High-performance liquid chromatographic assay for 3'-amino-3'-deoxythymidine in plasma, with 9-fluorenyl methyl chloroformate as the derivatization agent.
We have developed a sensitive high-performance liquid chromatographic assay for the determination of the zidovudine metabolite 3'-amino-3'-deoxythimidine (AMT) using fluorescence and sensitivity in the picomolar range. Plasma was diluted with 0.05 M sodium phosphate buffer pH 7.2 and subsequently prepared for analysis using solid-phase extraction. AMT was derivatized with 9-fluorenyl methylchloroformate and chromatographed using a reversed-phase system. The mobile phase consisted of acetonitrile-0.01 M potassium phosphate buffer (pH 7) (32.68, v/v). The fluorescence of the column effluent was monitored at 262 nm (excitation) and 306 nm (emission). Good resolution of AMT from endogenous plasma components was obtained. Within- and between-day variability was less than 10%. The limit of quantitation was 0.9 microgram/l. The assay was successfully applied to the determination of AMT in human plasma and in plasma of mice treated with zidovudine.
Nattouf
H
H
Département de Pharmacie Clinique, Faculté de Pharmacie, Châtenay-Malabry, France.
Davrinche
C
C
Sinet
M
M
Farinotti
R
R
D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D015224:Dideoxynucleosides / Q000097:blood*; D005449:Fluorenes; D015658:HIV Infections / Q000097:blood; D006801:Humans; D051379:Mice; D008811:Mice, Inbred DBA; D011786:Quality Control; D013050:Spectrometry, Fluorescence; D015215:Zidovudine / Q000493:pharmacokinetics
D015224:Dideoxynucleosides; D005449:Fluorenes; D015215:Zidovudine; C019252:3'-aminothymidine; C054007:1-(9-fluorenyl)methyl chloroformate