CN105699478B - A kind of fast method of identification sugar - Google Patents
A kind of fast method of identification sugar Download PDFInfo
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Abstract
The present invention relates to the methods of PMP (1 phenyl, 3 methyl, 5 pyrazolone) derivatization coupled ion mobility tandem mass spectrum (IM MS/MS), which solve in existing identification oligosaccharide isomers technology, can not simply, fast and accurately analyze the problem of sugar-like product actually mixed, while solve the problems, such as using mass spectrum means can not determine sugar monosaccharide residue form.The present invention compared the effect that PMP and phenylhydrazine (PHN) are identified the isomers of sugar for derivatization reagent combination IM MS/MS methods, and theoretically collision cross-section (CCS) value of derivatization product is calculated, it further confirms that successfully Qualitative Identification can be carried out to the mixed extract of sugar with PMP derivatization combination IM MS/MS methods, and can identify connection, configuration and the composition of sugar simultaneously.
Description
Technical field
The invention belongs to analytical chemistry fields, are related to PMP（1-phenyl-3-methyl-5-pyrazolones ketone）Derivatization combine from
Sub- mobility tandem mass spectrum（IM-MS/MS）The method for identifying sugar, in particular to sugared Structural Identification in natural extracts
PMP derivatization coupled ion mobility tandem mass spectrums method.
Background technology
Oligosaccharides is information substance important in organism, and sugar plays an important role in a variety of bioprocess, as anti-freezing is lived
Property, immunological regulation, it is antitumor, hypoglycemic, inhibit pathogenic microorganisms, anti-oxidant, anti-inflammatory activity etc.（X. Zhang. Curr.
Med. Chem. 2006, 13, 1141-1147; S. Hakomori. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 225-232.）.Sugar connection, configuration, monosaccharide residue composition be always sugar structural research difficult point, sugar chain knot
The complexity of structure and the limitation of technological means much lag behind the research to its function, seriously to the analysis of the structure of sugar chain
Constrain sugar chain structure and emic research.And the difficulty that the sample of this biological source often isolates and purifies is also very big,
Very micro mixture is often obtained, there is presently no any type analytical technologies can solve all the problems above, compels to be essential
Establish the mixed isomers of fast and effectively method analysis sugar.
The method of traditional analysis oligosaccharides includes nuclear magnetic resonance（NMR）, gas chromatography-mass spectrum（GC/MS）And efficient liquid phase
Chromatography（HPLC）Combination（H. A. Currie, C. C. Perry. J. Chromatogr., A 2006, 1128, 90-
96.）.But NMR sensitivity is low, requires height to sample purity, is unable to get extensive use in the analysis of Natural Samples at present.
HPLC methods than relatively time-consuming, and in terms of distinguishing isomers nor especially effectively.Has many advantages, such as quick, sensitive mass spectrography
It plays an important role in terms of the structural analysis of sugar, the especially appearance of IM-MS.It is that one kind being based on ion in drift pipe
In with collision cross-section when buffer gas collisions（CCS）Difference, the technology that ion can be detached with shape by size.IM-MS
Maximum difference lies in increase an ion drift tube before mass spectrographic mass analyzer with traditional mass spectrum.In this drift tube
Filled with inert gas such as nitrogen or helium, when ion moves in drift tube, moving ion meeting and inert gas
Molecular collision, the cross section when ion and inert gas that shape, size are different even if institute is electrically charged identical, and quality is similar collide
Product also differs, i.e., " resistance " of ion motion is different so that ion is different in size by the time of drift tube, reaches and passes through shape
Shape distinguishes the purpose of ion.Its quick, unique gaseous ion separating capacity makes it become the ideal of the sugared isomers of analysis
Tool, be successfully used for sugar structure identification.In the negative ion mode by adding anion, combined using IM-MS/MS
HPLC can be distinguished preferably from the isolated sugared isomers of ox submaxillary gland mucin（H. Li, B. Bendiak, W. F.
Siems, D. R. Gang and H. H. Hill. Anal. Chem. 2015, 87, 2228−2235.）, but analysis pair
As for connection more than trisaccharide, configurational isomer, structural difference is apparent.Not yet using the smaller sugar of IM-MS separation molecular weight
It is completely separable（H. Li, K. Giles, B. Bendiak, K. Kaplan, W. F. Siems, H. H. Hill.
