CN101531557A - Derivatization method of qualitative or quantitative analysis for polyhydroxy compound - Google Patents
Derivatization method of qualitative or quantitative analysis for polyhydroxy compound Download PDFInfo
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Abstract
The invention discloses a derivatization method for polyhydroxy compound. In the method, a sugar-containing sample is disposed in a reaction system using 1-methylimidazole as catalyst and acetic anhydride as derivatization reagent to have acetylation derivatization reaction. The method in the invention can carry out acetylation derivatization on the basis of maintaining native conformation of sugar, single derivatization product is obtained after derivatization, and qualitative or quantitative analysis can be accurately carried out on various simple sugars.
Description
Technical field
The present invention relates to a kind of derivatization method of polyol, particularly a kind of acetylize derivatization method that is used for the qualitative or quantitative analysis of polyol.
Background technology
The monose that occurring in nature exists can be divided into neutral monose (aldose and ketose), acid monose (uronic acid), sugar alcohol, osamine and sulfo-sugar etc., and the existence owing to its hydroxyl when with vapor-phase chromatography it being analyzed need be carried out derivatize.The method that is usually used in the monose derivatize at present mainly contains methyl-monosilaneization (trimethyl silicone hydride), acylations (sugared oxime acetylize and sugar alcohol acetylize) and method such as methylate.In the monose derivatization method commonly used at present, Silicane Method is because its derivatization reagent and derivatize product chance water molecules very easily decompose, and therefore strict waterless operation environment just can prevent the failure of an experiment.In this method, monose is dissolved in the solvent pyridine, also isomery can take place, this will directly influence the qualitative and quantitative analysis of this monose; Sugar oxime acetylize method can't solve the problem that ketose detects; The sugar alcohol acetylize then because aldose and ketose all are reduced into corresponding sugar alcohol, causes its result and naturally occurring sugar alcohol overlapping, finally influences these sugared qualitative, quantitatives.Ye Fang very waits ((2005) utilization variances such as Ye Fangting, Yan Xiaojun derive Synchronization Analysis aldose and ketose, analytical chemistry, o. 11th, the 33rd volume, 1569～1572) once utilized two kinds of methods of silanization of sugared oxime acetic ester derivatize and monose to carry out the purpose that the difference derivatize reaches analysis ketose aldose.But this method needs twice different derivatize experiment just can reach the purpose of analysis, has increased the operation easier of experiment and the intensity of work undoubtedly.We can keep the characteristics of unique cyclic configuration according to the monose crystal in specific solvent, only by acetylize, and can be with qualitative and quantitative analysis synchronously such as the aldose of native configurations, ketose, sugar alcohol.
One, the problem of the existence of derivatization method aspect derivatize mechanism commonly used
1, the problem of sugared oxime acetylize derivatize mechanism existence:
The aldehyde radical of monose is first with acetylize again (as Fig. 1) after the oxammonium hydrochloride oximate.This method can be used for aldose and the analysis of sugar alcohol derivatize preferably, but for ketose, as fructose etc., effective oximate derivatize then, reason is because oxammonium hydrochloride oximate ketone group fully, does not cause the derivatize product single and produce the voluminous thing phenomenon (as Fig. 2) that coexists and cause this method can't detect ketose.
2, sugar alcohol acetylize derivatize mechanism existing problems
This method has acetylize derivatization preferably to sugar alcohol, goes derivatize behind the corresponding sugar alcohol again but aldose and ketose then needed earlier with sodium borohydride it to be reduced into, the sample when causing accurately analyzing and to measure ketose, aldose and alcohol sugar and existing simultaneously.
(1), aldose problem: utilize aldehyde radical or ketone group reduction the becoming hydroxyl and then the acetylize of sodium borohydride with sugar.Just ketose and aldose are reduced into sugar alcohol, and then its hydroxyl is carried out the ethanoyl derivatize, so derivatize is complete, product is single, chromatographic peak profile is better.(Blakeney A.B., Harris P.J., Henry R.J.and StoneB.A. (1983) A simple and rapid preparation of alditol acetates for monosaccharideanalysis.Carbohydrate Research 113,291-299.) but this method shortcoming be, aldose is reduced to sugar alcohol (as shown in Figure 3 and Figure 4), and (Kong Fanzuo writes (2005), carbohydrate chemistry (modern chemistry basis book series 05), Science Press, the 2nd chapter, the 112nd page).These are by the sugar alcohol that aldose and ketose reduction back generate, and are most of in the occurring in nature existence, and have important physiological significance.Because aldose reductive sugar alcohol has been covered the sugar alcohol with important physiological function of nature necessary being, has not only caused and can't accurately measure natural aldose content, also directly causes these naturally occurring sugar alcohols to detect.
(2), ketose problem one this method can't directly detect ketose.The straight chain ketose because the C-2 epimer exists, generate two kinds of isomers products, and these products also is the common sugar alcohol of occurring in nature usually behind sodium borohydride reduction.For example, fructose generates N.F,USP MANNITOL and sorbitol (sorbyl alcohol) (shown in Fig. 5, Fig. 6, table 1) after reducing, and (Kong Fanzuo writes (2005), carbohydrate chemistry (modern chemistry basis book series 05), Science Press, the 2nd chapter, the 112nd page) and just contain these two kinds of sugar alcohols in the cell usually.In addition, seminose and glucose also can correspondingly generate N.F,USP MANNITOL and sorbitol (sorbyl alcohol) behind sodium borohydride reduction.Original respective components compound is overlapped in reduzate that this method produces and the sample, therefore uses this method to carry out the verity that the monose analysis usually can influence detected result.
Table 1. fructose generates the ratio of N.F,USP MANNITOL and sorbitol after reducing
| Kind | N.F,USP MANNITOL | Sorbyl alcohol |
| Ratio (%) | 48.28％ | 51.72％ |
Two, the problem of derivatization method aspect the derivative reaction condition commonly used---temperature requirement height, the time is long, reaction solvent is improper
1, derivatization method commonly used is to the requirement of temperature
1) Silicane Method need be at 70-90 ℃, even finish under the higher temperature condition, otherwise easily cause derivative reaction not exclusively ((2005) utilization variances such as Ye Fangting, Yan Xiaojun derive Synchronization Analysis aldose and ketose, analytical chemistry, o. 11th, the 33rd volume, 1569～1572).
2) in the sugared oxime acetylize method, oximation reaction need be finished at 80 ℃, otherwise easily causes derivative reaction not exclusively ((2005) utilization variances such as Ye Fangting, Yan Xiaojun derive Synchronization Analysis aldose and ketose; analytical chemistry; o. 11th, the 33rd volume, 1569～1572).
3) in the sugar alcohol acetylize method; need finish at 40 ℃ in the sodium borohydride reduction reaction; otherwise easily cause not exclusively (Blakeney A.B. of derivative reaction; Harris P.J.; Henry R.J.and Stone B.A. (1983) Asimple and rapid preparation of alditol acetates for monosaccharide analysis.Carbohydrate Research 113,291-299.).
2, derivatization method commonly used is to the requirement in reaction times
1) Silicane Method needs reaction at high temperature at least 1 hour, more time-consuming ((2005) utilization variances such as Ye Fangting, Yan Xiaojun derive Synchronization Analysis aldose and ketose, analytical chemistry, o. 11th, the 33rd rolls up 1569～1572).
2) in the sugared oxime acetylize method, oximation reaction needs reaction at high temperature at least 5 minutes, reactions steps more (sugar and sugar alcohol acetylize and capillary gas chromatography in Hu Lei etc. (2004) plant tissue, BULLETIN OF BOTANY Vol., 21 (6), 689～699).
3) in the sugar alcohol acetylize method; sodium borohydride reduction reaction needed 60-90 minute at least; reactions steps and chemical reagent are more; (the Blakeney A.B. that wasted the quality time; Harris P.J.; Henry R.J.and Stone B.A. (1983) A simple and rapid preparation of alditol acetates for monosaccharide analysis.Carbohydrate Research 113,291-299.).
3, use the problem of the selected solvent of derivatization method always
Need be in Silicane Method with anhydrous pyridine as reaction solvent, isomery (as Fig. 7) can take place behind the pyridine and the monose crystal is dissolved in, the phenomenon of a plurality of chromatographic peaks of a kind of monose appears when chromatographic separation, and influence is to the qualitative and quantitative analysis of monose.
