Optimisation of gradient HPLC analysis of phenolic compounds and flavonoids in beer using a coularray detector

A method was developed for simultaneous analysis of natural antioxidants in beer using multichannel electrochemical detection with a CoulArray detector, which enables selective and sensitive antioxidant detection in gradient HPLC and facilitates the identification of analytes based on the ratios of signals recorded at different potentials applied to the detection cells arranged in series. The separation conditions were optimised for 27 phenolic compounds including derivatives of benzoic and cinnamic acids, flavones, and a few related glycosides identified in beer samples. Separation selectivities of 11 columns with different stationary phase chemistries were compared, and the pH and gradient programs were optimised for the individual columns to provide best resolution and high number of resolved peaks, using the window-diagram approach. The effects of pH on the sensitivity of electrochemical coulometric detection were considered in the optimisation approach. The optimised conditions were applied to the analysis of real beer samples.     

Determination of the antioxidant activity of phenolic compounds by coulometric detection

The combination of reversed-phase high-performance liquid chromatography with coulometric detection allows the detection of phenolic antioxidants in complex matrices like plant extracts with a high degree of sensitivity and selectivity. According to their voltammetric behaviour, phenolic acids and tocopherols show maximal detector response at low potentials (100-450 mV) while flavonoids show optimal response at two different potential values (one at 0-300 mV and one at 600-900 mV). The potential corresponding to maximal detector response (MDRP) of phenolic acids was shown to be inversely proportional to their antioxidant efficiency as determined in a lipidic model system under strong oxidizing conditions (110 degrees C, intensive oxygenation) or by the DPPH() test. However, such a relationship was not observed for flavonoids.     

Multidimensional LC x LC analysis of phenolic and flavone natural antioxidants with UV-electrochemical coulometric and MS detection

A comprehensive 2-D LC x LC system was developed for the separation of phenolic and flavone antioxidants, using a PEG-silica column in the first dimension and a C(18) column with porous-shell particles or a monolithic column in the second dimension. Combination of PEG and C18 or C8 stationary phase chemistries provide low selectivity correlations between the first dimension and the second dimension separation systems. This was evidenced by large differences in structural contributions to the retention by -COOH, -OH and other substituents on the basic phenol or flavone structure. Superficially porous columns with fused core particles or monolithic columns improve the resolution and speed of second dimension separation in comparison to a fully porous particle C(18) column. Increased peak capacity and high orthogonality in different 2-D setups was achieved by using gradients with matching profiles running in parallel in the two dimensions over the whole 2-D separation time range. Multi-dimensional set-up combining the LC x LC separation on-line with UV and multi-channel coulometric detection and off-line with MS/MS technique allowed positive peak identification. The Coularray software compensates for the effects of the baseline drift during the gradient elution and is compatible with parallel gradient comprehensive LC x LC technique. Furthermore, it provides significant improvement in the sensitivity and selectivity of detection in comparison to both UV and MS detection. The utility of these systems has been demonstrated in the analysis of beer samples.     

Analysis of selected stilbenes in Polygonum cuspidatum by HPLC coupled with CoulArray detection

The roots of three varieties of Polygonum cuspidatum were analyzed for resveratrol and its analogs. The powder of the dried roots was extracted with aqueous ethanol (60% v/v) and the extracts obtained were analyzed using RP HPLC with coulometric detection. A simple HPLC method with a multichannel CoulArray detector was developed for the determination of four stilbenes: resveratrol, its glucoside piceid, piceatannol, and its glucoside astringin. Analyses were carried out on a LiChrospher C18 (125 x 4.6 mm id, particle size 5 microm) column with a mobile phase of ammonium acetate (pH 3) and ACN in gradient mode. Four compounds were monitored by a CoulArray electrochemical detector. Potentials of eight electrochemical cells in series were set in the range of 200-900 mV. Optimization of the mobile phase pH was performed. Calibration curves showed good linearity with correlation coefficients (r(2))--more than 0.9975.     

