CN103592386A - Method for detecting monosaccharide and disaccharide and method for preparing derivatization reagents - Google Patents
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Abstract
The invention belongs to the field of medicine, and relates to a method for detecting monosaccharide and disaccharide and a method for preparing derivatization reagents. The detection method comprises the steps as follows: (1), monosaccharide or disaccharide is derived by the derivatization reagents; (2), an LC-MS (liquid chromatography-mass spectrometry) is adopted for analyzing a derived sample; and the derivatization reagents comprise D3PMP (1-phenyl-3-deuterated methyl-5-pyrazolone), D5PMP (1-deuterated phenyl-3-methyl-5-pyrazolone), D8PMP (1-deuterated phenyl-3-deuterated methyl-5-pyrazolone) and PMP (1-phenyl-3-methyl-5-pyrazolone). The three deuterated derivatization reagents can be combined with the common PMP for use and can also be combined arbitrarily for use. A known quantity of standard samples and unknown samples are derived by different derivatization reagents and analyzed by the LC-MS, so that qualification and quantification can be performed on microscale monosaccharide and glycosaminoglycan enzymolysis disaccharide. With the adoption of the methods, four types of samples can be analyzed simultaneously, three types of an unknown quantity of samples can be quantified simultaneously, the dosage of detected samples is small, and the time consumed in the operation is short.
Description
Technical field
The invention belongs to field of medicaments, relate to a kind of method of monose and disaccharides and preparation method of derivative reagent of detecting.
Background technology
1-phenyl-3-methyl-5-pyrazolones ketone (being called for short PMP), well-known with cerebral protective agent " Edaravone " again, be mainly used in being clinically used for the treatment of the DPN that cerebral infarction causes, in sugared structural research as a kind of derivative preparation, by Honda in 1989 derivative for sugar chain at first, PMP is under gentle condition, to carry out quantitative reaction with reduction sugar chain as the advantage of derivative preparation, do not lose sialic acid, product does not have steric isomer, uv absorption is strong, sugar chain is after PMP is derivative, when its hydrophobicity improves, derivative products is electrically charged, be easy to ionization, can to it, analyze by different kinds of ions pattern, with it as derivative reagent, can carry out the composition analysis of monose, detectability can reach pmol, simultaneously also can be for separation and the purifying of oligonucleotide chain, can also improve the ground substance assistant laser of sugar chain and resolve the sensitivity that flight mass spectrum detects.Itself and carbohydrate reaction mechanism are, 1, there is α-hydrogen in 4 carbon of the pyrazolone reagent of 3 replacements, under alkaline environment, leave away and become enol form negative ion, carry out Aldol condensation reaction with the reducing end of glucide, dehydration under heating condition, formation has α, the unimolecule derivant of alpha, beta-unsaturated ketone structure, this unimolecule derivant can continue to carry out Michael1 with the enol form negative ion of another molecule pyrazolone reagent, 4 addition reactions, the last two PMP derivative products with strong uv absorption that quantitatively generate.The method has been widely used in having the carbohydrate analysis of reducing end, as patent CN101118230 discloses the monose composition that detects Cordyceps sinensis polysaccharide with 0.5mol/L PMP methanol solution.Patent CN200810013642 discloses the derivative content in conjunction with quinoline promise sugar in liquid-phase chromatographic analysis sample with PMP.Patent CN200810016622 discloses the derivative content that detects sea cucumber polysaccharide in cucumber product in conjunction with high performance liquid chromatography with PMP.
Liquid chromatography mass coupling (LC-MS) be again LC-MS-MS, it usings liquid chromatography as piece-rate system, mass spectrum is as detection system, liquid chromatography mass coupling has embodied the complementation of chromatogram and mass spectrum advantage, high separating power by chromatogram to complex sample, there is high selectivity, high sensitivity with mass spectrum and the advantages of relative molecular mass and structural information can be provided, in many fields such as Pharmaceutical Analysis, food analysis and environmental analyses, be widely used.
Isotope has identical chemical property, ionization ability is consistent, retention time in liquid phase is also identical, sample by the derivative unknown content of isotope reagent and known quantity, can be at a plurality of samples of inner analysis at the same time, use known quantity sample to carry out accurate quantitative analysis to unknown sample, thereby mass spectroscopy is become, a kind ofly can carry out to sample the method for accurate quantification.
