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1.
High-voltage capillary zone electrophoresis (CZE) has been used for the first time in the analysis of non-, mono-, di-, and trisulfated disaccharides derived from chondroitin sulfate, dermatan sulfate, and hyaluronic acid. These glycosaminoglycans are first depolymerized using polysaccharide lyases. The resulting unsaturated disaccharide products can be detected by their ultraviolet absorbance at 232 nm. Different retention times were obtained for each unsaturated disaccharide analyzed by CZE. The application of a constant voltage across a 70-cm fused silica capillary using a single, simple buffer system resolved an eight-component mixture within 40 min. Quantitation of disaccharides derived from chondroitin sulfate using chondroitin ABC lyase (EC 4.2.2.4) and mixtures of unsaturated disaccharide standards was possible requiring only picogram quantities of sample. The disaccharides examined had a net charge of from -1 to -4 and were resolved primarily on the basis of net charge and secondarily on the basis of charge distribution. Two unsulfated disaccharides both containing the same unsaturated uronic acid residue were analyzed. One was from chondroitin having an N-acetylgalactosyl residue and one from hyaluronate having an N-acetylglycosyl residue. Despite the fact that they differed only by the chirality at one center, these disaccharides were resolved by CZE. CZE is a fast and simple method that represents a powerful new tool for analysis and separation of acidic disaccharide components of glycosaminoglycans.  相似文献   

2.
SB-424323 is a new, orally active anti-thrombotic agent presently in phase-II clinical development, with limited hemorrhagic risk and a unique mechanism of action involving the induction of glycosaminoglycans (GAGs) biosynthesis. The objective of the present study was to develop a simple and rapid high performance liquid chromatography (HPLC) method for determination of endogenous GAGs derived disaccharides in plasma samples from a phase-II clinical study of SB-424323. Sample preparation was a simple heat treatment of the diluted plasma followed by digestion of endogenous GAGs with chondroitinase ABC to yield unsaturated disaccharides, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (DeltaDi-0S), 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (DeltaDi-4S), and 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (DeltaDi-6S). These disaccharides were recovered and purified using centrifugal filtration through a filter with 3000 molecular weight cut-off along with externally added internal standard 2-acetamido-2-deoxy-3-O-(2-O-sulfo-beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (DeltaDi-UA2S). A gradient reverse phase HPLC separation was developed on a Waters Symmetry C(18) column (4.6 mm x 150 mm, 5 microm) with a gradient mobile phase system consisting of 0.8 mM tetrabutylammonium hydrogen sulfate and 2mM sodium chloride and acetonitrile at a flow rate of 1.0 mL/min. The eluate was monitored with an ultraviolet detector set at 230 nm. Plasma standard curves were linear (r(2)> or =0.994) in the concentration range 1.0-20 microg/mL with a lower limit of quantification (LLOQ) of 1.0 microg/mL for each of the disaccharide. The mean measured quality control (QC) concentrations for the disaccharides deviated from the nominal concentrations in the range of -8.92 to 5.61% and -16.3 to 16.7%, for inter and intra-day, respectively. The inter and intra-day precision in the measurement of QC samples, were in the range of 3.21 to 18.2% relative standard deviation (R.S.D.) and 0.32 to 20.9% R.S.D., respectively. The inter and intra-day precision in the measurement of endogenous GAGs derived disaccharides in human control plasma, were in the range of 5.8 to 15.9% R.S.D. and 1.17 to 7.74% R.S.D., respectively. Stability of the processed samples was confirmed up to 48 h in the auto-sampler. The method is simple, reliable, and easily adaptable to analysis of large number of samples under logistics of a clinical study. The present method has been used to investigate the GAGs levels in the plasma of patients in a phase II clinical study of SB-424323.  相似文献   

