Fast separation and determination of carnosic acid and rosmarinic acid in different rosemary (Rosmarinus officinalis) extracts by capillary zone electrophoresis with ultra violet-diode array detection

Summary Carnosol, carnosic acid, rosmarinic acid and other not identified phenolic compounds were separated by capillary zone electrophoresis (CZE) using a 40-cm long capillary and a 20 mM tetraborate buffer (pH 9.0), within 3 min. A UV-diode array detector was employed to collect spectra of phenolic compounds. The effect of some separation parameters on peak resolution and migration time of phenolic species present in a refined rosemary extract was studied. The repeatability of the method was also investigated: the intraday relative standard deviation on total peak area was less than 4%, while the intraday relative standard deviation on migration time was less than 0.6%. Moreover the CZE method showed good sensitivity (0.0007 μg mL¹ for carnosic acid and rosmarinic acid). Carnosic acid and rosmarinic acid have been quantified in different commercial extracts of rosemary. Finally, the optimized method was also applied to evaluate the recovery of these two compounds when different organic solvents were employed during the extraction procedure.     

Determination of Phenolic Compounds by Capillary Zone Electrophoresis–Mass Spectrometry

A CZE-MS method was developed for the determination of several phenolic compounds (phenolic acids, flavonoids). Since the analysis of these components necessitates the application of basic conditions for CZE separation and negative ionization mode for MS detection, the simplest choice was to use 0.5 M NH₄OH and IPA:water (1:1 v/v%) as the background electrolyte and sheath liquid, respectively. The LOD values ranged between 0.004–1.9 mg/L showing that there are relatively large differences in the ionization (and chemical) features of these compounds. The precision data were better than 0.75 RSD% for migration times and were between 5–8 RSD% for peak areas. In order to test the applicability of the developed method, a honey sample was analyzed.     

Determination of Phenolic Acids in Herba Epilobi by ITP–CE in the Column-Coupling Configuration

The combination of capillary isotachophoresis (ITP) and capillary zone electrophoresis (CZE) in the column-coupling configuration has been optimized in a mode in which the background electrolyte employed in the CZE step was different from the leading and terminating electrolytes of the ITP step. The optimum composition of the electrolyte system was 0.01 M HCl, 0.02 M IMI, 0.2% HEC, pH 7.2 (leading electrolyte), 0.01 M HEPES, pH 8.2 (terminating electrolyte), and 25 mM MES, 50 mM TRIS, 30 mM boric acid, 0.2% HEC, pH 8.3 (background electrolyte). All solutions contained 20% methanol. The timing of the transfer of isotachophoretically stacked analyte zones into the CZE column was also optimized. An ITP–CZE method with UV detection at 270 nm was developed for separation of nine phenolic acids (protocatechuic, syringic, vanillic, cinnamic, ferulic, caffeic, ρ-coumaric, chlorogenic, and gentisic acids) in a model mixture and used for assay of some of these acids in a methanolic extract of herba epilobi. Application of ITP–CZE resulted in 100-fold better sensitivity than conventional CZE; limits of detection ranged between 10 and 60 ng mL⁻¹. When MES–TRIS–borate-based buffer, pH 8.3, was used in the CZE separation step the linearity of the ITP–CZE response was satisfactory (correlation coefficients were from 0.9937 to 0.9777). Repeatability was also satisfactory (RSD values ranged between 0.77% and 1.28% for migration times and between 1.65% and 13.69% for peak area). Revised: 23 March and 27 April 2006     

Comparison of Phenolic Profile of Balsamic Vinegars Determined Using Liquid and Gas Chromatography Coupled with Mass Spectrometry

Balsamic vinegar is one of the best known and most popular types of vinegar, and it is a rich source of polyphenolic compounds. The quality of balsamic vinegar as well as the content of phenolic substances vary depending on the production method. In the present work, we have developed a method for comprehensive characterization of the content of phenolic compounds in balsamic vinegars based on the combination of gas chromatography (GC) and high-performance liquid chromatography (HPLC) coupled with mass spectrometric detection in single mode (MS) and tandem mode (MS/MS). In total, 14 samples of different types of balsamic vinegar were analyzed without difficulty in sample preparation. The separation conditions and detection parameters of HPLC-MS/MS were optimized and used for the determination of 29 phenolic compounds and 6 phenolic acids. The profile of phenolic compounds was completed by semi-quantitative analysis of volatile organic compounds using GC-MS after optimized headspace solid-phase microextraction. Gallic acid, protocatechuic acid, caffeic acid, and p-coumaric acid have been identified as the major phenolic compounds in balsamic vinegars.     

