CN106248773B - It is a kind of quickly to measure vitamin B in multidimensional piece1With ascorbic method - Google Patents
It is a kind of quickly to measure vitamin B in multidimensional piece1With ascorbic method Download PDFInfo
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- CN106248773B CN106248773B CN201610569813.1A CN201610569813A CN106248773B CN 106248773 B CN106248773 B CN 106248773B CN 201610569813 A CN201610569813 A CN 201610569813A CN 106248773 B CN106248773 B CN 106248773B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
- G01N27/622—Ion mobility spectrometry
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention discloses vitamin Bs in a kind of quickly measurement multidimensional piece1With ascorbic method, include the following steps：(1) sample treatment：Sample obtains filtrate after the processing of the pre-treatment steps such as dissolution, extraction, centrifugation, filtering；(2) sample analysis：It is analyzed using the ionization device in situ that is sprayed, volume aspirated ratio is 1:1 filtrate and methanol, filling to the borosilicate glass capillary tube drawn, are inserted into metal microelectrode, are placed on ionic migration spectrometer injection port front end, apply spray voltage, and sample is ionized, into migration tube after separation with the detection of Faraday cup detector.Vitamin B in quick measurement multidimensional piece of the present invention1With ascorbic method is easy to operate, low in cost, speed is fast for analysis, specificity is good and it is quantitative accurately, and since ion mobility spectrometry instrument is small in size, move, mobility strong, it will be expected to realize the scene quickly analysis measurement of sample, there is more wide application prospect.
Description
Technical field
The present invention relates to detection method fields, more particularly to vitamin B in a kind of quickly measurement multidimensional piece1And vitamin
The method of C.
Background technique
Vitamin B1Also known as thiamine or anti-nerve element, are to maintain heart, nervous system and digestive system normal function institute
A kind of required vitamin, is primarily present in meat and vegetables.In vivo, it is important biomolecule catalyst --- carboxylation
The main component of coenzyme, when shortage, will lead to a series of illnesss, such as athlete's foot, polyneuritis.Vitamin C, alias L-
Ascorbic acid, can promote the metabolism of tyrosine and tryptophan, extend the human body service life, be constitute Skin Cell interstitial must be at
Point, it is distributed widely in all kinds of fresh vegetables and fruits, when shortage is susceptible to suffer from the diseases such as scurvy.
With the quickening of today's society rhythm, the nutrient prime replenisher of convenient and efficient is increasingly had been favored by people, more
Tieing up piece is exactly to supplement B needed by human1With a kind of compound preparation of the electrolytes and minerals such as C.Vitamin B1And vitamin C
Belong to human body and maintain a kind of trace organic substance of normal physiological function, but a series of side effects can be generated when overdose, such as
Internal vitamin B1It is excessive, it will lead to that body is shaken, bleb, edema, heartbeat are speeded and allergy etc.；Excessive use vitamin C can
Generate certain adverse reactions, such as diarrhea, skin rash and calculus urate.Therefore, it establishes a kind of simple and direct efficient, low-cost
Detection method is had important practical significance with preferably monitoring health care Safety of Food Quality.
Summary of the invention
Quickly, in easy, quantitative accurately measurement multidimensional piece life is tieed up the technical problem to be solved in the present invention is to provide a kind of
Plain B1With ascorbic method.
It is a kind of quickly to measure vitamin B in multidimensional piece1With ascorbic method, include the following steps：
(1) sample treatment：Sample obtains filtrate after the processing of the pre-treatment steps such as dissolution, extraction, centrifugation, filtering；
(2) sample analysis：It is analyzed using the ionization device in situ that is sprayed, volume aspirated ratio is 1:1 filtrate and methanol,
Filling is inserted into microelectrode, is placed on ionic migration spectrometer injection port front end, applies to the borosilicate glass capillary tube drawn
Add spray voltage, sample is ionized, into migration tube after separation with the detection of Faraday cup detector.