3.
A system capable of resolving all the known unsaturated nonsulfated, mono- and disulfated disaccharides derived from chondroitin sulfate samples, dermatan sulfate, and hyaluronic acid after their derivatization with dansylhydrazine and separation by HPLC and fluorimetric detection is reported. This method was found superior to others in that unsaturated disaccharides can be separated with good resolution in about 50 min in an isocratic solvent with a sensitivity greater than about 50 pmol (approx 20-30 ng) and linearity from 50 to 500 pmol. The system was applied to the analysis of various chondroitin sulfate samples, including highly sulfated species and dermatan sulfate, and also to a defructosylated polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41. Excellent agreement was obtained with traditional compositional analysis performed by anion-exchange HPLC separation and UV absorption at 230 nm.  相似文献   

4.
Nuclear Overhauser effects, with preirradiation of glycoside bond anomeric protons, coupling constants 3J C3, H1' and 3J C1', H4 and linkage optical rotations A were measured for L-Rha beta 1-3-L-Rha alpha 1-OMe and L-Rha alpha 1-3-L-Rha alpha 1-OMe which are the models of the disaccharide units of the Pseudomonas cepacia polysaccharide. Theoretical conformational analysis was carried out in terms of a mechanical molecular model approximation. The spatial structures of these disaccharides as well as of D-Rha alpha 1-2-D-Rha beta 1-OMe in aqueous solutions were discussed basing on the obtained results.  相似文献   

5.
In this report we describe a system capable of resolving all of the known unsaturated disaccharides derived from the chondroitin sulphates, dermatan sulphate and hyaluronic acid by chondroitinase digestion. This system is superior to others in that the non-sulphated and mono-, di- and tri-sulphated disaccharides can be separated with good resolution in approximately 40 min in an isocratic solvent. The system employs an amino-cyano silica gel column (Whatman Partisil 5 PAC, 25 cm) and is eluted with an isocratic solvent consisting of 48% (v/v) acetonitrile, 14% (v/v) methanol and 38% (v/v) aqueous buffer. This aqueous buffer contains 0.5 M Tris-HCl, 0.1 M boric acid, 23.4 mM sulphuric acid, pH 8.0. UV absorption is monitored at 229 nm and for most disaccharides as little as 150 ng can be reliably determined. The addition of boric acid to the eluent is essential for good resolution of all components and the addition of low concentrations of sulphuric acid is used to control the elution times of various components. The system was applied to the analysis of glycosaminoglycan standards and excellent agreement with previous compositional analyses was obtained.  相似文献   

6.
A high-performance liquid chromatography (HPLC) method for determining sophoridine in rat plasma was developed for application in the pharmacokinetic studies. The plasma was deproteinized with acetonitrile that contained an internal standard (ephedrine) and was separated from the aqueous layer by adding sodium chloride and sodium carbonate. The HPLC assay was carried out using a YMC-ODS column. The mobile phase was methanol-ethanol-0.01 moll(-1) ammonium acetate buffer-triethylamine (10:0.5:89.5:0.03, v/v/v/v) (pH 6.80). The flow rate was 0.8 ml min(-1). The detection wavelength was set at 210 nm. The method was used to determine the concentration-time profiles of sophoridine in the plasma following oral administration or injection of sophoridine aqueous solution. The fractions of sophoridine reaching the systemic circulation were estimated for the first time by a deconvolution method.  相似文献   

7.
Selective enzymatic hydrolysis of the peracetylated disaccharides, namely cellobiose, lactose, maltose and melibiose, with lipase from Asperilligus niger in aqueous buffer and organic solvent for 30 min afforded exclusively the corresponding heptaacetates with a free hydroxyl group at C-1 in high yield. Prolonged reaction of the β-1,4 linked cellobiose and lactose peracetates afforded selectively their hexaacetates with free hydroxyl groups at C-1,2, whereas the α-1,4 linked disaccharides maltose and melibiose peracetate gave a complex mixture of products. The reaction of 2-acetamido-2-deoxy-1,3,4,6-tetra-O-acetylglucopyranose (11) for 22 h afforded as the major product the diacetate 12 with free hydroxyl groups at C-1,4.  相似文献   