Separation and analysis of cis-diol-containing compounds by boronate affinity-assisted micellar electrokinetic chromatography

Cis-diol-containing compounds (CDCCs) are usually highly hydrophilic compounds and are therefore difficult to separate by conventional reversed-phase-based micellar electrokinetic chromatography (MEKC) due to poor selectivity. Here, we report a new method, called boronate affinity-assisted micellar electrokinetic chromatography (BAA-MEKC), to solve this issue. A boronic acid with a hydrophobic alkyl chain was added to the background electrolyte, which acted as a modifier to adjust the selectivity. CDCCs can covalently react with the boronic acid to form negatively charged surfactant-like complexes, which can partition into micelles formed with a cationic surfactant. Thus, CDCCs can be separated according to the differential partition constants of their boronic acid complexes between the micellar phase and the surrounding aqueous phase. To verify this method, eight nucleosides were employed as the test compounds and their separation confirmed that the combination of boronate affinity interaction with MEKC can effectively enhance the separation of CDCCs. The effects of experimental conditions on the separation were investigated. Finally, the BAA-MEKC method was applied to the separation and analysis of nucleosides extracted from human urine. BAA-MEKC exhibited better selectivity and improved separation as compared with conventional MEKC and CZE. Successful quantitative analysis of urinary nucleosides by BAA-MEKC was demonstrated.     

Combined microwave-assisted isolation and solid-phase purification procedures prior to the chromatographic determination of phenolic compounds in plant materials

A fast, sensitive and selective procedure employing a combination of microwave-assisted extraction (MAE) and solid phase extraction (SPE) was applied prior to liquid chromatographic identification and quantification of phenolic compounds in plant materials. MAE has been tested and optimized for the isolation of phenolic acids (gallic, protocatechuic, p-hydroxybenzoic, chlorogenic, vanilic, caffeic, syringic, p-coumaric, ferulic, sinapic, benzoic, m-coumaric, o-coumaric, rosmarinic, cinnamic acids) and 3,4-dihydroxybenzaldehyde, syringaldehyde, p-hydroxybenzaldehyde, and vanillin in various plants. The effects of experimental conditions on MAE efficiency, such as solvent composition, temperature, extraction time, have been studied. The extraction efficiencies were compared with those obtained by computer-controlled, two-step Soxhlet-like extractions. Plant extracts were purified and phenolic compounds were pre-concentrated using SPE on polymeric RP-105 SPE sorbent prior to HPLC analysis. Chromatographic separation was carried out on a Hypersil BDS C₁₈ column using a mobile phase consisted of 0.3% (v/v) acetic acid in water (solvent A) and methanol (solvent B) at flow rate 0.6 ml min⁻¹ and column temperature 30 °C with gradient elution.     

1-Hexadecyl-3-methylimidazolium Ionic Liquid as a New Cationic Surfactant for Separation of Phenolic Compounds by MEKC

The ionic liquid 1-hexadecyl-3-methylimidazolium bromide ([C₁₆MIm]Br) has been used as a novel cationic surfactant for separation of phenolic compounds, including quinol, phloroglucinol, resorcinol, phenol, p-cresol, and m-nitrophenol, by micellar electrokinetic capillary chromatography (MEKC). The effects of buffer concentration and pH, concentration of [C₁₆MIm]Br, and applied potential were studied. Use of the optimized buffer (25 mmol L^?1 NaH₂PO₄), 10 mmol L^?1 [C₁₆MIm]Br, and an applied potential of ?15 kV enables optimum separation with regard to resolution and migration time. The phenolic compounds were detected at 214 nm. The micelle of this long-alkyl-chain imidazolium ionic liquid acts as a pseudo-stationary phase in this MEKC separation.     

