Determination of key flavonoid aglycones by means of nano‐LC for the analysis of dietary supplements and food matrices

A method for the analysis of flavonoids (myricetin, quercetin, naringenin, hesperitin, and kaempferol), with interesting bioactivity, has been developed and validated utilizing nano‐LC technique. In order to find optimal conditions, capillary columns (75 μm id × 10 cm) packed with different types of stationary phases, Kinetex® C18 core–shell (2.6 μm particle size), Hydride‐based RP‐C18 (sub‐2 μm particle size), and LiChrospher® 100 RP‐18 endcapped (5 μm particle size) were evaluated. The method was validated using Hydride‐based RP‐C18 stationary phase, with sub‐2 μm particle size. A good chromatographic performance, expressed in terms of repeatability (RSD, in the range 1.63–4.68% for peak area), column‐to‐column reproducibility (RSD not higher than 8.01% for peak area), good linearity and sensitivity was obtained. In particular limit of detection values between 0.07 and 0.31 μg/mL were achieved with on column focusing technique. The method was applied to the determination of studied flavonoids in dietary supplements as well as in food matrices. The amount of quercetin found in the first analyzed dietary supplement, was in agreement to the labeled content. In the other samples, where the content of flavonoids was not labeled, most of the studied flavonoids were determined in amounts somewhere comparable to those reported in literature.     

Preparation and evaluation of open‐tubular capillary column combining a metal–organic framework and a brush‐shaped polymer for liquid chromatography

In this work, an open‐tubular capillary liquid‐phase column was prepared by modifying chain polymer on the inner surface of capillary and chemical bonding of metal organic frameworks, NH₂‐UiO‐66, to the brushes of chain polymer (poly(glycidyl methacrylate)). Besides advantages of facial preparation and good permeability, the chain polymer effectively increases the modification amount of NH₂‐UiO‐66 nanoparticles to increase the phase ratio of open‐tubular capillary column and enhance the interactions with analytes. The results of scanning electron microscope energy‐dispersive X‐ray spectra indicated that NH₂‐UiO‐66 nanoparticles were successfully bonded to the chain polymer. Because of the hydrophobic interaction and hydrogen bonding interaction between the analytes and the ligand of NH₂‐UiO‐66, different analytes were well separated on the NH₂‐UiO‐66‐modified poly(glycidyl methacrylate) capillary (1.12 m × 25 μm id × 365 μm od) with the high absolute column efficiency reaching 121 477 plates, benefiting from an open‐tubular column and low mass transfer resistance provided by polymer brush and metal–organic framework crystal. The relative standard deviations of the retention time for run‐to‐run, day‐to‐day, and column‐to‐column (n = 3) runs are below 4.28%, exhibiting good repeatability. Finally, the column was successfully applied to separation of flavonoids in licorice.     

High‐resolution peptide separations using nano‐LC at ultra‐high pressure

We report on the optimization of nano‐LC gradient separations of proteomic samples that vary in complexity. The gradient performance limits were visualized by kinetic plots depicting the gradient time needed to achieve a certain peak capacity, while using the maximum system pressure of 80 MPa. The selection of the optimal particle size/column length combination and corresponding gradient steepness was based on scouting the performance of 75 μm id capillary columns packed with 2, 3, and 5 μm fully porous silica C18 particles. At optimal gradient conditions, peak capacities up to 500 can be obtained within a 120 min gradient using 2 μm particle‐packed capillary columns. Separations of proteomic samples including a cytochrome c tryptic digest, a bovine serum albumin tryptic digest, a six protein mix digest, and an Escherichia coli digest are demonstrated while operating at the kinetic‐performance limit, i.e. using 2‐μm packed columns, adjusting the column length and scaling the gradient steepness according to sample complexity. Finally, good run‐to‐run retention time stability (RSD values below 0.18%) was demonstrated applying ultra‐high pressure conditions.     

