Rat dried blood spot analysis of (R,S)-(-)- and (S,R)-(+)- enantiomers of emtricitabin on immobilized tris-(3,5-dimethylphenyl carbamate) amylose silica as a chiral stationary phase.

An enantioselective high performance liquid chromatography method has been developed and validated by evaluating the suitability of newly introduced immobilized polysaccharide chiral stationary phases, the effect of different organic modifiers and temperature including the entropy and enthalpy on resolution of the (R,S)-(-) & (S,R)-(+) emtricitabine enantiomers on rat dried blood spots. Both the enantiomers were extracted from dried blood spots using ethanol: methanol (80:20 v/v) mixture and separated on an immobilized amylose tris-(3,5-dimethyl phenyl carbamate) chiral stationary phase using n-hexane:ethanol (65:35 v/v) as a mobile phase at a flow rate of 0.8mL/min. The detection was carried out at 280nm using photo diode array detector connected to a polarimeter in series to determine their order of eluton. The method was validated with respect to limits of detection and quantification, linearity, accuracy and precision. The calibration curves were linear over the concentration range of 0.5-500μg/mL for both enantiomers and the correlation coefficient (r(2)) was >0.998. The overall recovery of (R,S)- & (S,R)-enantiomers of emtricitabin from DBS were 90.4 and 90.6%, respectively. The limits of detection and quantification of enantiomers were 0.26, 0.30 and 0.85, 0.92μg/mL for (R,S)- and (S,R)-emtricitabin enantiomers, respectively. The assay was specific and precise (RSD <10%). The stability of emtricitabin was also performed and the results were found to be well within the limits. The effect of hematocrit on extraction of emtricitabin enantiomers from dried blood spots was evaluated and no interference from endogenous substances was observed.