Determination of phenolic compounds using high-performance liquid chromatography with Ce-Tween 20 chemiluminescence detection

A novel method for the simultaneous determination of phenolic compounds such as salicylic acid, resorcinol, phloroglucinol, p-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, and m-nitrophenol by high-performance liquid chromatography (HPLC) coupled with chemiluminescence (CL) detection was developed. The procedure was based on the chemiluminescent enhancement by phenolic compounds of the cerium(IV)-Tween 20 system in a sulfuric acid medium. The separation was carried out with an isocratic elution or with a gradient elution using a mixture of methanol and 1.5% acetic acid. For six phenolic compounds, the detection limits (3σ) were in the range 1.40-5.02 ng/ml and the relative standard deviations (n=11) for the determination of 0.1 μg/ml compounds were in the range 1.9-2.9%. The CL reaction was well compatible with the mobile phase of HPLC, no baseline drift often occurred in HPLC-CL detection was observed with a gradient elution. The method has been successfully applied to the determination of salicylic acid and resorcinol in Dermatitis Clear Tincture and p-hydroxybenzoic acid in apple juices.     

Analysis of non-anthocyanin phenolic compounds in wine samples using high performance liquid chromatography with ultraviolet and fluorescence detection

A simple and rapid HPLC method with UV and fluorescence detection (FLD) for the separation of ten phenolic compounds including gallic acid, catechin, epicatechin, caffeic acid, coumaric, trans-piceid, cis-piceid, trans-resveratrol, cis-resveratrol and quercetin is reported. The UV and fluorescence detector in series provided a high selectivity for the determination of these compounds. Precisions, recoveries and LODs achieved for all the analytes were satisfactory. The proposed method was applied to the determination of these compounds in commercially available red wines.     

Identification of phenolic compounds in artichoke waste by high-performance liquid chromatography-tandem mass spectrometry

A new fast and efficient method combining liquid chromatography coupled to ionspray mass spectrometry in tandem mode with negative ion detection is described for the qualitative analysis of artichoke waste. Forty-five phenolic compounds were identified on the basis of their mass spectra in full scan mode, mass spectra in different MS-MS modes, and retention times compared with those of available reference substances. The major compounds were found to be both caffeoylquinic and dicaffeoylquinic acids, luteolin glucuronide, luteolin galactoside, quercetin, and some quercetin glycosides.     

Automated on-line-solid-phase extraction—high-performance liquid chromatography-diode array detection of phenolic compounds in sherry wine

A new analytical method has been developed for sample preconcentration and analysis of phenolic compounds in sherry wine using on-line solid-phase extraction(SPE)-HPLC-diode array detection. The samples of wine were injected and adsorbed onto polystyrene divinylbenzene cartridges; a robotic semiflexible system was used to automate the SPE stage. Chromatographic separation was carried out in a Symmetry C₁₈ steel cartridge, with a two-step elution gradient. Peaks were identified by comparing their UV spectra with the library of spectra compiled by the authors.     

Direct identification of phenolic constituents in Boldo Folium (Peumus boldus Mol.) infusions by high-performance liquid chromatography with diode array detection and electrospray ionization tandem mass spectrometry

A very simple and direct method was developed for the qualitative analysis of polyphenols in boldo (Peumus boldus Mol., Monimiaceae) leaves infusions by high-performance liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionization tandem mass spectrometry (HPLC-MSⁿ). The phenolic constituents identified in infusions of the crude drug Boldo Folium were mainly proanthocyanidins and flavonol glycosides. In the infusions, 41 compounds were detected in male and 43 compounds in female leaf samples, respectively. Nine quercetin glycosides, eight kaempferol derivatives, nine isorhamnetin glycosides, three phenolic acids, one caffeoylquinic acid glycoside and twenty one proanthocyanidins were identified by HPLC-DAD and ESI-MS for the first time in the crude drug. Isorhamnetin glucosyl-di-rhamnoside was the most abundant flavonol glycoside in the male boldo sample, whereas isorhamnetin di-glucosyl-di-rhamnoside was the main phenolic compound in female boldo leaves infusion. The results suggest that the medicinal properties reported for this popular infusion should be attributed not only to the presence of catechin and boldine but also to several phenolic compounds with known antioxidant activity. The HPLC fingerprint obtained can be useful in the authentication of the crude drug Boldo Folium as well as for qualitative analysis and differentiation of plant populations in the tree distribution range.     

