Automated processing of whole blood samples for the determination of immunosuppressants by liquid chromatography tandem-mass spectrometry

Background: Liquid chromatography tandem-mass spectrometry
(LC-MS/MS) is an efficient technology for routine determination of
immunosuppressants in whole blood; however, time-consuming manual
sample preparation remains a significant limitation of this
technique. Methods: Using a commercially available robotic
pipetting system (Tecan Freedom EVO), we developed an automated
sample-preparation protocol for quantification of tacrolimus in
whole blood by LC-MS/MS. Barcode reading, sample resuspension,
transfer of whole blood aliquots into a deep-well plate, addition
of internal standard solution, mixing, and protein precipitation by
addition of an organic solvent is performed by the robotic system.
After centrifugation of the plate, the deproteinized supernatants
are submitted to on-line solid phase extraction, using column
switching prior to LC-MS/MS analysis. The only manual actions
within the entire process are decapping of the tubes, and transfer
of the deep-well plate from the robotic system to a centrifuge and
finally to the HPLC autosampler. Whole blood pools were used to
assess the reproducibility of the entire analytical system for
measuring tacrolimus concentrations. Results: A total coefficient
of variation of 1.7% was found for the entire automated analytical
process (n=40; mean tacrolimus concentration, 5.3 mu g/L). Close
agreement between tacrolimus results obtained after manual and
automated sample preparation was observed. Conclusions: The
analytical system described here, comprising automated protein
precipitation, on-line solid phase extraction and LC-MS/MS
analysis, is convenient and precise, and minimizes hands-on time
and the risk of mistakes in the quantification of whole blood
immunosuppressant concentrations compared to conventional methods.