Anal. Chem. 2012, 84, 3231-3239.）.Researchers in a document on nature are published in the recent period to utilize
IM-MS identifies connection and the anomeric carbon of 6 trisaccharide isomers, but forms isomers and can not distinguish（J. Hofmann,
H. S. Hahm, P. H. Seeberger, K. Pagel. Nature 2015, 526, 241-244.）, it is seen that determine sugar
Monosaccharide residue composition be strictly difficult point.
As it can be seen that larger technical difficulty is always existed to the Structural Identification of sugar using IM-MS at present, even if spreading out to sugar
After biochemistry, the close sugar of structure is from being also difficult to effective differentiation.Researcher expects to find to sugared similar in structure always
Quickly and effectively analysis means.
PMP is originally by Honda etc.（S. Honda, E. Akao, S. Suzuki, M. Okuda, K. Kakehi
and J. Nakamura, Anal. Biochem. 1989, 180, 351–357.）Report that the label as reducing sugar tries
Agent is mainly used to improve the detection sensitivity of sugar, and since this reaction has many advantages, such as quick, mild, easy to operate, PMP is wide
The general derivatization reagent for being used as sugar.It yet there are no the isomers of PMP derivatization coupled ion mobility tandem mass spectrums identification sugar
Report is also had not been reported using IM-MS identifications structure especially closely related a large amount of monosaccharide and disaccharides isomers.Existing research
In method main successful analysis structure comparison in difference big sugar, and the present invention relates to sugared structure it is very close, including
Connect isomers（Such as maltose（α1→4Glc-Glc）And isomaltose（α1→6Glc-Glc））, configurational isomer（Such as malt
Sugar（α1→4Glc-Glc）And cellobiose（β1→4Glc-Glc））, composition isomers（Such as lactose（β1→4Gal-Glc）And fibre
Tie up disaccharides（β1→4Glc-Glc））And epimer（Such as galactolipin and mannose）.Due to the isomers of monosaccharide and disaccharides by
Smaller in molecular weight, poor specificity accomplishes that Qualitative Identification is the most difficult in IM-MS analytic processes.The present invention can be to structure
Especially close sugar realizes identification, can also be readily used for the identification of polysaccharide and oligosaccharide and its isomers.
Invention content
The present invention uses PMP derivatization coupled ion mobility tandem mass spectrums, provide a kind of rapidly and efficiently identification monosaccharide and
The method of disaccharides isomers can be used for monosaccharide and disaccharides Structural Identification in natural extracts.Which solve existing identification widows
In sugared isomery body technique, can not simply, fast and accurately analyze the problem of sugar-like product actually mixed, while solving utilization
Mass spectrum means can not determine the problem of monosaccharide residue composition of sugar.
The PMP derivatizations of monosaccharide and disaccharides isomers of the present invention refer to that monosaccharide and disaccharides isomers derive through PMP
Change and show difference so as to cause the CCS of derivatization product, to distinguish isomers.
IM-MS/MS methods of the present invention refer to utilizing the result of ionic mobility separation under the Mass Spectrometry Conditions of optimization
Isomers is identified jointly with the series connection result of the analyte to cannot be distinguished in first mass spectrometric.
The present invention provides a kind of recognition methods of sugar, which is characterized in that using derivatization coupled ion mobility mass spectrum into
Row analysis.
Further, derivatization reagent used in the present invention is 1-phenyl-3-methyl-5-pyrazolones ketone.
Further, the sugar being analyzed in the present invention is monosaccharide and/or disaccharides and its isomers.
Further, analyzed sugar is sucrose, lactose, kojibiose, nigerose, maltose, isomaltose, Chinese scholartree
It is one or more in sugar, laminaribiose, cellobiose, gentiobiose, glucose, galactolipin, mannose, fructose.
Further, intermediate ion mobility mass spectrum of the present invention uses tandem mass spectrum.
In certain embodiments, the mass spectrographic testing conditions of the ionic mobility are as follows：It is measured, is wriggled using ESI-IM-MS
Pumping sample introduction speed is：5μL/min；ESI ion source conditions are as follows：Positive ion mode, capillary voltage：2.8 kV, sample taper hole
Voltage：50 V extract orifice potential：4 V, source temperature：120 °C, desolvation temperature：350 °C, taper hole gas velocity：30 L/
h；IM-MS conditions：T-wave rates：550-2500 m/s, T-wave height：40 V, drift（Nitrogen）Flow velocity：90 mL/min.
