|*Kerstin Yvonne Rauch, *Klaus Resch, *Volkhard Kaever, #Tania A. Sasaki, #Lisa Sapp |
*Institute of Pharmacology, Hannover Medical School, Germany,
#Applied Biosystems, Foster City, CA
|Introduction: In multi-dose drug regimens, drug-drug interactions (DDI) can affect biological drug concentrations. Person-to-person metabolism differences (e.g. diet, enzyme polymorphisms) introduce additional variability that can alter the efficacious dose. These potential problems should be considered especially for critical dose drugs like immunosuppressants, which are given to transplant recipients in combination with several agents, such as antifungals. These two drug classes can compete for the same enzymatic pathway (e.g. CYP3A4). An LC/MS/MS method was developed for the simultaneous quantitation of immunosuppressants and identification of antifungals. The selectivity and specificity of LC/MS/MS is a major advantage over traditional techniques, such as immunoassay, where cross-reactivity can artificially increase measured concentrations.|
Methodology: The LC/MS/MS method was developed to detect four commonly administered immunosuppressants and five antifungals. The immunsuppressants are: cyclosporin A, tacrolimus, sirolimus, and everolimus. Antifungals included are: ketoconazole, itraconazole, fluconazole, voriconazole, and posaconazole.
EDTA blood samples were manually processed by methanol/zinc sulphate deproteinization. After centrifugation, the supernatants were directly injected into the LC/MS/MS system. Chromatographic separation was achieved using an Oasis HLB extraction column and a Zorbax Extend-C18 analytical column protected by an octadecyl security guard column. Gradient elution with H2O/acetonitrile (95/5 to 5/95, v/v) + ammonium formate and formic acid was performed at 60°C with a total run time of 20 min. The hybrid triple quadrupole/linear ion trap mass spectrometer was operated in positive ESI mode with defined multiple reaction monitoring (MRM) and information dependent acquisition (IDA) of enhanced product ion (EPI) scan criteria.
Validation: Results from the LC/MS/MS method correlated well with data obtained using our routine, validated method with LOD (LLOQ) values of about 1 (2) ng/ml for tacrolimus, sirolimus, and everolimus and about 10 (10) ng/ml for cyclosporin A. The antifungals were also detected in clinically relevant concentrations and confirmed by library search of EPI spectra showing fit factors >65%.
Conclusion: The developed LC/MS/MS method was successfully applied for detection and confirmation of additional drugs in human blood samples simultaneously to the quantitation of immunosuppressants, allowing detection of potential drug interactions leading to unwanted changes in immunosuppressant levels to be easily observed. The ability to acquire MRM data for quantitation along with full scan EPI spectra for qualitative library search may therefore be ideal for construction of a more general “drug interaction” LC/MS/MS spectral library. This library can be used to detect and confirm the presence of additional drugs that may have DDI effects with immunosuppressants.