α-hydroxyprednisolone and its impurities.

[0042] As a preferred embodiment, the detection wavelength of the detector is 245 ± 10 nm. The ± 10 nm setting range is based on a comprehensive consideration of factors such as error tolerance, methodological superiority, and practical application requirements. This setting range helps ensure the reliability of the detection results, improves the repeatability and flexibility of the measurement, and meets the requirements of specific experiments.

[0043] As the most preferred option, the detection wavelength of the detector is 245 nm.

[0044] As a preferred embodiment, the components in the composition can be identified according to their relative retention times, wherein the components of the composition, in ascending order, are: impurity HYR-Z. 4h Impurities HYR-Z 4d Impurities HYR-Z 4a 16α-hydroxyprednisolone, impurity HYR-Z 4g Impurities HYR-Z 4e Impurities HYR-Z 4j Impurities HYR-Z 3c Impurity HYR-Z3, Impurity HYR-Z 4b Impurities HYR-Z 3e Impurity HYR-Z2, Impurity HYR-Z1.

[0045] Relative retention time (R0) typically describes the relative retention of different components in a mixture on a chromatographic column. It is the ratio of the retention time of a particular component to the retention time of a reference component (usually the main peak or a known component). This ratio reflects the relative retention performance of different components on the column and is an important parameter used for localization, qualitative, and quantitative analysis in chromatographic analysis. R0 is calculated by dividing the retention time of the target component (tR) by the retention time of the reference component (tR0).

[0046] As a preferred method, 16α-hydroxyprednisolone was used as a reference peak; the relative retention time was 0.38, which was determined to be the impurity HYR-Z. 4h The relative retention time was 0.64, indicating it was an impurity (HYR-Z). 4d The relative retention time was 0.96, indicating it was an impurity (HYR-Z). 4a The relative retention time was 1.09, indicating it was an impurity, HYR-Z. 4g The relative retention time was 1.24, indicating it was an impurity (HYR-Z). 4e The relative retention time was 1.45, indicating it was an impurity (HYR-Z). 4j The relative retention time was 1.52, indicating it was an impurity (HYR-Z). 3c The relative retention time was 1.59, indicating it was impurity HYR-Z3; the relative retention time was 1.65, indicating it was impurity HYR-Z. 4b The relative retention time was 1.67, indicating it was an impurity, HYR-Z. 3e The relative retention time was 1.69, which was identified as impurity HYR-Z2; the relative retention time was 1.75, which was identified as impurity HYR-Z1; the relative retention times of each component fluctuated within ±20%.

[0047] Besides relative retention time, retention time can also be used to identify components. Retention time refers to the time required for a sample to travel from entering the chromatographic column to being detected by the detector. This time is calculated based on the migration speed of the component on the chromatographic column, that is, the time interval from the start of injection to the chromatographic peak (maximum concentration) of a certain component. It is mainly used to determine the elution order and position of each component in the sample and is one of the basic data in chromatographic analysis. In quality control, changes in retention time can reflect factors such as the state of the chromatographic column, the stability of the mobile phase, and the performance of the instrument.

[0048] The third objective of this invention is to provide a method for determining the content of 16α-hydroxyprednisolone and its impurities, which can complete the identification and content determination of multiple substances in a short time.

[0049] To achieve the above objectives, the technical solution of the present invention is as follows:

[0050] A method for determining the content of 16α-hydroxyprednisolone and its impurities is described above, in which 16α-hydroxyprednisolone and its impurities are separated and identified to obtain chromatograms; based on the obtained chromatograms, the content of each impurity is calculated using the principal component self-comparison method multiplied by a correction factor.

[0051] Content determination can further be used to determine whether the content of 16α-hydroxyprednisolone and its impurities is up to standard. If impurities HYR-Z1, HYR-Z2, HYR-Z3, and HYR-Z are present... 3c Impurities HYR-Z 3e Impurities HYR-Z 4a Impurities HYR-Z 4b Impurities HYR-Z 4d Impurities HYR-Z 4e Impurities HYR-Z 4g Impurities HYR-Z4 h Impurities HYR-Z 4j If the peak area of ​​any one or more impurities in the solution is larger than the peak area of ​​the corresponding impurity in the control solution, it indicates that the impurity content is unqualified; conversely, if the peak area of ​​impurity HYR-Z1, impurity HYR-Z2, impurity HYR-Z3, or impurity HYR-Z... 3c Impurities HYR-Z 3e Impurities HYR-Z 4a Impurities HYR-Z 4b Impurities HYR-Z 4d Impurities HYR-Z 4e Impurities HYR-Z 4g Impurities HYR-Z4 h Impurities HYR-Z 4j If the peak area of ​​any one or more impurities in the solution is not greater than the peak area of ​​the corresponding impurity in the control solution, it indicates that the impurity content is qualified.

[0052] The aforementioned judgment method can serve as a drug quality assessment model and further as an indispensable key module in intelligent production processes. This model, through precise control of parameters such as mobile phase composition, flow rate, and column temperature, achieves accurate separation and quantitative analysis of active ingredients, impurities, and degradation products in drugs, providing a scientific basis for comprehensive drug quality assessment. In intelligent production systems, this model is seamlessly integrated, capable of receiving raw data from the production line in real time, automatically executing analysis tasks, and rapidly providing judgment results based on preset quality standards.

