Simmitecan (L-P) is an anticancer
ester prodrug, which involves activation to
chimmitecan (L-2-Z). In the current study, a liquid chromatography/tandem mass spectrometry-based method was developed for simultaneous determination of L-P and L-2-Z in various plasma samples. Because L-P is rapidly converted to L-2-Z by blood
carboxylesterase during and after sampling, which hampers accurate determination of L-P and L-2-Z in the
biological samples, different
carboxylesterase inhibitors were tested. As a result,
bis(4-nitrophenyl)phosphate gave the best results with respect to inhibitory capability,
hemolysis, and matrix effects and was used to deactivate blood
carboxylesterases when sampling. The plasma samples were precipitated with
acetonitrile and the resulting supernatants were separated using a pulse gradient method on a C18 column.
Irinotecan and
camptothecin were used as internal standards for quantification of L-P and L-2-Z, respectively. Protonated L-P, L-2-Z and their internal standards were generated by electrospray ionization and their precursor-product ion pairs (m/z 599→124, 405→361, 587→195, and 349→305, respectively) were used for measurement. The newly developed bioanalytical assay processed favorable accuracy and precision with lower limits of quantification of 2.1 nM for L-P and 3.4 nM for L-2-Z, and was successfully applied to pharmacokinetic studies in
tumor-bearing nude mice, rats, and dogs. There are substantial species differences in the
ester activity. The experimental strategies illustrated in our report may be adopted for measurement of other
prodrugs (including
irinotecan) or drugs subject to
ester hydrolysis, as well as their metabolites, in
biological matrices.