Biological analysis method for detecting concentration of hydrocortisone butyrate in plasma

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a biological analysis method for detecting the concentration of hydrocortisone butyrate in blood plasma, which is used for analyzing a compound to be detected in the blood plasma through an LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) method. According to the detection method, a liquid-liquid extraction pretreatment method is preferably adopted, finasteride serves as an internal standard substance, a Shim-pack Velox SP-C18 chromatographic column is adopted, gradient elution and electrospray ionization source tandem mass spectrometry detection are carried out, and the detection method is high in sensitivity, precise, accurate, high in analysis speed, high in sensitivity, high in sensitivity, high in sensitivity, high in sensitivity, high in sensitivity, high in sensitivity, high in sensitivity, high in sensitivity and high in sensitivity. The sample analysis requirement in the clinical research of the hydrocortisone butyrate is met.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a bioanalysis method for detecting the concentration of hydrocortisone butyrate in plasma. Background Art

[0002] Hydrocortisone butyrate is a new generation of glucocorticoid drugs, which has significant therapeutic effects in the treatment of inflammatory skin diseases such as contact dermatitis, atopic dermatitis, and eczema.

[0003] Few existing documents or patents describe methods for detecting hydrocortisone butyrate blood concentrations, particularly quantitative analysis methods that comply with GLP standards (i.e., complete methodological validation studies in accordance with Pharmacopoeia 9012 and ICH M10). CN102680617A discloses a method for detecting adrenal hormones (including hydrocortisone butyrate), using a whole blood volume of 0.5 mL / 1 mL. The limit of detection for hydrocortisone butyrate is 0.021 ng / mL, and the lowest level of hydrocortisone butyrate detected in the examples is 32 ng / mL. This method has the following defects: 1. The whole blood test involved is not suitable for pharmacokinetic, bioequivalence / safety studies. The pharmacological activity of a drug in the body is usually driven by its free state concentration. Plasma, as the main liquid medium for drug distribution, directly reflects the concentration and dynamic changes of unbound active drugs in the circulatory system, and can avoid interference introduced by blood cell binding or retention in whole blood. 2. The sample detection time is 8 minutes, and the operation cycle is long, which cannot adapt to the large-scale sample (>4000 samples / study) high-throughput detection requirements required for clinical research. 3. Although the detection limit of hydrocortisone butyrate is clearly 0.021 ng / mL, or 21 pg / mL, the quantitative limit of hydrocortisone butyrate that can be detected by this method is not clear. Due to the characteristics of the transdermal route of administration, the systemic exposure of the drug is extremely low (usually <1% of the administered dose), and the detection sensitivity (including the quantitative limit) of the analytical method is required to be high.

[0004] In summary, existing technologies cannot currently meet the requirements of sensitivity, analysis speed, and plasma dosage, which is not conducive to the accurate analysis of large quantities of samples in clinical research. In order to accelerate the clinical application of the method for detecting the blood concentration of hydrocortisone butyrate, a simple, accurate, rapid and highly sensitive bioanalytical method is needed. Summary of the Invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a bioanalytical method for detecting the concentration of hydrocortisone butyrate in plasma.

[0006] The present invention adopts the following technical solutions:

[0007] A bioanalytical method for detecting the concentration of hydrocortisone butyrate in plasma, the method being liquid chromatography-tandem mass spectrometry, wherein the liquid chromatography employs the following conditions:

[0008] Chromatographic column: Shim-pack Velox SP-C18,

[0009] Monitor: DAD,

[0010] Mobile phase A: distilled water: 1M NH4F, v:v = 1000:1

[0011] Mobile phase B: methanol: acetonitrile, v:v = 50:50,

[0012] Flow rate: 0.42 mL / min, column temperature: 35°C,

[0013] Automatic sampler temperature: 5°C, elution gradient:

[0014]

[0015] In some embodiments, the liquid chromatography column has a specification of 2.1×100 mm and a diameter of 1.8 μm.

[0016] In some embodiments, the mass spectrometry employs the following conditions: electrospray ion source, positive ion detection, multiple reaction monitoring monitoring mode, spray voltage 3000 V, and ion source temperature 500°C.

