Determination of adriamycin and its metabolites in plasma by high performance liquid chromatography using automated sample clean-up system

Direct injection of plasma sample onto the precolumn of an automated sample clean-up system for the RP-HPLC determination of adriamycins is proposed. After injection of plasma sample onto the precolumn of protein-coated ODS, the intial aqueous solution eluted out plasma proteins and hydrophilic components during the trapping of adriamycins on the precolumn. Then, the precolumn was connected, in a back flush mode, to the analytical ODS column to allow adriamycins to be separated through the analytical column. The elution system was changed depending on the detection method. With fluorometry (Ex. 475 nm/ Em. 580 nm), it was possible to determine the adriamycins in plasma with simple two steps elution system. However, with UV-spectrophotometry (UV 290 nm), two points were important, the first point was to wash out the interfering compounds from the precolumn using three steps washing solvents, the second was to elute adriamycinol from the analytical column after the buffer change artifacts. The latter condition might offer some concept for applying the present method for setting up a method for the determination of non-fluorecent drugs with UV adsorbing characteristics. In view of simplicity and accuracy for the determination of total (free + bound to plasma proteins) adriamycins in plasma, the present method was superior to the conventional HPLC method which involved deproteinization step with 5% TCA or 50% acetonitrile. The recovery of adriamycin, adriamycinol and adriamycinone spiked in rabbit plasma (5 × 10-6 M each) was almost quantitative (100±2%) with good reproducibility(R.S.D. less than 2%, n= 5), when 1 00 p.1 of the plasma was analyzed, indicating that the method did not always need an internal standard. Adriamycins in a pure aqueous solution (below ca. 10-6 M) have trends to be adsorbed to the glass wall, however, the present method was almost free from this trouble. Levels of adriamycins in rabbit plasma after intravenous administration of adriamycin (3.8 mg/kg) could be followed by the present method.