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Method for quantitative determination of content of insulin degludec in biological sample by UPLC-MS/MS

A technology for insulin degludec and biological samples, applied in the field of quantitative measurement of insulin degludec, can solve the problems of long detection process, false negatives, increased time cost and economic cost of detection, etc.

Inactive Publication Date: 2018-10-23
重庆美莱德生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method requires a long time of incubation for the specific binding of antigens and antibodies, as well as the binding of secondary antibodies, and the entire detection time takes a long time; and the specific monoclonal or polyclonal antibodies required for detection need to be purchased or specially prepared , which increases the time cost and economic cost of detection
Furthermore, due to the nature of the monoclonal or polyclonal antibody itself and the characteristics of solid-phase detection, the test results are prone to false positives and false negatives.
When testing a large number of samples, due to the long testing process, the workload of testing personnel will also be greatly increased

Method used

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  • Method for quantitative determination of content of insulin degludec in biological sample by UPLC-MS/MS
  • Method for quantitative determination of content of insulin degludec in biological sample by UPLC-MS/MS
  • Method for quantitative determination of content of insulin degludec in biological sample by UPLC-MS/MS

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1: sample preparation

[0046] Simulated plasma samples containing varying amounts of insulin degludec were prepared by tracing insulin degludec in simulated plasma (in blank plasma with 0.1% aprotinin) at various concentrations to assess linear response (500-50000 ng / mL) .

Embodiment 2

[0047] Example 2: Enrichment of insulin degludec before mass spectrometry analysis

[0048] The samples were pretreated by protein precipitation and solid phase extraction (SPE) column. The SPE cartridge retains insulin degludec while allowing other serum proteins and macromolecules to flow through, and introduces 50 μL of the sample into the Waters H Class With methanol: water: acetic acid (6:3:1, v / v / v) solution, insulin degludec was eluted from the extraction column to the analytical column (Waters, ACQUITY UPLC Peptide BEHC18 ). A UPLC gradient was applied to the analytical column to separate the insulin degludec from other analytes contained in the sample. Mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. The UPLC gradient started with a 15% organic gradient and increased to 85% in about 192s. The insulin degludec enriched samples were then subjected to MS / MS to quantify insulin degludec.

Embodiment 3

[0049] Example 3: Detection and quantification of insulin degludec by tandem MS

[0050] MS / MS was performed using a Waters TQ-S MS / MS system. All from Waters UNIFI 1.8.2.169 or newer were used in the examples described herein. ESI source interface to the MS / MS analyzer for the liquid solvent / analyte stream leaving the analytical column. The solvent / analyte mixture is converted to vapor in the heated tubing of the interface. In positive ion mode, analytes are ionized by ESI under acidic conditions. Ions enter the first quadrupole (Q1). Several possible insulin degludec precursor ions were observed at Q1. Figure 1-3 It is a typical spectrum of 4+, 5+, 6+ insulin degludec ion pairs detected by UPLC-MS / MS. Fragmentation studies were performed on multiply charged insulin degludec precursor ions at m / z of about 1527.27±0.50 (4+ ions), 1222.06±0.50 (5+ ions) and about 1018.57±0.50 (6+ ions).

[0051] Table 1. Optimal collision energies (positive polarity - acidic conditions) ...

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Abstract

The invention provides a method for quantitative determination of the content of insulin degludec in a biological sample by UPLC-MS / MS. The method adopts a mass spectrometric method combined with a tandem mass spectrometry technology purification method for detecting and quantifying insulin degludec in the biological sample, wherein the method includes the steps: the sample is subject to solid phase extraction (SPE) and ultra-high performance liquid chromatography (UPLC) to obtain a fraction rich in insulin degludec derived from the sample; (b) the enriched insulin degludec is treated by an ionization source under conditions suitable for production of one or more insulin ions that can be detected by the mass spectrometric method; and (c) the amount of one or more insulin degludec ions is detected by the tandem mass spectrometric method, wherein the sample is not subjected to immunological purification before ionization. Compared with an ELISA method of the conventional detection technology, the whole detection process has the advantages of short time consuming, relatively low cost, good specificity, extremely low generation of false positive and false negative probability, and labor amount saving, and is more suitable for clinical application.

Description

technical field [0001] The present invention relates to the quantitative measurement of insulin degludec. In a specific aspect, the present invention relates to a method for the quantitative determination of the content of insulin degludec in a biological sample by UPLC-MS / MS. Background technique [0002] Insulin degludec is made by changing an amino acid of the human insulin molecule, that is, removing the 30th amino acid of its B chain, and then connecting a side chain of a 16-carbon fatty diacid to the B29 position through a glutamic acid linker. The molecular weight of insulin degludec is about 6103.97 Da. The A chain has 21 amino acids and the B chain has 29 amino acids. Insulin degludec has an ultra-long-acting time, which can not only effectively lower blood sugar, but also reduce the incidence of hypoglycemia. On the premise of once-daily dosing, insulin degludec can provide more flexible injection time options, solve the problem that existing basal insulin needs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/72
CPCG01N30/02G01N30/72
Inventor 刘颜王娅张玉东周万威李宗晟袁雪梅任亮林琴牟道华
Owner 重庆美莱德生物医药有限公司
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