[0017] Preferably, in the mass spectrometry conditions, gas 1 is 55.0 psi, gas 2 is 60.0 psi, curtain gas is 35 psi, collision gas is 8 psi, and residence time is 200 ms; the quantitative analysis ion pair of hydrocortisone butyrate to be measured is m / z 433.3 / 120.9, the collision energy is 34 eV, the declustering voltage is 50 V, the entrance voltage is 10 V, and the collision cell exit voltage is 11 V; the quantitative analysis ion pair of internal standard finasteride is m / z 373.1 / 305.2, the collision energy is 40 eV, the declustering voltage is 50 V, the entrance voltage is 10 V, and the collision cell exit voltage is 10 V.

[0018] In some embodiments, before using liquid chromatography-tandem mass spectrometry for sampling and detection, the sample must be pretreated. The pretreatment method is: water is added to the plasma sample, vortexed to mix, and then the internal standard working solution is added. After vortexing to mix, ethyl acetate is added for liquid-liquid extraction. After vortexing to mix, the supernatant is centrifuged and diluted with a diluent to obtain the test sample; the internal standard working solution is a methanol and acetonitrile solution of finasteride.

[0019] In some embodiments, the concentration of hydrocortisone butyrate to be tested in the plasma sample is 15.00 pg / mL-300.0 pg / mL.

[0020] In some embodiments, the concentration of finasteride in the internal standard working solution is 200.0 pg / mL.

[0021] In some embodiments, the diluent is acetonitrile:water:formic acid, and the volume ratio of acetonitrile:water:formic acid is 500:500:1.

[0022] The present invention has at least the following advantages:

[0023] 1. The detection method described in the present invention is one of the few methods currently disclosed for detecting the blood concentration of hydrocortisone butyrate in plasma. The LC-MS / MS method has high analytical efficiency and can be used to support non-clinical and clinical research work on this drug.

[0024] 2. The detection method described in the present invention improves the recovery rate and reduces the loss of analytes in the pretreatment method of LC-MS / MS analysis by diluting the plasma sample and then adding the internal standard working solution for liquid-liquid extraction, thereby achieving more accurate and reliable results, thereby supporting higher-quality scientific research discoveries or stricter regulatory compliance;

[0025] 3. The detection method described in the present invention has high sensitivity. When the plasma sample volume is only 200 μL, the quantitative lower limit of the test hydrocortisone butyrate is 15 pg / mL. The detection limit is calculated to be 3.500 pg / mL based on a signal-to-noise ratio of 3. This can reduce the subject's medication dosage and the amount of whole blood collected, which is beneficial to increasing the safety of clinical research and subject compliance. In addition, the linear range is reasonably selected (15.00-300.0 pg / mL), which can accurately determine the concentration of the test compound in plasma. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the product ion scanning mass spectrum of hydrocortisone butyrate to be measured;

[0027] Figure 2 This is the product ion scan mass spectrum of finasteride;

[0028] Figure 3 : This is the MRM chromatogram of the tested hydrocortisone butyrate and finasteride in the blank plasma sample, wherein the left side is the MRM chromatogram of the tested hydrocortisone butyrate, and the right side is the MRM chromatogram of finasteride;

[0029] Figure 4 This is the MRM chromatogram of the quantification limit sample for hydrocortisone butyrate and finasteride, wherein the left side is the MRM chromatogram of the quantification limit sample, and the right side is the MRM chromatogram of finasteride;

[0030] Figure 5 This is the chromatogram when the chromatographic column in 1.1 of Example 1 is Agilent ZORBAX Eclipse Plus C18;

[0031] Figure 6 This is the chromatogram when the chromatographic column in 1.1 of Example 1 is Waters XBridge C18;

[0032] Figure 7 This is the chromatogram when the chromatographic column in 1.1 of Example 1 is Shim-pack Velox SP-C18 1.8 μm, 2.1×100 mm;

[0033] Figure 8 This is the chromatogram when the aqueous phase uses distilled water:formic acid (v:v, 1000:1) and the organic phase uses methanol:acetonitrile (v:v, 50:50) in 1.2 of Example 1;

[0034] Figure 9 This is a chromatogram obtained when the aqueous phase in step 1.2 of Example 1 is composed of distilled water:1M NH4F (v:v, 1000:1) and the organic phase is composed of methanol:acetonitrile (v:v, 50:50);

[0035] Figure 10 This is the chromatogram when elution gradient 1 is used in 1.4 of Example 1;

[0036] Figure 11 This is the chromatogram when elution gradient 2 is used in 1.4 of Example 1;

[0037] Figure 12 This is the chromatogram when elution gradient 3 is used in 1.4 of Example 1;

[0038] Figure 13 This is a drug concentration-time curve diagram of the in vivo pharmacokinetic study of Example 5. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail with reference to the following specific examples and accompanying drawings. The protection content of the present invention is not limited to the following examples. Without departing from the spirit and scope of the inventive concept, any changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims. The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art and are not particularly limited in the present invention.

