Analysis of Femtomole Concentrations of α-Ketoisocaproic Acid in Brain Tissue by Precolumn Fluorescence Derivatization with 4,5-Dimethoxy-1,2-diaminobenzene
✍ Scribed by R.E. Keen; C.H. Nissenson; J.R. Barrio
- Book ID
- 102966404
- Publisher
- Elsevier Science
- Year
- 1993
- Tongue
- English
- Weight
- 552 KB
- Volume
- 213
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
An extremely sensitive fluorimetric HPLC method used to determine in vivo cerebral (\alpha)-ketoisocaproic acid (KIC) concentration with less than (2 \mathrm{~g}) of tissue (e.g., a single rat brain, (\sim 1.8 \mathrm{~g}) ) is reported. Removal of unwanted lipids and amino acids and isolation of (\alpha)-ketoacids in an optimal derivatization buffer are achieved by (\mathbf{C 1 8}) solid-phase extraction of the acid-soluble fraction of brain tissue. Quantitation of KIC to the femtomole level is accomplished by reversed-phase HPLC using 4,5-dimethoxy-1,2-diaminobenzene precolumn fluorescence derivatization and on-line fluorescence detection. These techniques are applicable to femtomole quantitation of other (\alpha)-ketoacids in various tissue and blood matrices. Combination of this fluorimetric method with simple nonchromatographic procedures to measure in vivo cerebral (\left[1-{ }^{14} \mathrm{C}\right] \alpha)-ketoisocaproic acid radioactivities in tissue provides estimates of specific activities. c 1993 Academic Press. Inc.