Diastereoisomeric resolution of a pronucleotide using solid phase extraction and high performance liquid chromatography: application to a stereoselective decomposition kinetic in cell extracts.
A stereospecific HPLC methodology has been developed for the diastereoisomeric resolution of a mononucleotide prodrug in cell extracts. This method involves the use of solid phase extraction on a C18 cartridge. Diastereoisomers and internal standard resolutions were performed on a cellulose based chiral column (Chiralcel OD-H) used in the normal phase mode. The method was validated in terms of specificity, recovery, linearity (diasteroisomers mixture concentration: 3-60 micromol L(-1)), precision and accuracy and detection limit (1.67 and 1.33 micromol L(-1) for first and second eluted diastereoisomer). This method was applied to the determination of the apparent rate constants of disappearance and half-lives of each stereoisomers. This permits to conclude to the stereoselectivity of the enzymatic activity involved in the decomposition pathway of 2.
Foulon
C
C
Laboratoire de Chimie Analytique, EA 4034, Faculté des Sciences Pharmaceutiques et Biologiques, Université de LILLE 2, BP 83, 3 rue du Pr. Laguesse, 59006 Lille Cedex, France.
Vaccher
C
C
Villard
A-L
AL
Puy
J-Y
JY
Lefebvre
I
I
Perigaud
C
C
Bonte
J P
JP
Goossens
J F
JF
D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007700:Kinetics; D011355:Prodrugs / Q000032:analysis*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013237:Stereoisomerism; D015215:Zidovudine / Q000031:analogs & derivatives* / Q000032:analysis*
D011355:Prodrugs; D015215:Zidovudine