8.
Capillary liquid chromatography-mass spectrometry using graphitised carbon stationary phase and ion trap mass spectrometry was shown to be a powerful technique for analysing glycosaminoglycans digested with endoglycosidases. Commonly found disaccharides from heparin/heparan sulphate digests at sub nanomole levels were found to be separated by mass and/or retention time and detected by negative ion electrospray mass spectrometry predominantly as [M-H]- ions using a standard electrospray interface and flow rate between 6-10 microL/min. Graphitised carbon liquid chromatography-fragmentation mass spectrometry provided sequence data of disaccharides and oligosaccharides. Sequence information was obtained from either collision of the [M-H]- ions (low sulphated disaccharides) or of the [M+Na-2H]- ions (highly sulphated disaccharides). This separation and identification method of endoglycosidase digestion and sample preparation using a combination of cation exchange and graphitised carbon, was used to successfully analyse digests of keratan sulphate (keratanase) and heparin (heparinase) standards, and hyaluronic acid (hyaluronidase) from synovial fluid samples.  相似文献   

9.
In order to throw light on the mechanisms responsible for the enzyme-dependent absorption of disaccharides membrane hydrolysis of maltose and trehalose and the absorption of glucose (free and that derived from disaccharides) were studied in isolated loops (20 cm) of the rat small intestine in chronic experiments. The rates of glucose absorption were 0.26-0.81 micromol x min(-1) x cm(-1) when the loop was perfused with a 12.5 to 75.0 mmol/l free glucose solution, which is only insignificantly higher than the rates observed during perfusion with equivalent maltose solutions. The coupling coefficient (the ratio of glucose absorption rate to the rate of disaccharide hydrolysis) decreased from 0.90 to 0.60 with the increasing maltose concentrations in the infusate from 6.25 to 37.5 mmol/l, but remained unchanged (approximately 0.95) within the same range of trehalose concentrations. The permeability of the pre-epithelial barrier was equivalent to that of unstirred water layer of less than 40 microm thickness. Fluid absorption was within the range of 0.73-2.55 microl x min(-1) x cm(-1), and it showed a correlation with the rates of glucose absorption. The results agree with a model developed on the assumption that free glucose and that released from disaccharides share the same membrane transporters. It could be concluded that a close coupling of disaccharide hydrolysis with derived glucose absorption in chronic experiments is achieved mainly due to a high activity of glucose transporters, which are presumably not associated with membrane disaccharidases. The transcellular active transport is a predominant mechanism of disaccharide-derived glucose absorption under conditions close to physiological.  相似文献   

10.
A novel highly sensitive ion-pairing reversed-phase high performance liquid-chromatography/electrochemical detection method for simultaneous determination of l-ascorbic acid, aminothiols, and methionine in biological matrices was developed, optimized, and validated. Reduced forms of the analytes were extracted from the sample matrices with 10% meta-phosphoric acid solution((aqueous)). To determine the total vitamin C, the total aminothiols, and the total methionine, samples were treated with tris(2-carboxyethyl)phosphine solution in 0.05% trifluoroacetic acid solution((aqueous)) subsequent to deproteination to reduce the oxidized forms of these compounds. Various analytes were separated on a C(18) (250 × 4.6 mm, 5 μm) analytical column using methanol-0.05% trifluoroacetic acid solution((aqueous)) (05/95, v/v), containing 0.1mM 1-octane sulphonic acid as the ion-pairing agent) as the isocratic mobile phase pumped at a flow rate of 1.5 mL min(-1) at room temperature. The column eluents were monitored at a voltage of 0.85 V. These analytes were efficiently resolved in less than 20 min using n-acetyl cysteine as the internal standard. The present method was specific for the analysis of these analytes and demonstrated acceptable values for linearity (r(2)>0.999 in the range of 0.2-10,000 ng mL(-1) for all the analytes), recovery (>96%), precision (%RSD ≤ 2.0), and sensitivity (on column limit of detection: 250-400 fg and limit of quantification: 0.8-1.25 pg), indicating that the proposed method could be efficiently used for determination of these analytes in the context of clinical research.  相似文献   

