CN103808856A - Method for extracting diethylstilbestrol from euphausia superba and efficient thin layer chromatography scanning and detecting method - Google Patents

Abstract

The invention discloses a method for extracting diethylstilbestrol from a euphausia superba and an efficient thin layer chromatography scanning and detecting method. The method for extracting the diethylstilbestrol from the euphausia superba comprises the following steps: extracting the euphausia superba as a raw material with methyl alcohol; steaming a filtrate to dry through a rotary evaporation instrument after filtering, so as to obtain a crude extract containing the diethylstilbestrol. The diethylstilbestrol of the euphausia superba is detected by using an efficient thin layer chromatography scanning method. A developing solvent is a mixture with a volume ratio of carbon tetrachloride to ethyl acetate to acetone to glacial acetic acid to methylbenzene to cyclohexane of (78-80) to (9-11) to (9-11) to (2-4) to 3d to 2d; a coloring agent is a 10% sulfuric ethanol solution; the content of the diethylstilbestrol can be obtained through calculation of the absorbance of a detection sample product and the absorbance of a standard product by carrying out scanning detection with an absorption wavelength of 400nm by using a CAMAG thin layer chromatography scanner III. The method has the advantages of being simple to operate, fast, accurate and reliable.

Description

A kind of method and HPTLC scanning method detection method of extracting diethylstilbestrol from krill

Technical field

The present invention relates to a kind of method and HPTLC scanning method detection method of extracting diethylstilbestrol from krill.

Background technology

Diethylstilbestrol molecular formula is C ₁₈h ₂₀o ₂, molecular weight is 268.35 dalton, claims again Benovocylin, Diethylstilbestrol etc., belongs to estrogenic chemicals, has identical pharmacology and therapeutic action with natural estradiol, can be used for treating gynecological disease, promotes growth of animal etc.

Krill (Euphausia superba), is the krill of one way of life in waters, the Antarctica, lives in the mode of trooping, and every cubic metre of 10000-30000 of density reachable only sometimes.They are using small phytoplankton as food, the about 6cm of height, and heavily about 2g, MaLS can reach 6 years.They are key species of Antarctic ecosystems, if with biomass energy, they may be the most successful living species on the earth.Huge because of krill reserves, add that the life of colony is easily fished for, so attract a lot of countries to develop it.At present, the main purposes of krill is as fish meal.Krill shrimp sauce is extracted as health products to approximately 2000 yuan/kg of price in the state such as Norway, Canada.Because krill contains a large amount of fluorine (about 2400mg kg ^-1), can not directly eat.Thereby take krill as raw material, exploitation natural drug, to its comprehensive utilization, has broad application prospects.

The detection of diethylstilbestrol mainly adopts the method for the immune detection such as enzyme linked immunosorbent detection, fluoroimmunoassay, radioreceptor assay and gas chromatography, high performance liquid chromatography, bioanalysis to carry out, but these detection methods have following weak point: testing process length consuming time, expense is high, may cause environmental pollution etc.

Summary of the invention

Technical matters to be solved by this invention is to provide a kind of detection method of krill diethylstilbestrol simple to operate, quick, accurate, reliable.

The present invention is achieved by the following technical solutions:

A HPTLC scanning method detection method of analyzing diethylstilbestrol, step is as follows:

(1) get sample liquid to be detected and 1.0mg mL ^-1titer 5 μ L points in same GF ₂₅₄on efficient thin-layer silicon offset plate, take volume ratio as phenixin: ethyl acetate: acetone: glacial acetic acid: toluene: the developping agent of cyclohexane=78-80:9-11:9-11:2-4:3d:2d, in expansion cylinder, launch, exhibition, apart from being 9.5cm, is dried panel after having opened up;

(2) with 10% ethanol solution of sulfuric acid dyeing, be then placed under 100 ℃ of-110 ℃ of conditions and toast 5-10 minute;

(3), with the scanning of CAMAG thin-layer chromatogram scanner-III, scanning wavelength 400nm, calculates the content of diethylstilbestrol by detecting sample and standard items absorbance.

Sample liquid is by being dissolved in diethylstilbestrol crude extract in 5mL methyl alcohol and making.

The extracting method of diethylstilbestrol crude extract, comprises the following steps:

(1) get dry krill (w) and methyl alcohol (v) with the ultrasonic extraction under 35 ℃, the condition of 350w, 40kHz of the ratio of 1:8-11, filter extract;

(2) above-mentioned filtrate being placed in revolved and steam instrument evaporate to dryness, obtain the crude extract of diethylstilbestrol.

