Separation of antiviral nucleoside phosphoramidate diastereomers by analytical supercritical fluid chromatography

GRAPHICAL ABSTRACT ABSTRACT Two amino acid phosphoramidates with high antiviral activity were used as model compounds and separated using supercritical fluid chromatography on an achiral Hypersil BDS cyano column (250 × 4.6 mm, 5 µm). Supercritical CO2 was used as a mobile phase with different co-solvents, including methanol, ethanol, and 2-propanol. Several key processing parameters were evaluated, including the type and concentration of alcohol modifier (5 to 15%), backpressure (100 to 200 bar), and column temperature (30 to 45 ˚C) in terms of their impact on retention factor, diastereoselectivity, and resolution. The optimized chromatographic conditions allowed for a complete separation of mixture of two diastereomers with good resolution over a short time (5–9 min). At a backpressure of 150 bar and a temperature of 33°C, the diastereomers of phenylalanine methyl ester phenyl 5′-phosphoamidate of d4T (d4T-P-N-PheOMe) and alanine methyl ester phenyl 5′-phosphoamidate of d4T (d4T-P-N-AlaOMe) were separated with resolutions of 4.43 and 2.55 using 10.0% and 5.0% (v/v) methanol, respectively.