CN105699478A - Method for quickly identifying sugar - Google Patents
Method for quickly identifying sugar Download PDFInfo
- Publication number
- CN105699478A CN105699478A CN201610160329.3A CN201610160329A CN105699478A CN 105699478 A CN105699478 A CN 105699478A CN 201610160329 A CN201610160329 A CN 201610160329A CN 105699478 A CN105699478 A CN 105699478A
- Authority
- CN
- China
- Prior art keywords
- pmp
- sugar
- derivatization
- sample
- isomer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
- G01N27/68—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode using electric discharge to ionise a gas
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
Abstract
The invention relates to a method of combining PMP (1-Phenyl-3-Methyl-5-Pyrazolone) derivatization and IM-MS/MS (Ion Mobility Tandem Mass Spectrometry), which solves the problem that an actually mixed sugar sample cannot be analyzed easily, quickly and accurately in the prior art for identifying oligosaccharide isomers, and meanwhile, solves the problem that the compositions of monosaccharide residues of the sugar cannot be determined by utilizing a mass spectroscopy means. According to the method for quickly identifying the sugar, the effects on identifying sugar isomers by taking PMP and PHN (Phenylhydrazine) as derivatization reagents and combining the IM-MS/MS method are compared, the CCS (Collision Cross Section) value of a derivatization product is calculated theoretically for further proving that mixed extracts of the sugar can be qualitatively identified successfully by combining the PMP derivatization and the IM-MS/MS method, and meanwhile, the connection, structures and compositions of the sugar can be identified.
Description
Technical field
The invention belongs to analytical chemistry field, relate to PMP(1-phenyl-3-methyl-5-pyrazolone) derivatization coupled ion mobility tandem mass spectrum (IM-MS/MS) identify sugar method, in particular to the method for the PMP derivatization coupled ion mobility tandem mass spectrum of sugar Structural Identification in natural extracts。
Background technology
Oligosaccharide is information substance important in organism, sugar plays an important role in multiple bioprocess, such as anticoagulating active, immunomodulating, antitumor, blood sugar lowering, suppression pathogenic microorganism, antioxidation, 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.)。The connection of sugar, configuration, monosaccharide residue form the difficult point being always up sugared structural research, the complexity of sugar chain structure and the limitation of technological means, the analysis of the structure of sugar chain is lagged behind far away the research to its function, seriously constrains sugar chain structure and emic research。And difficulty that the sample of this biogenetic derivation often separates purification is also very big, often obtain the mixture of very trace, there is presently no any analytical technology and can solve all the problems above, in the urgent need to setting up the mixed isomers of methods analyst sugar fast and effectively。
Traditional method analyzing oligosaccharide includes the combination (H.A.Currie, C.C.Perry.J.Chromatogr., A2006,1128,90-96.) of nuclear magnetic resonance, NMR (NMR), gas chromatography-mass spectrum (GC/MS) and high performance liquid chromatography (HPLC)。But NMR sensitivity is low, sample purity is required height, cannot be used widely in the analysis of Natural Samples at present。HPLC method ratio is relatively time-consuming, and neither be effective especially in distinguishing isomer。The mass spectrography with the advantage such as quick, sensitive plays an important role in the structural analysis of sugar, the particularly appearance of IM-MS。It is that a kind of collision cross-section (CCS) based on ion when drift Guan Zhongyu buffer gas collisions is different, the technology that ion can be easily separated with shape by size。IM-MS with tradition mass spectrum is maximum be distinctive in that mass spectrographic mass analyzer before add an ion drift tube。This drift tube is filled with noble gas such as nitrogen or helium, when ion moves in drift tube, moving ion can with Inert gas molecule collision, even if institute is electrically charged identical, quality is similar and when ion that shape, size are different and noble gas collision cross-sectional area also differ, namely " resistance " of ion motion is different so that ion is different in size by the time of drift tube, reaches to be distinguished the purpose of ion by shape。Its quick, unique gaseous ion separating power makes it become the desirable instrument analyzing sugar isomer, is successfully used for the qualification of the structure of sugar。In the negative ion mode by adding anion, utilize IM-MS/MS can distinguish preferably in conjunction with HPLC and separate, from cattle submaxillary gland mucin, sugared isomer (H.Li, the B.Bendiak that obtain, W.F.Siems, D.R.GangandH.H.Hill.Anal.Chem.2015,87,2228?2235.), but analyzing object is trisaccharide the connections above, configurational isomer, structural difference is obvious。IM-MS is utilized to separate the sugar not yet completely separable (H.Li, K.Giles, B.Bendiak, K.Kaplan, W.F.Siems, H.H.Hill.Anal.Chem.2012,84,3231-3239.) that molecular weight is smaller。Being published on nature one section of document researcheres in the recent period utilizes IM-MS connection and the anomeric carbon identifying 6 trisaccharide isomers, but composition isomer cannot be carried out distinguishing (J.Hofmann, H.S.Hahm, P.H.Seeberger, K.Pagel.Nature2015,526,241-244.), it is seen that determine that the monosaccharide residue composition of sugar is strictly a difficult point。
Visible, be there is bigger technical difficulty by currently used IM-MS in the Structural Identification of sugar always, even if after sugar is performed the derivatization, the close sugar of structure is from being also difficult to effective differentiation。Research worker is expected to find the sugar analysis means fast and effectively that structure is close always。
