Desalting and Freeze-Drying Techniques to Prepare Plant Derived Neutral Sugar Extracts for Quantitative HPLC

Abstract

Desalting and sample concentration are frequent prerequisites for the quantitative HPLC analysis of sugars and polyhydric alcohols in extracts of biological material. However, desalting using Dowex mixed-bed resins and sample concentration by conventional freeze-drying adversely affect the recovery of the carbohydrates being analysed, making quantitation almost impossible. In the present paper we report modified sample preparation procedures which overcome this technical problem. Efficient desalting was achieved by batchwise deionisation with an Amberlite mixed-bed deionisation resin, incorporating thymolphthalein to provide a sensitive indicator of resin exhaustion. Complete recovery of neutral sugars was possible provided the resin was subsequently washed with water. Concentration of dilute extracts with full recovery of neutral sugars was achieved by freeze-concentration using a Savant Speed-Vac centrifugal freeze-concentrator. The combination of these desalting and freeze-concentration procedures gave excellent recoveries of the neutral sugars together with dramatic improvements in HPLC column life and chromatographic resolution.     

A Sensitive Gas Chromatographic Method for the Determination of Metoprolol in Human Plasma

Abstract

A sensitive method for the determination of metoprolol in plasma has been developed. The procedure is based on gas chromatographic measurements of derivatized metoprolol, using 9-bromophenanthrene as internal standard. Metoprolol is derivatized with pentafluoropropionic anhydride. The resulting derivative gives a four-fold increase in sensitivity compared to the published methods where trifluoroacetic anhydride was used for derivatization.     

High-Performance Liquid Chromatography of Arginine-Containing Neuropeptides by Precolumn Derivatization and Fluorimetric Detection

Abstract

A previously developed fluorescence method for determining leupeptin and the angiotensins is shown to be applicable to assaying other small arginine-containing peptides. In this procedure arginine residues are derivatized by reaction with benzoin. Femtomole sensivity is obtainable with this method. However, it cannot be used with larger arginine-containing peptides because those fragment upon derivatization.     

Gas Chromatographic Analysis of Cyclophosphamide in Plasma and Tissues Using Nitrogen-Phosphorus Detection

Abstract

A gas chromatographic method for the analysis of cyclophosphamide in plasma, blood, and organ tissues is described. This method involves extraction of aliquots of plasma or tissue homo-genate in alkaline condition with ether. The extracted drug is derivatized with heptafluorobutyric anhydride followed by gas chromatographic separation via a glass column of 183 cm × 2 mm i. d. packed with 3% SE-30 on chromosorb W-HP. The derivatized cyclophosphamide and isophosphamide, an added internal standard, are detected by a nitrogen-phosphorus detector. The sensitivity limit of this method is 10 ng per gm or ml of sample and gives linearity over 100-fold of concentration range.     

Neutral Lipid Class Fractionation and Further Separation of Simple Neutral Glycolipids by OPTLC

Abstract

In lipid preparation from biological sources extraction and chromatographic methods are widely used. The use of the OPTLC method is introduced in the present paper. The method is suitable for the class separation of the neutral fraction of a total lipid extract with a single isocratic run. With a step or an exponential gradient the simple neutral glycolipids can be separated. Either elution can be performed on a 10 × 20 cm plate on 12 parallel samples. The chromatograms were evaluated by densitometric scanning after staining with orcinol-H²SO₄ reagent.     

An HPLC Assay for a Prostacyclin Analogue,Ciprostene Calcium,in Human Plasma

Abstract

A sensitive assay has been developed for the quantification of the prostacyclin analogue, ciprostene calcium, in human plasma. The method involves solid phase extraction of ciprostene calcium and internal standard, carbacyclin, from a small volume of human plasma. The extract is derivatized with 4-bramamethyl-7-acetoxycoumarin, and the derivatized product extracted with a polar solid phase cartridge and concentrated by evaporation. The final extract is separated by reversed phase HPIC and measured by a fluorimetric detector following post-column alkaline hydrolysis. The overall extraction efficiency is better than 75%, and the assay is linear over the concentration range studied (5–20 ng/ml). The limit of quantification was approximately 5 ng/ml. Ultimate sensitivity was limited by interfering peaks endogenous to the biological matrix. Coefficients of variation at mid-range concentrations are less than 10%.     

