CN108693257A - The method for directly detecting tealeaves glucosides bound state aroma precursor substance - Google Patents
The method for directly detecting tealeaves glucosides bound state aroma precursor substance Download PDFInfo
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- CN108693257A CN108693257A CN201810179791.7A CN201810179791A CN108693257A CN 108693257 A CN108693257 A CN 108693257A CN 201810179791 A CN201810179791 A CN 201810179791A CN 108693257 A CN108693257 A CN 108693257A
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Abstract
The invention discloses a kind of methods that directly can detect tealeaves glucosides bound state aroma precursor substance simultaneously, as sample to be tested, are followed the steps below successively in product or sample tea using fresh tea leaves, tea:1), glucosides bound state aroma precursor extracts in sample to be tested, 2), glucosides bound state aroma precursor UPLC-MS/MS analyses in extract, 3), tealeaves glucosides bound state aroma precursor substance content calculation:To be designated as reference in 4- nitrobenzophenones-β-D-Glucose glycosides, obtain benzyl alcohol primrose glucosides, gaultherolin primrose glucosides, benzyl carbinol primrose glucosides, along 3- hexenol primroses glucosides, the content of material of linalool oxide I primroses glucosides, linalool oxide II primroses glucosides, linalool oxide primrose glucosides III, linalool primrose glucosides, geraniol primrose glucosides this 9 kinds of glucosides bound state aroma precursors.This method is high to glucosides bound state aroma precursor substance detection efficiency.
Description
Technical field
The present invention relates to the detection method of glucosides bound state aroma precursor substance in a plant tea, specifically a kind of utilization
Ultrahigh pressure liquid phase chromatography-mass spectroscopy/mass spectrum (UPLC-MS/MS) multiple-reaction monitoring pattern (MRM) directly exists to fresh leaves of tea plant, tealeaves
Product and the glucosides bound state aroma precursor substance of dry tea carry out quick, efficient detection method.
Background technology
Fragrance is important the tea leaf quality factor.Fragrance component in tea includes mainly alcohols, aldehydes, ketone, esters, miscellaneous
Cycle compound etc., wherein alcohol compound are mostly important fragrance components.Tealeaves alcohols aroma substance can be divided into again terpenol,
Aromatic alcohol and fatty alcohol three classes compound, and terpenol and aromatic alcohol have the flavouring essence quality of tealeaves often with there is floral character
Significant contribution.In tealeaves common terpenol have linalool, linalool oxide (I, II, III, IV), geraniol, nerol,
Nerolidol etc., aromatic alcohol have benzyl alcohol and benzyl carbinol etc., fatty alcohol to have suitable 3- hexenols, enanthol, octanol etc..Existing research card
Real, sample tea fragrance mainly has 4 kinds of sources, one to be glucosides reference state precursor hydrolysis, release volatile aroma, secondly for length
Chain fatty acid is catalyzed by lipoxygenase or free-radical oxidation approach forms volatile short chain fatty alcohol, thirdly in hyther
It reacts to form the heterocyclic compounds such as pyrazine, pyrans by carbonyl ammonia down, four be both already present Free aroma object in fresh tea leaves
Matter, wherein glucosides reference state precursor hydrolysis are the most critical approach that tea aroma is formed.Therefore glucosides reference state precursor is tealeaves
The important characterization ingredient of fragrance potentiality, the rapid extraction of glucosides bound state aroma precursor and accurate detection method are established to tea tree product
Kind flavouring essence quality potentiality objective evaluation and high-aroma tea cultivation process mating technological development are significant.
Although glucosides reference state precursor substance contributes significantly tea aroma, this substance is in fresh tea leaves or sample tea
Middle abundance is very low, detach and purify it is extremely difficult, and this substance UV absorption it is weak, even without UV absorption, thus it is more difficult
Directly, accurately qualitative and quantitative.Currently, the detection of tealeaves glucosides bound state aroma precursor substance is mainly using enzymolysis and gas
Phase-mass spectrometry (GC-MS) be combined method or glucosides bound state aroma it is precursor-derived with GC-MS be combined method or
Using progress such as ultrahigh pressure liquid phase chromatography-mass spectroscopy (UPLC-MS) single ion monitoring pattern (SIM) methods.Wherein, enzymolysis and GC-
MS detection techniques, need by tea sample free state and glucosides bound state aroma precursor water or buffer solution be leached out, simultaneously
The aroma substance for removing free state is extracted repeatedly with ether equal solvent, by the extracting solution containing glucosides bound state aroma precursor and Portugal
The mixing such as polyglycoside enzyme or pectase hydrolyzes and discharges aroma substance, then with solid phase microextraction (SPME) or Simultaneous distillation-extraction
(SDE) etc. modes are enriched with aroma substance, and then qualitative and quantitative, this measurement is carried out to the aroma substance of extraction by GC-MS
Method can only estimate the aglycon property of reference state glycosidic precursors indirectly, cannot obtain the information of glycosyl, and glucosides digest with
And often there is also hydrolysis to be not thorough, is enriched with the problems such as incomplete in the fragrance enrichment process of release.Before glucosides bound state aroma
Syntaxy and GC-MS binding analysis methods, before main process is the glucosides bound state aroma for extracting and purifying in the samples such as tealeaves
Body, and mixed with trifluoroacetamide reagent or silylating reagent after fully drying and carry out derivatization treatment introducing volatility acetyl group
Or silane group, the precursor substance after derivative convert is carried out through GC-MS analyses qualitative and quantitative;This method can obtain glucosides
The more complete structural information of bound state aroma precursor, but pure precursor substance process is cumbersome, and also it is follow-up to analyze data
Processing is complicated because GC-MS obtain be it is derivative after glycoside matter information, it is also necessary to the original that the reduction of the processes such as deconvolution obtains
Beginning glycosidic precursors information.When detecting glucosides bound state aroma precursor using the SIM of UPLC-MS, need the glucosides knot in tealeaves
Close state Aroma precursor be leached out and by means such as column chromatographies after purification, detached through UPLC, according to glucosides reference state precursor point
Sub- amount feature carries out signal acquisition to single target object using SIM and quantifies;Although this method can directly measure glucosides knot
State Aroma precursor substance is closed, still, precursor substance extraction and purification process are cumbersome, need that as much as possible to remove interference miscellaneous
Matter, because often there is the similar interfering substance of molecular weight in precursor extract;And this analysis method flux is low, every time
A kind of glucosides bound state aroma precursor substance can only be detected, a sample is needed could be by contained precursor substance by repeated detection
It detected completely.Up to the present, still lack quickly the directly efficient detection of tealeaves glucosides bound state aroma precursor substance
Method.
Invention content
The technical problem to be solved in the present invention is to provide one kind can detecting 9 kinds of glucosides bound state aroma precursors of tealeaves simultaneously
The method of substance.This method have many advantages, such as amount of samples is few, extraction operation is simple, analysis rapidly and efficiently, it is applied widely.
