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

• Published in: Chromatographia Vol. 54; no. 3-4; pp. 163 - 167
• Main Authors: Meyer, J, Karst, U
• Format: Journal Article
• Language: English
• Published: Oxford Vieweg Verlag 01.08.2001; Springer
• Subjects: acetaminophen; Analysis; Biological and medical sciences; catalytic activity; derivatization; detection limit; fluorescence; General pharmacology; hydrogen peroxide; Medical sciences; peroxidase; Pharmacology. Drug treatments; reversed-phase high performance liquid chromatography; urine; wavelengths; Urine; Biological fluid; Fluorescence detector; Chemical analysis; Hydrogen peroxide; Enzyme; Antimigrainous agent; HPLC chromatography; Derivatization; Postcolumn; Reversed phase chromatography; Paracetamol; Analgesic; Peroxidases; Antipyretic; Oxidation; Peroxidase; Oxidoreductases; Quantitative analysis; Enzymatic reaction
• ISSN: 0009-5893; 1612-1112
• DOI: 10.1007/BF02492237

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.