CN109828057A - A kind of sugarcane neurotoxin efficiently extracts and its liquid chromatography detecting method - Google Patents
A kind of sugarcane neurotoxin efficiently extracts and its liquid chromatography detecting method Download PDFInfo
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- CN109828057A CN109828057A CN201910235116.6A CN201910235116A CN109828057A CN 109828057 A CN109828057 A CN 109828057A CN 201910235116 A CN201910235116 A CN 201910235116A CN 109828057 A CN109828057 A CN 109828057A
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- sugarcane
- acid
- neurotoxin
- nitropropionic acid
- nitropropionic
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Abstract
It is disclosed by the invention to belong to commodity detection technique field, specially a kind of sugarcane neurotoxin efficiently extracts and its liquid chromatography detecting method, the sugarcane neurotoxin Efficient extraction method are as follows: S1: select commercially available sugarcane, S2: sugarcane is removed the peel, sonic oscillation 20min, oscillation 10min is centrifuged 10min under the conditions of 10,000r/min, takes supernatant to be measured；S3: 20 μ g/mL of sample spiked levels, column is crossed by MISPE loading respectively, this kind of sugarcane neurotoxin efficiently extracts and its liquid chromatography detecting method, MISPE has specific adsorption to 3-nitropropionic acid, 3-nitropropionic acid that can fast and effeciently in separation and concentration sugarcane, improves the efficiency of sample pre-treatments, the sample after extracting directly carries out HPLC measurement, it highly shortened sample analysis time, the trace analysis of 3-nitropropionic acid suitable for food.
Description
Technical field
The present invention relates to commodity detection technique field, specially a kind of sugarcane neurotoxin efficiently extracts and its liquid chromatogram
Detection method.
Background technique
Commodity inspection is exactly commodity inspection in brief, and it is indispensable one that commodity inspection, which is the product of development of international trade,
Content, commodity inspection embody country variant and implement quality control to import-export commodity, thus production of exports, sale and
Import is played a positive role by established condition buying etc..
Wherein liquid chromatogram is a kind of separation and analytical technology, its main feature is that using liquid as mobile phase, stationary phase can be with
There are many forms, such as paper, thin plate and packed bed.Is during chromatographic technique development in order to distinguish various methods, according to solid
The form for determining phase produces respective name, such as paper chromatography, thin-layer chromatography and column liquid chromatographic, needs to cooperate.
During existing sugarcane extraction and liquid chromatographic detection, there are more impurity, inspections for the sugarcane sample of extraction
Survey process agents useful for same is more, also at least needs 1.5h, and extraction step is cumbersome, and sample analysis time is long, affects 3- in food
The trace analysis efficiency of nitropropionic acid.
Summary of the invention
It is efficiently extracted the purpose of the present invention is to provide a kind of sugarcane neurotoxin and its liquid chromatography detecting method, with solution
During existing sugarcane extraction certainly mentioned above in the background art and liquid chromatographic detection, the sugarcane sample of extraction exists
More impurity, detection process agents useful for same is more, also at least needs 1.5h, and extraction step is cumbersome, and sample analysis time is long, shadow
The problem of having rung the trace analysis efficiency of 3-nitropropionic acid in food.
To achieve the above object, the invention provides the following technical scheme: a kind of sugarcane neurotoxin Efficient extraction method, is somebody's turn to do
Sugarcane neurotoxin Efficient extraction method are as follows:
S1: selecting commercially available sugarcane, and the 3-nitropropionic acid standard solution for being 1mg/mL with trifluoroacetic acid aqueous solution compound concentration is placed in
It is saved in 4 DEG C of constant refrigeration refrigerator；
S2: sugarcane is removed the peel, and is carried out homogeneous using homogeneous instrument, is weighed 1.0g sample and be placed in 50mL centrifuge tube, is added
1g sodium chloride is added in 10.0mL acetonitrile, sonic oscillation 20min, and oscillation 10min is centrifuged 10min under the conditions of 10,000r/min,
Take supernatant to be measured；
S3: 20 μ g/mL of sample spiked levels, column is crossed by MISPE loading respectively, is extracted using the SPE condition of optimization
It takes, finally crosses 0.22 μm of filter membrane, measured through HPLC, calculate the rate of recovery (n=3) to get neurotoxin.