In order to solve the technical problem, the present invention provides a kind of direct detection tealeaves glucosides bound state aroma precursor substance
Method (that is, the method for detecting 9 kinds of glucosides bound state aroma precursor substances simultaneously using UPLC-MS/MS MRM patterns), it is fresh with tea
Leaf or tea, as sample to be tested, follow the steps below successively in product or sample tea:
1), glucosides bound state aroma precursor extracts in sample to be tested:
The sample to be tested that will be equivalent to 0.05-0.1g dry weights is extracted using 20mL extractants, and extracting solution is obtained through centrifugation
Supernatant;The extractant is that the volumetric concentration of precooling (- 5 DEG C) is 90-95% methanol;
3 μ g 4- nitrobenzophenones-β-D-Glucose glycosides is added in supernatant as internal standard, and 0.1-0.2g is added through super
The preswollen insoluble polyvinylpyrrolidone PVPP of pure water is mixed well (with precipitate polyphenolics), and (time of repose is centrifuged after standing
10 ± 2min is centrifuged for 60min, 10000 ± 2000r/min), the supernatant for centrifuging gained is concentrated to dryness, and obtains medicinal extract (to contain glucosides
The medicinal extract of bound state aroma precursor substance);Medicinal extract is dissolved in 1-2mL ultra-pure waters, and crosses 0.22 μm of miillpore filter, obtains tealeaves sugar
Glycosides bound state aroma precursor extract;
2), glucosides bound state aroma precursor UPLC-MS/MS is analyzed in extract:
With UPLC HSST3 chromatographic columns (150mm × 2.1mm, 1.8 μ) for analytical column, 35 DEG C of column temperature, sample size 5ul;With first
Acid/water (0.05/99.95, v/v) is mobile phase A, carries out gradient elution by Mobile phase B of acetonitrile, and flow velocity 0.3ml/min is washed
De- time-program(me) is that 0-3min B phases keep 10%, 3.01-15min B phases to increase to 20%, 15.01-20min B phases from 10%
From 20% increase to 35%, 20.01-22min B phases from 35% increase to 90%, 22.01-24min B phases keep 90%,
24.01-26min B phases keep 10%;
MS analysis conditions are:Electron spray ESI negative electricity is from pattern, capillary voltage 3KV, two level orifice potential 3V, lens electricity
Press 0.2V, 150 DEG C of ion source temperature, 500 DEG C of desolvation temperature, desolventizing gas flow 800L/h, taper hole throughput 60L/h,
Orifice potential 25V, collision gas argon gas flow velocity 0.25mL/min, scanning mass charge ratio range are 100~700m/z, data acquisition time
For 26min;
In the corresponding channels 4-9min 1 with ion pair 346/138 detect internal standard 4- nitrobenzophenones-β-D-Glucose glycosides,
Benzyl alcohol primrose glucosides is detected with ion pair 447/269, gaultherolin primrose glucosides is detected with ion pair 491/293, in 7-
In the corresponding channels 13min 2 with ion pair 461/149 detect benzyl carbinol primrose glucosides, with ion pair 439/149 detect along 3- oneself
Enol primrose glucosides detects linalool oxide (I and II) primrose glucosides with ion pair 509/331, corresponding in 13-17min
Linalool oxide III primrose glucosides is detected with ion pair 509/331 in channel 3, in the corresponding channels 17-23min 4 with from
Son pair 493/161 detects linalool primrose glucosides, detects geraniol primrose glucosides with ion pair 493/149;
Specifically, under above-mentioned UPLC separation and MS analysis conditions, 4- nitrobenzophenones-β-D-Glucose glycosides can detect
Internal standard and tealeaves benzyl alcohol primrose glucosides, gaultherolin primrose glucosides, benzyl carbinol primrose glucosides, suitable 3- hexenol primeveroses
The glucosides such as glycosides, linalool oxide (I, II, III) primrose glucosides, linalool primrose glucosides and geraniol primrose glucosides combine
The clear signal of state Aroma precursor.Wherein, 4- nitrobenzophenones-β-D-Glucose glycosides mother ion mass-to-charge ratio is m/z 346 and 300,
M/z 346 is the Ou Helizi [ of the compound and formate;M+HCOO]-, m/z 300 is that the compound removes hydrogen ion;M-
H]-, and 346 signals of mother ion mass-to-charge ratio m/z are significantly stronger than m/z 300, the two level daughter ion of 346 parent ions of m/z is m/z
300,138;Benzyl alcohol primrose glucosides mother ion mass-to-charge ratio be m/z 447 (i.e.;M+HCOO]-) and 401 (i.e. [M-H]-), and it is female from
Sub- 447 signals of mass-to-charge ratio m/z are significantly stronger than m/z 401, and the two level daughter ion of 447 parent ions of m/z is m/z 401,269 and
161;Gaultherolin primrose glucosides mother ion mass-to-charge ratio be m/z 491 (i.e.;M+HCOO]-), 445 (i.e. [M-H]-), and it is female from
Sub- 491 signals of mass-to-charge ratio m/z are significantly stronger than m/z 445, the two level daughter ion of parent ion m/z 491 be m/z 293,233,
191,149 and 131;Benzyl carbinol primrose glucosides mother ion mass-to-charge ratio be m/z 461 (i.e.;M+HCOO]-), 415 (i.e. [M-H]-), and
461 signals of mother ion mass-to-charge ratio m/z are significantly stronger than m/z 415, the two level daughter ion of parent ion m/z 461 be m/z 415,
283,191 and 149;Along 3- hexenol primrose glucosides mother ion mass-to-charge ratios be m/z 439 (i.e.;M+HCOO]-), 393 (i.e. [M-
H]-), and 439 signals of mother ion mass-to-charge ratio m/z are significantly stronger than m/z 393, the two level daughter ion of parent ion m/z 439 is m/z
393,261,161 and 149;Three kinds of linalool oxide (I, II, III) primrose glucosides isomer mother ion mass-to-charge ratios are
509 (i.e. [ of m/z;M+HCOO]-), 463 (i.e. [M-H]-), and 509 signals of mother ion mass-to-charge ratio m/z are significantly stronger than m/z 463, it is female
The two level daughter ion of ion m/z 509 is m/z 463,331 and 161;Linalool primrose glucosides mother ion mass-to-charge ratio is m/z 493
(i.e. [M+HCOO]-), 447 (i.e. [M-H]-), and 493 signals of mother ion mass-to-charge ratio m/z are significantly stronger than m/z 447, parent ion m/z
493 two level daughter ion is m/z 447,315 and 161;The glucosides mother ion mass-to-charge ratios such as geraniol primrose glucosides are m/z 493
(i.e. [M+HCOO]-), 447 (i.e. [M-H]-), and 493 signals of mother ion mass-to-charge ratio m/z are significantly stronger than m/z 447, parent ion m/z
493 two level daughter ion is m/z 447,293 and 149.Parent ion is established with the pairing of corresponding daughter ion including internal standard
10 kinds of compounds multiple-reaction monitoring MRM ion pairs, can be realized under MRM patterns to being examined while above-mentioned glycoside matter
It surveys;Preferably, parent ion and daughter ion strong with characteristic, that signal-to-noise ratio is high are matched, and according to the UPLC colors of each substance
4 channels are established in spectrum behavior, i.e., detect internal standard 4- nitrobenzophenone-β-D- grapes in the channels 4-9min 1 with ion pair 346/138
Glucosides detects benzyl alcohol primrose glucosides with ion pair 447/269, detects gaultherolin primrose glucosides with ion pair 491/293,
Benzyl carbinol primrose glucosides is detected with ion pair 461/149 in the channels 7-13min 2, is detected along 3- hexenes with ion pair 439/149
Alcohol primrose glucosides, with ion pair 509/331 detect linalool oxide (I and II) primrose glucosides, in the channels 13-17min 3 with
Ion pair 509/331 detects linalool oxide III primrose glucosides, is detected with ion pair 493/161 in the channels 17-23min 4
Linalool primrose glucosides detects geraniol primrose glucosides with ion pair 493/149, and above-mentioned 10 kinds can be achieved at the same time under this condition
Effective detection (such as Fig. 1) of compound.