Vitamin B in quick measurement multidimensional piece of the present invention1With ascorbic method, wherein the Ion transfer
The analysis condition of spectrometer is as follows：
Electron spray voltage：Positive ion mode, vitamin B1：2000V；Negative ion mode, vitamin C：2100V；
Migrate tube voltage：8000V；
Migration tube temperature：180℃；
Gas preheater temperature：180℃；
Migrate spectrum width：26ms；
Ion gatewidth：70μs；
Ion gate voltage：40V；
Data acquisition rate：200,000s-1；
Acquisition time：20s；
Drift about gas velocity：1.5L/min；
Exhaust pump pumping speed：0.66L/min.
Vitamin B in quick measurement multidimensional piece of the present invention1With ascorbic method, wherein the filter of absorption
The volume of liquid and methanol is 5 μ L.
Vitamin B in quick measurement multidimensional piece of the present invention1With ascorbic method, wherein the Ion transfer
Spectrometer is used for instrumental correction before use, tryptophan and citric acid are configured to the solution that concentration is 10 μ g/mL with methanol,
Wherein, it is corrected under positive ion mode with tryptophan solution, is corrected under negative ion mode with citric acid solution.
Vitamin B in quick measurement multidimensional piece of the present invention1With ascorbic method, wherein step (1) is specific
It comprises the following processes：
Sample treatment：Sample is fully ground to powdered, precision weighs 0.1g into 10mL volumetric flask, 2mL water is added,
Then vortex 1min is settled to 10mL scale with water；It is vortexed again for 1min, sample is fully transferred in centrifuge tube, 8000rpm
It is centrifuged 5min, takes supernatant to cross 0.45 μm of miillpore filter, obtains filtrate.
Vitamin B in quick measurement multidimensional piece of the present invention1With ascorbic method, wherein for using in situ
The positive sample of spraying ionization device detection, is further confirmed using liquid chromatography-quadrupote mass spectrometry.
Vitamin B in quick measurement multidimensional piece of the present invention1With ascorbic method, wherein the liquid phase color
Spectrum-Quadrupole Mass Spectrometry condition is as follows：
Liquid phase chromatogram condition：
Chromatographic column：Waters ACQUITY UPLC HSS T3,100mm × 2.1mm, 1.8 μm；
Mobile phase A：01% aqueous formic acid, Mobile phase B：01% formic acid methanol solution, gradient elution, gradient elution program
It is shown in Table 1.
1 gradient elution program of table
Flow velocity：0.3mL/min；
Column temperature：30℃；
Sample volume：5μL；
Mass Spectrometry Conditions：
Ion source：Electric spray ion source；
Ionization mode：Positive ion mode；
Capillary voltage：3.0kV；
Extract orifice potential：4.0V；
Ion source temperature：150℃；
Desolvation temperature：500℃；
Data acquisition modes：Multiple-reaction monitoring pattern；
2 vitamin B of table1With ascorbic mass spectral analysis parameter
When carrying out Specimen Determination, sample liquid is suitably diluted, is surveyed by the condition of the liquid chromatography-quadrupote mass spectrometry
Sample solution and standard working solution are determined, if selected ion occurs, and selected ion ratio and standard substance
Relative abundance it is consistent, tolerance be no more than table 2 as defined in range, then contain vitamin B in judgement sample1And vitamin C.
The present invention quickly measures vitamin B in multidimensional piece1It is with ascorbic method difference from prior art：
The present invention quickly measures vitamin B in multidimensional piece1With ascorbic method using ionization in situ-portable ion
Migration spectral technology establishes vitamin B in multidimensional piece1With ascorbic rapid assay methods, solves common detection methods behaviour
Make cumbersome, time-consuming tediously long problem.It is measured in experiment with the sample of different spiked levels and methodological study, as the result is shown
This method is easy to operate, low in cost, analysis speed is fast, specificity is good and quantitative accurate, can be used for vitamin B in multidimensional piece1
It moves, mobility strong with ascorbic quantitative detection and since ion mobility spectrometry is small in size, will be expected to realize product
Live quickly analysis measurement, has more wide application prospect.