While expansion in expansion cylinder, first by developping agent pre-equilibration 35-45 minute at ambient temperature, put into again efficient thin layer plate, continue to launch at ambient temperature, in the time that exhibition distance reaches 4.5cm, take out thin layer plate, under room temperature, dry 10 minutes, again thin layer plate is put into expansion cylinder, the exhibition that is deployed into is apart from being 9.5cm.

Production standard curve:

(1) precision takes 1.0000mg diethylstilbestrol standard items (National Institute for Food and Drugs Control) and is dissolved in 1.0mL methyl alcohol, and obtaining concentration is 1.0mg mL ^-1titer;

(2) get the above-mentioned diethylstilbestrol standard solution configuring 2 μ L, 3 μ L, 4 μ L, 7 μ L, 8 μ L, point sample is in same GF ₂₅₄efficient thin-layer silicon offset plate, launches by the HPTLC scanning method detection method condition of diethylstilbestrol, dries;

(3) with after 10% ethanol solution of sulfuric acid dyeing, by CAMAG thin-layer chromatogram scanner-III, the scanning of 400nm absorbing wavelength, obtains regression equation Y=-285.7233+643.4958X through thin-layer chromatogram scanner software wincats1.4.1 statistics, relative coefficient r=0.99946, RSD=2.57%.

The present invention detects the efficient thin layer scanning detection method of diethylstilbestrol, its precision, repeatability, stability, recovery situation and developping agent formula are determined, dyeing time and dyeing temperature to determine, detect determining of wavelength as follows:

1. precision is measured

The accurate sample liquid of drawing, horizontal point sample 5 μ l on the efficient thin-layer silicon offset plate of same, launch, dry, with using CAMAG thin-layer chromatogram scanner-III after 10% ethanol solution of sulfuric acid dyeing, scan to such an extent that peak area is respectively 3234.1 with 400nm absorbing wavelength, 3301.3,3256.4,3199.2,3260.1, RSD=1.15%.

2. repeatability is measured

The accurate sample liquid of drawing, difference point sample 5 μ l on five blocks of efficient thin-layer silicon offset plates, launch, dry, with using CAMAG thin-layer chromatogram scanner-III after 10% ethanol solution of sulfuric acid dyeing, peak area with the scanning of 400nm absorbing wavelength is respectively 3234.1,3310.2,3320.3,3299.3,3298.5, RSD=1.03%.

3. Stability Determination

Draw sample liquid 5 μ L point sample on efficient thin layer plate, launch, dry, after 10% ethanol solution of sulfuric acid dyeing, by CAMAG thin-layer chromatogram scanner-III, scan with 400nm absorbing wavelength, later every 10 minutes run-downs, record each peak area, calculate corresponding RSD value, test of many times proves, more stable in 20-60 minute after dyeing, average RSD=1.64%.Concrete data are as shown in table 1:

Table 1

20min	30min	40min	50min	60min	Average RSD
						1428.9	1425.8	1394.7	1390.1	1378.7	1.60%
1470.3	1463.5	1431.5	1426.9	1417.0	1.64%
						1492.4	1489.1	1454.9	1446.8	1439.1	1.68%

4. recovery test

Accurate absorption 6 μ L sample liquid 3 times, add respectively 2.853 μ g, 5.706 μ g, 7.838 μ g diethylstilbestrol standard items, point sample, launch, dry, with using CAMAG thin-layer chromatogram scanner-III after 10% ethanol solution of sulfuric acid dyeing, scan with 400nm absorbing wavelength, calculating average recovery rate is 102.3%, RSD=0.87%.Data are as shown in table 2:

Table 2

Sample size/μ g	Add standard items amount/μ g	The amount of recording/μ g	Recovery %
				5.706	2.853	8.610	101.8
5.706	5.706	11.602	103.3
				5.706	8.559	14.410	101.7

5. developping agent formula is determined

In the time that developping agent is set as different formulas, each combination, has all carried out ratio debugging repeatedly, and separating effect is as shown in table 3:

Table 3

6. detect determining of wavelength

Scan image from the detection sample of Fig. 1-Fig. 4 and standard items when the different wave length, in the time that wavelength set is 400nm, sample and standard items length scanning image are the most identical.

Beneficial effect of the present invention:

The present invention is take krill as material extraction diethylstilbestrol, and krill biomass is large, with its exploitation natural drug, can extract research by strengthening its radix amount for the material of content pettiness.