In one embodiment of the invention, using PMP as derivatization reagent, using IM-MS/MS methods to special in structure
Closely related 4 monosaccharide isomers and 10 disaccharides isomers distinguish, and theoretically to the CCS values of derivatization product
It is calculated, with PMP derivatization combination IM-MS/MS methods to Aqueous soluble ginseng's monosaccharide（WGOS1）And disaccharides（WGOS2）'s
Mixed extract has carried out qualitative analysis.In level-one（It does not connect）Be concatenated can be with for undivided PMP derivatizations product in mass spectrum
It obtains single PMP and derives compound, their drift time（tD）Be worth it is variant, comprehensive first mass spectrometric and tandem mass spectrum as a result, sugar
Isomers can distinguish.And the product ion that undivided PHN derivatizations product is concatenated in first mass spectrometric is shown
Identical tDValue does not work to the isomers differentiation of sugar after series connection, therefore ideal effect is not achieved to the separation of sugared isomers
Fruit.
Technical scheme is as follows：
Ammonia water-methanol solution is prepared
NH3·H2O, which is added in methanol, fully to be vibrated, and the ammonia water-methanol solution of a concentration of 0.75-0.9 mol/L is obtained.
PMP derives liquid and prepares
PMP is configured to the solution of a concentration of 0.0349 mol/L, is stored in spare in 4 °C of refrigerators.
The extracting and developing of WGOS1 and WGOS2, enrichment
Dry ginseng is crushed to 50 ~ 80 mesh, is stayed overnight at room temperature with distilled water immersion, 2 ~ 3 are extracted under 70 °C
H, four layers of filtered through gauze, residue extract 2 times repeatedly again.Merge No. 3 extracting solutions and discard precipitation after 3500 rpm centrifuge 15 min,
Merge supernatant.With that is, by ginseng is obtained after supernatant rotary evaporation, freeze-drying.Thick oligosaccharide sample.By thick oligosaccharide sample with
Water is with mass ratio 7：1000 mixing carry out ultrafiltration classification, filter liquor phenol-with the ultrafiltration membrane that molecular cut off is 3000 Da
Sulfuric acid process monitors in real time, until not having color.It collects filter liquor and the oligomeric sugar-like of ginseng is lyophilized to obtain after rotary evaporation concentration
Product（WGOS）.
Gained sample is dissolved in the water, after 10000 rpm centrifuge 5 min, by supernatant up to the Bio-gel balanced
P-2 gel chromatography columns（1.0 cm × 100 cm）.Then be washed with deionized water it is de-, flow velocity be 0.06 mL/min, collect elution
Liquid.0.2 mL eluents are taken out from every pipe, measure sugared content therein with phend-sulphuric acid, and draw tomographic map.According to washing
De- curve collects the eluent of WGOS1 and WGOS2.
The PMP derivatization processes of standard items and extract
Standard items：It is 200 μ L, the monosaccharide of 2.5 mmol/L or two saccharides to react total volume, and deriving liquid with PMP mends
Foot.
Extract：Reaction total volume is 200 μ L, and WGOS1 the and WGOS2 mixtures of final concentration of 10 g/L are spread out with PMP
Raw liquid is supplied.
Standard items and extract are centrifuged in 10000 rmp, take supernatant, and 1 ~ 2 h is reacted in 60 ~ 80 °C of tepidarium.Instead
Ying Hou, centrifuging and taking supernatant high-purity N2Drying, after adding Milli-Q water fully to dissolve, 10000 rmp centrifuge 10 min, take
Clear liquid.In order to remove excessive PMP derivatization reagents in solution, add CCl3It fully vibrates, sub-cloud solution is removed after standing, repeatedly
Repeatedly, until lower layer is without color, the derivatization product of sample is finally obtained, is placed in 4 °C of refrigerators.
PHN derivatization processes
5 μ L phenylhydrazines are taken, the aqueous solution of 250 μ L sugar is added to（2.5μmol）In, react 1 h in 80 °C of stirred in water bath.
Reactant is extracted with ethyl acetate, and combined ethyl acetate layer is simultaneously washed with water, and combining water layer is placed in 4 °C of refrigerators.
Mass Spectrometry Conditions
Mass spectrograph：Waters Synapt G2 mass spectrographs, ion source：Electron spray ionisation source（ESI）Positive ion mode, capillary
Tube voltage：2.8 kV, sample orifice potential：50 V extract orifice potential：4 V, source temperature：120 °C, desolvation temperature：
350 °C, taper hole gas velocity：30 L/h, T-wave rate：450 ~ 5000 m/s, T-wave height：38 ~ 40 V, drift gas
For nitrogen, flow velocity：90 mL/min, collision gas are argon gas, trap collision energies：35~40 V.