3), tealeaves glucosides bound state aroma precursor substance content calculation:
To be designated as reference in 4- nitrobenzophenones-β-D-Glucose glycosides, benzyl alcohol primrose glucosides, gaultherolin primrose are obtained
Glucosides, benzyl carbinol primrose glucosides, suitable 3- hexenol primroses glucosides, linalool oxide I primroses glucosides, linalool oxide II cherries
This 9 kinds of glucosides reference states of careless glucosides, linalool oxide primrose glucosides III, linalool primrose glucosides, geraniol primrose glucosides are fragrant
The content of material of gas precursor.
Remarks explanation:
Tea refers to just the withering leaf after product, such as booth are green in each stage during tea processing, kills in product
Water-removing leaves after young worker's sequence are rubbed and rub leaf etc. after process;
Sample tea refers to just dry tea, and moisture content is generally≤7%;
The improvement of the method for directly detection tealeaves glucosides bound state aroma precursor substance as the present invention:
The formula of the step 3) is:Internal standard concentration × internal standard volume × target compound peak area/internal standard peak area ×
Extract volume/Tea Samples dry weight.
The further improvements in methods for directly detecting tealeaves glucosides bound state aroma precursor substance as the present invention:
The preparation method through the preswollen insoluble polyvinylpyrrolidone PVPP of ultra-pure water is:According to ultra-pure water:
PVPP=2.8~3.2:1 (preferably 3:1) PVPP is immersed in 2 ± 0.2h of standing in ultra-pure water by mass ratio;
After standing, the moisture (removal surface moisture can be filtered with filter paper) on the surfaces removal PVPP obtains ultra-pure water and is pre-dissolved
Swollen PVPP.
The further improvements in methods for directly detecting tealeaves glucosides bound state aroma precursor substance as the present invention:
One, when sample to be tested be fresh tea leaves or tea in product when:
The sample to be tested for being equivalent to 0.05-0.1g dry weights is weighed, after being mixed with 10mL extractants, 10- is homogenized with refiner
20min is then centrifuged for, respectively supernatant and residue for the first time;
After being mixed with 10mL extractants with residue, ultrasound extraction 10~20min, centrifugation, draw supernatant, and with supernatant for the first time
Liquid is used as supernatant after merging;
It is described to centrifuge as 5000 ± 500r/min, 4 ± 1 DEG C of 5 ± 1min of centrifugation;
Two, when sample to be tested is sample tea (dry tea):
Sample to be tested is crushed into (60 mesh sieve can be crossed by being crushed to), the sample to be tested powder of 0.05-0.1g dry weights is taken to be added
10mL extractant ultrasounds extract 20 ± 5min, are then centrifuged for, respectively supernatant and residue for the first time;
After residue is mixed with 10mL extractants ultrasound extraction 10~20min, centrifugation, draw supernatant, and with supernatant for the first time
Liquid is used as supernatant after merging;
It is described to centrifuge as 5000 ± 500r/min, 4 ± 1 DEG C of 5 ± 1min of centrifugation.
The further improvements in methods for directly detecting tealeaves glucosides bound state aroma precursor substance as the present invention:
In the step 1), 4- nitrobenzophenones-β-D-Glucose glycosides of 3.0 a concentration of 1mg/mL of μ L is added in supernatant
Solution is as internal standard.
In the present invention:
The multiple-reaction monitoring ion pair information for detecting 9 kinds of glucosides bound state aroma precursor substances is set, is only existed
Under the conditions of most suitable ion pair, the highest signal-to-noise ratio of object could be obtained;
The time-program(me) of gradient elution is set, only under suitable time-program(me), various target substances can obtain
Good baseline separation is very beneficial for the qualitative of substance and quantifies, in this way especially for parent ion and daughter ion information phase
As it is particularly important for isomer substance;If elution time program is improper, it may occur however that overlap of peaks, it can not be accurate
It is quantitative.
The present invention's utilizes UPLC-MS/MS MRM mode detections fresh tea leaves or tea in product or dry tea glucosides reference state
The method of Aroma precursor substance, has the following advantages:
1), direct efficient detection glucosides bound state aroma precursor substance, need not be derived or be hydrolyzed to target substance;
2), high to glucosides bound state aroma precursor substance detection sensitivity, amount of samples is few, and sample dry weight can be as little as
0.05g;
3), high to glucosides bound state aroma precursor substance detection high specificity, signal-to-noise ratio, when target substance extracts in sample
The pretreatment process such as column purification were not needed, it is easy to operate, time-consuming short;
4), high to glucosides bound state aroma precursor substance detection efficiency, the 9 kinds of glucosides that can be detected simultaneously in tea sample combine
State Aroma precursor.
5), applied widely to the detection of glucosides bound state aroma precursor substance, it can be used for fresh tea leaves analysis, it can also be used to tea
It is analyzed in product or various sample teas, is equally applicable to tea extract or tea beverage sample analysis.
Description of the drawings
The specific implementation mode of the present invention is described in further detail below in conjunction with the accompanying drawings.
Fig. 1 is glucosides bound state aroma precursor substance analysis result in tea under MRM patterns;
Note:In channel 1, peak 1 is 4- nitrobenzophenones-β-D-Glucose glycosides (internal standard), peak 2 is benzyl alcohol primrose glucosides, peak 3
For gaultherolin primrose glucosides;In channel 2, peak 4 is benzyl carbinol primrose glucosides, peak 5 is along 3- hexenol primroses glucosides, peak 6
It is linalool oxide II primrose glucosides for linalool oxide I primroses glucosides, peak 7;In channel 3, peak 8 is linalool oxide
III primrose glucosides;In channel 4, peak 9 is linalool primrose glucosides, peak 10 is geraniol primrose glucosides.
Specific implementation mode
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This:
Embodiment 1, a kind of method directly detecting tealeaves glucosides bound state aroma precursor substance, carry out following step successively
Suddenly:
1), glucosides bound state aroma precursor substance extracts in fresh tea leaves:
' 2 leaf young sprout of Zhejiang agriculture 113 ' spring, 1 bud, is beaten with the card punch of a diameter of 0.5cm on the young sprout blade of harvesting for harvesting
Hole samples, and weighs the leaf disk for being equivalent to 0.1g dry weights, is placed in homogenizer, and it is mixed that 10mL precooling (- 5 DEG C) 95% methanol is added
After conjunction, it is homogenized under the rotating speed of 1200-2000r/min (slow-speed of revolution can be used in the tender sample of children, high rotating speed then can be used in thick old sample)
Homogenate is transferred in 50mL centrifuge tubes by 10min, 5000r/min, 4 DEG C of centrifugation 5min, draw whole supernatants be transferred to newly from
Heart pipe;Residue is mixed with the pre- cold methanols of 10mL (- 5 DEG C) again, and ultrasonic (40W) extracts 20min, 5000r/min, 4 DEG C from
Heart 5min draws whole supernatants.Merge 2 supernatants, and adds 3.0 μ L 4- nitrobenzophenones-β-D-Glucose glycosides wherein
(1mg/mL) is used as internal standard, and 0.1g is added and (is by mass ratio through the preswollen insoluble polyvinylpyrrolidone PVPP of ultra-pure water
3/1 ultra-pure water mixes well with PVPP, stands 2h, filter paper filtering removal surface moisture, and as ultra-pure water is preswollen
PVPP), mix well with precipitate polyphenolics, after standing 60min, 10000r/min centrifuges 10min, transfer supernatant to new centrifuge tube
In, and it is concentrated to dryness that (rotary evaporation (30-60r/min) is concentrated to dryness at room temperature or to advertise nitrogen mode using room temperature dense
It is reduced to dry), obtain the medicinal extract of the bound state aroma precursor substance of glucosides containing fresh tea leaves;Medicinal extract is dissolved in 2.0mL ultra-pure waters, and with
0.22 μm of filtering with microporous membrane, filtrate are fresh tea leaves glucosides bound state aroma precursor extract.
