Analysis method for determining concentrations of methylprednisolone aceponate and metabolites thereof in blood plasma

Abstract

The invention belongs to the field of biological analysis of medicines, and particularly relates to a biological analysis method for determining concentrations of methylprednisolone aceponate and metabolites thereof in blood plasma, and the method is a liquid chromatography-tandem mass spectrometry method. The method for simultaneously detecting the plasma concentration of methylprednisolone aceponate and the metabolite 6 alpha-methylprednisolone 17-propionate thereof in the plasma is the only disclosed method at present, and can be used for supporting non-clinical and clinical research work of the medicine.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical bioanalysis and relates to a bioanalytical method for determining the concentration of methylprednisolone acetate and its metabolites in plasma. Background Technology

[0002] Prednisolone acetate cream, with methylprednisolone acetate as its active ingredient, is a corticosteroid used to treat inflammation. It is mainly suitable for various types of eczema, including endogenous eczema, contact eczema, degenerative eczema, and polyeczema.

[0003] During the use of propylprednisolone cream, propylprednisolone is metabolized in the epidermis and dermis to its main metabolite, 6α-methylprednisolone-17-propionate. In generic drug clinical trials, to investigate the pharmacokinetic characteristics and safety of the drug, it is necessary to accurately measure the concentrations of propylprednisolone and 6α-methylprednisolone-17-propionate in the plasma of subjects or patients.

[0004] Currently, there are no publicly available methods for simultaneously detecting the blood concentrations of methylprednisolone acetate and its main metabolite, 6α-methylprednisolone-17-propionate, especially quantitative analytical methods conforming to ICH M10 standards. Furthermore, given the characteristics of transdermal administration, systemic drug exposure is typically less than 1% of the administered dose, and the detection sensitivity (LLOQ > 1 ng / mL) of conventional liquid chromatography and mass spectrometry is relatively low, making it difficult to meet the precise detection requirements for pharmacokinetic and biosafety studies via transdermal administration.

[0005] Therefore, there is an urgent need in this field to develop a simple, accurate, rapid and sensitive bioanalytical method to accelerate the clinical application of this drug and provide a guarantee for the research and development and application of generic drugs. Summary of the Invention

[0006] To address the aforementioned technical problems, the present invention aims to provide an analytical method for determining the concentration of methylprednisolone acetate and its metabolites in plasma.

[0007] The present invention adopts the following technical solution:

[0008] An analytical method for determining the concentration of methylprednisolone acetate and its metabolites in plasma, wherein the method is liquid chromatography-tandem mass spectrometry, and the liquid chromatography is performed under the following conditions:

[0009] Chromatographic column: ChromCore 120C 183μm, 2.1×100mm

[0010] Monitor: MS

[0011] Mobile phase A: Distilled water: 1M NH4F

[0012] Mobile phase B: acetonitrile, elution gradient:

[0013]

[0014] The metabolite is 6α-methylprednisolone-17-propionate.

[0015] In some embodiments, the volume ratio of distilled water to 1M NH4F in the mobile phase A is 100000:5.

[0016] In some embodiments, the liquid chromatography uses a flow rate of 0.45 mL / min, a column temperature of 40°C, and an autosampler temperature of 5°C.

[0017] In some embodiments, the mass spectrometer uses the following conditions: electrospray ionization source, positive ion detection, multiple reaction monitoring mode, and ion source temperature of 400°C.

[0018] In some embodiments, the mass spectrometry conditions include 50.0 psi for gas 1, 55.0 psi for gas 2, 35.0 psi for curtain gas, and 9.0 psi for collision gas.

[0019] In some embodiments, the mass spectrometry conditions include a mass spectrometry acquisition time of 3.5 min; the ion pair for quantitative analysis of the analyte methylprednisolone is 473.4 / 381.3 with a collision energy of 18 eV, a declustering voltage of 60 V, an inlet voltage of 10 V, and a collision chamber outlet voltage of 15 V; the ion pair for quantitative analysis of the analyte 6α-methylprednisolone-17-propionate is 431.4 / 280.3 with a collision energy of 28 eV, a declustering voltage of 60 V, an inlet voltage of 10 V, and a collision chamber outlet voltage of 15 V; and the ion pair for quantitative analysis of the internal standard finasteride is 373.1 / 305.2 with a collision energy of 30 eV, a declustering voltage of 100 V, an inlet voltage of 10 V, and a collision chamber outlet voltage of 15 V.