Three, the problem of derivatization reagent commonly used and derivatize product stability aspect
Moisture is to the influence of derivative reaction and product thereof
1) methyl-monosilaneization is that the hydroxyl on the monose is by methyl-monosilaneization with reactions such as monose and derivatization reagent hexamethyldisilazane, trimethylchlorosilanes.This derivatization method needs to finish in water-less environment, finally generates the stronger methyl-monosilane derivative of volatility.This method is subjected to moisture effects bigger, need under strict anhydrous condition, to operate, otherwise methyl-monosilane reagent and derivatize product thereof are very easily degraded by water, impact analysis result's true (Marie-christineet. (1997) Determination of Carbohydrates in two ferrllitic soil:analysis by capiliary gas chromatography after derivatization by silylation.SoilBiol.Biochem.Vol.29, No.9/10, pp.1585-1589).
2) in the sugar alcohol acetylize method, the easy moisture absorption of sodium borohydride is rotten, and preservation condition is had relatively high expectations.
Four, derivatization method commonly used is in the problem of the monose kind being carried out exist aspect the qualitative, quantitative
1, in the Silanization reaction, owing to adopt pyridine as solvent, and mutarotation can take place in monose in pyridine, produce multiple isomer.This will certainly cause when detecting plant sample, owing to adopting pyridine to cause naturally occurring monose generation isomer in the plant sample as solvent, both influenced the accurate qualitative and quantitative analysis of this monose, the quality time that can take chromatographic separation again because of the chromatographic peak that isomer produces influences separation (Marie-christine et. (1997) the Determination of Carbohydrates in two ferrllitic soil:analysis by capiliary gas chromatography after derivatization by silylation.SoilBiol.Biochem.Vol.29 of other monose chromatographic peak, No.9/10, pp.1585-1589).
2, in sugar alcohol acetylize method, because being reduced to sugar alcohol, all aldoses and ketose detect, cause the sugar alcohol that can't qualitative, quantitative has important physiological significance, reduced the sensing range of monose and derivative thereof.
3, in sugared oxime acetylize method, oxammonium hydrochloride can't be with the ketone group oximate of ketose, causes this method can't effectively detect ketose ((2005) utilization variances such as Ye Fangting, Yan Xiaojun derive Synchronization Analysis aldose and ketose; analytical chemistry; o. 11th, the 33rd volume, 1569～1572).
4, to the detection of acid monose, osamine
1) only can use Silicane Method to carry out derivatize to acid monose such as uronic acid, osamine such as glucosamine at present, but because Silanization reaction conditional request harshness, the silanization product is met the water capacity and is easily decomposed, and causes the reliability of its qualitative, quantitative to be had a greatly reduced quality.
2) sugar alcohol acetylize and sugared oxime acetylize are more or less freely with respect to the condition of Silanization reaction; and acetylate is more stable under the exsiccant condition; can prolonged preservation; but these two kinds of methods again can not be with acid monose and the effective derivatize of osamine; limit use range (Marie-christine et. (1997) the Determinationof Carbohydrates in two ferrllitic soil:analysis by capiliary gas chromatography afterderivatization by silylation.Soil Biol.Biochem.Vol.29 of these methods; No.9/10, pp.1585-1589).
Five, derivatization method commonly used is to the problem of test material demand
The analysis each time of traditional monose analytical technology needs biological specimen material 0.5-1.0g usually, and this can cause very big damage undoubtedly for micro-material of research or biology in imminent danger.
Six, derivatization method commonly used is to the influence of environment
1, in Silicane Method, use pyridine as solvent.The pyridine environmental pollution is more serious, and is bigger to human health damage: intense stimulus is arranged; Can anaesthetize central nervous system.The eye and the upper respiratory tract there is hormesis.After high density sucked, the lighter had glad or sensation of asphyxia, the appearance depression, myasthenia, the vomiting that continue; Weight person's loss of consciousness, gatism, tonic spasm, blood pressure drops.Wrongly take and to cause death.Chronic influence: dizziness, headache, insomnia, instability of gait and tract function disorder appear in long-term the suction.Hepatorenal damage can take place.Can cause polyneuropathy.Skin there is pungency, can causes dermatitis, photosensitive dermatitis is arranged sometimes.Fire danger: pyridine is inflammable, the tool strong and stimulating.The pyridine hazard property: its steam and air can form explosive mixture, meet naked light, high thermoae easy firing blast.Contact fierce reaction with oxygenant.Decompose during high temperature, disengage the oxides of nitrogen gas of severe toxicity.With vigorous reactions such as sulfuric acid, nitric acid, chromic acid, oleum, chlorsulfonic acid, MALEIC ANHYDRIDE, silver perchlorates, explosion hazard is arranged.Flow velocity is too fast, is easy to generate and gathers static.Its steam is heavier than air, and can be diffused into place quite far away in the lower, and the chance burning things which may cause a fire disaster can catch fire and strile-back.If meet high heat, press in the container to increase, the danger of cracking and blast is arranged.
2, sugared oxime acetylize uses oxammonium hydrochloride as oximate reagent, but the oxammonium hydrochloride severe toxicity has pungency to skin.Its mouse oral LD50 is 400mg/kg
3, utilize sodium borohydride as reductive agent in the sugar alcohol acetylize method, and sodium borohydride is serious to human health damage: intense stimulus mucous membrane, the upper respiratory tract, eyes and skin.After the suction, can be because of larynx and bronchial spasm, inflammation and oedema, chemical pneumonitis and pulmonary edema and cause death.Oral corrosion digestive tube.Acute toxicity: LD5018mg/kg (in the film of rat chamber).Sodium borohydride hazard property: meet water, damp atmosphere, acids, oxygenant, Gao Re and naked light and can cause burning.
As seen from the above analysis, commonly used derivatize solvent, oximate reagent, going back original reagent all can be to environment, HUMAN HEALTH is caused serious harm, and therefore excavating a kind of toxicity is little, derivative reaction is easy, derivatize is effective derivatization reagent has very that important use is worth.
Summary of the invention
The derivatization method that the purpose of this invention is to provide a kind of polyol.
Polyol derivatization method provided by the present invention is to be solvent with containing that the polyol sample places with the dimethyl sulfoxide (DMSO), is catalyzer with the 1-Methylimidazole, and diacetyl oxide is in the reaction system of derivatization reagent, carries out the acetylize derivative reaction.
The interpolation volume parts ratio of described dimethyl sulfoxide (DMSO), 1-Methylimidazole and diacetyl oxide can be (dimethyl sulfoxide (DMSO)): (diacetyl oxide): (1-Methylimidazole)=(5000～100): (5000～10): (2000～1), be preferably 50:3-24:2, be preferably 25:3:1 especially.
The described ratio that contains polyol sample and described dimethyl sulfoxide (DMSO) is 10-1g:1000-10ml, is preferably 1g:10ml.The mol ratio of described polyol hydroxyl mole number and diacetyl oxide is 1:5-40; Be preferably 1:10.
Above-mentioned polyol comprises alcohols (as tonquinol) and sugar and sugared derivative, is preferably the derivative of sugar and/or sugar;
Wherein, sugar is meant: the arbitrary mixing of one or more in disaccharide, monose, trisaccharide and the tetrose.
Described monose is one or more the arbitrary mixing in neutral monose (aldose and ketose) and the acid monose (uronic acid); Described aldose is one or more the arbitrary mixing in 2-deoxidation-ribose, wood sugar, rhamnosyl, Fucose, pectinose, semi-lactosi, the glucose.Described ketose is a fructose.
The derivative of described sugar is one or more the arbitrary mixing in sugar alcohol, uronic acid and the osamine.
Described sugar alcohol is one or more arbitrary mixing of erythritol, 2-deoxidation-ribose, rhamnitol, ribitol, fucitol, arabitol, Xylitol, inositol, N.F,USP MANNITOL, sorbyl alcohol, melampyrum.
Described uronic acid is one or more arbitrary mixing of glucuronic acid, galacturonic acid.
Described osamine is N-acetyl-glucosamine.
Operation for convenience, above-mentioned dimethyl sulfoxide (DMSO), 1-Methylimidazole and diacetyl oxide can be formed a test kit, can three reagent independent packagings, also can be divided into two reagent packings, promptly be divided into reagent A (DMSO+1-Methylimidazole), reagent B (DMSO+ diacetyl oxide), reagent A is the mixed solution of DMSO and 1-Methylimidazole, reagent B is the mixed solution of DMSO and diacetyl oxide, during packing, wherein DMSO, diacetyl oxide and 1-Methylimidazole volume ratio are (5000～100): (5000～10): (2000～1), be preferably 50:3-24:2, be preferably 25:3:1 especially.
