RP-HPTLC fingerprinting of secondary metabolites from Nephrolepis exaltata and Cycas revoluta

This study aims to develop a quantitative fingerprinting between two evolutionary close plant species Nephrolepis exaltata (L.) Schott and Cycas revoluta Thunb. The crude plant extracts were prepared in 1:1:1, v/v/v: Ethyl Acetate: n-Hexane: Methanol (AR) solvent system through cold extraction for 24 Hrs. The plant extracts were diluted (10 mg/mL) with a similar solvent system spotted (10 μL) on silica gel 60 F²⁵⁴ thin-layer chromatography plates. Five mobile phases (i) tetrahydrofuran (THF): toluene: formic acid: water (16:8:2:1) (ii) toluene: ethyl acetate: diethylamine (7:2:1) (iii) toluene: ethyl acetate: formic acid (7:3:0.1) (iv) n-hexane: ethyl acetate (7.2:2.9) (v) toluene: methanol (9:1) were used. The results were scanned at 254 nm, 366 nm, and in visible white light. This study gave minimum compact spots between Rf 0.06 to 0.051 corresponding to terpenoids, simultaneously for flavonoids spots were found between Rf 0.004 to 0.910. Total eight phenolic bands were found in Cycas revoluta among which five was found in leaf extract with Max RF of 0.033, 0.093, 0.273, 0.901 and 0.964 whereas three bands were found in stem extract with Max Rf of 0.007, 0.897 and 0.954. The presence of fifteen different types of flavonoids was determined by the flavonoids profile at Rf 0.004, 0.027, 0.047, 0.073, 0.134, 0.230, 0.303, 0.369, 0.369, 0.427, 0.514, 0.631, 0.703, 0.757, 0.846 and 0.910 for Nephrolepis exaltata. HPTLC fingerprint has shown several peaks with different Rf for different mobile phases. The fingerprinting would be helpful in the identification, and authentication of these species ecologically and therapeutically.     

A validated high‐performance thin‐layer chromatography method for the identification and simultaneous quantification of six markers from Platanus orientalis and their cytotoxic profiles against skin cancer cell lines

Betulinic acid ( 1 ), betulinic acid‐3‐acetate ( 2 ), 3‐acetylbetulinaldehyde ( 3 ), oleanolic acid‐3‐acetate ( 4 ), 3‐β‐hydroxy‐28,19‐β‐olenolide ( 5 ), and β‐sitosterol ( 6 ) were isolated from Platanus orientalis and a high‐performance thin‐layer chromatography method was developed for their simultaneous quantification. The markers were first derivatized on the chromatogram with ceric ammonium sulfate and then high‐performance thin‐layer chromatography densitometry was carried out. Chromatographic separation of these markers was carried out on silica gel 60 plates using a ternary solvent system n‐hexane/toluene/acetone (6:3.5:1 v/v/v) as a mobile phase. For marker 1 , a deuterium (D₂) lamp and wavelength of 420 nm was used. A tungsten (W) lamp was used for markers 2 and 3 at 550 nm and for 4 – 6 at 500 nm. The method was validated for accuracy, precision, LOD, and LOQ. All calibration curves showed a good linear relationship (r > 0.9919). The precision evaluated by an intra‐ and interday study showed RSDs < 2.51% and accuracy validation recovery between 95.54 and 99.33% with RSDs < 1.55%. The successful application of the validated method showed 1 as the most abundant component (4.63%) and 5 (0.017%) the least. The markers displayed a significant cytotoxic effect against human keratinocyte, mouse melanoma, and human skin epithelial carcinoma cancer cells by using a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay.     

