BACKGROUND: Thiopurine metabolites, 6-thioguanine (6TG) and 6-methylmercaptopurine (6MMP), are monitored to aid therapeutic management of thiopurine drugs. At Manchester University NHS Foundation Trust (MFT), thiopurine metabolites are measured by high performance liquid chromatography with ultraviolet detection (HPLC-UV). Whole blood samples are lysed and subjected to hydrolysis with derivatisation of 6MMP before HPLC-UV detection at 304 nm for the 6MMP-derivative and 342 nm for 6TG. For some samples, 6MMP cannot be reported due to a chromatographic interference at 304 nm co-eluting with the 6MMP peak. An investigation was performed to identify the interfering compound. METHODS: Patient medication histories were examined to identify candidate compounds for the interference. Candidate compounds were spiked into blood at supraphysiological concentrations and tested on the assay. RESULTS: Metronidazole was identified as being prescribed to all patients whose samples demonstrated the interference. Metronidazole and its metabolite, hydroxymetronidazole, were spiked into blood. HPLC-UV analysis of spiked blood demonstrated similar UV absorbance patterns to those seen in patient samples with the interference. Hydroxymetronidazole co-eluted with 6MMP causing interference in the measurement. CONCLUSION: Metronidazole and its major metabolite can interfere with 6MMP measurement by HPLC-UV analysis at 304 nm.