[0020] In some embodiments, before sampling and detection using liquid chromatography-tandem mass spectrometry, the sample is pretreated. The pretreatment method is as follows: water is added to the plasma sample, the mixture is vortexed, an internal standard working solution is added, the mixture is vortexed again, ethyl acetate is added for liquid-liquid extraction, the mixture is vortexed, the supernatant is collected by centrifugation, diluted with a diluent, and the sample is obtained by vortexing. The internal standard working solution is a 50% acetonitrile aqueous solution.

[0021] In some embodiments, the volume of the plasma sample is 200 μL, the volume ratio of the plasma sample to the internal standard working solution is 4:1, the concentration of the methylprednisolone acetonide to be tested in the plasma sample is 10.00 pg / mL to 5000 pg / mL, and the concentration of the 6α-methylprednisolone-17-propionate to be tested is 30.00 pg / mL to 15000 pg / mL.

[0022] In some implementations, the concentration of finasteride in the internal standard working solution is 5.000 ng / mL.

[0023] In some embodiments, the diluent is acetonitrile:water, with a volume ratio of acetonitrile:water of 50:50.

[0024] The present invention has at least the following advantages:

[0025] 1. The detection method described in this invention is currently the only disclosed method for simultaneously detecting the plasma concentrations of methylprednisolone acetate and its metabolite 6α-methylprednisolone-17-propionate. The LC-MS / MS method has high analytical efficiency and can be used to support non-clinical and clinical research on this drug.

[0026] 2. The detection method described in this invention uses distilled water and 1M NH4F as mobile phase A, which significantly improves the sensitivity of the two analytes.

[0027] 3. The detection method described in this invention has high sensitivity. With a plasma sample volume of only 200 μL, the lower limit of quantification for the methylprednisolone acetonide is 10 pg / mL, and the lower limit of quantification for the 6α-methylprednisolone-17-propionate is 30 pg / mL. This can reduce the dosage and whole blood collection volume of the subjects, which is beneficial to increasing the safety of clinical research and the compliance of the subjects. Moreover, the linear range is reasonably selected, which can accurately determine the concentration of the test compounds in the plasma. Attached Figure Description

[0028] Figure 1 This is an ion scanning mass spectrum of methylprednisolone acetate;

[0029] Figure 2 This is the ion scanning mass spectrum of finasteride;

[0030] Figure 3 This is the ion scanning mass spectrum of 6α-methylprednisolone-17-propionate;

[0031] Figure 4 This is the ion scanning mass spectrum of finasteride;

[0032] Figure 5This is the chromatogram of the column selected during the column screening in Example 1: Waters ACQUITY UPLC BEH C18 (100mm × 2.1mm, particle size 1.7μm). The left side is the chromatogram of methylprednisolone acetate, and the right side is the chromatogram of 6α-methylprednisolone-17-propionate.

[0033] Figure 6 This is the chromatogram of Phenomenex Lura Omega (100mm × 2.1mm, particle size 1.7μm) column used in Example 1 column screening. The left side is the chromatogram of methylprednisolone acetate, and the right side is the chromatogram of 6α-methylprednisolone-17-propionate.

[0034] Figure 7 This is the chromatogram of Phenomenex Kinetex (100mm × 2.1mm, particle size 1.7μm) used as the chromatographic column during the column screening in Example 1. The left side is the chromatogram of methylprednisolone acetate, and the right side is the chromatogram of 6α-methylprednisolone-17-propionate.

[0035] Figure 8 This is the chromatogram of ChromCore 120C18 (100mm×2.1mm, particle size 3μm) column selected during the column screening in Example 1. The left side is the chromatogram of methylprednisolone acetate, and the right side is the chromatogram of 6α-methylprednisolone-17-propionate.

[0036] Figure 9 This is a chromatogram of the mobile phase selected in Example 1 when 0.1% acetic acid aqueous solution was used as mobile phase A and acetonitrile was used as mobile phase B. The left side is the chromatogram of methylprednisolone acetate, and the right side is the chromatogram of 6α-methylprednisolone-17-propionate.