HPLC of basic drugs using non‐aqueous ionic eluents: evaluation of a 3 μm strong cation‐exchange material

HPLC columns packed with 3 μm particle size HPLC Technology Techsphere SCX (propylsulfonic acid‐modified) silica offer considerable advantages over 5 μm SCX packings in the analysis of basic drugs using 100% methanol eluents containing an ionic modifier such as ammonium perchlorate. The basic drugs studied included clozapine and norclozapine, olanzapine, quinine and quinidine, and amitriptyline, nortriptyline, imipramine and desipramine. The 3 μm column was not only more efficient for a given column length compared with 5 μm materials, but also elution times were less, a phenomenon observed in reversed‐phase systems. The high efficiencies and excellent peak shapes obtained with the 3 μm SCX‐modified packing together with the relatively low back‐pressures attained show that such materials deserve serious consideration by laboratories involved in the analysis of basic drugs. Manufacturers should offer such packings as a matter of routine. Alternative ionic modifiers such as ammonium acetate are available for use with mass spectrometric detection if required. Copyright © 2009 John Wiley & Sons, Ltd.     

Preparation,characterization and application of a new 25,27‐bis‐[2‐(5‐methylthiadiazole)thioethoxyl]‐26,28‐dihydroxy‐para‐tert‐butyl calix[4]arene stationary phase for HPLC

A new para‐tert‐butylcalix[4]arene column containing thiadiazole functional groups was prepared and used for the separation of polycyclic aromatic hydrocarbons, phenolic compounds, aromatic amines, benzoic acid and its derivatives by high‐performance liquid chromatography (HPLC). The effect of organic modifier content in the mobile phase on retention and selectivity of these compounds were investigated. The results indicate that the stationary phase behaves like reversed‐phase packing. However, hydrogen bonding, π–π and inclusion interactions seem to be involved in the separation process. The column has been successfully employed for the analysis of clenbuterol in pork and pig casing; the limit of detection and the limit of quantitation for this method by HPLC‐UV detection was 0.03 and 0.097 μg/mL, respectively; the method is demonstrated to be suitable and a competitive alternative analytical method for the determination of clenbuterol.     

HPLC‐DAD methodology for the quantification of organic acids,furans and polyphenols by direct injection of wine samples

This article proposes a simple and sensitive HPLC method with photo‐diode array detection for the analysis of organic acids, monomeric polyphenols and furanic compounds in wine samples by direct injection. The chromatographic separation of 8 organic acids, 2 furans and 22 phenolic compounds was carried out with a buffered solution (pH 2.70) and acetonitrile as mobile phases and a difunctionally bonded C18 stationary phase, Atlantis dC18 (250×4.6 mm, 5 μm) column. The elution was performed in 12 min for the organic acids and in 60 min for the phenolic compounds, including phenolic acids, stilbenes and flavonoids. Target compounds were detected at 210 nm (organic acids, flavan‐3‐ols and benzoic acids), 254 nm (ellagic acid), 280 nm (furans and cinnamic acid), 315 nm (hydroxycinnamic acids and trans‐resveratrol) and 360 nm (flavonoids). The RSD for the repeatability test (n=5) of peak area and retention times were below 3.1 and 0.3%, respectively, for phenolics and below 1.0 and 0.2% for organic acids. The RSDs expressing the reproducibility of the method were higher than for the repeatability results but all below 9.0%. Method accuracy was evaluated by the recovery results, with averaged values between 80 and 104% for polyphenols and 97–105% for organic acids. The calibration curves, obtained by triplicate injection of standard solutions, showed good linearity with regression coefficients higher than 0.9982 for polyphenols and 0.9997 for organic acids. The LOD was in the range of 0.07–0.49 mg/L for polyphenols (cinnamic and gallic acids, respectively) and 0.001–0.046 g/L for organic acids (oxalic and lactic acids, respectively). The method was successfully used to measure and assess the polyphenolic fingerprint and organic acids profile of red, white, rosé and fortified wines.     

A 2‐D‐HPLC‐CE platform coupled to ESI‐TOF‐MS to characterize the phenolic fraction in olive oil

A 2‐D‐HPLC/CE method was developed to separate and characterize more in depth the phenolic fraction of olive oil samples. The method involves the use of semi‐preparative HPLC (C18 column 250×10 mm, 5 μm) as a first dimension of separation to isolate phenolic fractions from commercial extra‐virgin olive oils and CE coupled to TOF‐MS (CE‐TOF‐MS) as a second dimension, to analyze the composition of the isolated fractions. Using this method, a large number of compounds were tentatively identified, some of them by first time, based on the information concerning high mass accuracy and the isotopic pattern provided by TOF‐MS analyzer together with the chemical knowledge and the behavior of the compounds in HPLC and CE. From these results it can be concluded that 2‐D‐HPLC‐CE‐MS provides enough resolving power to separate hundreds of compounds from highly complex samples, such as olive oil. Furthermore, in this paper, the isolated phenolic fractions have been used for two specific applications: quantification of some components of extra‐virgin olive oil samples in terms of pure fractions, and in vitro studies of its anti‐carcinogenic capacity.     