Aforesaid method can be used for the qualitative and quantitative detection of polyol in each plant tissues of different growth phases such as carbohydrate; have simple, save time; environmental protection; and use method of the present invention that carbohydrate such as monose etc. is carried out the acetylize derivatize; can on the basis of the native conformation of keeping sugar, carry out derivatize, specifically can produce following advantage:
One, solved derivatization method commonly used existing problem aspect derivatize mechanism
1, the solution of the problem that sugared oxime acetylize derivatize mechanism is existed
In sugared oxime acetylize method, oxammonium hydrochloride is the oximate ketone group effectively, causes this method can't detect ketose.After the ketose crystal directly is dissolved in the DMSO solvent,, therefore solved the problem that in sugared oxime acetylize method oxammonium hydrochloride can't the oximate ketone group because ketone group participates in forming cyclic configuration.
2, to the in-problem solution of sugar alcohol acetylize derivatize mechanism
In sugar alcohol acetylize method, aldose is reduced to sugar alcohol, and the straight chain ketose causes producing two kinds of sugar alcohols owing to have epimer on the C-2 in reduction process.These are by the sugar alcohol that aldose and ketose reduction back generate, and are most of in the occurring in nature existence, and have important physiological significance.Sugar alcohol acetylize method directly causes these naturally occurring sugar alcohols to detect.
Solvent of the present invention is dimethyl sulfoxide (DMSO) (DMSO), and this organic solvent polarity (7.2) slightly is weaker than water (10), and is comparatively approaching with the polarity of monose molecule, the monose crystal is dissolved in this solvent after, can keep single ring type structure under the monose crystalline state.Under ring type aldose state,, thereby reduced the polarity of aldose molecule because the aldehyde radical of aldose forms the ring texture of hemiacetal; under this state; directly it is carried out acetylize, just can form the peculiar acetylate of this aldose, and will the aldose reduction not become the sugar alcohol form.
After the ketose crystal directly is dissolved in this solvent; it is same because ketone group participates in forming cyclic configuration; and can't form under the straight chain state with C-2 is the epimer of new symmetry centre; therefore both solve the problem that in sugared oxime acetylize method oxammonium hydrochloride can't the oximate ketone group, and avoided in sugar alcohol acetylize method straight chain ketose to be reduced the result of the sugar alcohol that is called two kinds of correspondences again.
Two, the invention solves derivatization method existing problem aspect the derivative reaction condition commonly used
1, in the derivatization method commonly used to the solution of the requirement of temperature
Silicane Method, sugared oxime acetylize method, sugar alcohol acetylize all need comparatively high temps just can make derivative reaction complete; is that catalyzer, diacetyl oxide are acetylation reagent and utilize dimethyl sulfoxide (DMSO) (DMSO) for reaction solvent, with the 1-Methylimidazole, just can finish derivative reaction down in room temperature (18-25 ℃).Its reason is that this method only has acetylize, has saved sodium borohydride reduction reaction higher than temperature requirement in difficult oximation reaction of grasping and the sugar alcohol acetylize method in the sugared oxime acetylize method.
2, in the derivatization method commonly used to the solution of the requirement of time
Silicane Method, sugar alcohol acetylize all need the long period, and (about 1 hour) just can make derivative reaction complete; because utilizing dimethyl sulfoxide (DMSO) (DMSO) is that catalyzer, diacetyl oxide are acetylation reagent for reaction solvent, with the 1-Methylimidazole; acetylize is only arranged; saved in the sugared oxime acetylize method difficult grasp oximation reaction and sugar alcohol acetylize method in the sodium borohydride reduction reaction long to time requirement, therefore only needed five minutes can finish whole derivative reactions.
3, the present invention has avoided in the derivatization method commonly used solvent to select problem improperly
Silicane Method, sugared oxime acetylize method often use pyridine as solvent, and the monose crystal is dissolved in the pyridine, and mutarotation also can take place, and produce isomer.After this will cause Silanization reaction, produce the isomery peak of monose, take the quality time of chromatographic separation and influence the separation of other monose chromatographic peak.Present method adopts dimethyl sulfoxide (DMSO) as solvent; this organic solvent polarity (7.2) slightly is weaker than water (10); comparatively approaching with the polarity of monose molecule, through reference with experiment showed, repeatedly monose is dissolved in this solvent; temperature is in the time of 18-25 ℃; mutarotation can not take place, and the ring texture during each monose crystal is kept directly acetylize in this solvent; each monose chromatographic peak type is unique, and no isomery peak occurs.
Three, the present invention is to the solution of derivatization reagent commonly used and the existing problem in derivatize product stability aspect
1, in Silicane Method, silylating reagent and silanization product are met water molecules and are very easily decomposed, and cause derivatization reagent to lose efficacy and the derivative reaction failure.
2, in the sugar alcohol acetylization reaction, going back the original reagent sodium borohydride also needs to keep away the water preservation in case sex change.Used reagent A in present method (DMSO+1-Methylimidazole), the two chemical property of reagent B (DMSO+ diacetyl oxide) is stable, does not all need particular surroundings to preserve.
3, the monose ring type acetylate Stability Analysis of Structures that present method generated after drying can prolonged preservation.
Four, present method has solved derivatization method commonly used in a difficult problem of monose being carried out exist aspect the qualitative, quantitative
1, Silicane Method adopts pyridine as solvent, and monose can produce isomer in pyridine, and silylating reagent and silanization product meet water and easily decompose, and therefore influences the verity of chromatogram detected result.Present method selects for use DMSO as the derivatize solvent, and monose isomery can not take place in this solvent, and acetylate can not meet water decomposition, therefore can carry out qualitative and quantitative analysis more accurately to the monose in the natural biological sample.
2, sugared oxime acetylize can't detect ketose, the sugar alcohol acetylize with the reduction of all aldoses and ketose for sugar alcohol, therefore can't be accurately the quantitative content of original sugar alcohol in the organism.Present method solvent DMSO has kept various aldoses and the unique cyclic configuration of ketose crystal, therefore behind acetylization reaction, also can keep its exclusive ring type structure, during chromatographic separation, can be not identical because of structure, and chromatographic peak is overlaped.This method has overcome the shortcoming of preceding two kinds of acetylize methods, has realized first detecting aldose, ketose and sugar alcohol simultaneously with the acetylize method.
Though 3, Silicane Method can detection of acidic monose (saccharic acid) and alkaline monose osamine, because the strict water-less environment of this method, its use is subjected to restriction to a certain degree.Acid monose such as glucuronic acid, galacturonic acid and alkaline monose such as glucose grapes glucosamine are dissolved among present method solvent DMSO; can keep the annular lactone structure of acid monose and the ring texture of alkaline monose osamine; and to its direct acetylize; can generate the acetylate that is used for stratographic analysis, so present method also is applicable to the analyzing and testing to acid monose and alkaline osamine.
Five, the invention solves the problem of derivatization method commonly used to the test material demand
The analysis each time of traditional monose analytical technology needs material 0.5-1.0g usually, and this can cause very big damage undoubtedly for micro-material of research or biology in imminent danger.Present method is owing to adopt DMSO that biological sample is directly extracted; extract the back and in DMSO solution, directly carry out acetylize; steps such as the centre concentrates have been reduced; thereby reduced the loss of monose; therefore the biological material specimens consumption can be reduced to below the 50mg; also can directly get one or a few drip body juice is directly used in this method and carries out the derivatize analysis; this methods and results is compared with traditional method; dimethyl sulfoxide (DMSO) and common method are extracted contents of monosaccharides and kind otherness statistical study experimental result is found out from testing; the statistical study no significant difference; present method has reduced the amount of samples to biomaterial greatly, has reduced the infringement to biology.
Six, the present invention has reduced the influence (comparing with derivatization method commonly used) to environment
The present invention's dimethyl sulfoxide (DMSO) is that solvent (a kind of transparent, colourless, odorless, is the liquid of little bitter taste, the extremely low and eco-friendly water miscible compound of toxicity), can dissolve most organic compound, be mainly used in anhydrous organic chemistry building-up reactions and medicine and intermediate synthesis reaction solvent, use this solvent can improve chemical reaction velocity, improve the rate of recovery of reactant, shorten the reaction times, make building-up reactions become more smooth.Compare poisonous or highly toxic substances such as present method non-pyridine, no hydrochloric acid azanol, no sodium borohydride reduction reagent, so present method environmental protection the most with silanization, sugared oxime acetylize, sugar alcohol acetylize.
