Several biochemical processes are accompanied by the formation of volatile carbonyl products. Identification and quantitation of these compounds from ambient air samples or exhaled breath have often been of interest. For example, measurement of acetaldehyde formed by the enzymatic oxidation of ethanol has been employed for metabolic flux analysis in fermentation experiments [1
]. Analysis of the metabolome for volatile carbonyl compounds may also be informative for the physiological state of an individual, as well as for exposure to various drugs and environmental chemicals [2
]. Notably, increased breath acetone levels are highly correlated with diabetes and have been successfully used for the non-invasive diagnosis/monitoring of diabetic patients [3
]. In general, volatile carbonyls in the exhaled breath representing lipid peroxidation end-products have often been considered as potential biomarkers of oxidative stress and metabolic status [4
]. Another important process that generates carbonyl compounds is dealkylation (heteroatom release) mediated by cytochrome P450 (CYP) during drug metabolism [6
]. CYP enzymes represent the main drug metabolizing system in mammals and they also catalyze the oxidation of various endogenous (e.g., bile acids, steroids, and cholesterol) and exogenous (e.g., drugs, pollutants, and dietary components) chemicals. Since oxidative dealkylation of a drug containing an ether, thioether or alkylamino functional group produces the corresponding aldehyde in addition to the dealkylated drug (), measurement of an exhaled carbonyl compound may allow for non-invasive assessing of in vivo
drug metabolism [7
]. Due to their high volatility and reactive nature, determination of carbonyl metabolites is usually performed after derivatization to fix their concentration at a given time and/or to afford improved detection [9
]. The classically used derivatization method to detect carbonyls has been the use of 2,4-dinitrophenylhydrazine (DNPH) to form the corresponding hydrazones.
Fig. 1 Flecainide dealkylation by cytochrome P450 .
Sampling of aldehyde or ketone from vapors requires impingers or bubblers [10
], and the usual sample preparation procedure involves extraction with large amount of organic solvent that needs to be removed before analysis [9
]. To overcome these limitations, a method to capture volatile carbonyl compounds by DNPH-coated cartridges has been developed that allows solvent-free collections and simultaneous derivatization of the carbonyl compound(s) of interest [11
]. Identification of the generated hydrazones is usually done by gas chromatography (GC) using flame ionization [12
] or electron capture [13
] and mass spectrometric (MS) detection [15
]. With the latter, negative-ion detection has been found to be the most advantageous [9
]. In particular, GC–MS with negative-chemical ionization (NCI) has been useful due to its high selectivity and sensitivity [17
]. In addition, HPLC alone [18
] or coupled with MS, or tandem MS (MS/MS) [9
] have been utilized for carbonyl determination. In any case, an internal standard (IS) is required for accurate quantitative analysis of these volatile molecules. Although deuterium labeled hydrazones have been considered as ISs in the DNPH derivatization strategy [9
], a typical obstacle is that the labeled analytes are not always available commercially or can be easily obtained by in-house synthesis. To overcome these limitations, deuterium labeled DNPH (d3
-DNPH) has been used to synthesize the corresponding d3
-labeled IS suitable for quantitation by isotope-dilution mass spectrometry [21
]. An isotope effect resulting in shorter retention time for the deuterated hydrazones compared to that of corresponding unlabeled counterparts has, however, been observed upon LC or GC separation [22
]. It has also been shown that such a deuterium isotope effect may be significant enough to change the analyte to IS peak area ratios and, therefore, to influence the accuracy of quantitations [23
]. To eliminate possible isotope effects brought about by the use of deuterium labeled ISs, we chose 15
-labeled DNPH for synthesizing appropriate ISs for quantitative measurements of carbonyl compounds. We have unequivocally by stable-isotope dilution MS coupled with chromatography.
The utility of this approach was tested by assembling a simple apparatus to generate and capture trifluoroacetaldehyde (TFAA) formed in vitro by CYP-catalyzed dealkylation of an antiarrhythmic agent, flecainide (N-(2-piperidylmethyl)-2,5-bis(2,2,2-trifluoroethoxy)benzamide monoacetate), in pooled human liver microsomes.