Versatile online SPE-HPLC method for the analysis of Irinotecan and its clinically relevant metabolites in biomaterials

Monitoring levels of Irinotecan and its metabolites during cancer therapy could help link broad interpatient variations in antitumor activity and toxicity to the patient's metabolic status. We have developed and validated a versatile and highly sensitive method for the simultaneous determination of Irinotecan and its clinically relevant metabolites 7-ethyl-10-hydroxy-camptothecin (SN-38) and SN-38 glucuronide. Sample clean-up involves precipitation by acetone/methanol/0.5 M trichloroacetic acid at 4:4:2 v/v followed by extraction of the metabolites on an SPE column by 20% methanol in 25 mM KH ₂PO₄ pH 2.9. Online transfer to an analytical μBondapak C18 column, elution with 24% acetonitrile (ACN) in 0.1 M KH₂PO ₄ pH 2.9 and fluorescence detection with excitation at 375 nm and emission at 430 nm for SN-38 glucuronide and Irinotecan or 540 nm for SN-38 results in high sensitivity (1-2 pg) and short (∼10 min) run times. The method was used to determine the degree of SN-38 glucuronidation in mice after Irinotecan administration and in cultured cancer cells exposed to SN-38. The method may be used to better understand Irinotecan metabolism, personalize therapy, and develop Irinotecan-based tumor targeting therapies.