Description of drawings
Fig. 1 is the oximation reaction process
Fig. 2 is acetylate total ion current figure after fructose (Fructose) oximate
Fig. 3 is that glucose (Glucose) is the molecule equation of sorbyl alcohol (Sorbitol) through sodium borohydride reduction
Fig. 4 is glucose (Glucose) acetylizad again total ion current figure after sodium borohydride reduction is sorbyl alcohol (Sorbitol)
Fig. 5 is fructose (Fructose) generates sorbyl alcohol (Sorbitol) and N.F,USP MANNITOL (Mannitol) under sodium borohydride reduction a molecule equation
Fig. 6 be fructose (Fructose) behind sodium borohydride reduction again acetylize generate the total ion current figure of sorbyl alcohol (Sorbitol) and N.F,USP MANNITOL (Mannitol)
Fig. 7 is that glucose (Glucose) is dissolved in behind the pyridine total ion current figure that directly acetylize generates d glucofuranose, β glucofuranose
Fig. 8 is ring type fructose (Fructose ketose) acetylization reaction (directly acetylize among the DMSO)
Fig. 9 is ring type fructose (Fructose ketose) acetylate (directly acetylize among the DMSO) total ion current figure
Figure 10 is ring type glucose (Glucose aldose) acetylization reaction (directly acetylize among the DMSO)
Figure 11 is ring type glucose (Glucose aldose) acetylate (directly acetylize among the DMSO) total ion current figure
Figure 12 is ring type fructose (Fructose) acetylization reaction (directly acetylize among the DMSO)
Figure 13 is ring type fructose (Fructose) acetylate (directly acetylize among the DMSO) total ion current figure
Figure 14 is galacturonic acid annular lactone (uronic acid) acetylate (directly acetylize among the DMSO)
Figure 15 is N-acetylglucosamine (osamine) ring-type acetylate (directly acetylize among the DMSO)
Figure 16 is 24 kinds of fried sugar spectrograms, and promptly GC-DSQ-MS detects 24 kinds of sugared total ion current figure.
Figure 17 is ribodesose acetylate mass spectrum and standard mass spectral database ring type monose acetylize standard spectrogram comparison diagram spectrum, and this figure has proved this monose in dimethyl sulfoxide (DMSO), has kept the cyclic configuration under the monose crystalline state.
Figure 18 is pectinose acetylate mass spectrum and standard mass spectral database ring type monose acetylize standard spectrogram comparison diagram spectrum, and this figure has proved this monose in dimethyl sulfoxide (DMSO), has kept the cyclic configuration under the monose crystalline state.
Figure 19 is Fucose acetylate mass spectrum and standard mass spectral database ring type monose acetylize standard spectrogram comparison diagram spectrum, and this figure has proved this monose in dimethyl sulfoxide (DMSO), has kept the cyclic configuration under the monose crystalline state.
Figure 20 is rhamnosyl acetylate mass spectrum and standard mass spectral database ring type monose acetylize standard spectrogram comparison diagram spectrum, and this figure has proved this monose in dimethyl sulfoxide (DMSO), has kept the cyclic configuration under the monose crystalline state.
Figure 21 is wood sugar acetylate mass spectrum and standard mass spectral database ring type monose acetylize standard spectrogram comparison diagram spectrum, and this figure has proved this monose in dimethyl sulfoxide (DMSO), has kept the cyclic configuration under the monose crystalline state.
Figure 22 is semi-lactosi acetylate mass spectrum and standard mass spectral database ring type monose acetylize standard spectrogram comparison diagram spectrum, and this figure has proved this monose in dimethyl sulfoxide (DMSO), has kept the cyclic configuration under the monose crystalline state.
Figure 23 is glucose acetylate mass spectrum and standard mass spectral database ring type monose acetylize standard spectrogram comparison diagram spectrum, and this figure has proved this monose in dimethyl sulfoxide (DMSO), has kept the cyclic configuration under the monose crystalline state.
Figure 24 is fructose acetylate mass spectrum and standard mass spectral database ring type monose acetylize standard spectrogram comparison diagram spectrum, and this figure has proved this monose in dimethyl sulfoxide (DMSO), has kept the cyclic configuration under the monose crystalline state.
Figure 25 is inositol acetylate mass spectrum and standard mass spectral database ring type monose acetylize standard spectrogram comparison diagram spectrum, and this figure has proved this monose in dimethyl sulfoxide (DMSO), has kept the cyclic configuration under the monose crystalline state.
Figure 26 is osamine acetylate mass spectrum and standard mass spectral database ring type monose acetylize standard spectrogram comparison diagram spectrum, and this figure has proved this monose in dimethyl sulfoxide (DMSO), has kept the cyclic configuration under the monose crystalline state.
Figure 27 is galacturonic acid acetylate mass spectrum and standard mass spectral database ring type monose acetylize standard spectrogram comparison diagram spectrum, and this figure has proved this monose in dimethyl sulfoxide (DMSO), has kept the cyclic configuration under the monose crystalline state.
To be DMSO, 1-Methylimidazole mix influence to derivative reaction with diacetyl oxide to Figure 28 in varing proportions
Embodiment
Method in the following method, if no special instructions, equal ordinary method.
One, the screening of monose derivatization method
The variety of problems qualitative at the described present carbohydrate of background technology (as ketose, aldose, pure sugar etc.) or quantitative analysis exists, the present inventor determines the acetylize derivatization method, to solve present existing problems.Carry out the derivatize experiment with monose in following 24 and derivative thereof, and analyze its effect, screening derivatize technical scheme.Concrete grammar is as described below:
1, laboratory apparatus and material
Vacuum concentration instrument, vacuum lyophilization instrument, Finnigan gaschromatographic mass spectrometry logotype instrument, microsyringe etc.24 kinds of monose standard specimens are all bought the company from Sigma (USA), are respectively: ribodesose (purity〉99.0%), rhamnosyl (purity〉98.0%), Fucose (purity〉99.0%), wood sugar (purity〉99.0%), pectinose (purity〉99.0%), semi-lactosi (purity〉99.0%), glucose (purity〉99.0%), erythritol (purity〉99.0%), ribodesose alcohol (purity〉99.0%), rhamnitol (purity〉99.0%), fucitol (purity〉99.0%), Xylitol (purity〉99.0%), arabitol (purity〉99.0%), melampyrum (purity〉99.0%), sorbitol (purity〉99.0%), N.F,USP MANNITOL (purity〉99.0%), ribitol (purity〉99.0%), inositol (purity〉99.0%), fructose (purity〉99.0%), galacturonic acid (purity〉99.0%), glucuronic acid (purity〉99.0%), the N-acetylglucosamine (purity〉99.0%), sucrose (purity〉99.0%), trehalose (purity〉99.0%).Dimethyl sulfoxide (DMSO) (chromatographically pure), 1-Methylimidazole (analytical pure), diacetyl oxide (analytical pure), sodium borohydride (analytical pure), ethyl acetate (chromatographically pure) are all bought from internal reagent company.
2,24 kinds of sugared derivatize experiments
Select for use dimethyl sulfoxide (DMSO) (DMSO) as solvent; the 1-Methylimidazole is a catalyzer; diacetyl oxide is an acetylation reagent; to above-mentioned 7 kinds of aldose (ribodesoses; rhamnosyl; Fucose; wood sugar; pectinose; semi-lactosi; glucose); 11 kinds of sugar alcohol (erythritols; ribodesose alcohol; rhamnitol; fucitol; Xylitol; arabitol; melampyrum; sorbitol; N.F,USP MANNITOL; ribitol; inositol); 1 kind of ketose (fructose); 2 kinds of uronic acid (galacturonic acids; glucuronic acid); 1 kind of osamine (N-acetylglucosamine); 2 kinds of disaccharide (sucrose; trehalose) carried out the derivatize experiment; carry out the making of qualitative analysis and 24 kinds of sugared total ion current figure with the derivatize product, concrete grammar is:
Take by weighing each sugared standard specimen of 10mg respectively, add the 1ml dimethyl sulfoxide (DMSO), preserve as mother liquor.During to 24 kinds of monose qualitative analyses, respectively getting the monose mother liquor adds respectively in 24 2ml centrifuge tubes, add 350ul dimethyl sulfoxide (DMSO), 60ul diacetyl oxide, 20ul 1-Methylimidazole then respectively and make DMSO: diacetyl oxide: 1-Methylimidazole=25:3:1, making sugared concentration make the monose hydroxyl value and the ratio of diacetyl oxide mole number is 1:10.
After above-mentioned reaction system at room temperature reacted 5 minutes respectively, add 500ul ethyl acetate and 500ul water respectively, spiral is after 5 minutes, gets that GC/MS detects on the 1ul organic phase, makes 24 kinds of sugared ion flow graphs and 24 kinds of sugared total ion current figure.When making 24 kinds of monose total ion current figure, get the above-mentioned 24 kinds of monose organic phases of 100ul respectively and be mixed in 1 test tube, after low temperature is drained, redissolve, get 1ul and advance the GC/MS detection, more than handle being three repetitions with the 100ul ethyl acetate.Wherein, chromatogram and mass spectroscopy condition are:
The U.S.'s thermoelectric Finnigan gaschromatographic mass spectrometry logotype instrument, chromatographic condition: DB-17 post (30m * 0.318mmi.d, 0.25um film thickness), 250 ℃ of injector temperatures, carrier gas is high-purity He of 99.999%, flow velocity 1ml/min, press 26.3kPa before the initial post, 100 ℃ of column temperature (keeping 3.5min), 15 ℃/min to 160 ℃ (keeping 20min), 15 ℃/min to 200 ℃ (keeping 15min), 20 ℃/min to 280 ℃ (keeping 5min).Split stream sampling 1 μ l (splitting ratio 10).