Since punching and sampling, the above process is repeated 2 times, obtain the extraction of fresh tea leaves glucosides bound state aroma precursor respectively
2nd and the 3rd repeating sample of object.
2), glucosides bound state aroma precursor UPLC-MS/MS is analyzed in extract:
With UPLC HSST3 chromatographic columns (150mm × 2.1mm, 1.8 μ) for analytical column, 35 DEG C of column temperature, 5 μ L of sample size;With first
Acid/water (0.05/99.95, v/v) is mobile phase A, carries out gradient elution by Mobile phase B of acetonitrile, and flow velocity 0.3ml/min is eluted
Time-program(me) be 0-3min B phases keep 10%, 3.01-15min B phases from 10% increase to 20%, 15.01-20min B phases from
20%, which increases to 35%, 20.01-22min B phases, increases to 90%, 22.01-24min B phases from 35% and keeps 90%, 24.01-
26min B phases keep 10%.
MS analysis conditions are:Electron spray ESI negative electricity is from pattern, capillary voltage 3KV, two level orifice potential 3V, lens electricity
Press 0.2V, 150 DEG C of ion source temperature, 500 DEG C of desolvation temperature, desolventizing gas flow 800L/h, taper hole throughput 60L/h,
Orifice potential 25V, collision gas argon gas flow velocity 0.25mL/min, scanning mass charge ratio range are 100~700m/z, data acquisition time
For 26min.
Under above-mentioned UPLC separation and MS analysis conditions, can detect 4- nitrobenzophenones-β-D-Glucose glycosides internal standard and
Tealeaves benzyl alcohol primrose glucosides, gaultherolin primrose glucosides, benzyl carbinol primrose glucosides, suitable 3- hexenol primroses glucosides, fragrant camphor tree
Before the glucosides bound state aromas such as alcohol oxide (I, II, III) primrose glucosides, linalool primrose glucosides and geraniol primrose glucosides
Body clear signal.
Wherein:
4- nitrobenzophenones-β-D-Glucose glycosides mother ion mass-to-charge ratio be m/z 346 and 300, m/z 346 be the compound and
The Ou Helizi [ of formate;M+HCOO]-, m/z 300 is that the compound removes hydrogen ion;M-H]-, and mother ion mass-to-charge ratio m/z
346 signals are significantly stronger than m/z 300, and the two level daughter ion of 346 parent ions of m/z is m/z 300,138;
Benzyl alcohol primrose glucosides mother ion mass-to-charge ratio be m/z 447 (i.e.;M+HCOO]-) and 401 (i.e. [M-H]-), and it is female from
Sub- 447 signals of mass-to-charge ratio m/z are significantly stronger than m/z 401, and the two level daughter ion of 447 parent ions of m/z is m/z 401,269 and
161;
Gaultherolin primrose glucosides mother ion mass-to-charge ratio be m/z 491 (i.e.;M+HCOO]-), 445 (i.e. [M-H]-), and
491 signals of mother ion mass-to-charge ratio m/z are significantly stronger than m/z 445, the two level daughter ion of parent ion m/z 491 be m/z 293,
233,191,149 and 131;
Benzyl carbinol primrose glucosides mother ion mass-to-charge ratio be m/z 461 (i.e.;M+HCOO]-), 415 (i.e. [M-H]-), and it is female from
Sub- 461 signals of mass-to-charge ratio m/z are significantly stronger than m/z 415, and the two level daughter ion of parent ion m/z 461 is m/z415,283,191
With 149;
Along 3- hexenol primrose glucosides mother ion mass-to-charge ratios be m/z 439 (i.e.;M+HCOO]-), 393 (i.e. [M-H]-), and
439 signals of mother ion mass-to-charge ratio m/z are significantly stronger than m/z 393, the two level daughter ion of parent ion m/z 439 be m/z 393,
261,161 and 149;
Three kinds of linalool oxide (I, II, III) primrose glucosides isomer mother ion mass-to-charge ratios are m/z 509
(i.e. [M+HCOO]-), 463 (i.e. [M-H]-), and 509 signals of mother ion mass-to-charge ratio m/z are significantly stronger than m/z 463, parent ion m/z
509 two level daughter ion is m/z 463,331 and 161;
Linalool primrose glucosides mother ion mass-to-charge ratio be m/z 493 (i.e.;M+HCOO]-), 447 (i.e. [M-H]-), and it is female from
Sub- 493 signals of mass-to-charge ratio m/z are significantly stronger than m/z 447, and the two level daughter ion of parent ion m/z 493 is m/z 447,315 and
161;
The glucosides mother ion mass-to-charge ratios such as geraniol primrose glucosides be m/z 493 (i.e.;M+HCOO]-), 447 (i.e. [M-H]-),
And 493 signals of mother ion mass-to-charge ratio m/z are significantly stronger than m/z 447, the two level daughter ion of parent ion m/z 493 be m/z 447,
293 and 149.
The pairing of parent ion and corresponding daughter ion is established the multiple-reaction monitoring MRM that includes internal standard in interior 10 kinds of compounds from
Son is right, is can be realized under MRM patterns to being detected while above-mentioned glycoside matter;Preferably, it is strong with characteristic, signal-to-noise ratio is high
Parent ion and daughter ion matched, and 4 channels are established according to the UPLC chromatographic behaviors of each substance, i.e., in the channels 4-9min
Internal standard 4- nitrobenzophenones-β-D-Glucose glycosides is detected with ion pair 346/138 in 1, benzyl alcohol cherry is detected with ion pair 447/269
Careless glucosides, with 491/293 gaultherolin primrose glucosides of ion pair, in the channels 7-13min 2 with ion pair 461/149 detect
Benzyl carbinol primrose glucosides is detected along 3- hexenol primroses glucosides, with ion pair 509/331 with ion pair 439/149 and detects fragrant camphor tree
Alcohol oxide (I and II) primrose glucosides detects linalool oxide III cherries in the channels 13-17min 3 with ion pair 509/331
Careless glucosides is detected linalool primrose glucosides with ion pair 493/161 in the channels 17-23min 4, is detected with ion pair 493/149
Effective detection of above-mentioned 10 kinds of glycoside matters including internal standard can be achieved at the same time in geraniol primrose glucosides under this condition.
It is respectively 1089.3,1069.6,1061.8 to repeat internal standard peak area three times;
Repeating target compound peak area three times is respectively:Benzyl alcohol primrose glucosides 323.2,310.2,322.1, salicylic acid
Methyl esters primrose glucosides 365.3,357.2,368.1, benzyl carbinol primrose glucosides 2337.6,2320.3,2268.0, along 3- hexenol cherries
Careless glucosides, 98.0,92.7,99.8, linalool oxide I primroses glucosides 327.5,319.5,326.3, linalool oxide II cherries
Careless glucosides 204.8,206.1,191.1, linalool oxide III primroses glucosides 3518.4,3466.9,3413.3, linalool cherry
Careless glucosides 3395.0,3316.5,3321.3, geraniol primrose glucosides 8084.1,7900.8,7911.1.