Standard items and extract are analyzed by above-mentioned Mass Spectrometry Conditions；
The calculating of CCS
CCS is ion average available cross-section, that is, collision cross-section in drift Guan Zhongyu buffer gas collisions（Ǻ2）, molecule
Collision cross-section is an important feature, directly related with the three-dimensional construction in its chemical constitution and gas phase, the charge of ion, matter
Lotus ratio is the important parameter for influencing CCS.Different from parameters such as retention times, CCS values are not by different instrument and chromatographic process
It influences, can differentiation more reliably be made to compound.As molecular weight, allow scientist that can further investigate chemical constitution or more
A molecule is confirmed well.We are using the compound with known CCS values as correction object, in a variety of different T-wave rates
With the CCS values for obtaining determinand under the conditions of T-wave height（Average value under the conditions of multiple）.Collision by calculating ion is cut
Face can improve our to sugar structure understanding, according to CCS values theoretically can objectively judge isomers whether the areas Neng Bei
It separates.
CCS values are calculated according to following formula：
t′ D = tD – [(c)/1000] ①
Ω′ = Ω/[c × (1/μ)1/2] ②
μ= m× mgas/ (m+mgas) ③
lnΩ′ = X × lnt′ D + lnA ④
In formula, tDFor drift time, c is institute's charge number, and m/z is mass-to-charge ratio, and Ω is CCS values, mgasFor the quality of nitrogen
Number.Under the conditions of a certain specific T-wave rates and T-wave height to correction object be measured, according to formula 1. -4. find out
X and lnA, determinand are also measured under corresponding conditions, find out Ω.Finally find out in different T-wave rates and T-wave
The average value of Ω under the conditions of height is the CCS values of the determinand.
Preferably, step（1）In, a concentration of 0.82 mol/L of the ammonia water-methanol solution.
Preferably, step（2）In, the PMP, which derives liquid, to be formulated by the ammonia water-methanol of 0.82 mol/L.
Preferably, step（4）In, the standard items and extract react 2 h in 70 °C of tepidarium.
Preferably, step（6）In, the T-wave rates are 550 ~ 2500 m/s, 450 ~ 5000 m/s and 800
M/s, T-wave height are 40 V.
The present invention provides the method that PMP derivatization combinations IM-MS/MS identifies all kinds of sugared isomers, in particular to
The method of the PMP derivatization coupled ion mobility tandem mass spectrums of sugared Structural Identification in natural extracts.The present invention spreads out PMP
Biochemistry combines in the identification of the sugared isomers of IM-MS/MS methods application, and connection, configuration and composition isomers can be by accurate, quick
Identification.The CCS values for calculating related substances simultaneously, can also distinguish isomers, and theoretically confirm using CCS
The size of collision cross-section is to influence the principal element of separating degree.Another advantageous effect of the present invention is can be to the mixture of sugared isomery
It directly and accurately analyzes, the present invention, which can organize sugar to learn, generates important influence.
Description of the drawings
Fig. 1 is the ionic mobility mass spectrogram of 10 kinds of disaccharides isomers and 4 kinds of monosaccharide isomers original shapes in embodiment 1.
Fig. 2 is the scatter plot that the CCS of monosaccharide and disaccharides isomers changes before and after PHN and PMP derivatizations in embodiment 2.
Fig. 3 is the ionic mobility mass spectrogram of PHN derivatizations monosaccharide and disaccharides isomers in embodiment 3.
Fig. 4 is the ionic mobility mass spectrogram of PMP derivatizations monosaccharide and disaccharides isomers in embodiment 4.
Fig. 5 is the ionic mobility tandem mass spectrum figure of 6 PMP derivatization disaccharides isomers in embodiment 4.The columns a：Protonation
Disaccharides-PMP derivatization products as a result, the columns b：The result of the disaccharides-PMP derivatization products of sodium ion.
Fig. 6 is the ionic mobility mass spectrogram of PMP derivatizations monosaccharide and disaccharides isomers in embodiment 5.
Fig. 7 is the ionic mobility tandem mass spectrum figure of 5 PMP derivatization disaccharides isomers in embodiment 5.The columns a：Protonation
Disaccharides-PMP derivatization products as a result, the columns b：The result of the disaccharides-PMP derivatization products of sodium ion.
Fig. 8 is the ionic mobility mass spectrogram of PMP derivatizations monosaccharide and disaccharides isomers in embodiment 6.