[0040] In the present invention, unless otherwise specified, the percentages and ratios mentioned in the reagents are weight percentages and weight ratios.

[0041] In the present invention, the unit "M" used represents mole per liter (mol / L), and "v / v" and "v / v / v / v" used represent volume ratios.

[0042] The instruments used in this invention are:

[0043]

[0044] Sources of reagents used in the present invention:

[0045] Methanol (HPLC grade), acetonitrile (HPLC grade), and isopropanol (HPLC grade) were purchased from Sigma. Formic acid (HPLC grade) and ammonium formate (HPLC grade) were purchased from ACS. Deionized water (18.2 mΩ, TOC ≤ 50 ppb) was prepared using a Milli-Q ultrapure water system. All other raw materials and reagents not listed were purchased from reputable commercial sources.

[0046] Example 1 Screening of chromatographic conditions

[0047] 1.1 Screening of chromatographic columns

[0048] In order to determine the best chromatographic column, we screened columns of different fillers and brands. The specific steps are as follows:

[0049] Packing Type Screening: We selected different types of packing materials, including C18, C8, C1, and phenyl, to evaluate their separation performance for the target compounds. By comparing the resolution, peak shape, and retention time of each packing material, we preliminarily determined that C18 packing material had the best separation performance.

[0050] Brand Screening: After determining the packing material, we further compared C18 columns from different brands, including Shim-pack Velox SP-C18, Agilent ZORBAX Eclipse Plus C18, and Waters XBridge C18. Experimentation revealed that the Shim-pack Velox SP-C18 column was optimal. To achieve better peak shape, sensitivity, and separation, we ultimately selected the Shim-pack Velox SP-C18 1.8μm, 2.1×100mm column.

[0051] 1.2 Screening of mobile phase and column temperature

[0052] In order to determine the optimal mobile phase, we screened a variety of aqueous and organic phases. The specific steps are as follows:

[0053] Aqueous Phase Screening: We used ammonium formate, ammonium acetate, formic acid, acetic acid, and ammonium fluoride as aqueous phases and compared their performance in separating the target compounds. The results showed that 1M ammonium fluoride (v:v, 1000:1) provided the best separation and peak shape for the target compounds.

[0054] Organic Phase Screening: We used methanol and acetonitrile as the organic phase and compared their separation performance. The results showed that the separation and peak shape of the target compound were best when methanol and acetonitrile (v:v, 50:50) were used as the organic phase.

[0055] Column temperature screening: We optimized the column temperature by conducting experiments at 30°C, 35°C, 40°C, and 45°C. The experimental results showed that the separation and peak shape of the target compound were optimal at a column temperature of 35°C, so 35°C was ultimately selected.

[0056] 1.3 Screening of flow rate and injector temperature

[0057] Flow rate screening: We optimized the flow rate and conducted experiments at 0.3 mL / min, 0.42 mL / min, and 0.5 mL / min. The results showed that the separation and peak shape of the target compound were best at a flow rate of 0.42 mL / min, so the final flow rate was 0.42 mL / min.

[0058] Injector temperature screening: We optimized the injector temperature and conducted experiments at 5°C, 10°C, and 15°C. The experimental results showed that when the injector temperature was 5°C, the compound treatment solution was more stable and less likely to volatilize.

[0059] 1.4 Screening of elution gradient

[0060] To determine the optimal elution gradient, we screened various gradients, adjusting the starting conditions and gradient variations to observe the separation of the target compound. The final gradient conditions were: 35% A, 65% B at 0.80 min; 5% A, 95% B at 2.00 min; 5% A, 95% B at 2.80 min; and 35% A, 65% B at 2.90 min. Elution was terminated at 4.00 min. Results showed that the analyte peak was baseline-separated from the impurity peaks, demonstrating optimal separation of the target compound under this gradient.

