The Rapid Determination of Neutral Sugars in Biological Samples by High-Performance Liquid Chromatography

Abstract

A procedure for the analysis of neutral sugars in biological specimens is described. The method entails acid hydrolysis of the sample to liberate monosaccharides, which are subsequently derivatized with dansyl hydrazine. The sugar-dansyl hydrazones are separated and quantitated by hplc on a 5μ C18 RadialPak column with a gradient of acetonitrile in 10mM ammonium sulfate at pH 7. Fluorescent detection of the derivatized sugars permits 100-fold increased sensitivity compared to previously published glc methods.

This procedure was applied to the neutral sugar analysis of a glycoprotein of known composition (thyroglobulin) and to hard keratin fibers. The latter substance served as a model to critically evaluate the method on a highly resistant biological matrix containing low concentrations of neutral sugars.     

Determination of hydroxy and peroxy acid derivatives of uroporphyrin in the plasma of patients with congenital erythropoietic porphyria by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic method is described for the determination of hydroxy and peroxy acid derivatives of uroporphyrin in the plasma of patients with congenital erythropoietic porphyria. The porphyrins were extracted from the plasma with 20% trichloroacetic acid-dimethyl sulphoxide (1:1, v/v). The supernatant after centrifugation was chromatographed on a Hypersil-ODS column by gradient elution with 9% (v/v) acetonitrile in 1 M ammonium acetate buffer (pH 5.16) (solvent A) and 10% (v/v) acetonitrile in methanol (solvent B) as the gradient mixture. The method was also suitable for the preparative isolation of the porphyrins.     

Thermal stability of glucose and other sugar aldoses in normal phase high performance liquid chromatography

Analysis of glucose and other simple sugars are often performed by use of normal phase HPLC methods with acetonitrile as major eluent. The present results clearly show that column temperature plays an important role with respect to chromatographic performance and detection limits of glucose when using a specific carbohydrate column. A change in column temperature from 25 to 45 degrees C reduced the detection of glucose (with ELSD) by more than 41%, whereas the detection of other sugar aldoses (galactose, xylose and rhamnose) were suppressed even more. By increase of column temperature to 70 degrees C the detector signal of glucose was found to be less than 2% compared to that obtained at 20 degrees C. Neither fructose nor sucrose showed similar correlation between column temperature and detection. The rate of decreased response is not dependent on sample concentration or the ELSD settings. The results express the importance of accurate temperature control in the analysis of sugar aldoses, and also values low column temperatures for samples with low concentrations of sugar aldoses in order to improve detection.     

Effects of elevated temperature and mobile phase composition on a novel C18 silica column

A novel polydentate C18 silica column was evaluated at an elevated temperature under acidic, basic, and neutral mobile phase conditions using ACN and methanol as the mobile phase organic modifier. The temperature range was 40-200 degrees C. The mobile phase compositions were from 0 to 80% organic-aqueous v/v and the mobile phase pH levels were between 2 and 12. The maximum operating temperature of the column was affected by the amount and type of organic modifier used in the mobile phase. Under neutral conditions, the column showed good column thermal stability at temperatures ranging between 120 and 200 degrees C in methanol-water and ACN-water solvent systems. At pH 2 and 3, the column performed well up to about 160 degrees C at two fixed ACN-buffer compositions. Under basic conditions at elevated temperatures, the column material deteriorated more quickly, but still remained stable up to 100 degrees C at pH 9 and 60 degrees C at pH 10. The results of this study indicate that this novel C18 silica-based column represents a significant advancement in RPLC column technology with enhanced thermal and pH stability when compared to traditional bonded phase silica columns.     

Detection of neutral and aminosugars from glycoproteins and polysaccharides as their alditol acetates

A new method for the preparation and separation of alditol acetates from neutral sugars has been applied to aminosugars. Reduced aminosugars were rapidly acetylated using 1-methylimidazole as the catalyst without removal of borate formed during reduction. The alditol acetates were separated by glass capillary gas chromatography on Silar 10C. The alditol acetates of aminosugars had retention times much longer than those of neutral sugars. However, the alditol acetates of the deamination products of aminosugars had shorter retention times and were resolved from those of neutral sugars. This method was used for the simultaneous detection of neutral and aminosugars in acid hydrolysates of chitin and the glycoproteins, ovalbumin and peroxidase.     

