Fast separation of antiviral nucleoside phosphoramidate and H-phosphonate diastereoisomers by reversed-phase liquid chromatography

Department of Chemistry and The Key Laboratory for Chemical Biology of Fujian Province, College of Chemistry and Chemical Engineering, Xiamen University, 422 Siming Nan Lu, Xiamen 361005, China.
Journal of Chromatography A (Impact Factor: 4.17). 03/2011; 1218(10):1416-22. DOI: 10.1016/j.chroma.2011.01.046
Source: PubMed


The nucleoside-based antiviral phosphoramidates and H-phosphonates were synthesized and separated using reversed-phase liquid chromatography on bridged ethane hybrid (BEH) C₁₈ column packed with 1.7 μm particles of non-chiral stationary phase. The influences of the composition of mobile phase and column temperature have been investigated to optimize the diastereoisomeric separation. Complete separations of the phosphoramidate and H-phosphonate prodrugs with good resolution (R(S)=1.99-2.77) were achieved within a short time (5-9 min). The validation study of the optimized method including linearity, accuracy, repeatability and detection limit has revealed it is better performance versus conventional HPLC method. In addition, HPLC was combined with high resolution electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS), which enabled the exact mass measurement and high sensitivity. Using MS as detection, the limits of detection and limits of quantification of the studied pronucleotide diastereoisomers were determined in the range of several nmol L⁻¹ level.

4 Reads
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: CE methods have been developed for the chiral analysis of new types of six acyclic nucleoside phosphonates, nucleotide analogues bearing [(3-hydroxypropan-2-yl)-1H-1,2,3-triazol-4-yl]phosphonic acid, 2-[(diisopropoxyphosphonyl)methoxy]propanoic acid or 2-(phosphonomethoxy)propanoic acid moieties attached to adenine, guanine, 2,6-diaminopurine, uracil and 5-bromouracil nucleobases, using neutral and cationic cyclodextrins as chiral selectors. With the exception of the 5-bromouracil-derived acyclic nucleoside phosphonate with 2-(phosphonomethoxy)propanoic acid side chain, the R and S enantiomers of the other five acyclic nucleoside phosphonates were successfully separated with sufficient resolutions, 1.51-2.94, within a reasonable time, 13-28 min, by CE in alkaline BGEs (50 mM sodium tetraborate adjusted with NaOH to pH 9.60, 9.85 and 10.30, respectively) containing 20 mg/mL β-cyclodextrin as the chiral selector. A baseline separation of the R and S enantiomers of the 5-bromouracil-derived acyclic nucleoside phosphonate with 2-(phosphonomethoxy)propanoic acid side chain was achieved within a short time of 7 min by CE in an acidic BGE (20/40 mM Tris-phosphate, pH 2.20) using 60 mg/mL quaternary ammonium β-cyclodextrin chiral selector. The developed methods were applied for the assessment of the enantiomeric purity of the above acyclic nucleoside phosphonates. The preparations of all these compounds were found to be synthesized in pure enantiomeric forms. Using UV absorption detection at 206 nm, their concentration detection limits were in the low micromolar range.
    Journal of Separation Science 02/2014; 37(3). DOI:10.1002/jssc.201301092 · 2.74 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: This review covers recent progress in the preparation of H-phosphonate mono- and diesters, basic studies on mechanistic and stereochemical aspects of this class of phosphorus compounds, and their fundamental chemistry in terms of transformation of P-H bonds into P-heteroatom bonds. Selected recent applications of H-phosphonate derivatives in basic organic phosphorus chemistry and in the synthesis of biologically important phosphorus compounds are also discussed.
    Topics in current chemistry 11/2014; 361. DOI:10.1007/128_2014_562 · 4.46 Impact Factor