Determination of Plant Thiols by Liquid Chromatography Coupled with Coulometric and Amperometric Detection in Lettuce Treated by Lead(II) Ions

The main aim of this paper is to utilize high performance liquid chromatography with electrochemical detection for determination of thiols content in plants tissues of lettuce treated with lead(II) ions (0, 0.5 and 1 mM). We used two HPLC‐ED instruments: HPLC coupled with one channel amperometric detector and HPLC coupled with twelve channel coulometric detector to detect simultaneously twelve thiols. The detection limits of thiols measured by CoulArray detector were about two magnitudes lower in comparison to those measured by Coulochem III detector and were from tens to hundreds pM. Under the optimal conditions, we utilized HPLC‐CoulArray detector for analysis of tissues from lettuce plants. In addition, distribution and accumulation of lead ions with high spatial resolution was monitored using laser induced breakdown spectroscopy.     

Benefits and Pitfalls of HPLC Coupled to Diode-Array,Charged Aerosol,and Coulometric Detections: Effect of Detection on Screening of Bioactive Compounds in Apples

The new screening method for rapid evaluation of major phenolic compounds in apples has been developed. Suitability of coupling HPLC/UHPLC separation with the diode-array detection and universal charged aerosol detection with respect to the presence of interfering substances was tested. Characteristics of both detection techniques were compared and method linearity, limits of detection and quantitation, and selectivity of them determined. Student t-test based on slopes of calibration plots was applied for the detailed comparison. The diode-array detection provided the best results regarding sensitivity and selectivity of the developed method in terms of evaluation of phenolics profiles. The response of the charged aerosol detector was negatively affected by co-eluting substances during rapid-screening analyses. Coulometric detection was used for advanced characterization of extracts in terms of antioxidant content and strength to obtain more complex information concerning sample composition. This detection also allowed evaluation of unidentified compounds with antioxidant activity. HPLC/UHPLC separation using a combination of diode-array and coulometric detectors thus represented the best approach enabling quick, yet complex characterization of bioactive compounds in apples.     

Two-dimensional and serial column reversed-phase separation of phenolic antioxidants on octadecyl-, polyethyleneglycol-, and pentafluorophenylpropyl-silica columns

The separation selectivity of octadecyl-silica (C18) and of bonded pentafluorophenylpropyl-silica (F5) and PEG-silica columns was compared for natural phenolic antioxidants. The separation selectivities for phenolic antioxidants on C18 and F5 columns are strongly correlated, but low selectivity correlation indicating strong differences in the retention mechanism was observed between the C18 and PEG columns. Hence, the combination of a C18 and a PEG column is useful for separation of phenolic antioxidants that are not fully separated on single columns. Two-dimensional comprehensive liquid chromatography using a short PEG-silica column in the first dimension and a conventional C18-silica in the second dimension has the advantage of on-column focusing of the fractions transferred onto the C18 column in the second dimension, as a weaker mobile phase is used in the first dimension than in the second dimension. However, a stop-flow set-up in the first dimension system is necessary after the transfer of each fraction to the second dimension. Peak capacity is considerably larger but the separation time is much longer than with serially coupled PEG and C18 columns, which were employed for separation of beer and hop extract samples in connection with coulometric detection.     

Comprehensive two-dimensional liquid chromatography with parallel gradients for separation of phenolic and flavone antioxidants

Various combinations of PEG-silica, phenyl-silica and C18 columns in a single-column or serial (tandem) arrangement in the first dimension and a monolithic Chromolith column in the second dimension were tested for comprehensive two-dimensional (2D) LCxLC separation of phenolic and flavone natural antioxidants. The combinations of different stationary phase chemistries provided low selectivity correlations between the first-dimension and the second-dimension separation systems. Improvement in system orthogonality, bandwidths suppression, more regular band distribution over the whole 2D retention plane and increased peak capacity in different 2D setups was achieved by using gradients with matching profiles running in parallel in the two dimensions over the whole 2D separation time range. Instead of two sampling loops, two alternating trapping XTerra columns were used for sample fraction transfer from the first-dimension column to the second dimension. Stronger retention on the XTerra columns in comparison to PEG-silica or phenyl-silica columns in the first dimension allowed using focusing of sample fractions in narrow zones on the top of a trapping column and back-flushing into the second dimension in a very low volume of the mobile phase. This fraction transfer modulation provided significant bandwidth suppression in the second dimension. 2D systems with optimized stationary phase selectivity, parallel gradients and fraction transfer modulation using two trapping columns were applied for the analysis of natural antioxidants in beer and wine samples.     