[0037] Figure 10 This is the chromatogram of mobile phase A (distilled water: 10M ammonium acetate (v:v, 100000:5) and mobile phase B (acetonitrile) used in Example 1. The left side is the chromatogram of methylprednisolone acetate, and the right side is the chromatogram of 6α-methylprednisolone-17-propionate.

[0038] Figure 11 This is the chromatogram of the mobile phase screening in Example 1, where distilled water: 1M ammonium fluoride (v:v, 100000:5) was used as mobile phase A and acetonitrile was used as mobile phase B. The left side is the chromatogram of methylprednisolone acetate, and the right side is the chromatogram of 6α-methylprednisolone-17-propionate.

[0039] Figure 12 This is the chromatogram of the mobile phase gradient screening in Example 1 when gradient 1 was used. The left side is the chromatogram of methylprednisolone acetate, and the right side is the chromatogram of 6α-methylprednisolone-17-propionate.

[0040] Figure 13 This is the chromatogram of gradient 2 when using mobile phase gradient screening in Example 1. The left side is the chromatogram of methylprednisolone acetate, and the right side is the chromatogram of 6α-methylprednisolone-17-propionate.

[0041] Figure 14 This is the chromatogram of the mobile phase gradient screening in Example 1 when gradient 3 was selected. The left side is the chromatogram of methylprednisolone acetate, and the right side is the chromatogram of 6α-methylprednisolone-17-propionate.

[0042] Figure 15 The chromatograms are those obtained under the mass spectrometry conditions of Example 2 with a source temperature of 600°C. The left side shows the chromatogram of methylprednisolone acetate, and the right side shows the chromatogram of 6α-methylprednisolone-17-propionate.

[0043] Figure 16 The chromatograms are those obtained under the mass spectrometry conditions of Example 2 with a source temperature of 400°C. The left side shows the chromatogram of methylprednisolone acetate, and the right side shows the chromatogram of 6α-methylprednisolone-17-propionate.

[0044] Figure 17 The image shows the MRM chromatograms of the methylprednisolone and finasteride to be tested in the blank plasma sample of Example 4. The left side shows the MRM chromatogram of the methylprednisolone to be tested, and the right side shows the MRM chromatogram of finasteride. Figure 18 The image shows the MRM chromatograms of 6α-methylprednisolone-17-propionate and finasteride in the blank plasma sample of Example 4. The left side shows the MRM chromatogram of 6α-methylprednisolone-17-propionate, and the right side shows the MRM chromatogram of finasteride.

[0045] Figure 19 This is the MRM chromatogram of the methylprednisolone and finasteride to be tested in the sample with the lower limit of quantitation in Example 4. The left side is the MRM chromatogram of the methylprednisolone to be tested, and the right side is the MRM chromatogram of finasteride. Figure 20 This is the MRM chromatogram of 6α-methylprednisolone-17-propionate and finasteride in the sample with the lower limit of quantitation in Example 4. The left side is the MRM chromatogram of 6α-methylprednisolone-17-propionate, and the right side is the MRM chromatogram of finasteride. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. The scope of protection of the present invention is not limited to the following embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention and are protected by the appended claims. The processes, conditions, reagents, experimental methods, etc., for implementing the present invention, except as specifically mentioned below, are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0047] In this invention, unless otherwise stated, percentages and ratios mentioned in the reagents are weight percentages and weight ratios.

[0048] In this invention, the unit “M” represents moles per liter (mol / L), and “v / v” and “v / v / v / v” represent volume ratios.

[0049] Example 1: Screening of Liquid Chromatography Conditions

[0050] 1.1 Column Screening

[0051] Chromatographic conditions:

[0052] Mobile phase A: 0.1% formic acid in water;

[0053] Mobile phase B: Acetonitrile;

[0054] Flow rate: 0.3 mL / min;

[0055] Column temperature: 40℃

[0056] The elution gradient is:

[0057]

[0058] Corticosteroids are weakly polar substances, so reversed nonpolar columns are preferred. Four different brands and models of C18 columns were compared: Waters ACQUITY UPLC BEH C18 (100mm × 2.1mm, 1.7μm particle size), Phenomenex Lura Omega (100mm × 2.1mm, 1.7μm particle size), Phenomenex Kinetex (100mm × 2.1mm, 1.7μm particle size), and ChromCore 120C18 (100mm × 2.1mm, 3μm particle size). The results showed that for the two analytes tested, the ChromCore 120C18 column provided the best separation, exhibiting high resolution, smooth and symmetrical peak shapes, high response values, and a wide applicable pH range.

