Determination of paracetamol (acetaminophen) by HPLC with post-column enzymatic derivatization and fluorescence detection

Summary A novel method is described for the determination of paracetamol (acetaminophen;N-acetyl-4-aminophenol) in urine. After reversed-phase HPLC separation, paracetamol is oxidized by H₂O₂ with horseradish peroxidase catalysis. Detection is performed fluorimetrically at an excitation wavelength of 329 nm and an emission wavelength of 435 nm. Urine samples were spiked with paracetamol, diluted, and injected directly without further pretreatment. Under these conditions, the limit of detection was 2×10⁻⁸ molL⁻¹, and the limit of quantification was 7×10⁻⁸ molL⁻¹. The method was validated by two different approaches based on HPLC with UV-Vis detection.