Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
7615065 | Journal of Chromatography B | 2018 | 9 Pages |
Abstract
Altered levels of polyamines in biological specimens have been suggested as potential biomarkers for cancer. Difluoromethylornithine (DFMO, an irreversible inhibitor of ornithine decarboxylase) is reported to modulate polyamines to potentially attenuate proliferation of neuroblastoma cells. A clinical trial is being conducted to evaluate DFMO in various cancers. To determine the pharmacodynamics effect of DFMO, an analytical assay is needed to accurately measure the changes in polyamines in cancer cells. In this study, a novel pH gradient LC-ESI-MS/MS method was developed and validated for the quantitation of polyamines (putrescine, spermidine and spermine) in cancer cells. To separate polar and basic polyamines, a multi-mode column composed of ODS and weak ionic ligands was used. The pH gradient was generated from pHâ¯5.3 to pHâ¯2.7 with 2â¯mM ammonium acetate and 0.4% acetic acid in 10% acetonitrile as mobile phase. The detection of polyamines was performed utilizing multiple reaction monitoring on electrospray ionization mass spectrometry operated in positive ion mode. A pH gradient method increased resolution and decreased peak width of conventional analytical assays, resulting in 10-250-fold higher detection limits. Mobile phases without ion-pairing reagents were LC-MS compatible and eliminated possible signal suppression and MS contamination. The developed method was successfully applied to the analysis of polyamines in neuroblastoma and leukemia cells treated with DFMO. Putrescine levels were significantly (pâ¯<â¯0.001) decreased in CCRF-CEM (3.68 vs 1.23â¯ng/mg protein), SK-N-BE(2) (1.98 vs 1.31â¯ng/mg protein) and CHLA-20 (2.06 vs 0.90â¯ng/mg protein) cells treated with DFMO relative to vehicle control. The assay will provide a useful tool in determining the pharmacodynamic effect of DFMO in cancer clinical trials.
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Hwang Eui Cho, Min H. Kang,