CN107064340A - A kind of method of GABA content in detection tealeaves - Google Patents
A kind of method of GABA content in detection tealeaves Download PDFInfo
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Abstract
The present invention provides a kind of method for detecting GABA content in tealeaves, and this method uses LC-MS, GABA is quantitatively detected.Comprise the following steps successively：Sample treatment, UPLC MS/MS analyses, quantified by external standard method.A certain amount of tealeaves is weighed, with 1:10 ratio adds water immersion, and takes supernatant after ultrasonic 1 4h in Ultrasound Instrument, the sample introduction after membrane filtration.Sample after processing is used into triple level Four bar LC-MS instrument, analyzed under rigid condition.The quantified by external standard method, using testing concentration as abscissa, the peak area of determinand is ordinate, and the linear equation tried to achieve, as working curve calculate the drug concentration in Tea Samples extract solution using standard curve.The beneficial effects of the invention are as follows GABA content in UPLC MS/MS detection methods detection tealeaves, simple and fast, sensitivity is high, and strong antijamming capability, the value for measurement tealeaves provides reliable basis.
Description
Technical field
The invention belongs to technical field of food detection, it is related to a kind of to a kind of method of GABA content detection in tealeaves.
Background technology
γ-aminobutyric acid (abbreviation GABA) is a kind of naturally occurring nonprotein amino acid, in being central nervous system
Critically important inhibitory neurotransmitter, with extremely important physiological function, it can promote the reactivity of brain, and brain tonic and intelligence development resists
Epilepsy, promotes sleep, beauty moisturizing delays brain aging function, can supplement human body inhibitory neurotransmitter, with good drop blood
Press effect.Promote kidney Fitness improvement and protective effect.Suppress fatty liver and obesity, activate liver function.Daily iron supplement is micro
γ-aminobutyric acid is conducive to the alleviation of heart and brain blood pressure, can promote the balance of amino acid metabolism in human body again, adjusts immunologic function.
γ-aminobutyric acid (GABA) is a kind of active skull cap components, is distributed widely in animal and plant body.Tealeaves has GABA
The biosynthesis basis of synthesis, is GABA natural origin, meanwhile, tealeaves is also a kind of natural healthy plant beverage, its
With promoting the production of body fluid to quench thirst, the improving eyesight that clears away heart-fire, diuresis heat-clearing, the effects such as remove fatigue, drink for a long time can prevent cardiovascular and cerebrovascular disease, it is anti-
Cancer, fat-reducing etc..
Therefore set up by extract obtain γ-aminobutyric acid method, using LC-MS-MS analysis methods to γ in tealeaves-
The content of aminobutyric acid is quantified, and has good application effect to the exploitation of tea value.
There is GABA detection method in existing tealeaves：Radioimmunoassays [RIA], paper electrophoresis, enzyme-linked fluorescence method
[ELISA], paper chromatography, amino-acid analyzer method, thin layer chromatography scanning, high performance liquid chromatography, gas chromatography.Height therein
Effect liquid phase chromatogram method is because its sensitivity is high, precision is good, and accuracy is high and is widely used.Because GABA is a kind of organic acid,
Polarity is strong, thus typically to have column front derivation and post-column derivation by deriving, and uses column front derivation, this method is cumbersome time-consuming.
The content of the invention
The purpose of the present invention be overcome prior art defect and it is not enough there is provided it is a kind of efficiently, quick, Accurate Determining tealeaves
The detection method of middle GABA content, is that the value of tealeaves is utilized there is provided guarantee.
Technical scheme：
A kind of method of GABA content in detection tealeaves, this method uses LC-MS, GABA is quantitatively detected.
The method of GABA content, comprises the following steps successively in a kind of detection tealeaves：Sample treatment, UPLC-MS/MS points
Analysis, quantified by external standard method.
The sample treatment is：
The extraction of tealeaves：A certain amount of tealeaves is weighed, with 1:10 ratio adds water immersion, and the ultrasound 1- in Ultrasound Instrument
Supernatant is taken after 4h, the sample introduction after membrane filtration.
