TY - JOUR
T1 - Capture of the volatile carbonyl metabolite of flecainide on 2,4-dinitrophenylhydrazine cartridge for quantitation by stable-isotope dilution mass spectrometry coupled with chromatography
AU - Prokai, Laszlo
AU - Szarka, Szabolcs
AU - Wang, Xiaoli
AU - Prokai-Tatrai, Katalin
N1 - Funding Information:
We thank Ms. Shastazia White for her excellent technical assistance with the synthesis and purification of internal standards. This work was supported in part by the National Institutes of Health (grant number AG025384 ) and the Robert A. Welch Foundation (endowment BK-0031).
PY - 2012/4/6
Y1 - 2012/4/6
N2 - Carbonyl compounds are common byproducts of many metabolic processes. These volatile chemicals are usually derivatized before mass spectrometric analysis to enhance the sensitivity of their detections. The classically used reagent for this purpose is 2,4-dinitrophenylhydrazine (DNPH) that forms the corresponding hydrazones. When DNPH is immobilized on specific cartridges it permits solvent-free collection and simultaneous derivatization of aldehydes and ketones from gaseous samples. The utility of this approach was tested by assembling a simple apparatus for the in vitro generation of trifluoroacetaldehyde (TFAA) and its subsequent capture on the attached DNPH cartridge. TFAA was generated via cytochrome P450-catalyzed dealkylation of flecainide, an antiarrhythmic agent, in pooled human liver microsomes. Stable-isotope dilution mass spectrometry coupled with GC and LC using negative chemical ionization (NCI) and electrospray ionization (ESI) was evaluated for quantitative analyses. To eliminate isotope effects observed with the use of deuterium-labeled DNPH, we selected its 15N 4-labeled analog to synthesize the appropriate TFAA adduct, as internal standard. Quantitation by GC-NCI-MS using selected-ion monitoring outperformed LC-ESI-MS methods considering limits of detection and linearity of the assays. The microsomal metabolism of 1.5μmol of flecainide for 1.5h resulted in 2.6±0.5μg TFAA-DNPH, corresponding to 9.3±1.7 nmol TFAA, captured by the cartridge.
AB - Carbonyl compounds are common byproducts of many metabolic processes. These volatile chemicals are usually derivatized before mass spectrometric analysis to enhance the sensitivity of their detections. The classically used reagent for this purpose is 2,4-dinitrophenylhydrazine (DNPH) that forms the corresponding hydrazones. When DNPH is immobilized on specific cartridges it permits solvent-free collection and simultaneous derivatization of aldehydes and ketones from gaseous samples. The utility of this approach was tested by assembling a simple apparatus for the in vitro generation of trifluoroacetaldehyde (TFAA) and its subsequent capture on the attached DNPH cartridge. TFAA was generated via cytochrome P450-catalyzed dealkylation of flecainide, an antiarrhythmic agent, in pooled human liver microsomes. Stable-isotope dilution mass spectrometry coupled with GC and LC using negative chemical ionization (NCI) and electrospray ionization (ESI) was evaluated for quantitative analyses. To eliminate isotope effects observed with the use of deuterium-labeled DNPH, we selected its 15N 4-labeled analog to synthesize the appropriate TFAA adduct, as internal standard. Quantitation by GC-NCI-MS using selected-ion monitoring outperformed LC-ESI-MS methods considering limits of detection and linearity of the assays. The microsomal metabolism of 1.5μmol of flecainide for 1.5h resulted in 2.6±0.5μg TFAA-DNPH, corresponding to 9.3±1.7 nmol TFAA, captured by the cartridge.
KW - 2,4-Dinitrophenylhydrazine (DNPH) cartridge
KW - GC-MS
KW - LC-MS
KW - N-labeled internal standard
KW - Volatile carbonyl metabolite
UR - http://www.scopus.com/inward/record.url?scp=84862782024&partnerID=8YFLogxK
U2 - 10.1016/j.chroma.2012.01.067
DO - 10.1016/j.chroma.2012.01.067
M3 - Article
C2 - 22342210
AN - SCOPUS:84862782024
SN - 0021-9673
VL - 1232
SP - 281
EP - 287
JO - Journal of Chromatography A
JF - Journal of Chromatography A
ER -