The mass spectrum condition: analyze with electron-bombardment (electron impact EI) source, electron energy is 70eV, 260 ℃ of ion source temperatures, 250 ℃ of interface temperature.When choosing full fragment ion scan pattern, the mass scanning scope is 50-600, solvent delay 3.5min.
24 kinds of sugared total ion current figure as shown in figure 16; part monose ion flow graph such as Fig. 9; Figure 11; shown in Figure 13; the color atlas result as shown in figure 16,7 kinds of aldoses; 1 kind of ketose; 1 kind of sugar alcohol; 1 kind of osamine and a kind of uronic acid acetylate mass spectrum (being positioned at the figure of top) and standard mass spectral database ring type monose acetylize standard spectrogram (being positioned at the figure of below) comparison diagram are shown in Figure 17-27: ribodesose (Figure 17); pectinose (Figure 18); Fucose (Figure 19); rhamnosyl (Figure 20); wood sugar (Figure 21); semi-lactosi (Figure 22); glucose (Figure 23); fructose (Figure 24); inositol (Figure 25); osamine (Figure 26); galacturonic acid (Figure 27).
With acetylize method behind the sodium borohydride reduction of the oximate of following fructose and fructose, glucose etc. in contrast:
The oximate working method of fructose is as follows, getting 10mg fructose standard specimen is dissolved in the 1ml water and is made into mother liquor, get the 10ul mother liquor and place glass capillary, after vacuumizing drying, (the 1-Methylimidazole dissolves, 100mg/ml) to add 10ul oxammonium hydrochloride solution, 80 ℃ of water-baths 5 minutes, add the 10ul diacetyl oxide then, mixing reacted 5 minutes.Add 10ul water and 10ul ethyl acetate, vibrate after 5 minutes, leave standstill and get organic phase 1ul and advance GC/MS.Make the ion flow graph.The result as shown in Figure 2.
The acetylize method is as follows behind the sodium borohydride reduction of fructose, glucose etc.: get 10mg monose standard specimen and be dissolved in the 1ml water and be made into mother liquor; get the 200ul mother liquor; (the dimethyl sulfoxide (DMSO) configuration 0.02g/ml), was reacted 90 minutes in 40 ℃ of shaking tables behind the vibration mixing to add the 2ml sodium borohydride solution.Add the 0.2ml Glacial acetic acid, mixing reaction 5 minutes adds the 0.4ml1-Methylimidazole with above solution, adds the 4ml diacetyl oxide more gradually, stirred 10 minutes, and added the 8ml ethyl acetate, and wash three times with 20ml, get organic phase and be spin-dried for, redissolve with the 1ml ethyl acetate at last, get 1ul and advance the GC/MS detection.Make the ion flow graph.Result such as Fig. 4, shown in Figure 6.
The above results shows; under ring type aldose state; because the aldehyde radical of aldose forms the ring texture of hemiacetal; thereby reduced the polarity of aldose molecule; under this state; directly it is carried out acetylize, just can form the peculiar acetylate of this aldose, and will the aldose reduction not become the sugar alcohol form.After the ketose crystal directly is dissolved in this solvent; it is same because ketone group participates in forming cyclic configuration; and can't form under the straight chain state with C-2 is the epimer of new symmetry centre; therefore both solve the problem that in sugared oxime acetylize method oxammonium hydrochloride can't the oximate ketone group, and avoided in sugar alcohol acetylize method straight chain ketose to be reduced the result of the sugar alcohol that is called two kinds of correspondences again.Specific as follows described:
1) to using the solution that sugared oxime acetylize derivatize can't detect the problem of ketose always
As far back as nineteen sixty-eight, Pigman and Isbell discover that the crystallization of every kind of sugar has single conformation.Dimethyl sulfoxide (DMSO) (DMSO) can be kept the ring type configuration of monose.The present invention is directed to existing problem in the current monose derivatization method, utilize DMSO can keep these characteristics of monose crystal state ring type configuration, in DMSO, directly carry out the acetylize derivatize.
After the ketose crystal directly is dissolved in the DMSO solvent; because ketone group participates in forming cyclic configuration; and can't form under the straight chain state with C-2 is the epimer of new symmetry centre, has therefore solved the problem that in sugared oxime acetylize method oxammonium hydrochloride can't the oximate ketone group.The ketose reaction formula as shown in Figure 8 in the DMSO solvent; total ion current figure as shown in Figure 9 for ring type fructose acetylate (directly acetylize among the DMSO); the result shows; because the ring type structure of fructose crystalline state is well kept in DMSO; there is not the appearance at isomery peak; therefore after the acetylize when chromatographic separation, the peak type is unique, retention time in advance.
2) to the in-problem solution of sugar alcohol acetylize derivatize mechanism
Adopting dimethyl sulfoxide (DMSO) (DMSO) is solvent, and this organic solvent polarity (7.2) slightly is weaker than water (10), and is comparatively approaching with the polarity of monose molecule, the monose crystal is dissolved in this solvent after, can keep single ring type structure under the monose crystalline state.Under ring type aldose state; because the aldehyde radical of aldose forms the ring texture of hemiacetal; thereby reduced the polarity of aldose molecule; under this state; directly it is carried out acetylize (reaction formula as shown in figure 10); just can form the peculiar acetylate of this aldose, and will the aldose reduction not become sugar alcohol form (as shown in figure 11).Figure 11 shows, the glucose crystal is dissolved in the DMSO solution, and the crystalline ring texture is kept, and during stratographic analysis, the peak type is unique, with sorbyl alcohol (sorbitol, Fig. 4) chromatographic peak zero lap.
After the ketose crystal directly is dissolved in dimethyl sulfoxide (DMSO) (DMSO); it is same because ketone group participates in forming cyclic configuration; and can't form under the straight chain state with C-2 is the epimer (Figure 12) of new symmetry centre; therefore both solve the problem that in sugared oxime acetylize method oxammonium hydrochloride can't the oximate ketone group, and avoided in sugar alcohol acetylize method straight chain ketose to be reduced result's (shown in Fig. 5, Fig. 6, table 1) of the sugar alcohol that is called two kinds of correspondences again.Shown in Figure 12,13 because the ring type structure of fructose crystalline state is well kept, do not have the appearance at isomery peak in DMSO, so after the acetylize when chromatographic separation, the peak type is unique.
3) though Silicane Method can detection of acidic monose (saccharic acid) and alkaline monose osamine, because the strict water-less environment of this method, its use is subjected to restriction to a certain degree.Acid monose such as glucuronic acid, galacturonic acid and alkaline monose such as glucose grapes glucosamine are dissolved among present method solvent DMSO; can keep the annular lactone structure of acid monose and the ring texture of alkaline monose osamine (as Figure 14, Figure 15) and to its direct acetylize; can generate the acetylate that is used for stratographic analysis, so present method also is applicable to the analyzing and testing to acid monose and alkaline osamine.
Two, the typical curve and the lowest detectable limit of monose derivatize of the present invention qualitative analysis
Getting organic phase that 24 kinds of monose that the step 2 of 100ul step 1 obtains obtain after with dimethyl sulfoxide (DMSO), diacetyl oxide and 1-Methylimidazole acetylpyridine derivativeization respectively is mixed in 1 test tube and (establishes 3 repetitions altogether), after low temperature is drained, redissolve with the 100ul ethyl acetate, stepwise dilution is 100ng/ul, 50ng/ul, 25ng/ul, 12.5ng/ul, 6.25ng/ul, 1ng/ul, 0.5ng/ul, 0.25ng/ul, 0.1ng/ul then.Each concentration is got 1ul respectively and is advanced the GC/MS detection from low to high.Typical curve is got 100ng/ul, 50ng/ul, 25ng/ul, 12.5ng/ul respectively, six points of 6.25ng/ul, 1ng/ul are done, each some triplicate.Lowest detectable limit is respectively the concentration of each monose in three times of signal to noise ratios of GC/MS detection baseline, and minimum detection by quantitative limit is respectively each monose detects ten times of signal to noise ratios of baseline at GC/MS concentration.Wherein, chromatogram and mass spectroscopy condition are as described in the step 2 of step 1, the result is as shown in table 2,24 kinds of sugared resolution are good as can be seen from Table 2, resolution between 24 kinds of sugar is all greater than 1.5, and minimum (theory) detectability can reach the fmol level, and lowest detectable limit reaches 10-15fmol, linear range is good, and is good in the scope internal linear of 0.05-50mg/L.