PMP is originally by (S.Honda such as Honda, E.Akao, S.Suzuki, M.Okuda, K.KakehiandJ.Nakamura, Anal.Biochem.1989,180,351 357.) report is as the labelled reagent of reducing sugar, is mainly used to improve the detection sensitivity of sugar, owing to this reaction has the advantages such as quick, gentle, easy and simple to handle, PMP is widely used as the derivatization reagent of sugar。Yet there are no the report of the isomer of PMP derivatization coupled ion mobility tandem mass spectrum identification sugar, the closely-related especially substantial amounts of monosaccharide of structure and disaccharide isomer also have no report to utilize IM-MS to identify。The sugar that method main successful analysis in existing research structure comparison in difference is big, and the sugared structure that the present invention relates to is closely, including connecting isomer (such as maltose (α 1 → 4Glc-Glc) and dextrinose (α 1 → 6Glc-Glc)), configurational isomer (such as maltose (α 1 → 4Glc-Glc) and cellobiose (β 1 → 4Glc-Glc)), composition isomer (such as lactose (β 1 → 4Gal-Glc) and cellobiose (β 1 → 4Glc-Glc)) and epimer (such as galactose and mannose)。Owing to the isomer of monosaccharide and disaccharide is due to molecular weight, poor specificity, in IM-MS analysis process, accomplish that Qualitative Identification is the most difficult。The sugar that structure is close especially can be realized identifying by the present invention, also can be readily used for the identification of polysaccharide and oligosaccharide and isomer thereof。
Summary of the invention
The present invention uses PMP derivatization coupled ion mobility tandem mass spectrum, it is provided that a kind of method rapidly and efficiently identifying monosaccharide and disaccharide isomer, can be used for monosaccharide and disaccharide Structural Identification in natural extracts。Which solve in existing identification oligosaccharide isomers technology, it is impossible to the problem analyzing the sugar-like product of reality mixing simply, fast and accurately, solve simultaneously and utilize mass spectrum means cannot determine the problem that the monosaccharide residue of sugar forms。
The PMP derivatization of monosaccharide of the present invention and disaccharide isomer refer to monosaccharide and disaccharide isomer through PMP derivatization thus cause derivatization product CCS show difference, thus distinguishing isomer。
IM-MS/MS method of the present invention refers under the Mass Spectrometry Conditions optimized, and utilizes result that ionic mobility separates and the series connection result common identification isomer to the analyte that cannot be distinguished by first mass spectrometric。
The invention provides the recognition methods of a kind of sugar, it is characterised in that use derivatization coupled ion mobility mass spectrum to be analyzed。
Further, derivatization reagent used by the present invention is 1-phenyl-3-methyl-5-pyrazolones ketone。
Further, analyzed in present invention sugar is monosaccharide and/or disaccharide and isomer thereof。
Further, analyzed sugar is one or more in sucrose, lactose, 2-O-alpha-D-Glucopyranosyl-D-glucose., nigerose, maltose, dextrinose, sophorose, 3-O-beta-D-Glucopyranosyl-D-glucose, cellobiose, gentiobiose, glucose, galactose, mannose, fructose。
Further, intermediate ion mobility mass spectrum of the present invention adopts tandem mass spectrum。
In certain embodiments, the mass spectrographic testing conditions of described ionic mobility is as follows: using ESI-IM-MS to measure, peristaltic pump sample introduction speed is: 5 μ L/min；ESI ion source condition is as follows: positive ion mode, capillary voltage: 2.8kV, sample taper hole voltage: 50V, extracts taper hole voltage: 4V, source temperature: 120 ° of C, desolventizing temperature: 350 ° of C, taper hole gas velocity: 30L/h；IM-MS condition: T-wave speed: 550-2500m/s, T-wave height: 40V, drift about (nitrogen) flow velocity: 90mL/min。
In one embodiment of the invention, with PMP for derivatization reagent, utilize IM-MS/MS method that 4 monosaccharide isomers closely-related especially in structure and 10 disaccharide isomers are made a distinction, and theoretically the CCS value of derivatization product has been calculated, in conjunction with IM-MS/MS method, the mixed extract of Aqueous soluble ginseng's monosaccharide (WGOS1) and disaccharide (WGOS2) is carried out qualitative analysis with PMP derivatization。In one-level (series connection) mass spectrum, undivided PMP derivatization product is concatenated obtaining the derivative compound of single PMP, their (t drift timeD) value is variant, the result of comprehensive first mass spectrometric and tandem mass spectrum, the isomer of sugar can distinguish。And the product ion that in first mass spectrometric, undivided PHN derivatization product is concatenated obtaining shows identical tDValue, distinguishes inoperative to the isomer of sugar after series connection, therefore the separation of sugar isomer is not reached desirable effect。
Technical scheme is as follows:
Ammonia water-methanol solution is prepared
NH3·H2O joins in methanol and fully vibrates, and obtains the ammonia water-methanol solution that concentration is 0.75-0.9mol/L。
The derivative liquid preparation of PMP
PMP is configured to the solution that concentration is 0.0349mol/L, is stored in 4 ° of C refrigerators standby。
The extracting and developing of WGOS1 and WGOS2, enrichment