Fig. 9 is the ionic mobility tandem mass spectrum figure of 5 PMP derivatization disaccharides isomers in embodiment 6.The columns a：Protonation
Disaccharides-PMP derivatization products as a result, the columns b：The result of the disaccharides-PMP derivatization products of sodium ion.
Figure 10 is the ionic mobility mass spectrogram of WGOS1 and WGOS2 in embodiment 7（a-d）Spread out with the PMP of WGOS1 and WGOS2
The ionic mobility mass spectrogram of biochemical products（e-h）.Dotted line representative is in wherein e and hm/z673.27 the tandem mass spectrum of ion
Figure.
Specific implementation mode
Embodiment 1
（1）The preparation of standard items
With 50% methanol-water（v/v）By sucrose, lactose, kojibiose, nigerose, maltose, isomaltose, sophorose,
It is 2 μM that laminaribiose, cellobiose, gentiobiose, glucose, galactolipin, mannose and fructose standard items, which are made into concentration,
Solution.
（2）The IM-MS of standard items is detected
The standard items prepared directly carry out ESI-IM-MS measurement, and peristaltic pump sample introduction speed is：5μL/min.ESI ion sources
Condition is as follows：Positive ion mode, capillary voltage：2.8 kV, sample orifice potential：50 V extract orifice potential：4 V, source temperature
Degree：120 °C, desolvation temperature：350 °C, taper hole gas velocity：30 L/h.IM-MS conditions：T-wave rates：550-2500
M/s, T-wave height：40 V, drift（Nitrogen）Flow velocity：90 mL/min.
The result is shown in Figure 1 compares the ionic mobility mass spectrogram of monosaccharide and disaccharides isomers, according to tD, find there was only sucrose（tD
= 3.36 ms）It can be distinguished with other disaccharides isomers, only glucose（tD= 2.17 ms）It can be different with other monosaccharide
Structure body distinguishes, and can not be distinguished between other monosaccharide and disaccharides isomers.
Embodiment 2
（1）Correct the preparation of object and standard substance to be measured
With 50% methanol-water（v/v）Object will be corrected（Sucrose, lactose, cellobiose, maltose, melibiose, trehalose, pine
Trisaccharide, raffinose and maltotriose）With standard substance to be measured（Kojibiose, nigerose, isomaltose, sophorose, kelp two
Sugar, gentiobiose, glucose, galactolipin, mannose and fructose）It is made into the solution that concentration is 2 μM.
（2）Ammonia water-methanol solution is prepared
Take the NH of 500 μ L a concentration of 28%3·H2O is added in 9.5 mL methanol and fully vibrates, obtains
The ammonia water-methanol solution of a concentration of 0.82 mol/L.
（3）PMP derives liquid and prepares
The PMP for weighing 60.67 mg is put in 10 mL volumetric flasks, constant volume after fully being dissolved with ammonia water-methanol solution, fully
Oscillation, is configured to a concentration of 0.0349 mol/L, is stored in spare in 4 °C of refrigerators.
（4）PMP derivatization processes
Weigh 4 kinds of monosaccharide standards（Each 0.17 mg）With 8 kind of two saccharide（Each 0.34 mg）, it is separately added into 200 μ L
Derivatization reagent is dissolved in the ep pipes of 1 mL, and 10000 rmp centrifuging and taking supernatants react 2 h in 70 °C of tepidarium.Instead
Ying Hou, centrifuging and taking supernatant high-purity N2Drying, after adding 1 mL Milli-Q water fully to dissolve, 10000 rmp centrifuge 10 min,
Take supernatant.In order to remove excessive PMP derivatization reagents in solution, add CCl31 mL fully vibrates, and sub-cloud is removed after standing
Solution, it is repeated multiple times, until lower layer is without color, the derivatization product of sample is finally obtained, is placed in 4 °C of refrigerators.
（5）PHN derivatization processes
5 μ L phenylhydrazines are taken, the aqueous solution of 250 μ L sugar is added to（2.5μmol）In, react 1 h in 80 °C of stirred in water bath.
Reactant is extracted with ethyl acetate（250 L × 3 time μ）, combined ethyl acetate layer is simultaneously washed with water（100 L × 3 time μ）, merge
Water layer is placed in 4 °C of refrigerators.