Glycosaminoglycan is the linear polymeric polysaccharide that a class is comprised of the disaccharide unit repeating (aminohexose and hexose uronic acid), is divided into heparin, Heparan sulfate, chondroitin sulfate, hyaluronic acid, keratan sulfate etc.Linear polymeric glycosaminoglycan just can obtain glycosaminoglycan enzymolysis disaccharides with enzyme degraded, as by heparinase degradeds such as Heparinase I, Heparinase I I, Heparinase I II for heparin, chondroitin sulfate is obtained to corresponding uronic acid dehydration disaccharides with chondroitinase ABC degraded.The disaccharides repetitive that different types of glycosaminoglycan has is different, if the repetition disaccharide unit of heparin and Heparan sulfate is iduronic acid (IdoA) or glucuronic acid (GlcA) and gucosamine (GlcN); The disaccharides repetitive of chondroitin sulfate is glucuronic acid (GlcA) and galacturonic acid (GalA).Different types of glycosaminoglycan degree is different, and sulphation position is also different, as higher in the N of gucosamine sulphation (GlcNS) degree than Heparan sulfate in heparin, and acetylation (GlcNAc) degree is low; Chondroitin sulfate A (CSA) is in 4 sulphations (GalNAc4S) of galactosamine, and chondroitin sulfate C is in 6 sulphations (GalNAc6S) of galactosamine.Glycosaminoglycan has multiple biologically active, if heparin is the anticoagulant of clinical first-selection; Heparan sulfate is the important molecule of the signal conduction of cell surface; Chondroitin class, hyalomitome acids has relevant medicine or health products to go on the market.2008, pollute heparin event and caused at least 149 people dead, trace it to its cause, be to having defect in the polysaccharide researches technology of this class formation complexity of glycosaminoglycan and method, so determine that monose and the disaccharides of glycosaminoglycan are very important.
At present conventional detection method is in various shortcomings, as protonated in must be first carried out with analytical reagent composition sample, and this process is subject to external environment, salinity in sample, and instrument state many factors, cannot be for quantitative test.Although PMP is derivative, in conjunction with high performance liquid chromatography, can carry out quantitative test to sample, need to first draw the typical curve of each monose, and then calculate the concentration of testing sample, detect limit for height, sample consumption is large, once can only detect a sample, length consuming time.The separation sugar PMP derivant obtaining with common PMP derivative reagent is subject to UV-detector sensitivity and the less restriction of the difference of monose own, need standard items and more sugared sample ability accurate analysis, complex steps not only, and for precious biological sample, be also a kind of waste.The multipotency of common deriving method and reagent (as aniline and deuterated aniline) carries out the analysis of 2 kinds of samples simultaneously, and multipotency is quantitative to a kind of unknown sample.When sample type is more, loaded down with trivial details with traditional method detecting step, sample consumption is large, detection time is long, and traditional method can not be satisfied the demand, and therefore needs badly and finds a kind of operation steps simple, save time, sample consumption is little, highly sensitive detection method.
Summary of the invention
One of object of the present invention is to provide a kind of method that detects monose and disaccharides, and the method can be carried out qualitative and quantitative analysis to monose and glycosaminoglycan enzymolysis disaccharides.
Another object of the present invention is to provide three kinds of derivative reagent 1-phenyl-3-deuterated methyl-5-pyrazolones (D3PMP), the preparation method of the deuterated methyl-5-of the deuterated phenyl-3-of 1-pyrazolone (D8PMP).
The technical scheme that realizes foregoing invention is:
1. a method that detects monose and disaccharides, comprises the steps:
(1) monose or disaccharides is derivative with derivative reagent;
(2) with LC-MS, analyze the sample after deriving;
Described derivative reagent comprises the deuterated methyl-5-of 1-phenyl-3-pyrazolone (D3PMP), 1-deuterated phenyl-3-methyl-5-pyrazolone (D5PMP), the deuterated methyl-5-of the deuterated phenyl-3-of 1-pyrazolone (D8PMP) and 1-phenyl-3-methyl-5-pyrazolones ketone (PMP);
Any one coupling in described derivative reagent PMP and D3PMP, D8PMP;
Described derivative reagent PMP and D3PMP, D5PMP, any two or three coupling of DSPMP;
Any several couplings of described derivative reagent D3PMP, D5PMP, D8PMP.
