Journal
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume 769, Issue 1, Pages 97-106Publisher
ELSEVIER
DOI: 10.1016/S1570-0232(01)00633-X
Keywords
doxorubicin
Funding
- NIGMS NIH HHS [R01-GM61969-01A1, 5-T32-GM08347] Funding Source: Medline
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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 muM 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. (C) 2002 Elsevier Science B.V. All rights reserved.
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