Reversed-Phase Ion-Pairing Liquid Chromatographic Separation and Fluorimetric Detection of Polyamines

Abstract

A rapid and specific reversed-phase ion-pairing high performance liquid chromatographic procedure for putrescine, spermidine and spermine is reported. The ion-pairing reagent, heptanesulfonate, was employed and o-phthalaldehyde and 2-mercaptoethanol were used for on-line post-column derivatization and subsequent fluorescence detection. Experiments were carried out to determine the effects of several variables such as pH, concentration of the aqueous buffer, counter-ion concentration, and the percentage of organic modifier in the moving phase. The minimum detection limits for the polyamines ranged from 120 pmoles for spermine to 12 pmoles for putrescine. The method includes a gradient program which provides complete separation from amino acids and specificity for the three polyamines. The procedure was applied successfully to urine and serum samples.     

Automated Determination of Urinary Polyamines

Abstract

A system for the determination of urinary polyamines by ion exchange chromatography with fluorescence detection is presented. It provides a sensitive and specific assay of putrescine, cadaverine, spermidine and spermine in urine hydrolyzates. An internal standard is used, which makes the automated determination of series of samples easy.     

High Performance Liquid Chromatographic Determination of Amino Acids in Biological Samples by Precolumn Derivatization with O-Phthaldialdehyde

Abstract

A suitable gradient system has been developed for rapid analysis of amino acids in biological samples using O-phthaldialdehyde as a precolumn derivatizing agent and fluorescence detection. Resolution of 21 amino acids has been accomplished with 3 μm Ultrasphere ODS column by using a multi-step gradient system of two solvents (0.1M sodium acetate, pH 7.2/methanol:tetrahydrofuran) in less than 1 hour. Within-assay and between-assay coefficients of variation of retention times and fluorescence yield show good reproducibility. The fluorometric detection response is linear from 25 to 500 pmoles with a minimum detection limit of less than 1 pmol. High resolution, rapid analysis and high sensitivity of this method facilitates amino acid analysis in samples of less than 1 mg of tissue.     

High-Performance Liquid Chromatography of Domoic Acid,a Marine Neurotoxin,with Application to Shellfish and Plankton

Abstract

A recent outbreak of poisoning resulting from the consumption of cultured blue mussels (Mytilus edulis L.) from a localized area in Eastern Canada has been attributed to the presence of domoic acid (1), a relatively rare neurotoxic amino acid, previously found only in some algae of the family Rhodomelaceae. Studies on aqueous extracts of shellfish tissue indicated that the toxin and several of its isomers could be separated (and isolated in sufficient amounts for subsequent structural identification) by reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) diode array detection (DAD). Aqueous acetonitrile containing 0.1% v/v trifluoroacetic acid was used as mobile phase. As the retention time and characteristic UV absorption spectrum of 1 (λ_max = 242 nm) permit unequivocal identification, the HPLC-DAD procedure was refined with a microbore column to provide a rapid (5 min), sensitive (0.3 ng detection limit) and reproducible assay method for the determination of 1 in shellfish tissue. Extraction was accomplished by boiling homogenized shellfish tissue for 5 min with distilled water. Extracts were taken through an octadecylsilica solid phase extraction clean-up prior to HPLC. This method has been applied to a variety of shellfish and phytoplankton samples.

BRIEF

Reversed-phase HPLC with ultraviolet diode array detection was used to analyze shellfish tissue and phytoplankton extracts for domoic acid. A rapid (5 min) and sensitive (0.3 ng detection limit) assay is presented.     

Spectral Assignment of Phenanthrene Derivatives Based on 6H-Dibenzo[C,E][1,2] Oxaphosphinine 6-Oxide by NMR and Quantum Chemical Calculations

Abstract

Organophosphorus compounds such as 6H-dibenzo[c,e][1,2]oxaphosphinine 6-oxide (DOPO, 1) and its derivatives are important and versatile compounds for a broad field of applications. However, a thorough spectral assignment is often subordinate to its chemical properties. This article presents and unambiguously attributes the ¹H and ¹³C NMR spectra of DOPO (1), selected products yielded from the Atherton–Todd reaction (2–4), DOPO-HQ (5) as well as sulfur derivatives (6–7) via a set of 1D- and 2D-NMR experiments. The complex P-C and P-H coupling patterns are discussed and compared with the derivatives possessing different chemical environments around the phosphorus atom. In addition, we compared our results with density functional theory calculations. Even though the prediction of NMR data of organophosphorus compounds via molecular modeling is limited, this study presents a method that yields good results for this class of heterocycles. This knowledge should help to quickly assign NMR spectroscopic data of other DOPO (1) derivatives and can be extrapolated to organophosphorus compounds in general.