2. detect a method for monose and disaccharides, described monose is animal, plant and microbe-derived polysaccharide and the basic composition unit of oligosaccharides, and described disaccharides is the disaccharides of enzymolysis glycosaminoglycan gained.
3. the method for preparing derivative reagent D3PMP, comprises the steps:
(1) get deuterated acetic acid, add ethanol, after stannous chloride shakes up, 70-90 ℃ adds hot reflux 1-5h, distills out liquid, generates deuterated ethyl acetate, by the saturated CaCl of deuterated ethyl acetate
2and dried over mgso, remove ethanol, acetic acid, water, obtains dry deuterated ethyl acetate;
(2) in dried deuterated ethyl acetate, add sodium metal, condensing reflux, then add 50% acetic acid, and regulate pH to 6-7, use saturated sodium-chloride stratification, transfer supernatant liquid, is dried with anhydrous sodium sulfate, obtains deuterated ethyl acetoacetate;
(3) get phenylhydrazine and add in ethanol, then add the deuterated ethyl acetoacetate of step (2) gained, under 70-85 ℃ of condition, react 10h, cold filtration obtains D3PMP;
Described deuterated acetic acid and ethanol volume ratio are 1:2-5;
Described phenylhydrazine and deuterated ethyl acetoacetate amount of substance are than being 1:0.2-0.8.
Chemical equation is as follows:
4. the method for preparing derivative reagent D8PMP, comprises the steps:
(1) get deuterated acetic acid, add ethanol, after stannous chloride shakes up, 70-90 ℃ adds hot reflux 1-5h, distills out liquid, generates deuterated ethyl acetate, by the saturated CaCl of deuterated ethyl acetate
2and dried over mgso, remove ethanol, acetic acid, water, obtains dry deuterated ethyl acetate.
(2) in dried deuterated ethyl acetate, add sodium metal, condensing reflux, then add 50% acetic acid, and regulate pH to 6-7, use saturated sodium-chloride stratification, transfer supernatant liquid, is dried with anhydrous sodium sulfate, obtains deuterated ethyl acetoacetate.
(3) get 30% hydrochloric acid and water, drip deuterated aniline, under 0-5 ℃ of condition, drip 71.8% sodium nitrite solution, stir and keep 0-5 ℃ of reaction 3-5h, obtain the deuterated benzene of diazotising.In the deuterated benzene of diazotising, add sodium bisulfite, 25% ammoniacal liquor and water, be warming up to 70～85 ℃, and back flow reaction 4h puts into 4 ℃ of Temperature drop in refrigerators, filters to obtain deuterated hydrazinobenzene hydrochloride salt.In deuterated hydrazinobenzene hydrochloride salt, add water, be heated to 50 ℃, the NaOH that drips 10M regulates pH to 10-11, stirs 30min, adds ethyl acetate extraction 3 times, and decompression distillation obtains deuterated phenylhydrazine.
(4) the deuterated phenylhydrazine of step (3) gained is dissolved in ethanol, adds the deuterated ethyl acetoacetate of step (2) gained, under 70-85 ℃ of condition, react 10h, cold filtration, obtains D8PMP.
Described deuterated acetic acid and ethanol volume ratio are 1: 2;
Described deuterated aniline is 1:1 with the ratio of sodium nitrite amount of substance;
Described deuterated phenylhydrazine is 1:0.7 with the ratio of the amount of substance of deuterated ethyl acetoacetate.
Chemical equation is as follows:
The present invention compared with prior art has the following advantages:
Three kinds of deuterated derivative reagents of the present invention can with the coupling of common PMP combination in any, also can any several couplings, the standard items of known quantity are derivative with different derivative reagents respectively with unknown sample, with LC-MS, analyze, can carry out quantitative and qualitative analysis to micro-monose and glycosaminoglycan enzymolysis disaccharides.The method can be analyzed 4 kinds of samples simultaneously, can to 3 kinds of unknown quantity samples, carry out quantitatively, greatly reducing the consumption of the sample of knowing clearly simultaneously, has saved the detection time of sample quantitative and qualitative analysis.4 kinds of derivative reagents differ larger at the mass spectrum number of improving quality, and sample is easy to be arrived by Mass Spectrometer Method after derivative by 4 kinds of larger derivative reagents of mass number, have improved sensitivity.This method of simultaneously carrying out Multi-example analysis increases the accuracy of result and reliability.