3) fresh tea leaves glucosides bound state aroma precursor substance content calculation:
To be designated as reference in 4- nitrobenzophenones-β-D-Glucose glycosides, using internal standard concentration 1mg/mL × internal standard dosage
0.003mL (i.e. 3.0 μ L) × target compound peak area/internal standard peak area × extract volume (the present embodiment 2.0mL)/tea
Leaf sample quality (the present embodiment is 0.1g dry weights) × 1000 (unit conversion is scaled μ g/g from mg/g) formula carries out sample sugar
Glycosides bound state aroma precursor substance content (unit is μ g/g dry weights) calculates.
It is calculated according to the formula, ' agriculture 113 ' fresh leaves of tea plant in Zhejiang repeats in 1 sample, benzyl alcohol primrose glucosides, gaultherolin
Primrose glucosides, benzyl carbinol primrose glucosides, suitable 3- hexenol primroses glucosides, linalool oxide I primroses glucosides, linalool oxide
II primroses glucosides, linalool oxide III primroses glucosides, linalool primrose glucosides and geraniol primrose Glycosides Contents are in terms of dry weight
(μ g/g dry weights) is respectively 17.80,20.12,128.76,5.40,18.04,11.28,193.80,187.00 and 445.28;Weight
In multiple 2 samples, benzyl alcohol primrose glucosides, gaultherolin primrose glucosides, benzyl carbinol primrose glucosides, suitable 3- hexenol primeveroses
Glycosides, linalool oxide I primroses glucosides, linalool oxide II primroses glucosides, linalool oxide III primroses glucosides, fragrant camphor tree
Alcohol primrose glucosides and geraniol primrose Glycosides Contents in terms of dry weight (μ g/g dry weights) be respectively 17.40,20.04,130.16,
5.20,17.92,11.56,194.48,186.04 and 443.20;It repeats in 3 samples, benzyl alcohol primrose glucosides, gaultherolin
Primrose glucosides, benzyl carbinol primrose glucosides, suitable 3- hexenol primroses glucosides, linalool oxide I primroses glucosides, linalool oxide
II primroses glucosides, linalool oxide III primroses glucosides, linalool primrose glucosides and geraniol primrose Glycosides Contents are in terms of dry weight
(μ g/g dry weights) is respectively 18.20,20.80,128.16,5.64,18.44,10.80,192.88,187.68 and 447.04.
According to repeating mean value computation three times, benzyl alcohol primrose glucosides, gaultherolin primrose in ' Zhenong 139 ' fresh leaf
Glucosides, benzyl carbinol primrose glucosides, suitable 3- hexenol primroses glucosides, linalool oxide I primroses glucosides, linalool oxide II cherries
Careless glucosides, linalool oxide III primroses glucosides, linalool primrose glucosides and geraniol primrose Glycosides Contents (the μ g/ in terms of dry weight
G dry weights) it is respectively 17.80 ± 0.40,20.32 ± 0.42,129.03 ± 1.03,5.41 ± 0.22,18.13 ± 0.27,11.21
± 0.38,193.72 ± 0.80,186.91 ± 0.82 and 445.17 ± 1.92.It can be seen that not only can to measure tea simultaneously fresh for present aspect
9 kinds of glucosides bound state aroma precursor substances of leaf, and the measurement coefficient of variation of 9 kinds of substances is respectively less than 5%, it is reproducible.
Embodiment 2, a kind of method directly detecting tealeaves glucosides bound state aroma precursor substance, carry out following step successively
Suddenly:
1), green tea glucosides bound state aroma precursor substance extracts:
Maofeng green tea sample is ground with sample grinding machine, crosses 60 mesh sieve, weigh be equivalent to the samples of 0.05g dry weights in 50mL from
In heart pipe, after 10mL precooling (- 5 DEG C) 90% methanol ultrasonic (40W) extractions 20min is added, with 5000r/min, 4 DEG C of centrifugations
5min, supernatant are fully transferred in new centrifuge tube;90% methanol ultrasonic (40W) is pre-chilled with 10mL again in residue and extracts 10min,
And centrifuge, it draws whole supernatants, merge 2 supernatants;3.0 μ L 4- nitrobenzophenones-β-D-Glucose is added in supernatant after merging
Glycosides (1mg/mL) be used as internal standard, be added 0.2g through the preswollen insoluble polyvinylpyrrolidone PVPP of ultra-pure water, mix well
With precipitate polyphenolics, after standing 60min, 10000r/min centrifuges 10min, shifts in supernatant to new centrifuge tube, and be concentrated to dryness, obtains
The medicinal extract of Maofeng green tea glucosides bound state aroma precursor substance must be contained;Medicinal extract is dissolved in 1.0mL ultra-pure waters, and micro- with 0.22 μm
Hole membrane filtration, filtrate are Maofeng green tea glucosides bound state aroma precursor extract.Since sample grind and sampling, then weigh
The multiple above process 2 times, obtains the 2nd and the 3rd repeating sample of green tea glucosides bound state aroma precursor extract respectively.
2), glucosides bound state aroma precursor UPLC-MS/MS is analyzed in extract:
Hair is analyzed using identical UPLC-MS/MS conditions in embodiment 1 and 4 10 kinds of channel compound MRM ion pairs
Internal standard in peak green-tea extract and glucosides bound state aroma precursor are detected that is, in the channels 4-9min 1 with ion pair 346/138
Internal standard 4- nitrobenzophenones-β-D-Glucose glycosides, benzyl alcohol primrose glucosides is detected with ion pair 447/269, with ion pair 491/293
Gaultherolin primrose glucosides detects benzyl carbinol primrose glucosides, with ion pair in the channels 7-13min 2 with ion pair 461/149
439/149 detection detects linalool oxide (I and II) primrose glucosides along 3- hexenol primroses glucosides, with ion pair 509/331,
Linalool oxide III primrose glucosides is detected with ion pair 509/331 in the channels 13-17min 3, in the channels 17-23min 4
Linalool primrose glucosides is detected with ion pair 493/161, geraniol primrose glucosides is detected with ion pair 493/149.In the condition
Under effective detections of 10 kinds of glycoside matters including above-mentioned internal standard can be achieved at the same time.
3) green tea glucosides bound state aroma precursor substance content calculation:
To be designated as reference in 4- nitrobenzophenones-β-D-Glucose glycosides, using internal standard concentration 1mg/mL × internal standard dosage
0.003mL (i.e. 3.0 μ L) × target compound peak area/internal standard peak area × extract volume (the present embodiment 1.0mL)/tea
Leaf sample quality (being 0.05g dry weights in the present embodiment) × 1000 (unit conversion is scaled μ g/g from mg/g) formula carries out sample
Product glucosides bound state aroma precursor substance content (μ g/g dry weights) calculates.