Determination of phenolic compounds in water samples by on-line solid-phase extraction—supercritical-fluid chromatography with diode-array detection

Summary The eleven Environmental Protection Agency (EPA) priority phenolic compounds have been determined by solid-phase extraction (SPE) coupled on-line to supercritical-fluid chromatography (SFC) with diodearray detection. The variables affecting chromatographic separation were optimized and the analytes were separated at 40 °C in two diol columns connected in series; a gradient of methanol, as modifier, and CO₂ was used as mobile phase. Under these conditions, all the compounds studied were separated to baseline in less than 13 min. PLRP-S and LiChrolut EN were tested as sorbents in a 10×3 mm i.d. laboratory-packed precolumn for solid-phase extraction. An ion-pair reagent, tetrabutylammonium bromide (TBA), was used in the extraction process to increase break-through volumes. The performance of the method was checked with tap and river waters and the pre-concentration of 20 mL of sample in a PLRP-S pre-column enabled phenolic compounds to be determined at low μg L⁻¹ levels with limits of detection ranging between 0.4 and 2 μg L⁻¹. The repeatability and reproducibility between days (n=3) for real samples spiked at 10 μg L⁻¹ were lower than 10%.     

Ultrasound-assisted extraction of phenolic compounds from strawberries prior to liquid chromatographic separation and photodiode array ultraviolet detection

Ultrasound-assisted extraction was used for the determination of phenolic compounds present in strawberries. The optimization study of the extraction was carried out using spiked samples (100 mg/kg). The sample immersed in an aqueous solution containing hydrochloric acid (0.4 M) was sonicated for 2 min (duty cycle 0.2 s, output amplitude 20% of the nominal amplitude of the converter, applied power 100 W with the probe placed 1cm from the bottom of the water bath and 5 cm from the walls of the precipitate glass). Subsequent separation was carried out by liquid chromatography (LC) with photodiode array UV detection. Calibration curves using the standard addition in green strawberries typically gave linear dynamic ranges of 2-300 mg/l for all analytes; R(2) values exceeded 0.996 in all cases. The method was applied to two types of strawberries to demonstrate the applicability of the proposed method, which is much faster and produces less analyte degradation than methods as solid-liquid, subcritical water and microwave-assisted extraction.     

Graphene oxide reinforced polymeric ionic liquid monolith solid‐phase microextraction sorbent for high‐performance liquid chromatography analysis of phenolic compounds in aqueous environmental samples

A graphene oxide reinforced polymeric ionic liquids monolith was obtained by copolymerization of graphene oxide doped 1‐(3‐aminopropyl)‐3‐(4‐vinylbenzyl)imidazolium 4‐styrenesulfonate monomer and 1,6‐di‐(3‐vinylimidazolium) hexane bihexafluorophosphate cross‐linking agent. Coupled to high‐performance liquid chromatography, the monolith was used as a solid‐phase microextraction sorbent to analyze several phenolic compounds in aqueous samples. Under the optimized extraction and desorption conditions, linear ranges were 5–400 μg/L for 3‐nitrophenol, 2‐nitrophenol, and 2,5‐dichlorophenol and 2–400 μg/L for 4‐chlorophenol, 2‐methylphenol, and 2,4,6‐trichlorophenol (R² = 0.9973–0.9988). The limits of detection were 0.5 μg/L for 3‐nitrophenol and 2‐nitrophenol and 0.2 μg/L for the rest of the analytes. The proposed method was used to determine target analytes in groundwater from an industrial park and river water. None of the analytes was detected. Relative recoveries were in the range of 75.5–113%.     

