Development and validation of an ultra-high performance LC-MS/MS assay for intracellular SN-38 in human solid tumour...

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 R(2)=0.9993±0.0016. The lower limit of detection (LOD) and lower limit of quantification (LOQ) for SN-38 were 0.1 and 0.3ng/ml, respectively. CPT recovery was 98.5±13% and SN-38 recoveries at low quality control (LQC, 5ng/ml) and high quality control (HQC, 500ng/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.