[0061] Table 1 Elution gradient 1

[0062]

[0063] Table 2 Elution gradient 2

[0064]

[0065] Table 3 Elution gradient 3

[0066]

[0067] Example 2 Screening of mass spectrometry conditions

[0068] Source temperature significantly influences the ionization efficiency and ion transport of a compound. We gradually adjusted the source temperature, starting from room temperature and increasing it to 500°C, while monitoring the signal intensity and stability of the target compound. Results showed that at 500°C, the signal intensity of the target compound reached its maximum and remained stable, leading us to settle on a source temperature of 500°C. Higher source temperatures promote the volatilization and ionization of the compound, but excessively high temperatures can lead to thermal decomposition. 500°C represents a balance point, ensuring effective ionization without causing thermal decomposition.

[0069] We gradually adjusted the gas parameters and monitored the signal intensity and stability of the target compound. The results showed that the signal intensity of the target compound reached its maximum and stable state under the following parameters. Therefore, the final gas parameters were determined to be CUR 35.0psi, CAD 8.0psi, Gas 155.0psi, and Gas 260.0psi.

[0070] Example 3 Screening of pre-treatment process

[0071] 3.1. Screening of sample preparation process

[0072] Take 200.0 μL each of the mixed standard curve sample, quality control sample, blank matrix sample, STD0 (blank sample spiked with internal standard only, without the analyte), test sample, equilibrium sample, and SST (system suitability) sample and add them to 2 mL EP tubes. Then, add 50.0 μL of the internal standard working solution to each sample. Perform liquid-liquid extraction, centrifugation, nitrogen drying, and dilution according to the steps in Table 4.

[0073] Take 200 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. Add 200 μL of ultrapure water to each sample and mix thoroughly. Then, add 50 μL of the internal standard working solution to each sample. Perform liquid-liquid extraction, centrifugation, nitrogen drying, and dilution according to the steps in Table 4.

[0074] Table 4 Processing steps for plasma samples

[0075]

[0076] Table 5 Extraction results after adding 200.0 μL ultrapure water

[0077]

[0078] By comparing the experimental results of the two groups, the recovery rate of the group without water addition was only 20% to 30%, and it was found that the recovery rate of the group with water addition was greatly improved. Therefore, in subsequent experiments, water was added before adding the internal standard working solution.

[0079] 3.2 Screening of internal standard working solution configuration methods

[0080] Internal standard working solutions of varying concentrations and compositions were prepared, such as finasteride solutions of varying concentrations and acetonitrile-ultrapure water mixtures of varying proportions. Comparison of the experimental results from various internal standard working solution configurations revealed that the optimal internal standard working solution configuration was a solution with a finasteride concentration of 200.0 pg·mL-1.

[0081] 3.3 Screening of extraction solvent

[0082] Different extraction solvents such as ethyl acetate, dichloromethane, and n-hexane were selected. Samples were processed according to the conditions in Table 6, and different extraction solvents were added. Liquid-liquid extraction, centrifugation, nitrogen drying, and dilution were performed. The experimental results of different extraction solvents were compared and their impact on the experimental results was evaluated. By comparing the experimental results of different extraction solvents, it was found that when ethyl acetate was used as the extraction solvent, the experimental results were the most ideal. The data on recovery rate and matrix effect met the regulatory requirements. Therefore, ethyl acetate was selected as the extraction solvent.

[0083] Table 6 Processing steps for plasma samples

[0084]

[0085] 3.4 Screening of the ratio of plasma sample to internal standard working solution

[0086] Different ratios of plasma sample and internal standard working solution were prepared, such as 1:1, 2:1, and 4:1. Samples were processed according to the steps in the final Table 7, and different ratios of internal standard working solution were added. Liquid-liquid extraction, centrifugation, nitrogen drying, and dilution were performed. The experimental results of different ratios were compared to evaluate their impact on the experimental results. By comparing the experimental results of different ratios, it was found that the optimal ratio of plasma sample to internal standard working solution was 4:1, so this ratio was selected for subsequent experiments.

[0087] Table 7 Processing steps for plasma samples

[0088]

[0089] Example 4 Methodology Verification Example

[0090] 1. Preparation of solutions and samples

[0091] Standard Series Samples: Accurately weigh an appropriate amount of each reference substance (hydrocortisone butyrate) and dissolve it in methanol:DMSO (50:50, v:v) to a constant volume to prepare a stock solution of approximately 2.000 mg / mL of hydrocortisone butyrate. Accurately pipette an appropriate amount of each stock solution and serially dilute them with methanol:water (50:50, v:v) to prepare the standard working solutions. Finally, prepare the standard plasma samples using blank plasma. The hydrocortisone butyrate concentration range is 15.00-300.0 pg / mL.

