MSACL 2023 Abstract

Background:
Therapeutic drug monitoring (TDM) of immunosuppressants in whole blood is critical for clinical follow-up of transplant patients to obtain the optimum balance between therapeutic efficacy and the occurrence of adverse effects. Immunosuppressants are routinely quantified using LC-MS/MS in whole blood which translates to frequent phlebotomy visits for patients for the purpose of venous blood collection. Alternatively, dry blood spot (DBS) measurements have the potential to reduce the cost of sample collection and shipping, while simultaneously increasing the convenience of at home collection for patients. However, the analytical implications of DBS measurements must be carefully assessed to ensure the results are sufficient to fulfill the clinical need.

Objective:
To evaluate the analytical applicability of a DBS method for monitoring of immunosuppressants in venous blood samples using an automated extraction platform.

Methods:
Calibrators and QC were made by spiking immunosuppressants (Cyclosporine A, Tacrolimus, Sirolimus and Everolimus) into bovine whole blood. Residual venous blood samples previously analyzed for Cyclosporine A, Tacrolimus, Sirolimus and/or Everolimus on validated LC-MS/MS methods were utilized for this study. Calibrators, QC, and patient samples were spotted (25 μL) on PerkinElmer 226 Bioanalysis RUO Card with Ahlstrom 226 grade paper. Internal standards (IS) included Cyclosporin A-[15N]11 (Cerilliant), FK-506-[13C]-D2 (Cayman Chemical Company), Rapamycin D3 (Cambridge Isotope Labs Inc), and Everolimus-D4 (Cambridge Isotope Labs Inc). Immunosuppressants were extracted using a fully automated Thermo Scientific™ Transcend™ DSX-1 UHPLC system that performs internal standard addition, analyte extraction, 2-D LC matrix cleanup and analyte separation without any manual intervention. Sample analysis was performed on a Thermo Scientific™ TSQ Altis™ MD mass spectrometer and data was analyzed using TraceFinder™ LDT software.

Results:
Extraction, LC separation, and quantification of immunosuppressants using the Transcend™ DSX-1 system was accomplished using a 10.95-minute method. This method includes extensive wash and equilibration steps to minimize carryover. The intra-run imprecision of four different concentrations of QC for the four immunosuppressants measured was ±12% (n=9). The signal-to-noise ratio for all samples was greater than ten. Patient results from the Transcend™ DSX-1 were compared to established LC-MS/MS testing methods that utilized osmotic shock lysis by water followed by protein precipitation with methanolic zinc sulfate. Linear regression comparison indicated less than or equal to 10% bias and R² greater than 0.85 for all analytes tested.

Conclusion:
Preliminary studies suggest that the Transcend™ DSX-1 platform has the potential to provide a fully automated extraction of DBS for immunosuppressants. This sample matrix enables convenient sample collection for patients for therapeutic drug monitoring of immunosuppressants. Although our initial results are promising, additional studies are required to demonstrate if this platform can achieve the necessary analytical performance characteristics and determine if simultaneous venous whole blood collection and DBS of patients undergoing immunosuppressant therapy produce clinically equivalent results.