CN106338570A - Improved method for determination of sodium cyclamate in aquatic processed product - Google Patents
Improved method for determination of sodium cyclamate in aquatic processed product Download PDFInfo
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- CN106338570A CN106338570A CN201610670763.6A CN201610670763A CN106338570A CN 106338570 A CN106338570 A CN 106338570A CN 201610670763 A CN201610670763 A CN 201610670763A CN 106338570 A CN106338570 A CN 106338570A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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Abstract
The invention discloses an improved method for determination of sodium cyclamate in an aquatic processed product. An aquatic processed product sample is extracted by a methanol aqueous solution, is centrifuged, makes the volume constant, and is determined through an ultra-high performance liquid chromatography-mass spectrometry method. Through optimization of pretreatment and chromatographic conditions, sodium cyclamate has a good linear relationship in a range of 0.5-50 ng/mL, the correlation coefficient is 0.996 or more, the average recovery rate of the aquatic processed product sample is 81%-109%, the relative standard deviation is 2.2%-9.7%, and the quantitative detection limit is 0.01 mg/kg; the method is efficient, sensitive and accurate, and can be widely applied in determination of sodium cyclamate in the aquatic processed product.
Description
Technical field
The present invention relates to aquatic products detection technique field, particularly to honey element in one kind improved mensure fishery technology
Method.
Background technology
Honey element also known as sodium cyclohexylsulfamate, soluble in water, it is a kind of artificial synthesis edulcorant low in calories, do not have
Have any nutritive value, but its sugariness can reach 50～60 times of white sugar, have both has fruit-like flavour simultaneously, with its cheap price and
The sugariness of remote ultrawhite sugar, is widely used in cake, flavouring, beverage, preserved fruit etc..The market circulation food of relevant department in recent years
Product monitoring finds, the honey element in fishery technology is often detected.Studies have reported that discovery, honey element excessive amount can simultaneously
Can have carcinogenicity, can metabolism be cyclohexylamine etc. in vivo, these materials can produce toxic work to cardiovascular system and testis
With, therefore in the import and export banned list of Japan and other countries, clearly having forbidden that honey element to use as food additives.I
A range of interpolation is allowed to use in related limitation regulation gb 2760 of state's food additives, state is often because of honey element still
Meet with technology barriers, enterprises' loss is serious.
Sodium cyclamate detection method has ultraviolet spectrophotometry, the chromatography of ions, gas chromatography, liquid chromatography etc..Its
In, gas chromatography is now widely used method, gb/t 5009.97-2003 " sodium cyclohexyl sulfamate in food
Measuring " the wherein first method is gas chromatography, the method needs to carry out the derivatization step of complexity, and pre-treatment step is loaded down with trivial details, makes
With substantial amounts of n-hexane reagent, detection efficiency is relatively low.Derivatization in gas chromatography there is also interference phenomenon simultaneously, in analysis
During rich in albumen, the matrix of alcohols, be especially rich in protein and lipid fishery technology when, derivatization interference is more tight
Weight, testing result occurs false positive.
Content of the invention
It is an object of the invention to provide a kind of improved method measuring honey element in fishery technology is it is not necessary to spread out
Raw, pre-treatment step is simple and convenient, greatly shortens detection cycle, improves detection efficiency.
The technical solution adopted for the present invention to solve the technical problems is:
A kind of improved method measuring honey element in fishery technology, comprises the steps:
(1) sample extraction: weigh 2.00g sample, be placed in 25ml centrifuge tube, add 10ml methanol aqueous solution, fully mixed
Co oscillation vortex 1min, is positioned over 60 DEG C of water-baths 15 minutes, then ultrasonic 10min, centrifugation, pours out supernatant, and residue uses
10ml methanol aqueous solution repeats aforesaid operations and extracts, is centrifuged 1 time, merges supernatant, is accurately settled to 25ml, obtains sample extraction
Liquid；
(2) liquid chromatography-mass spectrography detection: take sample extracting solution 1ml, through 0.22 μm of organic membrane filtration to sample injection bottle
In, carry out liquid chromatography-mass spectrography detection, then draw standard working curve, quantified by external standard method, according to peak area, honey element is entered
Row is quantitative.
Preferably, parameter of noncentricity is described in step (1): 6000r/min centrifugation 5min.
Preferably, the volumetric concentration of methanol aqueous solution is 50% in step (1).
