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Method for detecting in-vitro exposed hemoglobin adduct for evaluating acrylamide and application

A technology of acrylamide and hemoglobin, which is applied in measurement devices, instruments, scientific instruments, etc., can solve the problems of limited detection sensitivity, lengthy processing operations, unfavorable for the detection and detection of large quantities of biological samples, etc., and achieve high precision and high sensitivity. Effect

Active Publication Date: 2017-08-18
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above-mentioned methods involve lengthy pretreatment operations, low derivation efficiency and purification efficiency, and the detection sensitivity of the gas chromatography method is very limited, which is not conducive to the detection of large quantities of biological samples and the detection of samples with low levels of dietary exposure

Method used

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  • Method for detecting in-vitro exposed hemoglobin adduct for evaluating acrylamide and application
  • Method for detecting in-vitro exposed hemoglobin adduct for evaluating acrylamide and application
  • Method for detecting in-vitro exposed hemoglobin adduct for evaluating acrylamide and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Example 1. A synchronous detection method for evaluating hemoglobin adducts exposed in the body for a long time in dietary acrylamide, the detection object is rat blood, and the following steps are performed in sequence:

[0113] 1), prepare dry hemoglobin powder

[0114] Centrifuge 5 mL of the whole blood sample in the anticoagulation tube at 3500 rpm for 5 min, remove the supernatant (plasma) and the middle layer (white membrane), and add the red blood cells remaining in the lower layer to 3 mL of PBS solution or normal saline. After pipetting the dropper evenly, aspirate the supernatant, centrifuge at 3000 rpm for 5 min, and repeat washing 3 times to obtain 2 mL of red blood cells.

[0115] Take 1 mL of red blood cells, transfer to a centrifuge tube, add pure water (5 ml) to dilute the red blood cells 6 times, vortex for 10 minutes, and put them in a -80°C refrigerator for 2 hours. After that, it was thawed in a 37°C water bath to obtain hemolysis. Add 20mL of acidified i...

Embodiment 2

[0141] Example 2. To evaluate the method for simultaneous detection of hemoglobin adducts exposed to long-term in vivo in dietary acrylamide, the detection object is rat blood, and the following steps are performed in sequence:

[0142] 1). Preparation of dry hemoglobin powder:

[0143] Refer to Example 1 for the method of preparing red blood cells.

[0144] Take 0.8 mL of the prepared red blood cells, transfer them to a centrifuge tube, add pure water to dilute the red blood cells 5 times, vortex for 10 minutes, and put them in a -80°C refrigerator for 1.5 hours. After that, it was thawed in a 37°C water bath to obtain hemolysis. Add 16 mL of acidified isopropanol solution to the hemolysis, and centrifuge at 4500 rpm for 5 min. Transfer the dark red supernatant to another centrifuge tube, add 16 mL of ethyl acetate, vortex for 8 min, place in a refrigerator at 4° C. for 3.5 h to precipitate the protein, then centrifuge at 4500 rpm for 8 min, and discard the supernatant. Add 16 mL...

Embodiment 3

[0157] Example 3. To evaluate the synchronous detection method of hemoglobin adducts exposed in the body for a long time in dietary acrylamide, the detection object is human blood, and the following steps are performed in sequence:

[0158] 1) Preparation of dry hemoglobin powder

[0159] Collecting human blood in an anticoagulation tube, preparing red blood cell pretreatment and preparing hemoglobin dry powder processing methods are equivalent to step 1) of Example 1.

[0160] 2) Derivation and purification

[0161] This example is a human blood sample, 20 μL of mixed isotope internal standard is added, the concentration of d8-AAVal-PTH and d8-GAVal-PTH are both 1 μg / mL, and the rest are equivalent to step 2) of Example 1.

[0162] 3) Chromatographic conditions

[0163] It is equivalent to step 3) of Example 1.

[0164] 4) Mass spectrometry conditions

[0165] It is equivalent to step 4) of Example 1.

[0166] 5) Results

[0167] The sample was quantitatively analyzed by the standard curve ...

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Abstract

The invention discloses a method for synchronously detecting in-vitro long-term exposed hemoglobin adduct for evaluating dietary acrylamide. The method comprises the chromatographic conditions and mass spectrum conditions of a blood (rat blood and human body blood) pretreatment method and a detection method. The method has the advantages that a UHPLC-MS / MS (ultra performance liquid chromatography-tandem mass spectrometry) method of isotope dilution is adopted, two types of hemoglobin adducts of the acrylamide are quantified, and the analysis time is greatly shortened, and AAVal-PTH and two types of space isomers of GAVal-PTH can be well separated at the same time; the synchronous detection is realized, and the internal exposure amount of the acrylamide can be more comprehensively evaluated.

Description

Technical field [0001] The invention relates to a synchronous detection method for evaluating hemoglobin adducts exposed in the body for a long time in dietary acrylamide and its application, and belongs to the field of food safety. Background technique [0002] In 2002, researchers from the Swedish National Food Administration and Stockholm University jointly announced that high levels of acrylamide (AA) were found in fried foods and baked foods, and subsequent studies proved that acrylamide in hot processed foods The formation is derived from Maillard reaction (Swedish National Food Administration, Information about acrylamide in food, 2002, 4), which has attracted great attention from the international community. Acrylamide is a kind of colorless and odorless white crystal small molecule substance. It is an important chemical raw material and has a wide range of industrial applications. Human body intake of acrylamide mainly comes from foods with high carbohydrate content, su...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/062
Inventor 章宇王桥陈信宇
Owner ZHEJIANG UNIV
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