Columns and optimum gradient conditions for fast second-dimension separations in comprehensive two-dimensional liquid chromatography

Gradient elution provides significantly higher peak capacity in comparison to the isocratic elution mode, hence it is very useful in online comprehensive two-dimensional liquid chromatography (LC). We compared suitability of five commercial core-shell columns and one monolithic column for fast gradients in the second LC dimension, where the time of separation is strictly limited by the fraction cycle time. In two-dimensional reversed-phase systems with partially correlated retention, the resolution, the peak capacity, and the regularity of coverage of the second-dimension retention space can be improved by appropriate adjusting the gradient time and the gradient range to suit the sample properties. We developed a new strategy for adjusting the gradient mobile phase composition range in the second-dimension, employing the retention data of representative sample standards characterizing the sample properties, which can be calibrated using the reference alkylbenzene series. Optimized second-dimension gradients with single-step or segmented profiles covering two or more fraction ranges, employed for the separation of subsequent fractions from the first-dimension, improve significantly the resolution, the separation time, and the regularity of coverage of the two-dimensional retention plane. The approach was applied to the two-dimensional comprehensive separation of phenolic acids and flavonoid compounds occurring as natural antioxidants.     

Separation and determination of flavonoids and other phenolic compounds in cranberry juice by high-performance liquid chromatography

A HPLC method was developed for the separation and determination of flavonoid and phenolic antioxidants in cranberry juices. Free flavonoid and phenolic compounds were fractionated into neutral and acidic groups by means of a solid-phase extraction method, followed by subsequent HPLC separations. Combined flavonoids and phenolics were hydrolyzed by acid before HPLC analysis. This developed method provides a fast and high resolution of individual flavonoid and phenolic compounds. In cranberry fruit, flavonoids and phenolic acids exist predominantly in combined forms, such as glycosides and esters. A total of 400 mg of total flavonoids and phenolic compounds/l of sample was found in a freshly squeezed cranberry juice, which was distributed as about 44% of phenolic acids and 56% of flavonoids. Benzoic acid was the major phenolic compound. Major flavonoids in the freshly squeezed cranberry juice were quercetin and myricetin.     

Temperature effects on separation on zirconia columns: applications to one- and two-dimensional LC separations of phenolic antioxidants

The effects of temperature and mobile phase on LC chromatographic separation of phenolic antioxidants on zirconia-based columns were investigated. Unlike silica-based materials, zirconia columns show excellent thermal stability over a wide range of temperatures and enable high-temperature separations. Enthalpic and entropic contributions to the retention of phenolic compounds on ZR-Carbon and ZR-Carbon C18 columns were determined from retention versus temperature plots in order to elucidate the retention mechanism of sample compounds over the temperature range up to 14 degrees C. High-temperature liquid chromatography on ZR-Carbon columns was used for comprehensive LC x LC two-dimensional separation systems based on the different selectivity of a Zorbax SB micro-column used in the first dimension and a ZR-Carbon column used in the second dimension. Two-dimensional LC x LC systems employing a setup with two alternately operated parallel ZR-Carbon columns in the second dimension were used for the analysis of phenolic antioxidants in beer and wine samples.     

Optimization of separation in two-dimensional high-performance liquid chromatography by adjusting phase system selectivity and using programmed elution techniques

The overall peak capacity in comprehensive two-dimensional liquid chromatographic (LC x LC) separation can be considerably increased using efficient columns and carefully optimized mobile phases providing large differences in the retention mechanisms and separation selectivity between the first and the second dimension. Gradient-elution operation and fraction-transfer modulation by matching the retention and the elution strength of the mobile phases in the two dimensions are useful means to suppress the band broadening in the second dimension and to increase the number of sample compounds separated in LC x LC. Matching parallel gradients in the first and second dimension eliminate the necessity of second-dimension column re-equilibration after the independent gradient runs for each fraction, increase the use of the available second-dimension separation time and can significantly improve the regularity of the coverage of the available retention space in LC x LC separations, especially with the first- and second-dimension systems showing partial selectivity correlations. Systematic development of an LC x LC method with parallel two-dimensional gradients was applied for separation of phenolic acids and flavone compounds. Several types of bonded C18, amide, phenyl, pentafluorophenyl and poly(ethylene glycol) columns were compared using the linear free energy relationship method to find suitable column combination with low correlation of retention of representative standards. The phase systems were optimized step-by-step to find the mobile phases and gradients providing best separation selectivity for phenolic compounds. The optimization of simultaneous parallel gradients in the first and second dimension resulted in significant improvement in the utilization of the available two-dimensional retention space.     

