基于氨基酸同位素稀释质谱法分析——以血清淀粉样蛋白A为例

Analysis Based on Amino Acid Isotope Dilution Mass Spectrometry—A Case Study of Serum Amyloid A

摘要: 同位素稀释质谱法是小分子及蛋白质绝对定量的金标准，是微量、痕量和超痕量元素检测的权威方法，目前已成为国内外计量机构用于蛋白质定量和相关标准物质研制的首选方法。血清淀粉样蛋白A (SAA)是炎症相关疾病的关键标志物，其准确测定对临床检测标准化及标准物质的研制具有重要意义。本研究建立了一种基于氨基酸同位素稀释质谱法的SAA纯度分析方法，系统优化了水解条件、液相色谱分离条件及质谱检测参数，实现了SAA的绝对定量。实验选取Phe、Pro和Ile三种氨基酸作为特征氨基酸，以¹³C标记同位素为内标，通过重量法配制标准溶液，最终确定最优实验条件为：8 mol/L盐酸中水解40 h，采用ZORBAX Eclipse Plus C18柱等度洗脱(流动相为97% 0.1%甲酸水溶液与3% 0.1%甲酸乙腈溶液)，并优化了锥孔电压(200 V)及碰撞能(Phe/¹³C₉-Phe 54 eV、Pro/¹³C₅-Pro 78 eV、Ile/¹³C₆-Ile 73 eV)。采用括号法计算SAA纯度，通过系统评估氨基酸标准物质纯度、水解效率、称量误差、峰面积比、分子量及测量重复性等不确定度分量，测得重组人SAA纯度为86.40 μg/g，扩展不确定度为1.34% (k = 2)，表明方法重复性良好，且测量结果可溯源至SI国际单位。本研究构建的分析方法准确、可靠、具备溯源性，为SAA纯度标准物质研制及参考测量程序建立提供了坚实的技术支持。

Abstract: Isotope Dilution Mass Spectrometry (IDMS) is recognized as the gold standard for the absolute quantification of small molecules and proteins, as well as an authoritative method for the detection of trace, micro, and ultratrace elements. It has become the preferred method for protein quantification and the development of reference materials in metrological institutions worldwide. Serum Amyloid A (SAA), a key biomarker for inflammation-related diseases, requires accurate determination for the standardization of clinical assays and the production of certified reference materials. This study established a novel purity analysis method for SAA based on amino acid IDMS. The method involved systematic optimization of hydrolysis conditions, liquid chromatography separation, and mass spectrometric detection parameters, enabling the absolute quantification of SAA. Three amino acids, Phe, Pro, and Ile, were selected as characteristic analytes. Using weight-based preparation of standard solutions with ¹³C-labeled isotopic analogues as internal standards, the optimal experimental conditions were determined as follows: hydrolysis in 8 mol/L HCl for 40 hours, isocratic elution on a ZORBAX Eclipse Plus C18 column (mobile phase: 97% 0.1% formic acid in water/3% 0.1% formic acid in acetonitrile), with optimized cone voltage (200 V) and collision energies (54 eV for Phe/¹³C₉-Phe, 78 eV for Pro/¹³C₅-Pro, and 73 eV for Ile/¹³C₆-Ile). The purity of recombinant human SAA was calculated using the bracketing method. By systematically evaluating uncertainty components, including the purity of amino acid reference materials, hydrolysis efficiency, weighing errors, peak area ratios, molecular weight, and measurement repeatability, the SAA purity was determined to be 86.40 μg/g with an expanded uncertainty of 1.34% (k = 2), demonstrating good repeatability. The measurement result is traceable to the International System of Units. The established analytical method is accurate, reliable, and metrologically traceable, providing robust technical support for the development of SAA purity reference materials and the establishment of reference measurement procedures.