[0059] 1.2 Mobile phase screening

[0060] After determining the chromatographic column, the mobile phase was screened, as it significantly affects the peak height and shape of the compounds. For aqueous phase screening, we used ammonium formate, ammonium acetate, formic acid, acetic acid, and ammonium fluoride as aqueous phases and compared their separation effects on the target compounds. The results showed that using distilled water:1M ammonium fluoride (v:v, 100000:5) as the aqueous phase resulted in the best resolution and peak shape for the target compounds. This may be because, in ESI positive ion mode, ammonium fluoride reduces the formation of adducts (such as +Na, +K) between the target analyte and basic cation adducts (after the target analyte binds to Na+ / K+, the number of molecules or fragments bound to H+ will decrease).

[0061] Organic phase screening: We used methanol and acetonitrile as organic phases and compared their separation effects. The experimental results showed that acetonitrile provided the best separation degree and peak shape for the target compounds.

[0062] 1.3 Optimization of mobile phase gradient and flow velocity

[0063] After determining the chromatographic column and mobile phase, we screened the mobile phase gradient and flow rate. We optimized the flow rate, conducting experiments at 0.3 mL / min, 0.4 mL / min, 0.45 mL / min, and 0.5 mL / min. The results showed that at a flow rate of 0.45 mL / min, the column pressure was moderate, the peak elution time was short, the peak shape was sharp, and the resolution met the requirements. We further optimized the gradient elution program, determining the optimal mobile phase ratio and gradient conditions (elution gradient 3) for high resolution, short retention time, and symmetrical, sharp, and smooth peak shapes. All peaks could be separated within 3.5 minutes, achieving accurate quantification.

[0064] Table 1 Elution gradient 1 data table

[0065]

[0066] Table 2 Elution gradient 2 data table

[0067]

[0068]

[0069] Table 3 Elution gradient 3 data table

[0070]

[0071] Example 2: Screening of mass spectrometry conditions

[0072] Source temperature significantly affects the ionization efficiency and ion transport of compounds. We monitored the signal intensity and stability of the target compound at a source temperature of 400℃ by gradually increasing the source temperature from room temperature to 600℃. The results showed that the signal intensity of the target compound reached its maximum and remained stable at 400℃, thus confirming 400℃ as the optimal source temperature. Higher source temperatures promote the volatilization and ionization of the compound, but excessively high temperatures may lead to thermal decomposition. 400℃ represents a balance point, ensuring effective ionization without inducing thermal decomposition.

[0073] We monitored the signal intensity and stability of the target compound by gradually adjusting the gas parameters. The results showed that the signal intensity of the target compound reached its maximum and remained stable under the following parameter conditions, therefore the final gas parameters were determined to be CUR 35.0 psi, CAD 9.0 psi, Gas 150.0 psi, and Gas 255.0 psi.

[0074] Example 3: Screening of the pretreatment process

[0075] Take 200.0 μL each of the mixed standard curve sample, quality control sample, blank matrix sample, STD0, test sample, equilibration sample, and SST sample, and add them to 2 mL EP tubes respectively. Then add 50.0 μL of internal standard working solution to each sample. Perform liquid-liquid extraction, centrifugation, nitrogen drying, and dilution according to the steps in Table 1.

[0076] Take 200.0 μL each of the mixed standard curve sample, quality control sample, blank matrix sample, STD0, test sample, equilibrium sample, and SST sample, and add them to 2 mL EP tubes respectively. Mix thoroughly, and then add 50.0 μL of internal standard working solution to each sample. Perform liquid-liquid extraction, centrifugation, nitrogen drying, and dilution according to the steps in Table 1. However, the step of adding the extractant and shaking in step 1 is changed to shaking for 5 min, sonicating for 5 min, shaking for 5 min, and the step of vortexing and mixing after reconstitution for 3 min in step 3 is changed to mixing for 3 min, sonicating for 3 min, mixing for 3 min, and then centrifuging for 10 min (5℃, 4600g). Take 120 μL of the supernatant and transfer it to a new 96-well plate. It was found that after adding the sonication step in the pretreatment steps, the method recovery rate increased to 80%, and the coefficient of variation of samples of the same concentration decreased. The samples were cleaner, and the results were more stable and accurate. Therefore, in subsequent experiments, the sonication step was added during shaking and mixing.

