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  • Author: Katarína Hroboňová x
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HPLC separation and determination of dicoumarol and other simple coumarins in sweet clover

Abstract

Dicoumarol is a mycotoxin, that acts as a blood anticoagulant, is formed during the microbial action of molds and fungi in spoiled hay or silage containing high-coumarin plant. A HPLC-DAD method for determination of coumarins, including dicoumarol, coumarin, and 4-hydroxycoumarin was developed. Methanol and acetic acid were used as mobile phase with gradient elution. The simultaneous separation was performed using C18 type of stationary phase. The recoveries were 88.6 – 92.6 %, 91.8 – 95.0 %, and 89.7 – 94.1 % (evaluated for three concentration levels) for dicoumarol, coumarin, and 4-hydroxycoumarin respectively. The parameters of system suitability (repeatability of retention times and peak areas) were determined for evaluation of the method. The method showed a good linearity in the concentration range 0.7 – 100 μg.mL−1 for dicoumarol, 0.05 – 100 μg.mL−1 for coumarin and 4-hydroxycoumarin with correlation coefficients higher than 0.9885. Extracts of sweet clover herb, hay, and spoiled hay were subjected to HPLC-DAD analysis. The most abundant compound in sweet clover herb and hay extracts was coumarin. In spoiled sweet clover hay extract the 4-hydroxycoumarin was detected in addition. The formation of 4-hydroxycoumarin was also observed in the synchronous fluorescence spectra recorded at the wavelength difference of 90 nm (difference between emission and excitation wavelength).

Open access
Application of solid-phase extraction for isolation of coumarins from wine samples

Abstract

Coumarins can be as a result wine storage and aging in wood drums and they can affect organoleptic characteristics of wine. The aim of this work was to determine the content of coumarins in wine samples originated from Slovak Tokaj wine region. The HPLC method with high specificity, accuracy, precision, and recovery was proposed. SPE sorbents of C18 type, styrene-divinylbenzene copolymer and molecularly imprinted polymers were compared for extraction of six coumarins, coumarin, aesculin, scoparone, scopoletin, 4-methylumbelliferone, and herniarin. Higher recoveries (above 89 %; except aesculin – recoveries higher from 68 %, RSDs less than 6 %) were obtained with selective polymeric sorbent laboratory prepared by molecularly imprinted technology. The results showed that content of coumarins in wine samples are in ng.mL−1 concentration levels and depend on the age and wine puttony.

Open access
HPLC Enantioseparation of Phenylcarbamic Acid Derivatives by Using Macrocyclic Chiral Stationary Phases

Abstract

The HPLC by using chiral stationary phases based on macrocyclic antibiotics, dimethylphenyl carbamate cyklofructan 7 and β-cyclodextrin in terms of polar-organic separation mode (mobile phase methanol/acetonitrile/acetic acid/triethylamine) were used for enantioseparation of alkoxy derivatives of phenylcarbamic acid. The effect of the analyte structures on the efficiency of enantioseparation was investigated. The most suitable stationary phase was teicoplanin aglycone, where the separations of the enantiomers were obtained (the resolution value from 0.65 to 2.90, depending on the structure of the analyte). Significant effect on the resolution of the enantiomers has position of alkoxy substituent in the hydrophobic part of the molecule. The enantiorecognition was achieved for 3-alkoxysubstituted derivatives.

Open access
Examination of the Chemical Composition of Propolis IX. Solid Phase Extraction of Coumarins from Propolis by Using Molecularly Imprinted Polymer

Abstract

The group selective molecularly imprinted polymers (MIPs) for coumarins, including umbelliferone, herniarin, 4-methylumbelliferone, scoparone were developed. Using umbelliferone as the template molecule, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as linking agent, chloroform as porogen and bulk polymerization as synthetic method, the MIPs were synthesized and characterized with rebinding experiments. The characteristics of MIPs were evaluated by chromatographic method and frontal analysis, and demonstrating good selectivity and high binding capacity (269 μg of umbelliferone per 100 mg of polymer). The group selective MIP was used as sorbent for the SPE pretreatment of coumarins from propolis extracts prior to HPLC analysis. Analysis of the samples showed good recoveries (>70 %). The limits of quantitation (LOQs) for studied compounds were 0.3-10 ng.mL-1 (determined for fluorescence detection).

Open access
HPLC separation of panthenol enantiomers on different types of chiral stationary phases

Abstract

Panthenol is a biologically active compound closely related to vitamin B5 (pantothenic acid). This work deals with the separation of panthenol enantiomers using high performance liquid chromatography. Different types of chiral stationary phases (β-cyclodextrin, isopropyl carbamate cyclofructan 6, amylose tris(3,5-dimethylphenylcarbamate)) were tested in normal phase separation mode. Effect of mobile phase composition on the resolution and retention of enantiomers was studied. Two types of detectors, low-wavelength UV and polarimetric, were used. The optimal chromatographic system includes a chiral stationary phase based on amylose and a mobile phase of hexane/ethanol (60/40, v/v) where the resolution of enantiomers reached the value R s = 2.49. Suitable chromatographic conditions were applied for the determination of panthenol enantiomers in samples of pharmaceutical preparations with the obtained recovery of more than 92 %. Linearity of the high performance liquid chromatography method with spectrophotometric detection was from 1.0 × 10−3 to 1.3 mg mL−1 (R 2 = 0.998), with the limit of detection of 0.3 × 10−3 mg mL−1 for both enantiomers.

Open access
HIGH – PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR ENANTIOSEPARATION OF UNDERIVATIZED α-AMINO ACIDS USING CYCLOFRUCTAN – BASED CHIRAL STATIONARY PHASES

Abstract

Chromatographic columns with chiral stationary phases based on chemically - bonded derivatized cyclofructans were evaluated for HPLC enantioresolution of underivatized α-amino acids. The analytical study of chiral separation of these analytes was focused on the selection of suitable chiral stationary phase and mobile phase. Using isopropyl carbamate cyclofructan 6 as a chiral stationary phase, α- amino acid optical isomers were separated. The retention and enantioseparation of chiral amino acids were also influenced by a mobile phase composition. The mixture methanol/acetonitrile/acetic acid/triethylamine (75/25/0.3/0.2 v/v/v/v) was found to be the most effective mobile phase for HPLC separation of studied compounds. HPLC enantioresolution of chiral amino acids was thermodynamically studied. Based on the enthalpy and entropy contribution values calculated from the van´t Hoff equation, HPLC enantioseparation under chosen chromatographic conditions was found to be an enthalpically driven.

Open access
Separation Of Methionine Enantiomers By Using Teicoplanin And Cyclofructan Columns

Abstract

Methionine is a naturally occurring amino acid. Its enantiomeric separation by using high performance liquid chromatography on various types of chiral stationary phases was studied. The effect of mobile phase composition on enantioselectivity and retention was considered. The separation of the enantiomers was attained in different separation modes – reversed phase mode for the macrocyclic antibiotic chiral stationary phases (teicoplanin, teicoplanin aglycone), normal phase and polar organic phase modes for the isopropyl carbamate cyclofructan 6 chiral stationary phase. It was shown that the hydrogen bonding, dipole interactions, steric effects between methionine molecules and stationary phases play an important role in the separation of enantiomers.

Open access