Simultaneous quantification of busulfan, clofarabine and F-ARA-A using isotope labelled standards and standard addition in plasma by LC–MS/MS for exposure monitoring in hematopoietic cell transplantation conditioning

Abstract

In allogeneic hematopoietic cell transplantation (HCT) it has been shown that over- or underexposure to conditioning agents have an impact on patient outcomes. Conditioning regimens combining busulfan (Bu) and fludarabine (Flu) with or without clofarabine (Clo) are gaining interest worldwide in HCT. To evaluate and possibly adjust full conditioning exposure a simultaneous analysis of Bu, F-ARA-A (active metabolite of Flu) and Clo in one analytical run would be of great interest. However, this is a chromatographical challenge due to the large structural differences of Bu compared to F-ARA-A and Clo. Furthermore, for the bioanalysis of drugs it is common to use stable isotope labelled standards (SILS). However, when SILS are unavailable (in case of Clo and F-ARA-A) or very expensive, standard addition may serve as an alternative to correct for recovery and matrix effects. This study describes a fast analytical method for the simultaneous analysing of Bu, Clo and F-ARA-A with liquid chromatography-tandem mass spectrometry (LC–MS/MS) including standard addition methodology using 604 spiked samples. First, the analytical method was validated in accordance with European Medicines Agency guidelines. The lower limits of quantification (LLOQ) were for Bu 10 μg/L and for Clo and F-ARA-A 1 μg/L, respectively. Variation coefficients of LLOQ were within 20% and for low medium and high controls were all within 15%. Comparison of Bu, Clo and F-ARA-A standard addition results correspond with those obtained with calibration standards in calf serum. In addition for Bu, results obtained by this study were compared with historical data analysed within TDM. In conclusion, an efficient method for the simultaneous quantification of Bu, Clo and F-ARA-A in plasma was developed. In addition, a robust and cost-effective method to correct for matrix interference by standard addition was established.