In this study, the test formulation was TA injection (40 mg/1 mL/ampule, lot no. 221027, expiration date September 2023), provided by Shenyang Everbright Pharmaceutical Co.Co., Ltd., whereas the reference formulation was TA injection (40 mg/1 mL/ampule, lot no. 133, expiration date November 2023), manufactured by Laboratorio Italiano Biochimico Farmaceutico Lisapharma S. p.A. Eighteen healthy male volunteers, ages between 20 and 27 years and weighing between 56 and 82 kg, were selected based on inclusion criteria. Participants signed informed consent forms, underwent comprehensive physical examinations at the First Clinical Hospital of China Medical University before the experiment, and completed blood, urine, liver, and kidney function tests that were all normal. The medical professionals who conducted the experiment closely monitored blood collection times, and two nurses were responsible for blood collection. The study was conducted at the National Institute for Drug Clinical Experiments, First Affiliated Hospital of China Medical University.

To explore the bioequivalence of the test and reference formulations of TA after intramuscular injection, an open, randomized, single-dose crossover experiment was conducted with 18 healthy volunteers divided into two groups of 9. Group T received the test formulation before the reference formulation, whereas Group R received the reference formulation first, followed by the test formulation. The administration of each formulation was separated by a 6-week interval. Before injection of either formulation, blood samples were taken from the participants, and at 7:00 a.m., an 80 mg test or reference formulation was administered intramuscularly. Blood samples were taken at regular intervals of 0.5, 1.0, 1.5, 2.0, 3.0, 5.0, 8.0, 12.0, 24.0, 48.0, 96.0, 192.0, 336.0, 504.0, and 720.0 h after administration, with 3– to 5 mL venous blood drawn from the upper limb and stored at −20°°C until analysis. A standard low-fat meal was given 4.5 h after administration. After the first test period was completed, the same procedure was repeated in the second period after a 6-week clean-out period.

The purity of TA (Figure 1A) and cortisone acetate (CA; Figure 1B) was 99.8% and 99.6%, respectively, which were obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Ultra-pure water was prepared from Millipore (Bedford, MA, USA), and HPLC-grade acetonitrile was obtained from Merck (Darmstadt, Germany). Analytical-grade ethyl acetate was obtained from Sinopharm Chemical Reagent Co.Co., Ltd. (Shenyang, China). The blank plasma used in the experiment was obtained from the General Hospital of Shenyang Military.

The UPLC-ESI-MS/MS equipment used in the experiment included an ACQUITY™ ultra-performance LC quaternary pump, an ACQUITY™ autosampler, and a Micromass^®® Quattro Premier XE ES-MS/MS equipped with an ESI ion source (Waters Corp., Milford, MA, USA). Data acquisition and analysis were performed using Masslynx version 4.1. UPLC separation was carried out on an ACQUITY™ BEH C18 column (50 × 2.1 mm, 1.7 μm, Waters Corp.) at 40°°C. The mobile phase was a mixture of acetonitrile/water containing 1% formic acid (55:45, v/v), and a constant mobile phase flow rate of 0.2 mL/min was employed throughout the analysis. The total run time was 2.0 min. Nitrogen was used to assist nebulization during UPLC-ESI-MS/MS, and MS detection was performed using an electrospray ion source in the positive ionization mode. The ion spray for TA was optimized at a spray voltage of 3000 V and a capillary temperature of 350°°C, with the nitrogen sheath, ion sweep, and auxiliary set at 45, 10, and 10 psi, respectively. Collision energies of 13 V were optimized for TA and CA, and multiple-reaction monitoring (MRM) was used for data acquisition. MRM fragmentation transitions were m/z 435.4→397.3 for TA and m/z 403.4→163.1 for CA, with a scan dwell time of 0.2 s set for each channel.

In this study, 500 µL plasma was taken, and 50 µL of 40% acetonitrile aqueous solution was added. Then, 50 µL of 500 ng/mL CA internal standard (I.S.) solution comprising of 40% acetonitrile-water mixture was added. The mixture was vortexed for 1 min before adding 3 mL ethyl acetate/hexane (4:1, v/v). The solution was vortexed for 3 min minutes and centrifuged at 4000 rpm for 10 min, then the supernatant 500 µL was taken and transferred to a separate tube. The residue was dried under a stream of nitrogen and dissolved with 100 µL mobile phase. The mixture was vortexed for 1 min at 15,000 rpm. After centrifugation for 3 min, 10 µL supernatant was injected for analysis.

