A kind of extraction and detection method of prednisolone, aldosterone, testosterone and estradiol in Antarctic krill

A detection method, the technology of Antarctic krill, applied in the fields of medicine, food and chemical industry, can solve the problems of complex radioimmunoassay operation, high false positive method, short separation time, etc., and achieve accurate and reliable peak area quantification, simple operation and high extraction rate high effect

Active Publication Date: 2021-07-20
SHANDONG NORMAL UNIV
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Problems solved by technology

Among the above substances, prednisolone and estradiol can be detected by radioimmunoassay, gas chromatography-mass spectrometry, high-performance liquid chromatography-mass spectrometry, ELISA, thin-layer chromatography, etc.; Complicated, expensive instruments are required, and silane methylation or acetylation reactions are required to convert volatile components into volatile components for GC-MS determination. ELISA has high sensitivity, but the method has high false positives and can only be used qualitatively Preliminary screening; thin-layer chromatography is a semi-quantitative detection method, which is quick to detect and easy to operate, but the detection limit is high and more materials are needed; ultra-high performance liquid chromatography-mass spectrometry is a new chromatographic method with Small particle packing, low system volume, rapid data collection, high column efficiency, short separation time, less solvent consumption, etc., is a detection method superior to high performance liquid chromatography-mass spectrometry

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  • A kind of extraction and detection method of prednisolone, aldosterone, testosterone and estradiol in Antarctic krill
  • A kind of extraction and detection method of prednisolone, aldosterone, testosterone and estradiol in Antarctic krill
  • A kind of extraction and detection method of prednisolone, aldosterone, testosterone and estradiol in Antarctic krill

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Embodiment 1

[0050] Embodiment 1: Exploration of ultra-high performance liquid chromatography-mass spectrometry conditions

[0051] The selection of the ultra-high performance liquid chromatography condition in the present invention has investigated methyl alcohol, water; Methyl alcohol, acetonitrile; Methyl alcohol, the elution effect of formic acid water as eluent, methyl alcohol, the separation effect of water is not good, adopts methyl alcohol, acetonitrile afterwards as flowing It was found that almost all hormones peaked within 3 minutes, and they were difficult to separate. Afterwards, methanol and 0.1% formic acid water were used as the mobile phase, and it was found that a gradient separation effect was not good. After continuously adjusting the mobile phase, it was found that methanol and 0.1 % formic acid water adopts the effect of gradient separation to be better, and can completely elute the peak within 9min, finally determined as the mobile phase of detecting prednisolone, ald...

Embodiment 2

[0059] Implementation Example 2: Optimal Screening of Extraction Conditions

[0060] 1. Optimal screening of extraction solvents

[0061] Take 10g of fresh Antarctic krill and freeze-dry it at -40°C. Freeze-dried Antarctic krill were ground and pulverized with a high-speed tissue grinder. Add 12mL concentration 2.0mol L -1 Add 50 μL of β-glucuronidase (10000 U / mL) to acetic acid-sodium acetate buffer solution (PH=5.2), and perform enzymatic hydrolysis in a constant temperature shaker at 37°C for 12 hours. Take out the sample and cool it to room temperature, add 10mL of extraction solvent, vortex extract for 2min, take the supernatant, repeat the extraction 3 times, and combine the supernatant. Extract with QuEChERS solid-phase extraction column, vortex for 2min to purify, centrifuge at 4000r / min for 5min, and take the supernatant. Rotary evaporate to dryness, the rotary evaporation temperature is 50°C, reconstitute with 1mL extraction solvent, and pass through a 0.2μm filt...

Embodiment 3

[0080] Take 10g of fresh Antarctic krill and freeze-dry it at -40°C. Freeze-dried Antarctic krill were ground and pulverized with a high-speed tissue grinder. Add 12mL concentration 2.0mol L -1 Add 50 μL of β-glucuronidase (10000 U / mL) to acetic acid-sodium acetate buffer solution (PH=5.2), and perform enzymatic hydrolysis in a constant temperature shaker at 37°C for 12 hours. Take out the sample and cool it to room temperature, add 10mL of acetonitrile, vortex extract for 2min, take the supernatant, repeat the extraction 3 times, and combine the supernatant. Extract with QuEChERS solid-phase extraction column, vortex for 2min to purify, centrifuge at 4000r / min for 5min, and take the supernatant. Rotary evaporate to dryness at 50°C, redissolve with 1 mL of acetonitrile, and pass through a 0.2 μm filter membrane. Obtain the crude extract of prednisolone from Antarctic krill. Take 10 μL of the bold matter and use the ultra-high performance liquid chromatography mass spectrom...

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Abstract

The invention discloses a method for extracting and detecting prednisolone, aldosterone, testosterone and estradiol in Antarctic krill. Fresh Antarctic krill is freeze-dried at -10°C to -50°C and then pulverized; Add acetic acid-sodium acetate buffer and β-glucuronidase to the pulverized Antarctic krill for enzymolysis; add acetonitrile or ethyl acetate to the enzymolysis sample and vortex extract, repeat the extraction 3 to 4 times, and combine Supernatant: extracted with QuEChERS solid-phase extraction column, vortex purified, rotary evaporated to dryness, redissolved with acetonitrile or ethyl acetate, passed through a 0.2 μm filter membrane to obtain prednisolone, aldosterone, and testosterone in Antarctic krill and estradiol extracts; detection by ultra-high performance liquid chromatography-mass spectrometry. The extraction process of the invention is simple in operation, short in time consumption, and can fully remove impurities affecting detection, and is a simple, feasible, short time-consuming and high extraction rate method.

Description

technical field [0001] The invention relates to an efficient extraction method of prednisolone, aldosterone, testosterone and estradiol in Antarctic krill and an ultra-high performance liquid chromatography-mass spectrometry detection method, belonging to the technical fields of medicine, food and chemical industry. Background technique [0002] Prednisolone, molecular formula C 21 h 28 o 5 , molecular weight is 360.44, density is 1.31g / cm 3 , with a melting point of 240°C and a boiling point of 570.6°C at 760mmHg. This product is white or off-white crystalline powder; odorless, slightly bitter taste; hygroscopic. It is mainly used for allergic and autoimmune inflammatory diseases, collagen diseases, such as rheumatism, rheumatoid arthritis, lupus erythematosus, severe bronchial asthma, nephrotic syndrome, thrombocytopenic purpura, neutropenia, acute lymphoid Leukemia, various adrenal insufficiency, exfoliative dermatitis, pemphigus, neurodermatitis, eczema, etc. Aldos...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/06G01N30/02
CPCG01N30/02G01N30/06G01N2030/062
Inventor 韩香凝刘代成
Owner SHANDONG NORMAL UNIV
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