A kind of liquid chromatography detecting method of sugarcane neurotoxin, the liquid chromatography detecting method packet of the sugarcane neurotoxin
Include following steps:
Step 1: silica gel is chosen: silica gel 3mol/L salt acid soak 18h, to remove a small amount of inorganic impurity and increase silica gel
The hydroxyl value on surface, wash away hydrochloric acid with distilled water until pH be it is neutral, be put into after 120 DEG C of dry 12h spare in drier；
Step 2: reaction: l0g activated silica gel and l00mL toluene is taken to be put into three necks of reflux condensing tube and snorkel circle
In the flask of bottom, the triethylamine of the APTES and 1mL of 4mL are added dropwise under nitrogen protection, magnetic agitation reflux l0h at 95 DEG C, after reaction
Silica gel filtering, after toluene, methanol are cleaned multiple times, 50 DEG C of vacuum drying 12h；
Step 3: grafting effect: silica gel and APTES are acted on, and amination grafting are carried out in Silica Surface, by 0.1mmol
Template molecule and a certain amount of AM are dissolved in 3mL acetonitrile solution, and ultrasound fusion 30min is sufficiently mixed the two；
Step 4: stirring: being added 1.0g silanized silica gel, 2.0mmo1EGDMA and 20mgAIBN, and ultrasound merges 30min,
Logical nitrogen 15min, tube sealing under ice bath under magnetic agitation, polymerize under 60 DEG C of water-baths and obtain reaction product for 24 hours；
Step 5: mixing rinsing: by reaction product 300mL methanol/acetic acid (7:3, similarly hereinafter) mixed liquor Soxhlet extraction
48h, removes template molecule, then with 300mL methanol rinse removing fine particle and remaining acetic acid, 50 DEG C of vacuum drying 12h to get
Molecular imprinted polymer on surface (SMIP), i.e. succinic acid molecular imprinted polymer on surface (SA-SMIP) and 3-nitropropionic acid surface
Molecularly imprinted polymer (NA-SMIP)；
Step 6: storage: being stored in spare in drier, and template is not added in the preparation of non-molecular imprinted polymer on surface (SNIP)
Molecule；
Step 7: concussion: the SMIP of 20mg is weighed, the 3-nitropropionic acid aqueous solution 3mL of 1mmol/L is added, vibrates respectively
The different time took out 0.45 μm of miillpore filter, is diluted to after suitable concentration and measures absorbance, unit of account in 210nm
The adsorbance of quality molecules imprinted polymer；
Step 8: measurement: pipetting supernatant, measures absorbance, is converted into 3-nitropropionic acid concentration, is calculated with minusing poly-
Object is closed to the adsorbance Q of 3-nitropropionic acid, is measured in parallel and takes mean value for 3 times, the calculation formula of adsorbance Q (mg/g) (Xia,
It Guoetal.2006) is Q=(C0-Ct) V/W, wherein C0For the initial concentration (mg/L) of 3-nitropropionic acid in solution, CtWhen for t
The concentration (mg/L) of 3-nitropropionic acid in etching solution, V are liquor capacity (mL), and W is polymer quality (mg)；
Step 9: absorption Dynamic testing: equivalent SMIP is weighed, appropriate concentration range is added in 0.1mmol/L-1.0mmol/
The 3-nitropropionic acid aqueous solution of L, constant temperature oscillation 12h took out 0.45 μm of miillpore filter, be diluted to after suitable concentration in
210nm measures absorbance, and the adsorbance of unit of account quality 3-nitropropionic acid molecularly imprinted polymer pipettes supernatant, measures
Absorbance is converted into 3-nitropropionic acid concentration, calculates polymer to the adsorbance Q of 3-nitropropionic acid with minusing, is measured in parallel 3
It is secondary to take mean value；
Step 10: specific adsorption detection: equivalent SMIP and SNIP are weighed, succinic acid, the L- paddy of 1mmol/L are separately added into
Propylhomoserin water, ASPARTIC ACID, oxalic acid, fumaric acid and 3-nitropropionic acid aqueous solution each 3mL, constant temperature oscillation 12h took out
0.45 μm of miillpore filter is diluted to after suitable concentration and measures absorbance in 210nm, draws respective standard curve respectively, count
Unit mass molecularly imprinted polymer is calculated to the adsorbance of each analyte.
Compared with prior art, the beneficial effects of the present invention are: this kind of sugarcane neurotoxin efficiently extracts and its liquid phase color
Spectrum detection method, MISPE have specific adsorption to 3-nitropropionic acid, 3- nitro that can fast and effeciently in separation and concentration sugarcane
Propionic acid improves the efficiency of sample pre-treatments, and the sample after extracting directly carries out HPLC measurement, highly shortened sample point
Analyse time, the trace analysis of 3-nitropropionic acid suitable for food.