The primary excimer ions generated by TA and CA under the ESI(+) mode were m/z 435.4→397.3 and 403.4→163.1, respectively. During quantitative analysis, they were used as product ions to monitor TA and CA. Blank plasma collected from six different individuals was processed according to the sample processing method, and a 10 µL sample was injected to obtain a typical chromatogram (Figure 2A). A certain concentration of the standard solution and I.S. was spiked into blank plasma, and the chromatogram was obtained using the same method (Figure 2B). Chromatograms were also obtained from plasma samples collected from healthy subjects after intramuscular injection (Figure 2C). The retention times of TA and CA were 0.92 and 1.09 min, respectively. Results indicated that endogenous substances in the blank plasma did not interfere with TA and CA determination.

As the UPLC/MS/MS detection method was based on soft ionization MS, the possible dielectric effects in the analysis process were investigated by preparing two samples: one with 500 µL blank plasma and the other with 500 µL deionized purified water. Then, 50 µL low, medium, and high concentrations (1.06, 5.30, and 21.20 ng/mL) of TA solutions were spiked, and three samples were analyzed for each concentration level. A 10 µL sample was injected, and the chromatogram was recorded to obtain the peak area response values of TA and CA. The matrix effect was determined by comparing the mean peak area of the sample prepared with blank plasma to that of the same concentration sample prepared with deionized purified water. The matrix effects were 95.788%, 100.954%, and 101.079% for the three concentrations, respectively, ranging from 85% to 115%. The I.S. matrix effect was 100.554% (Supplementary Table S1).

For each concentration point of the designed calibration curve, 500 μL blank plasma was taken, and a series of standard solutions were spiked to prepare TA plasma standard series samples corresponding to concentrations of 0.53, 1.06, 2.12, 5.30, 10.60, and 21.20 ng/mL. Following the “Plasma sample processing” method, each concentration was used for single sample analysis, and 10 µL was injected. The chromatogram was recorded, with TA blood concentration as the abscissa and TA and CA peak area ratio as the ordinate. The weighted least-squares method was used for regression analysis, and the obtained linear regression equation was the calibration curve. The results of the 3-day validation method and the calibration curve regression equations for each batch of the methodology are presented in Supplementary Table S2. Figure 3 shows a typical calibration curve. Supplementary Table S1 shows each concentration point of the standard curve. According to the calibration curve, the linear range of TA was 0.53– to 21.20 ng/mL, and LLOQ was 0.53 ng/mL. Supplementary Table S3 shows the accuracy and precision results of LLOQ.

For each concentration, 500 µL blank plasma was taken, and quality control (QC) samples with low, medium, high and TA concentrations (1.06, 5.30, and 21.20 ng/mL, respectively) were prepared according to the method described in “Calibration curve and LLOQ” and processed according to the “Plasma sample processing” method. Five samples were analyzed for each concentration level, and the continuum determination was performed over 3 days. The measured TA concentration in the QC sample was calculated based on the calibration curve of the same day. The accuracy and precision of this method were calculated based on the measured TA concentration in the QC sample. The intra- and interday precision and accuracy checks are presented in Supplementary Tables S4a, b, respectively.

For each sample, 500 µL blank plasma was taken, and TA QC plasma samples with low, medium, and high concentrations (1.06, 5.30, and 21.20 ng/mL) were prepared according to the method described in “Precision and accuracy” and processed according to the “Plasma sample processing” method. Three samples were analyzed for each concentration level. Another 500 µL blank plasma was taken for each sample and processed according to the “Plasma sample processing” method (without adding 50 µL of 40% acetonitrile aqueous solution). The obtained supernatant was spiked with 50 µL of the corresponding concentration standard solution, vortexed and mixed for 1 min, and blow-dried under a stream of nitrogen, and the residue was dissolved with 100 µL mobile phase. After vortexing for 1 min at 15,000 rpm, the solution was centrifuged for 3 min, and 10 µL supernatant was injected and analyzed to obtain the corresponding peak areas (average of three determinations). Extraction recovery was calculated as the ratio of the peak areas of the two treatments for each concentration. The recovery rates of low, medium, and high concentrations of TA (1.06, 5.30, and 21.20 ng/mL) were 77.406%, 80.110%, and 99.000%, respectively, and the recovery rate of the I.S. was 90.251% (Supplementary Table S5a). Using 500 µL blank plasma for each sample, 1.06, 5.30, and 21.20 ng/mL TA QC plasma samples were prepared according to the “Precision and accuracy” method and processed using the “Plasma sample processing” method. Three samples were analyzed for each concentration level, and the concentration of the QC samples was calculated based on the calibration curve of the day. Relative recovery was calculated as the ratio of the measured concentration to the spiked concentration for each concentration level. The relative recoveries of this method were 112.358%, 103.874%, and 102.898% at low, medium, and high concentrations of TA (1.06, 5.30, and 21.20 ng/mL, respectively; Supplementary Table S5b).