Preferably, liquid phase chromatogram condition is: chromatographic column: acquity uplc beh c18 chromatographic column, specification: 2.1mm
× 50mm, 1.7 μm of particle diameter；Mobile phase: 5mmol/l ammonium acetate solution-methanol solution；Isocratic elution: flow velocity 0.3ml/min, post
40 DEG C of temperature, 4 DEG C of sample room temperature, sample size 10 μ l.
Preferably, Mass Spectrometry Conditions: ion gun: electric spray ion source, anion scans；Detection mode: multiple reaction prison
Survey；Capillary voltage: 3.5kv；Taper hole voltage: 40v；Ion source temperature: 120 DEG C；Desolvation temperature: 380 DEG C；Desolventizing gas
Flow: 600l/hr；Taper hole air-flow: 50l/hr；Collision voltage: 30v；Collision gas flow: 0.22ml/min.
The invention has the beneficial effects as follows: honey element has good linear relationship, phase relation in the range of 0.5-50ng/ml
Number more than 0.996, between 81%-109%, relative standard deviation is in 2.2%- for the fishery technology sample average rate of recovery
Between 9.7%, quantitative detection is limited to 0.01mg/kg, and the method is efficient, sensitive, accurate, can be widely applied in fishery technology
The detection of honey element.
Brief description
Fig. 1 is different mobile phase chromatogram contrasts；Note: 1 is 5mm ammonium acetate solution-acetonitrile mobile phase, 2 is 5mm acetic acid
Ammonium salt solution-methanol as mobile phase.
Fig. 2 is the comparison of 3 kinds of extracting modes.
Fig. 3 is standard liquid chromatogram.
Fig. 4 blank sample mark-on chromatogram.
Specific embodiment
Below by specific embodiment, technical scheme is described in further detail.
In the present invention, if not refering in particular to, the raw material being adopted and equipment etc. are all commercially available or commonly used in the art.
Method in following embodiments, if no special instructions, is the conventional method of this area.
Embodiment:
A kind of improved method measuring honey element in fishery technology, comprises the steps:
(1) sample extraction: weigh 2.00g sample, be placed in 25ml centrifuge tube, (volume is dense to add 10ml methanol aqueous solution
Degree 50%), it is sufficiently mixed vibration vortex 1min, be positioned over 60 DEG C of water-baths 15 minutes, then ultrasonic 10min, centrifugation, pour out supernatant
Liquid, residue uses 10ml methanol aqueous solution to repeat aforesaid operations and extracts, is centrifuged 1 time, merges supernatant, is accurately settled to 25ml,
Obtain sample extracting solution；
(2) liquid chromatography-mass spectrography detection: take sample extracting solution 1ml, through 0.22 μm of organic membrane filtration to sample injection bottle
In, carry out liquid chromatography-mass spectrography detection, then draw standard working curve, quantified by external standard method, according to peak area, honey element is entered
Row is quantitative.
Liquid phase chromatogram condition is: chromatographic column: acquity uplc beh c18 chromatographic column, specification: 2.1mm × 50mm, grain
1.7 μm of footpath；Mobile phase: 5mmol/l ammonium acetate solution-methanol solution (40:60, v/v)；Isocratic elution: flow velocity 0.3ml/min,
40 DEG C of column temperature, 4 DEG C of sample room temperature, sample size 10 μ l.
Mass Spectrometry Conditions: ion gun: electric spray ion source, anion scans；Detection mode: multiple reaction monitoring；Capillary
Voltage: 3.5kv；Taper hole voltage: 40v；Ion source temperature: 120 DEG C；Desolvation temperature: 380 DEG C；Desolventizing gas flow:
600l/hr；Taper hole air-flow: 50l/hr；Collision voltage: 30v；Collision gas flow: 0.22ml/min.
Test portion:
1 materials and methods
1.1 instruments and reagent
Ultra high efficiency liquid phase-GC-MS (waters company of the U.S.)；Supercentrifuge (German Bake Man)；Vortex
Oscillator (German ika company)；0.22 μm of filter membrane (Town in Shanghai spectrum)；(Shanghai section leads the limited public affairs of ultrasonic instrument to ultrasonic cleaner
Department).
Honey element (purity >=99%, German dr.ehrensotorfer)；Ammonium acetate (purity 99%)；
Methyl alcohol (chromatographically pure, German merck company)；Wahaha ultra-pure water.