It is furthermore preferred that the extraction of tealeaves：2-4g tealeaves is weighed, with 1:10 ratio adds 20-40mL water and soaked, and in
Supernatant is taken after ultrasound 2h in Ultrasound Instrument, the sample introduction after membrane filtration.
The sample treatment of graticule：Precision weighs 1.84mg GABA standard items, is dissolved in water and is configured to 2mg/mL mother liquor,
Then be diluted to 500 step by step with water, 200,100,50, sample introduction is analyzed after 20ng/mL serial solution.
The UPLC-MS/MS analyses：Sample after processing is used into triple level Four bar LC-MS instrument, in following condition
It is lower to be analyzed：
Liquid phase process
Liquid-phase chromatographic column：HILIC, 2.1*100mm, 1.7 μm of column temperatures of ACQUITY UPLC BEH：35℃
Mobile phase：A：Water (contains 0.1% formic acid, v/v), B：Acetonitrile；Flow velocity：0.3mL/min；Column temperature：35℃；Sample size：
10μL；
Gradient elution, elution requirement is shown in Table 1.
The liquid phase gradient of table 1
| Time/min | 0 | 1.5 | 3 | 5 | 5.01 | 7 |
| A% | 10 | 10 | 90 | 90 | 10 | 10 |
Mass spectrometry method：
The mass spectrometry parameters of composition to be measured are as follows：
The MRM mode detection mass spectrometry parameters of table 2
| Target compound | Mode | Chan Reaction | Cone(voLts) | CoLLision(voLts) |
| GABA | ESI+ | 103.88>86.67 | 15 | 13 |
The quantified by external standard method, using testing concentration as abscissa, the peak area of determinand is ordinate, the straight line tried to achieve
Equation, as working curve:Y=217.375x-693.954, r=0.999, standard curve are shown in Fig. 1.Calculated using standard curve
The GABA concentration gone out in Tea Samples extract solution.
A kind of method of GABA content in detection tealeaves, the range of linearity is 20~500ng/mL, and lower limit of quantitation is 20ng/
ML, detection is limited to 1.5ng/mL, and linear relationship is good in the range of 20~500ng/mL.
Inspection method is 94.4% to the GABA rate of recovery, meets the requirement that GABA Concentration Testings are analyzed in tealeaves.
The present invention has the advantages and positive effects of：GABA content in UPLC-MS/MS detection methods detection tealeaves,
Simple and fast, sensitivity is high, strong antijamming capability, and the value for measurement tealeaves provides reliable basis.
Brief description of the drawings
Fig. 1 GABA standard curves.
Fig. 2 blank solvent chromatograms.
Fig. 3 quantitative limit 20ng/mL GABA standard liquid chromatograms.
Fig. 4 concentration is 500ng/mL GABA standard liquid chromatograms.
The chromatogram of sample 1 is detected in Fig. 5 embodiments 3.
Embodiment
The present invention is further elaborated with reference to specific embodiment and with reference to accompanying drawing, but does not limit the present invention.
The method of GABA content in the detection tealeaves of embodiment 1
1.1 sample treatment
The extraction of tealeaves：Appropriate tealeaves is weighed, with 1:10 ratio adds water immersion, and the ultrasound 1-4h in Ultrasound Instrument
After take, it is stand-by after 4 DEG C of preservations through membrane filtration.
The processing of graticule：Precision weighs 1.84mg GABA standard items, is dissolved in water and is configured to 2mg/mL mother liquor, then
Be diluted to 500 step by step with water, 200,100,50,20ng/mL serial solution it is stand-by after 4 DEG C of preservations.