11.
《Carbohydrate research》1988,173(1):89-99
Reactions of (1→4)- and (1→6)-linked disaccharides, mainly of maltose and isomaltose, with the Fenton reagent under physiological conditions were studied. Chemical characterization of oxidation products was conducted by g.l.c. and g.l.c.-m.s. of their trimethylsilyl derivatives, and the results demonstrated that (1→6)-linked disaccharides are more reactive with the hydroxyl radical (·OH) generated by the Fenton reagent than (1→4)-linked disaccharides. About 35–40% of (1→6)-and 15–20% of (1→4)-linked disaccharides were oxidatively degraded to smaller molecules after incubation for 24 h. Of the four disaccharides examined, namely, maltose, isomaltose, cellobiose, and gentiobiose, the α-(1→6)-linked disaccharide isomaltose exhibited the highest reactivity, whereas the β-(1→4)-linked disaccharide cellobiose showed the lowest. These results suggest the existence of a relationship between the configuration of the glycosidic linkage and the reactivity with ·OH in aqueous solution.  相似文献   

12.
A fast, simple and sensitive high performance liquid chromatographic (HPLC) method has been described for determination of acyclovir in human serum. Since acyclovir is a polar compound and soluble in aqueous medium and practically insoluble in most of organic solvents, its analysis in biological fluids in currently published HPLC methods, involve pre-treatment of acyclovir plasma sample including deproteinization or solid phase extraction. In present method liquid-liquid extraction of acyclovir and internal standard (vanillin) is achieved using dichloromethane-isopropyl alcohol (1:1, v/v) as an extracting solvent. Analysis was carried out on ODS column using methanol-phosphate buffer (0.05 M) containing sodium dodecyl sulfate (200 mg/L) and triethylamine (2 mL/L, v/v) as mobile phase (pH=2.3; 5:95, v/v) at flow rate of 2 ml/min. The method was shown to be selective and linear into the concentration range of 10-2560 ng/mL. Accuracy and precision of the method were also studied. The limit of quantitation was evaluated to be 10 ng/mL. This method was applied in bioequivalence study of two different acyclovir preparations after administration of 400mg in 12 healthy volunteers.  相似文献   

13.
A novel method is described for the preparation of sterile submicron unilamellar liposomes. The method is based on the lyophilization of double emulsions containing disaccharides as lyoprotectants in both the inner and outer aqueous phase. Using various phospholipids or mixtures of lipids as emulsifiers, the double emulsions can be prepared by a two-step emulsification, including hydrophilic agents in the inner aqueous phase or lipophilic agents in the oil phase. Then, the double emulsions are lyophilized after sterilization by passing them through a 0.22-μm pore filter. Rehydration of the lyophilized products results in liposomes with a relatively high encapsulation efficiency (for calcein, 87%; 5-fluorouracil, 19%; flurbiprofen, 93%) and a size below 200 nm measured by the dynamic light scattering technique (DLS) and the atomic force microscopy (AFM). The liposomes were found to be unilamellar from freeze-fracture electron micrographs and X-ray diffraction patterns. In addition, the liposomes can be reconstituted just before use by rehydration of the lyophilized products which are relatively stable. Thus, this reproducible and simple technique can be used to prepare sterilized, submicron unilamellar liposomes with a relatively high encapsulation efficiency, and excellent stability during long-term storage.  相似文献   

