Capillary electrophoresis with laser-induced fluorescence detection was used to separate and detect doxorubicin and at least five metabolites from NS-1 cells that were treated with 25 μM doxorubicin for 8 h. Using 10 mM borate, 10 mM sodium dodecyl sulfate (pH 9.3) as separation buffer, the 488-nm argon-ion laser line for fluorescence excitation, and a 635±27.5 nm bandpass filter for detection, the limit of detection (S/N=3) for doxorubicin is 61±13 zmol. This low limit of detection allows for the detection of a larger number of metabolites than previously reported. Two extraction procedures were performed: a bulk liquid-liquid extraction and an in-capillary single-cell lysis. While in the bulk liquid-liquid extraction procedure, recovery for doxorubicin range from 50 to 99%, in single cell analysis the recovery is expected to be complete. Furthermore performing lysis of a single cell inside the separation capillary prevents doxorubicin or metabolite loss or degradation during handling. Based on the bulk method the calculated metabolite abundance is in the sub-amol per cell range while it varies from 0.1 to 1.1 fmol per cell in single cell analysis confirming metabolite loss during handling. Each metabolite was found at a level less than 0.1% of the doxorubicin content in either method, suggesting a slow metabolism in the NS-1 cell system or effective removal of metabolites by the cell.
|Number of pages
|Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
|Published - Mar 25 2002
Bibliographical noteFunding Information:
Financial support from the National Institute of Health (R01-GM61969-01A1) and the Office of the Vice President for Research and Dean of the Graduate School of the University of Minnesota (Grant-in-aid) is greatly appreciated. A.A. thanks NIH support through a Biotechnology Training Grant (#5-T32-GM08347). Dieter Starke, University of Alberta, for construction of the capillary holder used for single cell analysis. Kathyrn Fuller and Nilhan Gunasekera for maintenance of cell cultures. Dr Antonio Suarato, Pharmacia, for his kind donation of 13-dihydrodoxorubicin (doxorubicinol).