CN106290636B - The method for measuring steviol glycoside content in milk-contained drink - Google Patents
The method for measuring steviol glycoside content in milk-contained drink Download PDFInfo
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- CN106290636B CN106290636B CN201610650343.1A CN201610650343A CN106290636B CN 106290636 B CN106290636 B CN 106290636B CN 201610650343 A CN201610650343 A CN 201610650343A CN 106290636 B CN106290636 B CN 106290636B
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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
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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
Abstract
The invention proposes the methods of steviol glycoside content in measurement milk-contained drink.It is mixed the described method includes: the milk-contained drink is carried out first with acetic acid lead solution by (1), and the first obtained mixed liquor is subjected to the first centrifugation, the precipitating of resulting separation and the first supernatant；(2) precipitating is carried out second with mobile phase to mix, and obtained second mixed liquor is subjected to the second centrifugation, collect the second supernatant；(3) merge first supernatant and the second supernatant, and with the mobile phase constant volume, and obtained third mixed liquor is filtered, obtain prepare liquid；And (4) detect the prepare liquid using chromatography, and determine steviol glycoside content in the milk-contained drink based on testing result.Method accuracy of the invention is strong, precision is high, stability is good, easy to operate.
Description
Technical field
The present invention relates to field of food.In particular it relates to the method for measuring steviol glycoside content in milk-contained drink.
Background technique
Steviol glycoside is a kind of natural non-nutritive high-intensity sweeteners, it is from original South America Paraguay and Brazil
Obtained from being extracted in the leaf of the perennial shrub plant STEVIA REBAUDIANA of composite family (stevia rebaudiana bertoni), sweet tea
Degree is 250~300 times of sucrose, bitter and herbaceous taste after having.Steviol glycoside has the characteristics such as high sugariness, low in calories, nontoxic, for a long time
Edible to make one to get fat, particularly suitable obesity, diabetes, hypertension, artery sclerosis, saprodontia patient use, and object
Reason, chemical property be stable, without Fermented, can extend the shelf-life of steviol glycoside product.Thus, it is widely used in food and medicine
In object industry, the third natural sugar source after sucrose, beet sugar is known as by reputation.Steviol glycoside replaces sucrose to meet modern
Requirement to health idea, therefore steviol glycoside is developed with extremely wide prospect.
Milk-contained drink refers to fresh milk (or milk powder) the fermented or azymic for primary raw material, and water and appropriate is added
The drink with corresponding flavor that auxiliary material (such as cocoa, coffee, fruit juice and sucrose) is process.
However, at present in milk-contained drink the detection method of steviol glycoside content still have it is to be developed.
Summary of the invention
The present invention is directed at least solve one of the technical problems existing in the prior art.For this purpose, the invention proposes measurements
The method of steviol glycoside content in milk-contained drink, this method accuracy is strong, precision is high, stability is good, easy to operate.
It should be noted that the present invention is the following discovery based on inventor and completes:
The detection method of steviol glycoside is mainly " the food safety place provincial standard method DBSS22/007-2012 at present
The measurement high performance liquid chromatography of steviol glycoside in standard food ", the pre-treating method is relatively complicated, and it is more complex to be not suitable for matrix
Milk-contained drink in steviol glycoside detection.And national standard GB8270-2014 " national food safety standard food additives stevioside
Glycosides " it is the extraction and detection method for simply describing aqueous samples, relative to matrix for more complex milk-contained drink
Be not suitable for.
In view of this, inventor has found by many experiments, milk-contained drink is mixed with acetic acid lead solution in advance, with precipitating
Precipitating is removed by centrifugation in the impurity such as fat, protein, obtained supernatant, recycles in the further extraction precipitation of mobile phase
Steviol glycoside, and by obtained prepare liquid carry out chromatography detection, so as to accurately determine sweet tea in milk-contained drink
Chrysanthemum Glycosides Contents.The method accuracy of steviol glycoside content is strong, smart in measurement milk-contained drink according to an embodiment of the present invention as a result,
Density is high, stability is good, easy to operate.
