Identification of human urinary metabolites of isbufylline by high‐performance liquid chromatography/thermospray mass spectrometry

Analysis of urinary metabolites of isbufylline (1,3‐dimethyl‐7‐(2‐methylpropyl)xanthine) in healthy male volunteers after oral administration of a single dose of 320 mg was undertaken by high‐performance liquid chromatography/mass spectrometry (HPLC/MS with thermospray ionization. Filtered urines were directed injected into the HPLC/MS system, equipped with a Lichrospher 100 RP 18 analytical column. The mobile phase was 0.1 M, pH 3.7 ammonium acetate in water and acetonitrile; the composition was varied linearly from 5% to 40% of the organic modifier in 40 min with a flow rate of 1 ml min −1 . Three more chromatographic peaks appeared in urines from treated subjects as compared to untreated ones; their probable quasi‐molecular ions were at m/z 253, 239 and 267 respectively, while the original drug, of 236 Da, was not present in appreciable quantity. The collisionally activated daughter ion spectra of the above ions allowed identification of 1,3‐dimethyl‐7‐(3‐hydroxy‐2‐methylpropyl)xanthine (D3OHMPX), 1‐methyl‐7‐(2‐hydroxy‐2‐methylpropyl)xanthine (M2OHMPX), and 1,3‐dimethyl‐(2‐carboxypropyl)xanthine (D2CMPX), the first one being present as glucuronic acid conjugate.