[0053] As a preferred embodiment, the impurity HYR-Z 4h The correction factor is 1.1; the impurity HYR-Z 4d The correction factor is 1.0; the impurity HYR-Z4j The correction factor is 1.0; the correction factor for impurity HYR-Z3 is 1.2; the correction factor for impurity HYR-Z 4a The correction factor is 1.3; the impurity HYR-Z 4g The correction factor is 1.0; the impurity HYR-Z 4e The correction factor is 1.0; the impurity HYR-Z 3c The correction factor is 1.2; the impurity HYR-Z 4b The correction factor is 0.61; the impurity HYR-Z 3e The correction factor for the impurity is 1.3; the correction factor for the impurity HYR-Z2 is 1.7; and the correction factor for the impurity HYR-Z1 is 1.0.

[0054] As a preferred option, the formula for calculating the impurity content is as follows:

[0055]

[0056] Among them, A 杂质 The area of ​​the impurity peak;

[0057] A 对照 This represents the peak area of ​​its own control solution;

[0058] f is a known impurity correction factor.

[0059] The beneficial effects of this invention are as follows:

[0060] This invention provides a method for separating and determining 16α-hydroxyprednisolone and its impurities based on high-performance liquid chromatography (HPLC). The impurities include impurity HYR-Z1, impurity HYR-Z2, impurity HYR-Z3, and impurity HYR-Z. 3c Impurities HYR-Z 3e Impurities HYR-Z 4a Impurities HYR-Z 4b Impurities HYR-Z 4d Impurities HYR-Z 4e Impurities HYR-Z 4g Impurities HYR-Z 4h Impurities HYR-Z 4jThis method can effectively separate 16α-hydroxyprednisolone and its 12 impurities within 70 minutes, exhibiting short separation time and good separation effect. Verification through limit of quantitation, limit of detection, and robustness experiments with chromatographic conditions demonstrates that the method of this invention is highly specific, sensitive, and robust, possessing advantages such as good reproducibility, strong practicality, simple and feasible operation, and accurate and reliable detection results. This invention solves the problem of separating and determining 16α-hydroxyprednisolone and its 12 known impurities that has not been addressed by existing technologies, and is of great significance for achieving quality control of 16α-hydroxyprednisolone finished products. Attached Figure Description

[0061] Figure 1 The chromatogram is for a blank solution;

[0062] Figure 2 The chromatogram of the test solution;

[0063] Figure 3 The chromatogram is of the mixed solution;

[0064] Figure 4 The chromatogram is for the control solution;

[0065] Figure 5 The chromatogram is for the solution at the limit of quantitation.

[0066] Figure 6 Chromatogram of the solution at the detection limit;

[0067] Figure 7 The chromatogram is of the mixed solution under the condition that the mobile phase flow rate is 0.9 ml / min in the durability test;

[0068] Figure 8 The chromatogram of the mixed solution under the condition of a mobile phase flow rate of 1.1 ml / min in the durability test;

[0069] Figure 9 The chromatogram of the mixed solution was obtained under the condition of column temperature of 18℃ during the durability test.

[0070] Figure 10 The chromatogram of the mixed solution was obtained under the condition of column temperature of 22℃ during the durability test.

[0071] Figure 11 The chromatogram of the mixed solution under the condition of an initial mobile phase ratio of 86:14 during the durability test;

[0072] Figure 12 The chromatogram of the mixed solution under the condition of an initial mobile phase ratio of 84:16 during the durability test;

[0073] Figure 13 The chromatogram of the mixed solution under the mobile phase gradient time of 50→58 during the durability test;

[0074] Figure 14 The chromatogram of the mixed solution under the mobile phase gradient time of 50→62 during the durability test;

[0075] Figure 15 This is a chromatogram of a mixed solution under different column conditions during a durability test. Detailed Implementation

[0076] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0077] Supplementary tables to the accompanying drawings in the specification. Included in this patent. Figures 1-15 Visual aids are provided for understanding and interpretation. If anything is unclear, users should refer to the corresponding numbered tables (Tables 1-15) for more detailed information. Conversely, when reviewing Tables 1-... Figure 15 If any information that may cause misunderstanding or ambiguity is found during the process, the content of the corresponding numbered figure shall prevail. The above guidelines are intended to ensure the correct interpretation of this document and the consistency of its information. Although some text overlaps in the spectra of this application, it is still clearly visible, and the specification details the integration results of each figure. Furthermore, the numbers in the spectra do not affect the scope of protection of the claims or the full disclosure of the technical solutions in the specification.