Utilize HPTLC scanning method detection method to analyze the content of oestrone, automaticity is high, and sensitivity is strong.Compare with other detection methods, High Performance Thin Layer Chromatography detection method needs reagent still less, and this has good protective effect to environment; Whole analyte detection process is very of short duration.

With volume ratio phenixin: the developping agent that the proportioning of ethyl acetate: acetone: glacial acetic acid: toluene: cyclohexane=78-80:9-11:9-11:2-4:3d:2d mixes, make diethylstilbestrol and other component separating in sample liquid clear, spot is round and clear, standard items point and sample spot are accurately corresponding, Rf value is 0.36, is that in krill, diethylstilbestrol launches the good formula separating.The dyeing of employing ethanol solution of sulfuric acid, this developer versatility is good, and is conveniently easy to get.This detection method is simple, quick, accurate, reliable, stable.

Accompanying drawing explanation

Fig. 1 sets to detect the scan image that detects sample and standard items when wavelength is 600nm;

Fig. 2 sets to detect the scan image that detects sample and standard items when wavelength is 500nm;

Fig. 3 sets to detect the scan image that detects sample and standard items when wavelength is 400nm;

Fig. 4 sets to detect the scan image that detects sample and standard items when wavelength is 300nm.

Embodiment

Below in conjunction with embodiment, the present invention is further illustrated.

Embodiment 1

Get dry krill 1g, add methyl alcohol 9mL, in 35 ℃, the supersonic wave cleaning machine of 350w, 40kHz, Ultrasonic Heating extracts, and extracts 15 minutes, filters extract, and filtrate being placed in revolved to steaming instrument evaporate to dryness, obtains the crude extract of diethylstilbestrol.

The crude extract of the above-mentioned diethylstilbestrol making is dissolved in 5mL methyl alcohol and makes sample liquid; Precision takes 1.0000mg diethylstilbestrol standard items (National Institute for Food and Drugs Control) and is dissolved in 1.0mL methyl alcohol, and obtaining concentration is 1mg mL ^-1titer.

Sample liquid and titer are put in same GF ₂₅₄efficient thin-layer silicon offset plate (10 × 10cm ²) upper, developping agent volume ratio is phenixin: ethyl acetate: acetone: glacial acetic acid: toluene: cyclohexane=78:10:10:3:3d:2d, adds expansion cylinder (10 × 12 × 15cm by developping agent ³) pre-equilibration 30 minutes at room temperature, then put into efficient thin layer plate, continue at room temperature to launch, when exhibition is when reaching 4.5cm, take out thin layer plate, under room temperature, dry 10 minutes, then thin layer plate is put into expansion cylinder, exhibition, apart from 9.5cm, after launching to finish, is taken out plate to dry.With 10% ethanol solution of sulfuric acid dyeing, be then placed at 105 ℃ and toast 10 minutes, the some purple that sample liquid and standard items are corresponding.By CAMAG thin-layer chromatogram scanner-III, with the absorbing wavelength scanning of 400nm, obtain Rf=0.36, be 3.20mg g with calculating diethylstilbestrol content in krill after testing ^-1.

Embodiment 2

Get dry krill 1g, add methyl alcohol 11mL, in 35 ℃, the supersonic wave cleaning machine of 350w, 40kHz, Ultrasonic Heating extracts, and extracts 25 minutes, by extracting liquid filtering, filtrate being placed in is revolved to steaming instrument evaporate to dryness, obtains the crude extract of diethylstilbestrol.

The crude extract of the above-mentioned diethylstilbestrol making is dissolved in to 5mL methyl alcohol and makes sample liquid, precision takes 1.0000mg diethylstilbestrol standard items (National Institute for Food and Drugs Control) and is dissolved in 1.0mL methyl alcohol, and obtaining concentration is 1mg mL ^-1titer.

Sample liquid and titer are put in same GF ₂₅₄efficient thin-layer silicon offset plate (10 × 10cm ²) upper, developping agent volume ratio is phenixin: ethyl acetate: acetone: glacial acetic acid: toluene: cyclohexane=80:10:10:2:3d:2d, adds expansion cylinder (10 × 12 × 15cm by developping agent ³) pre-equilibration 35 minutes at room temperature, then put into efficient thin layer plate, continue at room temperature to launch, when exhibition is when reaching 4.5cm, take out thin layer plate, under room temperature, dry 10 minutes, then thin layer plate is put into expansion cylinder, exhibition, apart from 9.5cm, after launching to finish, is taken out plate to dry.With 10% ethanol solution of sulfuric acid dyeing, be then placed at 105 ℃ and toast 8 minutes, the some purple that sample liquid and standard items are corresponding.By CAMAG thin-layer chromatogram scanner-III, with the absorbing wavelength scanning of 400nm, obtain Rf=0.36.Be 3.18mg g with calculating diethylstilbestrol content in krill after testing ^-1.

