Abstract: AIM: To develop a sensitive and reliable method for determination of kaempferol-3-O-rutinoside (NFR) in plasma.METHODS: The optimal ionization and fragmentation conditions, aswell as liquid chromato-graphic ones, to detect kaempferol-3-O-rutinoside were identified.Effective sample clean-up method for recover-ing the analyte from plasma was also studied.The newly developed analytical methodwas evaluated in terms of ac-curacy, precison, selectivity, sensitivity, and stability, which was further demonstrated its applicability in a pilot plasma pharmacokinetics study in Sprague-Dawley rats re-ceiving an intravenous dose of NFR at 30 mg·kg^-1.RESULTS: Positive ion electrospray ionization was found to effeciently generate precursor protonated moleculesof both the analyte and the internal standard (IS, l-Stepholidine) which was optimized and used to produce their character-istic and abundant fragment ions for MS/MS analysis in selected reaction monitoring mode with precursor-to-prod-uct ion transitions m/z 595→287 for NFR and m/z 328 →178 for IS.The chromatographic retention times were 2.3 and 2.2 min for NFR and IS, respectively.Liquid-liquid extraction using ethyl acetate provided good recov-ery for both the analyte and IS from HCl-acidified rat plasma samples demonstrating 58.5% -70.1% and 72.5% recovery, respectively.NFR and IS were stable during the whole process of the bioassay.Good linearity was achieved for regresion of NFR IS peak area ratio to nominal plasma concentration of the analyte demonstrating r value equal to 0.9999 (n =6×5), when the concen-trationwas between 0.192 and 600 ng·ml^-1.The within-run acurracy and presicion of the analytical method were quite good for plasma NFR, i.e., 92%-107% and 1.0%-5.7%, respectively and the between-run data were 94%-99 %and 1.5%-8.4%, respectively.The lower limit of quantification (LLOQ) of the method was 0.192 ng·ml^-1.Following a single intravenous dose of NFR at 30 mg·kg^-1 to Sprague-Dawley rats, NFRexhib-ited the elimination half-life (T_1/2) 1.27 h and mean res-ident time (MRT) 0.32 h.The clearance (CL) and dis-tribution volume at stable status (V_SS) of NFRwere 2.73 L·h·kg^-1 and 0.92 L·kg^-1, respectively.CONCLUSION: The first analytical method based on LC-MS/MS, providing simple, robust, sensitive, and rapid measure-ment of NFR in plasma, is presented in this communica-tion, which will prove useful for the future preclinical pharmacokinetic evaluation of NFR.

Key words: Kaempferol-3-O-rutinoside, LC-MS/MS, plasma concentration, pharmacokinetics