1.2 test method
1.2.1 instrument parameter
1.2.1.1 liquid phase chromatogram condition
Chromatographic column: acquity uplc beh c18 post, specification: 2.1mm × 50mm, 1.7 μm of particle diameter；Mobile phase: 5mm second
Acid ammonium solution-methyl alcohol (volume fraction) solution (40:60, v/v)；Isocratic elution: flow velocity 0.3ml/min, 40 DEG C of column temperature, sample room
4 DEG C of temperature, sample size 10 μ l.
1.2.1.2 Mass Spectrometry Conditions parameter
Ion gun: electric spray ion source, anion scans (esi-)；Detection mode: multiple reaction monitoring (mrm)；Capillary
Tube voltage: 3.5kv；Taper hole voltage: 40v；Ion source temperature: 120 DEG C；Desolvation temperature: 380 DEG C；Desolventizing gas flow:
600l/hr；Taper hole air-flow: 50l/hr；Collision voltage: 30v；Collision gas flow: 0.22ml/min.
1.2.2 sample extraction
Weigh 2.00g sample, be placed in 25ml centrifuge tube, add 10ml methanol aqueous solution (volumetric concentration 50%), fully
Mixing vibration vortex 1min, is positioned over 60 DEG C of water-baths 15 minutes, then ultrasonic 10min, and centrifugation pours out supernatant, residue uses
10ml methanol aqueous solution repeats aforesaid operations and extracts, is centrifuged 1 time, merges supernatant, is accurately settled to 25ml, obtains sample extraction
Liquid.
2 results and discussion
The optimization of 2.1 chromatographic conditions
Chromatographic column adopts acquity uplc beh c18 post 2.1mm × 1.7 μm of 50mm, and mobile phase compares 5mm acetic acid
Ammonium salt solution-methyl alcohol combination and 5mm ammonium acetate solution-Acetonitrile combinations, the analysis test of flow velocity 0.3ml/min isocratic elution, two kinds are not
Fig. 1 is shown in same mobile phase chromatogram contrast, and result shows when mobile phase elution requirement is 5mm ammonium acetate solution-methyl alcohol, hexamethylene
Base sulfamic acid sodium peak and Interference Peaks in matrix are kept completely separate best results, and separating degree and response are best.
The optimization of 2.2 Mass Spectrometry Conditions
Because honey element is sodium salt, so from the anion scan pattern of electron spray ionisation, the honey element mark of 5.0mg/l
Quasi- solution is not separated by liquid-phase chromatographic column, and directly using mass spectrum automatic injector direct injected, mass spectrometry parameters is carried out excellent
Change, because honey element structure is more stable, therefore need higher collision voltage when characteristic ion is found in analysis.Groping matter
During spectral condition, the characteristic ion of honey element only has m/z=178.0, selects characteristic ion peak to have to m/z=79.8 mono- simultaneously
Fragment ion.Final determination mass spectrometry parameters are negative ion mode full scan and selection ion analysis, capillary voltage 3.5kv, cone
Hole voltage 40v, ion source temperature: 120 DEG C, desolvation temperature: 380 DEG C, desolventizing gas flow 600l/hr, taper hole air-flow
50l/hr, collision voltage 30v, collision gas flow 0.22ml/min.Obtain characteristic ion peak [m-na]-, select characteristic ion peak
(quota ion and qualitative ion are same) is shown in Table 1.
Table 1 honey element quantitation and qualitative ion information
2.3 extracting modes and the selection of Extractant concentration
Because aquatic products sample is complex, different extracting modes has considerable influence to result of the test.This test is selected
Extracted with whirlpool concussion, ultrasonic wave extraction and the above two mixing extracting modes carry to honey element in fishery technology sample
Take, each extracting mode makees 3 identical samples.Result of the test shows, whirlpool concussion extract and ultrasonic wave extraction combine into
Row extracts, and extraction effect is optimal, and the rate of recovery preferably, is shown in Fig. 2.Therefore select whirlpool concussion to extract and ultrasonic wave extraction mixing is extracted
As sample extraction mode.
20%, 30%, 50%, 70%, 80% (v/v) methanol aqueous solution respectively is selected to carry out in the Extraction solvent of sample
Extract.Result shows, when in methanol aqueous solution, methanol content is more than 50%, the rate of recovery of honey element declines.Use 50% methyl alcohol
The aqueous solution is made Extraction solvent and can be efficiently reduced the extraction to other impurities, simultaneously and does not affect the rate of recovery, therefore selects 50%
Methanol aqueous solution is as Extraction solvent.
2.4 linear relationships, detection limit, the rate of recovery and precision
Prepare honey element standard substance concentration be 0.5,1.0,2.0,10,20,50ng/ml, enter under 1.2.1 chromatographic condition
Row measures, and standard liquid chromatogram is shown in Fig. 3.With peak response for y value, concentration of standard solution draws curve for x value.Linear pass side
Journey, coefficient correlation and detection limit (s/n=3) are shown in Table 2.
