Development of a simple and rapid method to measure the free fraction of tacrolimus in plasma using ultrafiltration and LC-MS/MS

BACKGROUND: Tacrolimus is an immunosuppressant mainly used in the prophylaxis of solid organ transplant rejection. Therapeutic drug monitoring of tacrolimus is essential for avoiding toxicity related to overexposure and transplant rejection from underexposure. Previous studies suggest that unbound tacrolimus concentrations in the plasma may serve as a better predictor of tacrolimus-associated nephro- and neurotoxicity compared to tacrolimus concentration in whole blood. Monitoring the plasma concentrations of unbound tacrolimus might be of interest in preventing tacrolimus-related toxicity. Therefore, the aim was to develop a method for the measurement of total and unbound tacrolimus concentrations in plasma. METHODS: The sample preparation for the determination of the plasma concentrations of unbound tacrolimus consisted of an easy-to-use ultrafiltration method followed by solid phase extraction. To determine the total concentration of tacrolimus in plasma, a simple method based on protein precipitation was developed. The extracts were injected into a Thermo Scientific HyPurity C18 column using gradient elution. The analytes were detected by liquid chromatography-mass spectrometry using a triple quadrupole with positive ionization. RESULTS: The method was validated over a linear range of 1.00-200 ng/L for unbound tacrolimus concentrations in plasma and 100-3200 ng/L for total plasma concentrations. The lower limit of quantification was 1.00 ng/L in ultrafiltrate and 100 ng/L in plasma.The inaccuracy and imprecision for the determination of unbound tacrolimus concentrations in ultrafiltrate and plasma showed a maximum CV of 11.7% and a maximum bias of 3.8%. CONCLUSION: A rapid and easy method based on ultrafiltration and LC-MS/MS was established to measure the total and unbound tacrolimus concentrations in plasma. This method can facilitate further investigations on the relationship between plasma concentrations of unbound tacrolimus and clinical outcomes in transplant recipients.