Analysis of Femtomole Concentrations of α-Ketoisocaproic Acid in Brain Tissue by Precolumn Fluorescence Derivatization with 4,5-Dimethoxy-1,2-diaminobenzene

An extremely sensitive fluorimetric HPLC method used to determine in vivo cerebral α-ketoisocaproic acid (KIC) concentration with less than 2 g of tissue (e.g., a single rat brain, ∼1.8 g) is reported. Removal of unwanted lipids and amino acids and isolation of α-ketoacids in an optimal derivatization buffer are achieved by C18 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 α-ketoacids in various tissue and blood matrices. Combination of this fluorimetric method with simple nonchromatographic procedures to measure in vivo cerebral [1-14C]α-ketoisocaproic acid radioactivities in tissue provides estimates of specific activities.