Characterization and classification of medicinal plants according to their antioxidant profile estimated by thin layer chromatography assisted by chemometric expertise

This study focuses on the characterization and classification of 42 medicinal plants extracts according to their antioxidant activity. Principal component analysis (PCA), cluster analysis (CA) and the combination of PCA with linear discriminant analysis (PCA-LDA) were used as multivariate exploratory techniques for chromatographic data analysis. For the separation of the compounds a mobile phase containing ethyl acetate: toluene: formic acid: water (30:1.5:4:3 v/v/v/v) and different HPTLC plates (Silica gel 60 and HPTLC Silica gel 60?F₂₅₄) were used. The chromatographic plates were evaluated using the images obtained after spraying the plates with 2-aminoethyldiphenylborate solution (NTS, 0.2% in ethanol) and also after their reaction with 2, 2-diphenyl-1-picrylhydrazyl solution (DPPH^?) (0.2% in ethanol). The score projection on the plane defined by first two components (PC1 and PC2) revealed two large groups of the investigated samples depicted according to their antioxidant capacity. A better classification of samples according to their antioxidant capacity was obtained using the CA and PCA-LDA methodology in all cases. The excellent results obtained in this study concerning the classification of medicinal plants according to their antioxidant capacity using the PCA-LDA methodology applied to TLC chromatograms might lead to a new paradigm in the field of medicinal plant holistic evaluation.     

Development of off-line layer chromatographic and total reflection X-ray fluorescence spectrometric methods for arsenic speciation

Rapid and low cost off-line thin layer chromatography–total reflection X-ray fluorescence spectrometry and overpressured thin layer chromatography–total reflection X-ray fluorescence spectrometry methods have been developed for separation of 25 ng of each As(III), As(V), monomethyl arsonic acid and dimethylarsinic acid applying a PEI cellulose stationary phase on plastic sheets and a mixture of acetone/acetic acid/water = 2:1:1 (v/v/v) as eluent system. The type of eluent systems, the amounts (25–1000 ng) of As species applied to PEI cellulose plates, injection volume, development distance, and flow rate (in case of overpressured thin layer chromatography) were taken into consideration for the development of the chromatographic separation. Moreover, a microdigestion method employing nitric acid for the As spots containing PEI cellulose scratched from the developed plates divided into segments was developed for the subsequent total reflection X-ray fluorescence spectrometry analysis. The method was applied for analysis of root extracts of cucumber plants grown in As(III) containing modified Hoagland nutrient solution. Both As(III) and As(V) were detected by applying the proposed thin layer chromatography/overpressured thin layer chromatography–total reflection X-ray fluorescence spectrometry methods.     

Rapid preparative isolation of erythrocentaurin from Enicostemma littorale by medium‐pressure liquid chromatography,its estimation by high‐pressure thin‐layer chromatography,and its α‐amylase inhibitory activity

Erythrocentaurin is a relatively simple natural product present among the members of Gentianaceae. A preparative method for the isolation of erythrocentaurin from the ethyl acetate fraction of Enicostemma littorale using medium‐pressure liquid chromatography has been reported. The method consisted of a simple step gradient from 10 to 20% ethyl acetate in n‐hexane. Using a 70 × 460 mm Si60 column, this method is capable of processing 20 g of material in <3 h (purity ≈ 97%). The recovery of erythrocentaurin was 87.77%. Estimation of erythrocentaurin in extracts and fractions based on high‐pressure thin‐layer chromatography was carried out on silica gel 60 F₂₅₄ plates with toluene/ethyl acetate/formic acid (80:18:2 v/v/v) as the mobile phase. The densitometric analysis was performed at 230 nm. A well‐separated compact band of erythrocentaurin appeared at R_f 0.54 ± 0.04. The analytical method showed good linearity in the concentration range of 200–1500 ng/band with a correlation coefficient of 0.99417. The limits of detection and quantification were found to be ≈60 and ≈180 ng/band, respectively. Erythrocentaurin exhibited a concentration‐dependent α‐amylase inhibition (IC₅₀ 1.67 ± 0.28 mg/mL). The outcome of the study should be considered for pharmacokinetic and biotransformation studies involving E. littorale.     

