Intact HbA1c LC-MS Workflow for Rapid, Reproducible Measurement
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Solution Overview
Problem
Existing methods for determining glycated hemoglobin A1c (HbA1c) by mass spectrometry are time-consuming and provide unsatisfactory, reproducible results with high variation coefficients, making them unsuitable for high-throughput measurements.
Innovation Solution
A method involving the enrichment of intact hemoglobin A molecules, particularly the α- and β-chains, using a hydrophobic solid phase and organic solvent elution, followed by mass spectrometry analysis, including a chromatographic step to enhance specificity and reproducibility.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If enzymatic digestion of hemoglobin is used to determine HbA1c, then sufficient cleavage products are obtained for stable measurement signals, but the measurement process becomes time-consuming requiring overnight incubation
Solution Approach 1:
The patent extracts only the necessary information (intact HbA1c molecules) without requiring complete enzymatic digestion. By using mass spectrometry to directly detect intact glycated hemoglobin molecules, the method eliminates the time-consuming overnight incubation step while maintaining measurement accuracy through direct molecular identification.
Solution Approach 2:
The patent replaces the mechanical/enzymatic digestion process with a direct mass spectrometric detection approach. Instead of using enzymes to break down hemoglobin over time, the method uses MS to directly identify and quantify intact HbA1c molecules based on their mass-to-charge ratio, dramatically reducing measurement time.
2Measurement precision
If conventional chromatography is used to separate peptide mixtures before MS detection, then separation of complex mixtures is achieved, but the process becomes time-consuming and unsuitable for high-throughput measurements
Solution Approach 1:
The patent extracts and detects only the specific intact HbA1c molecules of interest using mass spectrometry, rather than performing comprehensive separation of all peptide components. This targeted approach eliminates time-consuming chromatographic steps while maintaining the ability to accurately measure HbA1c levels.
Solution Approach 2:
The patent replaces conventional chromatographic separation with direct mass spectrometric analysis. The MS instrument separates and detects ions based on their mass-to-charge ratio in the gas phase, eliminating the need for physical chromatographic columns and significantly increasing throughput capability.
3Productivity
If direct MS analysis of whole blood samples is performed, then measurement speed is increased, but results show high variation coefficients and unsatisfactory reproducibility
Solution Approach 1:
The patent performs preliminary enrichment of intact HbA1c molecules from the complex blood matrix before MS analysis. This pre-concentration step ensures that sufficient target molecules are present for reliable detection while removing interfering substances, thereby improving reproducibility without sacrificing the speed advantage of direct MS analysis.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method allows for rapid and reliable determination of HbA1c with a coefficient of variation of 2.5% or less, enabling high-throughput analysis of up to 100 samples per hour.
Implementation Method 1
enriching intact hemoglobin A, in particular the α- and/or the β-chain of hemoglobin A, molecules in said sample
Implementation Method 2
organic solvent elution
Implementation Method 3
subjecting the sample comprising intact hemoglobin A, in particular the α- and/or the β-chain of hemoglobin A, molecules to an analysis by mass spectrometry (MS)
Data Source
AI summary
The present invention refers to a method for determining intact glycated hemoglobin A (HbA1c) by mass spectrometry (MS), a kit and a diagnostic system adapted for performing the method.