Simultaneous simple and fast quantification of three major immunosuppressants by liquid chromatography--tandem mass-spectrometry

Abstract

Objectives: The aim of the current study was to develop a simple, fast and universal method for quantification of any combination of the three major immunosuppressants sirolimus, tacrolimus and cyclosporin in whole blood, using a LC-tandem mass spectrometer (API-2000, SCIEX, Toronto, Canada).

Methods: 250 microL whole blood was spiked with internal standard (ritonavir), and protein precipitated with 350 microL acetonitrile. The sample was centrifuged and 30 microL aliquot was injected onto the HPLC column, where it underwent an online extraction with ammonium acetate. After that the automatic switching valve was activated, changing the mobile phase to methanol and thereby eluting the analytes into the tandem mass spectrometer. The high selectivity of a tandem mass analyzer allows determination of any combination of the three drugs within a 5 min run.

Results: Between-day precision was between 2.4% and 9.7% for all analytes at the concentrations tested. Accuracy ranged between 98.8% and 103.2% (n = 20). The method was linear over the measuring ranges of all analytes. Within-run precision was below %CV = 6% for all analytes. Good correlation with other analytical methods was observed.

Conclusions: The simplicity, universality and high throughput of the method make it suitable for application in a clinical laboratory. The method has been implemented in our laboratory for a routine use.