For this purpose, the invention proposes a kind of methods of steviol glycoside content in measurement milk-contained drink.Reality according to the present invention
Apply example, which comprises (1) milk-contained drink is carried out first with acetic acid lead solution and mixed, and obtain first is mixed
It closes liquid and carries out the first centrifugation, the precipitating of resulting separation and the first supernatant；(2) precipitating second is carried out with mobile phase to mix
It closes, and obtained second mixed liquor is subjected to the second centrifugation, collect the second supernatant；(3) merge first supernatant and
Second supernatant, and be filtered with the mobile phase constant volume, then by obtained third mixed liquor, obtain prepare liquid；And
(4) prepare liquid is detected using chromatography, and determines that steviol glycoside contains in the milk-contained drink based on testing result
Amount.
Inventors have found that milk-contained drink mesostroma is more complex, if being directly centrifuged, collecting supernatant, and to supernatant
It is detected, then matrix interference is larger, it is inaccurate to lead to testing result, and damage to instrument.In turn, inventors have found that
Organic solvent is advanced with by a large amount of Stromal Precipitations in milk-contained drink, the supernatant containing steviol glycoside is detected, energy
It is enough effectively prevented from matrix interference, to improve the accuracy of testing result, precision and stability.For this purpose, inventor passes through
Many experiments obtain optimal organic solvent-lead acetate, lead acetate can abundant substances such as precipitating proteins, fat, and stevioside
Glycosides is retained in supernatant, and obtained supernatant is relatively clarified, smaller to the interference of subsequent chromatography detection, thus larger journey
Improve to degree the accuracy of testing result.Steviol glycoside content in measurement milk-contained drink according to an embodiment of the present invention as a result,
Method accuracy it is strong, precision is high, stability is good, easy to operate.
According to an embodiment of the invention, in said determination milk-contained drink the method for steviol glycoside content can also have it is following
Additional technical feature:
According to an embodiment of the invention, the concentration of the acetic acid lead solution is 2 volume %.Implement according to the present invention as a result,
The method accuracy of steviol glycoside content is strong in the measurement milk-contained drink of example, precision is high, stability is good, easy to operate.
According to an embodiment of the invention, the dosage of the acetic acid lead solution is 0.5~1mL based on milk-contained drink described in 5g,
It is preferred that 0.5mL.The method accuracy of steviol glycoside content is strong, smart in measurement milk-contained drink according to an embodiment of the present invention as a result,
Density is high, stability is good, easy to operate.
According to an embodiment of the invention, based on milk-contained drink described in 5g, the dosage of the mobile phase is in step (2)
20mL.The method accuracy of steviol glycoside content is strong in measurement milk-contained drink according to an embodiment of the present invention as a result, precision
Height, stability are good, easy to operate.
According to an embodiment of the invention, the mobile phase is phosphoric acid-acetonitrile solution, acetonitrile in the phosphoric acid-acetonitrile solution
Volume ratio with phosphoric acid is 3:7.The method of steviol glycoside content is quasi- in measurement milk-contained drink according to an embodiment of the present invention as a result,
Really property is strong, precision is high, easy to operate.
According to an embodiment of the invention, the chromatography testing conditions are as follows: chromatographic column: Ai Jieer C18 chromatographic column
(250mm × 4.6mm, 5.0 μm)；Flow velocity: 1.0mL/min；Mobile phase described in 0.002mol/L；Sample volume: 20 μ L；Column temperature: 30
℃；Wavelength: 210nm.The method accuracy according to an embodiment of the present invention for measuring steviol glycoside content in milk-contained drink as a result,
By force, precision is high, stability is good, easy to operate.
According to an embodiment of the invention, it is described first mixing and second mixing separately include carry out ultrasound 5~
10min.The method accuracy of steviol glycoside content is strong in measurement milk-contained drink according to an embodiment of the present invention as a result, precision
Height, stability are good, easy to operate.