Accompanying drawing explanation
Fig. 1 is chromatogram (A) and the total ion current figure (B) of eight kinds of monose standard items.
Fig. 2 is the mass spectrogram of the derivative glucuronic acid of PMP, D3PMP.
Fig. 3 is the mass spectrogram of the derivative gucosamine of PMP, D5PMP, D8PMP.
Fig. 4 is the mass spectrogram of the derivative glucose of D3PMP, D8PMP.
Fig. 5 is the mass spectrogram of the derivative gucosamine of D3PMP, D5PMP, D8PMP.
Fig. 6 is the mass spectrogram of the derivative glucose of PMP, D3PMP, D5PMP and D8PMP.
Fig. 7 is total ion current figure and the mass spectrogram of the derivative glycosaminoglycan enzymolysis disaccharides of PMP, D3PMP, D5PMP, D8PMP.
Embodiment
Below in conjunction with concrete example, the present invention is described in further detail.These embodiment are in order to demonstrate the invention, but not limit the scope of the invention by any way.
The preparation of embodiment 1D3PMP
(1) in 50mL there-necked flask, add the deuterated acetic acid of 10mL, 20mL ethanol, 3.7g stannous chloride, after shaking up, 80 ℃ add hot reflux 2h, use distilling apparatus to have the liquid of happy fragrance to steam, obtain deuterated ethyl acetate mixture 18mL, by the saturated CaCl of deuterated ethyl acetate of gained
2and dried over mgso, remove ethanol, acetic acid, water, obtains dry deuterated ethyl acetate 7mL.
(2) in the dry deuterated ethyl acetate of 7mL, add 3g sodium metal, condensing reflux, then add the acetic acid of 15mL50%, and regulate pH to 6.5, with isopyknic saturated sodium-chloride stratification, shift supernatant liquid, with 1g anhydrous sodium sulfate, be dried, obtain the deuterated ethyl acetoacetate of 3mL.
(3) 3mL phenylhydrazine is added in 10mL ethanol, then add the deuterated ethyl acetoacetate of 3mL step (2) gained, under 80 ℃ of conditions, react 10h, cold filtration, obtains 1.9g yellow mercury oxide, i.e. D3PMP.
The preparation of embodiment 2D3PMP
(1) in 50mL there-necked flask, add the deuterated acetic acid of 10mL, 50mL ethanol, 3.7g stannous chloride, after shaking up, 80 ℃ add hot reflux 2h, use distilling apparatus to have the liquid of happy fragrance to steam, obtain deuterated ethyl acetate mixture 20mL, by the saturated CaCl of deuterated ethyl acetate of gained
2and dried over mgso, remove ethanol, acetic acid, water, obtains dry deuterated ethyl acetate 10mL.
(2) in the dry deuterated ethyl acetate of 10mL, add 8g sodium metal, condensing reflux, then add the acetic acid of 12mL50%, and regulate pH to 6.5, with isopyknic saturated sodium-chloride stratification, shift supernatant liquid, with 2g anhydrous sodium sulfate, be dried, obtain the deuterated ethyl acetoacetate of 2mL.
(3) 8.3mL phenylhydrazine is added in 10mL ethanol, then add the deuterated ethyl acetoacetate of 2mL step (2) gained, under 80 ℃ of conditions, react 10h, cold filtration, obtains 0.6g yellow mercury oxide, i.e. D3PMP.
