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

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 (RS = 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.