In LC-MS, derivatization is primarily used to improve ionization characteristics, especially for analytes that are not (efficiently) ionized by ESI or APCI such as aldehydes, sugars, and steroids. Derivatization strategies are then directed at the incorporation of a group with a permanent charge. A compound class that typically requires derivatization prior to LC-MS is the group of small aliphatic aldehydes that are, for instance, analyzed as the key biomarkers for lipid peroxidation in organisms. Here we report the development of a new tailor-made, highly sensitive, and selective derivatization agent 4-(2-(trimethylammonio)ethoxy)benzenaminium halide (4-APC) for the quantification of aldehydes in biological matrixes with positive ESI-MS/ MS without additional extraction procedures. 4-APC possesses an aniline moiety for a fast selective reaction with aliphatic aldehydes as well as a quaternary ammonium group for improved MS sensitivity. The derivatization reaction is a convenient one-pot reaction at a mild pH (5.7) and temperature (10 degrees C). As a result, an in-vial derivatization can be performed before analysis with an LC-MS/MS system. All aldehydes are derivatized within 30 min to a plateau, except malondialdehyde, which requires 300 min to reach a plateau. All derivatized aldehydes are stable for at least 35 h. Linearity was established between 10 and 500 nM and the limits of detection were in the 3-33 nM range for the aldehyde derivatives. Furthermore, the chosen design of these structures allows tandem MS to be used to monitor the typical losses of 59 and 87 from aldehyde derivatives, thereby enabling screening for aldehydes. Finally, of all aldehydes, pentanal and hexanal were detected at elevated levels in pooled healthy human urine samples.

Based on the template of a recently introduced derivatization reagent for aldehydes, 4-(2-(trimethylammonio)ethoxy)benzeneaminium dibromide (4-APC), a new derivatization agent was designed with additional features for the analysis and screening of biomarkers of lipid peroxidation. The new derivatization reagent, 4-(2-((4-bromophenethyl)dimethylammonio)ethoxy)benzenaminium dibromide (4-APEBA) contains a bromophenethyl group to incorporate an isotopic signature to the derivatives and to add additional fragmentation identifiers, collectively enhancing the abilities for detection and screening of unknown aldehydes. Derivatization can be achieved under mild conditions (pH 5.7, 10 °C). By changing the secondary reagent (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide instead of sodium cyanoborohydride), 4-APEBA is also applicable to the selective derivatization of carboxylic acids. Synthesis of the new label, exploration of the derivatization conditions, characterization of the fragmentation of the aldehyde and carboxylic acid derivatives in MS/MS, and preliminary applications of the labeling strategy for the analysis of aldehydes in urine and plasma are described.FigureStructure and MS/MS fragmentation spectrum of 4-APEBA reagents derivatized with octanoic acidElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-010-3575-1) contains supplementary material, which is available to authorized users.

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A high-resolution screening (HRS) technology is described, which couples 2 parallel enzyme affinity detection (EAD) systems for substrates and inhibitors of rat cytosolic glutathione-S-transferases (cGSTs) and purified human GST P1 to gradient reversed-phase high-performance liquid chromatography (HPLC). The cGSTs and GST P1 EAD systems were optimized and validated first in flow injection analysis (FIA) mode, and optimized values were subsequently used for HPLC mode. The IC 50 values of 8 ligands thus obtained online agreed well with the IC 50 values obtained with microplate reader-based assays. For ethacrynic acid, an IC 50 value of 1.8 ± 0.4 µM was obtained with the cGSTs EAD system in FIA mode and 0.8 ± 0.6 µM in HPLC mode. For ethacrynic acid with the GST P1 EAD system, IC 50 values of 6.0 ± 2.9 and 3.6 ± 2.8 µM were obtained in FIA and HPLC modes, respectively. An HRS GST EAD system, consisting of both the cGSTs and the GST P1 EAD system in HPLC mode in parallel, was able to separate complex mixtures of compounds and to determine online their individual affinity for cGSTs and GST P1. Finally, a small library of GST inhibitors, synthesized by reaction of several electrophiles with glutathione (GSH), was successfully screened with the newly developed parallel HRS GST EAD system. It is concluded that the present online gradient HPLC-based HRS screening technology offers new perspectives for sensitive and simultaneous screening of general cGSTs and specific GST P1 inhibitors in mixtures. (Journal of Biomolecular Screening 2007:396-405)

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