14.
High performance liquid chromatography was performed by an ion-pair reversed-phase method of six standard unsaturated disaccharides derived from heparan sulfate and heparin. Separation of delta Di-GlcNAc, delta Di-GlcN(2S), delta Di-GlcNAc(6S), delta Di-GlcN(2,6- or 2,2'-diS) and delta Di-GlcN(2,6,2'-triS) was achieved on a column of Jasco SC-02 with 10 mM tetrabutylammonium phosphate (pH 7.0) containing 30 or 47% methanol as a mobile phase. delta Di-GlcN(2,6-diS) and delta Di-GlcN(2,2'-diS) were separated on the same column with 35 mM triethylamine phosphate (pH 5.3). Four preparations (BL-1.0-1, BL-1.0-2, BL-1.0-3, and BL-1.25-1) separated from crude bovine lung heparan sulfate, a standard bovine lung heparan sulfate (BL-ST), bovine kidney heparan sulfate 1.0 M Fr and 1.25 M Fr (BK-1.0 and BK-1.25), and porcine kidney heparan sulfate 1.0 M Fr (PK-1.0) were digested with a mixture of heparinase, and heparitinases 1 and 2. The resulting foregoing unsaturated disaccharides in the digests were analyzed by the above HPLC procedures. The proportions of the unsaturated disaccharides in the digests of BL-1.25-1 and BL-ST were similar, but those of the others differed from each other. It is noteworthy that delta Di-GlcNAc plus delta Di-GlcNAc(6S) in the digest of BL-1.0-1 was approximately 95% of the total unsaturated disaccharides. Small amounts of delta Di-GlcN (2,6,2'-triS) were found in all the samples. It was found that delta Di-GlcN(2,2'-diS) was a prominent component in the disulfated unsaturated disaccharides from BL-1.25-1 and BK-1.25.  相似文献   

15.
Radicals induced by gamma-irradiation of sucrose, in the solid state at different temperatures and in aqueous solution, have been investigated by the spin trapping method. Electron spin resonance (ESR) combined with high performance liquid chromatography (HPLC), followed by spectral analysis with a simulation program (Voyons) revealed seven main radical species. A comparative study of the ESR signals from spin trapped gamma-induced radicals in some glycosides, disaccharides, 13C specifically labelled carbohydrates, as well as in several deoxysucroses and fructans, led to the assignment of a chemical structure to five out of the seven sucrose-nitroxide adducts previously evidenced. Sucrose is shown to be a conceivable model for the study of fructans gamma-radiolysis mechanism in aqueous solution.  相似文献   

16.
One of the first steps in characterizing heparan sulfate (HS) and its close relative heparin is to conduct disaccharide composition analysis. This provides an overall picture of the structure of the polysaccharide in terms of its constituent disaccharides. This is of importance, for example, in the initial characterization of spatially and temporally regulated structures. Two protocols for conducting disaccharide analysis are presented here, both exploiting exhaustive digestion of the polysaccharide, yielding constituent disaccharides, by bacterial heparin lyases. The first method, suitable for microgram quantities of material, relies on the separation of the disaccharides by high-performance liquid chromatography (HPLC) coupled to ultraviolet absorbance detection and can be performed in 2 d. The second exploits reducing end-labeling with the fluorophore BODIPY hydrazide, separation by HPLC, and subsequent fluorescence detection and quantitation. The latter is a high-sensitivity method that requires nanograms of starting material and has a detection limit in the low fmol range, and is thus the most sensitive method for disaccharide compositional analysis of HS yet reported. Fluorescence detection can be routinely carried out in 3 d.  相似文献   

17.
A direct injection high-performance liquid chromatography method is described for the determination of mitomycin C (MMC) in human plasma. The stationary phase consisted of hydrophilic and hydrophobic functional groups covalently bound to silicone-coated silica beads (CAPCELL PAK MF Ph-1, 150×4.6 mm I.D., 5 μm). A mobile phase using 100% water gave a better separation of MMC from endogenous interferences as compared to a mobile phase with 12.5% acetonitrile and 2.5 mM phosphate buffer (pH 6.9). Using water as the eluent (1 ml/min) and UV detection at 365 nm, MMC was found to elute at 5.0 min with a peak width of 0.3 min, whereas endogenous interferences eluted before 3 min. Total assay time per sample was 6 min. Internal standard was not required because the recovery of MMC was nearly complete, about 90% from 20 to 5000 ng/ml. The standard curve was linear from 20 to 5000 ng/ml in plasma, and the intra- and inter-day variation was between 3 to 6%. The lower detection limit was 5 ng/ml with a 25 μl sample, which represent a two- to four-fold improvement over the 10 ng/ml detection limit by previous methods using liquid-liquid extraction and comparable sample size. The simplicity of this method, i.e., no sample extraction, no internal standard, 100% aqueous mobile phase, isocratic elution and short analysis time (6 min/sample), makes it suitable for large scale routine sample analysis, whereas its small sample volume requirement and high sensitivity are useful for pharmacokinetic studies in small animals where limited sample is available.  相似文献   

