Identification and Characterization of Metabolites of Irinotecan in-vivo and in-vitro matrixes by HPLC/LC-MS

Abstract: In the present study metabolite identification and characterization has done by using HPLC and LC-MS. During method development various mobile phases have tried for identification of
metabolites. The matrixes selected for in- vivo study were urine because nearly all the metabolites of irinotecan were obtained in it. The extraction mixtures have selected to retain the maximum amount of analyte with less effort. During experiment four extraction solvents were used in six different concentrations out of which TBME suit our method. In-vitro study done by Human Liver microsomes by using Phosphate buffer (pH 7.4) and NADPH as co-factors for initiation of enzymatic reaction Irinotecan is a prodrug that is converted in the liver to an active metabolite, SN-38. It is eliminate in Bile and Faeces and thus its dose reduced in Hepatic Failure. Irinotecan act by inhibiting Topoisomerase-1.It is the enzyme which nicks, introduces negative supercoils and reseals the DNA strand. Conventionally, drug metabolite identification in the past has usually been based on the comparison of ultraviolet (UV) spectral data and high-performance liquid chromatography (HPLC) retention times of isolated ‘unknown’ metabolites with those of synthesised standards. Such a method of detecting and characterising drug metabolites is an uncertain, time-consuming and expensive process, as well as affording very limited structural information. Furthermore, Phase I metabolism of a drug candidate often results in only minor structural modification of the parent compound; these minor changes can make it particularly difficult to determine suitable chromatographic conditions to effect HPLC separation of metabolites. This study describes contemporary approach to identification and characterization of xenobiotic metabolites in complex biological fluids derived from drug metabolism studies. Keywords: HP LC/MS/MS, Irinotecan, Metabolites, in vitro/in vivo