高效液相色谱法测定婴幼儿营养强化奶粉中核苷酸含量

Determination of Nucleotides in Infant Nutrition Milk Powder by High Performance Liquid Chromatography

建立了测定婴幼儿营养强化奶粉中5种核苷酸含量的高效液相色谱法。样品经碱性磷酸酯酶水解,将核苷酸转化成相应的核苷,采用硼酸凝胶吸附净化,使用Boston pHlex ODS色谱柱(250 mm×4.6 mm,5.0μm)分离,流动相A为含2 mmol/L 1-己烷磺酸钠的20.9 mmol/L NH4Ac(pH为4.3);流动相B为6.0%乙腈-离子对试剂(含2 mmol/L 1-己烷磺酸钠的20.9 mmol/L NH4Ac,pH为4.3),梯度洗脱后,二极管阵列检测器检测,检测波长为260 nm,内标法定量。结果表明,5种核苷在线性范围内相关性好,相关系数R2大于0.999;5种核苷酸的回收率在80.00%~111.23%范围,相对标准偏差在1.8%~6.3%之间。检出限为0.4~1.0 mg/kg,定量限为1.0~3.0 mg/kg。该方法净化效果显著,分离效果好,是婴幼儿营养强化奶粉中5种核苷酸的有效检测方法。

A high performance liquid chromatographic method(HPLC) was established for the determination of five nucleotides(cytidine 5′-monophosphate,guanosine 5′-monophosphate,uridine 5′-monophosphate,adenosine 5′-monophosphate and inosine 5′-monophosphate) in infant nutrition milk powder.The nucleotides in sample were hydrolyzed by alkaline phosphatase to the corresponding nucleosides.The nucleosides were purified by selective boric acid solid phase extraction.The chromatographic separation was performed on a Boston pHlex ODS column(250 mm×4.6 mm,5.0 μm) and a gradient elution with a mixture of two solvents,which effectively separate five kinds of nucleosides by HPLC.The diode array detector acquired spectral data at 260 nm.All the compounds were calibrated by the internal standard method.To minimize the matrix effect,this experiment quantified by the matrix-matched calibration curve.The results showed that the linear ranges of the five nucleotides were 0.4-8.0 μg/mL and the correlation coefficients were above 0.999.The recoveries were 80.00%-111.23% with the relative standard deviations from 1.8% to 6.3%(n=6).The limits of detection were 0.4-1.0 mg/kg,and the limits of quantification were 1.0-3.0 mg/kg respectively.The developed method is sensitive and accurate for the simultaneous determination of nucleotides content in infant nutrition milk powder.