Quantitative determination of the cytidine deaminase inhibitor tetrahydrouridine (THU) in mouse plasma by liquid chromatography/electrospray ionization tandem mass spectrometry

Abstract

A number of anticancer drugs are cytidine analogues that undergo metabolic deactivation catalyzed by cytidine deaminase (CD). 3,4,5,6-Tetrahydrouridine (THU) is a potent inhibitor of CD, by acting as a transition-state analogue of its natural substrate cytidine. However, to date its pharmacokinetic properties have not been fully characterized, which has impaired its optimal preclinical evaluation and clinical use. We report a liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay for the sensitive, accurate and precise quantitation of THU in mouse plasma. Validation was performed according to FDA guidelines. The assay employed deuterated THU as the internal standard and an acetonitrile protein precipitation step. Separation, based on hydrophilic interaction chromatography, was achieved with an amino column and an isocratic mobile phase of 0.1% formic acid in acetonitrile and water followed by a wash. Chromatographic separation was followed by positive-mode electrospray ionization MS/MS detection in the multiple reaction monitoring (MRM) mode. The assay was accurate (92.5-109.9%) and precise (2.1-9.0%) in the concentration range of 0.2-50 microg/mL. Recovery from plasma was near-complete (92.9-119.3%) and ion suppression was negligible (-17.5 to -0.2%). Plasma freeze/thaw stability (93.1-102.1%), stability for 3 months at -80 degrees C (99.5-110.9%), and stability for 4 h at room temperature (92.1-102.4%) were all in order. This assay is currently being used to quantitate THU in ongoing pharmacokinetic studies. In addition, the assay is expected to be a useful tool in any future studies involving co-administration of THU with cytidine analogues.

Plasma pharmacokinetics and oral bioavailability of 3,4,5,6-tetrahydrouridine, a cytidine deaminase inhibitor, in mice

“…This has impeded the exploration of its full potential as a modifier of cytidine metabolism. Using a recently developed specific and sensitive analytical method for the quantitation of THU [38], we characterized THU plasma pharmacokinetics and oral bioavailability in mice.…”

“…THU concentrations in plasma were quantitated with a previously developed and validated hydrophilic interaction chromatography (HILIC) LC-MS/MS assay [38]. The assay fulfilled the FDA criteria for bioanalytical method validation [43] and was accurate and precise in the concentration range of 0.2-50 μg/ml.…”

“…At the end of the collection period, the cages were washed with 15 ml of water. Quantitation of THU in urine and cage wash was accomplished by diluting (at least 10-fold) an aliquot of each sample with control plasma and analyzing that diluted sample with the HILIC LC-MS/MS assay used for plasma samples [38].…”

“…We aimed to characterize THU pharmacokinetics and oral bioavailability in mice, using a recently developed specific and sensitive high performance liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) assay [38] that had been validated according to FDA guidelines [43]. Our goal was to generate data that would assist in planning subsequent pharmacokinetic studies of THU in humans.…”

Potential of HILIC‐MS in quantitative bioanalysis of drugs and drug metabolites

“…Amino stationary phases employed for HILIC separation [67,68] were traditionally used in normal phase HPLC. Among bare silica, ZIC, and amino columns, the amino column had the strongest retention for the acidic polar compounds due to a significant ion-exchange effect on the amino phase [83].…”