Table 2. monose and derived material SPECTROSCOPIC CHARACTERIZATION thereof
| Retention time (minute) | Kind | Response factor (mmu/fmol) | R 2 | Lowest detection line S/N=3 (fmol) | Linearity range (pmol) | Resolution |
| 10.03 | Erythritol | 746.47 | 0.9993 | 4.18 | 10 4 | |
| 10.24 | 2-deoxidation-ribose | 222.63 | 0.9988 | 12.75 | 10 4 | 2.211>1.5 |
| 12.47 | 2-deoxidation-ribitol | 61.70 | 0.9975 | 46.01 | 10 4 | 46.526>1.5 |
| 13.34 | Wood sugar | 392.47 | 0.9990 | 6.46 | 10 4 | 15.652>1.5 |
| 13.55 | Rhamnosyl | 34.89 | 0.9993 | 59.89 | 10 4 | 4.000>1.5 |
| 13.70 | Fucose | 885.33 | 0.9992 | 2.62 | 10 4 | 3.275>1.5 |
| 14.07 | Pectinose | 39.70 | 0.9978 | 63.89 | 10 4 | 9.441>1.5 |
| 14.30 | Glucuronic acid | 18.54 | 0.9907 | 96.77 | 10 4 | 4.425>1.5 |
| 15.09 | Rhamnitol | 32.73 | 0.9971 | 63.84 | 10 4 | 11.375>1.5 |
| 15.15 | Ribitol | 634.49 | 0.9987 | 99.61 | 10 4 | 2.449>1.5 |
| 15.27 | Fucitol | 19.14 | 0.9986 | 121.13 | 10 4 | 4.898>1.5 |
| 15.33 | Arabitol | 40.41 | 0.9967 | 62.76 | 10 4 | 2.667>1.5 |
| 15.39 | Galacturonic acid | 3.00 | 0.9997 | 597.50 | 10 4 | 2.479>1.5 |
| 15.54 | Xylitol | 226.60 | 0.9980 | 11.19 | 10 4 | 8.354>1.5 |
| 16.81 | Semi-lactosi | 61.11 | 0.9979 | 34.57 | 10 4 | 61.951>1.5 |
| 16.98 | Glucose | 68.35 | 0.9988 | 30.94 | 10 4 | 8.000>1.5 |
| 17.12 | Fructose | 27.06 | 0.9978 | 78.10 | 10 4 | 2.000>1.5 |
| 17.47 | Inositol | 885.33 | 0.9984 | 2.39 | 10 4 | 10.769>1.5 |
| 17.90 | N.F,USP MANNITOL | 761.39 | 0.9990 | 2.74 | 10 4 | 20.408>1.5 |
| 18.04 | Sorbyl alcohol | 667.88 | 0.9985 | 3.13 | 10 4 | 4.898>1.5 |
| 18.15 | Melampyrum | 60.43 | 0.9964 | 34.97 | 10 4 | 5.714>1.5 |
| 19.90 | N-acetyl-D-glucosamine | 24.28 | 0.9986 | 106.54 | 10 4 | 15.909>1.5 |
| 36.34 | Trehalose | 7.94 | 0.9988 | 126.66 | 10 4 | 109.251>1.5 |
| 37.04 | Sucrose | 6.57 | 0.9982 | 169.18 | 10 4 | 3.9>1.5 |
Three, recovery of standard addition
(vegetable material is taken from the about 10 years fresh blade of Cortex Populi Tomentosae of growth in the Beijing Forestry University campus for standard model, plant sample, liquid nitrogen flash freezer is standby immediately after taking off) and plant sample in add standard model and detect the peak area that obtains chromatographic peak through extracting the back with instrument (instrument and chromatograph mass spectrum analysis condition as the step 2 of step 1 as described in) respectively, calculate the sample size of these peak area correspondences respectively with the typical curve formula, be designated as S0, S1 and S2 respectively, the rate of recovery=(S2-S1)/S0 * 100%.Three concentration of 24 kinds of standard models are added in this experiment in the fresh blade powder of plant sample Cortex Populi Tomentosae, be respectively to add 24 kinds of sugared 5ng, 10ng, 20ng standard model in every milligram of plant sample respectively.Mixture (the DMSO: diacetyl oxide: 1-Methylimidazole=25:3:1) that adds dimethyl sulfoxide (DMSO), diacetyl oxide and 1-Methylimidazole then respectively, concrete operations are as described below: take by weighing nine parts of plant samples, every part of 50mg, add 24 kinds of sugared 5ng, 10ng, 20ng standard model respectively, three kinds of standard model concentration are divided equally three repetitions.(580ul of dimethyl sulfoxide (DMSO), diacetyl oxide and 1-Methylimidazole=25:3:1) extracts and derivatize 1 hour, adds 500ul ethyl acetate and 500ul water, and vibration mixing standing demix after 5 minutes is got organic phase 1ul and advanced GC/MS and analyze to add derivatization reagent then.
The result is as shown in table 3, adds monose or disaccharide, monose rate of recovery height, good reproducibility as can be seen from Table 3 in the plant sample.
Table 3 monose and derivative recovery of standard addition thereof and precision detect (n=3)
The result of above-mentioned steps one, step 2, step 3 shows; 24 kinds of sugar can obtain effective acetylize under dimethyl sulfoxide (DMSO), diacetyl oxide and 1-Methylimidazole derivatization method; the natural ring texture of aldose, ketose, uronic acid, osamine is kept; and when chromatographic separation; resolution between 24 kinds of sugar is all greater than 1.5; lowest detectable limit reaches 10-15fmol; scope internal linear at 0.05-50mg/L is good; three kinds of different concns mark-on average recovery rates are more than 90%, and relative standard deviation is between 0.01%-5%.The result shows that 24 kinds of sugared resolution are good, and linear range is good, and minimum (theory) detectability can reach the fmol level; 24 kinds of sugared rate of recovery height, good reproducibility.
Four, the composition of each reagent and proportioning thereof screening in the derivatization method of the present invention
With Glucose-Galactose-fructose is example, and the best proportioning of solvent DMSO, catalyzer 1-Methylimidazole and acetylation reagent diacetyl oxide is screened, and concrete grammar is as described below:
Laboratory apparatus and material
Vacuum concentration instrument, vacuum lyophilization instrument, Finnigan gaschromatographic mass spectrometry logotype instrument, microsyringe etc.With 3 kinds of common in plant sample monose is example, and glucose (purity〉99.0%), semi-lactosi (purity〉99.0%), fructose (purity〉99.0%) standard specimen is all bought the company from Sigma (USA).Dimethyl sulfoxide (DMSO) (chromatographically pure), 1-Methylimidazole (analytical pure), diacetyl oxide (analytical pure), ethyl acetate (chromatographically pure) are all bought from internal reagent company.
Experimental technique
With 3 kinds of simple sugar glucose, semi-lactosi, fructose common in the plant sample is example, gets three kinds of monose 0.5mg respectively, adds in the solution of four groups of different reagent proportionings to carry out derivatize, every group of three repetitions.In first group, three kinds of monose are total to 1.5mg, and its total hydroxyl value and diacetyl oxide ratio are 1:5, DMSO: diacetyl oxide: 1-Methylimidazole=25:1.5:1 adds 500ul, 30ul, 20ul respectively.In second group, three kinds of monose are total to 1.5mg, and its total hydroxyl value and diacetyl oxide ratio are 1:10, DMSO: diacetyl oxide: 1-Methylimidazole=25:3:1 adds 500ul, 60ul, 20ul respectively.In the 3rd group, three kinds of monose are total to 1.5mg, and its total hydroxyl value and diacetyl oxide ratio are 1:20, DMSO: diacetyl oxide: 1-Methylimidazole=25:6:1 adds 500ul, 120ul, 20ul respectively.In first group, three kinds of monose are total to 1.5mg, and its total hydroxyl value and diacetyl oxide ratio are 1:40, DMSO: diacetyl oxide: 1-Methylimidazole=25:12:1 adds 500ul, 240ul, 20ul respectively.Behind the room temperature reaction 5 minutes, to adding 500ul ethyl acetate and 500ul water respectively in four groups of experiments, spiral is after five minutes, leave standstill phase-splitting after, get organic phase 1ul and advance GC/MS and detect.Wherein, chromatogram and mass spectroscopy condition are as described in the step 2 of step 1.
