Determination of Adenosine Triphosphate and Its Related Products in Prawn by Reversed-Phase High Performance Liquid Chromatography with Ultrasonic-Assisted Extraction

A method for the determination of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosinic acid (IMP), inosine (HxR) and hypoxanthine (Hx) in prawn by reversed-phase high performance liquid chromatography with ultrasonic-assisted extraction was proposed, and the effect of storage condition on K value was studied. Crushed prawn (2.0 g) was added into 20 mL of 10% (φ) perchloric acid solution, homogenized in ice water bath for 1 min, extracted with ultrasound for 5 min, and centrifuged at 4℃ for 2 min. The above procedure was repeated with 20 mL of 5% (φ) perchloric acid solution, and the combined supernatant was made its volume up to 50 mL with water. An aliquot (5 mL) was adjusted its acidity to pH 6.0-6.4 with 5.0 mol·L^-1 and 2.5 mol·L^-1 sodium hydroxide solution, and then its volume was made up to 10 mL with 0.048 mol·L^-1 potassium dihydrogen phosphate solution (pH 5.7). Ultimate^® XB-C₄ reversed-phase column (250 mm×4.6 mm, 5 μm) was used as stationary phase, and a mixture of 0.048 mol·L^-1 potassium dihydrogen phosphate solution (pH 5.7) and methanol at different volume ratios was used as mobile phase for gradient elution, and quantitative analysis was performed by external standard method. It was shown that the linear ranges of the standard curves were 1.250×10^-1-1.000×10²mg·L^-1 for ATP, ADP and IMP, and 6.250×10^-2-5.000×10 mg·L^-1 for AMP, HxR and Hx, with detection limits in the range of 0.65-2.2 mg·kg^-1. RSDs (n=6) of the determined values of 6 target compounds in the mixed standard solution and test solution were ranged from 0.28% to 8.7%. Test for recovery was made on the actual sample by standard addition method, giving results in the range of 75.3%-114%. This method was applied to determine ATP, ADP, AMP, IMP, HxR and Hx in the prawn sample at different storage temperatures and time. The results showed that K value was increased with storage time extension. When the storage time was constant, the storage temperature was lower while the increase rate of K value was solwer, indicating that low temperature was conducive to the preservation of prawn.