Acid modified diatomaceous earth--a sorbent material for thin layer chromatography

Natural diatomaceous earth (DE) is modified by flux calcination and refluxing with acid. To characterize natural DE, modified DE's [flux calcinated (FC)DE and FCDE-I] and silica gel 60GF(254) (Si-60GF(254)) are analyzed microscopically, physically, and chemically by various techniques. FCDE-I and Si-60GF(254) are investigated for their usefulness in the stationary phase of thin layer chromatography (TLC) both individually and in composition. Sodium diethyldithiocarbamate (DEDTC) and ammonium pyrrolidinedithiocarbamate (PyDTC) are prepared as Co or Cu (M) complexes [M(DEDTC)(2) and M(PyDTC)(2), respectively]. These complexes and their mixtures are run on thin layers of Si-60GF(254) and FCDE-I individually, and on various FCDE-I and Si-60GF(254) mixtures. Pure toluene and various toluene-cyclohexane mixtures (3:1, 1:1, 1:2, 1:3, v/v) are used as mobile phases for the running the complexes. The best analytical separations of both M(DEDTC)(2) and M(PyDTC)(2) complexes are obtained when using pure toluene and toluene-cyclohexane (3:1, 1:1, v/v) as mobile phases on FCDE-I-Si-60GF(254) (1:3, 1:1, w/w) layers as stationary phases. This study shows that it is possible to qualitatively analyze and to satisfactorily separate a mixture Cu(2+) and Co(2+) cations on cited chromatographic systems.     

Chemical fingerprint analysis of phenolics of Albizia chinensis based on ultra-performance LC-electrospray ionization-quadrupole time-of-flight mass spectrometry and antioxidant activity

Albizia species have been shown to have anti-inflammatory and anti-allergic properties. However, efficient analytical methods for identification of their active constituents are still lacking. Ultra-performance liquid chromatography coupled with electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS) was used to study the phenolic composition of the ethanolic extracts of different parts (flowers, leaves, pods and bark) of A. chinensis. In addition, the antioxidant activity of the ethanolic extracts was evaluated by the 1,1-diphenyl-2-picryl hydrazyl (DPPH) free-radical and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical cation scavenging methods. Four compounds were isolated from the ethanolic extract of the flowers and characterized by 1H and 13C NMR spectroscopy as quercetin-3-O-rhamnoside, quercetin, quercetin-3-O-arabinofuranoside, and myricetin-3-O-rhamnoside. Separation and quantification of the phenolics was accomplished using a reversed-phase BEH C18 column with the mobile phase of methanol-water (0.05% formic acid), and detection wavelengths of 360 and 254 nm.     

Selective separation and determination of isoproterenol on thin layers of bismuth silicate ion-exchanger

A simple and sensitive method for the separation and determination of isoproterenol from other doping drugs has been developed on thin layers of bismuth silicate, a synthetic inorganic ion exchanger as adsorbent in thin layer chromatography (TLC). A mixture of methanol and 0.1 mol/L formic acid (3:7, v/v) was employed as the mobile phase. The development time was 32 min. The quantitative measurement were performed with a Camag TLC Scanner-3 at wavelength (λ) of 410 nm. The isoproterenol recovery in this procedure was 98.9%. The linear correlation coefficient was greater than 0.9871 and the relative standard deviation (RSD) was less than 0.94. The limit of detection (LOD) and limit of quantification (LOQ) were 7.7×10^-7mol/L and 3.85 ×10^-6mol/L, respectively. This method has been applied in the determination of isoproterenol in dosage forms and in biological fluids.     

Bioassay-guided isolation of antioxidants from Astragalus altaicus by combination of chromatographic techniques