According to the formula, Maofeng green tea repeats in 1 sample, benzyl alcohol primrose glucosides, gaultherolin primrose glucosides, benzene second
Alcohol primrose glucosides, suitable 3- hexenol primroses glucosides, linalool oxide I primroses glucosides, linalool oxide II primroses glucosides, virtue
Camphor tree alcohol oxide III primroses glucosides, linalool primrose glucosides and geraniol primrose Glycosides Contents in terms of dry weight (μ g/g dry weights) point
It Wei 28.12,12.57,51.30,4.19,8.48,21.49,245.80,114.12,551.48;It repeats in 2 samples, benzyl alcohol
Primrose glucosides, gaultherolin primrose glucosides, benzyl carbinol primrose glucosides, suitable 3- hexenol primroses glucosides, linalool oxide I cherries
Careless glucosides, linalool oxide II primroses glucosides, linalool oxide III primroses glucosides, linalool primrose glucosides and geraniol
Primrose Glycosides Contents (μ g/g dry weights) in terms of dry weight are respectively 27.48,12.88,51.14,4.55,8.21,21.08,245.12,
115.74,552.17;It repeats in 3 samples, it is benzyl alcohol primrose glucosides, gaultherolin primrose glucosides, benzyl carbinol primrose glucosides, suitable
3- hexenol primroses glucosides, linalool oxide I primroses glucosides, linalool oxide II primroses glucosides, linalool oxide III
Primrose glucosides, linalool primrose glucosides and geraniol primrose Glycosides Contents in terms of dry weight (mg/g dry weights) are respectively 28.57,
12.41,51.88,4.47,8.67,21.94,246.81,114.82,549.04.According to repeating mean value computation, hair three times
Benzyl alcohol primrose glucosides in peak green tea, gaultherolin primrose glucosides, benzyl carbinol primrose glucosides, along 3- hexenol primroses glucosides,
Linalool oxide I primroses glucosides, linalool oxide II primroses glucosides, linalool oxide III primroses glucosides, linalool cherry
Careless glucosides and geraniol primrose Glycosides Contents in terms of dry weight (μ g/g dry weights) be respectively 28.06 ± 0.55,12.62 ± 0.24,
51.44±0.39,4.40±0.19,8.45±0.23,21.50±0.43,245.91±0.85,114.89±0.81,
550.90±1.64.It can be seen that present aspect can not only measure 9 kinds of glucosides bound state aroma precursor substances of green tea, but also 9 simultaneously
The measurement coefficient of variation of kind of substance is respectively less than 5%, reproducible.
Embodiment 3, a kind of method directly detecting tealeaves glucosides bound state aroma precursor substance, carry out following step successively
Suddenly:
1), oolong tea is extracted in product glucosides bound state aroma precursor substance:
Leaf rotation leaf in oolong tea process is sampled, the leaf rotation leaf sample for being equivalent to 0.1g dry weights is weighed, sets
In refiner, after 10mL precooling (- 5 DEG C) 95% methanol homogenate 20min is added, with 5000r/min, 4 DEG C of centrifugation 5min, supernatant
It is fully transferred in centrifuge tube;95% methanol is pre-chilled with 10mL again in residue to mix, and ultrasonic (40W) extracts 20min, from
The heart draws whole supernatants, merges 2 supernatants;3.0 μ L 4- nitrobenzophenones-β-D-Glucose glycosides is added in supernatant after merging
(1mg/mL) be used as internal standard, be added 0.1g through the preswollen insoluble polyvinylpyrrolidone PVPP of ultra-pure water, mix well with
Precipitate polyphenolics, after standing 60min, 10000r/min centrifuges 10min, shifts in supernatant to new centrifuge tube, and be concentrated to dryness, and obtains
Containing oolong tea product leaf rotation leaf glucosides bound state aroma precursor substance medicinal extract;Medicinal extract is dissolved in 2.0mL ultra-pure waters, and with
0.22 μm of filtering with microporous membrane, filtrate are oolong tea in product leaf rotation leaf glucosides bound state aroma precursor extract.From sampling
Start with homogenate, repeat the above process 2 times, obtains oolong tea respectively and extracted in product leaf rotation leaf glucosides bound state aroma precursor
2nd and the 3rd repeating sample of object.
2), glucosides bound state aroma precursor UPLC-MS/MS is analyzed in extract:
Using identical UPLC-MS/MS conditions in embodiment 1 and 4 10 kinds of channel compound MRM ion pairs analysis crows
Internal standard and glucosides bound state aroma precursor of the imperial tea in product leaf rotation leaf extract, i.e., with ion pair in the channels 4-9min 1
346/138 detection internal standard 4- nitrobenzophenones-β-D-Glucose glycosides, detected with ion pair 447/269 benzyl alcohol primrose glucosides, with from
491/293 gaultherolin primrose glucosides of son pair detects benzyl carbinol primeverose in the channels 7-13min 2 with ion pair 461/149
Glycosides, detected along 3- hexenol primroses glucosides with ion pair 439/149, detected with ion pair 509/331 linalool oxide (I and
II) primrose glucosides detects linalool oxide III primrose glucosides, in 17- in the channels 13-17min 3 with ion pair 509/331
Linalool primrose glucosides is detected with ion pair 493/161 in the channels 23min 4, geraniol primeverose is detected with ion pair 493/149
Glycosides.Effective detection of 10 kinds of glycoside matters including above-mentioned internal standard can be achieved at the same time under this condition.
3), oolong tea is in product leaf rotation leaf glucosides bound state aroma precursor substance content calculation:
To be designated as reference in 4- nitrobenzophenones-β-D-Glucose glycosides, using internal standard concentration 1mg/mL × internal standard dosage
0.003mL (i.e. 3.0 μ L) × target compound peak area/internal standard peak area × extract volume (the present embodiment 2.0mL)/tea
Leaf sample quality (being 0.1g dry weights in the present embodiment) × 1000 (unit conversion is converted into μ g/g from mg/g) formula carries out sample
Glucosides bound state aroma precursor substance content (μ g/g dry weights) calculates.According to the formula, oolong tea repeats 1 sample in product leaf rotation leaf
In product, benzyl alcohol primrose glucosides, gaultherolin primrose glucosides, benzyl carbinol primrose glucosides, suitable 3- hexenol primroses glucosides, fragrant camphor tree
Alcohol oxide I primroses glucosides, linalool oxide II primroses glucosides, linalool oxide III primroses glucosides, linalool primeverose
Glycosides and geraniol primrose Glycosides Contents in terms of dry weight (μ g/g dry weights) be respectively 7.47,3.94,11.78,1.46,4.11,2.68,
44.43,61.31,113.39,250.12;It repeats in 2 samples, benzyl alcohol primrose glucosides, gaultherolin primrose glucosides, benzene second
Alcohol primrose glucosides, suitable 3- hexenol primroses glucosides, linalool oxide I primroses glucosides, linalool oxide II primroses glucosides, virtue
Camphor tree alcohol oxide III primroses glucosides, linalool primrose glucosides and geraniol primrose Glycosides Contents in terms of dry weight (μ g/g dry weights) point
It Wei 7.23,3.67,11.14,1.38,3.95,2.65,44.80,61.86,112.94 and 249.85;It repeats in 3 samples, benzene
Methanol primrose glucosides, gaultherolin primrose glucosides, benzyl carbinol primrose glucosides, suitable 3- hexenol primroses glucosides, linalool oxidation
Object I primroses glucosides, linalool oxide II primroses glucosides, linalool oxide III primroses glucosides, linalool primrose glucosides and perfume (or spice)
Leaf-alcohol primrose Glycosides Contents (μ g/g dry weights) in terms of dry weight are respectively 7.64,3.79,11.35,1.49,3.88,2.85,44.02,
61.53,113.75 and 249.10.According to repeating mean value computation, oolong tea benzyl alcohol primrose in product leaf rotation leaf three times
Glucosides, gaultherolin primrose glucosides, benzyl carbinol primrose glucosides, suitable 3- hexenol primroses glucosides, linalool oxide I primeveroses
Glycosides, linalool oxide II primroses glucosides, linalool oxide III primroses glucosides, linalool primrose glucosides and geraniol primrose
Glycosides Contents (mg/g dry weights) in terms of dry weight are respectively 7.45 ± 0.21,3.80 ± 0.14,11.42 ± 0.33,1.44 ± 0.06,
3.98 ± 0.12,2.73 ± 0.11,44.42 ± 0.39,61.57 ± 0.28,113.36 ± 0.41 and 249.69 ± 0.53.It can be seen that
This method can not only measure oolong tea in 9 kinds of glucosides bound state aroma precursor substances of product leaf rotation leaf, but also 9 kinds of objects simultaneously
The measurement coefficient of variation of matter is respectively less than 5%, reproducible.
