CN110286188A - A method of detection 2,5-dimethyl pyrazine - Google Patents
A method of detection 2,5-dimethyl pyrazine Download PDFInfo
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- CN110286188A CN110286188A CN201910492577.1A CN201910492577A CN110286188A CN 110286188 A CN110286188 A CN 110286188A CN 201910492577 A CN201910492577 A CN 201910492577A CN 110286188 A CN110286188 A CN 110286188A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
Abstract
The invention discloses a kind of methods for detecting 2,5- dimethyl pyrazine, belong to detection technique field.The present invention utilizes UPLC quantitative detection 2,5-DMP, realizes the efficient biological detection of 2,5-DMP.The present invention realizes the mg grades of detection ranges for being clipped to g rank, and sample treatment is simple, and detection time is short, only needs 10min, can efficiently realize the detection of 2,5-DMP in fermented sample.
Description
Technical field
The present invention relates to a kind of methods for detecting 2,5- dimethyl pyrazine, belong to detection technique field.
Background technique
Alkyl pyrazine is the nitrogen-containing heterocycle compound that a kind of branch has alkyl group, main as important flavor substance
Contribute the nut taste in food, barbecue taste and pain grill.Alkyl pyrazine has extremely low threshold value, tetramethyl in the soybean of fermentation
The threshold value of base pyrazine (2,3,5,6-Tetramethylpyrazine, TTMP) is 10ppm, 2,5- dimethyl pyrazines (2,5-
Dimethylpyrazine, 2,5-DMP) 1-2ppm is only added in food, so that it may play the role of apparent flavouring.Due to
Threshold value is low, and alkyl pyrazine can show strong odor characteristics, be China GB2760-86 provide to allow using essence object
Matter is mainly used as the food additives and some fragrance intermediates of seasoning in the food industry.
Alkyl pyrazine also has important value in terms of medicine, can be used as medicine other than with unique flavor value
Object or medicine intermediate.TTMP is found to can be used as drug therapy some diseases, such as apoplexy, myocardial cell injury, kneecap
Arthritis etc..In addition, TTMP is also used as the precursor origin of other biological drug, for example, 4- (2,3,5,6-TTMP-1)-
4'- Demethylepipodophyllotoxin is a kind of new compound with compared with powerful antitumor activity, and TTMP-2'O- sodium ferulate can provide mind
It is protected, prevents neuroinflamation and cerebral injury.2,5-DMP can be used as the important of antimicrobial DP finish 5-Methylpyrazine-2-carboxylic acid
Synthesis material.
The method of 2,5-DMP of detection mainly uses GC-MS at present, i.e., referring to granted patent: pyrrole in a kind of measurement white wine
The method (application number: CN200710019764.5) of piperazine class compound.However, 1. 1 GC-MS with autosampler are sold
Valence is million or more, it is difficult to be applied to common testing agency；The detection amplitude of 2.GC-MS is narrow, can only generally detect mM rank
Pyrazine compound.
Summary of the invention
In order to solve the problems, such as it is above-mentioned at least one, the present invention provides a kind of detection 2,5- dimethyl pyrazines (2,5-DMP)
Method, by utilize UPLC quantitative detection 2,5-DMP, realize the mg grades of detection ranges for being clipped to g rank, sample treatment
Simply, detection time is short, only needs 10min.
Detection 2 of the invention, the method for 5- dimethyl pyrazine are detected using UPLC；The UPLC is detected
Condition is:
Chromatographic column is WatersBEHC18；
Mobile phase A is 0.1% formic acid solution, Mobile phase B is hplc grade methanol；
Mobile phase gradient: initial, 31%B；0-3min, 31%-69%B；3-10min, 31%B；
Runing time is 10min；
Flow velocity is 0.20mlmin-1；
Ultraviolet detection wavelength is 275nm.
In one embodiment, the 1 μ L of applied sample amount that UPLC is detected.
In one embodiment, described to be detected as quantitative detection or qualitative detection.
In one embodiment, the quantitative detection is first to prepare 2,5-DMP standard items, is carried out using the UPLC
Detection, is fitted to obtain 2,5-DMP standard curve according to the variation of 2,5-DMP concentration and peak area；Then sample to be tested is used
The UPLC is detected, and the peak area of sample to be tested is substituted into 2,5-DMP standard curve to get into sample to be tested
2,5-DMP concentration.
In one embodiment, the sample to be tested is 2,5-DMP aqueous solution, or contains 2, in the fermentation of 5-DMP
Clear liquid.