Implementation and optimisation of a high‐temperature loading strategy of liquid standards in the quantification of volatile organic compounds using solid sorbents

High‐temperature liquid standard loading strategy onto solid sorbent traps for calibration of thermal desorption–GC–MS techniques for the analysis of volatile organic compounds is evaluated and optimised. With this proposed set‐up, volatilised liquid‐loaded standards interact in gas phase with solid sorbent particles. Response factor for 15 volatile compounds with different vapour pressures have been evaluated and compared with common strategies based on liquid matrix interactions. Using gas matrix strategy improves signal output in the range 10–700%. Average increase for benzene, toluene, ethylbenzene and xylenes is 480%. Reported systematic bias between liquid standards and gas samples are reduced, enhancing thermal desorption methodologies on one of its more important issues. In addition, the proposed system improves the average repeatability to a 3.2%, over 13 times some reported data. The use of an ultra‐thin GC capillary column of 150 μm id performs better peak resolution in about 60% the time with usual 250 μm id capillary columns. The usefulness of this proposed optimised procedure has been proved in real air matrix samples, through a large study with the reliable characterisation of 93 different volatile compounds in the ambient air of a municipal solid waste treatment area     

Rapid determination of polycyclic aromatic hydrocarbons in rainwater by liquid–liquid microextraction and LC with core‐shell particles column and fluorescence detection

Liquid–liquid microextraction coupled to LC with fluorescence detection for the determination of Environmental Protection Agency's 16 priority pollutant polycyclic aromatic hydrocarbons in rainwater has been developed. The optimization of the extraction method has involved several parameters, including the comparison between an ultrasonic bath and a magnetic stirrer as extractant apparatus, the choice of the extractant solvent, and the optimization of the extraction time. Liquid–liquid microextraction gave good results in terms of recoveries (from 73.6 to 102.8% in rainwater) and repeatability, with a very simple procedure and low solvent consumption. The reported chromatographic method uses a Core‐Shell technology column, with particle size <3 μm instead of classical 5‐μm particles column. The resulting backpressure was below 300 bar, allowing the use of a conventional HPLC system rather than the more expensive ultrahigh performance LC (UHPLC). An average decrease of 59% in run time and 75% in eluent consumption has been obtained, compared to classical HPLC methods, keeping good separation, sensitivity, and repeatability. The proposed conditions were successfully applied to the determinations of polycyclic aromatic hydrocarbons in genuine rainwater samples.     

Simultaneous determination of quinolones in fish by liquid chromatography coupled with fluorescence detection: Comparison of sub‐2 μm particles and conventional C18 columns

A simple and effective multi‐residue analysis method is presented for the extraction and determination of eleven quinolones (pipemidic acid, enoxacin, norfloxacin, ciprofloxacin, lomefloxacin, enrofloxacin, gatifloxacin, difloxacin, oxolinic acid, nalidixic acid and flumequine) in fish tissues. In this study, multi‐residue separations on four columns packed with 5 μm or sub‐2 μm particles were simultaneously developed for the purpose of comparison. Various gradients were optimized and best resolutions were achieved on each column. A short and sub‐2 μm particle‐sized HPLC column was chosen for its advantages in analysis time and column performance. Additionally, considering the matrix effect of the complex crude fish tissue, an effective extraction protocol was also established for sample pre‐treatment procedure. Good recoveries (71–98%) were obtained from samples fortified with a mix of eleven quinolones at three levels, with satisfactory relative standard deviations and limits of detection. As a result, the sub‐2 μm HPLC column and proposed analytical procedures have been evaluated and applied to the analysis of different fish tissues. Detectable residues were observed in 8 of 30 samples, at concentrations ranging from 4.74 to 23.27 μg/kg.     