（6）Correct the IM-MS conditions that the CCS of object, saccharide and derivatization product is measured
16 IM-MS conditions：1. T-wave rates：400 m/s, T-wave height：40 V；2. T-wave rates：400
M/s, T-wave height：38 V；3. T-wave rates：400 m/s, T-wave height：35 V；4. T-wave rates：300
M/s, T-wave height：38 V；5. T-wave rates：800 m/s, T-wave height：40 V；6. T-wave rates：800
M/s, T-wave height：38 V；7. T-wave rates：800 m/s, T-wave height：35 V；8. T-wave rates：450-
5000 m/s, T-wave height：40 V；9. T-wave rates：450-5000 m/s, T-wave height：38 V；⑩ T-wave
Rate：450-5000 m/s, T-wave height：35 V；Remaining is the same as embodiment 1.T-wave rates：500-2500 m/s, T-
Wave height：40 V；T-wave rates：500-2500 m/s, T-wave height：38 V；T-wave rates：500-2500
M/s, T-wave height：35 V；T-wave rates：550-2500 m/s, T-wave height：40 V；T-wave rates：
550-2500 m/s, T-wave height：38 V；T-wave rates：550-2500 m/s, T-wave height：35 V.Remaining
With embodiment 1.
（7）The calculating of CCS
The CCS values of derivatization product are calculated according to following formula：
t′ D = tD – [(c)/1000] ①
Ω′ = Ω/[c × (1/μ)1/2] ②
μ= m× mgas/ (m+mgas) ③
lnΩ′ = X × lnt′ D + lnA ④
In formula, tDFor drift time, c is institute's charge number, and m/z is mass-to-charge ratio, and Ω is CCS values, mgasFor the quality of nitrogen
Number.Using the compound with known CCS values as correction object, in a certain specific T-wave rates and T-wave height conditions
Under, according to formula 1. -4. find out X and lnA, determinand is also measured under corresponding conditions, finds out Ω.Finally find out in difference
T-wave rates and T-wave height under the conditions of（Actual conditions are as illustrated in step 6）The average value of Ω be the determinand
CCS values.
As a result see Fig. 2, it is seen that CCS values increase apparent after derivatization, and the CCS values between partial allosteric body show difference.I.e.
Result of calculation shows that the glucide after derivatization can be detached.
Embodiment 3
PHN derivatizations process is with embodiment 2, and the IM-MS testing conditions of derivatization product are the same as embodiment 1.
As a result see Fig. 3, compare the ionic mobility mass spectrogram of monosaccharide and disaccharides isomers PHN derivatization products, according to tD, hair
Existing nigerose PHN derivatives（tD= 4.34 ms）, laminaribiose PHN derivatives（tD= 4.45 ms）And cellobiose
PHN derivatives（tD= 5.15 ms）It can be distinguished with other disaccharides isomers PHN derivatives, galactolipin PHN derivatives（tD
= 2.71 ms）It can be distinguished with other monosaccharide isomers PHN derivatives, it can not be between other monosaccharide and disaccharides isomers
Row is distinguished.
Embodiment 4
PMP derivatizations process is with embodiment 2, and the IM-MS testing conditions of derivatization product are the same as embodiment 1.
As a result see Fig. 4, compare the ionic mobility mass spectrogram of monosaccharide and disaccharides isomers PMP derivatization products, according to tD, hair
Existing maltose PMP derivatives（tD= 8.73 ms）, isomaltose PMP derivatives（tD= 8.03 ms）Spread out with laminaribiose PMP
Biology（tD= 5.15 ms）It can be distinguished with other disaccharides isomers PMP derivatives, the PMP of other disaccharides isomers spreads out
It can not distinguish between biology, can be distinguished between the PMP derivatives of three monosaccharide.
It connects to carrying out the still undivided 3 pairs of disaccharides isomers of mobility separation after PMP derivatizations（Ionic mobility detaches
The series connection carried out before）Mass spectral analysis.Collision gas is argon gas, trap collision energies：40 V.As a result as shown in Figure 5, a is utilized
The concatemer production of column protonation（The disaccharides isomers of single PMP derivatizations）TDValue, isomers can distinguish completely two-by-two.
Utilize the t of the concatemer production of the columns b sodium ionDValue, isomers can be distinguished partly two-by-two.
Embodiment 5
For PMP derivatizations process with embodiment 2, the IM-MS testing conditions of derivatization product are 800 in addition to T-wave rates
M/s, other are the same as embodiment 1.
As a result see Fig. 6, compare the ionic mobility mass spectrogram of monosaccharide and disaccharides isomers PMP derivatization products, according to tD, hair
Existing maltose PMP derivatives（tD= 9.33 ms）, isomaltose PMP derivatives（tD= 8.79 ms）, laminaribiose PMP spreads out
Biology（tD= 9.49 ms）With gentiobiose PMP derivatives（tD= 8.9 ms）It can derive with other disaccharides isomers PMP
Object distinguishes, and can not be distinguished between the PMP derivatives of other disaccharides isomers, can be between the PMP derivatives of three monosaccharide
It distinguishes.