According to an embodiment of the invention, first centrifugation and the second centrifugation are separately in 3000~5000r/min
Revolving speed under carry out 5~10min, preferably carry out 5min under the revolving speed of 4000r/min.Survey according to an embodiment of the present invention as a result,
The method accuracy for determining steviol glycoside content in milk-contained drink is strong, precision is high, stability is good, easy to operate.
In addition, the invention proposes the methods of steviol glycoside content in another measurement milk-contained drink.It is according to the present invention
Embodiment, which comprises 1) weigh milk-contained drink described in 5g in 50mL centrifuge tube, 2 volume % acetic acid of 0.5mL is added
Lead water solution, after ultrasonic 10min, 4000r/min is centrifuged 5min, takes obtained first supernatant in 50ml volumetric flask, sinks
Shallow lake is retained in centrifuge tube；2) be added 10ml mobile phase (phosphoric acid-acetonitrile solution, wherein the volume ratio of acetonitrile and phosphoric acid be 3:
7) in Yu Suoshu centrifuge tube, after ultrasonic 5min, 4000r/min is centrifuged 5min, takes obtained second supernatant in the capacity
In bottle, precipitating is retained in the centrifuge tube；3) operation 2 times of step 2) are repeated, the dosage of each mobile phase is 5mL；
4) in Yu Suoshu volumetric flask, with the mobile phase constant volume, prepare liquid is obtained；5) steviol glycoside standard items, compound concentration point are weighed
Not Wei 5.0 μ g/mL, 10.0 μ g/mL, 20.0 μ g/mL, 50.0 μ g/mL, 100.0 μ g/mL steviol glycoside standard solution,
0.22nm organic membrane filter obtains series standard solution；6) molten to the prepare liquid and series standard respectively using liquid chromatogram
Liquid is detected, and the peak area of obtained series standard solution and solution concentration is drawn standard curve, and will be described to be measured
The peak area of liquid substitutes into the standard curve, to determine steviol glycoside content in the milk-contained drink, wherein chromatographic test strip
Part is as follows: chromatographic column: Ai Jieer C18 chromatographic column (250mm × 4.6mm, 5.0 μm)；Flow velocity: 1.0mL/min；Mobile phase:
0.002mol/L phosphoric acid-acetonitrile solution, the volume ratio of acetonitrile and phosphoric acid is 3:7 in phosphoric acid-acetonitrile solution；Sample volume: 20 μ L；Column
Temperature: 30 DEG C；Wavelength: 210nm.The method of steviol glycoside content is quasi- in measurement milk-contained drink according to an embodiment of the present invention as a result,
Really property is strong, precision is high, stability is good, easy to operate.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Detailed description of the invention
Above-mentioned and/or additional aspect of the invention and advantage will become from the description of the embodiment in conjunction with the following figures
Obviously and it is readily appreciated that, in which:
Fig. 1 shows the process of the method for steviol glycoside content in measurement milk-contained drink according to an embodiment of the invention
Schematic diagram.
Specific embodiment
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair
It is bright, and be not considered as limiting the invention.
It should be noted that term " first ", " second " are used for description purposes only, it is not understood to indicate or imply phase
To importance or implicitly indicate the quantity of indicated technical characteristic.Define " first " as a result, the feature of " second " can be with
Explicitly or implicitly include one or more of the features.Further, in the description of the present invention, unless otherwise saying
Bright, the meaning of " plurality " is two or more.
The invention proposes a kind of methods of steviol glycoside content in measurement milk-contained drink, will be retouched in detail to it below
It states.
According to an embodiment of the invention, method includes: to obtain the first supernatant S100 referring to Fig. 1；Obtain the second supernatant
S200；Obtain prepare liquid S300；And chromatography detects S400.As a result, in measurement milk-contained drink according to an embodiment of the present invention
The method accuracy of steviol glycoside content is strong, precision is high, stability is good, easy to operate.
S100 obtains the first supernatant
In this step, milk-contained drink and acetic acid lead solution first is carried out to mix, and by the first obtained mixed liquor into
Row first is centrifuged, the precipitating of resulting separation and the first supernatant.