Simultaneous Determination of Organic Acids in Wine Samples by Capillary Electrophoresis and UV Detection: Optimization with Five Different Background Electrolytes

A simple technique is described for the routine capillary electrophoretic determination of organic acids in wine samples. Several aromatic and non‐aromatic compounds, including phthalic acid, benzoic acid, sorbic acid, boric acid, and phosphate, were evaluated as background electrolytes in order to obtain the highest resolution and detection sensivity. Factors that affect capillary electrophoretic separation such as the concentration and pH of the background electrolyte (BGE), the concentration of the electroosmotic flow modifier (EOF), and methanol addition to the electrolyte were investigated systematically. Tartaric, malic, succinic, acetic, and lactic acids were determined simultaneously in approximately six minutes using an electrolyte containing 3 mM phosphate and 0.5 mM myristyltrimethylammonium bromide (MTAB) as electroosmotic flow modifier at pH 6.5. This method is quantitative, with recoveries in the 90–102% range and linear up to 50 mg L^–1. The precision is better than 1% and the procedure shows the appropriate sensibility, with detection limits between 0.015 and 0.054 mg L^–1. The proposed method was successfully employed for the determination of organic acids in wine samples by direct sample injection after appropriate dilution and filtration.     

Separation and Determination of D-Gluconic Acid Produced During Fermentation by Capillary Zone Electrophoresis

Abstract

A novel method was developed for separation and determination of D-gluconic acid produced during fermentation by capillary zone electrophoresis (CZE) with direct UV detection at 214 nm, using selected carrier electrolyte composed of 6 mM potassium biphthalate, 50 mM disodium hydrogen phosphate and 15% (v/v) acetonitrile. The effects of concentration of phthalate, phosphate and organic modifier (acetonitrile), as well as temperature for the separation were investigated. The method is simple, inexpensive and will make it very useful in the gluconic acid industry.     

Comparative study of capillary zone electrophoresis and micellar electrokinetic chromatography for the separation of twelve aromatic sulphonate compounds

Summary Two modes of capillary electrophoresis (CE), capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC), were investigated for the separation of 12 aromatic sulphonate compounds. In CZE, although the voltage applied, the buffer concentration and the pH were optimized for effective separation of the compounds studied, under the best conditions four of the five amino compounds coeluted, as did naphthalene-1-sulphonic acid and naphthalene-2-sulphonic acid. In MEKC, sodium dodecyl sulphate (SDS) and Brij 35 were chosen as the anionic and nonionic surfactants and the effect of the concentration of micelles was examined. The effect of adding methanol as the organic modifier was also investigated with each of these micellar systems. All the analytes, including the isomers, were completely separated by use of MEKC with Brij 35 but when SDS was used only 11 compounds were separated because two amino compounds coeluted.     

Simultaneous Determination of Inorganic Anions and Organic Acids in Environmental Samples by Capillary Zone Electrophoresis with Indirect UV Detection

Capillary zone electrophoresis (CZE) with indirect UV detection was developed for the simultaneous determination of inorganic anions and organic acids in environmental samples. Various aromatic acids (benzoic, phthalic, trimellitic, and pyromellitic acids) were evaluated as background electrolytes (BGEs) to give high resolution and detection sensitivity. Co-electroosmotic conditions such as the concentration of BGE, electrolyte pH, and EOF modifier were systematically investigated. Three inorganic anions and ten organic acids were determined simultaneously in 10 min using an electrolyte containing 10 mM phthalic acid, 0.5 mM myristyltrimethylammonium bromide (MTAB), and 5% methanol (MeOH) (v/v) at pH 5.60. Linear plots for the test solutes were obtained in the concentration range 0.01–1.0 mM with detection limits in the range 5–30 μM. The proposed method was successfully demonstrated for the determination of inorganic anions and organic acids in natural water, soil, and plant extracts after direct sample injection.     

Determination of Phenols,Inorganic Anions,and Carboxylic Acids in Kraft Black Liquors by Capillary Electrophoresis

Two methods are presented for the quantitative capillary electrophoretic (CE) determination of phenolic lignin degradation compounds as well as of inorganic anions and organic acids in Kraft black liquors. Important phenolic lignin degradation compounds can be rapidly separated by co-electroosmotic CE after acidification of the liquors and subsequent extraction of the compounds with chloroform. A capillary electrophoretic separation of phenolic compounds is performed by using a phosphate/borate electrolyte system and UV detection at 214 nm. In addition, a HPLC method using a gradient with water, methanol, and acetic acid is also developed. Inorganic ions which are of importance to the pulping process can be determined by simply diluting the black liquors after sampling and subsequent analysis with a chromate electrolyte system and indirect UV detection at 185 nm. In addition, the concentration of low molecular aliphatic carboxylic acids can be determined simultaneously within the same run. By method optimization it is possible to separate the anions within one minute and, at the same time, to increase the resolution of the solutes. The electrolyte systems for the CE separations were optimized by varying the pH value and by adding organic solvents. Short separation times are obtained by adding a polycationic EOF modifier (hexadimethrine bromide) to the electrolyte which reverses the electroosmotic flow. A migration of the anionic analytes in the same direction as the electroosmotic flow is thus established.     