Supplemental materials are available for this article. Go to the publisher's online edition of Phosphorus, Sulfur, and Silicon and the Related Elements for the following free supplemental resource: NMR Spectra of Compounds 1-7 (Figures S1 - S15).     

PREPARATION OF METHYL ESTER DERIVATIVES OF AMINO ACIDS BEARING HYDROLYSABLE N-PROTECTION

ABSTRACT

A convenient method for the esterification of amino acids bearing labile N-substituents is described. Three compounds, 2(a–c), have been synthesised with retention of an N-trifluoroacetamide or an enamine group.     

Flow Injection Analysis of Hydrogen Peroxide,Sulfite, Formaldehyde and Hydroxymethanesulfonic Acid in Precipitation Samples

Abstract

Hydroxymethanesulfonic acid (HMSA), the reaction product of sulfite and formaldehyde plays an important part in the aqueous phase conversion of sulfite to sulfate. HMSA is fairly stable under acidic conditions and in presence of hydrogen peroxide. Sulfite is unstable under these conditions.

A flow injection set-up was developed, which allows the determination of H₂O₂, sulfite, formaldehyde and hydroxymethanesulfonic acid.

H₂O₂ analysis by amperometric detection offers the possibility of a simple, robust field instrument. The detection limit is 5μg/l and the method is linear up to 5mg/l.

Based on the 4,4-dithiodipyridine/sulfite reaction selective and sensitive spectrophotometric detections were developed for sulfite, formaldehyde and hydroxymethanesulfonic acid. The detection limit of these compounds is 50μg/l and the method is linear up to 5mg/l.

A large fraction of S(IV) is present as HMSA in fog, dew and precipitation samples in The Netherlands.     

Condensation nucleation light scattering detection for biogenic amines separated by ion-exchange chromatography.

A sensitive method of analysis for biogenic amines, putrescine, cadaverine, histamine and an amino acid precursor, histidine is described herein using ion-exchange chromatography and condensation nucleation light scattering detection. The method was successfully used for the analysis of biogenic amines in fish samples. The method offers a number of advantages: fast elution of analytes with no need for mobile phase conductivity suppression, no derivatization and no electrochemical activity for the analyte's detection. The 3 sigma detection limits for these compounds were found to range from 8 to 20 ng/ml.     

Simultaneous Determination of Phenolic Compounds in Red Wines by HPLC‐UV

Abstract

Ten samples of commercially Italian red wines were analyzed in order to determine the phenolic content. Variations in wine types are largely due to differences in concentration and composition of these compounds. Polyphenolic compounds are a large and complex group of substances which constitute one of the most important quality parameters of wine. These constituents of red wine contribute to organoleptic characteristics and to antioxidant and anti‐inflammatory properties. Moderate wine consumption is associated with several beneficial physiological effects, which include anticancer activities, inhibition of platelet aggregation, and inhibition of LDL oxidation which constitutes the initial stage of the pathogenesis of arteriosclerosis.

For the analysis, reversed‐phase high performance liquid chromatography (HPLC) method coupled with UV‐Vis detection was used. The method uses a gradient elution to identify nine biologically active phenolic constituents: catechin; epicatechin; trans‐ and cis‐resveratrol; gallic, chlorogenic and caffeic acid; rutin and quercetin in red wine samples. The samples are injected directly without any pretreatment. The method is simple, fast, not expensive and shows good linearity for all constituents, and the detection limits ranged from 0.3–1.6 µg/ml for trans‐resveratrol and gallic acid, respectively. Moreover, the samples were analyzed in different times for estimation of stability of these compounds.     

