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Simultaneous determination of cyclophosphamide, ifosfamide, doxorubicin, epirubicin and daunorubicin in human urine using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry: bioanalytical method validation.

A reversed-phase high-performance liquid chromatography (rp-HPLC) system interfaced with an electrospray ionization (ESI) source coupled to tandem mass spectrometry (MS/MS) was developed and validated for the determination of cyclophosphamide (CP), ifosfamide (IF), daunorubicin (DNR), doxorubicin (DXR), and epirubicin (EPI) in human urine. The analysis of samples containing multiple analytes with a dissimilar range of polarities was carried out using a conventional reversed-phase chromatographic BDS Hypersil C8 column. The analytical run was 15 min. The triple quadrupole mass spectrometer was operated in positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over a concentration range of 0.2 to 4.0 microg.L(-1) for CP, IF, DXR, EPI and 0.15-2.0 microg.L(-1) for DNR in human urine. The lower limit of quantification (LLOQ) was 0.2 microg.L(-1) for CP, IF, EPI and was set at 0.3 and 0.15 microg.L(-1) for DXR and DNR, respectively. The relative standard deviations (RSD%) were <11.2% for inter- and intra-day precisions. The overall accuracy was also within 114.7% for all analytes at the concentrations of the quality control samples. The potential of ionization suppression resulting from the endogenous biological material on the rp-HPLC/MS/MS method was evaluated and measured. The feasibility of the proposed HPLC/ESI-MS/MS procedure was demonstrated by analyzing urine samples from pharmacy technicians and nurses working in hospitals or personnel employed in drug-manufacturing plants.