Confirmatory experiment:
Enzymolysis that the industry generally uses at present, that detection result is good and knot are used to the sample of 1~embodiment of embodiment 3
It closes GC-MS analytic approach to be verified, specially extracts the glucosides bound state aroma precursor of gained in 1~embodiment of embodiment 3
Object is splined in the micro-extraction pillar (bed volume 2mL) equipped with Amberlite XAD-2 adsorbents, first uses 5mL water washing columns
Bed, then with 5ml pentanes/dichloromethane (volume ratio 2/1) column scrubber bed, before finally eluting glucosides bound state aroma with 20mL methanol
Body dries up the meoh eluate of the precursor of bound state aroma containing glucosides of collection with nitrogen at room temperature, and again by dried object
It is dissolved in the citrate buffer solution (50mM, pH6.0) of 100 μ L glycosidases containing primrose and glucuroide, 37 DEG C of reaction 14h;
30mg sodium chloride and 100 μ L dichloromethane mixings are added, 12000r/min centrifuges 10min, transfer dichloromethane layer to new centrifugation
Pipe, and 100mg anhydrous sodium sulfates dehydration 2h is added, the dichloromethane containing aroma substance is transferred to new pipe, it is analyzed with GC-MS
In fragrance component;By the 4- nitrophenols of acquisition, benzyl alcohol, gaultherolin, benzyl carbinol, suitable 3- hexenols, linalool oxygen
Compound I, linalool oxide II, linalool oxide III, linalool and geraniol peak area are converted into internal standard 4- nitrobenzenes
Base glucoside and benzyl alcohol primrose glucosides, gaultherolin primrose glucosides, benzyl carbinol primrose glucosides, suitable 3- hexenol primroses
Glucosides, linalool oxide I primroses glucosides, linalool oxide II primroses glucosides, linalool oxide III primroses glucosides, virtue
Camphor tree alcohol primrose glucosides and geraniol primrose glucosides peak area, and within be designated as adopting by reference method meter in similar embodiment 1~3
Calculate various object contents (in terms of μ g/g dry weights).Wherein, gained benzyl alcohol primrose glucosides, gaultherolin cherry in embodiment 1
Careless glucosides, benzyl carbinol primrose glucosides, suitable 3- hexenol primroses glucosides, linalool oxide I primroses glucosides, linalool oxide II
Primrose glucosides, linalool oxide III primroses glucosides, linalool primrose glucosides and geraniol primrose glucosides (in terms of μ g/g dry weights)
Respectively:17.65±0.92,19.85±0.79,127.38±1.77,5.04±0.41,17.89±0.68,11.05±
0.51,191.05 ± 1.78,185.21 ± 2.56 and 443.85 ± 3.15;Benzyl alcohol primrose glucosides, salicylic acid first in embodiment 2
Ester primrose glucosides, benzyl carbinol primrose glucosides are aoxidized along 3- hexenol primroses glucosides, linalool oxide I primroses glucosides, linalool
Object II primroses glucosides, linalool oxide III primroses glucosides, linalool primrose glucosides and geraniol primrose glucosides are (dry with μ g/g
Restatement) be respectively:27.41±0.92,12.13±0.62,50.09±0.88,4.15±0.53,8.07±0.51,20.94±
0.68,243.33±2.26,112.42±1.64,547.36±2.80;Benzyl alcohol primrose glucosides, salicylic acid first in embodiment 3
Ester primrose glucosides, benzyl carbinol primrose glucosides are aoxidized along 3- hexenol primroses glucosides, linalool oxide I primroses glucosides, linalool
Object II primroses glucosides, linalool oxide III primroses glucosides, linalool primrose glucosides and geraniol primrose glucosides are (dry with μ g/g
Restatement) be respectively:7.02±0.75,3.32±0.43,10.92±0.67,1.30±0.25,3.58±0.49,2.55±
0.45,43.53 ± 0.72,60.83 ± 0.96,112.28 ± 1.79 and 247.57 ± 2.05.
Compare it is found that the surveyed Examples 1 to 3 result of the present invention generally uses at present with the existing industry, detection result
It is very close that the best way obtains result.
Comparative example 1,
The daughter ion of 1 step 2) target substance multiple-reaction monitoring ion pair of embodiment is modified, that is, be changed to:Channel 1
In internal standard 4- nitrobenzophenones-β-D-Glucose glycosides detected with ion pair 346/138, benzyl alcohol cherry detected with ion pair 447/161
Careless glucosides, with 491/191 gaultherolin primrose glucosides of ion pair, in the channels 7-13min 2 with ion pair 461/283 detect
Benzyl carbinol primrose glucosides is detected along 3- hexenol primroses glucosides, with ion pair 509/161 with ion pair 439/261 and detects fragrant camphor tree
Alcohol oxide (I and II) primrose glucosides detects linalool oxide III cherries in the channels 13-17min 3 with ion pair 509/161
Careless glucosides is detected linalool primrose glucosides with ion pair 493/315 in the channels 17-23min 4, is detected with ion pair 493/293
Geraniol primrose glucosides;Remaining condition is equal to embodiment 1.Benzyl alcohol primrose glucosides, gaultherolin primrose glucosides, benzyl carbinol
Primrose glucosides, suitable 3- hexenol primroses glucosides, linalool oxide I primroses glucosides, linalool oxide II primroses glucosides, fragrant camphor tree
Alcohol oxide III primroses glucosides, linalool primrose glucosides and geraniol primrose Glycosides Contents (with μ g/g dry weights) measurement result point
It is not:17.41±1.09,19.89±1,41,128.72±5.94,5.19±0.66,16.93±1.13,11.04±0.83,
190.07 ± 5.58,184.86 ± 6.60 and 442.88 ± 8.69.Comparison is it is recognized that while various glycosidic precursors substances in the result
Content sequence and embodiment 1 are consistent, but content is generally lower than embodiment 1, and the coefficient of variation measured increases, be 1.88~
The lower substance coefficient of variation of 12.72%, especially content increases apparent.
Comparative example 2,
Parent ion in the multiple-reaction monitoring ion pair of 2 step 2) target substance of embodiment is modified, i.e.,:Channel 1
In internal standard 4- nitrobenzophenones-β-D-Glucose glycosides detected with ion pair 346/138, benzyl alcohol cherry detected with ion pair 401/269
Careless glucosides, with 445/293 gaultherolin primrose glucosides of ion pair, in channel 2 with ion pair 415/149 detect benzyl carbinol cherry
Careless glucosides is detected along 3- hexenol primroses glucosides, with ion pair 463/331 with ion pair 393/149 and detects linalool oxide
(I and II) primrose glucosides detects linalool oxide III primrose glucosides, in channel 4 in channel 3 with ion pair 463/331
Linalool primrose glucosides is detected with ion pair 447/161, geraniol primrose glucosides is detected with ion pair 447/149;Remaining condition
It is equal to embodiment 2.Benzyl alcohol primrose glucosides, gaultherolin primrose glucosides, benzyl carbinol primrose glucosides, suitable 3- hexenol primroses
Glucosides, linalool oxide I primroses glucosides, linalool oxide II primroses glucosides, linalool oxide III primroses glucosides, virtue
Camphor tree alcohol primrose glucosides and geraniol primrose Glycosides Contents (with μ g/g dry weights) measurement result are respectively:26.48±1.49,11.06
±0.90,49.27±1.53,4.01±0.53,7.89±0.91,20.14±1.04,241.33±4.73,111.38±
3.77,545.69±9.70..Comparison is it is recognized that while various glycosidic precursors content of material sequence and embodiment 2 one in the result
It causes, but content is generally lower than embodiment 2, and each substance coefficient of variation measured increases, and is 1.78~13.22%, especially contains
The lower substance coefficient of variation is measured to increase obviously.
