Development and validation of high-performance liquid chromatographic method for quantification of irinotecan and its active metabolite SN-38 in colon tumor bearing NOD/SCID mice plasma samples: Application to pharmacokinetic study

Abstract

Irinotecan (IRT) is an antineoplastic agent widely used in the treatment of various cancers primarily in colorectal cancer. A new, simple and sensitive high-performance liquid chromatography (HPLC) method coupled with fluorescence detector was developed and validated to quantify IRT and its active metabolite SN38 in the plasma of non-obese diabetic/severe combined immune-deficient mice (NOD/SCID) mice bearing colon tumor. The plasma samples were extracted by precipitation method using acetonitrile with 0.1% formic acid. The chromatographic separation was achieved using mobile phase consisted of water and acetonitrile (57:43 v/v) pH 3 at the flow rate of 0.8 mL/min in C18 column (internal diameter, 250 × 4.6 mm; pore size, 5 μm). The method was validated according to the bioanalytical guidelines defined by Food and Drug Administration (FDA) and European Medicine Agency (EMA). A regression (R2 ) value of 0.999 and 0.997 for IRT and SN38 suggested the good linearity in the range of 0.1–10 μg/mL and 5–500 ng/mL, respectively. The calculated lower limit of quantification (LLOQ) and limit of detection (LOD) for IRT were 0.1 and 0.065 μg/mL, respectively. However, for SN38, LLOQ and LOD were 5 and 2 ng/mL, respectively. The intra-day and inter-day variations (coefficient of variance; % CV) observed during the validation were found to be within the set limit of 15%. Both accuracy and percentage recovery analyzed and calculated from the quality control samples were in the between the defined range of 85–115%. Plasma samples were found to be stable when stored at room temperature for 2 h, after 2 freeze–thaw cycles and at −80 °C for 2 months. The developed method was successfully applied to study the plasma elimination profile of IRT in NOD/SCID mice with tumor. The results from plasma concentration time profile and pharmacokinetic parameter analyzed suggested the rapid elimination of IRT and SN38 from the plasma of NOD/SCID mice.