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

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, 14N/15N-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 14N- and 15N-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.