Study On Quality And Stability Of Tacrolimus

Tacrolimus (FK506) is a new cytokine inhibitor synthesized after cyclosporine (CsA). Compared with CsA, immunocompetence of tacrolimus is 50～100 times more than that of CsA, which was confirmed in vivo and in vitro examination and it can prevent effectively acute rejection after renal transplantation in clinic. Tacrolimus was first isolated in 1985 and has gotten good evaluation since it was trial-used in clinic in 1989 and at present, it is taken as the first-line drug for prevention, treatment of rejection and autoimmune diseases.1. Study on tacrolimus qualityIn this thesis, the characteristics of tacrolimus were investigated through appearance, illumination test and moisture test and this product was proved to have no significant moisture. Physical constants such as melting point, solubility and specific rotation were determined and the melting point was compared with that measured by DSC.Tacrolimus is identified mainly by retention time of HPLC and infrared spectrum. Retention time of HPLC had good specificity, while infrared spectrum was proved to conform to the functional groups of the molecular structure and they are the effective methods to identify bulk drug.The quality of tacrolimus is affected with the complexity of fermentation process. The test of the related substances in tacrolimus has been investigated. At first, the test system of the related substances was investigated and optimized through the selection of column temperature and solvents of isocratic HPLC. It was proved that the higher column temperature can be used to detect tacrolimus and the related substances effectively. Acetonitrile as solvent can reduce the transformation of tacrolimus to its isomer. Then, to separate the related substances more effectively, the method was adjusted to gradient elution system. The composition of mobile phase, the proportion of gradient, column and detection wavelength were investigated. The conditions of HPLC for testing the related substances were optimized and the analytical method was validated. The validated parameters consist of specificity, precision, linearity and range, accuracy, solution stability and limit of detection (10 ng). The structure assignment of main related substances was studied by LC-MS. The results showed that the degradation substances in tacrolimus caused by the destroying acid, alkali, oxidation, illumination and high temperature can be separated from intermediates produced during manufacturing process by HPLC established in this thesis. Finally, the related substances in three batches of sample were detected and the contents of the related substances were less than 1.0%.Because ethanol and acetone were used as solvents in the synthesis process of tacrolimus, the residual solvents are tested by GC with headspace sampling. The method specificity, repeatability and sensitivity were validated. The residual organic solvents in three batches of sample were determined and the results met the pharmacopoeia requirements. The clarity and color of ethanol, crystalline water, residue on ignition, heavy metal, arsenic salt, sulfate and chloride were also tested.The assay for tacrolimus by HPLC was developed and the analytical method was validated including accuracy, linearity and range, precision and solution stability. The contents in three batches of tacrolimus are more than 98.5%.The methods established in this thesis have been employed for quality control to tacrolimus.2. Establishment of tacrolimus quality standardOn the basis of overall quality research and in combination with practice situation and operating-convenience, quality standard of tacrolimus was established including description, identification, detection and assay. The physical constants such as melting point and specific rotation were measured. Tacrolimus was identified by spectrum (IR) and chromatography (HPLC). The related substances were detected by HPLC and the residue of ethanol and acetone by GC with headspace sampling. The residue on ignition, heavy metal, arsenic salt and water content was also determined. Tacrolimus was assayed by RP-HPLC.3. Stability testBased on the quality standard, the stability of tacrolimus was tested including influence factor test, accelerated test and long-term stability test. The results show that the sample of tacrolimus is stable under the conditions of high moisture, high temperature, 6-month accelerated test and 12-month stability test, but it should be stored from light.