In one embodiment, the fermented supernatant fluid, be will produce 2,5-DMP microorganism carry out fermented and cultured after,
The supernatant that bacterium solution centrifugation is obtained.
In one embodiment, the fermented supernatant fluid is to be seeded to B.subtilis168 seed liquor to revive containing L-
It is obtained after fermented and cultured in the culture medium of propylhomoserin.
In one embodiment, the culture medium is the LB culture medium (formula of LB: 5g/L yeast powder, 10g/L albumen
Peptone, 10g/L sodium chloride).
In one embodiment, the culture is in 37 DEG C, 200rpm culture 2d.
Advantages of the present invention and effect:
The present invention utilizes UPLC quantitative detection 2,5-DMP, realizes the efficient biological detection of 2,5-DMP.Before this, alkane
The detection method of base pyrazine is generally GC-MS, and the method is generally used in sample the trace detection of alkyl pyrazine and qualitative point
Analysis, and detection time is long, and sample handling procedure very complicated, cost is higher, can only generally detect the pyrazine chemical combination of mM rank
Object, sample higher for a large amount of detectable concentrations are not a selection well.The present invention utilizes UPLC quantitative detection 2,
5-DMP has the advantages that (1) realizes the mg grades of detection ranges for being clipped to g rank, at least can be in 27mg/L-1725 mg/L
In the range of realize detection,；(2) sample treatment is simple, it is only necessary to by fermented sample centrifuging and taking supernatant filtering after directly into
Sample；(3) detection time is short, only needs 10min, can efficiently realize the detection of 2,5-DMP in fermented sample.
Detailed description of the invention
Fig. 1 is the chromatogram of 2,5-DMP standard items detection.
Fig. 2 is the 2,5-DMP standard curve according to the variation fitting of 2,5-DMP solution concentration and peak area.
Fig. 3 is the qualitative analysis of 2,5-DMP in B.subtilis168 fermentation liquid；Wherein, a, 2,5-DMP standard items；B, B.
Subtilis168 produces 2,5-DMP using L-threonine；C, B.subtilis168 utilize [U-13C, 15N]-L-threonine to produce 2,
5-DMP。
Fig. 4 utilizes the chromatogram of 2,5-DMP in UPLC quantitative detection fermentation liquid.
Specific embodiment
The model WatersUPLCI-classsystem of ultra performance liquid chromatography (UPLC), can be public purchased from Waters
Department.
The model WatersBEHC18column (100mm × 2.1mm, 1.7 μm of particle) and a UV- of chromatographic column
Detector can be purchased from Waters company.
Here is that the present invention is specifically described.
Embodiment 1: UPLC quantitative detection 2,5-DMP is utilized
Quantitative detection is carried out to 2,5-DMP using ultra performance liquid chromatography (UPLC), actual conditions are as follows:
Chromatographic column WatersBEHC18 (100mm × 2.1mm, 1.7 μm of particle) carries out liquid to the 2,5-DMP in sample
Mutually separate.Mobile phase A is 0.1% formic acid solution (percentage unless otherwise specified, being related to is volume fraction), Mobile phase B
For hplc grade methanol.Mobile phase gradient: initial, 31%B；0-3min, 31%-69%B；3-10min, 31%B；When operation
Between be 10min.Flow velocity is 0.20mlmin-1, ultraviolet detection wavelength is 275nm.1 μ L of applied sample amount.
As shown in Figure 1, for according to UPLC 2, the 5-DMP standard items under 27mg/L concentration are carried out with the chromatography of quantitative detection
Figure.2,5-DMP appearance as the result is shown, appearance time are 2.969min, and peak shape is good, imply that 2,5-DMP can be by party's standard measure
Detection.
Further, 2, the 5-DMP standard solution (being dissolved with distilled water) for preparing various concentration gradient, according to concentration and peak
The variation of area fits 2,5-DMP standard curve using Origin software, and then according to appearance time corresponding in sample to be tested
The calculated by peak area of (corresponding appearance time, specifically the 2.969th minute) goes out the content of 2,5-DMP.The results show that such as Fig. 2 institute
Show, using the method standard curve obtained be y=0.00004312*x-1.19126 (wherein, y represents 2,5-DMP concentration,
Unit is mg/L, and x represents peak area, and unit is μ v*s, R2For 1), the range of linearity of standard items detection: 27mg/L-1725 mg/
L。
Embodiment 2: the preparation of the fermentation liquid containing 2,5-DMP
Respectively in addition 1gL-1L-threonine or [U-13C,15N]-L-threonine to LB culture medium (formula of LB:
5g/L yeast powder, 10g/L peptone, 10g/L sodium chloride) in, 37 DEG C, 200rpm culture B.subtilis168 to 2d, centrifuging and taking
Fermented supernatant fluid.Qualitative analysis, total ion figure (TIC) of acquisition are carried out to 2,5-DMP in fermented supernatant fluid using GC-MS
It is as shown in Figure 3 with mass spectrogram (MS).