Dry Radix Ginseng being crushed to 50 ~ 80 orders, with distilled water immersion overnight at room temperature, extracts 2 ~ 3h, four layers of filtered through gauze under 70 ° of C, residue extracts 2 times more repeatedly。Merge 3 extracting solution, after the centrifugal 15min of 3500rpm, discard precipitation, merge supernatant。Immediately, after supernatant rotary evaporation, lyophilizing, Radix Ginseng is obtained。Thick oligosaccharide sample。Thick oligosaccharide sample is mixed with mass ratio 7:1000 with water, carries out ultrafiltration classification, filter liquor phend-sulphuric acid monitor in real time with the ultrafilter membrane that molecular cut off is 3000Da, until there is no color。Collecting filter liquor, after rotary evaporation concentration, lyophilizing obtains Radix Ginseng oligosaccharide sample (WGOS)。
Gained sample is dissolved in the water, after the centrifugal 5min of 10000rpm, the Bio-gelP-2 gel chromatography column (1.0cm × 100cm) that extremely will balance on supernatant。Then using deionized water eluting, flow velocity is 0.06mL/min, collects eluent。From often pipe, take out 0.2mL eluent, measure sugar content therein with phend-sulphuric acid, and draw tomographic map。The eluent of WGOS1 and WGOS2 is collected according to elution curve。
The PMP derivatization process of standard substance and extract
Standard substance: reaction cumulative volume is monosaccharide or two saccharide of 200 μ L, 2.5mmol/L, supply with the derivative liquid of PMP。
Extract: reaction cumulative volume is WGOS1 and the WGOS2 mixture of 200 μ L, final concentration of 10g/L, supplies with the derivative liquid of PMP。
Standard substance and extract are centrifuged at 10000rmp, take supernatant, react 1 ~ 2h in 60 ~ 80 ° of C tepidarium。After reaction, centrifuging and taking supernatant high-purity N2Drying up, add after Milli-Q water fully dissolves, 10000rmp is centrifuged 10min, takes supernatant。In order to remove PMP derivatization reagent excessive in solution, add CCl3Fully vibration, removes lower floor's solution after standing, repeated multiple times, until lower floor, without color, finally gives the derivatization product of sample, is placed in 4 ° of C refrigerators。
PHN derivatization process
Take 5 μ L phenylhydrazines, join in the aqueous solution (2.5 μm of ol) of 250 μ L sugar, react 1h in 80 ° of C stirred in water bath。Reactant is extracted with ethyl acetate, and combined ethyl acetate layer also washes with water, and combining water layer is placed in 4 ° of C refrigerators。
Mass Spectrometry Conditions
Mass spectrograph: WatersSynaptG2 mass spectrograph, ion source: electron spray ionisation source (ESI) positive ion mode, capillary voltage: 2.8kV, sample taper hole voltage: 50V, extracts taper hole voltage: 4V, source temperature: 120 ° of C, desolventizing temperature: 350 ° of C, taper hole gas velocity: 30L/h, T-wave speed: 450 ~ 5000m/s, T-wave height: 38 ~ 40V, drift gas is nitrogen, flow velocity: 90mL/min, collision gas is argon, trap collision energy: 35 ~ 40V。
Standard substance and extract are all analyzed by above-mentioned Mass Spectrometry Conditions；
The calculating of CCS
CCS be ion when drift Guan Zhongyu buffer gas collisions average available cross-section and collision cross-section (?2), the collision cross-section of molecule is a key character, and it is directly relevant with the three-dimensional structure in its chemical constitution and gas phase, and the electric charge of ion, mass-to-charge ratio are to affect the important parameter of CCS。Being different from the parameters such as retention time, CCS value is not by the impact of different instruments and chromatographic process, it is possible to more reliably compound is made differentiation。As molecular weight, scientist can be furtherd investigate chemical constitution or confirm a molecule better。We as correction thing, obtain the CCS value (meansigma methodss under multiple conditions) of determinand using the compound with known CCS value under multiple different T-wave speed and T-wave height condition。Our understanding to the structure of sugar can be improved by calculating the collision cross-section of ion, can judge whether isomer can be distinguished theoretically objectively according to CCS value。
CCS value calculates according to below equation:
t′ D=tD–[(c)/1000]①
Ω′=Ω/[c×(1/μ)1/2]②
μ=m×mgas/ (m+mgas)③
lnΩ′=X×lnt′ D+lnA④
In formula, tDFor drift time, c is electrically charged number, and m/z is mass-to-charge ratio, and Ω is CCS value, mgasMass number for nitrogen。A certain specific T-wave speed and T-wave height condition under to correction thing be measured, according to formula 1.-4. obtain X and lnA, determinand is also measured under corresponding conditions, obtains Ω。The meansigma methods finally obtaining the Ω under different T-wave speed and T-wave height condition is the CCS value of this determinand。
Preferably, in step (1), the concentration of described ammonia water-methanol solution is 0.82mol/L。
Preferably, in step (2), the derivative liquid of described PMP is formulated by the ammonia water-methanol of 0.82mol/L。
Preferably, in step (4), described standard substance and extract react 2h in 70 ° of C tepidarium。
Preferably, in step (6), described T-wave speed is 550 ~ 2500m/s, 450 ~ 5000m/s and 800m/s, T-wave is highly 40V。
The invention provides PMP derivatization in conjunction with the IM-MS/MS method identifying the isomer of all kinds of sugar, in particular to the method for the PMP derivatization coupled ion mobility tandem mass spectrum of sugar Structural Identification in natural extracts。PMP derivatization is applied in the identification of sugar isomer by the present invention in conjunction with IM-MS/MS method, and connection, configuration and composition isomer all can be identified accurately and rapidly。Calculate the CCS value of related substances simultaneously, utilize CCS also can isomer be made a distinction, and confirm that the size of collision cross-section is the principal element affecting separating degree theoretically。The present invention another provide the benefit that and the mixture of sugar isomery can be analyzed directly and accurately, the present invention can on the important impact of generation of sugar group。