Inventors have found that milk-contained drink mesostroma is more complex, if being directly centrifuged, collecting supernatant, and to supernatant
It is detected, then matrix interference is larger, it is inaccurate to lead to testing result, and damage to instrument.In turn, inventors have found that
Organic solvent is advanced with by a large amount of Stromal Precipitations in milk-contained drink, the supernatant containing steviol glycoside is detected, energy
It is enough effectively prevented from matrix interference, to improve the accuracy of testing result.For this purpose, inventor by many experiments obtain it is optimal
Organic solvent-lead acetate, lead acetate can abundant substances such as precipitating proteins, fat, and steviol glycoside is retained in supernatant
In, and obtained supernatant is relatively clarified, and it is smaller to the interference of subsequent chromatography detection, to largely improve detection
As a result accuracy, precision and stability.However, the sedimentation effect of other organic solvents is poor, for example, will lead to albumen
The species precipitates such as matter, fat are incomplete, and supernatant is more muddy, influence subsequent detection；Or it will lead to a large amount of steviol glycosides with egg
The substances such as white matter, fat precipitate together, and testing result is relatively low.Sweet tea in measurement milk-contained drink according to an embodiment of the present invention as a result,
The method accuracy of chrysanthemum Glycosides Contents is strong, precision is high, stability is good, easy to operate.
According to an embodiment of the invention, the concentration of acetic acid lead solution is 2 volume %.Inventor is it was unexpectedly observed that lead acetate
The concentration of solution influences the accuracy of testing result.In turn, obtaining optimal lead acetate solution concentration by many experiments is 2 bodies
Product % can sufficiently make the species precipitates such as protein, fat with this condition, and supernatant is relatively clarified, the accuracy of testing result
It is relatively strong.If concentration is too low, supernatant is more muddy, and unobvious with precipitating boundary, causes the accuracy of testing result lower；If dense
Height is spent, the steviol glycoside concentration that will lead to extraction is lower, and the peak area of subsequent chromatography detection is smaller, and error is larger, and
Waste drug.The method accuracy of steviol glycoside content is strong, accurate in measurement milk-contained drink according to an embodiment of the present invention as a result,
Degree is high, stability is good, easy to operate.
According to an embodiment of the invention, being based on 5g milk-contained drink, the dosage of acetic acid lead solution is 0.5~1mL, preferably
0.5mL.Inventor obtains the optimal dosage of above-mentioned lead acetate by many experiments, can sufficiently make protein, rouge with this condition
The species precipitates such as fat, supernatant are relatively clarified, and the accuracy of testing result is stronger.If dosage is excessively high, the stevioside of extraction will lead to
Glycosides concentration is lower, and the peak area of subsequent chromatography detection is smaller, and error is larger；If dosage is too low, it is unable to fully precipitating fat, egg
The substances such as white matter, supernatant is more muddy, and unobvious with precipitating boundary, causes the accuracy of testing result lower.Basis as a result,
The method accuracy of steviol glycoside content is strong in the measurement milk-contained drink of the embodiment of the present invention, precision is high, stability is good, operation
It is easy.
According to an embodiment of the invention, the first mixing is included in 5~10min of progress under ultrasound.Thereby, it is possible to sufficiently precipitate
The substances such as fat, protein prevent from interfering.According to another embodiment of the invention, the first centrifugation is in 3000~5000r/
5~10min is carried out under the revolving speed of min, preferably carries out 5min under the revolving speed of 4000r/min.Thereby, it is possible to effectively make precipitating and
Supernatant separation is convenient for subsequent extracted.The side according to an embodiment of the present invention for measuring steviol glycoside content in milk-contained drink as a result,
Method accuracy is strong, precision is high, stability is good, easy to operate.
S200 obtains the second supernatant
In this step, precipitating is carried out second with mobile phase to mix, and obtained second mixed liquor is carried out second
The second supernatant is collected in centrifugation.