Modifications in the alditol acetate method for analysis of muramic acid and other neutral and amino sugars by capillary gas chromatography-mass spectrometry with selected ion monitoring

Two alditol acetate methods for the gas chromatographic (GC) analysis of neutral and amino sugars were compared. Following sodium borohydride reduction, one method uses methylimidazole as an acetylation catalyst without prior removal of water or borate salts and the other method uses sodium acetate after removal of borate and water. Depending on the acetylation conditions, muramic acid produced different derivatives. With methylimidazole, reliable derivatization of muramic acid was not possible, although other sugars derivatized reliably. With sodium acetate, all sugars tested were reproducibly derivatized. The utility of the sodium acetate method is shown by the trace GC-mass spectrometric analysis of muramic acid and rhamnose derived from bacterial peptidoglycan-polysaccharide complexes in mammalian tissue.     

A Convenient,Highly Efficient One-Pot Preparation of Peracetylated Glycals From Reducing Sugars

ABSTRACT

A convenient, highly efficient, one-pot, three-step procedure has been developed for the synthesis of peracetylated glycal derivatives from various reducing sugars including D-glucose, D-galactose, L-rhamnose, L-arabinose, D-maltose, D-lactose, and maltotriose. This procedure involves peracetylation of the reducing sugars with acetic anhydride and HBr/acetic acid followed by the transformation of the anomeric acetates to the corresponding bromides with additional HBr/acetic acid and finally reductive elimination of the 1-bromo and 2-acetoxy groups with Zn/CuSO₄·5H₂O in acetic acid/water containing sodium acetate. The overall yields of purified peracetylated glycals from the corresponding sugars range from 50 - 98%.

     

Capillary Column Inlet System for the Gas Chromatography of Biological Samples

Abstract

A glass injection system consisting of a packed precolumn and gasphase splitter has been developed for the introduction of derivatized biological samples on glass capillary columns. The precolumn provides complete sample evaporation before the splitter zone, traps contaminating nonvolatiles, and prevents decomposition or adsorption of components. In addition, a carrier gas control system is described that permits stable and repeatable adjustment of split ratio and capillary inlet pressure.

The linearity of the splitter has been established using a hydrocarbon mixture covering the range of methylene unit (MU) values in which the steroid derivatives are eluted.     

Determination of Glutamine and Asparagine by Isocratic Elution Reverse Phase Liquid Chromatography with Fluorescent Detection

Abstract

A method was developed specifically for the determination of glutamine and asparagine in the presence or absence of other amino acids. The amino acids were derivatized by o-phthalaldehyde/ 2-mercaptoethanol and separated by isocratic elution with a mobile phase consisting of acetonitrile and sodium acetate buffer. An application of the method for the analysis of glutamine and asparagine in the enzymatic hydrolysate of cottonseed protein is described.     

Amino Acid Profiling of Protein Hydrolysates Using Liquid Chromatography and Fluorescence Detection

Abstract

A liquid chromatography procedure is described for separating the amino acids in protein hydrolysates. The proteins are hydrolyzed with hydrochloric acid and an aliquot of the hydrolysate is derivatized with dansyl chloride reagent. The derivatization procedure takes only 2 minutes using a reaction temperature of 100°C. The dansylated amino acids are chromatographed using a reversed-phase C₈ column and a multi-step, nonlinear gradient elution solvent program which is readily achieved using a microprocessor-controlled liquid chromatograph. Chromatography is complete in approximately 40 min. The procedure is useful for characterizing proteins and may also be used to analyse intact dansylated polypeptides. Chromatograms showing the amino acid profile of chymotrypsin, albumin and histone are given.     