Comparison of green extraction methods with conventional extraction method for extract yield,L-DOPA concentration and antioxidant activity of Mucuna pruriens seed

ABSTRACT

Mucuna pruriens is a plant of Fabaceae family. Seed of M. pruriens is considered as a rich source of levo-3,4 dihydroxyphenylalanine (L-DOPA), a non-protein phenolic amino acid. In the present study, three different extraction methods were compared for extract yield, concentration of bioactive compounds such as total phenol, L-DOPA and antioxidant capacity. Extracts were prepared using water acidified with hydrochloric acid (0.1?N) by conventional method of refluxing as well as two green methods namely ultrasound and microwave assisted solvent extraction. A rapid and qualified high-performance liquid chromatography method was also developed for quantification of L-DOPA in different extracts. Among the three extraction methods, microwave assisted extraction provided the best results for yield and quality of M. pruriens extract in much shorter time in comparison to refluxing method of extraction.     

Determination of Caffeine in Small Plasma Samples by Gas-Liquid Chromatography with Thin-Layer Chromatographic Sample Clean-Up

Abstract

A sensitive method for the determination of caffeine in small plasma samples (50 μ1) is described. The samples are extracted with chloroform and the extrects are freed from interfering compounds by thin-layer chronatography. The spots of caffeine and the internal standard, 7-ethyl theophylline, are scraped off the plates and both compounds are eluted from the scrapings by means of a glass capillary collector. The caffeine in the eluate is quantitated by gas-     

New Principles of Ion-Exchange Techniques Suitable to Sample Preparation and Group Separation of Natural Products Prior to Liquid Chromatography

Abstract

A Gentle method of group separation of low molecular weight hydrophilic natural products is reported. The method is based on separation of the compounds according to their net charge at different pH values using different types of ion-exchange columns connected in series. Precolumns retaining interfering compounds are used in some cases. Elution of the compounds retained on the columns is performed by use of volatile eluents. The elution principle for two of the ion-exchangers in question is removal of the charges on the column materials while for the third column the positive net charge on the compounds retained is removed. Thereby, the total amount of ions retained on the different columns is released and eluted into small volumes, which after evaporation leaves the ions as well defined salts. The method is experimentally simple and efficient to separation of natural products into groups suitable to direct use in sensitive methods of analysis as e.g. high-performance liquid chromatography and gas chromatography. Combinations of these column chromatographic methods have been adpated for micro or semimicro determinations of naturally occurring compounds, e.g., aromatic choline esters, amines, amino acids and esters of phenolic carboxylic acids. The methods seem to be general practicable for group separation of low molecular weight hydrophilic compounds.     

High Performance Liquid Chromatography Analysis of Sugar Cane Bagasse Hydrogenation Products. I. Group-Type Separation

Abstract

An HPLC Group-Type Separation was developed for the analysis of the liquefaction product of sugar cane wood fibers (bagasse). First, a liquid-liquid extraction technique was applied to separate the highly polar and polymerized aqueous fraction from the less polar organic fraction. This last fraction was then separated by HPLC into four classification groups: three hydrocarbons (saturates, olefins and aromatics) and a combination of polar compounds.

The application of these extraction and separation methods to the analysis of Brazilian sugar cane residue liquefaction products was very promising since the major group present in the extract is the aromatic hydrocarbons, suggesting its potential use as chemical feedstock, as well as a possible renewable source of energy. In addition, the method generates discrete and “clean” fractions for further detailed characterization when desired.     

High Pressure Liquid Chromatography of Selected Sulfur Compounds

Abstract

High pressure liquid chromatography was used to separate and determine quantitatively the following groups of sulfur compounds: thiols, sulfides, disulfides, sulfones, isothiocyanates, thioamides, and thioureas. Amperometric and UV detectors were compared; for thiols, thioureas, isothiocyanates, and thioamides, the former was generally more sensitive. With the exception of alkyl and cycloalkyl sulfides, the liquid chromatographic method can be used for the analysis of the investigated sulfur compounds below the ppm range. The method developed was compared to gas chromatography with flame photometric detection. The latter was found to be superior for the analysis of alkyl and cycloalkyl thiols, sulfides and disulfides of molecular weight below 200, whereas the former was more suitable for the analysis of aromatic thiols, sulfides and disulfides, as well as thioamides, isothiocyanates, and thioureas. Both methods were equivalent for the analysis of aromatic sulfones.     