HPTLC and FTIR Fingerprinting of Olive Leaves Extracts and ATR-FTIR Characterisation of Major Flavonoids and Polyphenolics

The aim of this study was to analyse the effect of spontaneous microbial maceration on the release and extraction of the flavonoids and phenolics from olive leaves. Bioprofiling based on thin-layer chromatography effect-directed detection followed by ATR-FTIR spectroscopy proved to be a reliable and convenient method for simultaneous comparison of the extracts. Results show that fermentation significantly enhances the extraction of phenolic compounds and flavonoids. The polyphenolic content was increased from 6.7 µg GAE (gallic acid equivalents) to 25.5 µg GAE, antioxidants from 10.3 µg GAE to 25.3 µg GAE, and flavonoid content from 42 µg RE (rutin equivalents) to 238 µg RE per 20 µL of extract. Increased antioxidant activity of fermented ethyl acetate extracts was attributed to the higher concentration of extracted flavonoids and phenolic terpenoids, while increased antioxidant activity in fermented ethanol extract was due to increased extraction of flavonoids as extraction of phenolic compounds was not improved. Lactic acid that is released during fermentation and glycine present in the olive leaves form a natural deep eutectic solvent (NADES) with significantly increased solubility for flavonoids.     

Analysis of phenolic acids and flavonoids in honey

Honey is rich in phenolic acids and flavonoids, which exhibit a wide range of biological effects and act as natural antioxidants. The analysis of polyphenols has been regarded as a very promising way of studying floral and geographical origins of honeys. This review surveys recent literature on determination of these active compounds in honey. The analytical procedure to determine individual phenolic compounds involves their extraction from the sample matrix, analytical separation and quantification. We pay particular attention to sample pre-treatment and separation techniques (e.g., high-performance liquid chromatography and electrophoresis).     

HPLC analysis of flavonoids and phenolic acids and aldehydes inEucalyptus spp.

Summary Standards of the polyphenols occurring in wood, bark and leaf extracts ofEucalyptus spp. (i.e. flavonoids and phenolic acids and aldehydes) have been analyzed by HPLC using reversed phase columns, gradient elution and diode-array detection. The conditions used are reported.     

Analysis of phenolic compounds by high performance liquid chromatography and ultra performance liquid chromatography

Two novel chromatographic methods both based on utilization of sub-2-micron particle columns were developed for the analysis of phenolic compounds in this work. An HPLC system was equipped with C(18) silica-based analytical column (50 mm x 4.6 mm, 1.8 microm) and a UPLC system with ethylene-bridged hybrid C(18) analytical column (100 mm x 2.1 mm, 1.7 microm). In total 34 phenolic substances were divided into groups of phenolic acids, flavonoids, catechins and coumarins and were analysed in sequence using different gradient methods. System suitability test data, including repeatability of retention time and peak area, mean values of asymmetry factor, resolution, peak capacity and the height equivalent of a theoretical plate were determined for each gradient method by 10 replicate injections. The developed methods were applied in the analysis of real samples (grape wines, teas).     

Improvement of the sensitivity of 2D LC-MEKC separation of phenolic acids and flavonoids natural antioxidants using the on-line preconcentration step

A 2D method was developed for separation of phenolic acids and flavonoids natural antioxidants combining LC with MEKC. The in-capillary preconcentration step was applied for the improvement of the sensitivity of 2D method before the second dimension MEKC analysis. The influence of first dimension LC mobile phase composition on migration times in the second MEKC dimension was evaluated. When gradient elution is applied in the first dimension of 2D LC-MEKC system, increasing concentration of organic solvent in the mobile phase and in fractions transferred from LC influences the electroosmotic flow, partitioning equilibria of samples in micelles and properties of the micelles, which results in shifts of migration times during the consecutive runs in the second MEKC separation dimension. The shifts of migration times caused by the influence of increasing concentration of ACN on MEKC separation in second dimension of 2D LC-MEKC system were compensated by aligning the time axis using electroosmotic flow and micellar marker migration times. The optimized LC-MEKC method was applied on the separation of natural antioxidants in the plant extracts samples.     