Embodiment 3

Get dry krill 1g, add methyl alcohol 10mL, in 35 ℃, the supersonic wave cleaning machine of 350w, 40kHz, Ultrasonic Heating extracts, and extracts 25 minutes, by extracting liquid filtering, filtrate being placed in is revolved to steaming instrument evaporate to dryness, obtains the crude extract of diethylstilbestrol.

The crude extract of the above-mentioned diethylstilbestrol making is dissolved in to 5mL methyl alcohol and makes sample liquid; Precision takes 1.0000mg diethylstilbestrol standard items (National Institute for Food and Drugs Control) and is dissolved in 1.0mL methyl alcohol, and obtaining concentration is 1mg mL ^-1titer.

Sample liquid and titer are put in same GF ₂₅₄efficient thin-layer silicon offset plate (10 × 10cm ²) upper, developping agent volume ratio is phenixin: ethyl acetate: acetone: glacial acetic acid: toluene: cyclohexane=80:9:9:2:3d:2d, adds expansion cylinder (10 × 12 × 15cm by developping agent ³) pre-equilibration 45 minutes at room temperature, then put into efficient thin layer plate, continue at room temperature to launch, when exhibition is when reaching 4.5cm, take out thin layer plate, under room temperature, dry 10 minutes, then thin layer plate is put into expansion cylinder, exhibition, apart from 9.5cm, after launching to finish, is taken out plate to dry.With 10% ethanol solution of sulfuric acid dyeing, be then placed at 105 ℃ and toast 5 minutes, the some purple that sample liquid and standard items are corresponding.By CAMAG thin-layer chromatogram scanner-III, with the absorbing wavelength scanning of 400nm, obtain Rf=0.36.Be 3.17mg g with calculating diethylstilbestrol content in krill after testing ^-1.

By reference to the accompanying drawings the specific embodiment of the present invention is described although above-mentioned; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various modifications that creative work can make or distortion still in protection scope of the present invention.

Claims (4)

1. a HPTLC scanning method detection method for diethylstilbestrol, is characterized in that, step is as follows:

(1) get sample liquid to be detected and 1.0mg mL ^-1titer 5 μ L points in same GF ₂₅₄on efficient thin-layer silicon offset plate, take volume ratio as phenixin: ethyl acetate: acetone: glacial acetic acid: toluene: the mixed liquor of cyclohexane=78-80:9-11:9-11:2-4:3d:2d is developping agent, in expansion cylinder, launch, exhibition, apart from being 9.5cm, is dried panel after having opened up;

(2) with 10% ethanol solution of sulfuric acid dyeing, be then placed in the 100 ℃ of lower baking of-110 ℃ of conditions 5-10 minute;

(3), with the scanning of CAMAG thin-layer chromatogram scanner-III, scanning wavelength 400nm, calculates the content of diethylstilbestrol by detecting the absorbance of sample and standard items.

2. the HPTLC scanning method detection method of diethylstilbestrol as claimed in claim 1, is characterized in that, described sample liquid is by diethylstilbestrol crude extract is dissolved in 5mL methyl alcohol and is made.

3. the HPTLC scanning method detection method of diethylstilbestrol as claimed in claim 1, it is characterized in that, described step 1) in while launching in expansion cylinder, first by developping agent pre-equilibration 35-45 minute at ambient temperature, then put into efficient thin layer plate, continue to launch at ambient temperature, in the time that exhibition distance reaches 4.5cm, take out thin layer plate, under room temperature, dry 10 minutes, again thin layer plate is put into expansion cylinder, the exhibition that is deployed into is apart from being 9.5cm.

4. a method of extracting diethylstilbestrol from krill, is characterized in that, comprises the following steps:

(1) get dry krill (w) and methyl alcohol (v) with the ultrasonic extraction under 35 ℃, the condition of 350w, 40kHz of the ratio of 1:8-11, filter extract;

(2) above-mentioned filtrate being placed in revolved and steam instrument evaporate to dryness, obtain the crude extract of diethylstilbestrol.

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