(–)-β-Homoarginine anhydride,a new antioxidant and tyrosinase inhibitor,and further active components from Trichosanthes truncata

Abstract

One new amino acid derivative, (–)-β-homoarginine anhydride 1, as well as nine known compounds were isolated from Trichosanthes truncata. The structures of the isolates were elucidated by spectroscopic methods. Among them, compounds 5 and 11 could notably dose-dependently inhibit ROS productions in HaCaT keratinocyte cells without cytotoxicity in the concentration range of 0.2–20?μM. In cell-free mushroom tyrosinase assay, compounds 1–5, 10 and 11 had more potential anti-tyrosinase activities with IC₅₀ values of 106.9–255.6?μM than arbutin that were similar to predicted values of binding affinity calculated by molecule docking. The most active 2 had hydrogen bonds (Ser77, Glu309, Phe454) and electrostatic charges (Glu309, Glu248) interactions with mushroom tyrosinase, respectively. Our data manifested that T. truncata and its components are potentially to be developed as anti-aging and whitening agents for skin disorders.     

Chemiluminescent Method for Flow Injection Analysis of Anions

Abstract

We present here the preliminary results on a new flow injection chemiluminescent method of analysis for anions. This technique is rapid and sensitive with detection limits on the order of 1 × 10^?6M for chloride and 30 samples/hr possible.     

Rapid Determination of Methotrexate and 7-Hydroxymethotrexate in Serum and Cerebrospinal Fluid by Radial Compression Liquid Chromatography

Abstract

A rapid, specific and sensitive radial compression reverse phase liquid chromatographic method for the analysis of methotrexate and 7-hydroxymethotrexate in serum and cerebrospinal fluid is reported. A mobile phase consisting of acetonitrile-methanol-pH 3 phosphate (8:15:77) at 6 ml/min flow rate was employed. The U.V. detector was set at 317 nm, and folic acid was used as an internal standard. A rapid extraction of methotrexate and 7-hydroxymethotrexate was performed using Sep-Pak cartridges with high extraction efficiency for both compounds. Patients serum and cerebrospinal fluid samples were analyzed by the described method and the concentrations of methotrexate were compared to those obtained by an enzyme immunoassay. No interference from other metabolites or anticancer drugs in the described assay was observed.     

A High-Performance Liquid Chromatographic Approach for Monitoring Ethofumesate in Plant,Soil and Water

Abstract

A simple quantitative HPLC-UV method for the analysis of ethofumesate in fodder beet, soil and water samples is described. This method is based on a simple extraction step, gel-chromatographic and/or minisilica gel column clean up step and a separation step on a reversed phase column with uv-detection. The method is sensitive down to 10 μg/L in water and 50 μg/kg in plant and soil.

This approach shows recoveries between 90% and 110% in almost all matrices. Preliminary tests show that this method is also applicable for the analysis of ethofumesate in other plant materials.     

High-resolution magic angle spinning nuclear magnetic resonance (HR-MAS-NMR) as quick and direct insight of almonds

Abstract

Almonds are the tasty seeds of Prunus dulcis plants globally appreciated for the pleasant palatability and remarkable nutritional value, therefore it is very spread as snack and as basic ingredient of the confectionery products. The HR-MAS-NMR is a simple spectroscopy able to directly and quickly explore the chemical composition of powdered seed samples dispersed in D₂O. ¹H spectra witness the remarkable presence of triglyceride fatty esters together with sucrose; other minor water soluble metabolites are also detectable. This very rough approach is effectively providing chemical profiles featuring almond samples. In this analysis we were able to statistically distinguish the “Avola” almonds from other marketed products submitted to the same analysis. This is just a first investigation based on the main compounds but it might pave the way toward the quantitative evaluation of many other compounds in the almond therefore implementing the HR-MAS-NMR knowledge of these precious seeds.     

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.