In this study,the IP-RPLC method for the determination of six ATP-related compounds(ATP,ADP,AMP,IMP,HxR,Hx)in aquatic product was established.The optimal conditions such as pH,flow rate and concentration of mobile phase were confirmed.The accuracy,precision and limit of quantitation were surveyed and evaluated.The method is based on an extraction of nucleotides from aquatic product with a solution of 10% perchloric acid followed by neutralization by the addition of KOH just prior to HPLC analysis.Chromatographic separations were performed using a Thermo ODS-2 Hypersil(250 mm?4.6 mm,5 μm)column,the column temperature was 25 ℃.The mobile phase consisted of 90% 0.05 mol/L sodium phosphate buffer,10% methanol and 9.5 mmol/L tetrabutylammonium hydroxide,and was adjusted to pH 6.5 with phosphoric acid,the mobile phase was delivered at a flow rate of 0.9 mL/min with equivalent elution,and the detection wave length was 254 nm.The six ATP-related compounds were separated in 13 min.The standard curves of six ATP-related compounds were linear within a range of 2-400 μg/mL,and the correlation coefficient deviations range from 0.999 4 to 1.000 0.The limits of detection(S/N=3)were between 0.1 μg/mL and 0.4 μg/mL.The average recovery of spiked sample deviations range from 82.2% to 110.0%,and the relative standard deviations(RSD)were between 4.2% and 9.5%.In conclusion,the method proved to be simple,rapid,reliableand with high accuracy and precision.The method has practical value for detecting six ATP-related compounds in aquatic product.