According to an embodiment of the invention, mobile phase is phosphoric acid-acetonitrile solution, acetonitrile and phosphoric acid in phosphoric acid-acetonitrile solution
Volume ratio is 3:7.First supernatant of collection is directly carried out constant volume it has furthermore been found that if being free of step S200 by inventor
Operation, the rate of recovery is lower, and go out peak position and the peak position that goes out of object in sample of standard items are deviated, to make to examine
Accuracy and the precision for surveying result are lower.Main cause may be, since milk-contained drink mesostroma is more complex, to utilize lead acetate
When precipitating proteins and fat, it is easy to bring a small amount of steviol glycoside in precipitating into, causes the rate of recovery relatively low.In turn, invention people's will
Other places discovery, utilizes the sweet tea in the mobile phase in subsequent chromatography detection process-further extraction precipitation of phosphoric acid-acetonitrile solution
Synanthrin glycosides can effectively improve the accuracy and precision of testing result.Then, inventors have found that selection acetonitrile, phosphoric acid are made
For mobile phase, and when the volume ratio of acetonitrile and phosphoric acid is 3:7, the accuracy and precision of testing result are further increased.Acetonitrile
Steviol glycoside can be effectively extracted from precipitating, phosphate plays the role of adjusting flowing phase pH value, to influence target
The appearance time of object.If the ratio of acetonitrile increases, object appearance time is longer, extends round of visits；If acetonitrile ratio is smaller,
Object then coincides with the miscellaneous peak of sample, influences to examine accuracy, and be unable to fully extract steviol glycoside from precipitating.By
This, the method accuracy of steviol glycoside content is strong in measurement milk-contained drink according to an embodiment of the present invention, precision is high, stability
It is good, easy to operate.
According to an embodiment of the invention, being based on 5g milk-contained drink in step S200, the dosage of mobile phase is 15~25mL,
It is preferred that 20mL.Inventor obtains the optimal dosage of above-mentioned mobile phase by many experiments, can sufficiently extract egg with this condition
The accuracy of steviol glycoside in white matter and adipopexis, testing result is stronger.If dosage is excessively high, the stevioside of extraction will lead to
Glycosides concentration is lower, and the peak area of subsequent chromatography detection is smaller, and error is larger；If dosage is too low, it is unable to fully mention from precipitating
Steviol glycoside is taken out, causes testing result relatively low.Steviol glycoside contains in measurement milk-contained drink according to an embodiment of the present invention as a result,
The method accuracy of amount is strong, precision is high, stability is good, easy to operate.
According to an embodiment of the invention, mobile phase carries out second with the obtained precipitating of S100 in two times in step S200
Mixing.Inventors have found that mobile phase is divided into two parts with arbitrary proportion, first a copy of it is mixed with precipitating, then by institute
Obtained mixed liquor centrifugation, collects supernatant；Another mobile phase is added into precipitating again and mixes centrifugation, collects supernatant.
Thus, it is possible to sufficiently extract steviol glycoside, to improve the accuracy of testing result.It is preferred that mobile phase is divided into two
Part.The method accuracy of steviol glycoside content is strong in measurement milk-contained drink according to an embodiment of the present invention as a result, precision is high,
Stability is good, easy to operate.
According to an embodiment of the invention, the second mixing is included in 5~10min of progress under ultrasound.Thereby, it is possible to sufficiently extract
Steviol glycoside.According to another embodiment of the invention, second centrifugation be under the revolving speed of 3000~5000r/min carry out 5~
10min preferably carries out 5min under the revolving speed of 4000r/min.Thereby, it is possible to effectively make precipitating and supernatant separation, after being convenient for
It is continuous to extract.The method accuracy of steviol glycoside content is strong in measurement milk-contained drink according to an embodiment of the present invention as a result, precision
Height, stability are good, easy to operate.
S300 obtains prepare liquid
In this step, merge the first supernatant and the second supernatant, and with mobile phase constant volume, then by obtained third
Mixed liquor is filtered, and obtains prepare liquid.It was unexpectedly observed that if carrying out constant volume with water, object and standard items go out inventor
Peak position is different, has offset, extension detection time, and the accuracy of testing result is relatively low.After mobile phase constant volume, target
The appearance position consistency of object and standard items, and the accuracy of testing result is stronger.Measurement according to an embodiment of the present invention contains as a result,
The method accuracy of steviol glycoside content is strong in milk beverage, precision is high, stability is good, easy to operate.
