Objective: To establish an in vitro release evaluation method for tacrolimus ointment, and compare the consistency of in vitro release behavior between domestic generic products and the reference product. Methods: An in vitro release method for tacrolimus ointment was established using immersion cell-Model A. The mixture of anhydrous ethanol and physiological saline (30 ∶ 70) was selected as the release medium, and 0.45 μm polytrafluoroethylene (PTFE) membrane was used as the artificial membrane, the rotation speed was set at 200 r · min-1. High-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) was adopted to determine the release amount and release rate of tacrolimus, using Agilent Zorbax SB C18 (150 mm×2.1 mm, 3.5 µm) column and gradient elution. 0.1% formic acid solution (containing 5 mmol · L-1 ammonium formate) (A) and methanol (B) were used as the mobile phase, with column temperature of 55 ℃, and an injection volumn of 10 μL. The release amounts of the generic and reference products of tacrolimus ointment were measured, and their release rates were calculated. Wilcoxon and Mann-Whitney U statistical methods were employed to calculate the 90% standard confidence interval of the ratio of release rates between the generic product and the reference product, evaluating the consistency of in vitro release. Results: The LOD of the established method was 0.026 ng · mL-1, and the recovery of released tacrolimus in the receptor solution (mass balance) was within 92.6%-99.3%. Among the two domestic generic tacrolimus ointments, the 90% standard confidence interval of the ratio of the release rate between generic product 1 and reference product was 97.92%-122.19% which fell within the acceptable range of 75.00%-133.33%, indicating consistency in in vitro release rate with the reference product. The 90% confidence intervals of the ratio of the release rate between the generic product 2 and the reference product were all out of range of 75.00%-133.33% in both the initial and repeat tests, suggesting inconsistency in in vitro release rate compared to the reference product. The results obtained using the immersion cell method were consistent with those obtained using the Franz diffusion cell method. Conclusion: The developed immersion cell method meets the requirements for evaluating the in vitro release of tacrolimus ointment and the results are reliable. The immersion cell in vitro release method can be used to evaluate the in vitro release behavior of tacrolimus ointment.
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