Detailed description of the invention
Fig. 1 is the non-molecule of the present invention, succinic acid molecules and 3-nitropropionic acid molecularly imprinted polymer schematic diagram；
Fig. 2 is loading solvent Percentage bound schematic diagram of the present invention；
Fig. 3 is leacheate of the present invention, eluent Percentage bound schematic diagram；
Fig. 4 is that mobile phase of the present invention separates gradient schematic diagram.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
The present invention provides a kind of technical solution: a kind of sugarcane neurotoxin Efficient extraction method, and the sugarcane neurotoxin is high
Imitating extracting process is；
S1: selecting commercially available sugarcane, and the 3-nitropropionic acid standard solution for being 1mg/mL with trifluoroacetic acid aqueous solution compound concentration is placed in
It is saved in 4 DEG C of constant refrigeration refrigerator；
S2: sugarcane is removed the peel, and is carried out homogeneous using homogeneous instrument, is weighed 1.0g sample and be placed in 50mL centrifuge tube, is added
1g sodium chloride is added in 10.0mL acetonitrile, sonic oscillation 20min, and oscillation 10min is centrifuged 10min under the conditions of 10,000r/min,
Take supernatant to be measured；
S3: 20 μ g/mL of sample spiked levels, column is crossed by MISPE loading respectively, is extracted using the SPE condition of optimization
It takes, finally crosses 0.22 μm of filter membrane, measured through HPLC, calculate the rate of recovery (n=3) to get neurotoxin；
A kind of liquid chromatography detecting method of sugarcane neurotoxin, it is characterised in that: the liquid phase color of the sugarcane neurotoxin
Spectrum detection method includes the following steps:
Step 1: silica gel is chosen: silica gel 3mol/L salt acid soak 18h, to remove a small amount of inorganic impurity and increase silica gel
The hydroxyl value on surface, wash away hydrochloric acid with distilled water until pH be it is neutral, be put into after 120 DEG C of dry 12h spare in drier；
Step 2: reaction: l0g activated silica gel and l00mL toluene is taken to be put into three necks of reflux condensing tube and snorkel circle
In the flask of bottom, the triethylamine of the APTES and 1mL of 4mL are added dropwise under nitrogen protection, magnetic agitation reflux l0h at 95 DEG C, after reaction
Silica gel filtering, after toluene, methanol are cleaned multiple times, 50 DEG C of vacuum drying 12h；
Step 3: grafting effect: silica gel and APTES are acted on, and amination grafting are carried out in Silica Surface, by 0.1mmol
Template molecule and a certain amount of AM are dissolved in 3mL acetonitrile solution, and ultrasound fusion 30min is sufficiently mixed the two；
Step 4: stirring: being added 1.0g silanized silica gel, 2.0mmo1EGDMA and 20mgAIBN, and ultrasound merges 30min,
Logical nitrogen 15min, tube sealing under ice bath under magnetic agitation, polymerize under 60 DEG C of water-baths and obtain reaction product for 24 hours；
Step 5: mixing rinsing: by reaction product 300mL methanol/acetic acid (7:3, similarly hereinafter) mixed liquor Soxhlet extraction
48h, removes template molecule, then with 300mL methanol rinse removing fine particle and remaining acetic acid, 50 DEG C of vacuum drying 12h to get
Molecular imprinted polymer on surface (SMIP), i.e. succinic acid molecular imprinted polymer on surface (SA-SMIP) and 3-nitropropionic acid surface
Molecularly imprinted polymer (NA-SMIP)；
Step 6: storage: being stored in spare in drier, and template is not added in the preparation of non-molecular imprinted polymer on surface (SNIP)
Molecule；
Step 7: concussion: the SMIP of 20mg is weighed, the 3-nitropropionic acid aqueous solution 3mL of 1mmol/L is added, vibrates respectively
The different time took out 0.45 μm of miillpore filter, is diluted to after suitable concentration and measures absorbance, unit of account in 210nm
The adsorbance of quality molecules imprinted polymer；
Step 8: measurement: pipetting supernatant, measures absorbance, is converted into 3-nitropropionic acid concentration, is calculated with minusing poly-
Object is closed to the adsorbance Q of 3-nitropropionic acid, is measured in parallel and takes mean value for 3 times, the calculation formula of adsorbance Q (mg/g) (Xia,
