AbstractA high‐performance liquid chromatography method which uses direct injection and a column‐switching valve for determination of mitoxantrone in plasma is described. After addition of internal standard, plasma was deproteinized by adding 5‐sulphosalicylic acid reagent. The supernatant was injected onto an enrichment precolumn flushed with washing solvent (methanol and water 5:95).Absorbed mitoxantrone was backflushed from the precolumn into an analytical column C18 Nucleosil 250 × 4 mm with a gradient elution (solvent A, ammonium formate buffer 1.6M, pH 4.3; solvent B, acetonitrile and water 40:60; linear gradient from 45 to 55% of B for 30 min was programmed) at a flow rate of 1.3 mL/min. Detection was carried out at 665 nm.This method showed obvious advantages over conventional extraction procedures in terms of speed and facility of sample ha