Automatic sample workflow for lc-ms based hba1c measurement on intact protein level

A sample, complete technology in the field of automated sample workflows for LC-MS based HbA1c measurements on intact protein levels, addressing the issue of not providing satisfactory and reproducible results

Pending Publication Date: 2021-08-31
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] However, the above methods have disadvantages as they do not provide satisfactory and reproducible results

Method used

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  • Automatic sample workflow for lc-ms based hba1c measurement on intact protein level
  • Automatic sample workflow for lc-ms based hba1c measurement on intact protein level
  • Automatic sample workflow for lc-ms based hba1c measurement on intact protein level

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Experimental program
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Embodiment

[0062] A first aspect of the invention relates to a method for determining the intact molecule of glycated hemoglobin A (HbA1c) in a sample, the method comprising:

[0063] (a) providing a sample comprising hemoglobin A (HbA) molecules, particularly intact hemoglobin A (HbA) molecules;

[0064] (b) enriching said sample for hemoglobin A molecules, in particular alpha chain and / or beta chain molecules; and

[0065] (c) Subjecting a sample comprising intact hemoglobin A molecules, in particular alpha and / or beta chain molecules, to analysis by mass spectrometry (MS), wherein the amount or concentration of HbA1c in the sample is determined.

[0066] Thereby, the amount or concentration of HbA1c in the sample can be determined, in particular the relative amount of HbA1c, ie the ratio of glycated HbA1c molecules to non-glycated HbA0 molecules or to total HbA molecules. When repeatedly determining the amount of HbA1c, the ratio of HbAlc / HbA or the ratio of HbAlc / HbA0 in a given sam...

example 1

[0167] Determination of intact HbA1c in whole blood

[0168] Three 100 μL whole blood samples were each diluted with 400 μL deionized water and mixed by vortexing for 10 s until a clear solution was obtained. Then, each hemolysate was diluted 20-fold in different buffers with pH 2, 6 or 10, respectively.

[0169] Three batches of hydrophobic magnetic beads (100 μL per batch) were equilibrated by mixing with 900 μL of each of the three different buffers described above. Subsequently, 900 μL of the supernatant was discarded and the equilibrated beads recovered.

[0170] Three 900 μL samples of whole blood hemolysate (which had been diluted in three different buffers as indicated above) were added to the corresponding batch of equilibrated beads to obtain loaded beads. The mixture was then shaken at 37 °C and 1200 rpm for 15 min.

[0171] A series of several fractions for LC-MS were collected from the loaded beads as follows:

[0172] First, collect 900 μL of supernatant from...

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Abstract

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.

Description

technical field [0001] The present invention relates to methods, kits and diagnostic systems suitable for performing the method for the determination of intact glycated hemoglobin A (HbA1c) molecules by mass spectrometry (MS). Background technique [0002] Impaired control of circulating blood glucose levels is an indicator of diabetes. Glucose can attach to lysine residues of polypeptides in a non-enzymatic statistical process, resulting in glycated polypeptides. In the case of hemoglobin A (HbA), a reaction occurs between glucose and the N-terminus of the beta chain. The resulting glycated hemoglobin Aβ chain was designated as HbA1c. [0003] HbA1c is the major glycated hemoglobin species in human blood. The comprehensive Diabetes Control and Complications Trial (DCCT) provides evidence that complications such as retinopathy, nephropathy and neuropathy are directly related to the degree of hyperglycemia in patients with insulin-dependent diabetes mellitus (IDDM) and sug...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/72
CPCG01N33/6848G01N33/723
Inventor U·科博尔德A·莱嫩巴赫I·米特拉H·布斯坎普
Owner F HOFFMANN LA ROCHE & CO AG
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