离子对反相高效液相色谱法同时检测水产品中6种ATP关联化合物

Simultaneous determination of six ATP-related compounds in aquatic product using IP-RPLC

建立了水产品中6种ATP关联化合物的离子对反相高效液相色谱(IP-RPLC)检测方法,确定了缓冲液的最佳pH、流速和浓度,并对方法的准确度、精确度和检测限进行了测定。采用10%的高氯酸分离提取样品,用KOH溶液中和后进行HPLC分析。采用Waters 2695色谱系统,Thermo ODS-2 Hypersil(250 mm×4.6 mm,5μm)色谱柱,柱温为25℃;以10%甲醇,90%0.05 mol/L磷酸盐溶液为流动相,磷酸盐溶液含9 mmol/L四丁基氢氧化氨(TBA),并用1 mol/L的磷酸调pH至6.5,采用等度洗脱,流速为0.9 mL/min;检测波长为254 nm。结果表明:6种ATP关联化合物在13 min内完全分离;在2～400μg/mL范围内线性关系良好,相关系数在0.999 4～1.000 0之间;方法最低检测限(信噪比S/N=3)在0.1～0.4μg/mL之间;加标平均回收率为82.2%～110.0%,相对标准偏差为4.2%～9.5%。本方法可用于水产品中ATP、ADP、AMP、IMP、Hx和HxR等6种ATP关联化合物的同时测定和分析。

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.