1.2 UPLC-MS/MS are analyzed
1.2.1 liquid phase process
Liquid-phase chromatographic column：ACQUITY UPLC BEH HILIC,2.1*100mm,1.7μm
Mobile phase：A：Water (contains 0.1% formic acid, v/v), B：Acetonitrile
Flow velocity：0.35mL/min；Column temperature：35℃；Sample size：10μL
Gradient elution, elution requirement see the table below
| Time/min | 0 | 1.0 | 2 | 3.5 | 3.51 | 5 |
| A% | 10 | 10 | 90 | 90 | 10 | 10 |
1.2.2 mass spectrometry method：
The mass spectrometry parameters of composition to be measured are as follows：
MRM mode detection mass spectrometry parameters
| Target compound | Mode | Chan Reaction | Cone(voLts) | CoLLision(voLts) |
| r-aminobutyric acid | ESI+ | 103.88>86.67 | 15 | 13 |
Instrument in the present embodiment：The triple level Four bar LC-MS instrument of Waters UPLC-Quattro Premier XE
(SoLvent containing Binary manager, SampLe manager, Quattro Premier XE MS, MassLynx V4.1
Chromatographic work station)；Turbine mixer, model：MX-S, the production of SCILOGEX companies of the U.S.；Supersonic cleaning machine, the new sesame in Ningbo is biological
Scientific ＆ technical corporation produces, model：HBA-M-090601-99.
Agents useful for same and mark product in the present embodiment：Formic acid:Chromatographically pure CNW lot numbers:L401K140；Acetonitrile:Chromatographically pure
Fisher lot numbers:071757；Water:MiLLi Q；GABA：Chromatographically pure chromaDex lot numbers：00001674-5QO, purity：
99.0%, room temperature preservation.
The Method validation of embodiment 2 is tested
2.1 selectivity are investigated
Selectivity refers in the case of there is interference component in the sample that analysis method accurately, can be determined exclusively point
The ability of thing is analysed, the selective effect of the experimental method is preferable, as a result such as Fig. 2 blank solvent chromatograms, Fig. 3 20ng/mLGABA
Standard liquid chromatogram, shown in the chromatogram of Fig. 5 samples 1, shows that matrix, without any interference, is somebody's turn to do to target compound GABA detection
Method choice is good.
2.2 detection limits are investigated
Determination to GABA test limits is determined according to signal to noise ratio method.The stock solution of concentration known is diluted to low dense
The signal (baseline noise) of the sample of degree, the signal measured and blank space is compared, and calculates what may reliably be detected
Least concentration or percentage.It is required that：Detection limit signal to noise ratio is not less than 3.
Detection limit is tested：Take GABA appropriate, it is accurately weighed, it is diluted with water the molten of the 20ng containing GABA in every 1mL is made respectively
Liquid, precision measures the solution 10 μ L, injects liquid chromatograph, records chromatogram, minimum detection is calculated with 3 times of baseline noise
Limit is about 1ng/mL, standard liquid is diluted to 0.5 respectively, 1,1.5, inject liquid chromatograph after 2ng/mL, record chromatogram,
Signal to noise ratio is calculated, concentration is that 1.5ng/mL is to meet the requirement that signal to noise ratio is more than or equal to 3.
Conclusion：That is GABA detection is limited to 1.5ng/mL.
2.3 quantitative limits are investigated
Determination to GABA quantitative limits is determined according to signal to noise ratio method.The stock solution of concentration known is diluted to low dense
The signal (baseline noise) of the sample of degree, the signal measured and blank space is compared, it is desirable to：Quantitative limit signal to noise ratio is not less than
10,2.0% should be not more than by determining the relative standard deviation of 6 secondary peak retention times, and the relative standard deviation of peak area should be not more than
5.0%.
Quantitative limit is tested：Take GABA appropriate, it is accurately weighed, it is diluted with water the molten of the 20ng containing GABA in every 1mL is made respectively
Liquid, precision measures the solution 10 μ L, injects liquid chromatograph, records chromatogram, parallel determination 6 times.As a result such as table 3.