An improved method for quantitative sugar analysis of glycoproteins

Although there are numerous methods available to hydrolyze glycans utilizing strong acids, it all requires lengthy steps to obtain quantitative yield. We have developed a new simple one-step method for analysis of amino and neutral monosaccharides of glycoproteins quantitatively. Free monosaccharides were found to be stable during hydrolysis of glycans with 6 N HCI at 80 degrees C up to 2 h. Using this condition, analysis of free monosaccharides hydrolyzed from the bovine fetuin showed sugar composition of Gal: Man: GlcN: GaIN = 13.2: 11.0: 15.5: 2.6, which is closely matched with the reported value of 12.4: 9.6: 17.2: 2.7 (Townsend et al., ABRF News 8: 14, 1997). This method was shown to be applicable to varieties of well-characterized glycoproteins, erythropoietin, fibrinogen and soybean agglutinin. The amounts of sugars released under the condition were very close to the experimental values by other procedures or to the theoretical ones. This condition was found to be suitable for direct sugar analysis of fetuin, which have been immobilized onto polyvinylidene difluoride membrane. Based on these results, it support that the 6 N HCl/80 degrees C/2 h is the simplest method for quantitative analysis of monosaccharide composition of glycoproteins.     

Examination of complex oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry on time-of-flight and magnetic sector instruments

Matrix-assisted laser desorption/ionization (MALDI) spectra of underivatized oligosaccharides of the type attached to asparagine in glycoproteins (N-linked oligosaccharides) were examined with linear time-of-flight (TOF) and magnetic sector instruments using 2,5-dihydroxybenzoic acid (2,5-DHB), α-cyano-4-hydroxycinnamic acid, sinapinic acid, 1,4-dihydroxynaphthalene-2-carboxylic acid or 2-(4-hydroxyphenylazo)benzoic acid (HABA) as the matrices. All compounds formed abundant [M + Na]⁺ ions with the strongest signals being obtained from 2,5-DHB after recrystallization of the initially dried sample spot from ethanol. Only traces of fragmentation were detected from neutral oligosaccharides on the TOF system but more abundant fragment ions (about 5% relative abundance) were present in the spectra from the magnetic sector instrument. Fragmentation was dominated by Y-type glycosidic cleavages (Domon and Costello nomenclature) between all sugar residues yielding sequence and branching information. Sialic acid-containing oligosaccharides generally produced the sodium adduct of the sodium salt and gave much weaker signals than the neutral sugars in the positive-ion mode. There was also considerable loss of the sialic acid moleties as the result of fragmentation on the magnetic sector instrument. The least fragmentation of both neutral and acidic sugars was caused by 2.5 DHB, which proved to be the most appropriate matrix for examination of oligosaccharide mixtures. Much better resolution of the oligosaccharides was obtained than by traditional methods such as the use of Bio-Gel P-4 gel filtration column chromatography. It is worth noting also that the measurements were considerably faster (a few minutes as opposed to about 16 h). In addition, no radiolabelling was necessary as required for detection on the P-4 columns. Mixtures of oligosaccharides from several glycoproteins (ribonuclease B, human immunoglobulin G (IgG) transferrin, bovine fetuin and chicken ovalbumin) were examined and the patterns of the identified oligosaccharides were found to agree closely with the known compositions of the sugar mixtures. The mass spectrometric resolution on the magnetic sector instrument was very much better (up to 3000, FWHM) than could be obtained with the linear TOF systems (200–400). The technique was used as a detection system for the products of exoglycosidase digestion in experiments to determine the detailed structure of the oligosaccharide chains from human IgG.     

Determination of propranolol concentration in small volume of rat plasma by HPLC with fluorometric detection.

A simple, rapid and sensitive fluorescence high performance liquid chromatographic method was developed to determine propranolol concentration in the small volume of rat plasma without the solvent extraction step using pronethanol as the internal standard. The analysis was accomplished using a 5 microm CAPCELL PAK analytical cyano column at room temperature and a mobile phase consisted of 1% aqueous acetic acid containing 0.2% triethylamine and acetonitrile (65:35, v/v; pH 3.8). The flow-rate was kept at 0.5 mL/min and column effluent was monitored with a fluorescence detector at an excitation wavelength of 230 nm and an emission wavelength of 340 nm. Retention times for pronethalol and propranolol were 8.5 min and 10.5 min, respectively. Linear regressions for the standard curves were linear in the range 2-800 ng/mL, giving correlation coefficients above 0.998. The detection limit was 1.34 ng/mL. No analytical interference was observed from endogenous components in rat plasma. This simple and sensitive assay method was feasibly applied to the pharmacokinetic study of propranolol after intravenous administration of 2 mg/kg of propranolol to normal and carbon tetrachloride-induced liver cirrhotic rats.     

