Interference of a methotrexate derivative with urinary oncopterin [N²-(3-aminopropyl)biopterin] measurement by high-performance liquid chromatography with fluorimetric detection

We previously reported a HPLC assay method using fluorimetric detection for the simultaneous determination of urinary N²-(3-aminopropyl)biopterin (oncopterin, a natural pteridine newly found in urine from cancer patients), biopterin and neopterin. We now have observed that an unknown substance, which may be derived from methotrexate, in urine from a patient with stomach cancer interfered with the assay of oncopterin and demonstrated that oncopterin could be completely separated from the unidentified substance by HPLC using a Nucleosil 100-5SA strong cation-exchange column. Furthermore, oncopterin was not detectable by this HPLC-fluorimetric method in urine samples from patients with stomach cancer who were not treated with methotrexate. The content of urinary oncopterin from cancer patients is supposed to be very low, with less than 1 μmol/mol creatinine. The present results indicate that the peak found with elution from the C₁₈ column was a methotrexate-derived compound and co-eluted with the analyte oncopterin.