Background: Our objective was to develop a high throughput, robust and simple method for quantification of any combination of immunosuppressants like sirolimus, tacrolimus, everolimus, and cyclosporin A by implementing APCI source and LC column multiplexing system.
Material/Methods: Blood samples were processed with a protein precipitation method and analyzed on 10-2.1 mm Hypersil C18 guard column with 2 min LC gradient method (1 min data collection time with two column switch method). Data was collected in MRM mode where ammoniated adducts of protonated molecules were used as precursor ions for all analytes. Ascomycin and cyclosporin D were used as internal standards. The whole blood calibrators and QC samples with concentrations across calibration range were prepared in house. The method was validated by processing and analyzing QC samples to obtain inter- and intra- assay variability. An interference study along with BioRad QC analysis was conducted to test method robustness. Extracted sample stability in autosampler rack at 8°C was obtained. Method was correlated with immunoassay by analyzing clinical samples.
Results: The quantification was performed with LOQ of 1 ng/mL and a linearity range 1-50 ng/mL for tacrolimus, sirolimus, everolimus and LOQ of 10 ng/mL and a linearity range of 10-2000 ng/mL for cyclosporin A. Intra and inter-assay CV's were all less than 10%. BioRad QC's results met supplier specification. No interferences were detected. Extracted samples were stable in 8°C for at least 24 hours.
Conclusions: Validation data demonstrates high throughput, robust method with a linearity range, accuracy and precision meeting industry standards.

Keywords: immunosuppressants, Everolimus, cyclosporin