Accompanying drawing explanation
Fig. 1 is the ionic mobility mass spectrum of 10 kinds of disaccharide isomers and 4 kinds of monosaccharide isomer original shapes in embodiment 1。
Fig. 2 is the scatterplot of the CCS change of monosaccharide and disaccharide isomer before and after PHN and PMP derivatization in embodiment 2。
Fig. 3 is the ionic mobility mass spectrum of PHN derivatization monosaccharide and disaccharide isomer in embodiment 3。
Fig. 4 is the ionic mobility mass spectrum of PMP derivatization monosaccharide and disaccharide isomer in embodiment 4。
Fig. 5 is the ionic mobility tandem mass spectrum figure of 6 PMP derivatization disaccharide isomers in embodiment 4。A hurdle: the result of the disaccharide-PMP derivatization product of protonation, b hurdle: the result of the disaccharide-PMP derivatization product of sodium ion。
Fig. 6 is the ionic mobility mass spectrum of PMP derivatization monosaccharide and disaccharide isomer in embodiment 5。
Fig. 7 is the ionic mobility tandem mass spectrum figure of 5 PMP derivatization disaccharide isomers in embodiment 5。A hurdle: the result of the disaccharide-PMP derivatization product of protonation, b hurdle: the result of the disaccharide-PMP derivatization product of sodium ion。
Fig. 8 is the ionic mobility mass spectrum of PMP derivatization monosaccharide and disaccharide isomer in embodiment 6。
Fig. 9 is the ionic mobility tandem mass spectrum figure of 5 PMP derivatization disaccharide isomers in embodiment 6。A hurdle: the result of the disaccharide-PMP derivatization product of protonation, b hurdle: the result of the disaccharide-PMP derivatization product of sodium ion。
Figure 10 is the ionic mobility mass spectrum (e-h) of the PMP derivatization product of the ionic mobility mass spectrum (a-d) of WGOS1 and WGOS2 in embodiment 7 and WGOS1 and WGOS2。That wherein in e and h, dotted line represents is the tandem mass spectrum figure of m/z673.27 ion。
Detailed description of the invention
Embodiment 1
(1) preparation of standard substance
With 50% methanol-water (v/v), sucrose, lactose, 2-O-alpha-D-Glucopyranosyl-D-glucose., nigerose, maltose, dextrinose, sophorose, 3-O-beta-D-Glucopyranosyl-D-glucose, cellobiose, gentiobiose, glucose, galactose, mannose and fructose standard substance are made into concentration and are the solution of 2 μMs。
(2) the IM-MS detection of standard substance
The standard substance prepared are made directly ESI-IM-MS and measure, and peristaltic pump sample introduction speed is: 5 μ L/min。ESI ion source condition is as follows: positive ion mode, capillary voltage: 2.8kV, sample taper hole voltage: 50V, extracts taper hole voltage: 4V, source temperature: 120 ° of C, desolventizing temperature: 350 ° of C, taper hole gas velocity: 30L/h。IM-MS condition: T-wave speed: 550-2500m/s, T-wave height: 40V, drift about (nitrogen) flow velocity: 90mL/min。
Result is shown in Fig. 1, compares the ionic mobility mass spectrum of monosaccharide and disaccharide isomer, according to tD, it has been found that only sucrose (tD=3.36ms) can distinguish with other disaccharide isomers, only glucose (tD=2.17ms) can distinguish with other monosaccharide isomers, cannot be carried out between other monosaccharide and disaccharide isomer distinguishing。
Embodiment 2
(1) preparation of correction thing and standard substance to be measured
Thing (sucrose, lactose, cellobiose, maltose, 6-(.alpha.-D-galactosido)-D-glucose., trehalose, melezitose, cottonseed sugar and maltotriose) will be corrected with 50% methanol-water (v/v) and standard substance to be measured (2-O-alpha-D-Glucopyranosyl-D-glucose., nigerose, dextrinose, sophorose, 3-O-beta-D-Glucopyranosyl-D-glucose, gentiobiose, glucose, galactose, mannose and fructose) will be made into concentration and is the solution of 2 μMs。
(2) ammonia water-methanol solution preparation
Taking 500 μ L concentration is the NH of 28%3·H2O, joins in 9.5mL methanol and fully vibrates, obtain
Concentration is the ammonia water-methanol solution of 0.82mol/L。
(3) the derivative liquid preparation of PMP
The PMP weighing 60.67mg is put in 10mL volumetric flask, and constant volume after fully dissolving with ammonia water-methanol solution fully vibrates, and being configured to concentration is 0.0349mol/L, is stored in 4 ° of C refrigerators standby。
(4) PMP derivatization process
Weigh 4 kinds of monosaccharide standard substance (each 0.17mg) and 8 kind of two saccharide (each 0.34mg), be separately added into 200 μ L derivatization reagents and be dissolved in the ep pipe of 1mL, 10000rmp centrifuging and taking supernatant, reacts 2h in 70 ° of C tepidarium。After reaction, centrifuging and taking supernatant high-purity N2Drying up, add after 1mLMilli-Q water fully dissolves, 10000rmp is centrifuged 10min, takes supernatant。In order to remove PMP derivatization reagent excessive in solution, add CCl31mL fully vibrates, and removes lower floor's solution after standing, repeated multiple times, until lower floor, without color, finally gives the derivatization product of sample, is placed in 4 ° of C refrigerators。
(5) PHN derivatization process
Take 5 μ L phenylhydrazines, join in the aqueous solution (2.5 μm of ol) of 250 μ L sugar, react 1h in 80 ° of C stirred in water bath。Reactant is extracted with ethyl acetate (250 μ L × 3 time), and combined ethyl acetate layer also washes (100 μ L × 3 time) with water, and combining water layer is placed in 4 ° of C refrigerators。
(6) the IM-MS condition that the CCS of correction thing, saccharide and derivatization product measures