The preparation of embodiment 3D5PMP
(1) get hydrochloric acid and the 20mL water of 30mL30%, drip deuterated aniline 10mL, under 0-5 ℃ of condition, drip 10mL71.8% sodium nitrite solution, stir and keep 0-5 ℃ of reaction 4h, obtain the deuterated benzene of diazotising.In the deuterated benzene of diazotising, add 30g sodium bisulfite, 10g25% ammoniacal liquor and 15mL water, be warming up to 80 ℃, and back flow reaction 4h puts into 4 ℃ of Temperature drop in refrigerators, filters to obtain deuterated hydrazinobenzene hydrochloride salt.In deuterated hydrazinobenzene hydrochloride salt, add water 20mL, be heated to 50 ℃, the NaOH that drips 10M regulates pH to 11, stirs 30min, adds ethyl acetate extraction 3 times, and decompression distillation obtains the deuterated phenylhydrazine of 6mL.
(2) get the deuterated phenylhydrazine of 6mL step (1) gained and add in 10mL ethanol, add 7mL ethyl acetoacetate, under 80 ℃ of conditions, react 10h, cold filtration, obtains 3.7g yellow mercury oxide, is D5PMP.
The preparation of embodiment 4D8PMP
(1) in 50mL there-necked flask, add the deuterated acetic acid of 10mL, 20mL ethanol, 3.7g stannous chloride, after shaking up, in 80 ℃, add hot reflux 2h, use distilling apparatus to have the liquid of happy fragrance to steam, obtain deuterated ethyl acetate mixture 18mL, by the saturated CaCl of deuterated ethyl acetate of gained
2and dried over mgso, remove ethanol, acetic acid, water, obtains dry deuterated ethyl acetate 7mL.
(2) in the dry deuterated ethyl acetate of 7mL, add 3g sodium metal, condensing reflux, then add the acetic acid of 15mL50%, and regulate pH to 6.5, with isopyknic saturated sodium-chloride stratification, shift supernatant liquid, with 1g anhydrous sodium sulfate, be dried, obtain the deuterated ethyl acetoacetate of 3mL.
(3) get hydrochloric acid and the 20mL water of 30mL30%, drip deuterated aniline 10mL, under 0～5 ℃ of condition, drip 10mL71.8% sodium nitrite solution, stir and keep 0-5 ℃ of reaction 4h, obtain the deuterated benzene of diazotising.In the deuterated benzene of diazotising, add 30g sodium bisulfite, 10g25% ammoniacal liquor and 15mL water, be warming up to 80 ℃, and back flow reaction 4h puts into 4 ℃ of Temperature drop in refrigerators, filters to obtain deuterated hydrazinobenzene hydrochloride salt.In deuterated hydrazinobenzene hydrochloride salt, add water 20mL, be heated to 50 ℃, the NaOH that drips 10M regulates pH to 11, stirs 30min, adds ethyl acetate extraction 3 times, and decompression distillation obtains the deuterated phenylhydrazine of 6mL.
(4) the deuterated phenylhydrazine of 3mL step (3) gained is dissolved in to 10mL ethanol, adds the deuterated acetoacetyl ethyl ester of 3mL step (2) gained, under 60 ℃ of conditions, react 10h, obtain yellow mercury oxide, cold filtration, obtains 1.75g D8PMP.
The product yield of embodiment 1-4 is in Table 1.
Table 1 yield
| ? | Yield (%) |
| |
7 |
| |
2 |
| |
20 |
| Embodiment 4 | 7 |
As shown in Table 1, the yield of preferred embodiment 1 is higher 2.5 times than embodiment 2.The yield of embodiment 3 is that the yield of 20%, embodiment 4 is 7%.
The derivative monose of embodiment 5 use embodiment 1,3,4 gained derivative reagents and LC-MS analyze
PMP/D3PMP/D5PMP/D8PMP is made into 0.5mol/L methanol solution, under ammoniacal liquor alkaline medium, under 70 ℃ of conditions, reacts 90min with glucose, glucuronic acid, gucosamine, mannose, wood sugar, galactose, galactosamine, arabinose.After completion of the reaction, use chloroform extraction three times, centrifugal, get supernatant, carry out LC-MS analysis.Liquid-phase condition: liquid phase post 0.3 * 250mm SB-C18 post (5um, Agilent), flow velocity 15uL/min, mobile phase A is 0.01mol/L ammonium acetate solution, and Mobile phase B is acetonitrile, and applied sample amount is 0.02uL; Mobile phase condition: mobile phase 17%B15min, 17%B30min linearity is increased to 21%B, and 21%B15min linearity is increased to 23%B, 17%B15min, UV-detector DAD245nm detects.Mass spectrum is electrospray ionization mass spectrum negative ion mode.The results are shown in Figure 1 and table 2.