[0077] Table 4. Procedures for processing plasma samples

[0078]

[0079] Table 5. Extraction and recovery rates of the analyte (methylprednisolone acetate).

[0080]

[0081] Table 6. Extraction recovery rate of the analyte (α-methylprednisolone-17-propionate)

[0082]

[0083]

[0084] Table 7 Extraction and recovery rates of internal standards

[0085]

[0086] Example 4: Methodological Validation Example

[0087] 1. Preparation of solutions and samples

[0088] Standard series samples: Accurately weigh appropriate amounts of each reference standard (propylprednisolone), dissolve and dilute to volume with DMSO to prepare a stock solution with a propylprednisolone concentration of approximately 2.000 mg / mL. Accurately pipette appropriate amounts of each stock solution and dilute stepwise with methanol:water (50:50, v:v) to obtain the standard curve working solution. Finally, prepare the standard curve plasma sample with a propylprednisolone concentration range of 10.00-5000 pg / mL using blank plasma.

[0089] Accurately weigh appropriate amounts of each reference standard (6α-methylprednisolone-17-propionate), dissolve and dilute to volume with methanol:DMSO (50:50, v:v) to prepare a stock solution with a 6α-methylprednisolone-17-propionate concentration of approximately 1.000 mg / mL. Accurately pipette appropriate amounts of each stock solution and serially dilute with methanol:water (50:50, v:v) to obtain the standard curve working solution. Finally, prepare the standard curve plasma sample with a 6α-methylprednisolone-17-propionate concentration range of 30.00-15000 pg / mL using blank plasma.

[0090] Quality control samples: Three concentration levels of mixed quality control samples of methylprednisolone acetate / 6α-methylprednisolone-17-propionate were prepared using methods similar to those used for the standard series samples. The lower limit of quantitation (LQC) concentrations were 10.00 / 30.00 pg / mL, the low quality control (LQC) concentrations were 30.00 / 90.00 pg / mL, the medium quality control (MQC) concentrations were 1500 / 4500 pg / mL, and the high quality control (HQC) concentrations were 3750 / 11250 pg / mL.

[0091] Internal standard working solution: Accurately weigh finasteride reference standard, dilute with methanol to volume, and prepare a solution with a concentration of approximately [missing value].

[0092] Internal standard stock solution of 1.000 mg / mL. Accurately pipette an appropriate amount of each of the above internal standard stock solutions and dilute with acetonitrile:water (50:50, v:v) to obtain internal standard working solution with finasteride concentration of 5.000 ng / mL.

[0093] 2. Plasma Sample Processing Steps

[0094] Table 8. Plasma Sample Processing Steps

[0095]

[0096] 3. Chromatographic and mass spectrometric conditions

[0097] Table 9 Chromatographic conditions

[0098]

[0099] Table 10 Mass Spectrometry Conditions

[0100]

[0101]

[0102] 4. Methodological Validation

[0103] The method was validated according to the guidelines of the 2020 edition of Chinese Pharmacopoeia Part 4, 9012 and ICH M10, including stability, selectivity, linearity, accuracy, precision, recovery, and matrix effects.

[0104] Selective

[0105] Six blank plasma samples from different sources, along with their respective prepared limit-of-quantitation (LOQ) samples, were processed and injected for analysis. The peak area of ​​the chromatographically eluted interfering substances must be less than 20% of the peak area of ​​the analyte at the LOQ and less than 5% of the peak area of ​​the internal standard.

[0106] Standard curve

[0107] A linear regression equation (weighting factor W = 1 / x²) was calculated using the theoretical concentration of the analyte as the x-axis and the peak area ratio of the analyte to the internal standard as the y-axis. Method validation involved two-sample analysis of each analytical batch against the standard curve.