Determination of phenolic compounds in dietary supplements and tea blends containing Echinacea by liquid chromatography with coulometric electrochemical detection

Analytical methodologies with ultrasonic extraction and liquid chromatography (LC) were developed for the determination of phenolic compounds in dietary supplements containing Echinacea. The phenolic compounds determined by these methods included caftaric acid, chlorogenic acid, cynarin, echinacoside, and cichoric acid. Samples from tablets, capsules, and bags of tea blends were extracted by sonication for < or = 30 min with methanol-water (60 + 40). The extracts were centrifuged and filtered, and the filtrates were diluted and analyzed by LC using a reversed-phase column and coulometric electrochemical (EC) detection. The mobile phase was acetonitrile-ammonium formate buffer, pH 3.5 (15.3 + 84.7) containing tetrabutyl ammonium hydrogen sulfate as an ion-pairing reagent. Extraction conditions (e.g., composition of the extraction solvent and sonication time) were optimized for different types of samples. Intra- and interday analytical variations were determined, and intraday analyses were performed by 2 independent analysts using 2 different LC systems. Results were generally comparable. The LC method with EC detection showed better sensitivity and selectivity when compared with LC with ultraviolet detection, although results were similar for the 2 methods for major compounds, i.e., caftaric acid, echinacoside, and cichoric acid. The identities of these major compounds found in samples were confirmed by LC/electrospray ionization mass spectrometry.     

New approach to the simultaneous analysis of catecholamines and tyrosines in biological fluids

New high-performance liquid chromatographic (HPLC) methods with amperometric-CoulArray detection were developed for simultaneous analyses of norepinephrine, epinephrine, L-DOPA, dopamine, 3-nitrotyrosine, m-, o-, and p-tyrosines. Overall, detection limit was in the low pmol range with amperometry, and in the low fmol range for the CoulArray method. Linear (r2 = 0.99) detector performances were observed in the ranges of 2-200 pmol with amperometry, and 0.2-20 pmol for the CoulArray method. Analytical precision values were better than 80 and 95% for HPLC-amperometry and HPLC-CoulArray method, respectively. These methods offer sensitivity, specificity, minimal sample requirement, and especially the HPLC-CoulArray method allows simultaneous assessment of various similar biomolecules.     

New zwitterionic polymethacrylate monolithic columns for one‐ and two‐dimensional microliquid chromatography

We prepared 0.53 and 0.32 mm id monolithic microcolumns by in situ copolymerization of a zwitterionic sulfobetaine functional monomer with bisphenol A glycerolate dimethacrylate (BIGDMA) and dioxyethylene dimetacrylate crosslinkers. The columns show a dual retention mechanism (hydrophilic‐interaction mode) in acetonitrile‐rich mobile phases and RP in highly aqueous mobile phases. The new 0.53 mm id columns provided excellent reproducibility, retention, and separation selectivity for phenolic acids and flavonoids. The new zwitterionic monolithic columns are highly orthogonal, with respect to alkyl silica stationary phases, not only in the hydrophilic‐interaction mode but also in the RP mode. The optimized monolithic zwitterionic microcolumn of 0.53 mm id was employed in the first dimension, either in the aqueous normal‐phase or in the RP mode, coupled with a short nonpolar core‐shell column in the second dimension, for comprehensive 2D LC separations of phenolic and flavonoid compounds. When the 2D setup with the sulfobetaine–BIGDMA column was used for repeated sample analysis, with alternating gradients of decreasing (hydrophilic‐interaction mode), and increasing (RP mode) concentration of acetonitrile on the sulfobetaine–BIGDMA column in the first dimension, useful complementary information on the sample could be obtained.