[0078] Table 1

[0079]

[0080] Table 2

[0081]

[0082] Table 3

[0083]

[0084] Table 4

[0085]

[0086] Table 5

[0087]

[0088] Table 6

[0089]

[0090] Table 7

[0091]

[0092] Table 8

[0093]

[0094] Table 9

[0095]

[0096] Table 10

[0097]

[0098] Table 11

[0099]

[0100] Table 12

[0101]

[0102] Table 13

[0103]

[0104] Table 14

[0105]

[0106] Table 15

[0107]

[0108] In Table 2 above, peak number 1 is named impurity HYR-Z. 4h Peak number 2 is named an unknown impurity, and peak number 3 is named impurity HYR-Z. 4d Peak number 4 is named 16α-hydroxyprednisolone, and peak number 5 is named impurity HYR-Z. 4j Peak number 6 is named impurity HYR-Z3.

[0109] In Table 4 above, peak number 1 is named 16α-hydroxyprednisolone.

[0110] In Tables 5 and 6 above, the name of peak number 1 is impurity HYR-Z. 4h The names of the two peaks are both impurity HYR-Z. 4d The names of the three peaks are all impurity HYR-Z. 4a Peak number 4 was named 16α-hydroxyprednisolone, and peak number 5 was named impurity HYR-Z. 4gThe names of the six peaks are all impurity HYR-Z. 4e The names of the seven peaks are all impurity HYR-Z. 4j The names of the eight peaks are all impurity HYR-Z. 3c Peak number 9 is named after impurity HYR-Z3, and peak number 10 is named after impurity HYR-Z. 4b The name of peak number 11 is impurity HYR-Z. 3e Peak number 12 is named as impurity HYR-Z2, and peak number 13 is named as impurity HYR-Z1.

[0111] In Tables 3 and 7 through 15 above, the name of peak number 1 is always impurity HYR-Z. 4h Peak number 2 is named as an unknown impurity, and peak number 3 is named as impurity HYR-Z. 4d The fourth peak is named after the impurity HYR-Z. 4a Peak number 5 was named 16α-hydroxyprednisolone, and peak number 6 was named impurity HYR-Z. 4g The names of the seven peaks are all impurity HYR-Z. 4e The names of the eight peaks are all impurity HYR-Z. 4j The names of the nine peaks are all impurity HYR-Z. 3c Peak number 10 is named after impurity HYR-Z3, and peak number 11 is named after impurity HYR-Z. 4b The names of the 12th peak are all impurity HYR-Z. 3e Peak number 13 is named as impurity HYR-Z2, and peak number 14 is named as impurity HYR-Z1.

[0112] To enhance understanding of the present invention, certain key technologies and scientific terms will be clearly defined below. Unless specifically defined herein, all other technical and scientific terms shall follow their generally accepted and understood meanings within the art to which this invention pertains. It should be emphasized that the scope of the present invention is not limited to the specific methods, reagents, compounds, compositions, reference standards, and test items described, but allows for reasonable variations and adjustments in these aspects. Furthermore, please understand that the terminology used herein is intended to illustrate specific embodiments and not to impose a limiting interpretation.

[0113] Furthermore, all references cited in this document, including but not limited to patents, patent applications, academic papers, textbooks, and further citations therein, are considered to be incorporated into this document in their entirety through citation, unless directly cited, as a reference. If there are any inconsistencies or conflicts between the content of these cited references or similar materials and this application, particularly regarding terminology definitions, usage, or technical descriptions, the content of this application shall prevail.

[0114] If any chromatographic conditions are not mentioned, refer to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, 0512) for determination.

[0115] the term

[0116] The limit of quantitation (LOQ) is the lowest amount of an analyte in a sample that can be quantitatively determined, and the measurement result should have a certain degree of accuracy and precision. In other words, the LQ is the lowest level at which an analytical method can accurately and reliably determine the concentration of the analyte in a sample. In HPLC, the determination of the LQ usually relies on the signal-to-noise ratio (S / N) method, that is, the concentration of the analyte corresponding to a certain level of signal-to-noise ratio is taken as the LQ. Determining the LQ is crucial for ensuring the accuracy and reliability of analytical results.

[0117] Chromatographic robustness refers to the ability of a chromatographic analysis system to maintain stable analytical performance and unaffected analytical results when measurement conditions are slightly changed. This robustness is crucial for ensuring the reliability, repeatability, and stability of analytical results.

[0118] The limit of detection (LOD) is the lowest concentration or amount of an analyte in a sample that can be detected. It reflects the sensitivity and noise level of the analytical method and instrument, and also indicates the level of the blank (background) value after sample processing.

[0119] A correction factor is a coefficient or parameter used to correct analytical results. It aims to improve data accuracy and reliability. In HPLC analysis, because the same detector responds differently to different substances, peak areas produced when the same mass of different substances passes through the detector may not be equal. To ensure that the peak area accurately reflects the content of the analyte, standard substances are used for correction, a correction factor is calculated, and this factor is applied to the measurement results of the sample.

[0120] The peak height to noise ratio (S / N, or signal-to-noise ratio) is used in high-performance liquid chromatography (HPLC) to evaluate the detection sensitivity and resolution of an instrument, and is an important indicator of instrument performance. Peak height refers to the signal value output by the detector when the analyte elutes from the column; noise refers to the fluctuation of the baseline signal, i.e., the signal value measured for a blank sample. The signal-to-noise ratio is the ratio of the signal measured for a sample of known concentration to the signal measured for a blank sample. A higher signal-to-noise ratio means that the instrument can more accurately separate and identify the target component when detecting samples, while also reducing interference from background noise.