Micellar and aqueous‐organic liquid chromatography using sub‐2 μm packings for fast separation of natural phenolic compounds

The objective of the present work was to investigate the chromatographic behavior of natural phenolic compounds in micellar and aqueous‐organic LC using a short column packed with 1.8 μm particles. Firstly, the effect of ACN and SDS on elution strength and selectivity was examined by isocratic submicellar (0–30% ACN/5% 1‐butanol/1–6 mM SDS) and micellar (0–30% ACN/5% 1‐butanol/40–60 mM SDS) systems. The varied concentrations of two modifiers in the mobile phases revealed different eluting power. Then, the application of organic modifier gradient was discussed in both submicellar and micellar LC using mobile phases of 4 mM SDS/5% 1‐butanol or 50 mM SDS/5% 1‐butanol containing ACN gradient from 0 to 30%, respectively. For micellar system, the separation was found to be better in gradient than isocratic elution. Additionally, the sensitivity of aqueous‐organic LC was examined. The mobile phase was a mixture of ACN and water employing gradient elution at a flow rate of 0.5 mL/min, with analysis time below 9 min. It was found that separation efficiency was significantly better compared with micellar LC. Besides, the aqueous‐organic LC has been applied to separation of various phenolic compounds in Yangwei granule or Radix Astragali samples.     

Development and validation studies for determination of phenylpropanoid‐substituted flavan‐3‐ols in semipurified extract of Trichilia catigua by high‐performance liquid chromatography with photodiode array detection

The phenolic compounds are the main phytochemical constituents of the bark of Trichilia catigua and are commonly used for medicinal purposes. An HPLC method for the simultaneous quantification of phenolic compounds (procyanidin B2 (PB2), epicatechin (EPC), chinchonains Ia, Ib, IIa, IIb, catechin, and chrologenic acid) in T. catigua extract was developed and validated. A suitable chromatographic system was selected, which uses a gradient elution with methanol/ACN (75:25), and water both with 0.05% TFA, as mobile phase, column Luna, 280 nm, and flow 0.4 mL/min. Validation of the analytical method was based on the parameters: linearity, precision, LODs and LOQs, accuracy, robustness, and stability. The method showed linearity for PB2 and EPC, in the range 10–120 μg/mL with good correlation coefficients (>0.996). For precision, the repeatability ranged from 1.89 to 3.23%, and the values for accuracy for PB2 and EPC were 95 and 89%, respectively. The LODs and LOQs for PB2 were 1.36 and 4.12 μg/mL, and for EPC were 2.18 and 6.61 μg/mL, respectively. The method was robust under the conditions employed. The proposed method could be employed for quality assessment of T. catigua, as well as pharmaceutical products.     

Analysis of free amino acids in Amur sturgeon by ultra‐performance liquid chromatography using pre‐column derivatization with 6‐aminoquinolyl‐carbamyl

A rapid, sensitive, and reliable ultra‐performance liquid chromatography (UPLC) coupled with photodiode array detection method was developed for the amino acid analysis of Amur sturgeon (Acipenser schrenckii Brandt). The method uses minimal sample volume and automated online precolumn derivitization of amino acids with fluorescent 6‐aminoquinolyl‐carbamyl reagent. The chromatographic separation was achieved by UPLC, which used a column with 1.7 μm particle packing that enabled higher speed of analysis, peak capacity, greater resolution, and increased sensitivity. Amino acid derivatives obtained under optimal conditions were separated on a Waters UPLC BEH C₁₈ column with Acetonitrile–acetate buffer as mobile phase. Matrix effects were investigated and good linearities with correlation coefficients better than 0.9949 were obtained over a wide range of 5–1000 μmol/L for all amino acids. The simple sample preparation and minimal sample volume make the method useful for the quantitation of 17 amino acids in Amur sturgeon samples. It is concluded that a rapid and robust platform based on UPLC was established, and a total of 17 amino acids of Amur sturgeon were tentatively detected. This method showed good accuracy and repeatability that can be used for the quantification of amino acids in real samples.     

