Improved LC-MS/MS method for the simultaneous quantification of tacrolimus and cyclosporine A in human blood and application to therapeutic drug monitoring.

ABSTRACT

In order to facilitate therapeutic drug monitoring of tacrolimus and cyclosporine A in clinical practice, a simple, rapid, robust, sensitive and specific LC-MS/MS assay was developed and validated for the simultaneous determination of tacrolimus and cyclosporine A in human whole blood. Erythrocytes were destroyed using internal standard solution with 10% (w/v) zinc sulfate in water. The analytes were extracted from 100 µl of whole blood by protein precipitation with acetonitrile. Chromatographic separation was conducted on a Kinetex PFP column (60°C) by a gradient elution with a flow rate of 0.450 ml/min in 2.5 min. Quantitative analysis was performed using electrospray ionization and multiple reaction monitoring in positive ionization mode. The method was fully validated as per current guidelines on bioanalytical methodologies of the US Food and Drug Administration and European Medicines Agency. The method developed was applied successfully in analyzing clinical samples from patients administered tacrolimus or cyclosporine A. The sample treatment procedure was rationalized and improved to fulfill the complete target extraction. The chromatography conditions were optimized to achieve rapid and accurate quantification of both analytes. This method may be beneficial as a constructive input for the therapeutic drug monitoring of tacrolimus and cyclosporine A in obtaining individualized therapy.