[0121] Solvent peaks are the elution peaks of the diluent in the chromatographic system. Gradient elution peaks are abnormal peaks that appear per unit time due to changes in the elution ratio of the mobile phase. These two types of chromatographic peaks are collectively referred to as solvent peaks and gradient elution peaks in analytical quality standards.

[0122] In the table of this invention, "——" represents none.

[0123] In this embodiment of the invention, information on 16α-hydroxyprednisolone and its related impurities is shown in Table A.

[0124] Table A. Compound Information Table

[0125]

[0126]

[0127] In this embodiment of the invention, the preparation of a 0.1% phosphoric acid aqueous solution (mobile phase A) is as follows: 1 ml of phosphoric acid is measured, added to 1000 ml of water, and mixed well to obtain the solution.

[0128] Example 1.16 Method for the separation and determination of α-hydroxyprednisolone and related substances

[0129] (1) Preparation of the test solution

[0130] Solvent: A mixed solution of mobile phase A and acetonitrile, wherein the volume ratio of mobile phase A to acetonitrile is 70:30.

[0131] Test solution: Take an appropriate amount of this product (16α-hydroxyprednisolone), accurately weigh it, add solvent, sonicate to dissolve and dilute to prepare a solution containing about 1 mg per 1 ml.

[0132] Control solution: Accurately measure 2 ml of the test solution and place it in a 100 ml volumetric flask. Dilute to the mark with solvent and shake well. Accurately measure 1 ml of the test solution and place it in a 10 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0133] System suitability solution: Take 16α-hydroxyprednisolone system suitability reference standard (containing 16α-hydroxyprednisolone, impurity HYR-Z3, and impurity HYR-Z) 4d Impurities HYR-Z 4h Impurities HYR-Z 4j Add an appropriate amount of solvent, sonicate to dissolve and dilute to prepare a solution containing approximately 1 mg per 1 ml.

[0134] System suitability solution 1: Take 16α-hydroxyprednisolone system suitability reference standard 1 (containing 16α-hydroxyprednisolone and impurity HYR-Z). 4a Impurities HYR-Z 4g Impurities HYR-Z 4e Impurities HYR-Z 4b Impurities HYR-Z 3c Impurities HYR-Z 3eAdd appropriate amounts of impurities HYR-Z2 and HYR-Z1, dissolve and dilute with solvent using ultrasound to prepare a solution containing approximately 1 mg per 1 ml.

[0135] (2) Chromatographic conditions

[0136] Octylsilane-bonded silica gel was used as the stationary phase (Phenomenex Kinetex C8, 4.6 mm × 150 mm, 2.6 μm, or a column with equivalent performance). A 0.1% aqueous phosphoric acid solution was used as mobile phase A, and acetonitrile as mobile phase B. Linear gradient elution was performed according to Table B. The flow rate was 1.0 mL / min; the column temperature was 20 °C; the detection wavelength was 245 nm; the injection volume was 10 μL; and a ghost peak column was used.

[0137] Table B. Gradient Elution Program Table

[0138]

[0139]

[0140] (3) Detection

[0141] Accurately measure the system suitability solution, system suitability solution 1, test solution and control solution, and inject them into the liquid chromatograph in sequence, and record the chromatograms.

[0142] System suitability requirements: In the system suitability solution chromatogram, the impurity HYR-Z is present. 4h Impurities HYR-Z 4d 16α-hydroxyprednisolone, impurity HYR-Z 4j Impurity HYR-Z3 eluted sequentially, and the 16α-hydroxyprednisolone peak was adjacent to that of impurity HYR-Z. 4d The resolution between peaks should meet the requirements. In the chromatogram of solution 1, the 16α-hydroxyprednisolone peak is adjacent to the impurity HYR-Z. 4g The separation between peaks should meet the requirements.

[0143] Limits: In the chromatogram of the test solution, excluding solvent peaks and gradient elution peaks, the limit is calculated using the principal component self-comparison method multiplied by a correction factor. The chromatogram of the control solution is shown below. Figure 4 As shown. The content of each impurity should comply with the limits specified in Tables C and D, and the total amount of impurities should not exceed 1.0%. Impurity peaks with a content of less than 0.05% are negligible.

[0144] The formula for calculating impurity content is as follows:

[0145]

[0146] In the above formula, A 杂质 Area of ​​the impurity peak; A 对照is the peak area of ​​the self-reference solution; f is the correction factor for known impurities.

[0147] Table C. Impurity Limits Table 1

[0148] Principal components and impurities Relative retention time Correction factor Impurity limit Impurity HYR-Z 4h ]] 0.38 1.1 0.15％ Impurity HYR-Z 4d ]]> 0.64 1.0 0.15％ 16 alpha-hydroxyprednisolone 1.00 —— —— Impurity HYR-Z 4g ]] 1.10 —— —— Impurity HYR-Z 4j ]]> 1.45 1.0 0.2％ Impurity HYR-Z3 1.59 1.2 0.15％ Other individual impurities —— —— 0.10％

[0149] Note: The relative retention times in the table above are for reference only. Peaks should be located based on the chromatogram of the system suitability solution.

