高效液相色谱-非特异性同位素稀释质谱联用技术定量分析全血中总血红蛋白含量

doi:10.7503/cjcu20200321

摘要/Abstract

摘要：

建立了基于同位素稀释(ID)的高效液相色谱(HPLC)-电感耦合等离子体质谱(ICP-MS)联用技术, 通过测量血红蛋白(HGB)中铁(Fe)元素含量实现了全血中总HGB准确定量的策略. 该策略包括2种方法, 第一种方法为在线HPLC-ID-ICP-MS定量技术: 全血中HGB经液相色谱分离后, 通过柱后在线添加浓缩⁵⁴Fe同位素稀释剂的方式, 与液相色谱洗脱液经三通混合后进入ICP-MS在线测量⁵⁴Fe/⁵⁶Fe的同位素比值, 并根据同位素稀释质谱法公式及蛋白中Fe的含量计算HGB的浓度. 针对全血样品中存在的其它主要含铁蛋白[如转铁蛋白(Tf)], 在实现色谱完全分离的基础上, 通过在线优化改变⁵⁴Fe稀释剂流速实现了HGB和Tf的同时定量. 在另一种方法中, 先对全血样品进行消解并通过ID-ICP-MS测定其中Fe的总量, 然后根据全血样品的HPLC-ICP-MS分析结果, 通过计算HGB质谱峰面积占总峰面积的比值从而得到HGB中Fe含量占总Fe含量的比例, 实现了其中HGB的准确定量. 2种定量方法得到的结果分别为(115.3±2.4) mg/g和(115.5±2.1) mg/g, 结果吻合良好, 方法的检出限为1.0×10^?7 mg/g, 方法精密度RSD均<3%. 2种方法均经过HGB标准物质IRMM/IFCC-467进行验证, 测量结果在标准值不确定度范围之内, 可作为临床检验领域HGB分析的参考测量方法.

关键词: 蛋白质定量, 高效液相色谱-电感耦合等离子体质谱, 同位素稀释, 血红蛋白, 转铁蛋白

Abstract:

A methodology based on isotope dilution-high performance liquid chromatography(HPLC) coupled with inductively coupled plasma mass spectrometry(HPLC-ID-ICP-MS) was established for the absolute quantification of haemoglobin(HGB) in whole blood by measuring content of iron, through two specific methods. The first method is online HPLC-ID-ICP-MS quantitative technology, following the separation of HGB by HPLC, the enriched ⁵⁴Fe isotope solution was continuously mixed with the eluate from HPLC via a three-way connection. The isotope ratio of ⁵⁶Fe/⁵⁴Fe was measured online by ICP-MS. The concentration of HGB was calculated by isotope dilution equation by the contents of iron in each protein. For Tf in the whole blood, we changed the flow rate of isotopically enriched ⁵⁴Fe spike online to simultaneous quantification of HGB and Tf on the basis of complete chromatographic separation. In the other method, the total Fe concentration was first measured by bulk ID-ICP-MS after digesting the whole blood sample. And then the ratio of Fe content from HGB in blood obtained by peak area after HPLC separation, which achieved accurate quantification of HGB. The quantification results obtained with the two methods are (115.3±2.4) mg/g and (115.5±2.1) mg/g, respectively, which are in good agreement and the RSDs are all within 3%. The methods can be used as the main reference method to quantify HGB in clinic. All the proposed methods were validated for the analysis of a human blood certified reference material(IRMM/IFCC-467), and it might be used as the main reference method for HGB quantitative analysis in clinical.

Key words: Protein quantification, HPLC-ICP-MS, Isotope dilution, Haemoglobin, Transferrin

中图分类号:

O657.63

TrendMD:

潘梦芸, 冯流星, 李红梅. 高效液相色谱-非特异性同位素稀释质谱联用技术定量分析全血中总血红蛋白含量. 高等学校化学学报, 2020, 41(9): 1983.

PAN Mengyun, FENG Liuxing, LI Hongmei. Absolute Quantification of Haemoglobin in Whole Blood by Species-unspecific Isotope-dilution Liquid Chromatography-inductively Coupled Plasma Mass Spectrometry. Chem. J. Chinese Universities, 2020, 41(9): 1983.

图/表 7

Fig.1 LC spectrum of IRMM/IFCC?467 reference material by SEC

Fig.2 ICP?MS spectra of IRMM/IFCC?467 via iron(A) HPLC?ID?ICP?MS; (B) chromatogram of mass flow of iron and the 56Fe/54Fe isotope ratio.

Table 1 Absolute quantitative results of IRMM/IFCC-467 reference materials(n=3)

Method	HGB concentration/(mg·g^-1)
Method	Measurement	RSD(%)	Certified value
HPLC?ID?ICP?MS	117.6± 1.3	1.1	119.7 ± 3.7
Bulk?ID?ICP?MS	116.0 ± 2.1	0.1	119.7 ± 3.7

Fig.3 LC spetrum of whole blood sample by SEC

Fig.4 Mass spectra of whole blood sample via iron(A) HPLC-ID-ICP-MS; (B) chromatogram of mass flow of iron and the 56Fe/54Fe isotope ratio.

Table 2 Absolute quantitative analysis of total HGB and Tf in whole blood(n=3)

Protein	Protein concentration /(mg·g^-1)
Protein	HPLC?ID?ICP?MS	RSD(%)	Reference range
Tf	4.4±1.4	0.4	2.2—4.0
HGB	115.3±2.4	1.0	100.0—150.0

Fig.5 Comparison results of HGB measured by bulk?ID?ICP?MS and HPLC?ID?ICP?MS method

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