It connects to carrying out the still undivided disaccharides isomers of mobility separation after PMP derivatizations（Ionic mobility detaches it
The series connection of preceding progress）Mass spectral analysis.Collision gas is argon gas, trap collision energies：40 V.As a result as shown in Figure 7, the columns a are utilized
The concatemer production of protonation（The disaccharides isomers of single PMP derivatizations）TDIt is worth, can be distinguished completely between isomers.It utilizes
The t of the concatemer production of the columns b sodium ionDIt is worth, can be partly distinguished between isomers.
Embodiment 6
For PMP derivatizations process with embodiment 2, the IM-MS testing conditions of derivatization product are 450- in addition to T-wave rates
5000 m/s, other are the same as embodiment 1.
As a result see Fig. 8, compare the ionic mobility mass spectrogram of monosaccharide and disaccharides isomers PMP derivatization products, according to tD, hair
Existing kojibiose PMP derivatives（tD= 7.22 ms）, nigerose PMP derivatives（tD= 7.32 ms）, maltose PMP derives
Object（tD= 7.49 ms）With laminaribiose PMP derivatives（tD= 7.70 ms）It can be with other disaccharides isomers PMP derivatives
It distinguishes, can not be distinguished between the PMP derivatives of other disaccharides isomers, it can be between the PMP derivatives of three monosaccharide
Row is distinguished.
It connects to carrying out the still undivided disaccharides isomers of mobility separation after PMP derivatizations（Ionic mobility detaches it
The series connection of preceding progress）Mass spectral analysis.Collision gas is argon gas, trap collision energies：40 V.As a result as shown in Figure 9, the columns a are utilized
The concatemer production of protonation（The disaccharides isomers of single PMP derivatizations）TDIt is worth, can be distinguished completely between isomers.It utilizes
The t of the concatemer production of the columns b sodium ionDIt is worth, can be partly distinguished between isomers.
Embodiment 7
（1）The extracting and developing of WGOS1 and WGOS2, enrichment
It takes dry ginseng to crush, weighs 500 g, stayed overnight at room temperature with 5 L distilled water immersions, 3 are extracted under 70 °C
H, four layers of filtered through gauze, residue extract 2 times repeatedly again.Merge No. 3 extracting solutions and discard precipitation after 3500 rpm centrifuge 15 min,
Merge supernatant.With that is, the thick oligosaccharide sample of ginseng will be obtained after supernatant rotary evaporation, freeze-drying.By thick oligosaccharide sample with
Water is with mass ratio 7：1000 mixing carry out ultrafiltration classification, filter liquor phenol-with the ultrafiltration membrane that molecular cut off is 3000 Da
Sulfuric acid process monitors in real time, until not having color.It collects filter liquor and the oligomeric sugar-like of ginseng is lyophilized to obtain after rotary evaporation concentration
Product（WGOS）.
Gained sample is weighed into 15 mg, is dissolved in 1 mL water, after 10000 rpm centrifuge 5 min, by supernatant up to flat
The Bio-gel P-2 gel chromatography columns to have weighed（1.0 cm × 100 cm）.Then it is eluted with 96 mL deionized waters, flow velocity is
0.06 mL/min, 1.2 mL eluents of often pipe collection.0.2 mL eluents are taken out from every pipe, it is measured with phend-sulphuric acid
In sugared content, and draw tomographic map.The eluent of WGOS1 and WGOS2 is collected according to elution curve.
（2）PMP derivatization processes
Weigh WGOS1 and WGOS2 mixtures（2 mg）, it is separately added into the ep pipes that 200 μ L derivatization reagents are dissolved in 1 mL
In, remaining is the same as embodiment 2.
（3）The IM-MS/MS of derivatization product is detected
The IM-MS testing conditions of derivatization product are the same as embodiment 1.Collision gas is argon gas, trap collision energies：40 V.
The results are shown in Figure 10, it is before and after Integrated comparative derivatization as a result, understand WGOS1 by fructose and glucose group at,
WGOS2 is made of sucrose and maltose.