An efficient method for bioassay-guided preparative isolation of antioxidants from the n-butanol extract of Astragalus altaicus Bunge was ingeniously developed by combination of silica gel column chromatography and high-speed counter-current chromatography. Under the bioassay-guidance of antioxidant activities, the antioxidants were gradually separated from the crude sample of Astragalus altaicus Bunge by silica gel column chromatography and high-speed counter-current chromatography. Silica gel column chromatography separation was performed with chloroform, chloroform-methanol (100:1~5:1, v/v) and chloroform-methanol-water (5:1:0.1~2:1:0.1, v/v/v). High-speed counter-current chromatography separation was performed with a two-phase solvent system composed of ethyl acetate-n-butanol-water (2:1:6, v/v/v), which was successfully selected by thin layer chromatography analysis, at a flow rate of 1.5 mL/min. As a result, isorhamnetin-3-gentiobioside (20.8 mg), rutin (82.0 mg), and narcissin (12.8 mg) were obtained for the first time from 200 mg of the crude sample, ABS-5 of Astragalus altaicus Bunge. The purities were all at over 95% by high-performance liquid chromatography analysis, and their structures were unambiguously identified by mass spectroscopy, (1) H, and (13) C nuclear magnetic resonance spectroscopy. Antioxidant activities of the three compounds were also assayed by in vitro ABTS [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) diamonium salt] radical cation scavenging activity. Among them, rutin possessed the highest antioxidant capacity with SC(50) value of 22.15 μg/mL.     

Dietary burden of phenolics per serving of "Mountain tea" (Sideritis) from Macedonia and correlation to antioxidant activity

This work was afforded from 2 points of view, phytochemical evaluation and relation to antioxidant activity and dietary burden of phenolics of a cup of "Mountain tea", a drink obtained by domestic infusion of Sideritis. Phytochemically, two extraction protocols using water and methanol as solvent were used for comparison. Methanol and boiling water extracts (by domestic infusion procedure) showed that extracts were rich in bound forms of phenolics such as hydroxycinnamic acids, phenylethanoid glycosides and flavonoid glycosides. The total phenolic content for Sideritis species ranged around 190 mg per serving (2 g infusion bag) for methanol extracts and around 72 mg per serving in water extracts. Among the two different Macedonian Sideritis species, Sideritis raeseri (wild growing) showed the highest phenolics content in both extracts (212 mg and 89 mg per serving, respectively). Concerning the phenolic content in the different aerial parts, leaf was the richest plant organ in phenolics followed by flower and stem with the lowest amount. The methanol extract from Sideritis raeseri (wild growing) showed the highest antioxidant capacity as shown by DPPH, ABTS and FRAP assays. The antioxidant capacity was linearly correlated with phenolic content. Nutritionally, the dietary burden of phenolics of a "Mountain tea" bag for domestic infusion (serving size) was established at 89 mg for an homogeneous and equal distribution of the different aerial parts (leaf, flower and stem). However, and according to our results a rate of 60% leaf and 40% flower would increase the content of bioavailable phenolics and also the total phenolics content of a serving bag of "Mountain tea".     

Examining the Performance of Two Extraction Solvent Systems on Phenolic Constituents from U.S. Southeastern Blackberries

Two common extraction solvent systems, namely acidified aqueous methanol and acidified aqueous acetone, were used to extract blackberry phenolics, and the antioxidant properties of the recovered extracts were compared. The crude extracts were fractionated into low- and high-molecular-weight phenolics by Sephadex LH-20 column chromatography. The hydrophilic-oxygen radical absorbance capacity (H-ORAC_FL), ferric reducing antioxidant power (FRAP), and the cellular antioxidant activity (CAA) assays were employed as indices to assess antioxidant capacity of the extracts and their respective fractions. The methanolic solvent system displayed a greater efficiency at extracting anthocyanin and flavonol constituents from the blackberries, while the acetonic solvent system was better at extracting flavan-3-ols and tannins. Anthocyanins were the dominant phenolic class found in the blackberries with 138.7 ± 9.8 mg C3G eq./100 g f.w. when using methanol as the extractant and 114.6 ± 3.4 mg C3G eq./100 g f.w. when using acetone. In terms of overall antioxidant capacity of blackberry phenolics, the acetonic solvent system was superior. Though present only as a small percentage of the total phenolics in each crude extract, the flavan-3-ols (42.37 ± 2.44 and 51.44 ± 3.15 mg/100 g f.w. in MLF and ALF, respectively) and ellagitannins (5.15 ± 0.78 and 9.31 ± 0.63 mg/100 g f.w. in MHF and AHF, respectively) appear to account for the differences in the observed antioxidant activity between the two solvent systems.     

