Absolute Quantification of Haemoglobin in Whole Blood by Species-unspecific Isotope-dilution Liquid Chromatography-inductively Coupled Plasma Mass Spectrometry

doi:10.7503/cjcu20200321

Abstract

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

CLC Number:

O657.63

TrendMD:

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[J]. Chem. J. Chinese Universities, 2020, 41(9): 1983.

Figures/Tables 7

References 23

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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

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

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

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

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