From Figure 1A chromatogram, eight kinds of monose standard items have obtained good separation by the inventive method; From Figure 1B total ion current figure, the derivant of these eight kinds of monosaccharide components all has the quasi-molecular ions apparently higher than background on mass spectrum.
Molecular weight after table 2 monose PMP is derivative
Result shows, the mass number of D3PMP derivant is than PMP derivant large 6, the mass number of D5PMP derivant is than PMP derivant large 10, the mass number of D8PMP derivant is than PMP derivant large 16, illustrate that PMP/D3PMP/D5PMP/D8PMP successfully reacts with the reducing end of neutral sugar, acid sugar, amino sugar, with a kind of monose, with different deuterated reagents, derive and on mass spectrum, can obtain one group of peak.
Embodiment 6 monose qualitative and quantitative analysis
Glucose, glucuronic acid, gucosamine, mannose, wood sugar, galactose, galactosamine, arabinose standard items (2nmol/uL) are derived with D3PMP, and testing sample derives with PMP, carries out LC-MS analysis.Deriving method and LC-MS analytical approach are with embodiment 5, and loading volume is 0.02uL.Result is as Fig. 2.
Experimental result demonstration, unknown sample contains glucose uronic acid.From the relative abundance of ions of Fig. 2, in testing sample, the content of glucuronic acid is 1.34nmol/uL.
The qualitative and quantitative analysis of 7 two kinds of unknown samples of embodiment
Glucose, glucuronic acid, gucosamine, mannose, wood sugar, galactose, galactosamine, arabinose standard items (2nmol/uL) are derived with D5PMP, and two kinds of testing samples derive with PMP, D3PMP respectively, carry out LC-MS analysis.Deriving method and LC-MS analytical approach are with embodiment 5, and loading volume is 0.02uL.Result is as Fig. 3.
Experimental result demonstration, two kinds of unknown samples contain glucose osamine.From the relative abundance of ions of Fig. 3, in two kinds of testing samples, the derivative glucose osamine content of D3PMP is 2.79nmol/uL, and the derivative gucosamine content of PMP is 4nmol/uL.
The qualitative and quantitative analysis of 8 one kinds of unknown samples of embodiment
Glucose, glucuronic acid, gucosamine, mannose, wood sugar, galactose, galactosamine, arabinose standard items (2nmol/uL) are derived with D5PMP, and testing sample derives with D8PMP, carries out LC-MS analysis.Deriving method and LC-MS analytical approach are with embodiment 5, and loading volume is 0.02uL.Result is as Fig. 4.
Experimental result demonstration, unknown sample contains glucose sugar.From the relative abundance of ions of Fig. 4, in testing sample, the derivative glucose sugar content of D8PMP is 4.17nmol/uL.
The qualitative and quantitative analysis of 9 two kinds of unknown samples of embodiment
Glucose, glucuronic acid, gucosamine, mannose, wood sugar, galactose, galactosamine, arabinose standard items (2nmol/uL) are derived with D5PMP, two kinds of testing samples derive with D3PMP, D8PMP respectively, carry out LC-MS analysis.Deriving method and LC-MS analytical approach are with embodiment 5, and loading volume is 0.02uL.Result is as Fig. 5.
Experimental result demonstration, two kinds of unknown samples contain glucose osamine.From the relative abundance of ions of Fig. 5, in two kinds of testing samples, the derivative glucose osamine content of D3PMP is 1.44nmol/uL, and the derivative gucosamine content of D8PMP is 1.04nmol/uL.
The qualitative and quantitative analysis of 10 3 kinds of unknown samples of embodiment
Glucose, glucuronic acid, gucosamine, mannose, wood sugar, galactose, galactosamine, 8 kinds of monose standard items of arabinose (2nmol/uL) are derived with D5PMP, 3 kinds of testing samples derive with PMP, D3PMP, D8PMP respectively, and LC-MS analyzes.Deriving method and LC-MS analytical approach are with embodiment 5, and loading volume is 0.02uL.Result is as Fig. 6.
