Simultaneous determination of adenine nucleotides, creatine phosphate and creatine in rat liver by high performance liquid chromatography–electrospray ionization-tandem mass spectrometry

► A simple and reliable HPLC–ESI-MS/MS method was developed for simultaneous determination of ATP, ADP, AMP, CP and creatine in liver tissues. ► The inorganic buffer and/or ion-pairing reagents were avoided and reasonable retention time for the analytes was achieved. ► The sample pre-treatment procedure was rapid, simple and well compatible with ESI-MS/MS analysis. ► The method was successfully applied to determination of ATP, ADP, AMP, CP and creatine in liver tissue. A high performance liquid chromatography–electrospray ionization-tandem mass spectrometric method (HPLC–ESI-MS/MS) was developed for simultaneous determination of adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP), creatine phosphate (CP), and creatine in rat liver. After extraction with pre-cooled (4°C) methanol/water (1:1, v/v), the analytes were separated on a porous graphitic carbon (Hypercarb) column (2.1mm×150mm, 5μm) using a programmed gradient elution with a mobile phase consisting of 2mmol/L ammonium acetate in water and 2mmol/L ammonium acetate in acetonitrile (pH=10.0). The analytes were detected in a way of multiple reaction monitoring (MRM) under negative scan mode by a triple quadrupole mass spectrometer with electrospray ionization (ESI). An external calibration method with linear ranges from 10 to 5000ng/mL for the five target compounds was used for quantification with a correlation coefficients≥0.9973. The limits of detection and limits of quantification for all analytes were in ranges from 0.50 to 1.5ng/mL and 1.6 to 0.5ng/mL, respectively. The average recoveries spiked in three levels were from 77.2% to 102% and precisions expressed in RSDs were from 0.2% to 4.8%. The established method was successfully applied to determination of ATP, ADP, AMP, CP and creatine in liver tissue.