The actual conditions of GC-MS are:
Using Agilent6890 gas-chromatography-Agilent5975 mass spectrometry (GC/MSD) to 2 in fermentation broth sample,
5-DMP carries out qualitative detection, is extracted using HS-SPME to sample.Chromatographic condition: using DB-FFAD chromatographic column (60m ×
0.25 mmi.d., 0.25 μm of filmthickness) 2,5-DMP in sample is separated, column carrier gas is helium, and flow velocity is
2 mL·min-1, do not shunt, 250 DEG C of injector temperature.Temperature program: 50 DEG C of holding 2min, with 6 DEG C of min-1Rise to 230
DEG C, keep 2min.Mass Spectrometry Conditions: use full scan mode, ionization energy 70eV, 230 DEG C of ion source temperature.The method for qualitative analysis
Detection for tagging laboratory sample.2) right using ultra high efficiency liquid phase-level four bars mass spectrometry (UPLC-TQ-S-MS)
2,5-DMP in pure enzymatic system is detected.Chromatographic column is WatersBEHC18 (100mm × 2.1mm, 1.7 μ
Mparticle), mobile phase A is 0.1% formic acid solution, and Mobile phase B is chromatographic grade acetonitrile.Mobile phase gradient: it is initial,
10%B；0-8.5min, 10%-50%B；8.6-10min, 100%B；10.1-12min 10%B；Runing time 12min.Stream
Speed is 0.15mlmin-1.Mass spectrometry method: positive ion mode, second order ms detection are used.MStune parameter: degassing rate
500L·h-1, 350 DEG C of degassing temperature, taper hole air-flow 50Lh-1, 150 DEG C of source temperature, capillary voltage 3.5KV, orifice potential
25V, collision voltage 25V, data collection time 0.3s, mass charge ratio range 40-400.It is obtained using Daughters detection pattern
The mass spectrum of 2,5-DMP standard items is chosen ion pair 109 → 56, is detected using MRM detection pattern to 2,5-DMP.
The results show that in addition L-threonine and [U-13C,15N]-L-threonine fermented sample in, in retention time
It is consistent (Fig. 3 a) with the retention time of standard items 2,5-DMP to have chromatographic peak appearance at 13.5 min (Fig. 3 b, Fig. 3 c).L- Soviet Union
The compound mass spectrum (Fig. 3 b) and 2,5-DMP standard items mass spectrum (Fig. 3 a) detected in propylhomoserin addition sample fits like a glove, and shows
The compound of the appearance time is 2,5-DMP in fermented sample.
More than, illustrate to be cultivated in the LB culture medium for being added to L-threonine using B.subtilis168, can obtain
To the fermentation liquid containing 2,5-DMP.
Embodiment 3: the 2,5-DMP in UPLC quantitative detection fermentation liquid is utilized
In the LB culture medium (formula of LB: 5g/L yeast powder, 10g/L peptone, 10g/L of the L-threonine of addition 1g/L
Sodium chloride) in, 37 DEG C, 200rpm culture B.subtilis168 to 2d, centrifuging and taking fermented supernatant fluid.
It is specific as follows using 2,5-DMP in UPLC quantitative detection fermentation liquid:
Quantitative detection, chromatographic column WatersBEHC18 are carried out to 2,5-DMP using ultra performance liquid chromatography (UPLC)
(100mm × 2.1mm, 1.7 μm of particle) carry out liquid phase separation to the 2,5-DMP in sample.Mobile phase A is 0.1% formic acid
Solution, Mobile phase B are hplc grade methanol.Mobile phase gradient: initial, 31%B；0-3min, 31%-69%B；3.10min
31% B；Runing time is 10min.Flow velocity is 0.20mlmin-1, ultraviolet detection wavelength is 275nm.
1 μ L of applied sample amount.
As shown in figure 4, it is apparent that 2,5-DMP may be implemented to efficiently separate in complicated fermentation liquid system.