Validated Assay for the Determination of Mitoxantrone in Pharmaceuticals Using Capillary Zone Electrophoresis

Abstract

The first capillary zone electrophoretic (CZE) method for the determination of mitoxantrone (MTX) in pharmaceutical formulations was developed. The influence of background electrolyte (BGE) species, pH, concentration (c _BGE), organic modifier, capillary temperature, applied voltage, and injection time was investigated. Optimum results were achieved with 25 mM ammonium acetate at an apparent pH value of 5.0 in 50% v/v acetonitrile, applied voltage of +30 kV, and capillary temperature of 25°C. The samples were introduced into the capillary hydrodynamically for 2 s at 33.5 mbar. Mitoxantrone was detected at a wavelength of 242 nm. Mitoxantrone and doxorubicin (DOX) (used as internal standard, ISTD) were completely separated in less than 7 min. The method was suitably validated with respect to linearity, limits of detection (LOD) and quantification (LOQ), accuracy, precision, selectivity, and robustness. The proposed method was applied successfully for the determination of MTX in its injectable pharmaceutical formulation.     

Speciation of inorganic and methylated arsenic compounds by capillary zone electrophoresis with indirect UV detection. Application to the analysis of alkali extracts of As2S2 (realgar) and As2S3 (orpiment)

A capillary zone electrophoresis (CZE) method with indirect UV detection was developed to simultaneously separate inorganic and organic arsenic compounds including arsenite (iAsIII), arsenate (iAsV), monomethylarsonate and dimethylarsenic acid (DMAV). 2,6-Pyridinedicarboxylic acid (PDC) and n-hexadecyltrimethylammonium hydroxide (CTAOH) were selected to compose a background electrolyte (BGE), where PDC was used as chromophore and CTAOH functioned as electroosmotic flow (EOF) modifier to reduce/eliminate EOF. The choice of detection wavelength, the optimization of BGE pH, and effects of applied electric field strength and temperature on separation were further investigated. The limits of detection for the targeted analytes were between 0.19 and 0.23 ppm as molecule. Good linearity of more than three orders of magnitude was obtained. Repeatability of migration times and peaks areas were 0.8-1.7 and 3.4-6.9% R.S.D.; whereas reproducibility were 1.2-2.2 and 3.6-7.1% R.S.D., respectively. The established CZE method was then applied to analyze the alkali extracts of realgar (As2S2) and orpiment (As2S3). The main components in both alkali extracts were identified to be iAsIII and iAsV.     

CZE and ESI-MS of Borate-Sugar Complexes

Capillary zone electrophoresis (CZE) with electrospray ionization (ESI) mass spectrometry (MS) was used to study borate (B^?1) and sugar (L) complexes (L _x B^?1). Boric acid was adjusted to pH 10 with ammonium hydroxide to create an ESI-MS compatible CZE background electrolyte. We show for the first time that the electrophoretic peaks for each injected sugar contained both the substrates (i.e., sugar and/or multimers) and products (i.e., L _x B^?1). The effects of sheath liquid, temperature, and borate concentration were studied. The molecular mass information obtained from the ESI-MS provided new evidence on the mechanisms of borate-sugar complexation. Direct infusion ESI-MS and CZE-ESI-MS experiments strongly suggest that the formation of L _x B^?1 was from the direct reaction of a sugar or sugar multimer (L _x ) and B^?1. Larger L _x B^?1, where x > 2 were observed. Separation in the CZE dimension allows for the simultaneous analysis of a sugar mixture and simplified the ESI-MS analysis of sugars of the same molecular mass. The increase in sugar electrophoretic mobility caused by the increase in borate concentration was discussed in terms of the formation of L _x B^?1 complexes. In addition, the separation of five nucleosides by CZE using a borate electrolyte and detection using ESI-MS is demonstrated.     

Use of eosin as a fluorophore in capillary electrophoresis with laser detection.