The present invention migrates spectral technology using the spraying coupled ion of extraction nanoliter, establishes vitamin B in multidimensional piece1It is given birth to dimension
The rapid assay methods of plain C.Sample is after the pre-treatments such as water vortex oscillation and centrifugation, and sample liquid is with 1 ︰ 1 (volume ratio) of methanol dilution
It is directly measured afterwards.Respectively to being moved in electrospray ionisation condition, sample solvent and ion mobility spectrometry in ionization in situ in experiment
It moves the key parameters such as tube temperature degree, drift gas velocity and has carried out emphasis optimization.The result shows that vitamin B1With ascorbic detection
It is limited to 0.1mg/g, is quantitatively limited to 0.2mg/g；In 1~10 μ g/mL and 1~100 μ g/mL concentration range, vitamin B1And dimension
Raw element C is in good linear relationship respectively, and related coefficient is greater than 0.99；The repeatability (being measured in parallel 6 times) of transit time is 0.6%
~2.9%；The instrument analysis time of singleton sample is no more than 20ms.This method is quick, and pretreatment process is easy and result is stablized
Reliably, it can be used for vitamin B in multidimensional piece1It is measured with ascorbic quick analysis.
With reference to the accompanying drawing to vitamin B in quick measurement multidimensional piece of the invention1Make with ascorbic method further
Explanation.
Detailed description of the invention
Fig. 1 is vitamin B in the embodiment of the present invention1With ascorbic ion transfer spectrogram.
The bilingual of all English occurred in attached drawing of the present invention is as follows：
Intensity:Signal strength；Drift time:Transit time；ms:Millisecond.
Specific embodiment
Embodiment 1
One, instrument and reagent
It is sprayed ionization device using in situ, specific structure is shown in the patent document application No. is 201510514854.6.
GA2100 spectrometric system (Excellims company of the U.S.)：It is controlled with electric spray ion source, ion fence gate
Device, air filter (sulfur acid calcium and molecular sieve), high-resolution Ion transfer analyzer, Faraday cup detector,
VisIon instrument controlling and data processing system are corrected with tryptophan and citric acid under positive and negative ion mode respectively using preceding
Instrument；Milli-Q Superpure water machine (Millipore company of the U.S.)；MS2 type turbula shaker (German IKA company)；P-1000 type
Microelectrode draws instrument, (U.S. Sutter is public for borosilicate glass capillary tube (outer diameter 1.50mm, internal diameter 0.86mm, length 10cm)
Department)；Axio Scope A1 microscope (German Zeiss company)；ESW-M15P type electrode jaw (U.S. Warner
Instruments company)；Suction pipette head (Bio-Rad company of the U.S.).
Vitamin B1(Sigma-Alarich company of the U.S.) and vitamin C (ChemService company of the U.S.) purity is big
In 95%, concentration is first configured to as the standard reserving solution of 1mg/mL with water, it is suitable for dense that when analysis is diluted to 90% methanol/water
Degree；Methanol (chromatographically pure) is purchased from U.S. Fisher company；Tryptophan and citric acid are purchased from Sigma-Aldrich, with
Methanol is made into 10 μ g/mL and carries out instrumental correction.
Two, sample treatment and analysis
Sample is fully ground to powdered, precision weighs 0.1g (being accurate to 0.01g) into 10mL volumetric flask, is added
Then 2mL water, vortex 1min are settled to 10mL scale with water；It is vortexed again for 1min, sample is fully transferred in centrifuge tube,
8000rpm is centrifuged 5min, and supernatant is taken to cross 0.45 μm of miillpore filter.
