Derivatization oriented strategy for enhanced detection of valproic acid and its metabolites in human plasma and detection of valproic acid induced reactive oxygen species associated protein modifications by mass spectrometry

• Chemical labeling was used to enhance sensitivity in detecting VA and its metabolites in plasma.
• Derivatives of VA and its major metabolite were quantitated by MALDI-TOF MS.
• Derivatives of VA and its metabolites were confirmed by nanoUPLC-LTQ Orbitrap.
• VA induced protein adducts and modifications were identified by nanoUPLC-LTQ Orbitrap.

Valproic acid (VA) is a branch chain fatty acid that is widely used to treat epilepsy and convulsion. Recent studies show that VA can also be used to treat migraine headaches, bipolar disorder, and other diseases such as Alzheimer disease. However, clinical treatment with VA may cause hepatotoxicity, bone marrow suppression, and hyperammonemic encephalopathy. Valproic acid is also a known human teratogen. Because of the potential cytotoxic effects of VA and its major metabolite, 2-propyl 4-pentenoic acid (4-ene VA), VA plasma concentrations must be closely monitored during clinical applications of VA in order to avoid severe side effects.This study developed a derivatization oriented strategy for increasing sensitivity in detecting VA in quantities as low as 20 μL and its metabolites in human plasma. After micro-scale liquid-liquid extraction (MLLE) and micro-scale derivatization, VA and 4-ene VA were quantitated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The linear ranges were 10-1000 μM for VA and 5-500 μM for 4-ene VA. All relative standard deviation (RSD) and relative error (RE) values obtained in intra- and inter-day analyses of VA and 4-ene VA were below 8%. The structures of VA and its metabolite derivatives were further identified by nano ultra performance liquid chromatographic system (nanoUPLC) coupled with tandem mass spectrometry (MS/MS). Since protein modifications induced by VA were also identifiable by nanoUPLC-MS/MS, these modifications may be useful biological indicators of a toxic reaction during clinical applications of VA.