Rapid determination of rat plasma uridine levels by HPLC-ESI-MS utilizing the Captiva™ filter plates for sample preparation

• Published in: Biomedical chromatography Vol. 17; no. 4; pp. 215 - 218
• Main Authors: Williams, Marta G., Palandra, Joe, Shobe, Eric M.
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01-06-2003
• Subjects: 5-fluorouracil toxicity; autistic behaviour; hypoxia; pyrimidine metabolism; seizures; uridine phosphorylase
• ISSN: 0269-3879; 1099-0801
• DOI: 10.1002/bmc.210

A rapid, accurate and precise HPLC‐ESI‐MS method for the determination of rat plasma uridine concentrations was developed and is described here. Sample preparation involves methanol precipitation of plasma proteins in a 96‐well Captiva™ protein precipitation filter plate. A clear extract is drawn through the filter plate with vacuum, followed by evaporation of the extract and subsequent reconstitution prior to chromatography on a reversed‐phase column with an aqueous mobile phase [0.1% (v/v) glacial acetic acid]. Detection was accomplished by positive‐ion electrospray ionization mass spectrometry. A calibration curve ranging in concentration from 0.78 to 25 µM was constructed by best‐fit, 1/x weighting linear regression analysis of the calibration standard concentrations vs peak height ratios of analyte with internal standard. The correlation coefficient was >0.995. The overall assay accuracy as shown by the back‐calculated concentrations of the calibration curve ranged from 96.6 to 103% with RSD ranging from 4.5 to 20%. While this assay method was developed for the determination of uridine in rat plasma, it could be readily adapted for determination of uridine in plasma from other species, such as human. Copyright © 2003 John Wiley & Sons, Ltd.