The quantitative limit testing result of table 3
| Sequence number | 1 | 2 | 3 | 4 | 5 | 6 | Mean | RSD |
| Retention time (min) | 2.37 | 2.36 | 2.37 | 2.36 | 2.36 | 2.37 | 2.37 | 0.232 |
| Peak area | 4703 | 4789 | 4667 | 4805 | 4687 | 4765 | 4736 | 1.22 |
Conclusion：Replication 6 times, GABA retention times RSD is 0.232%, and the RSD of peak area is 1.22%.Meet and test
(peak retention time RSD cannot be greater than 2.0% to the requirement of card scheme；5.0%) peak area RSD cannot be greater than
2.4 system precision tests
Take with a need testing solution, by the above-mentioned pin of chromatographic condition continuous sample introduction 6,6 measurement results will have good
Repeatability；The RSD of the peak area of 6 measurement results of reference substance solution must not cross 5.0%, and the RSD of retention time must not mistake
2.0%, carrying out authentication system has good precision.
It it is 2 years by above-mentioned sample treatment processing Tea Samples, precision measures 10 μ L injection liquid chromatographs, record
Chromatogram, repeats sample introduction 6 times.Each peak area and the RSD of retention time are calculated respectively, the results are shown in Table 3.
The precision testing result of table 4
| Sequence number | 1 | 2 | 3 | 4 | 5 | 6 | Mean | RSD |
| Retention time (min) | 2.33 | 2.37 | 2.36 | 2.36 | 2.36 | 2.33 | 2.35 | 0.732 |
| Peak area | 21571 | 20401 | 20793 | 20900 | 21343 | 21682 | 21115 | 2.36 |
Conclusion：System precision：Replication 6 times, GABA retention times RSD is 0.732%, and the RSD of peak area is
2.36%.Explanation system precision is good, meets proof scheme requirement.
2.5 linear test
Concentration is configured to dilution water to be ground for 20,50,100,200,500ng/mL GABA series standard solution
Study carefully.Response signal (peak area) of the linear relationship to measure using each analyte concentration as abscissa (x), is entered for ordinate (y)
Row linear regression analysis.It is required that the value of the correlation coefficient r of regression equation cannot be less than 0.990.
GABA standard items 1.84mg are dissolved in water for 2mg/mL storing solution, be then diluted to 500 step by step with water,
200th, 100,50,20ng/mL series standard solution, the results are shown in Table 5.
The standard curve of table 5
Conclusion：Linearly：GABA is in 20~500ng/mL concentration range, and peak area is with concentration in good linear pass
System, correlation coefficient r>0.990.
2.6 recovery test
The rate of recovery is 100% standard liquid by adding index in known test sample, and test sample is subtracted with measured value
Value is again divided by result measured by addition is rate of recovery value, is represented with percentage %, it is desirable to the rate of recovery 80.0%~
Between 115.0%, RSD is not higher than 5%, has the good degree of accuracy with substantive approach.
Tea Samples 2.89g is taken, with 1：10 ratios add the dissolving of 28.9mL pure water and extracted, then precision weighs GABA standard items
2.38ug, is mixed after ultrasound 2h in Ultrasound Instrument, takes supernatant, and filter membrane waits sample introduction, the results are shown in Table 6.
The rate of recovery result of table 6
Conclusion：In tealeaves between the GABA rate of recovery 93.3%~97.5%, average recovery rate is that 94.3%, RSD is
1.82, meet Method validation requirement, illustrate that this method degree of accuracy is high.
2.6 replica test
Replica test is that 100% is loaded each pin of solution sample introduction 1 of solution by preparing 6 100% sample-adding solution,
The RSD for the measured amount that 100% sample-adding solution 6 times is measured must not cross 5.0%, and carrying out substantive approach has good repeatability.
Precision measures the sample-adding μ L of solution 10 of recovery test 100%, injects liquid chromatograph, chromatogram is recorded, according to above-mentioned side
Method replication 6 times.As a result such as table 7.
