Fluorophore peak analysis

A prevailing problem with neuroactive steroid analysis by HPLC is the lack of sufficient chromaphores or fluorophores within their chemical structures to allow suitable spectrophotometric end-point detection such as with UV/VIS or fluorescence with adequate sensitivity. The multitude of structural isomers of the metabolites also decreases the applicability of RP-HPLC since the chromatographic profiles become very complex with co-eluting peaks. Due to these inherent problems, it is often necessary to derivatize this group of compounds prior to chromatographic separation and suitable end-point detection to allow their direct determination at physiological concentrations. 

The increase of carboxymethyllysine (batch 1) and pentosidine (batches 1 and 11) thus observed provided additional proof for fhe Maillard reaction in caries (fable 3, figs. 2, 3). The pentosidine level ranged from abouf equal fo a manifold of the level in sound dentin. The formation of pentosidine can only account for a fraction of the increase in 328/378 fluorescence, which is in accordance with a major contribution from a different fluorophore as stated above. Unfortunately, an increase of dify-rosine as expected from fhe gain in 317/407 fluorescence (fable 2) could not be substantiated unequivocally by HPLC analysis because dityrosine co-eluted with lysylpyridinoline. Even if we would consider dityrosine to originate the lysylpyridinoline peaks observed in HPLC of carious dentin, but not of sound dentin, only one quarter of the increase in 317/407 fluorescence would derive from difyrosine. 

The foremost advantage of the chemical assay is that the specificity of the analysis can be tested with the most rigid criteria. In the pituitary assay of oxytocin and vasopressin (Gruber et a/., 1976), specificity was demonstrated by the following four criteria (1) The oxytocin and vasopressin peaks were identified by their retention times. (2) Addition of synthetic oxytocin or vasopressin to the pituitary extract resulted in a quantitative increase in the height of the specific peak with no noticeable peak broadening. (3) After purification by reverse-phase chromatography, each of the nonapeptide fluorophors was collected, hydrolyzed in 6 N HCl, and then characterized... 

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