Development of an LC-MS/MS Method for Measurement of Irinotecan and Its Major Metabolites in Plasma: Technical Considerations

Abstract

Objective: Irinotecan (CPT-11) is an important drug used in the treatment of several solid tumor types. To minimize its toxicity, therapeutic drug monitoring of CPT-11 and its major metabolites (SN-38, SN-38-glucuronide [SN-38G], and APC) has been proposed. We aimed to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of CPT-11 and its major metabolites in plasma. Methods: Specimen preparation consisted of protein precipitation, evaporation, and reconstitution. Analyses were performed on a C18 column using reverse-phase gradient elution. Electrospray ionization and multiple reaction monitoring in positive mode were used for MS. The following heavy isotope-labeled internal standards were used: CPT-11 D10, SN-38 D3, SN-38G D3, and APC D3. Results: We found that CPT-11, SN-38G, and APC eluted at ~4.6 to 4.7 minutes, and SN-38 eluted at ~5.1 to 5.2 minutes. A second peak for SN-38 was detected at ~4.6 to 4.7 minutes. Given that the structure of SN-38 is found in CPT-11, SN-38G, and APC, and in the CPT-11 D10 used here, in-source fragmentation was the likely cause. In addition, we found that a low-level SN-38 impurity was present in CPT-11 D10 and to a lesser extent in SN-38 D3. Conclusion: When developing methods for CPT-11 and its metabolites, it is important to consider the effects of in-source fragmentation and the choice of internal standards.