Electrochemical detection of phenolic compounds using cylindrical carbon-ink electrodes and microchip capillary electrophoresis

A simple method to fabricate cylindrical carbon electrodes for use in capillary electrophoresis (CE) microchips is described. The electrodes were fabricated using a metallic wire coated with carbon ink. Several experimental variables were studied in order to establish the best conditions to fabricate the electrode. Finally, the electrodes were integrated in a poly(dimethylsiloxane) microchip and used for the analysis of phenolic compounds. Using the optimum conditions, the analysis of a mixture of dopamine, epinephrine, catechol, and 4-aminophenol was achieved in less than 240 s, showing good linear responses (R² = 0.999) in the 0.1-190 μM range, and limits of detection (without the use of stacking or a decoupler) of 140 and 105 nM for dopamine and epinephrine, respectively.     

Analyses of phenolic compounds by microemulsion electrokinetic chromatography

In this study, a microemulsion electrokinetic chromatography (MEEKC) method was developed to analyze and detect 13 phenolic compounds (syringic acid, p-cumaric acid, vanillic acid, caffeic acid, gallic acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, (+)-catechin, (-)-epigallocatechin, (-)-epicatechin gallate, (-)-epigallocatechin gallate, (-)-epicatechin, and (-)-gallocatechin), which are present in many plant-derived foods. The effects of cosurfactant, organic modifier, and oil were examined in order to optimize the separation of these phenolic compounds. The amounts of cosurfactant (cyclohexanol) and organic modifier (acetonitrile) were determined as the major influence on the separation selectivity, while the type of oil partially affected the separation resolution of the phenolic compounds. A highly efficient MEEKC separation method was achieved within 14 min by using a microemulsion solution of pH 2.0 containing 2.89% w/v SDS, 1.36% w/v heptane, 7.66% w/v cyclohexanol, and 2% w/v ACN. Furthermore, the present work could demonstrate that the nature of the oil phase has a significant influence on the separation selectivity of phenolic compounds.     

Fast separation of (poly)phenolic compounds from apples and pears by high-performance liquid chromatography with diode-array detection

Polyphenolic compounds in apples and pears were analysed by HPLC on C₁₈-modified silica. Gradient elution with phosphoric acid–methanol mixtures and phosphoric acid–acetonitrile mixtures gave complete separation of all polyphenolics of interest. The use of methanol as modifier was preferred because it provides a more rapid separation (20 min). Diode-array detection was used for the provisional identification of polyphenolic compounds not available as standards.     

A reliable quantitative method for the analysis of phenolic compounds in Brazilian propolis by reverse phase high performance liquid chromatography

A sensitive and reliable RP-HPLC method was developed using a C18 CLC-ODS (M) - 4.6x250 mm(2)column and gradient elution for the analysis of phenolic compounds in propolis raw material and its products. A procedure for the extraction of phenolic compounds using aqueous ethanol (90%) with the addition of veratraldehyde as the internal standard (IS) was developed allowing to quantify ten compounds: caffeic acid, coumaric acid, ferulic acid, cinnamic acid, aromadendrin-4'-methyl ether (AME), isosakuranetin, drupanin, artepellin C, baccharin, and 2,2-dimethyl-6-carboxyethenyl-2H-1-benzopyran acid (DCBEN). The developed method gave good detection response and linearity in the range of 20.83-533.33 microg/mL.     

Electrophoretic separation of environmentally important phenolic compounds using montomorillonite-coated fused-silica capillaries

This paper reports a simple procedure for coating fused-silica capillaries with poly(diallyldimethyl ammonium chloride) and montmorillonite. The coated capillaries were characterized by performing EOF measurements as a function of buffer pH, number of layers of coating, and number of runs (stability). The coated capillaries showed a highly stable mu(EOF) (run-to-run RSD less than 1.5%, n = 20), allowing continuous use for several days without conditioning. The coated capillaries were then used for the effective separation of nine environmentally important phenolic compounds showing a significant improvement in the resolution, when compared to bare fused-silica capillaries. The EOF of the coated capillaries was constant in alkaline solutions (pH > or = 7), allowing the optimization of the separation conditions of phenolic compounds without significantly affecting the mu(EOF).     