Gradient elution time-program(me) in 1 step 2) of embodiment is by comparative example 3:0-3min B phases keep 10%, 3.01-
22min B phases increase to 90%, 22.01-24min B phases from 10% and 90%, 24.01-26min B phases are kept to keep 10%;Its
Remaining condition is equal to embodiment 1.Under this condition, benzyl alcohol primrose glucosides, gaultherolin primrose glucosides, the benzyl carbinol of measurement
Primrose glucosides connects with embodiment 1 substantially along 3- hexenol primroses glucosides, linalool primrose glucosides and geraniol primrose Glycosides Contents
Closely, but linalool oxide I primroses glucosides, linalool oxide II primroses glucosides and linalool oxide III primrose glucosides
Baseline separation is can not achieve, can not accurately be measured.
Finally, it should also be noted that it is listed above be only the present invention several specific embodiments.Obviously, this hair
Bright to be not limited to above example, acceptable there are many deformations.Those skilled in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (5)
1. the method for directly detecting tealeaves glucosides bound state aroma precursor substance, it is characterized in that:With fresh tea leaves or tea product,
Or sample tea follows the steps below successively as sample to be tested:
1), glucosides bound state aroma precursor extracts in sample to be tested:
The sample to be tested of 0.05-0.1g dry weights is extracted using 20mL extractants, extracting solution obtains supernatant through centrifugation;Institute
It is 90-95% methanol to state the volumetric concentration that extractant is precooling;
3 μ g 4- nitrobenzophenones-β-D-Glucose glycosides is added in supernatant, and 0.1-0.2g is added through ultra-pure water as internal standard
Preswollen insoluble polyvinylpyrrolidone PVPP is mixed well, and is centrifuged after standing, and the supernatant for centrifuging gained is concentrated to dryness, and is obtained
Medicinal extract;Medicinal extract is dissolved in 1-2mL ultra-pure waters, and crosses 0.22 μm of miillpore filter, obtains the extraction of tealeaves glucosides bound state aroma precursor
Object;
2), glucosides bound state aroma precursor UPLC-MS/MS is analyzed in extract:
Using UPLC HSST3 chromatographic columns as analytical column, 35 DEG C of column temperature, sample size 5ul;It is with formic acid/water (0.05/99.95, v/v)
Mobile phase A carries out gradient elution, flow velocity 0.3ml/min by Mobile phase B of acetonitrile, and elution time program is 0-3min B phases
Keep 10%, 3.01-15min B phases from 10% increase to 20%, 15.01-20min B phases increase to 35% from 20%,
20.01-22min B phases increase to 90%, 22.01-24min B phases from 35% and 90%, 24.01-26min B phases are kept to keep
10%;
MS analysis conditions are:Electron spray ESI negative electricity is from pattern, capillary voltage 3KV, two level orifice potential 3V, lens voltage
150 DEG C of 0.2V, ion source temperature, 500 DEG C of desolvation temperature, desolventizing gas flow 800L/h, taper hole throughput 60L/h, cone
Hole voltage 25V, collision gas argon gas flow velocity 0.25mL/min, scanning mass charge ratio range are 100~700m/z, data acquisition time is
26min;
In the corresponding channels 4-9min 1 with ion pair 346/138 detect internal standard 4- nitrobenzophenones-β-D-Glucose glycosides, with from
Son pair 447/269 detects benzyl alcohol primrose glucosides, detects gaultherolin primrose glucosides with ion pair 491/293, in 7-13min
Benzyl carbinol primrose glucosides is detected with ion pair 461/149 in corresponding channel 2, is detected along 3- hexenols with ion pair 439/149
Primrose glucosides detects linalool oxide (I and II) primrose glucosides with ion pair 509/331, in the corresponding channels 13-17min 3
In linalool oxide III primrose glucosides detected with ion pair 509/331, with ion pair in the corresponding channels 17-23min 4
493/161 detection linalool primrose glucosides detects geraniol primrose glucosides with ion pair 493/149;
3), tealeaves glucosides bound state aroma precursor substance content calculation:
To be designated as reference in 4- nitrobenzophenones-β-D-Glucose glycosides, benzyl alcohol primrose glucosides, gaultherolin primeverose are obtained
Glycosides, benzyl carbinol primrose glucosides, suitable 3- hexenol primroses glucosides, linalool oxide I primroses glucosides, linalool oxide II primroses
Glucosides, linalool oxide primrose glucosides III, linalool primrose glucosides, geraniol primrose glucosides this 9 kinds of glucosides bound state aromas
The content of material of precursor.
2. the method according to claim 1 for directly detecting tealeaves glucosides bound state aroma precursor substance, it is characterized in that:
The formula of the step 3) is:Internal standard concentration × internal standard volume × target compound peak area/internal standard peak area × extraction
Object product/Tea Samples dry weight.
3. the method according to claim 1 or 2 for directly detecting tealeaves glucosides bound state aroma precursor substance, feature
It is that the preparation method through the preswollen insoluble polyvinylpyrrolidone PVPP of ultra-pure water is:According to ultra-pure water:PVPP=
2.8~3.2:PVPP is immersed in 2 ± 0.2h of standing in ultra-pure water by 1 mass ratio;
After standing, the moisture on the surfaces removal PVPP obtains the preswollen PVPP of ultra-pure water.
4. the method according to claim 1 or 2 for directly detecting tealeaves glucosides bound state aroma precursor substance, feature
It is:
One, when sample to be tested be fresh tea leaves or tea in product when:
The sample to be tested for weighing 0.05-0.1g dry weights after being mixed with 10mL extractants, is homogenized 10-20min, then with refiner
Centrifugation, respectively supernatant and residue for the first time;
After being mixed with 10mL extractants with residue, ultrasound 10~20min of extraction, centrifugation is drawn supernatant, and is closed with supernatant for the first time
And it is used as supernatant afterwards;
It is described to centrifuge as 5000 ± 500r/min, 4 ± 1 DEG C of 5 ± 1min of centrifugation;
Two, when sample to be tested is sample tea:
Sample to be tested is crushed, take 0.05-0.1g dry weights sample to be tested powder be added 10mL extractant ultrasounds extraction 20 ±
5min is then centrifuged for, respectively supernatant and residue for the first time;
10~20min of ultrasound extraction, centrifugation after residue is mixed with 10mL extractants are drawn supernatant, and are closed with supernatant for the first time
And it is used as supernatant afterwards;
It is described to centrifuge as 5000 ± 500r/min, 4 ± 1 DEG C of 5 ± 1min of centrifugation.
5. the method according to claim 1 or 2 for directly detecting tealeaves glucosides bound state aroma precursor substance, feature
It is:
In the step 1), 4- nitrobenzophenones-β-D-Glucose glycosides solution of 3.0 a concentration of 1mg/mL of μ L is added in supernatant
As internal standard.
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