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.

6 Reads
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cefprozil (CPZ) is a second-generation semi-synthetic cephalosporin antibiotic that commonly exists as the mixture of Z and E diastereoisomers, at the ratio of approximately 9:1. A novel reversed-phase HPLC method for the determination of CPZ in tablets was described. The separation of CPZ diastereoisomers and caffeine (internal standard) was carried out by applying the same analytical and instrumental conditions on two stationary phases, which have different surface chemistries. The columns used in the study were monolithic silica Merck Chromolith Performance RP-18e and conventional C(18) silica Phenomenex Synergi Hydro RP columns. In total, 10 μL aliquots of samples were injected into the system and eluted using water-acetonitrile (90:10, v/v) solution, which was pumped through the column at a flow rate of 1.0 mL/min. The analyte peaks were detected at 200 nm using diode array detector with high specificity. CPZ diastereoisomers and caffeine were measured within 13 min using the C(18) column, whereas <5 min was required for the monolithic one. Validation studies were performed according to official recommendations. Value of a monolithic column for the assay of diastereoisomers in pharmaceutical tablets was evaluated for the first time and found as a powerful alternative to highly efficient C(18) columns.
    No preview · Article · May 2011 · Journal of Separation Science
  • 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.
    Full-text · Article · Feb 2014 · Journal of Separation Science
  • 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. Graphical Abstract
    Full-text · Article · Nov 2014 · Topics in current chemistry