Strategies for quantitation of endogenous adenine nucleotides in human plasma using novel ion-pair hydrophilic interaction chromatography coupled with tandem mass spectrometry

• A novel and highly sensitive IP–HILIC–MS/MS method was reported for the quantitation of nucleotides.
• The lower limit of quantitation (LLOQ) of 2.00 ng/mL obtained for ATP.
• This novel chromatographic mechanism is first reported and named as IP–HILIC–MS/MS.

We present here a novel and highly sensitive ion-pair hydrophilic interaction chromatography–tandem mass spectrometry (IP–HILIC–MS/MS) method for quantitation of highly polar acid metabolites like adenine nucleotides. A mobile phase based on diethylamine (DEA) and hexafluoro-2-isopropanol (HFIP) and an aminopropyl (NH2) column were applied for a novel chromatographic separation for the determination of AMP, ADP and ATP in biological matrices. This novel IP–HILIC mechanism could be hypothesized by the ion-pairing reagent (DEA) in the mobile phase forming neutral and hydrophilic complexes with the analytes of polar organic acids. The IP–HILIC–MS/MS assay for adenine nucleotides was successfully validated with satisfactory linearity, sensitivity, accuracy, reproducibility and matrix effects. The lower limit of quantitation (LLOQ) at 2.00 ng/mL obtained for ATP showed a least 10-fold higher sensitivity than previous LC–MS/MS assays except nano-LC–MS/MS assay. In summary, this novel IP–HILIC–MS/MS assay provides a sensitive method for nucleotides bioanalysis and shows great potential to determine a number of organic acids in biological matrices.