[0108] Precision and accuracy

[0109] Method validation involved measuring six samples from each of the four concentration quality control samples in each analytical batch. For the limit of quantitation (LOQ), intra- and inter-batch precision (calculated as relative standard deviation (RSD)) was acceptable if less than 20%, and accuracy (calculated as relative deviation (RE)) was acceptable if between -20% and 20%. For the remaining QC samples at all concentration levels, intra- and inter-batch precision for each component was acceptable if less than 15%, and accuracy was acceptable if between -15% and 15%.

[0110] stability

[0111] To investigate the stability of each analyte in plasma samples, LQC and HQC were placed in different temperatures and environments, and six samples were analyzed after the placement was completed. A total of four placement conditions were investigated: 18 h in an ice-water bath, 47 h at 4°C after preparation, five freeze-thaw cycles (from -80°C to room temperature), and 80 days at -80°C.

[0112] Recovery rate

[0113] Take 200 μL of blank plasma, extract it (without adding internal standard working solution), add the analyte solution and internal standard working solution to make the final concentration the same as LQC, MQC, and HQC, and inject for analysis. Separately extract 6 aliquots each of LQC, MQC, and HQC, and inject for analysis. Calculate the extraction recovery rate based on the peak area ratio of the two processing methods.

[0114] Matrix effect

[0115] Six blank plasma samples from different sources were extracted (without internal standard working solution), and then analyte and internal standard working solution of the same concentration as those used in LQC and HQC were added. The mixture was vortexed and then analyzed. Water was used in place of plasma and processed according to the same method. The matrix factor was calculated using the ratio of peak areas obtained by the two methods. The matrix effect was assessed by the RSD of the matrix factor normalized to internal standard; a value less than 15% was acceptable.

[0116] 5. Results and Discussion

[0117] Method selectivity

[0118] like Figures 4 to 5 As shown, the retention times of methylprednisolone acetate, 6α-methylprednisolone-17-propionate, and finasteride were approximately 1.45, 1.09, and 1.15 min, respectively, with no co-eluent interference peaks at the retention times.

[0119] Standard curve

[0120] The linear range for methylprednisolone acetate in clinical study plasma samples was 10.00-5000 pg / mL; the linear range for 6α-methylprednisolone-17-propionate was 30.00-15000 pg / mL. The typical linear regression equations for the standard curves of the analytes were as follows:

[0121] 6α-Methylprednisolone acetate: y = 4.84x + 0.00337; 6α-Methylprednisolone-17-propionate: y = 1.12x ± 0.000461. Detection limit.

[0122] The concentrations of propylprednisolone / 6α-methylprednisolone-17-propionate in the samples with lower limits of quantitation were 10.00 / 30.00 pg / mL, with signal-to-noise ratios of 12.3 / 34.7, respectively. Based on a signal-to-noise ratio of 3, the limits of detection were calculated to be 2.440 / 2.594 pg / mL, respectively.

[0123] Precision and accuracy of the method

[0124] The precision and accuracy results all met the acceptance criteria. The results are shown in Table 11. Table 11 shows the precision and accuracy of the determination of hydrocortisone butyrate in rat plasma.

[0125] Table 11 Results of Precision and Accuracy Testing

[0126] Treatment recovery rate

[0127] LQC, MQC, and HQC concentration levels: Extraction recoveries of methylprednisolone acetate were 82.4%, 84.6%, and 88.3%; overall recovery was 85.1%, with an overall variation CV of 3.50%; extraction recoveries of α-methylprednisolone-17-propionate were 83.0%, 81.2%, and 81.4%; overall recovery was 81.9%, with an overall variation CV of 1.20%; matrix effect.

[0128] At LQC and HQC concentration levels, the normalized matrix factors for methylprednisolone acetate (MPA) were 110.0% and 103.0%, with RSDs of 4.55% and 12.62%, respectively; the normalized matrix factors for α-methylprednisolone-17-propionate (MPA) were 105.0% and 98.0%, with RSDs of 1.90% and 3.06%, respectively. These results indicate that the matrix effect does not interfere with the accuracy of analyte analysis.

