Femtomole quantitation of 7-ethyl-10-hydroxycamptothecine (SN-38) in plasma samples by reversed-phase high-performance liquid chromatography

7-Ethyl-10-hydroxycamptothecine (SN-38) is the active metabolite of the topoisomerase I inhibitor and antineoplastic agent, irinotecan (CPT-11). Here, we present a new and sensitive reversed-phase high-performance liquid chromatographic method for the determination of SN-38 in human plasma samples. Sample pretreatment involves a protein precipitation of 1-mL samples with 2 mL of acetonitrile, followed by a one-step solvent extraction with 5 mL of chloroform, with camptothecine used as internal standard. Chromatographic separation was achieved on an analytical column packed with Hypersil ODS material (100 x 4.6 mm i.d., 5 microm P.S.), and isocratic elution with a mixture of acetonitrile:0.1 M ammonium acetate containing 10 mM tetrabutylammonium sulfate (23:77, v/v), pH 5.3 (hydrochloric acid). The column effluent was monitored at excitation and emission wavelengths of 380 and 556 nm, respectively. The limit of quantitation of the method presented was at the low femtomole level ( approximately 8.4 fmol; equivalent to 5 pg/mL), with the standard curves being linear over nearly three orders of magnitude. Intraassay precision was9%, while interassay variations were between 2 and 5%. The extraction efficiency was concentration independent and averaged 88.0 +/- 14.3% (mean +/- standard deviation; n = 59). The described method will be used in future studies to assess the extent of enterohepatic recirculation of SN-38 in cancer patients following intravenous CPT-11 treatment.