Meanwhile it is in y=0.00004312*x-1.19126 that peak area, which is substituted into standard curve, the content that 2,5-DMP is calculated is
40 mg/L。
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (10)
1. a kind of method for detecting 2,5- dimethyl pyrazine, which is characterized in that the method is detected using UPLC；It is described
UPLC carries out testing conditions:
Chromatographic column is Waters BEH C18；
Mobile phase A is 0.1% formic acid solution, Mobile phase B is hplc grade methanol；
Mobile phase gradient: initial, 31%B；0-3min, 31%-69%B；3-10min, 31%B；
Runing time is 10min；
Flow velocity is 0.20mlmin-1；
Ultraviolet detection wavelength is 275nm.
2. the method according to claim 1, wherein the 1 μ L of applied sample amount that the UPLC is detected.
3. the method according to claim 1, wherein described be detected as quantitative detection or qualitative detection.
4. method according to claim 1 to 3, which is characterized in that the quantitative detection is first to prepare 2,5-DMP mark
Quasi- product are detected using the UPLC, are fitted to obtain 2,5-DMP standard song according to the variation of 2,5-DMP concentration and peak area
Line；Then sample to be tested is detected using the UPLC, the peak area of sample to be tested is substituted into 2,5-DMP standard curve
In to get 2, the 5-DMP concentration into sample to be tested.
5. the method according to claim 1, wherein the sample to be tested is 2,5-DMP aqueous solution, or containing
The fermented supernatant fluid of 2,5-DMP.
6. according to the method described in claim 5, it is characterized in that, the fermented supernatant fluid, is the microorganism that will produce 2,5-DMP
After carrying out fermented and cultured, by the supernatant of bacterium solution centrifugation acquisition.
7. the method according to claim 1, wherein the fermented supernatant fluid, is by 168 kinds of B.subtilis
Sub- liquid, which is seeded to, to be obtained after fermented and cultured in the culture medium containing L-threonine.
8. the method according to the description of claim 7 is characterized in that the culture medium is LB culture medium.
9. according to the method described in claim 8, it is characterized in that, the formula of LB: 5g/L yeast powder, 10g/L peptone, 10g/
L sodium chloride.
10. the method according to the description of claim 7 is characterized in that the culture is in 37 DEG C, 200rpm culture 2d.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105353045A (en) * | 2015-04-15 | 2016-02-24 | 贵州省产品质量监督检验院 | Detection method of pyrazine compounds in Jiang-flavour Chinese spirit |
| CN105842377A (en) * | 2016-05-18 | 2016-08-10 | 贵州省产品质量监督检验院 | High performance liquid chromatography detection method for pyrazine compounds in Baijiu |
| CN107247101A (en) * | 2017-06-15 | 2017-10-13 | 云南中烟工业有限责任公司 | A kind of detection of fragrance component for improving flue gas pleasant impression and decision method |
| CN108459131A (en) * | 2018-04-03 | 2018-08-28 | 江南大学 | A method of quantitative determination chocolate malt characteristic flavor object content |
-
2019
- 2019-06-06 CN CN201910492577.1A patent/CN110286188B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105353045A (en) * | 2015-04-15 | 2016-02-24 | 贵州省产品质量监督检验院 | Detection method of pyrazine compounds in Jiang-flavour Chinese spirit |
| CN105842377A (en) * | 2016-05-18 | 2016-08-10 | 贵州省产品质量监督检验院 | High performance liquid chromatography detection method for pyrazine compounds in Baijiu |
| CN107247101A (en) * | 2017-06-15 | 2017-10-13 | 云南中烟工业有限责任公司 | A kind of detection of fragrance component for improving flue gas pleasant impression and decision method |
| CN108459131A (en) * | 2018-04-03 | 2018-08-28 | 江南大学 | A method of quantitative determination chocolate malt characteristic flavor object content |
Non-Patent Citations (3)
| Title |
|---|
| CHRISTIAN LARROCHE ET AL: "High pyrazine production by Bacillus subtilis in solid substrate fermentation on ground soybeans", 《PROCESS BIOCHEMISTRY》 * |
| 刘丽娟 等: "产二甲苯单加氧酶菌株的筛选、鉴定及转化2,5-－二甲基吡嗪条件的优化", 《化学与生物工程》 * |
| 董棒 等: "UPLC-PDA法研究Liguzinediol在大鼠体内的排泄", 《南京中医药大学学报》 * |
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