The detection of S400 chromatography
In this step, prepare liquid is detected using chromatography, and sweet tea in milk-contained drink is determined based on testing result
Chrysanthemum Glycosides Contents.
According to an embodiment of the invention, chromatography testing conditions are as follows: chromatographic column: Ai Jieer C18 chromatographic column (250mm ×
4.6mm, 5.0 μm)；Flow velocity: 1.0mL/min；0.002mol/L mobile phase；Sample volume: 20 μ L；Column temperature: 30 DEG C；Wavelength:
210nm.The method accuracy of steviol glycoside content is strong in measurement milk-contained drink according to an embodiment of the present invention as a result, precision
Height, stability are good, easy to operate.
In addition, the invention proposes the methods of steviol glycoside content in another measurement milk-contained drink.It is according to the present invention
Embodiment, method include: 1) to weigh 5g milk-contained drink in 50mL centrifuge tube, and 2 volume % acetic acid lead solution of 0.5mL is added,
After ultrasonic 10min, 4000r/min be centrifuged 5min, take obtained first supernatant in 50ml volumetric flask, precipitating be retained in from
In heart pipe；2) mobile phase (phosphoric acid-acetonitrile solution, wherein the volume ratio of acetonitrile and phosphoric acid is 3:7) of 10ml is added in centrifuge tube
In, after ultrasonic 5min, 4000r/min is centrifuged 5min, takes obtained second supernatant in volumetric flask, and precipitating is retained in centrifugation
Guan Zhong；3) operation 2 times of step 2) are repeated, the dosage of each mobile phase is 5mL；4) it in volumetric flask, with mobile phase constant volume, obtains
To prepare liquid；5) steviol glycoside standard items are weighed, compound concentration is respectively 5.0 μ g/mL, 10.0 μ g/mL, 20.0 μ g/mL, 50.0
The steviol glycoside standard solution of μ g/mL, 100.0 μ g/mL, 0.22nm organic membrane filter obtain series standard solution；6) liquid is utilized
Phase chromatography respectively detects prepare liquid and series standard solution, by the peak area and solution of obtained series standard solution
Concentration draws standard curve, and the peak area of prepare liquid is substituted into standard curve, and to determine, steviol glycoside contains in milk-contained drink
Amount, wherein chromatographic test strip part is as follows: chromatographic column: Ai Jieer C18 chromatographic column (250mm × 4.6mm, 5.0 μm)；Flow velocity:
1.0mL/min；Mobile phase: 0.002mol/L phosphoric acid-acetonitrile solution, the volume ratio of acetonitrile and phosphoric acid is in phosphoric acid-acetonitrile solution
3:7；Sample volume: 20 μ L；Column temperature: 30 DEG C；Wavelength: 210nm.Sweet tea in measurement milk-contained drink according to an embodiment of the present invention as a result,
The method accuracy of chrysanthemum Glycosides Contents is strong, precision is high, easy to operate.
In the context of the present invention, all numbers being disclosed are approximation.The numerical value of each number has
It is possible that the differences such as 1%, 2%, 5%, 7%, 8% or 10%.Whenever disclosing a number with N value, any tool
There is the number within N+/- 1%, N+/- 2%, N+/- 3%, N+/- 5%, N+/- 7%, N+/- 8% or the value of N+/- 10% can be bright
It is really open, it adds deduct wherein " +/- " refers to.Whenever disclosing a lower limit in a numberical range, DL and upper limit,
DU, when, any numerical value within the scope of the disclosed can be specifically disclosed.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment
Part, it described technology or conditions or is carried out according to the literature in the art according to product description.Agents useful for same or instrument
Production firm person is not specified in device, and being can be with conventional products that are commercially available.