[0092] Quality control samples: A mixed quality control sample of hydrocortisone butyrate was prepared at three concentration levels using a method similar to that used for the standard series samples. The lower limit of quantification (LOQ) was 15.00 pg / mL, the low quality control (LQC) was 25.00 pg / mL, the medium quality control (MQC) was 100.00 pg / mL, and the high quality control (HQC) was 225.0 pg / mL.

[0093] Internal standard working solution: Accurately weigh the finasteride reference substance and dilute to a concentration of approximately

[0094] 1.000 mg / mL internal standard stock solution. Accurately pipette an appropriate amount of each internal standard stock solution and dilute it with acetonitrile:water (20:80, v:v) to obtain an internal standard working solution with a finasteride concentration of 200.0 pg / mL.

[0095] 2. Plasma Sample Processing Steps

[0096] Table 8 Plasma sample processing steps

[0097]

[0098] 3. Chromatographic and mass spectrometry conditions

[0099] Table 9 Chromatographic conditions

[0100]

[0101]

[0102] Table 10 Mass spectrometry conditions

[0103]

[0104] 4. Methodological Validation

[0105] The method was validated according to the Chinese Pharmacopoeia 9012 guidelines and ICH M10, including stability, selectivity, linearity, accuracy, precision, recovery, and matrix effect.

[0106] Selectivity

[0107] Six blank plasma samples from different sources and their respective prepared lower limit of quantitation samples were processed and analyzed. The peak area of the chromatographic coeluting interfering substance must be less than 20% of the peak area of the lower limit of quantitation analyte and less than 5% of the peak area of the internal standard.

[0108] Standard curve

[0109] The linear regression equation (weighting factor W = 1 / x2) was calculated using the theoretical concentration of the analyte as the abscissa (x) and the peak area ratio of the analyte to the internal standard as the ordinate (y). Method validation involved double analysis of the standard curve samples for each analytical batch.

[0110] Precision and accuracy

[0111] For method validation, six quality control samples at each of four concentration levels were measured for each analytical batch. Acceptable results were achieved when the intra- and inter-assay precision for the lower limit of quantification (LOQ) was less than 20%, calculated as the relative standard deviation (RSD). Accuracy, calculated as the relative error (RE), was between -20% and 20%. For each component of the remaining QC samples at each concentration level, acceptable results were achieved when the intra- and inter-assay precision was less than 15%, and the accuracy was between -15% and 15%.

[0112] stability

[0113] To investigate the stability of each analyte in plasma samples, LQC and HQC samples were subjected to various temperatures and environments, and six samples were analyzed after each exposure. Four storage conditions were investigated: 31 hours in an ice-water bath, 114 hours at 4°C after preparation, five freeze-thaw cycles (from -80°C to room temperature), and 43 days at -80°C.

[0114] Recovery rate

[0115] 200 μL of blank plasma was extracted (without the addition of internal standard working solution). The analyte solution and internal standard working solution were then added to achieve the same final concentration as that of the LQC, MQC, and HQC. Six aliquots of each of the LQC, MQC, and HQC were also extracted and injected. The extraction recovery was calculated based on the peak area ratio of the two treatment methods.

[0116] Matrix effects

[0117] Six blank plasma samples from different sources were extracted (without the addition of internal standard working solution). The analyte solution and internal standard working solution were added at the same concentrations as those used in the LQC and HQC samples, mixed by vortexing, and then measured. Water was used to replace the plasma and processed as described above. The matrix factor was calculated using the ratio of the peak areas obtained by the two methods. Matrix effects were assessed using the RSD of the matrix factor normalized to the internal standard; a value less than 15% was considered acceptable.

[0118] 5. Results and Discussion

[0119] Selectivity of method

[0120] like Figures 3 and 4 As shown, the retention times of hydrocortisone butyrate and finasteride are approximately 2.01 and 2.00 min, respectively, and there are no co-eluting interference peaks at these retention times.

[0121] Standard curve

[0122] The linear range for the determination of hydrocortisone butyrate in plasma samples was 15.00-300.0 pg / mL. The typical linear regression equations for the standard curves of the analytes were:

[0123] Hydrocortisone butyrate: y = 1.35x + -0.00177;

[0124] Detection limit

[0125] The quantification limit of hydrocortisone butyrate in the sample was 15.00 pg / mL, and the signal-to-noise ratio was 12.7. Based on a signal-to-noise ratio of 3, the detection limit was 3.500 pg / mL.

[0126] Precision and accuracy of the method

[0127] 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.