It Guoetal.2006) is Q=(C0-Ct) V/W, wherein C0For the initial concentration (mg/L) of 3-nitropropionic acid in solution, CtWhen for t
The concentration (mg/L) of 3-nitropropionic acid in etching solution, V are liquor capacity (mL), and W is polymer quality (mg)；
Step 9: absorption Dynamic testing: equivalent SMIP is weighed, appropriate concentration range is added in 0.1mmol/L-1.0mmol/
The 3-nitropropionic acid aqueous solution of L, constant temperature oscillation 12h took out 0.45 μm of miillpore filter, be diluted to after suitable concentration in
210nm measures absorbance, and the adsorbance of unit of account quality 3-nitropropionic acid molecularly imprinted polymer pipettes supernatant, measures
Absorbance is converted into 3-nitropropionic acid concentration, calculates polymer to the adsorbance Q of 3-nitropropionic acid with minusing, is measured in parallel 3
It is secondary to take mean value；
Step 10: specific adsorption detection: equivalent SMIP and SNIP are weighed, succinic acid, the L- paddy of 1mmol/L are separately added into
Propylhomoserin water, ASPARTIC ACID, oxalic acid, fumaric acid and 3-nitropropionic acid aqueous solution each 3mL, constant temperature oscillation 12h took out
0.45 μm of miillpore filter is diluted to after suitable concentration and measures absorbance in 210nm, draws respective standard curve respectively, count
Unit mass molecularly imprinted polymer is calculated to the adsorbance of each analyte.
In summary, this kind of sugarcane neurotoxin efficiently extracts and its liquid chromatography detecting method, MISPE is to 3- nitre
Base propionic acid has specific adsorption, 3-nitropropionic acid that can fast and effeciently in separation and concentration sugarcane, improves sample pre-treatments
Efficiency, the sample after extracting directly carries out HPLC measurement, highly shortened sample analysis time, the 3- suitable for food
The trace analysis of nitropropionic acid.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (2)
1. a kind of sugarcane neurotoxin Efficient extraction method, it is characterised in that: the sugarcane neurotoxin Efficient extraction method are as follows:
S1: selecting commercially available sugarcane, and the 3-nitropropionic acid standard solution for being 1mg/mL with trifluoroacetic acid aqueous solution compound concentration is placed in 4 DEG C
Constant refrigeration refrigerator in save；
S2: sugarcane is removed the peel, and is carried out homogeneous using homogeneous instrument, is weighed 1.0g sample and be placed in 50mL centrifuge tube, 10.0mL is added
1g sodium chloride is added in acetonitrile, sonic oscillation 20min, and oscillation 10min is centrifuged 10min under the conditions of 10,000r/min, takes supernatant
Liquid is to be measured；
S3: 20 μ g/mL of sample spiked levels, column is crossed by MISPE loading respectively, is extracted using the SPE condition of optimization,
0.22 μm of filter membrane is finally crossed, is measured through HPLC, calculates the rate of recovery (n=3) to get neurotoxin.
2. a kind of liquid chromatography detecting method of sugarcane neurotoxin as described in claim 1, it is characterised in that: sugarcane mind
Liquid chromatography detecting method through toxin includes the following steps:
Step 1: silica gel is chosen: silica gel 3mol/L salt acid soak 18h, to remove a small amount of inorganic impurity and increase Silica Surface
Hydroxyl value, wash away hydrochloric acid with distilled water until pH be it is neutral, be put into after 120 DEG C of dry 12h spare in drier；
Step 2: it reaction: takes l0g activated silica gel and l00mL toluene to be put into and is burnt with three neck round bottoms of reflux condensing tube and snorkel
In bottle, the triethylamine of the APTES and 1mL of 4mL are added dropwise under nitrogen protection, magnetic agitation reflux l0h at 95 DEG C, by the silicon after reaction
Glue filtering, after toluene, methanol are cleaned multiple times, 50 DEG C of vacuum drying 12h；
Step 3: grafting effect: silica gel and APTES are acted on, and amination grafting are carried out in Silica Surface, by 0.1mmol template
Molecule and a certain amount of AM are dissolved in 3mL acetonitrile solution, and ultrasound fusion 30min is sufficiently mixed the two；
