In this communication, we report the development of a new ultra-performance liquid chromatographic/tandem mass spectrometry (UPLC-MS-MS) assay for measurement of
amrubicin (an
anthracycline anti-
cancer agent) and its active metabolite,
amrubicinol, in plasma. The enhanced electrospray ionization signal intensity of the analytes achieved by modifying the mobile phase with
formic acid was associated with improvement in the lower limit of quantification. These favorable effects were
electrolyte concentration-dependent. In order to maximize assay throughput, we used
methanol protein precipitation to prepare the plasma samples, and simplified sample preparation by injecting 40 microL of the supernatant containing
methanol at 87.5% (v/v) directly onto the UPLC column without any intermediary
solvent evaporation step. The large-volume injection of highly organic supernatant sample increased matrix and elutropic effects, but these drawbacks were respectively overcome by using a 5mM
formic acid-modified mobile phase and a new pulse gradient method. To our knowledge, this is the first report successfully using large-volume injection of strong organic samples with UPLC-MS-MS bioanalysis. The pulse gradient elution also resulted in band compression and enhanced the robustness of the chromatography. The promising new approach illustrated herein is extremely straightforward to optimize, and may be used for UPLC-MS-MS bioanalytical assay of other compounds.