Cyclodextrin assisted enantiomeric recognition of emtricitabine by 19F NMR spectroscopy

Abstract

A simple, rapid, precise and selective ¹⁹F NMR spectroscopic method was developed and validated for the quantitative determination of (R,S)-(−)- and (S,R)-(+)-enantiomers of emtricitabine using cyclodextrins as chiral recognition agents. Different parameters including the type, temperature and concentration of the chiral selector affecting the enantioresolution of emtricitabine were optimized. Among the different chiral selectors studied, α-cyclodextrin displayed the highest resolution of ¹⁹F NMR signals of (R,S)-(−)- and (S,R)-(+)-emtricitabine enantiomers. The binding constants (K) of the complexes determined from ¹⁹F NMR titration curves indicated that the α-CD forms a stronger complex with (R,S)-(−)-EMT (6.09 M⁻¹) than with (S,R)-(+)-EMT (3.88 M⁻¹). The enantioselectivity (α = 1.57) also indicated that α-CD was more suitable for recognition of the enantiomers of (R,S)-(−)- and (S,R)-(+)-emtricitabine by ¹⁹F NMR spectroscopy. The complexation process of emtricitabine with α-CD in D₂O was also investigated by 2D ¹H ROESY NMR experiments. The method was validated in terms of accuracy, precision, linearity, LOD and LOQ. The linear ranges of the calibration curves were 0.0007 to 0.0135 mmol mL⁻¹ in D₂O for both the enantiomers. The limit of detection (LOD; S/N = 3) and the limit of quantification (LOQ; S/N = 10) were 0.099, 0.096 and 0.33, 0.32 μmol mL⁻¹ for (R,S)-(−)- and (S,R)-(+)-emtricitabine enantiomers respectively. The accuracy of the method was established by spiking with known amounts of enantiomers and the found values ranged between 95 and 103% with a relative standard deviation of <7%.