Choose and do not contain the fishery technology (crisp-fried little yellow croaker) of honey element, add standard liquid respectively, do 6 times parallel,
Carry out sample pre-treatments and chromatographic determination according to 1.2.1 and 1.2.2, calculate the rate of recovery and precision, the results are shown in Table 2, blank sample
Product mark-on chromatogram Fig. 4.
Table 2 linear equation, coefficient correlation, detection limit, the rate of recovery and precision (n=6)
3 conclusions
No matter the mensure of honey element in fishery technology, adopt gas phase spectrometry or liquid chromatography, loaded down with trivial details derivatization
Step is essential.The method that this test establishes honey element in Ultra Performance Liquid Chromatography-mass spectrometric determination fishery technology, water
Produce processed goods sample to extract through ultra-pure water, be centrifuged constant volume, through Ultra Performance Liquid Chromatography-mass spectrometric determination detection, pre-treatment step
Simple and convenient, greatly shorten detection cycle, improve detection efficiency, be applied in actual sample detection, achieved good
Effect, there is higher practical value.
Detect 50 parts of processing of aquatic products product using the method for the present invention, including 10 parts of siphonopods processed goods, shrimps processed goods
20 parts and 20 parts of fish processed goods, wherein 3 parts detect honey element: baked squid son, fish processed goods in siphonopods processed goods
Middle crisp-fried little yellow croaker and roast eel, measured value is respectively 0.12g/kg, 0.45g/kg, 0.31g/kg, in remaining 47 parts of product all not
Detection honey element.Consistent with the method testing result using gb/t5009.97-2003.
Embodiment described above is one kind preferably scheme of the present invention, not the present invention is made any pro forma
Limit, also have other variants and remodeling on the premise of without departing from the technical scheme described in claim.
Claims (5)
1. a kind of improved method measuring honey element in fishery technology is it is characterised in that comprise the steps:
(1) sample extraction: weigh 2.00g sample, be placed in 25ml centrifuge tube, add 10ml methanol aqueous solution, be sufficiently mixed and shake
Swing vortex 1min, be positioned over 60 DEG C of water-baths 15 minutes, then ultrasonic 10min, centrifugation, pour out supernatant, residue uses 10ml first
Alcohol solution repeats aforesaid operations and extracts, is centrifuged 1 time, merges supernatant, is accurately settled to 25ml, obtains sample extracting solution；
(2) liquid chromatography-mass spectrography detection: take sample extracting solution 1ml, through 0.22 μm of organic membrane filtration in sample injection bottle, enter
Row liquid chromatography-mass spectrography detects, then draws standard working curve, quantified by external standard method, it is fixed according to peak area, honey element to be carried out
Amount.
2. method according to claim 1 it is characterised in that: described in step (1), parameter of noncentricity is: 6000r/min from
Heart 5min.
3. method according to claim 1 and 2 it is characterised in that in step (1) volumetric concentration of methanol aqueous solution be
50%.
4. method according to claim 1 and 2 is it is characterised in that liquid phase chromatogram condition is: chromatographic column: acquity
Uplc beh c18 chromatographic column, specification: 2.1mm × 50mm, 1.7 μm of particle diameter；Mobile phase: 5mmol/l ammonium acetate solution-methyl alcohol is molten
Liquid；Isocratic elution: flow velocity 0.3ml/min, 40 DEG C of column temperature, 4 DEG C of sample room temperature, sample size 10 μ l.
5. method according to claim 1 and 2 is it is characterised in that Mass Spectrometry Conditions: ion gun: electric spray ion source, bear from
Son scanning；Detection mode: multiple reaction monitoring；Capillary voltage: 3.5kv；Taper hole voltage: 40v；Ion source temperature: 120 DEG C；
Desolvation temperature: 380 DEG C；Desolventizing gas flow: 600l/hr；Taper hole air-flow: 50l/hr；Collision voltage: 30v；Impinging air flows
Amount: 0.22ml/min.
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| CN108508128A (en) * | 2018-03-30 | 2018-09-07 | 南通市产品质量监督检验所 | The detection method of honey element in a kind of white wine |
| CN111983011A (en) * | 2020-08-30 | 2020-11-24 | 广东利诚检测技术有限公司 | Method for detecting sodium cyclamate in steamed bread based on four-way equal-difference induced potentials with different phases |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108508128A (en) * | 2018-03-30 | 2018-09-07 | 南通市产品质量监督检验所 | The detection method of honey element in a kind of white wine |
| CN111983011A (en) * | 2020-08-30 | 2020-11-24 | 广东利诚检测技术有限公司 | Method for detecting sodium cyclamate in steamed bread based on four-way equal-difference induced potentials with different phases |
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Application publication date: 20170118 |