Separation of 3′-azido-2′,3′-dideoxythymidine pronucleotide diastereoisomers in biological samples by CZE with cyclodextrin addition

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Abstract

The possibility of using capillary electrophoresis as an alternative technique to HPLC for the separation of pronucleotide diastereoisomers of AZT was investigated. In the pH range 6.2–7.2 where the analytes are stable, a chiral additive, carboxymethyl-β-CD, was found appropriate to enable the separation of the uncharged diastereoisomers. An experimental design strategy was used to study the influence of several parameters (CD and phosphate buffer concentration, methanol content of the electrolyte, injected volume, capillary length, electric field and separation temperature) on the separation and find suitable analytical conditions for monitoring the prodrugs in cell extracts. The diastereoisomers of the three tBuSATE phenylphosphotriester derivatives of AZT studied could be fully resolved within short analysis time (less than 10 min). Method validation results showed satisfactory results for linearity, accuracy and repeatability.

Introduction

Nucleoside analogues are an important class of therapeutic agents used in the treatment of viral infections [1]. Their action is based upon their intracellular conversion to their phosphorylated forms (nucleotides) which can interact with different viral enzymatic systems involved in the biosynthesis of nucleic acids. To overcome the poor affinity of the nucleoside analogues for the cell membrane, which limits the first phosphorylation step and may markedly impair their biological efficacy [2], [3], lipophilic prodrugs (pronucleotides) have been synthesized that penetrate the cell membrane and deliver the active monophosphorylated nucleoside directly into the cell [4], [5], [6], [7]. In this field, we recently reported [8], [9], [10] the potentialities of mononucleoside phosphotriester derivatives of 3′-azido-2′,3′-dideoxythymidine (AZT) bearing a S-pivaloyl-2-thioethyl (tBuSATE) group and an aryl residue as biolabile phosphate protections. Due to the presence of a phosphorus atom, SATE arylphosphotriester pronucleotides exist as a mixture of two diastereoisomers in which the R- or S-configuration around the chiral center may impact significantly on the in vitro antiviral activity, enzymatic recognition and pharmacokinetic profile, as observed in other series [11], [12]. Therefore, analytical methods are needed to independently monitor the diastereoisomers in biological matrices. Diastereomeric separation of some pronucleotides has to date been essentially carried out by HPLC on both achiral or chiral stationary phases. The former gave usually insufficient separation using C18 stationary phases [13], [14] while the latter gave satisfactory separation using polysaccharide stationary phases [15], [16].

The aim of this work was to evaluate the potential of capillary electrophoresis for the separation of pronucleotide diastereoisomers as an alternative technique to HPLC. Indeed CE present many advantages in the field of chiral separations such as a high efficiency, a broad variety of chiral selectors that can be added to the background electrolyte (BGE) and a low consumption of these selectors. The study has been carried out on tBuSATE phenylphosphotriester derivatives of AZT 13 (Fig. 1) where the SATE phosphate protection is characterized by the presence of hydroxyl functions. Due to their limited pH stability range, the diastereoisomers had to be analysed in their neutral form which required the addition of a charged additive (e.g. cyclodextrin) in the electrolyte to achieve their separation. This paper describes the experimental design strategy used to study the influence of several analytical parameters on the separation and the results of method validation study for monitoring the analytes in biological fluids.

Section snippets

Chemicals

tBuSATE phenylphosphotriester derivatives of AZT 13 were synthesized as diastereoisomeric mixtures following adapted procedures [8], [9]. O-p-tButylphenyl-3′-azido-2′,3′-dideoxythymidin-5′-yl phosphorothioate sodium salt 4 (internal standard for the study of pronucleotide 2) was obtained following an H-phosphonate approach [17] and 5′-tosyl-2′-deoxythymidine 5 (internal standard for the study of pronucleotide 3) was synthesized using a standard procedure [18]. Carboxymethyl-β-CD and

Results and discussion

Initial experiments were performed with the hydroxy tBuSATE phenylphosphotriester derivative of AZT 2.

Concluding remarks

This study has shown the potential of capillary electrophoresis to obtain efficient, rapid and reliable separations of pronucleotide diastereoisomers of AZT in biological samples. Separations could be achieved at a pH compatible with the stability of the compounds by the incorporation of CDs into the separation electrolyte. Suitable analysis conditions were rapidly determined using an experimental design strategy. Satisfactory results were obtained in terms of linearity, accuracy and

References (30)

  • E. De Clercq

    J. Clin. Virol.

    (2004)
  • N. Schlienger et al.

    Bioorg. Med. Chem. Lett.

    (1998)
  • C.J. Allender et al.

    Anal. Chim. Acta

    (2001)
  • N. Mesplet et al.

    J. Chromatogr. A

    (2003)
  • N. Mesplet et al.

    Bioorg. Chem.

    (2003)
  • Y. Vander Heyden et al.

    J. Pharm. Biomed. Anal.

    (2001)
  • Y. Vander Heyden et al.
  • H. Wan et al.

    J. Chromatogr. A

    (1997)
  • D.K. Bempong et al.

    J. Pharm. Biomed. Anal.

    (1993)
  • D.K. Bempong et al.

    J. Pharm. Biomed. Anal.

    (1996)
  • V. Sandor et al.

    J. Chromatogr. B

    (1995)
  • S. Zaugg et al.

    J. Pharm. Biomed. Anal.

    (2001)
  • S.A.C. Wren

    J. Chromatogr.

    (1993)
  • A. Pompon et al.

    Biochem. Pharmacol.

    (1992)
  • J. Balzarini

    Pharm. World Sci.

    (1994)
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