Preparation and evaluation of monodispersed,submicron, non‐porous silica particles functionalized with β‐CD derivatives for chiral‐pressurized capillary electrochromatography

Submicron, non‐porous, chiral silica stationary phase has been prepared by the immobilization of functionalized β‐CD derivatives to isocyanate‐modified silica via chemical reaction and applied to the pressurized capillary electrochromatography (pCEC) enantio‐separation of various chiral compounds. The submicron, non‐porous, cyclodextrin‐based chiral stationary phases (sub_μm‐CSP2) exhibited excellent chiral recognition of a wide range of analytes including clenbuterol hydrochloride, mexiletine hydrochloride, chlorpheniramine maleate, esmolol hydrochloride, and metoprolol tartrate. The synthesized submicron particles were regularly spherical and uniformly non‐porous with an average diameter of around 800 nm and a mean pore size of less than 2 nm. The synthesized chiral stationary phase was packed into 10 cm × 100 μm id capillary columns. The sub_μm‐CSP2 column used in the pCEC system showed better separation of the racemates and at a higher rate compared to those used in the capillary liquid chromatography mode (cLC) system. The sub_μm‐CSP2 possessed high mechanical strength, high stereoselectivity, and long lifespan, demonstrating rapid enantio‐separation and good resolution of samples. The column provided an efficiency of up to 170 000 plates/m for n‐propylbenzene.     

Analysis of anthocyanins in commercial fruit juices by using nano-liquid chromatography-electrospray-mass spectrometry and high-performance liquid chromatography with UV-vis detector

Nano-LC and conventional HPLC techniques were applied for the analysis of anthocyanins present in commercial fruit juices using a capillary column of 100 μm id and a 2.1 mm id narrow-bore C(18) column. Analytes were detected by UV-Vis at 518 nm and ESI-ion trap MS with HPLC and nano-LC, respectively. Commercial blueberry juice (14 anthocyanins detected) was used to optimize chromatographic separation of analytes and other analysis parameters. Qualitative identification of anthocyanins was performed by comparing the recorded mass spectral data with those of published papers. The use of the same mobile phase composition in both techniques revealed that the miniaturized method exhibited shorter analysis time and higher sensitivity than narrow-bore chromatography. Good intra-day and day-to-day precision of retention time was obtained in both methods with values of RSD less than 3.4 and 0.8% for nano-LC and HPLC, respectively. Quantitative analysis was performed by external standard curve calibration of cyanidin-3-O-glucoside standard. Calibration curves were linear in the concentration ranges studied, 0.1-50 and 6-50 μg/mL for HPLC-UV/Vis and nano-LC-MS, respectively. LOD and LOQ values were good for both methods. In addition to commercial blueberry juice, qualitative and quantitative analysis of other juices (e.g. raspberry, sweet cherry and pomegranate) was performed. The optimized nano-LC-MS method allowed an easy and selective identification and quantification of anthocyanins in commercial fruit juices; it offered good results, shorter analysis time and reduced mobile phase volume with respect to narrow-bore HPLC.     

Capillary electrochromatography‐mass spectrometry for the determination of 5‐nitroimidazole antibiotics in urine samples

The separation of eight antibiotics belonging to 5‐nitroimidazole family was carried out by means of CEC coupled with MS. Preliminary experiments were carried out with ultraviolet detection in order to select the proper stationary and mobile phase. Among the different stationary phases studied (namely Lichrospher C18, 5 μm particle size; Cogent^TM Bidentate C18, 4.2 μm; Pinnacle II? Phenyl, 3 μm; Pinnacle II? Cyano, 3 μm), Cogent? Bidentate C18 (4.2 μm) gave the best performance. For CEC‐MS coupling, a laboratory assembled liquid‐junction‐nano‐spray interface was used. In order to achieve a good sensitivity, special attention was paid to both optimization of the sheath liquid composition as well as selection of the injection mode. Under optimized CEC‐ESI‐MS conditions, the separation was accomplished within 22 min by using a column packed with a mixture of Bidentate C18:Lichrospher Silica‐60 (5 μm) 3:1 w/w, an inlet pressure of 11 bar, a voltage of 15 kV, and a mobile phase composed by 45:10:45 v/v/v ACN/MeOH/water containing ammonium acetate (5 mM pH 5). A combined hydrodynamic and electrokinetic injection of 8 bar, 15 kV, and 96 s was adopted. The method was validated in terms of repeatability and intermediate precision of retention times and peak areas, linearity, and LODs and LOQs. RSDs values were <2.9% for retention times and <16.1% for peak areas in both intraday and interday experiments. LOQ values were between 0.09 and 0.42 μg/mL for all compounds. Finally, the method was applied to the determination of three most employed 5‐nitroimidazole antibiotics (metronidazole, secnidazole, and ternidazole) in spiked urine samples, subjected to a SPE procedure. Recovery values in the 67–103% range were obtained. Furthermore, for the selected antibiotics, CEC‐MS² spectra were obtained providing the unambiguous confirmation of these drugs in urine samples.     

