Quantitative targeted biomarker assay for glycated haemoglobin by multidimensional LC using mass spectrometric detection.

The development of quantitative strategies for targeted biomarker analysis represents an urgent task especially in the field of clinical diagnosis. In this regard, the measurement of glycohaemoglobin (HbA(1c)) in blood has become the most specific way of monitoring long-term glycaemia in diabetic patients. Thus, there is an urgent need for methods that provide accurate and precise HbA(1c) results. A new method for the determination of HbA(1c) in blood samples based on the complementary use of multidimensional liquid chromatography (LC) and elemental (inductively coupled plasma mass spectrometry, ICP-MS) and molecular (electrospray-mass spectrometry, ESI-MS) MS techniques has been developed and validated. Different multidimensional separation possibilities by combining affinity and cation exchange chromatography have been explored for the adequate isolation of HbA(1c), which purity is addressed by ESI-MS. The workflow includes a final quantitative determination of HbA(1c) by elemental (Fe) isotope dilution analysis (IDA) with ICP-MS. For this purpose, the post-column addition of the isotopically labeled iron ((57)Fe) has been used to quantify the eluting Fe-species from the column. The IDA methodology has been validated by analyzing a certified reference material and several samples from patients whose HbA(1c) levels were determined by a standard reference method.