[0150] Table D. Impurity Limits Table 2

[0151]

[0152]

[0153] Example 2. Specificity Experiment

[0154] (1) Preparation of the test solution

[0155] Solvent (blank solution): A mixed solution of mobile phase A and acetonitrile, wherein the volume ratio of mobile phase A to acetonitrile is 70:30.

[0156] Solvent A: A mixed solution of mobile phase A and acetonitrile, wherein the volume ratio of mobile phase A to acetonitrile is 1:1.

[0157] Impurity HYR-Z1 stock solution: Weigh approximately 25 mg of impurity HYR-Z1 reference standard accurately, place it in a 25 ml volumetric flask, add solvent A to dissolve and dilute to the mark, and shake well.

[0158] Impurity HYR-Z2 stock solution: Weigh approximately 25 mg of impurity HYR-Z2 reference standard accurately, place it in a 25 ml volumetric flask, add solvent A to dissolve and dilute to the mark, and shake well.

[0159] Impurity HYR-Z3 stock solution: Weigh approximately 25 mg of impurity HYR-Z3 reference standard accurately, place it in a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, and shake well.

[0160] Impurities HYR-Z 3c Stock solution: Take impurity HYR-Z 3c Weigh approximately 25 mg of the reference standard accurately, place it in a 25 ml volumetric flask, add solvent A to dissolve and dilute to the mark, and shake well.

[0161] Impurities HYR-Z 3e Stock solution: Take impurity HYR-Z 3e Weigh approximately 25 mg of the reference standard accurately, place it in a 25 ml volumetric flask, add solvent A to dissolve and dilute to the mark, and shake well.

[0162] Impurities HYR-Z 4aStock solution: Take impurity HYR-Z 4a Weigh approximately 10 mg of the reference standard accurately, place it in a 10 ml volumetric flask, add solvent A to dissolve and dilute to the mark, and shake well.

[0163] Impurities HYR-Z 4b Stock solution: Take impurity HYR-Z 4b Weigh approximately 25 mg of the reference standard accurately, place it in a 25 ml volumetric flask, add solvent A to dissolve and dilute to the mark, and shake well.

[0164] Impurities HYR-Z 4d Stock solution: Take impurity HYR-Z 4d Weigh approximately 25 mg of the reference standard accurately, place it in a 25 ml volumetric flask, add solvent A to dissolve and dilute to the mark, and shake well.

[0165] Impurities HYR-Z 4e Stock solution: Take impurity HYR-Z 4e Weigh approximately 10 mg of the reference standard accurately, place it in a 10 ml volumetric flask, add solvent A to dissolve and dilute to the mark, and shake well.

[0166] Impurities HYR-Z 4g Stock solution: Take impurity HYR-Z 4g Weigh approximately 25 mg of the reference standard accurately, place it in a 25 ml volumetric flask, add solvent A to dissolve and dilute to the mark, and shake well.

[0167] Impurities HYR-Z 4h Stock solution: Take impurity HYR-Z 4h Weigh approximately 25 mg of the reference standard accurately, place it in a 25 ml volumetric flask, add solvent A to dissolve and dilute to the mark, and shake well.

[0168] Impurities HYR-Z 4j Stock solution: Take impurity HYR-Z 4j Weigh approximately 25 mg of the reference standard accurately, place it in a 25 ml volumetric flask, add solvent A to dissolve and dilute to the mark, and shake well.

[0169] Impurity HYR-Z1 positioning solution: Measure 0.1 ml of impurity HYR-Z1 stock solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0170] Impurity HYR-Z2 positioning solution: Measure 0.1 ml of impurity HYR-Z2 stock solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0171] Impurity HYR-Z3 positioning solution: Measure 0.1 ml of impurity HYR-Z3 stock solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0172] Impurities HYR-Z3c Positioning solution: Measure the impurity HYR-Z 3c Place 0.1 ml of the stock solution into a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0173] Impurities HYR-Z 3e Positioning solution: Measure the impurity HYR-Z 3e Place 0.1 ml of the stock solution into a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0174] Impurities HYR-Z 4a Positioning solution: Measure the impurity HYR-Z 4a Place 0.1 ml of the stock solution into a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0175] Impurities HYR-Z 4b Positioning solution: Measure the impurity HYR-Z 4b Place 0.1 ml of the stock solution into a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0176] Impurities HYR-Z 4d Positioning solution: Measure the impurity HYR-Z 4d Place 0.1 ml of the stock solution into a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0177] Impurities HYR-Z 4e Positioning solution: Measure the impurity HYR-Z 4e Place 0.1 ml of the stock solution into a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0178] Impurities HYR-Z 4g Positioning solution: Measure the impurity HYR-Z 4g Place 0.1 ml of the stock solution into a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0179] Impurities HYR-Z 4h Positioning solution: Measure the impurity HYR-Z 4h Place 0.1 ml of the stock solution into a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0180] Impurities HYR-Z 4j Positioning solution: Measure the impurity HYR-Z 4d Place 0.1 ml of the stock solution into a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0181] Test solution: Weigh approximately 25 mg of the test sample accurately, place it in a 25 ml volumetric flask, add solvent, sonicate to dissolve and dilute to the mark, and shake well.

