D-Serine was detected in dialysate collected from the rat striatum using an online microdialysis-CE-LIF instrument. Dialysate can be analyzed every 12.5 s using the online instrument, giving much better temporal resolution than previously possible for D-serine. Basal concentrations of D-serine (8 +/- 2 microM), glutamate (0.8 +/- 0.2 microM), GABA (0.11 +/- 0.04 microM), and L-serine (23 +/- 4 microM) were measured. Increases in the concentrations of these neurochemicals induced by the introduction of high-K+ aCSF were quantitated. Notably, an increase in D-serine concentration in response to high-K+ aCSF was observed for the first time. The identity of the D-serine peak was confirmed unambiguously using D-amino acid oxidase to selectively remove D-serine from a dialysate sample. The microdialysis-CE-LIF instrument was able to monitor this enzymatic reaction as it proceeded over a period of 60 min, demonstrating that online microdialysis-CE-LIF is not only useful in monitoring in vivo dynamics but can also be used to monitor other chemical systems.

Doxorubicin (DOX) treatment of NS-1 mouse hybridoma cells results in the formation of zeptomole amounts of metabolites per cell that are difficult to determine by confocal microscopy or HPLC. The native fluorescence of DOX and its metabolites together with laser-induced fluorescence detection (HF) has previously been used to detect a maximum of four components. In this study, we use capillary electrophoresis with postcolumn LIF (CE-LIF) to separate and detect 12 components attributed to DOX metabolism, resulting from treatment of NS-1 cells with 25 microM DOX for 8 h. The so-called metabolites 8 and 10 have been identified as doxorubicinone (DOXone) and 7-deoxydoxorubicinone (7-deoxyDOXone), respectively, by comigration with the corresponding synthetic standard. Due to comigration of DOX with doxorubicinol (DOXone), the presence of DOXone had to be determined separately by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The rest of the metabolites remain unidentified and are referred to by their number assignment. In comparison with the whole cell lysate, fractionation by differential centrifugation results in a better separation resolution of metabolites due to reduced amounts of metabolites in each fraction. This approach was chosen to compare the distribution of 13 metabolites in three subcellular fractions that form a pellet at< 1,400 g, 1,400-14,000 g, and > 14, 000 g and that generically are enriched in nuclei, organelles (mitochondria and lysosomes), and cytosolic components, respectively. The most abundant metabolite, DOXone, was estimated to be 90 +/- 15, 18 +/- 2, and 60 +/- 12 amol/cell (n = 5) in the nuclear-enriched, organelle-enriched, and cytosole-enriched fractions, respectively. In contrast, the total amount of other metabolites in a given fraction varied from 0 to 1,300 zmol. 7-DeoxyDOXone is the only metabolite that was present at similar levels in the three fractions. Other salient observations are metabolites 3, 7, and 11 are not detectable in the nuclear-enriched, organelle-enriched, and cytosole-enriched fractions, respectively; metabolite 9 and DOXone are more abundant in the nuclear-enriched fraction than in the other two fractions. The observations presented here suggest that subcellular fractionation followed by CE-LIF could be a powerful diagnostic for monitoring drug distribution, which is highly relevant to DOX cytoxicity studies.

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