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Abstract
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Topiramate is an antiepileptic agent, which is structurally different from the other anticonvulsants. The
drug has no UV–Vis absorption or emits fluorescence. Thus for its analysis using high performance liquid
chromatography (HPLC) with conventional UV or fluorescence detectors, the drug should be derivatized
with a suitable reagent. In previous study using fluorenylmethyl chloroformate (FMOC-Cl) and HPLC
coupled with fluorescence detector, we reported an analytical method for derivatization and analysis
of the drug in human serum. In this method, several factors including time and temperature of the
reaction, pH and concentration of the used buffer, ratio of organic phase in the medium and removal
of the reagent excess by glycine should be optimized to obtain maximum yield of the product. In HPLC
coupled with fluorescence detector, there is not any signal from intact topiramate and only the final product (FMOC-topiramate) is appeared. Thus to optimize the reaction conditions for obtaining the highest
derived yield, intensity of the final product peak is considered as a criteria for progression of the reaction.
In LC–MS/MS system however, both free and reacted topiramate are visible in observed spectra. In the
present study reaction of the drug with FMOC-Cl was re-optimized using LC–MS/MS technique on the
basis of reacted/free topiramate ratio as the new and more accurate index. The results showed that, ratio
of organic/aqueous phase has a dominant effect on the reaction, the most efficient temperature is 70 ?C
and the reaction is reversed following addition of the glycine.
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