An Aminated GDP-Fucose Analog Useful in the Fucosyltransferase Catalyzed Addition of Biological Probes onto Oligosaccharide Chains1

Abstract

An analog (1) of GDP-fucose, where C-6 is derivatized with an eight-atom spacer terminating in a primary amino group, was chemically synthesized. This amino group in sugar nucleotide 1 can be acylated using an N-hydroxysuccinimide ester of biotin and it can be coupled to another molecule that also contains an amino group using squaric acid diethyl ester as the coupling reagent. In this way, biotin and a blood group A-active trisaccharide were linked to C-6 of fucose in GDP-fucose. Both complex sugar nucleotides thus prepared were active as donors for a human milk fucosyltransferase, which transferred the derivatized α-linked fucose residue to a glycoside of N-acetyllactosamine, thus labeling this sequence with either biotin or the blood-group A trisaccharide. Compound 1 is proposed as a general and versatile reagent which should permit the addition of biological probes to the sugar chains of cell surface glycoproteins or glycolipids.     

Optimization of a Sample Preparation Procedure for the Speciation of Organotin Compounds in Sediment Samples Using GC-AED

Abstract

Results of a comprehensive study of all analytical steps involved in the sample preparation procedure for the speciation of butyl- and phenyltin compounds in sediments are presented. The proposed method is based on acid leaching (using aqueous acetic acid) and simultaneous extraction of the ionic species into an organic solvent (n-hexane/ethyl acetate) with the addition of a complexing agent (diethyl dithiocarbamic acid). After evaporation to dryness, the residue is derivatized with sodium tetraethylborate in an aqueous buffer solution (acetate buffer, 0.1 M, pH 5) and extracted into n-hexane. Cleanup is performed over basic alumina and the ethylated organotin species are analyzed with a gas chromatograph coupled to a microwave-induced helium plasma atomic emission detector (GC-AED). The optimized method was validated within an interlaboratory study for the certification of tributyltin, triphenyltin and their degradation products in a freshwater sediment, the BCR candidate reference material 646.     

Rapid HPLC Methods for the Separation and Quantitation of a Mono-, Di-, and Tri-Saccharides Mixture and Applications

Abstract

A simple, rapid method has been developed for the separation and quantitation of mono-, di-, and tri-saccharides. The method utilizes a 30cmx 3.9mm i.d. Microbondapak NH₂ column, refractive index detection and water-acetonitrile elution. Two chromatographic systems are described. The isocratic mode was necessary to develop a procedure. 20 Carbohydrate's retention times were evaluated. To optimize the separation of nine water-soluble sugars, a gradient mode flow-programming was used. Separation was achieved within 28 minutes. The low detection limit (4 micrograms) of the above chromatographic procedure and its different possibilities could be of great interest to the analyst. The method has been successfully applied to quantify the major carbohydrates found in two types of commercial honey.     

Isolation of Lipophilic Persistent Organic Pollutants From Human Breast Milk

Background: Lipid removal from biological samples can be achieved by addition of concentrated sulfuric acid. However, certain persistent organic pollutants (POPs) such as chlorophenols are decomposed by sulfuric acid treatment and, thus, a more gentle lipid reduction method is needed for extraction of many environmental contaminants from biological samples. Membrane dialysis extraction (MDE) is a non-disruptive method to extract POPs from biological matrices.

Methods: Human breast milk samples were spiked with radiolabelled p,p′-dichlorodiphenyl trichloroethane ([C-14]-DDT) as a POP proxy and extracted using solid phase extraction (SPE). The extracts obtained were dialyzed by MDE in low-density polyethylene tubings containing a mixture of n-hexane and dichloromethane for 24 h, 48 h, or 72 h.

Results: The lipid content was reduced by 86.2% after one dialysis cycle of 24 h using MDE, and 87.1% recovery of the [C-14]-DDT standard was obtained. The DDT recovery could be further increased up to 96.3% and 98.1% by repeating the dialyses for one or two more cycles, respectively. However, the increased [C-14]-DDT recovery includes a concomitant increase in lipid carryover from 13.8% with one dialysis cycle to 22.1% with three cycles.