[0129] Plasma stability study

[0130] The results of the plasma stability test are shown in Table 12. The results show that propylprednisolone and α-methylprednisolone-17-propionate are stable under the test conditions. Table 2 shows the stability of propylprednisolone and α-methylprednisolone-17-propionate in human plasma (n=6).

[0131] Table 12 Results of stability study in rat plasma

[0132]

[0133] Example 5: In vivo pharmacokinetic study

[0134] The validated method was used to analyze plasma concentrations of propylprednisolone and α-methylprednisolone-17-propionate to evaluate their pharmacokinetic characteristics. Plasma concentrations of propylprednisolone and α-methylprednisolone-17-propionate at different sampling times were determined using LC-MS / MS. Pharmacokinetic parameters were calculated using a non-compartmental model and statistical analysis was performed. The ratio of in vivo exposure of the test formulation to that of the reference formulation after topical administration was also studied to evaluate the safety of the test formulation. The sensitivity of the detection method is sufficient for the experimental detection of propylprednisolone and α-methylprednisolone-17-propionate.

[0135] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An analytical method for determining the concentration of propylprednisolone and its metabolites in plasma, characterized in that, The method is liquid chromatography-tandem mass spectrometry, wherein the liquid chromatography is performed under the following conditions: Chromatographic column: ChromCore 120C 183μm, 2.1×100mm Monitor: MS Mobile phase A: Distilled water: 1M NH4F Mobile phase B: Acetonitrile The elution gradient is: The metabolite is 6α-methylprednisolone-17-propionate.

2. The method according to claim 1, characterized in that, The volume ratio of distilled water to 1M NH4F in the mobile phase A is 100000:

5.

3. The method according to claim 1, characterized in that, The liquid chromatography was performed at a flow rate of 0.45 mL / min, a column temperature of 40 °C, and an autosampler temperature of 5 °C.

4. The method according to claim 1, characterized in that, The mass spectrometry was performed under the following conditions: electrospray ionization source, positive ion detection, multiple reaction monitoring mode, and ion source temperature of 400℃.

5. The method according to claim 4, characterized in that, In the mass spectrometry conditions, gas 1 is 50.0 psi, gas 2 is 55.0 psi, curtain gas is 35.0 psi, and collision gas is 9.0 psi.

6. The method according to claim 4, characterized in that, The mass spectrometry conditions were as follows: mass acquisition time 3.5 min; the ion pair for quantitative analysis of the methylprednisolone acetate was 473.4 / 381.3, collision energy 18 eV, declustering voltage 60 V, inlet voltage 10 V, and collision chamber outlet voltage 15 V; the ion pair for quantitative analysis of the 6α-methylprednisolone-17-propionate was 431.4 / 280.3, collision energy 28 eV, declustering voltage 60 V, inlet voltage 10 V, and collision chamber outlet voltage 15 V; the ion pair for quantitative analysis of the internal standard finasteride was 373.1 / 305.2, collision energy 30 eV, declustering voltage 100 V, inlet voltage 10 V, and collision chamber outlet voltage 15 V.

7. The method according to claim 1, characterized in that, Before using liquid chromatography-tandem mass spectrometry (LC-MS / MS) for sample injection and detection, the sample must be pretreated. The pretreatment method is as follows: water is added to the plasma sample, the mixture is vortexed and mixed, then internal standard working solution is added, the mixture is vortexed and mixed again, then ethyl acetate is added for liquid-liquid extraction, the mixture is vortexed and mixed, the supernatant is collected by centrifugation, the supernatant is diluted with diluent, and the sample is obtained by vortexing and mixing. The internal standard working solution is a 50% acetonitrile aqueous solution.

8. The method according to claim 7, characterized in that, The volume of the plasma sample is 200 μL, the volume ratio of the plasma sample to the internal standard working solution is 4:1, the concentration of the methylprednisolone acetonide to be tested in the plasma sample is 10.00 pg / mL to 5000 pg / mL, and the concentration of the 6α-methylprednisolone-17-propionate to be tested is 30.00 pg / mL to 15000 pg / mL.

9. The method according to claim 7, characterized in that, The concentration of finasteride in the internal standard working solution was 5.000 ng / mL.

10. The method according to claim 7, characterized in that, The diluent is acetonitrile:water, with a volume ratio of acetonitrile:water of 50:50.

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