Automated high‐speed CE system for multiple samples

A high‐speed CE system for multiple samples was developed based on a short capillary and an automated sample introduction device consisting of a commercial multi‐well plate and an x‐y‐z translation stage. The spontaneous injection method was used to achieve picoliter‐scale sample injection from different sample wells. Under the optimized conditions, a 40 μm‐long sample plug (corresponding to 78‐pL plug volume) was obtained in a 50 μm id capillary, which ensured both the high separation speed and high separation efficiency. The performance of the system was demonstrated in the separation of FITC‐labeled amino acids with LIF detection. Five FITC‐labeled amino acids including arginine, phenylalanine, glycine, glutamic acid, and asparagine were separated within 15 s with an effective separation length of 1.5 cm. The separation efficiency ranged from 7.96 × 10⁵/m to 1.12 × 10⁶ /m (corresponding to 1.26–0.89 μm plate heights). The repeatability of the peak heights calibrated with an inner standard for different sample wells was 2.4 and 2.7% (n = 20) for arginine and phenylalanine, respectively. The present system was also applied in consecutive separations of 20 different samples of FITC‐labeled amino acids with a whole separation time of less than 6 min.     

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.     

Preparation and evaluation of 3 m open tubular capillary columns with a zwitterionic polymeric porous layer for liquid chromatography

A 3 m zwitterionic polymeric porous layer open tubular column (3 m × 25 μm id × 375 μm od) with a polymeric porous layer thickness of 4 μm was fabricated by the copolymerization of [2‐(methacryloyloxy)ethyl] dimethyl‐(3‐sulfopropyl) ammonium hydroxide and N,N’‐methylenebis(acrylamide). The effects of the diameter of the capillary, reaction temperature, and polymerization time on the preparation of the open tubular column were investigated. Characterized by scanning electron microscopy, the zwitterionic layer was observed to be rough and throughout the fused‐silica capillary homogenously, which increased the phase ratio. The separation of neutral, basic, and acidic compounds demonstrates the strong hydrophilicity of the poly[2‐(methacryloyloxy)ethyl] dimethyl‐(3‐sulfopropyl) ammonium hydroxide coating. In addition, the poly[2‐(methacryloyloxy) ethyl] dimethyl‐(3‐sulfopropyl) ammonium hydroxide porous layer open tubular column was applied for the analysis of flavonoids from the rootstalk of licorice, revealing the potential in separating complex samples. The relative standard deviation of retention time for run‐to‐run (n = 5), day‐to‐day (n = 3), and column‐to‐column (n = 3) of toluene, N,N‐dimethylformamide, formamide, and thiourea were below 1.2%, exhibiting good repeatability.     

Separation of catechins and methylxanthines in tea samples by capillary electrochromatography

In this paper, the simultaneous separation of several polyphenols such as (+)‐catechin, (–)‐epicatechin, (–)‐epigallocatechin, theophylline, caffeine in green and black teas by capillary electrochromatography (CEC) was developed. Several experimental parameters such as stationary phase type, mobile phase composition, buffer and pH, inner diameter of the columns, sample injection, were evaluated to obtain the complete separation of the analysed compounds. Baseline resolution of the studied polyphenols was achieved within 30 min by using a capillary column (id 100 μm) packed with bidentate C₁₈ particles for 24.5 cm and a mobile phase composed of 5 mM ammonium acetate buffer pH 4 with H₂O/ACN (80:20, v/v). The applied voltage and the temperature were set at 30 kV and 20°C. Precision, detection and quantification limits, linearity, and accuracy were investigated. A good linearity (R² > 0.9992) was achieved over a concentration working range of 2–100 μg/mL for all the analytes. LOD and LOQ were 1 and 2 μg/mL, respectively, for all studied compounds. The CEC method was applied to the analysis of those polyphenols in green and black tea samples after an extraction procedure. Good recovery data from accuracy studies ranged between 90% and 112% for all analytes.