[0182] Mixed solution: Accurately weigh approximately 25 mg of the test sample and place it in a 25 ml volumetric flask. Measure the contents of the impurity HYR-Z1 stock solution, impurity HYR-Z2 stock solution, impurity HYR-Z3 stock solution, and impurity HYR-Z... 3c Stock solution, impurities HYR-Z 3e Stock solution, impurities HYR-Z 4a Stock solution, impurities HYR-Z 4b Stock solution, impurities HYR-Z 4d Stock solution, impurities HYR-Z 4e Stock solution, impurities HYR-Z 4g Stock solution, impurities HYR-Z 4h Stock solution, impurities HYR-Z 4j Place 0.25 ml of each stock solution in the same volumetric flask, add solvent, sonicate to dissolve and dilute to the mark, and shake well.

[0183] (2) Detection

[0184] Inject 10 μl each of the blank solution (solvent), each impurity localization solution, the test solution, and the mixed solution, and perform the chromatographic analysis according to the chromatographic conditions in Example 1, and record the chromatograms.

[0185] The measurement results are shown in Table E. Figures 1-3 Tables 1-3. Neither the blank solution nor the test solution interfered with the impurity HYR-Z. 4h Impurities HYR-Z 4d Impurities HYR-Z 4a Impurities HYR-Z 4g Impurities HYR-Z 4e Impurities HYR-Z 4j Impurities HYR-Z 3c Impurity HYR-Z3, Impurity HYR-Z 4b Impurities HYR-Z 3e Detection of impurities HYR-Z2 and HYR-Z1. The main peak and impurity HYR-Z1 in the mixed solution. 4a The peak separation was 1.61, and the main peak was separated from the impurity HYR-Z. 4g The peak resolution was 3.10, and the minimum resolution between known impurity peaks was 2.98. The method specificity met the requirements.

[0186] Table E. Results of Specificity Measurement

[0187]

[0188] Example 3. Limit of Quantitation and Limit of Detection Experiment

[0189] Solvent: A mixed solution of mobile phase A and acetonitrile, wherein the volume ratio of mobile phase A to acetonitrile is 70:30.

[0190] Limit of Quantitation Solution: Accurately measure the test solution, impurity HYR-Z1 stock solution, impurity HYR-Z2 stock solution, impurity HYR-Z3 stock solution, and impurity HYR-Z2 stock solution from "Example 2. Specificity Test". 3c Stock solution, impurities HYR-Z 3e Stock solution, impurities HYR-Z 4a Stock solution, impurities HYR-Z 4b Stock solution, impurities HYR-Z 4d Stock solution, impurities HYR-Z 4e Stock solution, impurities HYR-Z 4g Stock solution, impurities HYR-Z 4h Stock solution, impurities HYR-Z 4j Take 2.5 ml of each stock solution and place them in the same 50 ml volumetric flask. Dilute to the mark with acetonitrile and mix well. Then accurately measure 0.4 ml of each stock solution and place it in a 50 ml volumetric flask. Dilute to the mark with solvent and mix well. (0.04%)

[0191] Detection limit solution: Accurately measure 5 ml of the quantitation limit solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0192] The limit of quantitation solution was injected 6 times consecutively, and the limit of detection solution was injected 3 times consecutively. The detection was performed under the chromatographic conditions in Example 1. The chromatograms were recorded, and the ratio of the main peak height to the noise (signal-to-noise ratio) was calculated.

[0193] The results of the limit of quantitation detection are shown in Table F. Figure 5 Table 5. Data shows that the impurity HYR-Z 4h The limit of quantitation was 0.3988 μg / ml, the peak area RSD was 1.1% (N=6), the average S / N ratio was 56.7, and the concentration in the sample was expressed as 0.040%; impurity HYR-Z 4d The limit of quantitation was 0.4003 μg / ml, the peak area RSD was 1.9% (N=6), the average S / N ratio was 41.0, and the concentration in the sample was expressed as 0.040%; impurity HYR-Z 4a The limit of quantitation was 0.4044 μg / ml, the peak area RSD was 3.3% (N=6), the average S / N ratio was 24.9, and the concentration in the sample was expressed as 0.040%; impurity HYR-Z 4g The limit of quantitation was 0.3871 μg / ml, the peak area RSD was 3.4% (N=6), the average S / N ratio was 24.3, and the concentration in the sample was expressed as 0.039%; impurity HYR-Z 4eThe limit of quantitation was 0.3971 μg / ml, the peak area RSD was 2.9% (N=6), the average S / N ratio was 22.8, and the concentration in the sample was expressed as 0.040%; impurity HYR-Z 4j The limit of quantitation was 0.3982 μg / ml, the peak area RSD was 2.6% (N=6), the average S / N ratio was 111.3, and the concentration in the sample was expressed as 0.040%; impurity HYR-Z 3c The limit of quantitation (LOQ) concentration was 0.3878 μg / ml, the peak area RSD was 2.4% (N=6), the average S / N ratio was 92.2, and the concentration in the sample was expressed as 0.039%. The limit of quantitation (LOQ) concentration of impurity HYR-Z3 was 0.4010 μg / ml, the peak area RSD was 1.8% (N=6), the average S / N ratio was 105.2, and the concentration in the sample was expressed as 0.040%. Impurity HYR-Z... 4b The limit of quantitation was 0.4028 μg / ml, the peak area RSD was 0.3% (N=6), the average S / N ratio was 218.8, and the concentration in the sample was expressed as 0.040%; impurity HYR-Z 3e The limit of quantitation (LOQ) for each impurity was 0.3879 μg / ml, with a peak area RSD of 0.7% (N=6) and an average S / N ratio of 94.4, representing a concentration of 0.039% in the sample. The LQ for impurity HYR-Z2 was 0.3951 μg / ml, with a peak area RSD of 1.1% (N=6) and an average S / N ratio of 75.4, representing a concentration of 0.040% in the sample. The LQ for impurity HYR-Z1 was 0.3974 μg / ml, with a peak area RSD of 2.6% (N=6) and an average S / N ratio of 128.2, representing a concentration of 0.040% in the sample. The LQ for 16α-hydroxyprednisolone was 0.4030 μg / ml, with an average S / N ratio of 30.9. For all impurities and the test sample at a relative concentration of 0.04%, the signal-to-noise ratio was greater than the requirement of 10:1.