Eosin has been used to generate the background signal for indirect fluorimetric detection of inorganic and organic ions, simultaneously separated by capillary zone electrophoresis (CZE). This reagent provides constant fluorescence over the pH range of 5-10 and is compatible with the excitation by an argon ion laser at 488 nm with emission at 520 nm. The use of esosine as fluorophore, H3BO3, and Na2B4O7 as electrolyte and diethylentriamine as modifier of the electroosmotic flow in CZE were optimised. The analytical potential of the studied buffer was tested on a group of 12 anions, used as model compounds. Both, hydrodynamic and electrokinetic injection mode were optimised. The detection limits determined by the last injection mode, were in the range 0.008-0.037 mg l(-1). By using this method, the quantitation of the common anions in tap and mineral water has been carried out successfully.     

Determination of C4–C14 carboxylic acids by capillary zone electrophoresis: Application to the identification of diamide degradation products and partitioning studies

Capillary zone electrophoresis (CZE) was investigated for the determination of linear saturated carboxylic acid homologues ranging from C₄ to C₁₄. Separation conditions were optimised to overcome the problems of decreasing solubility and decreasing selectivity between successive homologues with increasing chain length. Separations were performed at 20°C, using a 20 kV separation voltage and a pH 8 electrolyte containing 30% methanol. A suitable chromophore (4-aminobenzoate) was added to ensure indirect UV detection of the analytes. Calibration curves and repeatability were established. Minimum detectable concentrations of 3·10⁻⁶ mol l⁻¹ were achieved. Resolution between successive homologues was better than 2. The electrophoretic mobility of each homologue (n=7–14) was assessed and a quasi-linear relationship between the mobility value and 1/n was observed. The quantitative analysis of a diamide degradation solution was performed and compared to potentiometric results. The CZE method was also applied to the determination of C₇–C₁₄ partitioning between an organic medium containing tributylphosphate in n-dodecane and different basic solutions. Their behaviour was established according to the chain length and the pH of the aqueous phase. For C₁₀–C₁₄ compounds, results were validated by comparison with gas chromatographic analysis of the organic phases.     

Polymeric ionic liquid as additive for the high speed and efficient separation of aromatic acids by co-electroosmotic capillary electrophoresis

A simple and reliable co-electroosmotic capillary electrophoresis system for the fast determination of aromatic acids has been developed by employing poly (1-vinyl-3-butylimidazolium bromide) as the background electrolyte modifier. The polymeric ionic liquid was synthesized by the conventional radical polymerization. The reversed electroosmotic flow was obtained by adding a small amount of the polymeric ionic liquid (0.0006%, w/v) to the electrolyte. To further improve the resolution of aromatic acids, conditions including the concentration of polymeric ionic liquid and pH of background electrolytes were optimized. All eight aromatic acids were baseline resolved in one measurement in a short time (less than 3.5 min) under optimized conditions, 100 mM NaH₂PO₄ buffer containing 0.006% (w/v) polymeric ionic liquid, pH 6.0. Separation efficiencies were in the range from 355,000 to 943,000 (plates/m). Satisfactory reproducibility on the basis of the migration time of analytes was achieved. RSDs (n = 3) were less than 0.33% except the p-aminobenzoic acid (0.9%). The applicability of the present method has been demonstrated for the determination of water-soluble aromatic acids in a common drug for external use.     

Determination of phenolic acids by capillary zone electrophoresis and HPLC

Selected phenolic acids are determined by capillary zone electrophoresis and HPLC, each using UV detection. The optimised CZE background electrolyte contained 50 mM acetic acid, 95 mM 6-aminocaproic acid, 0.1% polyacrylamide, 1% polyvinylpyrrolidone, and 10% methanol. Twelve phenolic acids (gallic, p-hydroxybenzoic, 3,4-dihydroxybenzoic, vanillic, syringic, o-coumaric, p-coumaric, caffeic, sinapic, ferulic, salicylic and chlorogenic) were separated within 10 minutes. Chromatographic separation of these phenolic acids was carried out on an Eclipse XBD C8 column using a mobile phase gradient (acetonitrile / methanol / water / 0.1% phosphoric acid); all were separated within 25 minutes. Electrophoretic and chromatographic determinations of ferulic and chlorogenic acids were compared on barley, malt, and potato samples. The methods’ characteristics were: linearity (1–20 mg ml and 0.2–4 mg ml⁻¹), accuracy (recovery 94 ± 5% and 96 ± 4%), intra-assay repeatability (4.1% and 3.5%), and detection limit (0.2 and 0.02 mg ml⁻¹).      

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.