Determination of phenolic acids and flavonoids of apple and pear by high-performance liquid chromatography

A new HPLC stationary phase has been applied to the analysis of phenolic acids and flavonoids with diode array and mass spectrometric detection. The separation of 26 standard compounds was achieved within 1 h. The stationary phase displayed excellent resolution especially of flavonol glycosides. The analytical system has been used for the determination of phenolic compounds in apple pomace and apple juice, and in extracts of pear fruits of different cultivars. Apple pomace was found to be a promising source of phenolics. However, yields are affected by the drying conditions applied. Furthermore, the applicability of the analytical system for the authenticity control of apple and pear juice was demonstrated by determination of characteristic quercetin and isorhamnetin glycosides, and dihydrochalcones, respectively. Since isorhamnetin-3-glucoside was present in all pear cultivars investigated, the usefulness of arbutin as a specific marker of pear products appears to be doubtful.     

Rapid qualitative and quantitative ultra high performance liquid chromatography method for simultaneous analysis of twenty nine common phenolic compounds of various structures

Twenty nine phenolic compounds comprising nine phenolic acids, sixteen flavonoids (including eight tea catechins, glycosides and aglycones), four coumarins plus caffeine were analysed within 20 min using ultra high performance liquid chromatography (UHPLC) with PDA detection. UHPLC system was equipped with C18 analytical column (100 mm × 2.1 mm, 1.7 μm), utilising 0.1% formic acid and methanol mobile phase in the gradient elution mode. The developed method was tested for the system suitability: resolution, asymmetry factor, peak capacity, retention time repeatability and peak area repeatability. The method was fully validated in the terms of linearity (r² > 0.9990 for all 30 compounds), range (typically 1-100 mg L⁻¹), LOD, LOQ, inter/intra-day precision (<3% and <9% respectively) and inter/intra-day accuracy (typically 100 ± 10%). Subsequently the method was applied to the identification (spectral information and peak purity calculations were profited) and quantification of phenolic compounds and caffeine present in tea infusions and extracts.     

Comprehensive two-dimensional liquid chromatography in the analysis of antioxidant phenolic compounds in wines and juices

A novel comprehensive two-dimensional liquid chromatographic (LC×LC) system was developed for the quantification of antioxidant phenolic compounds in wine and juice. The system allows parts of the sample that are well separated in the first column to pass directly to the detector after the first column, while the rest of the sample proceeds to the second column. The optimised LC×LC system employed a combination of two C18 columns, the latter column with an ion-pair reagent (tetrapentylammonium bromide). The relative standard deviations (RSD) for the retention times were better than 0.01% in the first dimension and on average 6.3% in the second. The RSD values of the peak volumes varied from 3% (protocatechuic acid) to 13% (caffeic acid, n = 3, 10 μg/ml).     

Improved extraction and identification by ultra performance liquid chromatography tandem mass spectrometry of phenolic compounds in burdock leaves

The simultaneous ultrasonic and microwave assisted extraction (UMAE) technique was first employed to obtain phenolics. The effects of UMAE variables including extraction time, microwave power, and solvent to solid radio on the yield of phenolics were investigated. The optimized conditions were as follows: solvent to solid ratio was 20:1 (ml/g), extraction time was 30 s, microwave power was 500 W and two times of extraction. Moreover, the phenolic yield of UMAE was higher than that by maceration, indicating a significant reduction of extraction time and an improvement of efficiency. The phenomenon is related to the strong disruption of leaf tissue structure by microwave induced expansion and ultrasonic shaking, which had been observed with the scanning electron microscopy. The phenolic compositions of the extract was then identified by ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS), 10 compounds had been characterized, providing a more complete identification of phenolic compounds in burdock leaves than previously reported. The occurrence of benzoic acid and p-coumaric acid is reported for the first time. This study suggests that UMAE is a good alternative for the extraction of phenolics, with a great potential for industrial application. Also, UMAE provides a new sample preparation technique for characterization of the phenolic compounds from plants.     