Conclusion: An SPE procedure for extracting POPs from breast milk and dialytic conditions for isolation of the extracted POP with minimal lipid carryover was established. The method is nondestructive and acceptable recoveries can be obtained within a single solvent shift as demonstrated by spiking standards. The lipid carryover was minimized, and the method may be considered for lipid removal before HPLC or GC analysis of environmental contaminants.     

High Performance Liquid Chromatographic Determination of Neomycin in Milk Using a Hisep Column

Abstract

A high performance liquid chromatographic (HPLC) method for the determination of neomycin in milk is described. Milk is passed directly through an amberlite CG-50 ion exchange resin column, and the neomycin which is retained on the column is derivatized with ortho-phthalaldehyde (OPA) reagent. The derivatized neomycin is eluted from the column with potassium borate buffer/methanol and analyzed by HPLC. A HISEP HPLC column with fluororoetric detection was used. Recoveries ranged from 94 to 102% in samples fortified between 0.1 to 5ppra levels. The detection limit is 50ppb.     

Development of an HPLC-Method for the Determination of E-2-Nonenal in Beer using Column Switching Techniques

Abstract

E-2-nonenal is an important compound formed during ageing of beer. It has a flavour threshold value of about 0.1 μg/l. A method is described for isolation of E-2-nonenal from beer by solid phase extraction. After extraction E-2-nonenal is derivatized with dansylhydrazine and analyzed with reversed phase HPLC using column switching techniques. The combination of on-line preconcentration of the derivative and heartcutting enables the analysis of E-2-nonenal in beer at natural levels.     

Monosaccharide composition analysis of immunomodulatory polysaccharides by on‐line hollow fiber microextraction with high‐performance liquid chromatography

The monosaccharide compositions of functional polysaccharides are essential for structure elucidation and biological activity determination. A sensitive method based on on‐line hollow‐fiber liquid‐phase microextraction with high‐performance liquid chromatography has been established for the analysis of ten monosaccharide compositions (two uronic acids, two amino sugars and six neutral sugars) of the immunomodulatory polysaccharides. After derivatization , the sample was injected into the lumen of a hollow fiber immersed in butyl ether and separated by liquid chromatography. Under optimized conditions, the calibration curves were linear (r ≥ 0.9996) in the range of 10–2000 μmol L^?1. The limits of detection were in the range of 0.04–1.58 μmol L^?1, and the recoveries were in the range of 92.1–99.6%, which shows that the method is applicable to the analysis of the monosaccharide composition of various polysaccharides.     

Determination of the Major Factors of Fermentation of the Nebramycin Complex by High Performance Liquid Chromatography

Abstract

For the determination of the major factors (tobramycin, kanamycin B, apramycin) from the fermentation broth a new method has been developed with combination of some earlier published method. In this method the protein content of the mixture was removed by treatment with tris-(hydroxymethyl)-aminomethane followed by centrifuging then the antibiotic content was derivatized by 1-fluoro-2,4-dinitro-benzene, The mixture was analysed on a reversed phase column.     

Tetracycline Sensitive Membrane Electrodes Based on Poly (Vinyl Chloride) Matrices and their use in Drug Analysis

ABSTRACT

Tetracycline hydrochloride-selective electrodes of both the coated wire and the conventional polymer membrane types have been prepared. They are based on incorporating the tetracycline-phosphotungstate ion-associate in plasticized poly(vinyl chloride) film. A Nernstian response is shown by these electrodes within certain concentration ranges depending on the type of electrode. The effect of pH of the test solution and time of soaking on the electrodes' performance are studied. The electrodes are highly selective for tetracycline with respect to several inorganic cations, sugars and some amino-acids of significant importance in biological fluids and pharmaceutical preparations. The standard cell potentials, E°, were determined at different temperatures and used to calculate the isothermal temperature coefficient of the cell. Tetracycline is determined successfully in pure solutions and in pharmaceutical preparation using the standard additions method and potentiometric titration.