| Fluoxetine(FLX), N-methyl-3-phenyl-3-(2,2,2-trifiuoro-p-tolyloxy) propylamine hydrochloride, C17H18F3NO HC1, MW = 345.8, is an antidepressant which differs structurally and pharmacologically from the tricyclic agents. It has been shown to selectively inhibit the reuptake of serotonin in presynaptic neurons. Besides as a tool to increase the level of serotonin in the synaptic junction for fundamental researches on the neurosciences, FLX has been prescribed widely for the therapy of mental depression, obsessive compulsive disorders and pain syndromes. FLX is extensively metabolized by demethylation in liver, to its primary active metabolite norfluoretine (NFLX), which is also a potent serotonin (5-HT) reuptake inhibitor.In order to sensitively determine FLX, NFLX and their enantiomers in biosamples, this paper successfully explored the applicability of 2,1,3-benzoxadiazole compounds to derivatize FLX and NFLX. 4-(N-Chloroformylmethyl-N-methyl) amino-7-nitro-2,l,3-benzoxadiazole (NBD-COC1) and 4-(N-chloroformethyl-N-methyl) amino-7-N,N-dimethylamino-sulfonyl-2,l,3-benzoxadiazole(DBD-COCl) were chosen as the fluorescent derivatizing reagents. The derivatives of FLX were resolved on Chiralpak AD-RH, Chiralpak AD and Chiralcel OD columns. The derivatization and resolution techniques were applied to pharmacokinetic studies on FLX and NFLX in rats. 1. Derivatization of FLX and NFLX with NBD-COC1 and DBD-COC1.As the fluorescent labeling reagents, NBD-COC1 and DBD-COC1, both of which has an acyl halide group, were selected to derivatize the amines. The temperature, time and other conditions were examined for the reaction and the fluoresceace yield of the derivatives were compared. Between two reagents, NBD-COC1 was better reagent with regard to the sensitivity and reactivity. NBD-COC1 reacted with FLX at 60℃ for 2h ,faster than DBD-COCI , to form strong fluorescent derivative (ex=478 2nm , em=537 2nm) while DBD-COCI reacting with FLX at 60 for 3h to form weaker fluorescent derivative ( ex=454 2nm, em=562 2nm). Like FLX, NBD-NFLX derivative was more fluorescence than DBD-NFLX derivative, and both NBD-COCI and DBD-COCI reacted with NFLX at 60 within 30min. To our knowledge, no paper has been published on derirvatizing amines with NBD-COCI . 2. Resolution of FLX fluorescent derivativesThe enantiomers of NBD-FLX and DBD-FLX were separated on Chiralpak AD-RH, Chiralpak AD and Chiralcel OD columns, but no resolution was observed for NFLX derivatives. On AD-RH, the best separation was obtained with CH3CN-CH3OH-IPA-H2O (52:23:5:20) as mobile phase at 35 for NBD-FLX and with CH3CN-H2O (54:46) as mobile phase at 38 for DBD-FLX. The mobile phase components and column temperature affected the resolution greatly in the reversed phase mode. Increasing column temperature favorites the sharping the peak to gain high column efficiency and resolution. More Rs and a values were for NBD-FLX other than DBD-FLX enantiomers. As regard with the elution order, S-isomes of the derivatives eluted first on AD-RH.The separation of NBD-FLX and DBD-FLX enantiomers was also performed on the Chiralpak AD column. Baseline separation was obtained for NBD-FLX with mobile phase of IPA-hexane (50:50) and nearly baseline separation for DBD-FLX with hexane-IPA(80:20) at 25 . The elution order of the two derivatives on the AD column was reversed compared to that on the AD-RH column as expected. Just like on the AD-RH column, better separation was obtained for NBD-FLX. It may be attributed to the structural difference between two compounds which leads to a different chiral recognition ability.Alcohol modifiers such as methanol, ethanol and IPA failed to resolve NBD- and DBD-FLX on OD column. So non-alcohol modifiers such as THF, acetonitrile were tested. As a result, better enantiomeric separations of the derivatives were reached by use of THF or THF and acetonitrile as modifiers. The optimal mobile phases were hexane-THF (75:25) for NBD-FLX on 15cm OD column and hexane-THF-acetonitrile (80:20:2) for DBD-FLX on 25cm OD column. Interestin...
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