5 μ L filtrates and 5 μ L methanol are drawn, filling to the borosilicate glass capillary tube drawn is inserted into microelectrode, is put
It is placed in ionic migration spectrometer injection port front end, applies spray voltage, sample is ionized, into migration tube after separation with farad
The detection of the detector.
The analysis condition of the ionic migration spectrometer is as follows：
Electron spray voltage：Positive ion mode, vitamin B1：2000V；Negative ion mode, vitamin C：2100V；
Migrate tube voltage：8000V；
Migration tube temperature：180℃；
Gas preheater temperature：180℃；
Migrate spectrum width：26ms；
Ion gatewidth：70μs；
Ion gate voltage：40V；
Data acquisition rate：200,000s-1；
Acquisition time：20s；
Drift about gas velocity：1.5L/min；
Exhaust pump pumping speed：0.66L/min.
For further using liquid chromatography-tandem quadrupole rod using the positive sample in situ for being sprayed ionization device detection
Mass spectrography is confirmed, and actual conditions are as follows：
Liquid phase chromatogram condition：
Chromatographic column：Waters ACQUITY UPLC HSS T3,100mm × 2.1mm, 1.8 μm；
Mobile phase A：0.1% aqueous formic acid, Mobile phase B：0.1% formic acid methanol solution, gradient elution；Gradient elution journey
Sequence is shown in Table 1.
1 gradient elution program of table
Flow velocity：0.3mL/min；
Column temperature：30℃；
Sample volume：5μL；
Mass Spectrometry Conditions：
Ion source：Electric spray ion source；
Ionization mode：Positive ion mode；
Capillary voltage：3.0kV；
Extract orifice potential：4.0V；
Ion source temperature：150℃；
Desolvation temperature：500℃；
Data acquisition modes：Multiple-reaction monitoring pattern；
2 vitamin B of table1With ascorbic mass spectral analysis parameter
When carrying out Specimen Determination, sample liquid is suitably diluted, is surveyed by the condition of the liquid chromatography-quadrupote mass spectrometry
Sample solution and standard working solution are determined, if selected ion occurs, and selected ion ratio and standard substance
Relative abundance it is consistent, tolerance be no more than table 2 as defined in range, then contain vitamin B in judgement sample1And vitamin C.
Three, analysis condition optimizes
1, the optimization of nanoliter electrospray ionisation condition is extracted
Vitamin B1Belong to water soluble vitamin with vitamin C, therefore, this experiment, which takes water as a solvent, prepares its standard inventory
Liquid.To enhance its response under electrospray mode, while being provided with methanol-water, alcohol-water, the acetonitrile-of different volumes concentration
Water is compared test.The result shows that vitamin B1With vitamin C respectively under positive ion mode and negative ion mode obtain compared with
Height response；The acetonitrile-water of same volume concentration is very nearly the same as solvent response intensity with methanol-water, but the former matrix is made an uproar
The a little higher than the latter of sound；When using 50% methanol-water as solvent, target analytes response intensity highest, and matrix noise phase
To lower；In addition, the spray voltage being affected to ionization is also optimized, the results showed that vitamin B1And vitamin C
Higher response intensity is obtained at 2000V (positive ion mode) and 2100V (negative ion mode) respectively.
2, the optimization of migration tube temperature
In Ion transfer spectrum analysis, the setting of migration tube temperature often has larger shadow to the response intensity of target analytes
It rings.In a certain range, migration tube temperature is improved, memory effect in pipe can be effectively reduced, to improve sensitivity；But migration tube
Temperature is excessively high, can generate a series of unstable factors, such as heat exchange acceleration, object are decomposed.150 are respectively set in this experiment
DEG C, 160 DEG C, 170 DEG C, 180 DEG C, 190 DEG C be used as migration tube temperature, the results showed that, vitamin B1With vitamin C at 180 DEG C and
Response intensity is higher and essentially identical at 190 DEG C, therefore, select 180 DEG C as Ion transfer spectrum analysis when migration tube temperature.