Instrument and equipment:
Balance: sensibility reciprocal 0.1mg, producer: METTLER TOLEDO, model: XP205
Liquid chromatograph: band UV detector, producer: Dai An, model: U-3000
Centrifuge: producer: Heal Force, model: Neofuge 23R
Supersonic wave cleaning machine: producer: flag beauty, model: QC10260
Embodiment 1
In this embodiment, the method for steviol glycoside content in milk-contained drink is measured in following manner:
1, reagent
Ultrapure water；
0.002mol/L phosphoric acid solution: precision weighs 0.196g phosphoric acid in 1000mL volumetric flask, and water is added to be settled to scale；
2% acetic acid lead solution: accurately weighing 20g lead acetate in 1L volumetric flask, and scale is dissolved and be settled to water, mixes
It is spare afterwards；
Acetonitrile: chromatographically pure.
2, determination step
(1) preparation of prepare liquid
(1-1) weighs sample about 5g (being accurate to 0.1mg) in 50mL centrifuge tube, and it is water-soluble that 2% lead acetate of 0.5mL is added
Liquid, after ultrasonic 10min, 4000r/min is centrifuged 5min, takes supernatant in 50ml volumetric flask, and precipitating is retained in centrifuge tube；
(1-2) be added 10ml mobile phase (phosphoric acid-acetonitrile solution, wherein the volume ratio of acetonitrile and phosphoric acid be 3:7) in
In 50ml centrifuge tube, after ultrasonic 5min, 4000r/min is centrifuged 5min, takes supernatant in above-mentioned 50ml volumetric flask, and precipitating retains
In centrifuge tube；
(1-3) repeats step (1-2) and operates 2 times, and the dosage of each mobile phase is 5mL；
(1-4) is settled to scale in 50mL volumetric flask, with mobile phase, obtains prepare liquid.
(2) steviol glycoside standard solution is prepared:
Steviol glycoside stock solution: precision weighs steviol glycoside standard items 10mg in 10mL volumetric flask, adds 1ml acetonitrile, molten
Xie Houjia water is settled to scale, obtains the steviol glycoside stock solution that steviol glycoside concentration is 1mg/mL.
The preparation of steviol glycoside series standard solution: precision draw steviol glycoside stock solution 0.05mL, 0.1mL, 0.2mL,
0.5mL, 1.0mL with flowing phase dilution and are settled to scale in 10mL volumetric flask, and obtaining concentration is 5.0 μ g/mL, 10.0 μ g/
The series standard solution of the steviol glycoside of mL, 20.0 μ g/mL, 50.0 μ g/mL, 100.0 μ g/mL.Take this series standard solution suitable
Amount crosses 0.22nm organic film, obtains series standard solution.
(3) using liquid chromatogram, the prepare liquid to step (1) and the obtained series standard solution of step (2) are carried out respectively
The peak area of obtained series standard solution and solution concentration are drawn standard curve by detection, and by the peak area of prepare liquid
Standard curve is substituted into, so that steviol glycoside content in milk-contained drink be calculated.
Wherein, chromatographic test strip part is as follows:
Chromatographic column: Ai Jieer C18 chromatographic column (250mm × 4.6mm, 5.0 μm)；
Flow velocity: 1.0mL/min；
Mobile phase: 0.002mol/L phosphoric acid-acetonitrile solution, the volume ratio of acetonitrile and phosphoric acid is 3 in phosphoric acid-acetonitrile solution:
7；
Sample volume: 20 μ L；
Column temperature: 30 DEG C；
Wavelength: 210nm.
2 accuracy of embodiment and precision measurement
In this embodiment, the rate of recovery is measured in following manner:
(1) milk-contained drink without steviol glycoside is mixed with steviol glycoside standard solution, obtains mark-on sample；
(2) steviol glycoside content in mark-on sample is measured according to the method for embodiment 1, and recycling is calculated based on following equation
Rate:
Steviol glycoside contains in the measured value of steviol glycoside content × 100/ mark-on sample in the rate of recovery (%)=mark-on sample
The theoretical value of amount.
The results are shown in Table 1, wherein the theoretical value of steviol glycoside content is " scalar quantity " as shown in Table 1 in mark-on sample.