Claims (5)
1. low in a kind of identification ginseng using 1-phenyl-3-methyl-5-pyrazolones one derivative coupled ion mobility tandem mass spectrum
The method of glycan, which is characterized in that include the following steps：
(1) ammonia water-methanol solution is prepared
NH3·H2O, which is added in methanol, fully to be vibrated, and the ammonia water-methanol solution of a concentration of 0.75-0.9mol/L is obtained；
(2) 1-phenyl-3-methyl-5-pyrazolones ketone derives liquid and prepares
1-phenyl-3-methyl-5-pyrazolones ketone is configured to the solution of a concentration of 0.0349mol/L, is stored in standby in 4 DEG C of refrigerators
With；
(3) extracting and developing of ginseng oligosaccharide sample, enrichment
Dry ginseng is crushed to 50~80 mesh, is stayed overnight at room temperature with distilled water immersion, 2~3h of extraction at 70 DEG C, four
Layer filtered through gauze, residue extract 2 times repeatedly again；Merge No. 3 extracting solutions and discard precipitation after 3500rpm centrifuges 15min, in merging
Clear liquid；The thick oligosaccharide sample of ginseng will be obtained after supernatant rotary evaporation, freeze-drying；By thick oligosaccharide sample and water with mass ratio 7：
1000 mixing, the ultrafiltration membrane for being 3000Da with molecular cut off carry out ultrafiltration classification, and filter liquor is supervised in real time with phend-sulphuric acid
Control, until not having color；It collects filter liquor and ginseng oligosaccharide sample is lyophilized to obtain after rotary evaporation concentration；By gained sample
It is dissolved in the water, it is after 10000rpm centrifuges 5min, supernatant is solidifying up to the 1.0cm × 100cm Bio-gel P-2 balanced
Glue chromatographic column；Then de-, flow velocity 0.06mL/min is washed with deionized water, collects eluent；0.2mL elutions are taken out from every pipe
Liquid measures sugared content therein with phend-sulphuric acid, and draws tomographic map；Ginseng oligosaccharide sample 1 is collected according to elution curve
With the eluent of ginseng oligosaccharide sample 2；
(4) the 1-phenyl-3-methyl-5-pyrazolones one derivative process of standard items and extract
Standard items：The monosaccharide or two saccharides that total volume is 200 μ L, 2.5mmol/L are reacted, with 1- phenyl -3- methyl -5- pyrroles
Oxazoline ketone derives liquid and supplies；
Extract：React the ginseng oligosaccharide sample 1 and ginseng oligosaccharide sample 2 that total volume is 200 μ L, final concentration of 10g/L
Mixture derives liquid with 1-phenyl-3-methyl-5-pyrazolones ketone and supplies；Standard items and extract are centrifuged in 10000rmp, are taken
Clear liquid reacts 1~2h in 60~80 DEG C of tepidarium；After reaction, centrifuging and taking supernatant high-purity N2Drying, adds Milli-Q water
Fully after dissolving, 10000rmp centrifuges 10min, and supernatant is taken to add CCl3It fully vibrates, sub-cloud solution is removed after standing, it is repeatedly more
It is secondary, until lower layer is without color, the derivatization product of sample is finally obtained, is placed in 4 DEG C of refrigerators；
(5) Mass Spectrometry Conditions
Mass spectrograph：Waters Synapt G2 mass spectrographs, ion source：Electron spray ionisation source positive ion mode, capillary voltage：
2.8kV, sample orifice potential：50V extracts orifice potential：4V, source temperature：120 DEG C, desolvation temperature：350 DEG C, taper hole gas
Flow velocity：30L/h, T-wave rate：550-2500m/s, T-wave height：38~40V, drift gas are nitrogen, flow velocity：
90mL/min, collision gas are argon gas, trap collision energies：35~40V；Standard items and extract press above-mentioned Mass Spectrometry Conditions into
Row analysis.
2. the method as described in claim 1, which is characterized in that ammonia water-methanol solution is a concentration of in the step (1)
0.82mol/L。
3. the method as described in claim 1, which is characterized in that 1-phenyl-3-methyl-5-pyrazolones in the step (2)
Ketone, which derives liquid, to be formulated by the ammonia water-methanol of 0.82mol/L.
4. the method as described in claim 1, which is characterized in that in step (4), the standard items and extract are in 70 DEG C of temperature
2h is reacted in water-bath.
5. the method as described in claim 1, which is characterized in that in step (5), the T-wave rates be 550~
2500m/s, T-wave height are 40V.
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| EP3559657A1 (en) * | 2016-12-23 | 2019-10-30 | Roche Diagnostics GmbH | Method for identifying a reagent during a process in an analysis system |
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