Experimental result demonstration, three kinds of unknown samples contain glucose sugar.From the relative abundance of ions of Fig. 6, in two kinds of testing samples, the derivative glucose sugar content of D3PMP is 1.41nmol/uL, and the derivative glucose sugar content of PMP is 2.35nmol/uL, and the derivative glucose sugar content of D8PMP is 1.06nmol/uL.
The qualitative and quantitative analysis of 11 two kinds of unknown glycosaminoglycan enzymolysis disaccharides of embodiment.
By chondroitin sulfate two saccharide UA-GalNAc6S, UA-GalNAc4S, each 10ug of UA2S-GalNAc, derivative with D5PMP.The chondroitin sulfate C S1 of unknown content, CS2, CS3 respectively adds 200uL enzymolysis liquid (50mmol/LTris-HCl damping fluid, pH7.3, containing 0.01% bovine serum albumin(BSA), 100mmol/LNaCl, 0.1u chondroitinase abc), enzymolysis 48h in 37 ℃ of water-baths, deactivation 5min in boiling water, the centrifugal 15min of ultrafiltration (MWCO3000), freeze-drying.CSl enzymolysis disaccharides product is derivative with PMP, and CS2 is derivative with D3PMP, and CS3 is derivative with D8PMP.CS1, CS2, CS3 and the standard items derivant got after same volume derives mix rear LC-MS analysis, and deriving method and LC-MS analytical approach are with embodiment 5.Liquid-phase condition: liquid phase post 0.3 * 250mm SB-C18 post (5um, Agilent), flow velocity 10uL/min, mobile phase A is 0.01mol/L ammonium acetate solution, and Mobile phase B is acetonitrile, and applied sample amount is 0.02uL; Mobile phase condition: mobile phase 15%B15min, 17%B30min linearity is increased to 20%B, and 20%B15min linearity is increased to 23%B, 17%B15min, UV-detector DAD245nm detects.Mass spectrum is Negative electrospray ionization pattern.Result is as Fig. 7.
As shown in the figure, A is the total ion current figure of four kinds of samples; B is the mass spectrogram at peak 1, and corresponding standard items are the derivative UA-GalNAc6S of D5PMP; C is the mass spectrogram at peak 2, and corresponding standard items are the derivative UA-GalNAc4S of D5PMP; D is the mass spectrogram at peak 3, and corresponding standard items are the derivative UA2S-GalNAc of D5PMP.In figure, m/z=788.24 is the mass number of the CS1 after deriving, and m/z=792.26 is the mass number of the CS2 after deriving, and m/z=798.27 is the mass number of standard items, and m/z=804.29 is the mass number of the CS3 after deriving.As seen from the figure, in CS1, contain 5ug UA-GalNAc6S and 9.1ug UA-GalNAc4S, in CS2, contain 3.2ug UA-GalNAc6S and 7.7ugUA-GalNAc4S, in CS3, contain 9.3ug UA-GalNAc6S and 3.2ug UA-GalNAc4S, in all samples, all do not contain UA2S-GalNAc.
The reaction equation of embodiment 5-11 monose or glycosaminoglycan enzymolysis disaccharides and PMP, D3PMP, D5PMP, D8PMP is as follows:
X=H or D; Y=H or D.
Glucose in reaction equation can be worked as X=H for other monose or glycosaminoglycan enzymolysis disaccharides, and during Y=H, reactant is PMP, works as X=D, and during Y=H, reactant is D3PMP, works as X=H, and during Y=D, reactant is D5PMP, works as X=D, and during Y=D, reactant is D8PMP.
Claims (5)
1. a method that detects monose and disaccharides, comprises the steps:
(1) monose or disaccharides is derivative with derivative reagent;
(2) with liquid chromatography mass coupling (LC-MS), analyze the sample after deriving;
It is characterized in that, described derivative reagent comprises the deuterated methyl-5-of 1-phenyl-3-pyrazolone (D3PMP), 1-deuterated phenyl-3-methyl-5-pyrazolone (D5PMP), the deuterated methyl-5-of the deuterated phenyl-3-of 1-pyrazolone (D8PMP) and 1-phenyl-3-methyl-5-pyrazolones ketone (PMP);
Any one coupling in described derivative reagent PMP and D3PMP, D8PMP;
Described derivative reagent PMP and D3PMP, D5PMP, any two or three coupling of D8PMP;
Any several couplings of described derivative reagent D3PMP, D5PMP, D8PMP.
