N-Dealkylation is a common metabolic step carried out by cytochromes P450 toward drugs, pollutants, and dietary molecules; the oxidative reaction ultimately yields an amine and an aldehyde.  The aldehydes are typically low molecular weight and thus not amenable to identification and quantitation by traditional analytical tools.  The common strategy is to label aldehydes (or more generally, carbonyls) through reaction with 2,4-dinitrophenylhydrazine to form the corresponding hydrazones.  This method relies on acidification with high levels of HCl, liquid-liquid extraction in glass reaction vials, and isolation of fractions containing hydrazones for dry down/resuspension for LC analysis using absorbance and/or MS detection.  Nevertheless, this approach requires harsh acidic conditions, labor-intensive liquid-liquid extraction, and multiple other steps that make it unsuitable for processing large numbers of samples.  As an alternative, we are developing high throughput labeling methods to identify and quantitate carbonyls including aldehydes using absorbance and fluorescence tags.  We are currently optimizing the labeling of a diverse set of alkyl, allyl, and aromatic aldehydes as standards and products of reactions with dansyl hydrazine (DNH).  In brief, samples are arranged in a 96 well plastic microplate.  The addition of a 10-fold excess of acetonitrile precipitates proteins and buffer, which are then removed by centrifugation.  Aldehydes are then labeled in 15 min upon DNH addition in the presence of acetic acid as a catalyst.  After 15 min reaction, samples are dried down and then resuspended in mobile for LC analysis measuring fluorescence and MS (due to increase in molecular weight form the label).  On-going studies have shown that DNH reacts almost stoichiometrically with aldehydes, so only low molar excess is necessary.  Only catalytic amounts of acid needed but does lead to formation of acid hydrazone, which is resolvable chromatographically or by MS.  Moreover, signal intensities for hydrazones depended on depended on aldehyde structure.  Current efforts are establishing the limits of detection and quantification for this method before exploring labeling of other carbonyls and other labels.  These seminal findings provide encouraging results for a more efficient, less costly yet sensitive strategy to identify and quantitate aldehydes such as those from P450 reactions.

Development of rapid, high throughout labeling methods for measuring aldehydes from P450 reactions

254th National Meeting (2017)

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