Simultaneous determination of creatine phosphate, creatine and 12 nucleotides in rat heart by LC-MS/MS.

A simple, rapid and sensitive LC-MS/MS method was developed and validated for simultaneous determination of creatine phosphate (CP), creatine (Cr) and 12 nucleotides in rat heart. The analytes, ATP, ADP, AMP, GTP, GDP, GMP, CTP, CDP, CMP, UTP, UDP, UMP, CP, Cr, were extracted from heart tissue with pre-cooled (0°C) methanol/water (1:1, v/v) and separated on a Hypersil Gold AQ C18 column (150mm×4.6mm, 3μm) using an isocratic elution with a mobile phase consisting of 2mmol/L ammonium acetate in water (pH 10.0, adjusted with ammonia). The detection was performed by negative ion electrospray ionization in selective reaction monitoring mode (SRM). In the assay, all the analytes showed good linearity over the investigated concentration range (r>0.99). The accuracy was between 80.7% and 120.6% and the precision expressed in RSD was less than 15.6%. This method was successfully applied to measure the concentrations of the 12 nucleotides, creatine phosphate and creatine in rat heart for the first time.