The repeated testing result of table 7
Conclusion：The sample solution retention time RSD of GABA 100% are 0.566%, and peak area RSD is 1.51%, the side of meeting
Science of law checking requires that repeatability is good.
2.7 solution stability testing
Room temperature is taken after need testing solution, processing to place, and sample introduction, note respectively at 0, after 0.5,2,4,6,8,12,24 hours
Peak area is recorded, the situation of change of chemicals peak area is investigated, the RSD of peak area is calculated.It is required that：The peak area RSD of compound should
No more than 5.0%.If meet proof scheme requirement, it is stable to illustrate that reference substance solution is placed within the period, be with
The time limit of test solution standing time provides foundation when detecting afterwards.
Weigh Tea Samples appropriate, it is accurately weighed, handled by sample treatment, after processing respectively at 0,0.5,2,
4th, 6,8,12, the μ L sample introductions of solution 10 are measured successively within 24 hours, inject liquid chromatograph, record chromatogram, experimental result is shown in Table 8.
The Detection of Stability result of table 8
Conclusion：GABA retention time RSD in 24h are 0.15% in tealeaves, and peak area RSD is 3.64%, meets method
Learn checking to require, show that sample solution places stable within this time, the phase of solution standing time is tested when being detected after being
Limit provides foundation.
The measure of the actual sample of embodiment 3
In buying different types of tealeaves respectively on the market, and by they be named as sample 1, sample 2, sample 3, sample 4,
Sample 5, sample 6, this experiment are quantitatively detected for these Tea Samples respectively：
Sample 1,3.01g is taken, with 1：10 ratios add 30.1mL water to dissolve, and mix the ultrasound 2h in Ultrasound Instrument, draw supernatant
Liquid, after membrane filtration, under above-mentioned chromatographic condition, sample introduction analysis；
Sample 2,3.52g is taken, with 1：10 ratios add 35.2mL water to dissolve, and mix in Ultrasound Instrument in ultrasound 2.5h, absorption
Clear liquid, after membrane filtration, under above-mentioned chromatographic condition, sample introduction analysis；
Sample 3,2.04g is taken, with 1：10 ratios add 20.4mL water to dissolve, and mix the ultrasound 1h in Ultrasound Instrument, draw supernatant
Liquid, after membrane filtration, under above-mentioned chromatographic condition, sample introduction analysis；
Sample 4,2.25g is taken, with 1：10 ratios add 22.5mL water to dissolve, and mix in Ultrasound Instrument in ultrasound 1.5h, absorption
Clear liquid, after membrane filtration, under above-mentioned chromatographic condition, sample introduction analysis；
Sample 5,3.33g is taken, with 1：10 ratios add 33.3mL water to dissolve, and mix in Ultrasound Instrument in ultrasound 3.5h, absorption
Clear liquid, after membrane filtration, under above-mentioned chromatographic condition, sample introduction analysis；
Sample 6,2.97g is taken, with 1：10 ratios add 29.7mL water to dissolve, and mix the ultrasound 4h in Ultrasound Instrument, draw supernatant
Liquid, after membrane filtration, under above-mentioned chromatographic condition, sample introduction analysis；As a result such as table 9 below.
The sample detection result of table 9
One embodiment of the present of invention is described in detail above, but the content is only the preferable implementation of the present invention
Example, it is impossible to be considered as the practical range for limiting the present invention.All equivalent changes made according to the present patent application scope and improvement
Deng within the patent covering scope that all should still belong to the present invention.
Claims (10)
1. a kind of method for detecting GABA content in tealeaves, it is characterised in that：This method uses LC-MS, and GABA is determined
Amount detection.
2. a kind of method for detecting GABA content in tealeaves according to claim 1, it is characterised in that：Include successively following
Step：Sample treatment, UPLC-MS/MS analyses, quantified by external standard method.
3. a kind of method for detecting GABA content in tealeaves according to claim 2, it is characterised in that：
The sample treatment is：
The extraction of tealeaves：A certain amount of tealeaves is weighed, with 1:10 ratio adds water immersion, and in Ultrasound Instrument after ultrasound 1-4h
Supernatant is taken, the sample introduction after membrane filtration.
