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

Abstract

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 microM 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.

Sample preparation for liquid chromatography-tandem mass spectrometry using functionalized ferromagnetic micro-particles

“…Good extraction recovery was observed even when applying very small quantities of the of magnetic particles; this is probably due to the intensive interaction of the extraction material which was dispersed into the sample, in contrast to a weaker interaction within packed extraction cartridges. Our results are of importance with respect to the goal of fully automated LC-MS/MS methods, since the micro particle technology offers substantial advantages with respect to automation compared to extraction protocols which involve "solid" extraction materials [4][5][6][7][8]: minimized handling of solid consumables (cartridges, plates); minimized volumes of extraction fluids; and no technically demanding application of vacuum or pressure to the extraction materials. After the "proof of principle" in this feasibility study, we are currently developing a fully automated sample preparation device which is on-line coupled to a LC-MS/MS instrument -corresponding to fully automated micro particle based immunoassay systems.…”

“…Protein precipitation as well as solid phase extraction protocols can potentially be subject of automation [3][4][5][6][7][8], even allowing a continuous work-flow from serum or plasma to MS-analysis [7]. Solid extraction materials packed in cartridges, however, require complex mechanical and/or pneumatic handling.…”

Liquid chromatographic method for the determination of uridine in human serum

“…In a recently published paper rat plasma uridine levels were detected by HPLC-ESI-MS, whereas cytidine, another endogenous nucleoside, was used as IS. So far, this MS method was not evaluated in humans [10]. In contrast the described method relies on standard HPLC conditions without MS and uses oxypurinol as a non-endogenous IS.…”

Functional and structural evidence for the catalytic role played by glutamate-47 residue in the mode of action of Mycobacterium tuberculosis cytidine deaminase

“…36 One enzyme unit is dened as the amount of enzyme which catalysis the deamination of 1 mmol of cytidine per minute at 25 C. Mixtures containing Tris-HCl 50 mM, pH 7.5, mutant enzyme (0.24 mg mL À1 ), and cytidine (50 mM to 1800 mM) were incubated at 25 C. At six timed intervals, aliquots (200 mL) were boiled for 3 min to stop reaction and centrifuged at 10 600Â g for 3 min.…”