[0128] Table 11 Results of precision and accuracy investigation of hydrocortisone butyrate

[0129]

[0130] Treatment recovery rate

[0131] The extraction recoveries of hydrocortisone butyrate at LQC, MQC, and HQC concentration levels were 84.5%, 85.0%, and 86.1%, respectively; the overall recovery was 85.2%, and the overall CV was 0.96%.

[0132] Matrix effects

[0133] The matrix factors for the internal standard normalized to hydrocortisone butyrate at the LQC and HQC concentration levels were 97.0% and 95.0%, respectively, with RSDs of 4.12% and 2.11%, respectively. These results indicate that matrix effects do not interfere with the accuracy of the analyte analysis.

[0134] Plasma stability study

[0135] The results of the plasma stability test are shown in Table 12, which show that hydrocortisone butyrate is stable under the conditions tested, wherein Table 12 shows the stability of hydrocortisone butyrate in rat plasma (n=6).

[0136] Table 12 Stability study results of hydrocortisone butyrate in rat plasma

[0137]

[0138] Example 5 In vivo pharmacokinetic study

[0139] The validated method was used to analyze hydrocortisone butyrate in plasma to evaluate the pharmacokinetic characteristics of hydrocortisone butyrate. A single-center, single-dose, randomized, open-label, two-period crossover design was used. Twelve healthy subjects were enrolled and received a single dose of the test preparation (T, strength: 0.1% (30g:30mg)) or the reference preparation (R, strength: 0.1%, trade name: ), the drug was administered to the back skin and then wiped off after 24 hours. The plasma concentration of hydrocortisone butyrate in plasma at different sampling times was determined by LC-MS / MS. The pharmacokinetic parameters were calculated using a non-compartmental model and statistically analyzed. The plasma drug concentration-time curve is shown in Figure 13 The sensitivity of the detection method can fully describe the pharmacokinetic characteristics of hydrocortisone butyrate.

[0140] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A bioanalytical method for detecting the concentration of hydrocortisone butyrate in plasma, characterized in that: The method is liquid chromatography-tandem mass spectrometry, wherein the liquid chromatography adopts the following conditions: Chromatographic column: Shim-pack Velox SP-C18, Monitor: DAD, Mobile phase A: distilled water: 1M NH4F, v:v = 1000:1 Mobile phase B: methanol: acetonitrile, v:v = 50:50, Flow rate: 0.42 mL / min, Column temperature: 35°C, Autosampler temperature: 5°C, The elution gradient is:

2. The method according to claim 1, characterized in that The specifications of the chromatographic column are 2.1×100 mm and 1.8 μm.

3. The method according to claim 1, characterized in that The mass spectrometry employed the following conditions: electrospray ion source, positive ion detection, multiple reaction monitoring mode, spray voltage 3000 V, and ion source temperature 500°C.

4. The method according to claim 3, characterized in that In the mass spectrometry conditions, gas 1 is 55.0 psi, gas 2 is 60.0 psi, curtain gas is 35 psi, collision gas is 8 psi, and dwell time is 200 ms; the quantitative analysis ion pair of hydrocortisone butyrate to be measured is m / z 433.3 / 120.9, the collision energy is 34 eV, the declustering voltage is 50 V, the entrance voltage is 10 V, and the collision cell exit voltage is 11 V; the quantitative analysis ion pair of internal standard finasteride is m / z 373.1 / 305.2, the collision energy is 40 eV, the declustering voltage is 50 V, the entrance voltage is 10 V, and the collision cell exit voltage is 10 V.

5. The method according to claim 1, wherein Before using liquid chromatography-tandem mass spectrometry for sampling and detection, the sample must be pretreated. The pretreatment method is as follows: water is added to the plasma sample, vortexed to mix, and then the internal standard working solution is added. After vortexing to mix, ethyl acetate is added for liquid-liquid extraction. After vortexing to mix, the supernatant is centrifuged and diluted with a diluent to obtain the test sample; the internal standard working solution is a methanol and acetonitrile solution of finasteride.

6. The method according to claim 5, 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, and the concentration of hydrocortisone butyrate to be measured in the plasma sample is 15.00 pg / mL-300.0 pg / mL.

7. The method according to claim 5, characterized in that The concentration of finasteride in the internal standard working solution was 200.0 pg / mL.

8. The method according to claim 5, characterized in that The diluent is acetonitrile:water:formic acid, and the volume ratio of acetonitrile:water:formic acid is 500:500:1.

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