title: 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.
abstract: 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.
authors.affiliation: Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London EC1M 6BQ, UK. Electronic address: e.a.ghazaly@qmul.ac.uk.
authors.forename: Essam
authors.initials: E
authors.lastname: Ghazaly
authors.affiliation: Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London EC1M 6BQ, UK.
authors.forename: Jackie
authors.initials: J
authors.lastname: Perry
authors.affiliation: Centre for Experimental Cancer Medicine, Barts Cancer Institute, Queen Mary University of London, London EC1M 6BQ, UK.
authors.forename: Christiana
authors.initials: C
authors.lastname: Kitromilidou
authors.affiliation: Centre for Experimental Cancer Medicine, Barts Cancer Institute, Queen Mary University of London, London EC1M 6BQ, UK.
authors.forename: Thomas
authors.initials: T
authors.lastname: Powles
authors.affiliation: Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London EC1M 6BQ, UK.
authors.forename: Simon
authors.initials: S
authors.lastname: Joel
mesh_terms: D000097:Acetonitriles; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000032:analysis; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003110:Colonic Neoplasms / Q000737:chemistry*; D004355:Drug Stability; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D042541:Intracellular Space / Q000737:chemistry; D000077146:Irinotecan; D016018:Least-Squares Analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000097:Acetonitriles; D000077146:Irinotecan; D002166:Camptothecin; C032159:acetonitrile
keywords: HPLC-fluorescence; Method comparison; Method validation; SN-38; Ultra-high performance LC–MS/MS