Carboxylic acids participate in many metabolic pathways including tricarboxylic acid (TCA) cycle. Therefore,
there have been ongoing attempts to develop sensitive liquid chromatography-mass spectrometry methods over
the last decades. Derivatization of the carboxylic acids with 3-nitrophenylhydrazine presents a well-established
methodology, and yet the derivatized species of polycarboxylic acids and their fragmentation in collisioninduced
dissociation have not been fully studied before. In our study, we elucidated how annotation of most
abundant 3-nitrophenylhydrazine derivatives and optimization of their fragmentation in multiple reaction
monitoring can boost the sensitivity, especially for polycarboxylic acids. Finally, the optimized liquid
chromatography-tandem mass spectrometry method allowed for low detection limits ranging from 10 pM for 2-
oxoglutaric acid to 800 pM for pyruvic acid. All TCA carboxylates were quantified in 20 μL of human plasma and
the targeted method was validated in the same matrix. The same methodology with a modified gradient elution
was also applied to untargeted screening of fatty acids by using high-resolution mass spectrometry enabling
identification of 29 medium- to long-chain fatty acids in human plasma. The TCA carboxylates were also
quantified in 105 of C2C12 mouse myuotube cells grown under different treatments to proof applicability of the
methodology to biological studies in a wider sense. However, unfortunately all the TCA carboxylates were also
found in the derivatized blanks in substantial amounts, which prevents from using the methodology for quantification
of the carboxylates in less than 105 cells.