[0194] Table F. Results of Limit of Quantitation Determination

[0195]

[0196]

[0197]

[0198] The results of the detection limit experiment are shown in Table G. Figure 6 As shown in Table 6, the data shows that the impurity HYR-Z 4h The detection limit was 0.1994 μg / ml, the average S / N ratio was 30.4, and the concentration in the sample was expressed as 0.020%; impurity HYR-Z 4dThe detection limit was 0.2001 μg / ml, the average S / N ratio was 21.3, and the concentration present in the sample was expressed as 0.020%; impurity HYR-Z 4a The detection limit was 0.2022 μg / ml, the average S / N ratio was 13.3, and the concentration present in the sample was expressed as 0.020%; impurity HYR-Z 4g The detection limit was 0.1936 μg / ml, the average S / N ratio was 12.7, and the concentration present in the sample was expressed as 0.019%; impurity HYR-Z 4e The detection limit was 0.1985 μg / ml, the average S / N ratio was 12.3, and the concentration present in the sample was expressed as 0.020%; impurity HYR-Z 4j The detection limit was 0.1991 μg / ml, the average S / N ratio was 58.2, and the concentration present in the sample was expressed as 0.020%; impurity HYR-Z 3c The detection limit concentration was 0.1939 μg / ml, the average S / N ratio was 47.9, and the concentration in the sample was expressed as 0.019%; the detection limit concentration of impurity HYR-Z3 was 0.2005 μg / ml, the average S / N ratio was 54.8, and the concentration in the sample was expressed as 0.020%; impurity HYR-Z 4b The detection limit was 0.2014 μg / ml, the average S / N ratio was 113.7, and the concentration in the sample was expressed as 0.020%; impurity HYR-Z 3e The detection limit concentration (LOC) was 0.1940 μg / ml, with an average S / N ratio of 48.8, representing a concentration of 0.019% in the sample. The LOC concentration for impurity HYR-Z2 was 0.1975 μg / ml, with an average S / N ratio of 39.5, representing a concentration of 0.020% in the sample. The LOC concentration for impurity HYR-Z1 was 0.1987 μg / ml, with an average S / N ratio of 65.8, representing a concentration of 0.020% in the sample. The LOC concentration for 16α-hydroxyprednisolone was 0.2015 μg / ml, with an average S / N ratio of 16.8. For all impurities and the test sample at a relative concentration of 0.02%, the signal-to-noise ratio was greater than 3:1.

[0199] Table G. Results of Limit of Detection

[0200]

[0201]

[0202] Example 4. Chromatographic condition durability

[0203] Take the mixed solution from "Example 2. Specificity Experiment" and test it under normal chromatographic conditions (the chromatographic conditions of Example 1), with different column temperatures, column flow rates, initial mobile phase ratios, intermediate mobile phase ratios, and different batches of chromatographic columns. After the instrument system stabilizes, test it separately, record the resolution between each peak, and examine the changes in the relative retention time and normalized content of each known impurity.

[0204] The test results are shown in Tables H to J. Figures 7-15 Tables 7-15. When chromatographic conditions fluctuate slightly, the resolution between the main component and each known impurity and the adjacent components is greater than 1.5, and the relative retention time, the normalized content of the main peak and each known impurity peak are basically consistent.

[0205] Table H. Results of relative retention time determination of impurities under chromatographic conditions variation test.

[0206]

[0207]

[0208] Table I. Results of Resolution Determination of Each Component in Chromatographic Condition Variation Test

[0209]

[0210] Table J. Results of Area-Normalized Content Determination of Each Component in Chromatographic Condition Variation Test

[0211]

[0212]

[0213] Note: In Tables 4-6 above, gradient time (minutes) 50→58 means that under the same gradient elution conditions, the percentages of mobile phase A (40%) and mobile phase B (60%) at 50 minutes are replaced with 42% and 58% at 50 minutes. Similarly, 50→62 means that the percentages of mobile phase A (38%) and mobile phase B (62%) at 50 minutes are the same.

