Analysis of Amino Acids and Biogenic Amines in Breast Cancer Cells by Micellar Electrokinetic Chromatography
Date Issued
2009
Date
2009
Author(s)
Kao, Yu-Yen
Abstract
We have demonstrated separation of the derivatives of amino acids and biogenic amines with naphthalene-2,3-dicarboxaldehyde (NDA) by micellar electrokinetic chromatography (MEKC) in conjunction with a purple light-emitting diode-induced fluorescence detection using poly(ethylene oxide) (PEO). The roles that the concentrations of PEO, sodium dodecyl sulfate (SDS), and Tris-borate (TB) at various pH values played in resolution and separation efficiency of the analytes have been investigated. After injecting the sample to a capillary filled with 0.5 M TB buffer (pH 10.2) containing 40 mM SDS, 0.1% PEO that has been prepared in 100 mM TB (pH 9.0) containing 35 mM SDS enters the capillary. This MEKC condition allows separation of 14 analytes within 16 minutes, with limits of detection (LODs) at signal-to-noise ratio (S/N) of 3 ranging from 2.06 nM (glycine, Gly) to 19.2 nM (serotonin, 5-HT). RSD values (n = 5) of the migration times for the analytes are all less than 0.5%. This approach has been validated for the analysis of human breast cancer cells (MCF-7) and normal epithelial cell line (H184B5F5/M10) (2-3 × 106 cells /mL). The amounts of 9 (MCF-7) or 10 (H184B5F5/M10) amino acids, including serine (Ser), glutamine (Gln), histidine (His), Gly, alanine (Ala), taurine (Tau), tyrosine (Tyr), glutamate (Glu), valine (Val), and aspartate (Asp) in one cell could be calculated, respectively. The amounts of some amino acids in MCF-7 and H184B5F5/M10 are significantly different, maybe because of proliferation of cancer cells which consume much energy. Besides, we calculated the amounts of amino acids in cells incubated at different time interval, and found that the consumption of some amino acids in cancer cells rather than normal cells was obvious. In order to further improve the sensitivity of the amino acids and biogenic amines, we developed an on-line concentration technique using similar MEKC conditions. Once high voltage is applied, the NDA derivatives enter PEO solution containing SDS and then become stacked due to decreases in electric field and increases in viscosity. We have also found that TB and sodium chloride in the sample zone play some roles in determining the stacking efficiency. This on-line concentration MEKC technique allows improvement in the detection of the analytes by factors ranging from 4.4 (His) to 38.7 (Glu), leading to their LODs ranging from 0.10 nM (Glu) to 3.63 nM (5-HT). This approach has been validated by the analysis of 0.284-μL MCF-7 cells (2 × 105 cells/mL), and the results were consistent with those described in the first part.
Subjects
Micellar electrokinetic chromatography
Light-emitting diode induced fluorescence detection
Amino acids
Biogenic amines
Sodium dodecyl sulfate
Poly(ethylene oxide)
SDGs
Type
thesis
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