Step 4: stirring: being added 1.0g silanized silica gel, 2.0mmo1EGDMA and 20mgAIBN, and ultrasound fusion 30min leads to nitrogen
Gas 15min, tube sealing under ice bath under magnetic agitation, polymerize under 60 DEG C of water-baths and obtain reaction product for 24 hours；
Step 5: mixing rinsing: reaction product 300mL methanol/acetic acid (7:3, similarly hereinafter) mixed liquor Soxhlet extraction 48h is removed
Template molecule is removed, then removes fine particle and remaining acetic acid with 300mL methanol rinse, 50 DEG C of vacuum drying 12h are to get surface point
Sub- imprinted polymer (SMIP), i.e. succinic acid molecular imprinted polymer on surface (SA-SMIP) and 3-nitropropionic acid surface molecular print
Mark polymer (NA-SMIP)；
Step 6: storage: being stored in spare in drier, and template point is not added in the preparation of non-molecular imprinted polymer on surface (SNIP)
Son；
Step 7: concussion: weighing the SMIP of 20mg, the 3-nitropropionic acid aqueous solution 3mL of 1mmol/L be added, and oscillation is different respectively
Time, took out 0.45 μm of miillpore filter, and be diluted to after suitable concentration and measure absorbance, unit of account quality in 210nm
The adsorbance of molecularly imprinted polymer；
Step 8: measurement: pipetting supernatant, measures absorbance, is converted into 3-nitropropionic acid concentration, calculates polymer with minusing
To the adsorbance Q of 3-nitropropionic acid, it is measured in parallel and takes mean value for 3 times, the calculation formula of adsorbance Q (mg/g) (Xia,
It Guoetal.2006) is Q=(C0-Ct) V/W, wherein C0For the initial concentration (mg/L) of 3-nitropropionic acid in solution, CtWhen for t
The concentration (mg/L) of 3-nitropropionic acid in etching solution, V are liquor capacity (mL), and W is polymer quality (mg)；
Step 9: absorption Dynamic testing: equivalent SMIP is weighed, appropriate concentration range is added 0.1mmol/L-1.0mmol/L's
3-nitropropionic acid aqueous solution, constant temperature oscillation 12h took out 0.45 μm of miillpore filter, surveyed after being diluted to suitable concentration in 210nm
Determine absorbance, the adsorbance of unit of account quality 3-nitropropionic acid molecularly imprinted polymer pipettes supernatant, absorbance is measured,
It is converted into 3-nitropropionic acid concentration, polymer is calculated to the adsorbance Q of 3-nitropropionic acid with minusing, is measured in parallel 3 times and takes
Value；
Step 10: specific adsorption detection: equivalent SMIP and SNIP are weighed, succinic acid, the Pidolidone of 1mmol/L are separately added into
Water, ASPARTIC ACID, oxalic acid, fumaric acid and 3-nitropropionic acid aqueous solution each 3mL, constant temperature oscillation 12h took out 0.45 μm
Miillpore filter is diluted to after suitable concentration and measures absorbance in 210nm, draws respective standard curve, unit of account respectively
Adsorbance of the quality molecules imprinted polymer to each analyte.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105136918A (en) * | 2015-07-15 | 2015-12-09 | 广州市谱尼测试技术有限公司 | High performance liquid chromatographic (HPLC) analytic and detecting method of 3-nitropropionic acid in food |
| CN106442483A (en) * | 2016-11-22 | 2017-02-22 | 暨南大学 | Luminous bacterium flow injection method for quickly detecting and warning food-borne toxin pollution and application of luminous bacterium flow injection method |
-
2019
- 2019-03-27 CN CN201910235116.6A patent/CN109828057A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105136918A (en) * | 2015-07-15 | 2015-12-09 | 广州市谱尼测试技术有限公司 | High performance liquid chromatographic (HPLC) analytic and detecting method of 3-nitropropionic acid in food |
| CN106442483A (en) * | 2016-11-22 | 2017-02-22 | 暨南大学 | Luminous bacterium flow injection method for quickly detecting and warning food-borne toxin pollution and application of luminous bacterium flow injection method |
Non-Patent Citations (3)
| Title |
|---|
| DAN TIAN 等: "A Microporous Luminescent Metal-Organic Framework for a Sensitive and Selective Fluorescence Sensing of Toxic Mycotoxin in Mouldy Sugarcane", 《ACS APPLIED MATERIALS & INTERFACES》 * |
| 哈兰: "果蔗中3-硝基丙酸分子印迹固相微萃取分离、纯化及高效液相色谱测定研究", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 * |
| 岳亚军 等: "高效液相色谱-串联质谱法测定甘蔗中的3-硝基丙酸含量", 《食品安全质量检测学报》 * |
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