4. a kind of method for detecting GABA content in tealeaves according to claim 3, it is characterised in that：
The extraction of tealeaves：2-4g tealeaves is weighed, with 1:10 ratio adds the immersion of 20-40mL water, and ultrasonic in Ultrasound Instrument
Supernatant is taken after 2h, the sample introduction after membrane filtration.
5. a kind of method for detecting GABA content in tealeaves according to claim 2, it is characterised in that：
The sample treatment of graticule：Precision weighs 1.84mg GABA standard items, is dissolved in water and is configured to 2mg/mL mother liquor, then
Be diluted to 500 step by step with water, 200,100,50, sample introduction is analyzed after 20ng/mL serial solution.
6. a kind of method for detecting GABA content in tealeaves according to claim 2, it is characterised in that：The UPLC-MS/
MS is analyzed：Sample after processing is used into triple level Four bar LC-MS instrument, analyzed under the following conditions：
Liquid phase process：
Liquid-phase chromatographic column：ACQUITY UPLC BEH HILIC,2.1*100mm,1.7μm；
Mobile phase：A：Water (contains 0.1% formic acid, v/v), B：Acetonitrile；Flow velocity：0.35mL/min；Column temperature：35℃；Sample size：10μ
L；
Gradient elution, elution requirement：
Mass spectrometry method：
The mass spectrometry parameters of composition to be measured：
MODE:ESI+；
CapiLLary(kV):3.2kV； Source Temperature(℃):120；
DesoLvation Temperature(℃):350； DesoLvation Gas FLow(L/Hr):600；
Cone Gas FLow(L/Hr):50； DweLL time(s)：0.1s；
MRM mode detection mass spectrometry parameters：
7. a kind of method for detecting GABA content in tealeaves according to claim 2, it is characterised in that：
The quantified by external standard method, using testing concentration as abscissa, the peak area of determinand is ordinate, the straight line side tried to achieve
Journey, as working curve, the GABA concentration in Tea Samples extract solution is calculated using standard curve.
8. a kind of method for detecting GABA content in tealeaves according to claim 2, it is characterised in that：This method detection
The range of linearity is 20~500ng/mL, and lower limit of quantitation is 20ng/mL, and detection is limited to 1.5ng/mL, in 20~500ng/mL models
Enclose interior linear relationship good.
9. a kind of method for detecting GABA content in tealeaves according to claim 1, it is characterised in that：Inspection method pair
The GABA rate of recovery is 94.3%, meets the requirement that GABA Concentration Testings are analyzed in tealeaves.
10. the method for GABA content in a kind of detection tealeaves according to claim any one of 1-9, it is characterised in that：It is right
All Tea Samples are applicable.
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| CN112451996A (en) * | 2020-11-10 | 2021-03-09 | 浙江大学 | Optimization method for capturing protein by multi-column continuous flow chromatography |
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| WO2011139914A1 (en) * | 2010-04-29 | 2011-11-10 | Wisconsin Alumni Research Foundation | Metabolic biomarkers of autism |
| CN106442766A (en) * | 2016-08-31 | 2017-02-22 | 安徽瑞思威尔科技有限公司 | Method for fast detecting gamma-aminobutyric acid in baijiu |
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| CN107976499A (en) * | 2017-11-27 | 2018-05-01 | 广州傲农生物科技有限公司 | A kind of method of alpha-aminobutyric acid content in efficiently binary liquid chromatography detection livestock and poultry organization |
| CN112451996A (en) * | 2020-11-10 | 2021-03-09 | 浙江大学 | Optimization method for capturing protein by multi-column continuous flow chromatography |
| CN112451996B (en) * | 2020-11-10 | 2021-09-24 | 浙江大学 | Optimization method for capturing protein by multi-column continuous flow chromatography |
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Application publication date: 20170818 |