16 IM-MS conditions: 1. T-wave speed: 400m/s, T-wave height: 40V；2. T-wave speed: 400m/s, T-wave height: 38V；3. T-wave speed: 400m/s, T-wave height: 35V；4. T-wave speed: 300m/s, T-wave height: 38V；5. T-wave speed: 800m/s, T-wave height: 40V；6. T-wave speed: 800m/s, T-wave height: 38V；7. T-wave speed: 800m/s, T-wave height: 35V；8. T-wave speed: 450-5000m/s, T-wave height: 40V；9. T-wave speed: 450-5000m/s, T-wave height: 38V；10. T-wave speed: 450-5000m/s, T-wave height: 35V；All the other are with embodiment 1。T-wave speed: 500-2500m/s, T-wave height: 40V；T-wave speed: 500-2500m/s, T-wave height: 38V；T-wave speed: 500-2500m/s, T-wave height: 35V；T-wave speed: 550-2500m/s, T-wave height: 40V；T-wave speed: 550-2500m/s, T-wave height: 38V；T-wave speed: 550-2500m/s, T-wave height: 35V。Remaining is with embodiment 1。
(7) calculating of CCS
The CCS value of derivatization product calculates according to below equation:
t′ D=tD–[(c)/1000]①
Ω′=Ω/[c×(1/μ)1/2]②
μ=m×mgas/ (m+mgas)③
lnΩ′=X×lnt′ D+lnA④
In formula, tDFor drift time, c is electrically charged number, and m/z is mass-to-charge ratio, and Ω is CCS value, mgasMass number for nitrogen。Using the compound with known CCS value as correction thing, a certain specific T-wave speed and T-wave height condition under, according to formula 1.-4. obtain X and lnA, determinand is also measured under corresponding conditions, obtains Ω。Finally obtain the meansigma methods of the Ω of (actual conditions is as illustrated in step 6) under different T-wave speed and T-wave height condition and be the CCS value of this determinand。
Result is shown in Fig. 2, it is seen that after derivatization, CCS value increases substantially, and the CCS value between partial allosteric body demonstrates difference。Namely result of calculation shows, the glucide after derivatization can be separated。
Embodiment 3
PHN derivatization process is with embodiment 2, and the IM-MS testing conditions of derivatization product is with embodiment 1。
Result is shown in Fig. 3, compares monosaccharide and the ionic mobility mass spectrum of disaccharide isomer PHN derivatization product, according to tD, it has been found that nigerose PHN derivant (tD=4.34ms), 3-O-beta-D-Glucopyranosyl-D-glucose PHN derivant (tD=4.45ms) and cellobiose PHN derivant (tD=5.15ms) can distinguish with other disaccharide isomer PHN derivants, galactose PHN derivant (tD=2.71ms) can distinguish with other monosaccharide isomer PHN derivants, cannot be carried out between other monosaccharide and disaccharide isomer distinguishing。
Embodiment 4
PMP derivatization process is with embodiment 2, and the IM-MS testing conditions of derivatization product is with embodiment 1。
Result is shown in Fig. 4, compares monosaccharide and the ionic mobility mass spectrum of disaccharide isomer PMP derivatization product, according to tD, it has been found that maltose PMP derivant (tD=8.73ms), dextrinose PMP derivant (tD=8.03ms) and 3-O-beta-D-Glucopyranosyl-D-glucose PMP derivant (tD=5.15ms) can distinguish with other disaccharide isomer PMP derivants, cannot be carried out between the PMP derivant of other disaccharide isomer distinguishing, can make a distinction between the PMP derivant of three monosaccharide。
Separate still undivided 3 pairs of disaccharide isomers connect (ionic mobility separate before the series connection that carries out) mass spectral analysis to carrying out mobility after PMP derivatization。Collision gas is argon, trap collision energy: 40V。Result as shown in Figure 5, utilizes the t of the concatemer production (the disaccharide isomer of single PMP derivatization) that a hurdle protonatesDValue, isomer between two can make a distinction completely。Utilize the t of the concatemer production of b hurdle sodium ionDValue, isomer between two can partly make a distinction。
Embodiment 5
PMP derivatization process is with embodiment 2, and the IM-MS testing conditions of derivatization product is 800m/s except T-wave speed, and other are with embodiment 1。
Result is shown in Fig. 6, compares monosaccharide and the ionic mobility mass spectrum of disaccharide isomer PMP derivatization product, according to tD, it has been found that maltose PMP derivant (tD=9.33ms), dextrinose PMP derivant (tD=8.79ms), 3-O-beta-D-Glucopyranosyl-D-glucose PMP derivant (tD=9.49ms) and gentiobiose PMP derivant (tD=8.9ms) can distinguish with other disaccharide isomer PMP derivants, cannot be carried out between the PMP derivant of other disaccharide isomer distinguishing, can make a distinction between the PMP derivant of three monosaccharide。
Separate still undivided disaccharide isomer connect (ionic mobility separate before the series connection that carries out) mass spectral analysis to carrying out mobility after PMP derivatization。Collision gas is argon, trap collision energy: 40V。Result as shown in Figure 7, utilizes the t of the concatemer production (the disaccharide isomer of single PMP derivatization) that a hurdle protonatesDValue, can make a distinction between isomer completely。Utilize the t of the concatemer production of b hurdle sodium ionDValue, can partly make a distinction between isomer。
Embodiment 6
PMP derivatization process is with embodiment 2, and the IM-MS testing conditions of derivatization product is 450-5000m/s except T-wave speed, and other are with embodiment 1。
Result is shown in Fig. 8, compares monosaccharide and the ionic mobility mass spectrum of disaccharide isomer PMP derivatization product, according to tD, it has been found that 2-O-alpha-D-Glucopyranosyl-D-glucose. PMP derivant (tD=7.22ms), 3-O-alpha-D-Glucopyranosyl-D-glucose PMP derivant (tD=7.32ms), maltose PMP derivant (tD=7.49ms) and 3-O-beta-D-Glucopyranosyl-D-glucose PMP derivant (tD=7.70ms) can distinguish with other disaccharide isomer PMP derivants, cannot be carried out between the PMP derivant of other disaccharide isomer distinguishing, can make a distinction between the PMP derivant of three monosaccharide。