Quantitative determination of seven chemical constituents and chemo‐type differentiation of chamomiles using high‐performance thin‐layer chromatography

A simple and rapid high‐performance thin‐layer chromatographic method was developed for the separation and determination of six flavonoids (rutin, luteolin‐7‐O‐β‐glucoside, chamaemeloside, apigenin‐7‐O‐β‐glucoside, luteolin, apigenin) and one coumarin, umbelliferone from chamomile plant samples and dietary supplements. The separation was achieved on amino silica stationary phase using dichloromethane/acetonitrile/ethyl formate/glacial acetic acid/formic acid (11:2.5:3:1.25:1.25 v/v/v/v/v) as the mobile phase. The quantitation of each compound was carried out using densitometric reflection/absorption mode at their respective absorbance maxima after postchromatographic derivatization using natural products reagent (1% w/v methanolic solution of diphenylboric acid‐β‐ethylamino ester). The method was validated for specificity, limits of detection and quantification, precision (intra‐ and interday) and accuracy. The limits of detection and quantification were found to be in the range from 6–18 and 16–55 ng/band for six flavonoids and one coumarin, respectively. The intra‐ and interday precision was found to be <5% RSD and recovery of all the compounds was >90%. The data acquired from high‐performance thin‐layer chromatography was processed by principal component analysis using XLSTAT statistical software. Application of principal component analysis and agglomerative hierarchial clustering was successfully able to differentiate two chamomiles (German and Roman) and Chrysanthemum.     

Optimization of the dopant for the trace determination of polycyclic aromatic hydrocarbons by liquid chromatography/dopant-assisted atmospheric-pressure photoionization/mass spectrometry

The composition of the dopant for the analysis of polycyclic aromatic hydrocarbons (PAHs) by liquid chromatography/dopant-assisted atmospheric-pressure photoionization/mass spectrometry under reversed-phase conditions was optimized to enhance the ionization efficiency for PAHs. The most suitable dopant was a toluene/anisole mixture (99.5:0.5, v/v) and it could improve limit of detections (LODs) to 0.79-168 ng mL(-1) (signal-to-noise (S/N)=3) for 16 common PAHs. The LODs are 3.8-40 times lower than those obtained with toluene alone and are comparable to those obtained using gas chromatography/mass spectrometry.     

Phenolic compounds and antioxidant activity of olive leaf extracts

The total phenolic content and antioxidant activities of olive leaf extracts were determined. Plant material was extracted with methanol and fractionated with solvents of increasing polarity, giving certain extracts. The qualitative changes in the composition of the extracts were determined after the storage of leaves for 22?h at 37°C, before the extraction. Total polyphenol contents in extracts were determined by the Folin-Ciocalteu procedure. They were also analysed by liquid chromatography-mass spectrometry. Their antioxidant activities were evaluated using the diphenyl picrylhydrazyl method and the β-carotene linoleate model assay. Moreover, the effects of different crude olive leaf extracts on the oxidative stability of sunflower oil at 40°C and sunflower oil-in-water emulsions (10% o/w) at 37°C, at a final concentration of crude extract 200?mg?kg(-1) oil, were tested and compared with butylated hydroxyl toluene.     

