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

The aim of this work was to develop a selective method for the simultaneous quantification of cyclosporine, tacrolimus, sirolimus and everolimus in whole blood. 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/H 2O (5/95 v/v), supplemented with formic acid 0.02% and sodium formate 1 μM. After column-switching, the analytes were transferred in the back-flush mode on a C18 column with MeOH/H 2O (65/35). The valve was then commuted to its initial position and the chromatographic separation was performed with a gradient of MeOH/H 2O (65/35–95/5). The sodium adducts [M+Na] + were monitored for quantification with an electrospray ionization-single quadrupole MS. The LC–MS assay was fully validated on a concentration range of 2.5–30 ng/mL for tacrolimus, sirolimus and everolimus and of 50–1500 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. This method provided a selective, rapid and automated procedure that can be easily used for routine quantification of immunosuppressive drugs in most clinical laboratories.