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A method for detecting n,n-diisopropylcarbodiimide in polypeptide

A technology of diisopropylcarbodiimide and mass spectrometry, which is applied in the detection of N,N-diisopropylcarbodiimide in polypeptides, and in the field of detection of carbodiimide in polypeptides, which can solve the problem of no conversion of carbodiimide Problems such as urea derivatives, to achieve accurate and obvious detection results, good effect, and high detection sensitivity

Active Publication Date: 2019-02-22
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In fact, there will still be a small amount of carbodiimide in the sample that has not been converted to its urea derivative

Method used

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  • A method for detecting n,n-diisopropylcarbodiimide in polypeptide
  • A method for detecting n,n-diisopropylcarbodiimide in polypeptide
  • A method for detecting n,n-diisopropylcarbodiimide in polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: the preparation of solution

[0048] 1mol / L hydrochloric acid solution containing 30% acetonitrile: take 120mL of hydrochloric acid, add 780mL of water and 360mL of acetonitrile and mix well;

[0049] 30% acetonitrile solution: Take 300mL of acetonitrile and add 700mL of water, mix well.

[0050] Mobile phase A: water.

[0051] Mobile Phase B: Acetonitrile

[0052] Need testing solution: take about 20mg of need testing, weigh accurately, put in 20mL volumetric flask, accurately add 2mL of 1mol / L hydrochloric acid solution containing 30% acetonitrile to dissolve the sample, sonicate for 1min, let stand at room temperature for 2 hours, Dilute with 30% acetonitrile solution, dilute to the mark, and shake well. Prepare 2 copies in parallel.

[0053] Reference substance stock solution: take 40 mg of DIC reference substance and put it in a 50 mL measuring bottle, add 20 mL of 1 mol / L hydrochloric acid solution containing 30% acetonitrile to dissolve the samp...

Embodiment 2

[0067] Embodiment 2, detection condition

[0068] 1. Liquid phase conditions

[0069] Chromatographic column: Agilent Eclipse C18 3.5μm 4.6×100mm, chromatographic column number: RD-152; UV detection wavelength: 210nm; column temperature: 25°C; liquid phase flow rate: 1.0ml / min; adjust the split ratio so that the flow rate into the mass spectrometer 0.3ml / min; injection volume: 10μL; mobile phase A: water; mobile phase B: acetonitrile; gradient elution program as follows:

[0070] time (minutes)

Mobile phase A(%)

Mobile phase B(%)

0

90

10

10

50

50

11

90

90

20

90

10

[0071] 2. Mass Spectrometry Conditions

[0072] Mass spectrometer detector: Thermo LTQXL; ion mode: positive ion mode; ion current data acquisition range: 144-146Da; ion source temperature: 200°C; ion transfer tube temperature: 320°C; sheath airflow: 30au; auxiliary airflow: 0au; Spray voltage: 3KV; deflection voltage: 80KV.

Embodiment 3

[0073] Embodiment 3, system adaptability

[0074] Take the reference substance solution and inject 6 needles continuously, the RSD of the liquid phase peak area and the ion flow peak area shall not exceed 20.0%, and the area RSD of the mass spectrometry ion flow shall not be greater than 20.0%. The test results are shown in Table 1 and Table 2.

[0075] Table 1 6 Injection results for standard products (liquid phase)

[0076]

[0077] Table 2 6 Injection results for standard samples (mass spectrometry)

[0078]

[0079] From the results in Table 1 and Table 2, it can be seen that the liquid phase peak area RSD is 0.53%, less than 20%, and the mass spectrometry ion flow peak area RSD is 2.76%, less than 20%, showing that the detection method of the present invention has good system adaptability.

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Abstract

The invention belongs to the field of pharmaceutical analysis chemistry, and discloses a method for detecting N,N'-diisopropylcarbodiimide in polypeptide. The method comprises the steps that a solution with pH smaller than or equal to 5 is used for dissolving a polypeptide sample and a N,N'-diisopropylcarbodiimide reference substance to obtain a test solution and a reference solution; a liquid chromatogram and mass spectrum combined method is adopted for detecting the content of N,N'-diisopropylcarbodiimide in the test solution. According to the detection method, the solution with pH smaller than or equal to 5 is used for dissolving a polypeptide sample, an acid environment is built, and DIC in polypeptide is completely converted into a urea derivative. The detection method is easy to operate, detection instruments and reagents are common, and the detection cost is low; detection time is short, and a detection result is accurate, obvious and easy to judge; detection sensitivity is high, the mass spectrometric detection limit can reach 0.8 ppm, and the method is widely suitable for detecting DIC in polypeptide drugs.

Description

technical field [0001] The invention belongs to the field of pharmaceutical analytical chemistry, and in particular relates to a method for detecting carbodiimide in polypeptides. In particular, it relates to a method for detecting N,N-diisopropylcarbodiimide in polypeptides. Background technique [0002] Carbodiimide (Carbodiimide), also known as carbodiimide, contains N=C=N functional groups and is a commonly used water-losing agent. Since the carbodiimide peptide synthesis method was developed in 1955, carbodiimide has been applied in many chemical fields. N,N-Diisopropylcarbodiimide, abbreviated as DIC, is a commonly used condensing agent. For example, DIC is used in the synthesis of nesiritide, and DIC is also used in the synthesis of polypeptides such as Atosiban, oxytocin, somatostatin, and vasopressin. However, the European Pharmacopoeia’s Structural Warning of Genotoxic Impurities clearly points out that DIC has a warning structure of genotoxic impurities, which ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/06
Inventor 何雄风唐洋明陶安进袁建成
Owner HYBIO PHARMA
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