高效液相色谱法测定水产品中ATP关联化合物

Determination of ATP-related compounds in aquatic products by high performance liquid chromatography

建立了水产品中三磷酸腺苷(ATP)、二磷酸腺苷(ADP)、腺苷酸(AMP)、肌苷酸(IMP)、次黄嘌呤核苷(HxR)和次黄嘌呤(Hx)6种ATP关联化合物的高效液相色谱检测方法。样品中的ATP关联物经高氯酸提取并用氢氧化钠调节pH值至6.0-6.4后,以AQ-C18(250 mm×4.6 mm,5μm)柱为分析柱,0.02 mol/L KH2PO41-0.02 mol/L K2HPO4(V/V=1/1)的缓冲液为流动相进行洗脱,在254 nm波长下用高效液相色谱进行检测,外标法定量。研究表明,ATP、ADP、AMP、IMP和HxR线性范围为0.2-40.0μg/ml,Hx线性范围为0.1-20.0μg/ml,相关系数均大于0.999 0;ATP、ADP、AMP、IMP和HxR检出限为5.00 mg/kg,Hx检出限为2.5 mg/kg(S/N=3);对真鲷、鱼师鱼、鲑鱼、凡纳滨对虾和梭子蟹5种水产品进行3个浓度水平的加标实验,回收率在85.5%-105%之间,相对标准偏差(RSD)小于12.9%。实验结果表明,该方法简便、准确,适合于水产品中ATP关联物的测定。

A high performance liquid chromatography （HPLC） method for determination of adenosine triphosphate （ATP） and its breakdown products,adenosine diphosphate （ADP）,adenosine monophosphate （AMP）,inosinic acid （IMP）,inosine （HxR） and hypoxanthine （Hx） was studied.The samples were extracted with perchloric acid,and pH value was adjusted to 6.0-6.4 with sodium hydroxide.The six ATP-related compounds were then separated on an AQ-C18 column （250 mm ×4.6 mm,5 μm） with 0.02 mol/L KH2PO4-0.02 mol/L K2HPO4 （V/V=1/1） buffer as the mobile phase.At wavelength of 254 nm,ATP-related compounds were detected and quantified by external standard method.The calibration curves were linear in the range of 0.2-40.0 μg/ml for ATP,ADP,AMP,IMP and HxR,and 0.1-20.0 μg/ml for Hx,with all correlation coefficients above 0.999.The detection limit for ATP,ADP,AMP,IMP,and HxR was 5.00 mg/kg,and was 2.50 mg/kg for Hx,with S/N =3.The average recoveries of ATP compounds in Pagrosomus major,Seriola quinqueradiata,Oncorhynchus keta,Litopenaeus vannamei and Portunus trituberculatus samples at three spiked levels varied from 85.5% to 105%,and the relative standard deviation （RSD） was less than 12.9%.The results indicated that this method is rapid and reliable,and it is suitable for simultaneous determination of the six ATP-related compounds in aquatic products.