NGHIÊN CỨU XÂY DỰNG VÀ THẨM ĐỊNH PHƯƠNG PHÁP ĐỊNH LƯỢNG TACROLIMUS TRONG MÁU NGƯỜI BẰNG PHƯƠNG PHÁP SẮC KÝ LỎNG KHỐI PHỔ

Abstract

Objectives: To develop and validate an LC-M/SMS method for the determination of tacrolimus (FK506) in whole blood in kidney transplant recipients. Subjects and methods: Tacrolimus and the internal standard were extracted from human whole blood using methanol/1.125M ZnSO₄ (66/34, v/v) and separated using gradient elution on an ACQUITY UPLC® BHE C18 column (2.1x100 mm; 1.7 µm) kept at 25°C with acetonitrile and 5 mM ammonium acetate as mobile phase. The flow rate was 0.2 mL/min, and a 5 µL aliquot of residues was injected into the LC-MS/MS. Detection was carried out by multiple reactions monitoring on a MS/MS Xevo TQD system. The method validation was performed according to FDA guidelines and applied to determine tacrolimus concentration in kidney transplant patients. The method comparison of LC-MS/MS vs chemiluminescent microparticle immunoassay (CLIA) was performed using a standardized major axis regression analysis and Bland-Altman plots. Results: The method had a total chromatographic run time of 5 minutes. The calibration curves were linear over the range of 1.0 - 100.0 ng/mL with a lower limit of quantification of 1 ng/mL. The intra-day and inter-day accuracy were within the range of 93.3 - 109.2% and 96.0 - 108.4%, respectively, and the intra-day and inter-day precision ranged from 0.8 - 9.4%. The mean extraction recoveries of tacrolimus ranged from 102.6 - 107.8%. The methods of accuracy, precision, and recovery meet the acceptance criteria according to FDA guidelines. The results of the correlation and bias in tacrolimus concentrations between the two methods show that the LC-MS/MS method is reliable for application in clinical tacrolimus concentration monitoring. Conclusion: A rapid, sensitive, selective, and reliable method for the determination of tacrolimus in human whole blood has been successfully developed, validated, and applied.