3, the selection of drift gas velocity
Under the conditions of the spray voltage of optimization and migration tube temperature, the flow of drift gas (nitrogen) is also optimized.
0.5,1.0,1.5,2.0L/min conduct drift gas velocity are chosen respectively, carry out parameter optimization.The experimental results showed that drift air-flow
Speed influences less the separating degree of two kinds of target analytes, but in 1.5mL/min, the two response intensity is compared with other flow velocity ends
Height, and under the drift gas velocity, vitamin B1Be respectively 11.5ms and 8.3ms with ascorbic transit time, peak shape compared with
It is good, and good separation can be obtained with Interference Peaks.Vitamin B1Fig. 1 is seen with ascorbic characteristic ion migration spectrogram.
4, the investigation of transit time repeatability
Ion mobility spectrometry mainly carries out the separation of ion and qualitative, therefore, the weight of transit time with the difference of transit time
Importance of the renaturation in Ion transfer spectral analysis method is self-evident.It is molten that sample substrate is obtained by treatment conditions of the invention
Then liquid adds the standard solution (2 parts of each spiked levels sample preparation, parallel testing 6 times) of various concentration, investigate dimension respectively
Raw element B1With the repeatability of vitamin C transit time under different spiked levels.Analysis condition according to the invention is measured,
Vitamin B1And ascorbic transit time relative standard deviation shows the migration of two kinds of substances between 0.6%~2.9%
Time repeatability under the analysis condition of foundation is preferable.
5, linear relationship and detection limit, quantitative limit
By a series of standard solution of different quality concentration successively sample introduction, line is carried out to mass concentration (x) with peak area (y)
Property return, draw standard working curve.Experimental result shows, vitamin B1With vitamin C respectively 1~10 μ g/mL and 1~
Linear good within the scope of 100 μ g/mL, equation of linear regression, related coefficient and detection limit, quantitative limit are shown in Table 3.
3 linear relationship of table, detection limit and quantitative limit
7, the measurement of mark-on sample
For the quantitative accuracy of verifying analysis method, this experiment be respectively set quantitative limit concentration (0.2mg/g), 0.4mg/g,
Tri- pitch-based spheres of 1.0mg/g, each concentration are prepared 3 parts (tables 4) in parallel, are quantified with standard curve external standard method.As a result it shows
Show, vitamin B1The accuracy (%, average value ± standard deviation) added under concentration level with vitamin C at three is between 98.6
Between ± 1.4 and 103.4 ± 1.3, shows that method accuracy is preferable, vitamin B in sample can be met1With it is ascorbic accurate
Measurement.
Result (%, average value ± standard deviation, parallel testing 3 times) is investigated in 4 method accuracy of table
Embodiment described above only describe the preferred embodiments of the invention, not to model of the invention
It encloses and is defined, without departing from the spirit of the design of the present invention, those of ordinary skill in the art are to technical side of the invention
The various changes and improvements that case is made should all be fallen into the protection scope that claims of the present invention determines.
Claims (5)
1. vitamin B in a kind of quickly measurement multidimensional piece1With ascorbic method, it is characterised in that：Include the following steps：
(1) sample treatment：Sample is fully ground to powdered, precision weighs 0.1g into 10mL volumetric flask, 2mL water is added,
Then vortex 1min is settled to 10mL scale with water；It is vortexed again for 1min, sample is fully transferred in centrifuge tube, 8000rpm
It is centrifuged 5min, takes supernatant to cross 0.45 μm of miillpore filter, obtains filtrate；
(2) sample analysis：It is analyzed using the ionization device in situ that is sprayed, volume aspirated ratio is 1:1 filtrate and methanol, filling
To the borosilicate glass capillary tube of drawing, it is inserted into microelectrode, is placed on ionic migration spectrometer injection port front end, applies spray
Mist voltage, sample is ionized, into migration tube after separation with the detection of Faraday cup detector；
The analysis condition of the ionic migration spectrometer is as follows：
Electron spray voltage：Positive ion mode, vitamin B1：2000V；Negative ion mode, vitamin C：2100V；
Migrate tube voltage：8000V；
Migration tube temperature：180℃；
Gas preheater temperature：180℃；
Migrate spectrum width：26ms；
Ion gatewidth：70μs；
Ion gate voltage：40V；
Data acquisition rate：200000s-1；
Acquisition time：20s；
Drift about gas velocity：1.5L/min；
Exhaust pump pumping speed：0.66L/min.
