A validated high performance liquid chromatography–diode array detector method for the quantification of mitomycin C in plasma, peritoneal fluid and urine

Mitomycin C (MMC) is a quinone-containing alkylating agent, that has been extensively studied in preclinical and clinical work due to its antitumor activity. A thoroughly validated high performance liquid chromatography-diode array detector method is provided to quantify MMC in plasma, peritoneal fluid and urine. Porfiromycin served as internal standard. The mobile phase for the plasma, peritoneal fluid and urine analysis consisted of 27% MeOH and 73% 20 mM ammoniumacetate buffer (pH 6.5) and 9% ACN and 91% 20 mM phosphate buffer (pH 6.5) respectively. The residue from 100 µL ACN deproteinated plasma was dissolved in 250 µL mobile phase. Peritoneal fluid and urine were diluted 10-fold in their respective mobile phases. UV detection was performed at 365 nm. Quantification of MMC was achieved over a linear range of 0.05–5 µg/mL and 5–50 µg/mL in plasma; 0.1–5 µg/mL and 5–100 µg/mL in peritoneal fluid; 0.25–5 µg/mL and 5–100 µg/mL in urine. The limit of quantification was 0.05 µg/mL in plasma, 0.1 µg/mL in peritoneal fluid and 0.25 µg/mL in urine. The method was further validated for selectivity, specificity, inter- and intraday precision and accuracy (=15%), extraction recovery and stability. MMC remains stable in the different biofluids for 20 days (short-term stability) at 4 °C, -27 °C and -80 °C and for 80 days (long-term stability) at -80 °C.