Separate still undivided disaccharide isomer connect (ionic mobility separate before the series connection that carries out) mass spectral analysis to carrying out mobility after PMP derivatization。Collision gas is argon, trap collision energy: 40V。Result as shown in Figure 9, utilizes the t of the concatemer production (the disaccharide isomer of single PMP derivatization) that a hurdle protonatesDValue, can make a distinction between isomer completely。Utilize the t of the concatemer production of b hurdle sodium ionDValue, can partly make a distinction between isomer。
Embodiment 7
(1) extracting and developing of WGOS1 and WGOS2, enrichment
Taking dry Radix Ginseng to pulverize, weigh 500g, with 5L distilled water immersion overnight at room temperature, extract 3h, four layers of filtered through gauze under 70 ° of C, residue extracts 2 times more repeatedly。Merge 3 extracting solution, after the centrifugal 15min of 3500rpm, discard precipitation, merge supernatant。Immediately, the thick oligosaccharide sample of Radix Ginseng will be obtained after supernatant rotary evaporation, lyophilizing。Thick oligosaccharide sample is mixed with mass ratio 7:1000 with water, carries out ultrafiltration classification, filter liquor phend-sulphuric acid monitor in real time with the ultrafilter membrane that molecular cut off is 3000Da, until there is no color。Collecting filter liquor, after rotary evaporation concentration, lyophilizing obtains Radix Ginseng oligosaccharide sample (WGOS)。
Gained sample is weighed 15mg, is dissolved in 1mL water, after the centrifugal 5min of 10000rpm, the Bio-gelP-2 gel chromatography column (1.0cm × 100cm) that extremely will balance on supernatant。Then with 96mL deionized water eluting, flow velocity is 0.06mL/min, and often 1.2mL eluent collected by pipe。From often pipe, take out 0.2mL eluent, measure sugar content therein with phend-sulphuric acid, and draw tomographic map。The eluent of WGOS1 and WGOS2 is collected according to elution curve。
(2) PMP derivatization process
Weighing WGOS1 and WGOS2 mixture (2mg), be separately added into 200 μ L derivatization reagents and be dissolved in the ep pipe of 1mL, remaining is with embodiment 2。
(3) the IM-MS/MS detection of derivatization product
The IM-MS testing conditions of derivatization product is with embodiment 1。Collision gas is argon, trap collision energy: 40V。
As shown in Figure 10, the result before and after Integrated comparative derivatization, it is known that WGOS1 is made up of fructose and glucose, WGOS2 is made up of result sucrose and maltose。
Claims (11)
1. recognition methods one kind sugared, it is characterised in that use derivatization coupled ion mobility mass spectrum to be analyzed。
2. recognition methods sugared as claimed in claim 1, it is characterised in that described derivatization reagent is 1-phenyl-3-methyl-5-pyrazolones ketone。
3. the recognition methods of sugar as described in any one of claim 1-2, it is characterised in that described sugar is monosaccharide and/or disaccharide and isomer thereof。
4. recognition methods sugared as claimed in claim 3, it is characterized in that, described sugar is one or more in sucrose, lactose, 2-O-alpha-D-Glucopyranosyl-D-glucose., nigerose, maltose, dextrinose, sophorose, 3-O-beta-D-Glucopyranosyl-D-glucose, cellobiose, gentiobiose, glucose, galactose, mannose, fructose。
5. the recognition methods of sugar as described in any one of claim 1-4, it is characterised in that described ionic mobility mass spectrum adopts tandem mass spectrum。
6. as described in any one of claim 1-5 sugar recognition methods, its feature with, the mass spectrographic testing conditions of ionic mobility is as follows: use ESI-IM-MS measure, peristaltic pump sample introduction speed is: 5 μ L/min；ESI ion source condition is as follows: positive ion mode, capillary voltage: 2.8kV, sample taper hole voltage: 50V, extracts taper hole voltage: 4V, source temperature: 120 ° of C, desolventizing temperature: 350 ° of C, taper hole gas velocity: 30L/h；IM-MS condition: T-wave speed: 550-2500m/s, T-wave height: 40V, drift about (nitrogen) flow velocity: 90mL/min。
7. one kind utilizes PMP(1-phenyl-3-methyl-5-pyrazolone) method of oligosaccharide in derivatization coupled ion mobility tandem mass spectrum identification Radix Ginseng, it is characterised in that comprise the following steps:
(1) ammonia water-methanol solution preparation
NH3·H2O joins in methanol and fully vibrates, and obtains the ammonia water-methanol solution that concentration is 0.75-0.9mol/L；
(2) the derivative liquid preparation of PMP
PMP is configured to the solution that concentration is 0.0349mol/L, is stored in 4 ° of C refrigerators standby；
(3) extracting and developing of Radix Ginseng oligosaccharide sample, enrichment
Dry Radix Ginseng being crushed to 50 ~ 80 orders, with distilled water immersion overnight at room temperature, extracts 2 ~ 3h, four layers of filtered through gauze under 70 ° of C, residue extracts 2 times more repeatedly；Merge 3 extracting solution, after the centrifugal 15min of 3500rpm, discard precipitation, merge supernatant；The thick oligosaccharide sample of Radix Ginseng will be obtained after supernatant rotary evaporation, lyophilizing；Thick oligosaccharide sample is mixed with mass ratio 7:1000 with water, carries out ultrafiltration classification, filter liquor phend-sulphuric acid monitor in real time with the ultrafilter membrane that molecular cut off is 3000Da, until there is no color；Collecting filter liquor, after rotary evaporation concentration, lyophilizing obtains Radix Ginseng oligosaccharide sample (WGOS)；Gained sample is dissolved in the water, after the centrifugal 5min of 10000rpm, the 1.0cm × 100cmBio-gelP-2 gel chromatography column that extremely will balance on supernatant；Then using deionized water eluting, flow velocity is 0.06mL/min, collects eluent；From often pipe, take out 0.2mL eluent, measure sugar content therein with phend-sulphuric acid, and draw tomographic map；The eluent of WGOS1 and WGOS2 is collected according to elution curve；
(4) the PMP derivatization process of standard substance and extract
