Development of a Simple and Rapid Method to Measure the Free Fraction of Tacrolimus in Plasma Using Ultrafiltration and LC-MS/MS.
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 nephrotoxicity 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.



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-tandem mass spectrometry with positive ionization.



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 coefficients of variation (CV) of 11.7% and a maximum bias of 3.8%.



A rapid and easy method based on ultrafiltration and liquid chromatography-tandem mass spectrometry 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.
Departments of *Clinical Pharmacy and †Intensive Care, University Medical Center Utrecht, Utrecht; and ‡Department of Clinical Pharmacy, Erasmus University Medical Center, Rotterdam, the Netherlands.
Nicolaas A
NA
Stienstra
Maaike A
MA
Sikma
Anouk L
AL
van Dapperen
Dylan W
DW
de Lange
Erik M
EM
van Maarseveen
D002853:Chromatography, Liquid / Q000379:methods*; D016903:Drug Monitoring / Q000379:methods*; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood*; D057230:Limit of Detection; D010949:Plasma / Q000737:chemistry*; D015203:Reproducibility of Results; D052616:Solid Phase Extraction / Q000379:methods; D016559:Tacrolimus / Q000097:blood*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014462:Ultrafiltration / Q000379:methods*
D007166:Immunosuppressive Agents; D016559:Tacrolimus