Five d-amino acids have been used for the first time to synthesize chiral derivatizing reagents (as variants of Marfey's reagent) by nucleophilic displacement of one of the fluorine atoms in 1,5-difluoro-2,4-dinitrobenzene as against the literature reports on application of only l-amino acids or their amides as chiral auxiliaries in dinitrobenzene (DNB) moiety. Five other DNB based reagents were also prepared by nucleophilic substitution of fluorine atom with the set of the same amino acids in l-configuration, as chiral auxiliaries. These reagents were characterized and used for synthesis of diastereomers of (RS)-Mexiletine spiked in human plasma. Diastereomers were synthesized employing microwave irradiation and were separated on reversed-phase C18 column. Performance of the two types of chiral derivatizing reagents was compared. The reagents containing d-amino acids provided enhanced separation of diastereomers than those containing l-amino acids. The best resolution was obtained using mobile phase consisting of acetonitrile and 0.1% trifluoroacetic acid in gradient mode. The detection was carried out at 340nm. The method so developed was validated for linearity, accuracy and precision. The limit of quantitation was found to be approximately 25.2ngmL(-1) in human plasma.