人转铁蛋白含量同位素稀释质谱测定方法研究

Determination on mass fraction of pure human transferrin through high performance liquid chromatography-isotope dilution mass spectrometry

建立一种人转铁蛋白(h TRF)纯品含量同位素稀释质谱测定方法。人转铁蛋白经酸水解后,以水(含0.8mmol/L全氟庚酸和0.1%三氟乙酸)和乙腈为流动相,以氨基酸国家标准物质为标准,同位素标记氨基酸为内标,采用高效液相色谱-同位素稀释联用法对水解液中的脯氨酸、缬氨酸和苯丙氨酸进行测定,并进一步计算得出蛋白纯品的含量。同时采用质量平衡法测定人转铁蛋白的含量,验证所建方法的可行性。在优化的条件下,人转铁蛋白含量测定结果为0.830 g/g,相对标准偏差为0.6%,扩展不确定度为0.014 g/g(k=2),检出限和定量限分别为3.78×10-5 g/g和1.26×10-4g/g。所建方法准确度高、溯源清晰,可作为人转铁蛋白纯品标准物质的定值方法,对于建立我国人转铁蛋白测定结果的量值溯源传递体系具有积极的意义。

This paper discusses a high performance liquid chromatography-isotope dilution mass spectrometry(HPLC-IDMS) method established to determine the mass fraction of pure human transferrin(h TRF). It comprises the following steps: first,hydrolyze the pure h TRF with acid and use water(containing 0.8 mmol/L of perfluoroheptanoic acid and 0.1% of trifluoroacetic acid) and acetonitrile as mobile phases; second, quantify the proline, valine and phenylalanine in the hydrolysate with the HPLC-IDMS method based on amino acids-national reference materials and isotope-labeled amino acids-internal standards; and third,calculate the mass fraction of pure h TRF according to the results of the above steps. In addition,the h TRF was also quantified by the mass balance method to verify the feasibility of the established method. Under optimized conditions,the h TRF had a mass fraction of 0.830 g/g,with a relative standard deviation(RSD) of 0.6% and an expanded uncertainty of 0.014 g/g(k=2). The detection and quantitation limits were 3.78×10-5 g/g and1.26×10-4g/g,respectively. Characterized by high accuracy and clear traceability,this method can be applied to determine the reference material amounts in pure h TRFs,playing a significant role in the establishment of valuation transfer and traceability systems for h TRF detection in China.