Result such as table 3 and shown in Figure 28 can be obtained by experimental result, when monose hydroxyl value and diacetyl oxide mol ratio are 1:10, and when DMSO, diacetyl oxide mix with 25:3:1 with the 1-Methylimidazole, the derivatize best results.
Table 3.DMSO, 1-Methylimidazole and diacetyl oxide mix the influence to derivative reaction in varing proportions
| Numbering | Glc-Gal-F ru total amount (mg) | Diacetyl oxide (uL) | Mol is than (monose hydroxyl value and diacetyl oxide) | 1-Methylimidazole (uL) | Methyl-sulphoxide (uL) | Trisaccharide total peak area (rAU) |
| 1 | 1.5 | 30 | 1∶5 | 20 | 500 | 37292840±5810450 |
| 2 | 1.5 | 60 | 1∶10 | 20 | 500 | 41709920±4601567 |
| 3 | 1.5 | 120 | 1∶20 | 20 | 500 | 35364329±3941964 |
| 4 | 1.5 | 240 | 1∶40 | 20 | 500 | 30239701±4728519 |
For easy to use, above-mentioned three kinds of reagent can be formed a test kit, three kinds of reagent independent packagings or divide two kinds of reagent packing to preserve in the test kit: reagent A (DMSO+1-Methylimidazole) and reagent B (DMSO+ diacetyl oxide); Reagent A is the mixed solution of DMSO and 1-Methylimidazole, and reagent B is mixed solution when packing of DMSO and diacetyl oxide, makes wherein DMSO, diacetyl oxide and 1-Methylimidazole with 25:3:1.
To the detection of solubility monose and derivative thereof in the fresh blade of Cortex Populi Tomentosae, concrete grammar is as follows to utilize the derivatization method (DMSO, diacetyl oxide mix with 25:3:1 with the 1-Methylimidazole) of embodiment 1:
1. laboratory apparatus and material
24 kinds of sugared standard specimens are all bought the company from Sigma (USA), are respectively: ribodesose (purity〉99.0%), rhamnosyl (purity〉98.0%), Fucose (purity〉99.0%), wood sugar (purity〉99.0%), pectinose (purity〉99.0%), semi-lactosi (purity〉99.0%), glucose (purity〉99.0%), erythritol (purity〉99.0%), ribodesose alcohol (purity〉99.0%), rhamnitol (purity〉99.0%), fucitol (purity〉99.0%), Xylitol (purity〉99.0%), arabitol (purity〉99.0%), melampyrum (purity〉99.0%), sorbitol (purity〉99.0%), N.F,USP MANNITOL (purity〉99.0%), ribitol (purity〉99.0%), inositol (purity〉99.0%), fructose (purity〉99.0%), galacturonic acid (purity〉99.0%), glucuronic acid (purity〉99.0%), the N-acetylglucosamine (purity〉99.0%), sucrose (purity〉99.0%), trehalose (purity〉99.0%).Methyl alcohol (analytical pure), chloroform (analytical pure), ethyl acetate (chromatographically pure) are all bought from internal reagent company deionized water, electronic balance.Dimethyl sulfoxide (DMSO) (chromatographically pure), 1-Methylimidazole (analytical pure), diacetyl oxide (analytical pure) independent packaging respectively, perhaps be divided into reagent A and reagent B preserves according to embodiment 1 is described, wherein be that methyl-sulphoxide (DMSO) is pressed 25:6 blended mixed solution with diacetyl oxide in the reagent A, reagent B is that methyl-sulphoxide (DMSO) is pressed 25:2 blended mixed solution with the 1-Methylimidazole.
Methyl-sulphoxide was used the potassium hydroxide processed before using.
Vegetable material is taken from the about 10 years fresh blade of Cortex Populi Tomentosae of growth in the Beijing Forestry University campus, and liquid nitrogen flash freezer is standby immediately after taking off.
The use equipment comprises: vacuum concentration instrument, vacuum lyophilization instrument, baking oven, Finnigan gaschromatographic mass spectrometry logotype instrument, microsyringe etc.
The preparation of 2 monose standardized solution
Take by weighing respectively in the molten 1ml methyl-sulphoxide of 10mg sugar standard specimen, be made into mother liquor.Use the back to be stored in-80 ℃ of refrigerators at once.During the configuration standard curve, directly mother liquor is made into the standardized solution derivatize of other concentration.
3, extraction and derivatization treatment method
Take by weighing the fresh Cortex Populi Tomentosae blade powder of six parts of low-temperature freeze dryinies; every part of 50mg; get wherein three parts and add mentioned reagent A and reagent B 580ul altogether, make wherein to add extracting solution DMSO500ul, acetylation reagent diacetyl oxide 60ul, catalyzer 1-Methylimidazole 20ul, vibration was extracted 1 hour.Add 500ul ethyl acetate and 500ul water then, spiral is after 5 minutes, centrifugal 5 minutes 5000 rev/mins, get organic phase 1ul and directly advance GC/MS and analyze.Other three parts add extracting solution 500ul (chloroform: methyl alcohol: water=12:5:3) respectively, vibration was extracted 1 hour, centrifugal 5 minutes 5000 rev/mins, the taking-up supernatant vacuumizes and is spin-dried for, and adds 60ul diacetyl oxide and 20ul1-Methylimidazole, behind the vibration mixing, reacted 5 minutes, add 500ul ethyl acetate and 500ul water then, spiral was got organic phase 1ul and is directly advanced the GC/MS analysis after 5 minutes minutes.
Other gets fresh Cortex Populi Tomentosae blade and takes by weighing three parts of 50mg, behind the liquid nitrogen flash freezer, as for grinding in the mortar, takes out the back and adds DMSO500ul, and vibration was extracted 1 hour, added diacetyl oxide 60ul, catalyzer 1-Methylimidazole 20ul then, behind the vibration mixing, and room temperature reaction 5 minutes.Add 500ul ethyl acetate and 500ul water, spiral is after 5 minutes, centrifugal 5 minutes 5000 rev/mins, get organic phase 1ul and directly advance GC/MS and analyze.
Utilize 13.0 pairs of three kinds of methods of SPSS software to carry out the check of t value.
4, chromatogram and mass spectroscopy condition
The U.S.'s thermoelectric Finnigan gaschromatographic mass spectrometry logotype instrument, chromatographic condition: DB-17 post (30m * 0.318mmi.d, 0.25um film thickness), 250 ℃ of injector temperatures, carrier gas is high-purity He of 99.999%, flow velocity 1ml/min, press 26.3kPa before the initial post, 100 ℃ of column temperature (keeping 3.5min), 15 ℃/min to 160 ℃ (keeping 20min), 15 ℃/min to 200 ℃ (keeping 15min), 20 ℃/min to 280 ℃ (keeping 5min).Split stream sampling 1 μ l (splitting ratio 10).
The mass spectrum condition: analyze with electron-bombardment (electron impact EI) source, electron energy is 70eV, 260 ℃ of ion source temperatures, 250 ℃ of interface temperature.When choosing full fragment ion scan pattern, the mass scanning scope is 50-600, solvent delay 3.5min.
5, result and discussion
The result is as shown in table 4, and the result shows, utilizes derivatization reagent of the present invention to carry out derivatize, and the sensing range of monose kind obtains extending to 24 kinds by about 10 kinds.Sugar oxime acetylize method all can't be compared with present method with sugar alcohol acetyl method; By detected result as can be seen, derivatization reagent of the present invention has well solved the problem of the ketose that sugared oxime acetylize and sugar alcohol acetylize can't directly detect.Can be analyzed by following table 4 results and to obtain, dimethylsulfoxide extraction monose kind is compared with traditional extraction process with content does not have significant difference.
Table 4. Different Extraction Method is formed and the statistical study of content influence otherness solubility monose and derivative thereof in Cortex Populi Tomentosae (Populus tomentosa) blade
Utilize the fresh Cortex Populi Tomentosae blade cell wall sugar component of derivatization method (DMSO, diacetyl oxide mix with 25:3:1 with the 1-Methylimidazole) of embodiment 1 screening to analyze, concrete grammar is as follows:
1, experiment material
The reagent that uses has methyl-sulphoxide (DMSO), diacetyl oxide, 1-Methylimidazole, ethyl acetate etc.Monose and sugar alcohol standard specimen are erythritol, ribodesose, Fucose, rhamnosyl, wood sugar, pectinose, semi-lactosi, glucose, fructose, sorbose, ribitol, Xylitol, inositol, N.F,USP MANNITOL, sorbyl alcohol, glucuronic acid, galacturonic acid, N-acetylglucosamine, trehalose, sucrose.Above reagent and standard monose are analytical pure.Methyl-sulphoxide was used the potassium hydroxide processed before using.
