Simultaneous quantification of cyclosporine, tacrolimus, sirolimus and everolimus in whole blood by liquid chromatography - electrospray mass spectrometry

Objectives: The aim of this work was to develop a selective method for the simultaneous quantification of cyclosporine, tacrolimus, sirolimus and everolimus in whole blood. Design and methods: An automated on-line solid-phase extraction system coupled with liquid chromatography-mass spectrometry (LC-MS) was used. After a simple protein precipitation, the supernatant was load on a C8 column with a mobile phase composed of MeOH/H2O (5/95 v/v), supplemented with formic acid 0.02% and sodium formate 1 mu M. After column-switching, the analytes were transferred in the back-flush mode on a C18 column with McOH/H2O (65/35). The valve was then commuted to its initial position and the chromatographic separation was performed with a gradient of MeOH/H2O (65/35-95/5). The sodium adducts [M+Na](+) were monitored for quantification with an electrospray ionization-single quadrupole MS. Results: The LC-MS assay was fully validated on a concentration range of 2.5-30 ng/mL for tacrolimus, sirolimus and everolimus and of 501500 ng/mL for cyclosporine, allowing a quantification of cyclosporine 2 h post-dose without sample dilution. Trueness, repeatability and intermediate precision were found to be satisfactory. Conclusion: This method provided a selective, rapid and automated procedure that can be easily used for routine quantification of immumosuppressive drugs in most clinical laboratories. (c) 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.