[0214] The initial mobile phase ratio AB 86:14 means that, under the gradient elution conditions with other conditions remaining unchanged, the ratio of "mobile phase A 85%, mobile phase B 15%" at 0, 25, 61, and 70 minutes is adjusted to "mobile phase A 86%, mobile phase B 14%". Similarly, the initial mobile phase ratio AB 84:16 means that the ratio of mobile phase A is 84%, mobile phase B is 16% at 0, 25, 61, and 70 minutes.

Claims

1. A method for separating 16α-hydroxyprednisolone and its impurities based on high performance liquid chromatography, characterized in that, The 16α-hydroxyprednisolone and the impurities together constitute the composition, the impurities including impurity HYR-Z. 4h Impurities HYR-Z 4d Impurities HYR-Z 4a Impurities HYR-Z 4g Impurities HYR-Z 4e Impurities HYR-Z 4j Impurities HYR-Z 3c Impurity HYR-Z3, Impurity HYR-Z 4b Impurities HYR-Z 3e Any one or more of the following: impurity HYR-Z2, impurity HYR-Z1; the structural formula of each component in the composition is as follows: In the high-performance liquid chromatography method, the mobile phase is: mobile phase A is an aqueous solution of phosphoric acid, and mobile phase B is acetonitrile; the stationary phase is: the chromatographic column uses octylsilane-bonded silica gel as the packing material; 16α-hydroxyprednisolone and its impurities are separated by linear gradient elution.

2. The method according to claim 1, characterized in that, The procedure for linear gradient elution is as follows: 。 3. The method according to claim 1, characterized in that, The procedure for linear gradient elution is as follows: 。 4. The method according to claim 1, characterized in that, The flow rate is 0.7-1.3 mL / min; the column temperature is 15-30℃.

5. A method for identifying 16α-hydroxyprednisolone and its impurities, characterized in that, The composition is separated by the method according to any one of claims 1-4 and detected by a detector to obtain a chromatogram; by comparing the chromatographic characteristics of the test sample and the reference sample, it is determined whether the test sample contains 16α-hydroxyprednisolone and its impurities.

6. The method according to claim 5, characterized in that, The detector has a detection wavelength of 245±10nm.

7. The method according to claim 5, characterized in that, The components of the composition can be identified according to their relative retention times. The components of the composition, in ascending order, are: impurity HYR-Z. 4h Impurities HYR-Z 4d Impurities HYR-Z 4a 16α-Hydroxyprednisolone, impurity HYR-Z4g, impurity HYR-Z4e, impurity HYR-Z4j, impurity HYR-Z3c, impurity HYR-Z3, impurity HYR-Z 4b Impurities HYR-Z 3e Impurity HYR-Z2, Impurity HYR-Z1.

8. The method according to claim 7, characterized in that, Using 16α-hydroxyprednisolone as a reference peak; the relative retention time was 0.38, indicating it was identified as the impurity HYR-Z. 4h ; The relative retention time was 0.64, indicating it was an impurity (HYR-Z). 4d ; The relative retention time was 0.96, indicating it was an impurity, HYR-Z4a. The relative retention time was 1.09, which was determined to be impurity HYR-Z4g; The relative retention time was 1.24, indicating it was an impurity, HYR-Z4e. The relative retention time was 1.45, indicating it was an impurity, HYR-Z4j. The relative retention time was 1.52, indicating it was an impurity, HYR-Z. 3c ; The relative retention time was 1.59, which was determined to be impurity HYR-Z3; The relative retention time was 1.65, indicating it was an impurity (HYR-Z). 4b ; The relative retention time was 1.67, indicating it was an impurity, HYR-Z. 3e ; The relative retention time was 1.69, which was determined to be impurity HYR-Z2; The relative retention time was 1.75, which was identified as impurity HYR-Z1; the relative retention times of each component fluctuated within ±20%.

9. A method for determining the content of 16α-hydroxyprednisolone and its impurities, characterized in that, The method described in any one of claims 5-8 is used to separate and identify 16α-hydroxyprednisolone and its impurities, and a chromatogram is obtained. Based on the obtained chromatogram, the content of each impurity is calculated by the principal component self-comparison method multiplied by a correction factor.

10. The method according to claim 9, characterized in that, The impurity HYR-Z 4h The correction factor is 1.1; the impurity HYR-Z 4d The correction factor is 1.0; the impurity HYR-Z 4j The correction factor is 1.0; the correction factor for impurity HYR-Z3 is 1.2; the correction factor for impurity HYR-Z 4a The correction factor is 1.3; the impurity HYR-Z 4g The correction factor is 1.0; the impurity HYR-Z 4e The correction factor is 1.0; the impurity HYR-Z 3c The correction factor is 1.2; the impurity HYR-Z 4b The correction factor is 0.61; the impurity HYR-Z 3e The correction factor for the impurity is 1.3; the correction factor for the impurity HYR-Z2 is 1.7; and the correction factor for the impurity HYR-Z1 is 1.0.

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