Vegetable material is taken from the about 10 years fresh blade of Cortex Populi Tomentosae of growth in the Beijing Forestry University campus, and liquid nitrogen flash freezer is standby immediately after taking off.
The use equipment comprises: low-temperature and high-speed whizzer, temperature control shaking table, vacuum lyophilization instrument, Finnigan gaschromatographic mass spectrometry logotype instrument, microsyringe etc.
2, the preparation of monose standardized solution
Take by weighing respectively in the molten 1ml dehydration of the 20mg sugar standard specimen methyl-sulphoxide, be made into mother liquor.Use the back to be stored in-80 ℃ of refrigerators at once.During the configuration standard curve, directly mother liquor is made into the standardized solution derivatize of other concentration.
3, be insoluble to the preparation of ethanol powder (alcohol-insoluble residue)
Take by weighing the fresh blade of the natural air drying that about 10g smashed, add the PBS of about 300ml, place about 1 hour of 42 ℃ of water-baths, centrifugal, abandon supernatant, repeat 3 twice, add 80% ethanol, the centrifugal supernatant of abandoning, repeat 6 times, do not have color, 70 ℃ to supernatant, 95% ethanol is given a baby a bath on the third day after its birth inferior, after last the cleaning, and 7000rpm, centrifugal 15min abandons supernatant, adds 100ml acetone and hangs, 7000rpm, centrifugal 15min abandons supernatant and gets final product.In 45 ℃ of oven dry of baking oven, wait to remove starch at last.
Remove starch, with Type I porcine a-amylase (Sigma-Aldrich 23u/mg), join concentration be about 20u/m (50mM Tris-HCL, PH7.0).After the amylase enzymolysis sample, give a baby a bath on the third day after its birth time sample of water.DdH2O adds about 100-150ml acetone and washes once after cleaning three times, 10000rpm, centrifugal 7min, remove supernatant after, 45 ℃ of oven dry of baking oven.Powder is waited to do classification and is decomposed.
4, the mensuration of cell walls total reducing sugar
Each repeats to take by weighing 20mgA.I.R (being insoluble to the ethanol powder) sample and places dry test-tube, adds 72% (mass ratio) sulfuric acid, and mixing immediately vibrates.Hydrolysis 60min in 25 ℃ of shaking tables 200 change.Add inositol as interior mark.The vibration mixing after in Autoclave 121 ℃ of hydrolysis 90min.The cooling back adds the strong aqua neutralization.
5, the preparation of cell walls classification component and hydrolysis
Take by weighing 6 repetitions of A.I.R powder 100mg.Decompose the A.I.R powder with polygalacturonase reaction soln (3ul/ml).24 ℃ of 200rpm 24 hours in the shaking table.Collect supernatant in the 50ml centrifuge tube.Triplicate is used 2ml ddH
2O washing and precipitating three times is collected supernatant and is crossed glass fiber filter.Add Na
2CO
3(50mM) and EGTA (5mM) solution 10ml in the precipitation in, in the shaking table 24 ℃ 12 hours.Supernatant is crossed glass fiber filter and is collected in the 50ml centrifuge tube, and triplicate is used 2ml ddH
2O washing and precipitating three times is collected the merging supernatant and is crossed glass fiber filter.Add 1MKOH and 1% NaBH
4(DMSO solution) 8ml in precipitation, behind the vortex settling, be put in the shaking table 25 ℃ 12 hours.Triplicate is used 2ml ddH
2O washing and precipitating three times is collected supernatant and is crossed glass fiber filter.Add 4MKOH and 1%NaBH
4(DMSO solution) 8ml in precipitation, behind the vortex settling, be put in the shaking table 25 ℃ 12 hours.Triplicate is used 2ml ddH
2O washing and precipitating three times is collected the merging supernatant and is crossed glass fiber filter.More than each component BaCO
3Neutralization, dialysis, hydrolysis.
6, derivatization treatment
Get each hydrolyzed solution low-temperature freeze drying of 200ul step 5 preparation, add embodiment 2 described reagent A 260ul and reagent B260ul 580ul altogether, make DMSO: diacetyl oxide: 1-Methylimidazole 25:3:1, react after 5 minutes, add 500ul ethyl acetate and 500ul water, behind the spiral 5 minutes, centrifugal 5 minutes 5000 rev/mins, get organic phase 1ul and directly advance GC/MS and analyze.
7, chromatogram and mass spectroscopy condition
The U.S.'s thermoelectric Finnigan gaschromatographic mass spectrometry logotype instrument, chromatographic condition: DB-17 post (30m * 0.318mmi.d, 0.25um film thickness), 250 ℃ of injector temperatures, carrier gas is high-purity He of 99.999%, flow velocity 1ml/min, press 26.3kPa before the initial post, 100 ℃ of column temperature (keeping 3.5min), 15 ℃/min to 160 ℃ (keeping 20min), 15 ℃/min to 200 ℃ (keeping 15min), 20 ℃/min to 280 ℃ (keeping 5min).Split stream sampling 1ul (splitting ratio 10).
The mass spectrum condition: analyze with electron-bombardment (electron impact EI) source, electron energy is 70eV, 260 ℃ of ion source temperatures, 250 ℃ of interface temperature.When choosing full fragment ion scan pattern, the mass scanning scope is 50-600, solvent delay 3.5min.
8. result and discussion
The measurement result such as the table 5 of cell walls total reducing sugar, cell walls classification numbers of constituent monosaccharides kind and Determination on content are as a result shown in the table 6, the result shows, adopt derivatization reagent of the present invention to carry out derivatize and detect and contain proportional higher fructose component in the cell walls, 1. this is significant to the effect of research ketose in cell walls.2. remove to detect and contain fructose in the cell walls; also can detect part sugar alcohols such as N.F,USP MANNITOL, sorbyl alcohol; if traditional sugar alcohol acetylize method is adopted in this explanation; to cause analytical results can not reflect each true contents of monosaccharides; reason is that the fructose reduction is for N.F,USP MANNITOL and sorbitol; and seminose and glucose also can be reduced to N.F,USP MANNITOL and sorbitol respectively, and natural fine cell wall itself also contains a spot of N.F,USP MANNITOL and sorbitol.This directly may cause the analytical results distortion.Adopt derivatization method of the present invention to carry out the cell wall sugar proximate analysis of derivatize to the fresh blade of Cortex Populi Tomentosae, find to contain in this cell walls the fructose of high level first, this is significant to the physiological action of further research ketose in cell walls.This method has remedied former monose and the defective of derivative in acetylize is analyzed thereof, and has realized the Synchronization Analysis of aldose, ketose, sugar alcohol, uronic acid, osamine and the disaccharide of native configurations.
Table 5. Cortex Populi Tomentosae (Populus tomentosa) blade cell wall total monosaccharide is formed and content
Each monose and monosaccharide derivatives content and each component proportion in table 6. Cortex Populi Tomentosae (Populus tomentosa) the blade cell wall classification component
Claims (10)
1, polyol derivatization method is to be solvent with containing that the polyol sample places with the dimethyl sulfoxide (DMSO), is catalyzer with the 1-Methylimidazole, and diacetyl oxide is in the reaction system of derivatization reagent, carries out the acetylize derivative reaction.
2, derivatization method according to claim 1 is characterized in that: in the described reaction system, the interpolation volume ratio of DMSO, diacetyl oxide and 1-Methylimidazole is (5000～100): (5000～10): (2000～1).
3, derivatization method according to claim 2 is characterized in that: in the described reaction system, the interpolation volume ratio of DMSO, diacetyl oxide and 1-Methylimidazole is 50:(3-24): 2.
4, derivatization method according to claim 3 is characterized in that: in the described reaction system, the interpolation volume ratio of DMSO, diacetyl oxide and 1-Methylimidazole is 25:3:1.
5, method according to claim 1 is characterized in that: the described ratio that contains polyol sample and described dimethyl sulfoxide (DMSO) is 10-1g:1000-10ml, is preferably 1g:10ml.
6, according to any described method among the claim 1-5, it is characterized in that: described polyol is sugar and/or sugared derivative.
7, method according to claim 6 is characterized in that: described sugar is monose and/or disaccharide and/or trisaccharide and/or tetrose; Be preferably monose; The derivative of described sugar is one or more the arbitrary mixing in sugar alcohol, uronic acid and the osamine.
8, the application in the qualitative and/or detection by quantitative of any described method polyol among the claim 1-7 to sample.
9, application according to claim 8 is characterized in that: described polyol is sugar and/or sugared derivative.
10, application according to claim 9 is characterized in that: described sugar is monose and/or disaccharide and/or trisaccharide and/or tetrose; Be preferably monose.
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