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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.

On-line coupling of reversed-phase liquid chromatography to a biochemical detection system based on receptor-ligand interactions is described. The receptor affinity detection is performed using a postcolumn reaction detection system with open-tubular reaction coils. In the first step, a solution containing the steroid binding domain of the human estrogen receptor is added to the LC effluent via a mixing union, and the mixture is allowed to react. In the second step, a fluorescent estrogenic ligand, coumestrol, is added to saturate the remaining free binding sites of the estrogen receptor. Both heterogeneous and homogeneous detection systems have been developed. In the former case, free and receptor-bound coumestrol were separated prior to detection by means of a short column containing a restricted-access reversed-phase support. Since free and receptor-bound coumestrol differ significantly in fluorescence intensity, the system can also be operated in the homogeneous mode without requiring a separation step. Using 5 nmol/L of human estrogen receptor, a detection limit of 5 nmol/L was obtained for compounds possessing a high affinity for the estrogen receptor, such as 17 beta-estradiol and diethylstilbestrol. Detection limits for weaker ligands amount to 20 nmol/L. Non-estrogenic steroids such as methyltestosterone or progesterone are not detected at all. The selectivity of the present method is demonstrated for the analysis of urine samples.

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