Quantitative gas-liquid chromatography of neutral sugars in human serum glycoproteins. Fucose, mannose, and galactose as predictors in ovarian and small cell lung carcinoma

A precise and accurate gas-liquid chromatographic (GLC) method has been developed for the quantitative analysis of the neutral sugars L-fucose (6-deoxygalactose), mannose, galactose, and glucose in ethanol precipitates of human serum proteins. The chromatographic conditions and sample preparation resulted in short analysis time (20 min per run) and made routine analyses practicable (twelve samples per day). The alditol acetate derivatization yielded single derivatives for each sugar. Complete separation was achieved on a 2.0 m X 2 mm I.D. column with 2.0% Silar-7CP on Chromosorb W AW 80--100 mesh. The results of hydrolysis showed that the release of fucose and galactose preceded the release of mannose. Hydrolysis with AG 50 W-X8 (H+) ion-exchange resin in 0.5 N HCl at 100 degrees for 7 h optimized glycosidic bond cleavage with only minimal destruction of fucose, mannose and galactose. A combination of strong cation- and anion-exchange resin columns was used to remove chromatographic background of peptides, amino acids, amino sugars, and inorganic ions. An average R.S.D. of less than 4% with recovery of greater than 86% for the three sugars was achieved. The homogeneity of the chromatographic peaks for the neutral sugars of normal human serum glycoproteins was confirmed by GLC--mass spectrometry. Significantly elevated ratios of fucose, galactose, and mannose to serum protein were observed for patients with small cell lung and ovarian carcinomas.     

Capillary gas chromatographic analysis of amino acids in blood and protein hydrolysates as tert.-butyldimethylsilyl derivatives

A method is given for a one-step derivatization and gas chromatography of amino acids in blood and protein hydrolysates. Blood samples are partially purified by solvent extraction. Protein hydrolysates are neutralized with a triethylamine solution. Then tert.-butyldimethylsilyl derivatives of the amino acids are prepared in a one-step procedure and separated on a 30-m fused-silica SE-30 capillary column. Except for tryptophan and cystine, amino acids are eluted within 30 min. Amino acids are derivatized more rapidly than their corresponding trimethylsilyl derivatives and do not degrade on the long fused-silica columns.     

Separation and identification of chiralN-acylcalix[4]arene amino acid derivatives by use of reversed-phase HPLC

Summary This work focuses on the separation and identification of p-tert butyl calix[4]arene derivatives. An isocratic mixture of 65% (v/v) acetonitrile and 35% (v/v) water with 0.1% (v/v) phosphoric acid was used as the mobile phase. A Bio-Rad Bio-Sil ODS-5S (250 mm×4 mm) column was used as the stationary phase with UV detection of the analytes at 274 nm. The reversed-phase high performance liquid chromatographic method which was developed provided baseline separation of five p-tertbutyl calix[4]arene derivatives in twenty minutes.     

Simultaneous determination of glucose, 1,5-anhydro-d-glucitol and related sugar alcohols in serum by high-performance liquid chromatography with benzoic acid derivatization

A new, simple and sensitive pre-column high-performance chromatographic method for the determination of diabetes marker d-glucose, 1,5-anhydro-d-glucitol and related compounds is reported. Sugars (d-glucose, d-galactose, d-mannose, sucrose and arabinose) were derivatized with benzoic acid (BA) at 80 degrees C for 60 min. l-Fucose, fructose, d-lactose, l-rhamnose, arabinose and ascorbic acid were not reacted. Sugar alcohols (xylitol, erythritol, mannitol, sorbitol myo-inositol) were also derivatized with BA at 80 degrees C for 60 min. The fluorescence derivatives were separated on a TSK amide 80 column (4.6 mm i.d. x 250 mm, 5 microm) with acetonitrile-50 mm acetate buffer (pH 5.6; 4:96, v/v) as the mobile phase. The detection wavelength of beizoic acid derivatives was lambda(ex) 275 nm and lambda(em) 315 nm. The detection limits of sugars were 10-80 microg/mL. The calibration graphs were linear up to 10 mg/mL. The relative standard deviations of 500 microg/mL sugars were 7.0-7.3%. The proposed method was compared with the enzymatic photometric glucose analysis method (Glucose B-Test II Wako). The correlation coefficient was 0.83 (n = 20) and y = 0.82x + 5.91, where y and x are concentrations in microg/mL obtained by the proposed pre-column HPLC and enzyme-photometric method, respectively. The detection limits of sugar alcohols were 100-1000 ng/mL. The calibration graphs were linear to 50 microg/mL and relative standard deviations of 10 microg/mL were 7.2-8.2%. The 1,5-AG data by the proposed method was also compared with the enzymatic photometric 1,5-AG analysis method (Rana AG 1,5-AG determination kit, Nihon Kayaku) and good correlation (r = 0.91, n = 20) was also obtained. The proposed method was applied to the simultaneous determination of d-glucose, 1,5-AG and related sugar alcohols in serum from healthy males.     

Fast HPLC method using ion-pair and hydrophilic interaction liquid chromatography for determination of phenylephrine in pharmaceutical formulations

A rapid procedure based on a direct extraction and HPLC determination with fluorescence detection of phenylephrine in pharmaceutical sachets that include a large excess of paracetamol (65 + 1, w/w), ascorbic acid (5 + 1, w/w), and other excipients (aspartame and sucrose) was developed and validated. The final optimized chromatographic method for ion-pair chromatography used an XTerra RP18 column, 3 microm particle size, 50 x 3.0 mm id. The mobile phase consisted of a mixture of acetonitrile and buffer (10 mM sodium octane-1-sulfonate, adjusted with H3PO4 to pH 2.2; 200 + 800, v/v), with a constant flow rate of 0.3 mL/min. The separation was carried out at 30 degrees C, and the injection volume was 3 microL. Fluorescence detection was performed at excitation and emission wavelengths of 275 and 310 nm, respectively. The mobile phase parameters, such as the organic solvent fraction (acetonitrile) in mobile phase as an organic modifier, the concentration of sodium octane-1-sulfonate as a counter-ion, temperature, and pH of mobile phase, were studied. As an alternative to ion-pair chromatography, hydrophilic interaction liquid chromatography (HILIC) was investigated using a Luna HILIC column, 3 microm, 100 x 4.6 mm id. The mobile phase consisted of acetonitrile and buffer (5 mM potassium dihydrogen phosphate, adjusted with H3PO4 to pH 2.5; 750 + 250, v/v) at a flow rate of 0.8 mL/min. The separation was carried out at 25 degrees C, and the injection volume was 5 microL. The proposed method has an advantage of a very simple sample pretreatment, and is much faster than the currently utilized HPLC methods using gradient elution and UV detection. Commercial samples of sachets were successfully analyzed by the proposed HPLC method.     

Application of dansyl derivatization to the high pressure liquid chromatographic identification of equine estrogens

The HPLC qualitative analysis of conjugated estrogens is accomplished by a two-step procedure involving the formation of the corresponding dansyl derivatives. The first step involves the acid hydrolysis of the conjugated estrogens, followed by dansyl derivatization and HPLC separation of these derivatives on a liChrosorb Si-60 column with 50% (v/v) chloroform-n-heptane as the mobile phase. All of the dansyl estrogens are well separated except for the 17-keto estrogens, estrone, equilin, and equilenin. The second step, designed to detect the three 17-keto estrogens, begins with the selective sodium borohydride reduction of the conjugated 17-keto estrogens to the corresponding 17-hydroxyl compounds (the beta-epimer being formed in vast predominance over the alpha-epimer), followed by acid hydrolysis, dansyl derivatization, and HPLC separation of the derivatives as in the first step. Detection of the 17-keto estrogens is possible by determining differences in peak heights between the chromatograms of the first and second analyses. The The proposed method is sensitive, the dansyl derivatives stable, and nine different estrogens can be readily identified.     

Indirect resolution of enantiomers of penicillamine by TLC and HPLC using Marfey's reagent and its variants

TLC and HPLC methods were developed for indirect chiral separation of penicillamine (3,3-dimethylcysteine) enantiomers after derivatization with Marfey's reagent (FDNP-Ala-NH(2)) and two of its structural variants, FDNP-Phe-NH(2) and FDNP-Val-NH(2). The binary mobile phase of phenol-water (3:1 v/v) and solvent combinations of acetonitrile and triethylamine phosphate buffer were found to give the best separation in normal and reversed-phase TLC, respectively. The diastereomers were also resolved on a reversed-phase C18 HPLC column with gradient elution of acetonitrile and 0.01 m trifluoroacetic acid. The results due to these three reagents were compared. The method was successful for checking the enantiomeric impurity of l-penicillamine in d-penicillamine and to check the enantiomeric purity of pharmaceutical formulations of d-penicillamine. The method was validated for linearity, repeatability, limit of detection and limit of quantification.     

Temperature-programmed packed capillary liquid chromatography coupled to evaporative light-scattering detection and electrospray ionization time-of-flight mass spectrometry for characterization of high-molecular-mass hindered amine light stabilizers

High-molecular weight-hindered amine light stabilizers (HMW-HALSs) are of utmost importance in modern polyolefin stabilization technology and in-depth knowledge about their chemical composition, particularly the oligomers, is essential for development of new and more efficient stabilizers. In the present study, the applicability of temperature-programmed packed capillary LC coupled to miniaturized ELSD and positive mode ESI-TOF-MS for analysis of HMW-HALSs is demonstrated through extensive characterization of two state-of-the-art stabilizers, i.e., HALS-1 and HALS-2. Both stabilizers were individually separated on a 320 microm i.d. x 35 cm long Hypersil 3 microm ODS-100 column using a temperature program from 30 to 120 degrees C and a quaternary mixture of ethylacetate, acetonitrile, triethylamine (TEA) and acetic acid (45.0:44.9:10.0:0.1 (v/v/v/v)) as the mobile phase. The effect of using various amounts of ethylacetate, acetonitrile and triethylamine in the mobile phase on the chromatographic separation is demonstrated. Furthermore, the LC-ESI-TOF-MS analyses revealed that HALS-1 (oligomeric) was highly complex and consisted of at least five different mass series, while HALS-2, which was assumed to be monomeric, contained two different mass series. Chemical structures for nearly all species of both stabilizers are proposed.     

Chloride attachment negative-ion mass spectra of sugars by combined liquid chromatography and atmospheric pressure chemical ionization mass spectrometry

A method has been developed for the rapid determination of sugars, including molecular weight measurements, using high-performance liquid chromatography coupled with negative-ion, atmospheric-pressure chemical-ionization mass spectrometry. The chromatography was carried out on a 250 x 4 mm I.D. column packed with 7 microns NH2-silica. The mobile phase consisted of a high percentage of methanol or acetonitrile with a small amount of chloroform. During the mass spectrometry, a strong base is formed from the polar solvent molecules by corona discharge, followed by ion-molecule reactions in the chemical ionization ion source (e.g. the methoxy anion is formed from methanol). This strong base reacts with the chloroform, generating chloride ions, which in turn react with the neutral sugar molecules as they elute from the chromatograph. The chloride ion and sugar interactions yield chloride-attachment ions, which are further stabilized by successive collisions. In this method, authentic monosaccharides and some oligosaccharides show dominant quasi-molecular ions, [M + Cl]-, with little fragmentation, and its particularly useful for the molecular weight determination of sugars.     

高效液相色谱法测定萨拉沙星

采用高效液相色谱法测定了萨拉沙星。色谱柱为μ－BondapakTMC18柱（3．9mm×300mm）,流动相为V（乙腈）：V（甲醇）：V（2mmol／L磷酸,用三乙胺调pH3．5）＝30：5：65,用二极管阵列检测器检测,检测波长278nm,得到了满意的分离效果。     

High resolution glass capillary columns for the gas chromatographic analysis of alditol acetates of neutral and amino sugars

The development of improved high resolulation glass capillary columns for separating the alditol acetates of neutral and amino sugars is described. Different capillary column treatments were evaluated for their activity toward amino sugar derivatives and for their effect on the efficiency of the completed column. The simple two-step procedure of leaching with aqueous hydrochloric acid and dynamic coating with SP-2330 was found to produce efficient and inert columns.     

Temperature-promoted large-volume solute enrichment in column-switching miniaturized liquid chromatography: determination of an antioxidant

A two-valve sub-ambient temperature-promoted reversed-phase packed-capillary liquid-chromatography column-switching system has been tailored for sensitive determination of hydrophobic compounds. Such compounds are not easily dissolved in solvent mixtures of non-eluting properties that traditionally are used for solute enrichment in reversed-phase liquid chromatography. Enrichment-column solute focusing of large sample volumes was promoted by use of sub-ambient temperatures only, allowing the use of sample solvents that were stronger or equal to the mobile phase solvent strength. Subsequent column switching and enrichment-column temperature increment provided efficient low-dispersion back-flushed enrichment-column solute desorption onto the analytical column, where the solute was subjected to temperature-programmed gradient action. The antioxidant, Irganox 1076 (octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate) extracted from low density polyethylene with 100% acetonitrile served as a hydrophobic model compound. The mobile phase consisted of acetonitrile containing 10 mM triethylamine and formic acid, and the 0.25 mm id enrichment-column and analytical column in lengths of 27 and 250 mm, respectively, were packed with 3.5 microm Kromasil C18 particles. Sample volumes of up to 500 microL were successfully focused on the enrichment column at 5 degrees C using loading flow rates of up to 40 microL min(-1) prior to temperature programming to 90 degrees C. The concentration limit of detection of Irganox 1076 was 6 ng mL(-1) when using an injection volume of 500 microL. The within-assay precision was in the range 3.5-6.8% (n = 6) while the between-day precision was 7.5% (n = 3) relative standard deviation. The method was linear within the investigated mass range 3-100 ng (R2 = 0.9993).