2. a kind of method that detects monose and disaccharides as claimed in claim 1, is characterized in that, described monose is animal, plant and microbe-derived polysaccharide and the basic composition unit of oligosaccharides, and described disaccharides is the disaccharides of enzymolysis glycosaminoglycan gained.
3. the method for the derivative reagent D3PMP of preparation as described in claim 1 or 2 any one, is characterized in that comprising the steps:
(1) get deuterated acetic acid, add ethanol, after stannous chloride shakes up, 70-90 ℃ adds hot reflux 1-5h, distills out liquid, generates deuterated ethyl acetate, by the saturated CaCl of deuterated ethyl acetate
2and dried over mgso, remove ethanol, acetic acid, water, obtains dry deuterated ethyl acetate;
(2) in dried deuterated ethyl acetate, add sodium metal, condensing reflux, then add 50% acetic acid, and regulate pH to 6-7, use saturated sodium-chloride stratification, transfer supernatant liquid, is dried with anhydrous sodium sulfate, obtains deuterated ethyl acetoacetate;
(3) get phenylhydrazine and add in ethanol, then add the deuterated ethyl acetoacetate of step (2) gained, under 70-85 ℃ of condition, react 10h, cold filtration obtains D3PMP;
Described deuterated acetic acid and the volume ratio of ethanol are 1:2-5;
The ratio of described phenylhydrazine and the amount of substance of deuterated ethyl acetoacetate is 1:0.2-0.8.
4. the method for the derivative reagent D3PMP of preparation as described in claim 1 or 2 any one, is characterized in that, described deuterated acetic acid and the volume ratio of ethanol are 1:2, and the ratio of the amount of substance of phenylhydrazine and deuterated ethyl acetoacetate is 1:0.8.
5. the method for the derivative reagent D8PMP of preparation as described in claim 1 or 2 any one, is characterized in that comprising the steps:
(1) get deuterated acetic acid, add ethanol, after stannous chloride shakes up, 70-90 ℃ adds hot reflux 1-5h, distills out liquid, generates deuterated ethyl acetate, by the saturated CaCl of deuterated ethyl acetate
2and dried over mgso, remove ethanol, acetic acid, water, obtains dry deuterated ethyl acetate;
(2) in dried deuterated ethyl acetate, add sodium metal, condensing reflux, then add 50% acetic acid, and regulate pH to 6-7, use saturated sodium-chloride stratification, transfer supernatant liquid, is dried with anhydrous sodium sulfate, obtains deuterated ethyl acetoacetate;
(3) get 30% hydrochloric acid and water, drip deuterated aniline, under 0-5 ℃ of condition, drip 71.8% sodium nitrite solution, stir and keep 0-5 ℃ of reaction 3-5h, obtain the deuterated benzene of diazotising.In the deuterated benzene of diazotising, add sodium bisulfite, 25% ammoniacal liquor and water, be warming up to 70～85 ℃, and back flow reaction 4h puts into 4 ℃ of Temperature drop in refrigerators, filters to obtain deuterated hydrazinobenzene hydrochloride salt.In deuterated hydrazinobenzene hydrochloride salt, add water, be heated to 50 ℃, the NaOH that drips 10M regulates pH to 10-11, stirs 30min, adds ethyl acetate extraction 3 times, and decompression distillation obtains deuterated phenylhydrazine;
(4) the deuterated phenylhydrazine of step (3) gained is dissolved in ethanol, adds the deuterated ethyl acetoacetate of step (2) gained, under 70-85 ℃ of condition, react 10h, cold filtration, obtains D8PMP;
Described deuterated acetic acid and the volume ratio of ethanol are 1:2;
Described deuterated aniline is 1:1 with the ratio of the amount of substance of sodium nitrite;
Described deuterated phenylhydrazine is 1:0.7 with the ratio of the amount of substance of deuterated ethyl acetoacetate.
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| CN107831227A (en) * | 2017-10-12 | 2018-03-23 | 青岛力腾化工医药研究有限公司 | A kind of assay method of glucan side chain degree |
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