Separation of phenolic compounds and corresponding glucuronides by coupled-column micellar reversed-phase liquid chromatography

Summary An isocratic HPLC technique for separation of phenolic compounds and corresponding glucuronides in urine is developed. Sample pre-treatment, often a tedious and rate-limiting factor, was eliminated by use of a coupled column system. Spiked urine samples were injected directly into a C₄ precolumn and a selected fraction was transferred on-line from the precolumn to a silanized C₁₈ analytical column in the backflush mode. Analyte peak enrichment was attained by employing mobile phases of different elution strengths. The weaker mobile phase (7% v/v acetonitrile) was used to strongly retain the analytes on the precolumn while most of the polar endogenous compounds were washed to waste. Elution and transfer of the trapped analytes from the precolumn to the analytical column was achieved by introducing a stronger mobile phase (20% v/v acetonitrile) with the aid of a switching valve. The use of cetyltrimethylammonium bromide as counter ion and micellar agent in the mobile phase involved a high selectivity for the analytes relative to the urine matrix components and allowed simultaneous analysis of the glucuronides and parent compounds without the need of gradient elution. The system demonstrated a good repeatability on spiked urine samples. Who passed away July 21, 1996     

Electrochemical detection of phenolic estrogenic compounds at carbon nanotube-modified electrodes

The use of a carbon nanotube-modified glassy carbon electrode (CNT-GCE) for the LC-EC detection of phenolic compounds with estrogenic activity is reported. Cyclic voltammograms for phenolic endocrine disruptors and estrogenic hormones showed, in general, an enhancement of their electrochemical oxidation responses at CNT-GCE attributable to the electrocatalytic effect caused by CNTs. Hydrodynamic voltammograms obtained under flow injection conditions lead to the selection of +700 mV as the potential value to be applied for the amperometric detection of the phenolic estrogenic compounds, this value being remarkably less positive than those reported in the literature using other electrode materials. Successive injections of these compounds demonstrated that no electrode surface fouling occurred. A mobile phase consisting of a 50:50 (v/v) acetonitrile:0.05 mol l⁻¹ phosphate buffer of pH 7.0 was selected for the chromatographic separation of mixtures of these compounds, with detection limits ranging between 98 and 340 nmol l⁻¹. Good recoveries were obtained in the analysis of underground well water and tap water samples spiked with some phenolic estrogenic compounds at a 14 nmol l⁻¹ concentration level.     

A general analytical strategy for the characterization of phenolic compounds in fruit juices by high-performance liquid chromatography with diode array detection coupled to electrospray ionization and triple quadrupole mass spectrometry

In the present study, a methodology based on liquid chromatography with diode array detection (HPLC/DAD) coupled to an electrospray ionization (ESI) interface and a triple quadrupole mass spectrometer for the simultaneous identification of phenolic compounds in fruit juices has been developed. 72 available phenolic compound standards from diverse families present in fruits have been studied in order to analyze their fragmentation pattern. As a result, a general strategy for the characterization of unknown phenolic compounds in fruit juices was designed: (i) taking into account its UV–visible spectrum and elution order, assign the unknown polyphenol to a polyphenol class, (ii) identify the quasi-molecular ion using positive and negative MS spectra, being supported by adducts generated with solvent or sodium and molecular complexes, (iii) determinate the pattern of glycosylation in positive mode using ESI(+)-CID MS/MS product ion scan experiments, selecting the quasi-molecular ion as precursor ion, and finally, (iv) study the identity of the aglycone through ESI(+)-CID MS/MS product ion spectra from the protonated aglycone, [Y₀]⁺. This strategy was successfully employed for the characterization of known and unknown phenolic compounds in juices from 17 different fruits.