Determination of glucuronides of molecules of toxicological interest by liquid chromatography negative-ion mass spectrometry with atmospheric pressure chemical ionization

A new chromatographic method for the direct determination of metabolites (g lucuronide-conjugates) of molecules of toxicological relevance in biologica l media with the minimum sample pre-treatment has been developed. A high pe rformance liquid chromatography-atmospheric pressure chemical ionization-ma ss spectrometry (LC-APCI-MS) system was used for this purpose. The separati on of four glucuronides Aminophenylglucuronide (APhG), Phenylglucuronide (P hG), p-Nitrophenylglucuronide (NPhG) and alpha-Naphthylglucuronide (NG) was obtained under ion-suppressed reversed-phase chromatography conditions, by using high-speed (3 cm, 3 mu m) columns and formic acid (2 mM) as the acid modifier in the mobile phase. Different C-18 stationary phases (partially endcapped and non-endcapped) were evaluated in order to obtain retention fo r these very polar, water soluble molecules. The ionization of the analytes was obtained in negative-ion (NI) mode. Detection limits were in the range 1-5 mg L-1 and calibration curves were linear over two order of magnitude. Intra-day and inter-day precision were in the range 2.9-10.6 % for all the compounds. The method was successfully applied for the determination of Ph G in a urine sample of a European Quality Assurance Programme for Organic S olvent Metabolites.