As can be seen that the rate of recovery, in the range of 90%~100%, RSD value illustrates the present invention in the range of 1.34%~1.68%
The preci-sion and accuracy of method is higher.
The preci-sion and accuracy of the invention of table 1 (n=6) test
| Serial number | Scalar quantity (mg/kg) | Testing result (mg/kg) | Rate of recovery range (%) | RSD (%) |
| 1 | 4.0 | 3.83 | 92.12~96.42 | 1.34 |
| 2 | 8.0 | 7.68 | 93.59~96.87 | 1.52 |
| 3 | 20.0 | 19.1 | 93.35~97.57 | 1.68 |
Comparative example 1
It in the comparative example, is detected according to the method for embodiment 1, difference is, lead acetate solution concentration is 1%.
The results are shown in Table 2.As can be seen that accuracy, precision and stability that 2% acetic acid lead solution is added all compare
The effect of 1% acetic acid lead solution is good.
The testing result of table 2 embodiment 1 and comparative example 1
Comparative example 2
In the comparative example, being detected according to the method for embodiment 1, difference is, step (1-2) and (1-3) are free of,
Supernatant is directly subjected to constant volume.
The results are shown in Table 3.As can be seen that being extracted using mobile phase than not having to accuracy, the precision that mobile phase is extracted
And high stability.
The testing result of table 3 embodiment 1 and comparative example 2
Comparative example 3
It in the comparative example, is detected according to the method for embodiment 1, difference is, carries out constant volume using water.
With the direct constant volume extracting solution of water, the appearance time of object and the appearance time of standard items are partially away from, and are joined
4 are shown in Table, the accuracy of documents 3 is poor.
The testing result of table 4 embodiment 1 and comparative example 3
Comparative example 4
It in the comparative example, is detected according to the method for embodiment 1, difference is, substitutes lead acetate with 5mL acetonitrile.
The results are shown in Table 5.As can be seen that using acetonitrile precipitation protein and fat, the rate of recovery and precision are lower.
The testing result of 5 comparative example 4 of table
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples
It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replacement and variant.
Claims (1)
1. a kind of method of steviol glycoside content in measurement milk-contained drink characterized by comprising
1) milk-contained drink described in 5g is weighed in 50mL centrifuge tube, and 0.5mL2 volume % acetic acid lead solution, ultrasonic 10min is added
Afterwards, 4000r/min is centrifuged 5min, takes obtained first supernatant in 50ml volumetric flask, and precipitating is retained in the centrifuge tube
In；
2) mobile phase of 10ml is added in the centrifuge tube, after ultrasonic 5min, 4000r/min is centrifuged 5min, takes obtained
In the volumetric flask, precipitating is retained in the centrifuge tube second supernatant；
3) operation 2 times of step 2) are repeated, the dosage of each mobile phase is 5mL；
4) in Yu Suoshu volumetric flask, with the mobile phase constant volume, prepare liquid is obtained；
5) steviol glycoside standard items are weighed, compound concentration is respectively 5.0 μ g/mL, 10.0 μ g/mL, 20.0 μ g/mL, 50.0 μ g/
The steviol glycoside standard solution of mL, 100.0 μ g/mL, 0.22nm organic membrane filter obtain series standard solution；
6) prepare liquid and series standard solution are detected respectively using liquid chromatogram, obtained series standard is molten
The peak area and solution concentration of liquid draw standard curve, and the peak area of the prepare liquid is substituted into the standard curve, so as to
Determine steviol glycoside content in the milk-contained drink,
Wherein, chromatographic test strip part is as follows:
Chromatographic column: Ai Jieer C18 chromatographic column, specification be 250mm × 4.6mm, 5.0 μm；
Flow velocity: 1.0mL/min；
Mobile phase: 0.002mol/L phosphoric acid-acetonitrile solution, the volume ratio of acetonitrile and phosphoric acid is 3 in the phosphoric acid-acetonitrile solution:
7；
Sample volume: 20 μ L；
Column temperature: 30 DEG C；
Wavelength: 210nm.
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