title: 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.
authors.affiliation: Molecular Therapeutics/Drug Discovery Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Parise
authors.forename: Merrill J
authors.initials: MJ
authors.lastname: Egorin
authors.forename: Julie L
authors.initials: JL
authors.lastname: Eiseman
authors.forename: Erin
authors.initials: E
authors.lastname: Joseph
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Covey
authors.forename: Jan H
authors.initials: JH
authors.lastname: Beumer
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D003564:Cytidine Deaminase / Q000037:antagonists & inhibitors*; D004355:Drug Stability; D004791:Enzyme Inhibitors / Q000097:blood* / Q000737:chemistry; D005615:Freezing; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D015394:Molecular Structure; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013047:Specific Pathogen-Free Organisms; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013696:Temperature; D013767:Tetrahydrouridine / Q000097:blood* / Q000737:chemistry; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D013767:Tetrahydrouridine; D003564:Cytidine Deaminase