Validation of a high-performance liquid chromatographic-mass spectrometric method for the measurement of 5-chloro-2',3'-dideoxy-3' -fluorouridine (935U83) in human plasma.
An isocratic reversed-phase LC-MS method for measuring concentrations of 5-chloro-2',3'-dideoxy-3'-fluorouridine (935U83; I) directly and its 5'-glucuronide metabolite (5-chloro-2',3'-dideoxy-5'-O-beta-D-glucopyranuronosyl-3'-fluorour idine) indirectly in human plasma was developed, validated, and applied to a Phase I clinical study. The pyrimidine nucleoside, I, was extracted from human plasma by using anionic solid-phase extraction. The concentration of the glucuronide conjugate was determined from the difference between the molar concentration of I in a sample hydrolyzed with beta-glucuronidase and the nonhydrolyzed sample. Recovery of I from human plasma averaged 90%. The bias of the assay for I ranged from -5.5 to 7.1% during the validation and from -6.0 to 1.4% during application of the assay to the Phase I single-dose escalation study. The intra- and inter-day precision was less than 8% for I and its glucuronide conjugate. The lower and upper limits of quantitation for a 50-microliters sample were 4 ng/ml and 3000 ng/ml, respectively. No significant endogenous interferences were noted in human plasma obtained from drug-free volunteers nor from predose samples of HIV-infected patients.
Dunn
J A
JA
Division of Bioanalysis and Drug Metabolism, Glaxo Wellcome Inc., Research Triangle Park, NC 27709, USA.
Savina
P M
PM
D000998:Antiviral Agents / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015224:Dideoxynucleosides / Q000097:blood*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000998:Antiviral Agents; D015224:Dideoxynucleosides; C059694:raluridine