Quantitative determination of an extremely polar compound allantoin in human urine by LC-MS/MS based on the separation on a polymeric amino column.
Quantitative determination of allantoin in biological matrix poses a challenge to bioanalysis due to the extreme polarity of allantoin. The molecule exhibits virtually no retention on any of the available hydrophobic HPLC packing materials. In this study, an assay was developed for the LC-MS/MS quantitation of allantoin in human urine using a polymeric amino column to achieve the necessary separation. The urine samples were prepared by a single-step solid phase extraction (SPE) procedure. The extracted samples were analyzed on a jordi-gel DVB polyamine column (operated under an acetonitrile/water gradient with water as the stronger mobile phase) interfaced with a Finnigan TSQ 7,000 mass spectrometer. Negative electrospray ionization (ESI) was employed as the ionization source. Allantoin and its internal standard (13C1, 15N1-allantoin) were detected by use of single reaction monitoring (SRM) mode. The method was validated in the concentration range of 14.6-213 microg/ml(-1), with within and between run accuracy and precision both < 7%. The method has been successfully applied to clinical sample analysis.