For each sample, 500 µL blank plasma was used to prepare TA QC plasma samples with low, medium, and high concentrations (1.06, 5.30, and 21.20 ng/mL) as described in “Precision and accuracy,” with three samples per concentration level. QC samples were processed according to “Plasma sample processing” and left at room temperature for 24 h for determination. In Supplementary Table S6a, the treated TA plasma samples were stable for 24 h at room temperature. QC samples were prepared and stored at −20°°C for 120 days, processed separately using the “Plasma sample processing” method, and determined. In Supplementary Table S6b, TA plasma samples stored at −20°°C were stable for 120 days. Furthermore, QC samples were prepared and subjected to three freeze-thaw cycles, with the concentration calculated after each cycle. After processing using the “Plasma sample processing” method, TA plasma samples were stable after three freeze-thaw cycles (Supplementary Table S6c). To evaluate their stability, the TA stock solution (200 ng/mL) and the I.S. stock solution (500 ng/mL) were stored at 4°°C for 30 days. Afterward, 10 µL was injected and analyzed to obtain the corresponding peak areas (Supplementary Table S7). Results showed that TA and I.S. stock solutions were stable within 30 days at 4°°C. Overall, the analytical method met the requirements of relevant regulations and could be used to determine TA plasma concentrations in pharmacokinetic studies.

The TA plasma concentrations in healthy subjects were determined at each time point after intramuscular injection of the reference and test formulations (Supplementary Tables S2, S3, respectively). The corresponding plasma concentration-time curves are displayed in Figures 4, 5. The mean plasma concentration-time curve for all 18 healthy subjects is shown in Figure 6.

The plasma drug concentration-time points of all 18 healthy subjects after intramuscular injection were collected and entered into a Masslynx 4.1 workstation for data analysis. DAS 2.0 was used to calculate the pharmacokinetic parameters and bioequivalence. The trapezoidal method was applied to calculate AUC_0-720 and AUC_0-∞ using the concentration-time curves. Furthermore, t _1/2 was calculated from the concentration-time point of the elimination phase using the semilogarithmic mapping method. The measured T _max and C _max and the obtained pharmacokinetic parameters are presented in Supplementary Tables 8, 9. The relative bioavailability (Fr) was calculated using the AUC_0-720 of the test and reference formulations with the formula: Fr = (AUC test formulation / AUC reference formulation) × 100% (Supplementary Table S10).

After intramuscular injection of 80 mg test and reference formulations in healthy subjects, the pharmacokinetic parameters obtained were as follows: C _max was 8.616 ± 1.232 and 8.285 ± 1.218 ng/mL, T _max was 1.833 ± 0.243 and 1.861 ± 0.230 h, t _1/2 was 181.249 ± 78.585 and 201.782 ± 83.551 h, AUC_0-720 was 835.642 ± 297.209 and 830.684 ± 331.168 ng h/mL, and AUC_0-∞ was 991.859 ± 355.939 and 1018.665 ± 420.769 ng h/mL, respectively. The average relative bioavailability of TA was 105.4% ± 26.9%, as calculated by AUC_0-720. The 90% confidence interval (CI) of the test formulation AUC_0-720 was 92.8%– to 113.4% of the corresponding parameters of the reference formulation, and the 90% CI of the test formulation C _max was 99.1%– to 109.1% of the corresponding parameters of the reference formulation. The 90% CIs of AUC_0-∞ of the test formulation was 89.7%– to 110.9% of the corresponding parameters of the reference formulation (Supplementary Tables S11–S13). The evaluation of bioequivalence was performed according to the protocol requirements, indicating that if the 90% confidence limit of AUC_0-∞ and C _max for the test formulation were within 80%– to 125% and 70%– to 143%, respectively, compared to the reference formulation, the test formulation would be considered bioequivalent. In this study, the test formulation met these criteria ([National Medical Products Administration and Center for Drug Evaluation, 2009)], indicating that it was bioequivalent to the reference formulation.

No adverse effects were reported for the 18 healthy subjects during the bioequivalence experiment, indicating that both formulations were well tolerated at the doses tested. After the experiment, four subjects (2, 4, 15, and 20) reported cough, hoarseness, runny nose, and other symptoms during follow-up physical examinations. The doctor’s diagnosis concluded that the symptoms were due to influenza, likely unrelated to the experimental drug. The study period coincided with a particularly cold winter season, which may have contributed to the onset of cold symptoms. All subjects fully recovered from the cold symptoms, as confirmed by telephone follow-up after the end of the study. The comprehensive evaluation of clinical examination and doctor’s consultation indicated that the experimental drug did not contribute to the reported symptoms.