Standard substance: reaction cumulative volume is monosaccharide or two saccharide of 200 μ L, 2.5mmol/L, supply with the derivative liquid of PMP；
Extract: reaction cumulative volume is WGOS1 and the WGOS2 mixture of 200 μ L, final concentration of 10g/L, supplies with the derivative liquid of PMP；Standard substance and extract are centrifuged at 10000rmp, take supernatant, react 1 ~ 2h in 60 ~ 80 ° of C tepidarium；After reaction, centrifuging and taking supernatant high-purity N2Drying up, add after Milli-Q water fully dissolves, 10000rmp is centrifuged 10min, takes supernatant and adds CCl3Fully vibration, removes lower floor's solution after standing, repeated multiple times, until lower floor, without color, finally gives the derivatization product of sample, is placed in 4 ° of C refrigerators；
(5) Mass Spectrometry Conditions
Mass spectrograph: WatersSynaptG2 mass spectrograph, ion source: electron spray ionisation source (ESI) positive ion mode, capillary voltage: 2.8kV, sample taper hole voltage: 50V, extracts taper hole voltage: 4V, source temperature: 120 ° of C, desolventizing temperature: 350 ° of C, taper hole gas velocity: 30L/h, T-wave speed: 450 ~ 5000m/s, T-wave height: 38 ~ 40V, drift gas is nitrogen, flow velocity: 90mL/min, collision gas is argon, trap collision energy: 35 ~ 40V；Standard substance and extract are all analyzed by above-mentioned Mass Spectrometry Conditions。
8. the method for a kind of PMP derivatization coupled ion mobility tandem mass spectrum identification monosaccharide as claimed in claim 7 and disaccharide isomer, it is characterised in that in described step (1), the concentration of ammonia water-methanol solution is 0.82mol/L。
9. method as claimed in claim 7, it is characterised in that in described step (2), the derivative liquid of PMP is formulated by the ammonia water-methanol of 0.82mol/L。
10. method as claimed in claim 7, it is characterised in that in step (4), described standard substance and extract react 2h in 70 ° of C tepidarium。
11. method as claimed in claim 7, it is characterised in that in step (5), described T-wave speed is 550 ~ 2500m/s, 800m/s and 450-5000m/s, and T-wave is highly 40V。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610160329.3A CN105699478B (en) | 2016-03-21 | 2016-03-21 | A kind of fast method of identification sugar |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610160329.3A CN105699478B (en) | 2016-03-21 | 2016-03-21 | A kind of fast method of identification sugar |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105699478A true CN105699478A (en) | 2016-06-22 |
| CN105699478B CN105699478B (en) | 2018-07-24 |
Family
ID=56232209
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610160329.3A Expired - Fee Related CN105699478B (en) | 2016-03-21 | 2016-03-21 | A kind of fast method of identification sugar |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105699478B (en) |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106248833A (en) * | 2016-09-26 | 2016-12-21 | 中国农业科学院农产品加工研究所 | Lac Bovis seu Bubali oligosaccharide assay method |
| CN109416343A (en) * | 2016-06-30 | 2019-03-01 | 沃特世科技公司 | The chemical classes composition of collision cross-section fragment ion |
| CN110114667A (en) * | 2016-12-23 | 2019-08-09 | 豪夫迈·罗氏有限公司 | Method for identifying reagent during the process in analysis system |
| CN110455963A (en) * | 2019-09-13 | 2019-11-15 | 广东省生物工程研究所（广州甘蔗糖业研究所） | A kind of crop source discrimination method of sucrose |
| CN110554114A (en) * | 2019-09-13 | 2019-12-10 | 广东省生物工程研究所（广州甘蔗糖业研究所） | Method for analyzing oligomeric isomaltose and isomers thereof in yoghourt |
| CN112461913A (en) * | 2020-11-06 | 2021-03-09 | 华南农业大学 | Method for improving identification capability of isomer compound |
| CN113433198A (en) * | 2020-03-20 | 2021-09-24 | 南京理工大学 | Non-denaturing mass spectrometry methods for membrane protein complexes using hexaethyleneglycol mono-octaalkyl ethers |
| CN113433196A (en) * | 2020-03-20 | 2021-09-24 | 南京理工大学 | Non-denaturing mass spectrometry of membrane protein complexes using nonaethylene glycol monododecyl ether |
| CN113433199A (en) * | 2020-03-20 | 2021-09-24 | 南京理工大学 | Non-denaturing mass spectrometry of membrane protein complexes using octaethylene glycol monododecyl ether |
| CN113433197A (en) * | 2020-03-20 | 2021-09-24 | 南京理工大学 | Non-denaturing mass spectrometry of membrane protein complexes using tetraethylene glycol monodecaalkyl ethers |
| CN113433195A (en) * | 2020-03-20 | 2021-09-24 | 南京理工大学 | Non-denaturing mass spectrometry methods for membrane protein complexes using pentaethyleneglycol mono-octaalkyl ethers |
| CN113834871A (en) * | 2021-09-18 | 2021-12-24 | 北京中医药大学 | Method for rapidly analyzing low-molecular-weight sugar based on paper spray mass spectrum and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101502546A (en) * | 2009-02-24 | 2009-08-12 | 姜瑞芝 | Ginseng (root) oligosacchride preparation and novel use thereof |
| CN102512439A (en) * | 2011-10-28 | 2012-06-27 | 焦丽丽 | Ginseng oligosaccharide composition as well as preparation method and application thereof |
| CN104820009A (en) * | 2014-01-31 | 2015-08-05 | 安捷伦科技有限公司 | Fast method for measuring collision cross section of ions utilizing ion mobility spectrometry |
-
2016
- 2016-03-21 CN CN201610160329.3A patent/CN105699478B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101502546A (en) * | 2009-02-24 | 2009-08-12 | 姜瑞芝 | Ginseng (root) oligosacchride preparation and novel use thereof |
| CN102512439A (en) * | 2011-10-28 | 2012-06-27 | 焦丽丽 | Ginseng oligosaccharide composition as well as preparation method and application thereof |
| CN104820009A (en) * | 2014-01-31 | 2015-08-05 | 安捷伦科技有限公司 | Fast method for measuring collision cross section of ions utilizing ion mobility spectrometry |
Non-Patent Citations (3)
| Title |
|---|
| HONGLI LI等: "Determining the Isomeric Heterogeneity of Neutral Oligosaccharide-Alditols of Bovine Submasillary Mucin Using Negative Ion Traveling Wave Ion Mobility Mass Spectrometry", 《ANALYTICAL CHEMISTRY》 * |
