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Hemoglobin adduct assay method and application for evaluating acrylamide exposure in vivo

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: 2019-04-30
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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  • Hemoglobin adduct assay method and application for evaluating acrylamide exposure in vivo
  • Hemoglobin adduct assay method and application for evaluating acrylamide exposure in vivo
  • Hemoglobin adduct assay method and application for evaluating acrylamide exposure in vivo

Examples

Experimental program
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Effect test

Embodiment 1

[0112] Embodiment 1. A method for synchronous detection of hemoglobin adducts for evaluating long-term in vivo exposure to dietary acrylamide. The detection object is rat blood, and the following steps are performed in sequence:

[0113] 1), preparation of dry hemoglobin powder

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

[0115] Take 1 mL of red blood cells, transfer them to a centrifuge tube, add pure water (5 ml) to dilute the red blood cells 6 times, vortex and shake for 10 min, and put them in a -80°C refrigerator for 2 h. Afterwards, hemolysate was obtained by thawing in a water bath at 37°C. Add 20 mL of acid...

Embodiment 2

[0141] Example 2. A method for synchronous detection of hemoglobin adducts in the long-term in vivo exposure of dietary acrylamide. The detection object is rat blood, and the following steps are performed in sequence:

[0142] 1), preparation of dry hemoglobin powder:

[0143] The method for preparing red blood cells refers to Example 1.

[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 and shake for 10 min, and put them into a -80°C refrigerator for 1.5 h. Afterwards, hemolysate was obtained by thawing in a water bath at 37°C. 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 protein, then centrifuge at 4500 rpm for 8 min, and discard the supernatant. Add 16mL of ...

Embodiment 3

[0157]Example 3. A method for synchronous detection of hemoglobin adducts in the long-term in vivo exposure of dietary acrylamide. The detection object is human blood, and the following steps are performed in sequence:

[0158] 1) Preparation of dry hemoglobin powder

[0159] The blood of the crowd was collected in anticoagulant tubes, and the red blood cell pretreatment and dry hemoglobin powder were prepared. The processing method was the same as step 1) of Example 1.

[0160] 2) Derivation and purification

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

[0162] 3) Chromatographic conditions

[0163] Identical to step 3) of embodiment 1.

[0164] 4) Mass spectrometry conditions

[0165] Identical to step 4) of embodiment 1.

[0166] 5) Results

[0167] The samples were quantified by the standard cu...

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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 method for synchronous detection of hemoglobin adducts for evaluating medium and long-term in vivo exposure of dietary acrylamide and an application thereof, belonging to the field of food safety. Background technique [0002] In 2002, the Swedish National Food Administration and Stockholm University researchers jointly announced that high levels of acrylamide (Acrylamide, AA) were found in fried foods and baked foods, and subsequent studies proved that acrylamide in heat-processed foods Formed from the Maillard reaction (Swedish National Food Administration, Information about acrylamide in food, 2002, 4), it has aroused great concern from the international community. Acrylamide is a colorless, odorless white crystalline small molecular substance, which is an important chemical raw material and has a wide range of industrial uses. Human intake of acrylamide mainly comes from foods with high carbohydrate content, such as pota...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/06
CPCG01N30/02G01N30/06G01N2030/062
Inventor 章宇陈信宇王桥
Owner ZHEJIANG UNIV
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