2. vitamin B in quick measurement multidimensional piece according to claim 11With ascorbic method, it is characterised in that：Institute
Ionic migration spectrometer is stated before use, tryptophan and citric acid, which are configured to concentration with methanol, is the solution of 10 μ g/mL, and is used for
Instrumental correction, wherein corrected under positive ion mode with tryptophan solution, corrected under negative ion mode with citric acid solution.
3. vitamin B in quick measurement multidimensional piece according to claim 11With ascorbic method, it is characterised in that：It inhales
The volume of the filtrate and methanol that take is 5 μ L.
4. vitamin B in quick measurement multidimensional piece according to claim 11With ascorbic method, it is characterised in that：It is right
In using the positive sample in situ for being sprayed the detection of ionization device coupled ion migration spectrum, liquid chromatography-tandem four is further used
Pole bar mass spectrography is confirmed.
5. vitamin B in quick measurement multidimensional piece according to claim 41With ascorbic method, it is characterised in that：Institute
The condition for stating liquid chromatography-quadrupote mass spectrometry is as follows：
Liquid phase chromatogram condition：
Chromatographic column：Waters ACQUITY UPLC HSS T3,100mm × 2.1mm, 1.8 μm；
Mobile phase A：0.1% aqueous formic acid, Mobile phase B：0.1% formic acid methanol solution, gradient elution, gradient elution program are shown in
Table 1；
1 gradient elution program of table
Flow velocity：0.3mL/min；
Column temperature：30℃；
Sample volume：5μL；
Mass Spectrometry Conditions：
Ion source：Electric spray ion source；
Ionization mode：Cation or negative ion mode；
Capillary voltage：3.0kV；
Extract orifice potential：4.0V；
Ion source temperature：150℃；
Desolvation temperature：500℃；
Data acquisition modes：Multiple-reaction monitoring pattern；
2 vitamin B of table1With ascorbic mass spectral analysis parameter
When carrying out Specimen Determination, sample liquid is suitably diluted, measures sample by the condition of the liquid chromatography-quadrupote mass spectrometry
Product solution and standard working solution, if selected ion occurs, and the phase of selected ion ratio and standard substance
Consistent to abundance, tolerance is no more than range as defined in table 2, then contains vitamin B in judgement sample1And vitamin C.
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| CN108732235B (en) * | 2018-08-14 | 2020-11-06 | 青岛农业大学 | Method for rapidly detecting nisin content in chicken based on ion mobility spectrometry |
| CN110927277A (en) * | 2019-12-05 | 2020-03-27 | 合肥谱佳医学检验实验室有限公司 | Method for rapidly detecting concentrations of vitamin B1 and vitamin C based on liquid chromatography-tandem mass spectrometry technology |
| CN111398439A (en) * | 2020-03-06 | 2020-07-10 | 权丽 | Liquid chromatography-tandem mass spectrometry detection method for B vitamins in serum |
| CN112162028A (en) * | 2020-09-29 | 2021-01-01 | 中国农业科学院农业质量标准与检测技术研究所 | Mass spectrum imaging method for vitamin C in strawberry tissue |
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| Ion mobility spectrometry for the rapid analysis of over-the-counter drugs and beverages;Roberto Fernández-Maestre 等;《Journal for Ion Mobility Spectrometry》;20090701;第12卷(第3期);第91页 * |
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