| 杨洪梅等: "利用离子淌度串联质谱结合PMP衍生化识别单糖及二糖异构体", 《中国化学会第二届全国质谱分析学术报告会》 * |
| 齐鹏: "人参发状根低聚糖分离及活性初步研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109416343A (en) * | 2016-06-30 | 2019-03-01 | 沃特世科技公司 | The chemical classes composition of collision cross-section fragment ion |
| CN109416343B (en) * | 2016-06-30 | 2022-10-28 | 沃特世科技公司 | Chemical-based composition of collision cross-section fragment ions |
| CN106248833A (en) * | 2016-09-26 | 2016-12-21 | 中国农业科学院农产品加工研究所 | Lac Bovis seu Bubali oligosaccharide assay method |
| CN110114667A (en) * | 2016-12-23 | 2019-08-09 | 豪夫迈·罗氏有限公司 | Method for identifying reagent during the process in analysis system |
| CN110554114B (en) * | 2019-09-13 | 2022-06-28 | 广东省生物工程研究所（广州甘蔗糖业研究所） | Method for analyzing oligomeric isomaltose and isomers thereof in yoghourt |
| CN110455963A (en) * | 2019-09-13 | 2019-11-15 | 广东省生物工程研究所（广州甘蔗糖业研究所） | A kind of crop source discrimination method of sucrose |
| CN110554114A (en) * | 2019-09-13 | 2019-12-10 | 广东省生物工程研究所（广州甘蔗糖业研究所） | Method for analyzing oligomeric isomaltose and isomers thereof in yoghourt |
| CN113433198A (en) * | 2020-03-20 | 2021-09-24 | 南京理工大学 | Non-denaturing mass spectrometry methods for membrane protein complexes using hexaethyleneglycol mono-octaalkyl ethers |
| CN113433199A (en) * | 2020-03-20 | 2021-09-24 | 南京理工大学 | Non-denaturing mass spectrometry of membrane protein complexes using octaethylene glycol monododecyl ether |
| CN113433197A (en) * | 2020-03-20 | 2021-09-24 | 南京理工大学 | Non-denaturing mass spectrometry of membrane protein complexes using tetraethylene glycol monodecaalkyl ethers |
| CN113433195A (en) * | 2020-03-20 | 2021-09-24 | 南京理工大学 | Non-denaturing mass spectrometry methods for membrane protein complexes using pentaethyleneglycol mono-octaalkyl ethers |
| CN113433196A (en) * | 2020-03-20 | 2021-09-24 | 南京理工大学 | Non-denaturing mass spectrometry of membrane protein complexes using nonaethylene glycol monododecyl ether |
| CN112461913A (en) * | 2020-11-06 | 2021-03-09 | 华南农业大学 | Method for improving identification capability of isomer compound |
| CN113834871A (en) * | 2021-09-18 | 2021-12-24 | 北京中医药大学 | Method for rapidly analyzing low-molecular-weight sugar based on paper spray mass spectrum and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105699478B (en) | 2018-07-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105699478A (en) | Method for quickly identifying sugar | |
| Zhao et al. | Simultaneous determination of ginkgo flavonoids and terpenoids in plasma: ammonium formate in LC mobile phase enhancing electrospray ionization efficiency and capacity | |
| Pozo et al. | Comparison between triple quadrupole, time of flight and hybrid quadrupole time of flight analysers coupled to liquid chromatography for the detection of anabolic steroids in doping control analysis | |
| CN107247105B (en) | A kind of method that Solid Phase Extraction-high performance liquid chromatography-tandem mass method detects perchlorate in tealeaves | |
| CN106093261B (en) | A kind of method that starch syrup is mixed in discriminating honey | |
| He et al. | An online field-amplification sample stacking method for the determination of β2-agonists in human urine by CE-ESI/MS | |
| Hou et al. | Studies on the interactions between ginsenosides and liposome by equilibrium dialysis combined with ultrahigh performance liquid chromatography-tandem mass spectrometry | |
| CN105510480A (en) | Detection method and application of 2-methylimidazole and 4-methylimidazole in baked food | |
| CN105136957A (en) | Detection method for simultaneously measuring OXC in human plasma and metabolite MHD and MHD-G | |
| CN107102079A (en) | Detect the application of Liquid Chromatography-Tandem Mass Spectrometry method and the clinical pharmacokinetics research of Atorvastatin and metabolin in human plasma | |
| CN110988193B (en) | Method for detecting advanced glycosylation end products in aquatic products | |
| CN104515821B (en) | The rapid assay methods of fumonisin in a kind of corn kernel | |
| CN104833761B (en) | Method for quickly analyzing carbohydrates in samples | |
| CN102955008A (en) | Method for detecting sulfonamide residue in eel by pressurized capillary electrochromatography | |
| CN103575827B (en) | Method for detecting monosaccharide and preparation method for derivative reagent | |
| CN105527368A (en) | Method for detecting 8-hydroxydeoxyguanosine and 8-hydroxyguanosine in urine by high-performance liquid chromatography tandem mass spectrometry technology | |
| CN103207256B (en) | Method for detecting floridoside and isofloridoside contents in porphyra haitanensis | |
| CN105548402A (en) | Kit for detecting 8-hydroxyl deoxyguanosine and 8-hydroxyl guanosine in urine through high performance liquid chromatography tandem mass spectrometry technology | |
| Liu et al. | Fast quantitative analysis of ginsenosides in Asian ginseng (Panax ginseng CA Mayer) by using solid‐phase methylation coupled to direct analysis in real time | |
| CN108426965A (en) | The detection method of isomaltose, Isomaltotriose, maltose, panose in yellow rice wine | |
| KR101159064B1 (en) | Quantification of active androgens in urine and blood by isotope dilution-mass spectrometry | |
| CN110726787B (en) | LC-MS/MS negative ion mode detection method for analyzing seven mogrosides | |
| Huo et al. | Simultaneous and sensitive determination of eight coumarins in rat bile and urine after oral administration of Radix Angelicae dahuricae extract by liquid chromatography-electrospray ionization-mass spectrometry | |
| CN106970163B (en) | aceglutamide metabolite identification and detection method | |
| CN104914205B (en) | Segregation analysis method for heparan disaccharide sulfate containing FlcNH3+ |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180724 Termination date: 20210321 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |