Determination of the absolute configuration of the sugar residues of complex polysaccharides by circular dichroism spectroscopy

Circular dichroism spectroscopy is used to determine the absolute configuration of the constituent sugar residues of bacterial polysaccharides from two strains of Streptococcus sanguis which are important receptors in bacterial coaggregation in the formation of dental plaque. A high-pressure liquid chromatographic method for carbohydrate analysis of glycoproteins, glycolipids, and polysaccharides has been extended to sugar chirality determination by collection of the eluted HPLC fractions and subsequent measurement of their circular dichroism spectra. The method involves methanolysis of the polysaccharide followed by formation of O-benzoyl derivatives and HPLC on reverse-phase columns. Circular dichroism spectra of the collected derivatives in acetonitrile solution in the region 230 nm show large ellipticity bands resulting from "chiral exciton" interaction among the O-benzoyl chromophores which are sensitive to the orientation of hydroxyl groups in the parent sugars. The sensitivity of the method is in the submicrogram range for the absolute configuration of single sugar residues. The circular dichroism of the intact polysaccharide in aqueous solution shows CD bands from the amide chromophore in the region 180 to 220 nm which are sensitive to the chirality of 2-acetamido sugar residues.     

Determination of ganglioside non-hydroxy fatty acid and long chain base by analysis of perbenzoylated derivatives

The non-hydroxy fatty acid and long chain base compositions from as little as 2.7 nmol of ganglioside were ascertained from perbenzoylated ganglioside derivatives. Non-hydroxy fatty acids were determined by mild alkaline methanolysis of the derivatives, followed by gas-liquid chromatography (GLC) of the methyl esters. N-acyl and N-benzoyl "gangliosides" that were generated by the methanolysis were hydrolyzed by a standard procedure that utilized aqueous acetonitrile-HCl, followed by high performance liquid chromatography (HPLC) determination of the biphenylcarbonyl derivatives with ultraviolet (UV) detection at 280 nm. A critical aspect of this procedure is a modified workup for the isolation of the biphenylcarbonyl derivatives which eliminates by-products that otherwise interfere with their separation by HPLC, especially when high sensitivity is required.     

An o-toluidine method for detection of carbohydrates in protein hydrolysates

The o-toluidine high-performance thin-layer chromatography (HPTLC) method for detection of reducing sugars has been demonstrated to be a facile method for composition analysis of protein hydrolysates with a maximum sensitivity range of 50-100 pmol. The solution phase reaction of o-toluidine with reducing sugars has been previously used for spectrophotometric detection of glucose at 480-630 nm. In contrast, the heterogeneous reaction of o-toluidine with reducing sugars resolved by thin-layer chromatography produces chromophoric derivatives which have a broad absorbance at 295 nm. Detection of these chromophoric derivatives is achieved by uv diffuse reflectance scanning densitometry. It is demonstrated that detection limits of less than 10 ng can be achieved by using HPTLC plates and is therefore equal or more sensitive for some sugars than recently reported high-pressure liquid chromatography methods using amperometric or fluorescence detection.     

Methanolysis of D-galacturonic acid: dimethyl acetals

Dimethyl acetals of D-galacturono-6,3-lactone and methyl D-galacturonate have been detected during methanolysis of D-galacturonic acid. The products of methanolysis were studied by ion-exchange chromatography and by g.l.c. of the trimethylsilyl (TMS) derivatives. Structural determinations were made from the mass spectra of the TMS derivatives. The course of methanolysis was monitored by g.l.c.     

The stability of the o-phthalaldehyde/2-mercaptoethanol derivatives of amino acids: an investigation using high-pressure liquid chromatography with a precolumn derivatization technique

The investigation of the stabilities of o-phthalaldehyde/2-mercaptoethanol derivatives of amino acids using a precolumn reaction technique and a high-pressure liquid chromatographic procedure is reported. The amino acid derivatives are shown to be stable on the high-pressure liquid chromatography column. Optimal conditions for the development of these derivatives for their separation using this technique are recommended.     

Rapid determination of doxorubicin and its fluorescent metabolites by high pressure liquid chromatography.

An original method for the separation and quantitation of doxorubicin (DOX) and its metabolites by high-pressure liquid chromatography and fluorometry is described. Doxorubicin and its derivatives are extracted from biological samples in a rapid, non-destructive manner, with a recovery close to 100%. The different compounds are rapidly separated by high-pressure liquid chromatography using an eluant system containing magnesium chloride, and detected quantitatively by fluorometry down to a concentration of 1.5 ng/ml in less than 5 min. Using this method, we have determined doxorubicin and its metabolites in plasma and urine, after an intravenous injection into DBA₂ and NMRI mice.     

Analysis of lectin properties with membrane ultrafiltration and high-pressure liquid chromatography.

A convenient method for the analysis of the binding properties of lectin with fluorogenic sugar chains is described. A lectin (concanavalin A or Datura stramonium agglutinin) was mixed with pyridylaminated sugar chains in buffer and the free chains obtained were isolated by membrane ultrafiltration. The amount of free sugar chains in the filtrate was measured by high-pressure liquid chromatography. The binding constants with the sugar chains, reaction kinetics, and other properties of these lectins were easily investigated. The method is simple and could be used to study the characteristics of any lectin in native form.     

Bile acids: XLVIII. Separation of conjugated bile acids by high-pressure liquid chromatography

Major conjugated bile acids of human bile have been resolved by high-pressure liquid chromatography. The elutions are carried out in two stages on Corasil II or μPorasil columns; first, an alkaline solvent system (2-propanol/ethyl acetate/water/7n ammonium hydroxide, 260:600:50:3) was used for separation into groups: tauro-dihydroxy derivatives, taurocholate, glyco-dihydroxy derivatives, and glycocholate. The fraction containing glyco-dihydroxy conjugates was separated by rechromatography in acetonitrile/acetic acid, 400:10, and the fraction containing tauro-dihydroxy conjugates could be partially resolved by rechromatography in acetonitrile/acetic acid/formic acid (97%)/water, 500:10:5:10. Three samples of prepared human bile have been similarly treated.     

Optimized isocratic conditions for the simultaneous determination of serotonin precursors and metabolites by reversed-phase high-pressure liquid chromatography with electrochemical detection

We describe an analytical procedure for the simultaneous determination of 5-hydroxyindole derivatives using high-pressure liquid chromatography with electrochemical detection. The procedure clearly resolves 5-hydroxytryptophan, 5-hydroxytryptamine, 5-hydroxytryptophol, and 5-hydroxyindole-3-acetic acid. The C-18 extraction column methodology and high-pressure liquid chromatography-electrochemical detection parameters have been developed to provide a rapid, sensitive, and reproducible quantitative determination of these 5-hydroxyindoles with picogram sensitivity. Chromatograms obtained from the analysis of whole normal mouse brain by the present technique clearly resolve the 5-hydroxyindoles and appear to be uncomplicated by interfering substances.     

Biological activity of 1,25-dihydroxyvitamin D2 in the chick.

1,25-Dihydroxyvitamin D2 has been prepared from 25-hydroxyvitamin D2 using rachitic chick kidney mitochondria. This metabolite was highly purified by Sephadex LH-20 chromatography and by preparative high-pressure liquid chromatography. Its purity was assessed by analytical high-pressure liquid chromatography which revealed no other 254-nm absorbing material and by mass spectrometry. The concentration of dilute solutions of 1,25-dihydroxyvitamin D2 was determined by high-pressure liquid chromatography and deflection of the 254-nm column monitor. The 1,25-dihydroxyvitamin D2 was then shown to be 1/5 to 1/10 as active as 1,25-dihydroxyvitamin D3 in the chick while it had previously been shown to be equal in activity in the rat. Thus, discrimination against the vitamin D2 side chain by the chick persists in the metabolically active 1,25-dihydroxyvitamin D compounds.     

High-performance liquid chromatography and gas chromatography-mass spectrometry determination of specific lipid peroxidation products in vivo

Peroxidation of membrane lipids has been implicated in the toxicity of reactive oxygen intermediates and of several hepatotoxins, but the specific products of this peroxidation in vivo have not been chemically identified. A method for the isolation, identification, and quantitation of specific lipid hydroperoxy and hydroxy acids formed in vivo has been developed. Hydroxylated derivatives of linoleic, arachidonic, and docosahexaenoic acids formed in mouse liver phosphatidylcholines following carbon tetrachloride administration were isolated by high-pressure liquid chromatography and identified as the trimethylsilyl ether methyl ester derivatives by gas chromatography-mass spectrometry. This methodology should be important for the investigation of the role of lipid peroxidation in a variety of normal physiologic and pathologic processes.     

A modified system for thiazolinone conversion to thiohydantoin derivatives and their separation by high-pressure liquid chromatography

An improved and very simple procedure for thiazolinone conversion to thiohydantoin derivatives and their separation by reverse-phase high-pressure liquid chromatography is described. Trifluoroacetic acid (10%) in ethyl acetate has been employed as a conversion reagent to circumvent the deamidation of acid amides and methylation of acidic amino acids, with a concomitant increase in the detection limits of these residues. Additionally, a very simple procedure has been developed for the separation of phenylthiohydantoin (PTH) derivatives of amino acids. The system takes advantage of the computer-controlled precise mixing of the solvents A and B to achieve accurate pH and thus avoid the necessity of pH adjustment of a buffer. The procedure is simple and highly reproducible, and separates all the 20 known PTH amino acids. The efficiency of the method has been examined on synthetic and natural proteins/peptides, in manual and autoconversion systems, over a period of more than 18 months.     

High-pressure liquid chromatography of polycyclic aromatic hydrocarbons and some of their derivatives.

The separation of polycyclic aromatic hydrocarbons and their derivatives by means of high-pressure liquid chromatography on Permaphase ODS is described. The method consists of the (isocratic) elution of compounds from the column with a methanol-water mixture of constant composition and is particularly suited to the identification of metabolic products of polycyclic hydrocarbons.     

Amino acid analysis by dinitrophenylation and reverse-phase high-pressure liquid chromatography

The determination of amino acids has been achieved by reverse-phase high-pressure liquid chromatography of their dinitrophenyl derivatives. The methods developed permit the quantitation of all amino acids commonly encountered in a protein hydrolysate and the effect of various parameters on this separation was systematically evaluated. The procedure eliminates the need for specialized postcolumn equipment as employed in conventional amino acid analysis and can be obtained by a simple gradient high-pressure chromatograph. The sensitivity obtained is comparable to that available by methods in common usage, being able to determine amino acids quantitatively in the low picomole range.     

Structural characterization of oligosaccharides by high-performance liquid chromatography, fast-atom bombardment-mass spectrometry, and exoglycosidase digestion

A method for structural characterization of oligosaccharides after preparing uv-absorbing derivatives is described. The derivatives can be rapidly analyzed and purified by high-performance liquid chromatography, with separation of various structures determined primarily by size and sugar composition. Derivatization requires as little as 0.5-1.0 nmol of oligosaccharide, and detection of down to 50 pmol of oligosaccharide is possible by monitoring absorbance at 229 nm. In addition, the carbohydrate portion of the derivative was found to retain its sensitivity to exoglycosidases, allowing sequential enzymatic digestions for determination of sugar sequence and anomerity to be performed. The derivatives also possessed a site of potential positive charge, making them amenable to analysis by fast-atom bombardment-mass spectrometry. Permethylation of the derivatives permitted their separation by capillary gas chromatography, thus allowing investigation of their structures by gas chromatography-mass spectrometry. The combination of these techniques will allow almost the complete structure of small amounts of oligosaccharides to be determined.     

Anaerobic degradation of uric acid via pyrimidine derivatives by selenium-starved cells of Clostridium purinolyticum

Clostridium purinolyticum decomposed uric acid via pyrimidine derivatives under selenium starvation conditions. Products were acetate, formate, glycine, ammonia, and CO₂. 4,5-Diaminouracil could be identified as an intermediate after converting the labile substance into 6,7-dimethyllumazine. The breakdown of uric acid was inhibited by EDTA. High-pressure liquid chromatography methods have been developed for the simultaneous determination of uric acid, 4,5-diaminouracil, and 6,7-dimethyllumazine. The significance of the new pathway is discussed.Abbreviation HPLC high-pressure liquid chromatography     

N-terminal sequence analysis of polypeptide at the picomole level.

This paper describes a manual method for N-terminal sequence analysis of polypeptides at subnanomole sensitivity. The polypeptide is degraded stepwise by using the dimethylaminoazobenzene isothiocyanate/phenyl isothiocyanate double-coupling method, and the released dimethylaminoazobenzenethiohydantoins of amino acids were identified by reversed-phase high-pressure liquid chromatography. The dimethylaminoazobenzenethiohydantoins are coloured compounds and can be detected in the visible region with the sensitivity limit of 1 pmol (signal-to-baseline noise ratio 5). A high-pressure liquid-chromatographic method was developed for complete analysis of all amino acid dimethylaminoazobenzenethiohydantoin derivatives, including the by-products of serine and threonine. Thus, without use of an automatic sequenator or radioactive materials, it is possible to determine the complete sequence of peptides and N-terminal sequence of proteins with less than 1 nmol of material.     

An improved procedure for high-sensitivity microsequencing: Use of aminoethyl aminopropyl glass beads in the beckman sequencer and the Ultrasphere ODS column for PTH amino acid identification

An improved procedure for high-sensitivity microsequencing is described. The method employs (i) aminoethyl aminopropyl glass beads for in situ purification of quadrol by absorbing α-amino group reactive impurities commonly found even in highly purified quadrol and (ii) an Altex 5-μm Ultrasphere ODS column and highly purified methanol which is available commercially to identify phenylthiohydantoin derivatives of amino acids by high-pressure liquid chromatography. The efficiency of the method has been demonstrated by the results of sequence analyses of peptides obtained by acid and cyanogen bromide digestions of viral proteins. A significant increase in the initial and repetitive yields has been consistently observed by this procedure.     

The use of high-pressure liquid chromatography in the preparation of tritium-labeled chloramphenicol and its analogs.

The 1-hydroxy epimers of chloramphenicol and thiamphenicol formed from the reduction of the respective 1-oxo derivatives with [³H]NaBH₄ have been separated preparatively by high-pressure liquid chromatography on a μBondapak C₁₈ column. This separation procedure permits the facile and rapid preparation of the 1-³H-labeled derivatives of chloramphenicol and its analogs.     

Design and operation of a completely automated Beckman microsequencer

A unique, efficient, and inexpensive system has been designed and built for the automatic conversion of anilinothiazolinone derivatives extracted from a Beckman spinning-cup sequencer with subsequent on-line high-pressure liquid chromatography separation of the phenylthiohydantoin derivatives. The Auto Converter-Auto Sampler system is controlled by a tape programmer or microprocessor and operates by transfer of the sample from the conversion vial into an HPLC injection loop by nitrogen pressure. Incorporation of a minor programming change on the sequencer allows the introduction of nitrogen vapor into the spinning cup during phenylisothiocyanate coupling. These modifications have resulted in a completely automated subnanomole protein sequencer.