Simultaneous quantitation of alpha-ketoglutaric acid and 5-hydroxymethylfurfural in plasma by HPLC with UV and fluorescence detection.

Alpha-ketoglutaric acid (KG) and hydroxymethylfurfural (HMF) are currently being investigated in clinical trials as an approach in targeted cancer therapy. Hence, a method for the simultaneous determination of KG and HMF in plasma has been developed. Due to the strongly discriminative chemical properties of KG and HMF, SPE purification is performed using an ion-exchange cartridge to separate KG, and a hydrophobic polymeric cartridge to separate HMF. The cartridges are connected together for several steps, thus resulting in a quicker approach for the purification of plasma samples. The derivatization step is based on the reaction of the carbonyl groups of KG and HMF with dansylhydrazine (DNSH) catalyzed by trifluoroacetic acid. The formed derivatives could be separated by reversed-phase LC on a C8-column, and analyzed by UV and fluorescence detection in a single run using a gradient program. The obtained results show good reproducibility, specificity, and detection limits down to the low picomole range.