18.
High-speed countercurrent chromatography (HSCCC) has been applied for the separation of theaflavins and catechins. The HSCCC run was carried out with a two-phase solvent system composed of hexane-ethyl acetate-methanol-water-acetic acid (1:5:1:5:0.25, v/v) by eluting the lower aqueous phase at 2 ml/min at 700 rpm. The results indicated that pure theaflavin, theaflavins-3-gallate, theaflavins-3'-gallate and theaflavin-3,3'-digallate could be obtained from crude theaflavins sample and black tea. The structures of the isolated compounds were positively confirmed by (1)H NMR and (13)C NMR, MS analysis, HPLC data and TLC data. Meanwhile, catechins including epigallocatechin gallate, gallocatechin gallate, epicatechin gallate and epigallocatechin were isolated from the aqueous extract of green tea by using the same solvent system. This study developed a modified method combined with enrichment theaflavins method by using HSCCC for separation of four individual theaflavins, especially for better separation of theaflavins monogallates.  相似文献   

19.
A sensitive and selective HPLC method for the determination of the disaccharides of chondroitin sulfate in horse and dog plasma was validated. Chondroitin sulfate is degraded by chondroitinase ABC to three primary unsaturated disaccharides, (1) 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose, (2) 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose, and (3) 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose, when treated with chondroitinase. Plasma samples (0.5 ml) were treated with 50 mU of chondroitinase ABC in 50 microl of 1 mM sodium phosphate buffer (pH 7.0) at 37 degrees C for 6 h. The samples were extracted with 25% trifluoroacetic acid in ethanol. The resultant samples were derivatized with 1% dansylhydrazine in ethanol at 40 degrees C for 3 h. The chromatographic conditions consisted of fluorescence detection (excitation at 350 nm and emission at 530 nm), mu-Bondapack NH(2) (300 x 3.9 mm), and mobile phase of acetonitrile:100 mM acetate buffer, pH 5.6 (76:24), pumped at 1.0 ml/min. The standard curves for each chondroitin disaccharide showed linearity over the selected concentration range (r > or = 0.99). The intraday percentage relative standard deviation was < or =9.5% and the interday precision was < or =6.9% or less. The relative intraday and interday error ranged from -7.3 to 6.6% for each chondroitin disaccharide in the plasma. The extraction recovery was found to be in the range of 90-96%. The validated method accurately quantitated the disaccharides of chondroitin sulfate after administration to dogs and horses.  相似文献   

20.
Dumville JC  Fry SC 《Planta》2003,216(3):484-495
Two neutral disaccharides, gentiobiose [beta- D-Glc p-(1-->6)- D-Glc] and nigerose [alpha- D-Glc p-(1-->3)- D-Glc], were detected in tomato ( Lycopersicon esculentum Mill.) pericarp and locule. Gentiobiose was present in the locule of green fruit and ripe fruit at 0.88 and 5.8 micro mol (kg fresh weight)(-1), respectively. When vacuum-infiltrated into green tomato fruit, exogenous gentiobiose (50 or 200 micro g per fruit) hastened the initiation of ripening (as judged by colour change) by 1-3 days relative to fruit that were infiltrated with glucose or isomaltose. Nigerose plus gentiobiose was particularly effective, but nigerose alone had no significant effect. The endogenous disaccharides were found to be present in the apoplastic fluid of the fruit, compatible with a proposed intercellular signalling role. The origin and metabolic fate of the disaccharides were investigated. Phenolic esters of these disaccharides were not detectable in tomato fruit and it is therefore unlikely that the free disaccharides were formed from a pool of such esters. An alternative possible biosynthetic origin, via transglycosylation, is discussed. When [(14)C]gentiobiose was vacuum-infiltrated into unripe or ripe fruit, the disaccharide remained intact for at least 1 h but was largely degraded within 24 h. The results suggest that gentiobiose is a new, naturally occurring oligosaccharin with a rapid turnover rate.  相似文献   

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