Development and validation of an ultra-high performance LC–MS/MS assay for intracellular SN-38 in human solid tumour cell lines: Comparison with a validated HPLC-fluorescence method

• An UPLC–MS/MS method was developed for measuring intracellular SN-38 concentrations.
• SN-38 was separated using a pentafluoryl phenyl UPLC column.
• Fast analysis and good peak shape obtained without the need for ion-pairing agents.
• Method was used to investigate SN-38 efflux in colorectal cancer cell lines.
• The method results were compared to a validated HPLC-fluorescent method.

A simple and rapid ultra-high performance liquid chromatography–mass spectrometry/mass spectrometry (UPLC–MS/MS) method has been developed for measuring intracellular concentrations of the anticancer agent 7-ethyl-10-hydroxycamptothecin (SN-38) in tumour cells using camptothecin (CPT) as internal standard. SN-38 extraction was carried out using acidified acetonitrile. SN-38 and CPT were separated on a PFP column using gradient elution with acidified water and acetonitrile. SN-38 and CPT were quantified using a triple quadrupole mass spectrometry system. Least square regression calibration lines were obtained with average correlation coefficients of R2 = 0.9993 ± 0.0016. The lower limit of detection (LOD) and lower limit of quantification (LOQ) for SN-38 were 0.1 and 0.3 ng/ml, respectively. CPT recovery was 98.5 ± 13% and SN-38 recoveries at low quality control (LQC, 5 ng/ml) and high quality control (HQC, 500 ng/ml) were 89 ± 6% and 95 ± 8%, respectively. The intra- and inter-day imprecision for LQC was 5.8 and 8.5%, and for HQC was 6.3 and 4.4%, respectively. The method was compared to a validated high performance liquid chromatography-fluorescent method. In addition, the method has been successfully applied to determine the intracellular accumulation of SN-38 investigating the transport through ABCB1 (P-gp) and ABCG2 (BCRP) efflux pumps in colorectal cancer cell lines.