Novel Isotope-Coded Derivatization Method for Aldehydes Using ¹⁴N/¹⁵N-Ammonium Acetate and 9,10-Phenanthrenequinone

Abstract

Isotope-coded derivatization (ICD) is used as a promising alternative approach to isotope internal standards in order to overcome matrix effects caused by coexisting substances that often occur while analyzing metabolites by LC-MS/MS. ICD introduces two different mass tags to every analyte via the use of heavy and light forms of the derivatization reagents. Herein, we report the first ICD approach for aldehydes that uses commercially available reagents avoiding the need for expensive and tedious multisteps synthetic procedures. The method is based on the reaction of the safe and stable derivatizing agent, 9,10-phenanthrenequinone, and the cheap and commercially available ICD reagent, 14 N/ 15 N-ammonium acetate, with aldehydes followed by LC/ESI + -MS/MS. Multiple reaction monitoring is done at the transitions m/z [M + H] + → m/z [Product ion A] and m/z [M + 2 + H] + → m/z [Product ion A + 2] for 14 N-and 15 N-labeled analytes, respectively. Among lipid peroxidation products, 4-hydroxy-2-nonenal (HNE) and 4-hydroxy-2-hexenal (HHE) are considered the most toxic produced aldehydes as they contain additional two reactive functional groups, the unsaturated bond and the hydroxyl group, besides the aldehyde one. Thus, they were chosen as representative analytes in this study. The developed method was able to detect HHE and HNE in human serum with very high sensitivity down to LOQ of 0.2 and 0.05 nM, respectively, employing an expedient salting out liquid−liquid extraction method. The developed method was able to differentiate between the levels of HHE and HNE in serum samples of healthy subjects and diabetic, rheumatic, and cardiac disorder patients.

Current trends in isotope‐coded derivatization liquid chromatographic‐mass spectrometric analyses with special emphasis on their biomedical application

“…Such a method was applied for the determination of two highly toxic lipid peroxidation-related aldehydes, namely, 4-hydroxy-2-nonenal and 4-hydroxy-2-hexenal in the serum of diabetic, rheumatic, and cardiac disorder patients as well as a control group. The levels of aldehydes were found to be elevated in all the studied pathological conditions compared with the healthy group (El-Maghrabey, Kishikawa, et al, 2018a).…”

“…Regarding the abundance of the natural isotope, a similar experiment but in a reverse manner could be conducted through injecting the light form of the reagent and simultaneously monitoring the normalized chromatograms of the heavy and light forms. Absence of substantial detected peaks for the heavy labeled form proves the selectivity and reliability of the ICD reagent (El‐Maghrabey, Kishikawa, et al, ). Based on this we can conclude that the low percentage of heavy isotope content, along with low chemical purity, could result in misinterpretation of the results and thus they should be carefully studied by the analyst.…”

“…Furthermore, the heavy isotope contents in the ICD reagent should be large enough to overcome the effect of chemical purity and abundance of the natural isotope of the reagents (El‐Maghrabey, Kishikawa, et al, ; Niedenführ et al, ). The effect of these two factors was recently studied clearly by El‐Maghrabey et al Regarding the chemical purity of the heavy form of the reagents, the heavy labeled analytes were injected into the LC–MS system and the normalized chromatograms of the heavy and light forms are simultaneously monitored.…”

“…Furthermore, the heavy isotope contents in the ICD reagent should be large enough to overcome the effect of chemical purity and abundance of the natural isotope of the reagents (El-Maghrabey, Kishikawa, et al, 2018a;Niedenführ et al, 2016). The effect of these two factors was recently studied clearly by El-Maghrabey et al…”

“…• Isotope contents in the ICD reagent should be large enough to overcome these problems and their effect should be tested in detail as performed by El-Maghrabey, Kishikawa, et al (2018a). 6) False-positive results in metabolomics owing to the reagent impurities and by-products, as well as the absence of adaptation to the compounds having non-reactive functional groups.…”