Detection of antioxidant butylated hydroxytoluene (BHT) in Antarctic krill (Euphausia superba Dana)

In this study, butylated hydroxytoluene (BHT) was separated and prepared from Antarctic krill. BHT was separated from the volatile oil of Antarctic krill using high performance thin layer chromatography (HPTLC) with petroleum ether/ethyl acetate/hexane (4:1:0.5, v/v/v) was used as the developing solvent. The content of BHT in volatile oil was 9.20?mg/g and the content of BHT in dried Antarctic krill was 0.35?mg/g (0.070?mg/g in frozen whole Antarctic krill). The linearity, accuracy, stability, and recovery of the analysis showed that HPTLC is the most suitable method for the determination of BHT in Antarctic krill. The BHT crude sample was obtained by scraping the separated spot in the thin layer chromatography plate, which was then analyzed using high performance liquid chromatography (HPLC). The resulting sample was identified with 96.05% purity based on the HPLC analysis. The structure of BHT was determined using gas chromatography–mass spectrometry (GC–MS). The results of the HPLC and GC–MS analysis validated the HPTLC method. BHT is a widely used antioxidant in food, pharmaceuticals, and in industrial production. The exploitation and utilization of BHT in Antarctic krill is of great economic value.     

Effects of pH, sample size, and solvent partitioning on recovery of soluble phenolic acids and isoflavonoids in leaves and stems of red clover (Trifolium pratense cv. Kenland)

Several extraction parameters were tested to determine optimal conditions for extracting phenolics from leaves and stems of red clover (Trifolium pratense L. cv. Kenland), with the goal of using extracts in bioassays and in assessment of phenolic profiles. HPLC-UV profiles were compared before and after partitioning a methanolic extract of soluble phenolics with ethyl acetate-ethyl ether (1:1, v/v). The effect of extract pH on the partitioning of phenolics into the ethyl acetate-ethyl ether (EtOAc-Et2O) phase was evaluated, and several tissue weights were extracted to determine a minimum amount that could be extracted without loss of information. HPLC profiles of soluble phenolics were similar in the methanolic extracts and the partitioned EtOAc-Et2O extracts. However, recoveries in unpartitioned extracts were 2- to 4-fold greater than in the acidified, partitioned extracts. Also, recovery was considerably affected by the pH to which extracts were adjusted prior to partitioning. In extracts acidified to pH 2, recoveries were 2- to 7-fold higher than in extracts partitioned at pH 6. In extracts prepared from 250, 120, or 60 mg of tissue, peak areas of methanolic extracts were directly proportional to the amount of tissue extracted.     

Quantitative analysis of D-24851, a novel anticancer agent, in human plasma and urine by liquid chromatography coupled with tandem mass spectrometry

The development of a liquid chromatography/tandem mass spectrometric assay for the quantitative analysis of the novel tubulin inhibitor D-24851 in human plasma and urine is described. D-24851 and the deuterated internal standard were extracted from 250 microL of plasma or urine using hexane/ether (1:1, v/v). Subsequently, 10-microL aliquots of reconstituted extracts were injected onto an Inertsil ODS analytical column (50 x 2.0 mm i.d., 5 microm particle size). An eluent consisting of methanol/5 mM ammonium acetate, 0.004% formic acid in water (80:20, v/v) was pumped at a flow rate of 0.2 mL/min. An API 365 triple quadrupole mass spectrometer was used in the multiple reaction monitoring mode for sensitive detection. For human plasma a dynamic range of 1-1000 ng/mL was validated, and for human urine a range of 0.25-50 ng/mL. Validation was performed according to the most recent FDA guidelines and all results were within requirements. The assay has been successfully applied to support a phase I clinical trial with orally administered D-24851.     

Capillary electrophoresis,high‐performance liquid chromatography,and thin‐layer chromatography analyses of phenolic compounds from rapeseed plants and evaluation of their antioxidant activity

Rapeseed plants, known for oil production, are also known to contain phenolic compounds such as phenolic acids and flavonoids, with potential antioxidant and anticancer activities. The separation and identification of 11 phenolic acids in rapeseed extracts (including leaves, flowers, Chinese seeds, Belgian seeds, and cake) by capillary electrophoresis were investigated. The results were compared with those obtained with high‐performance liquid chromatography and thin‐layer chromatography and showed that the capillary electrophoresis technique offers several advantages for the identification of phenolic compounds in various rapeseed extracts. The antioxidant activity of rapeseed extracts and reference compounds was evaluated using four different approaches, namely, 2,2′‐azinobis‐ (3‐ethylbenzohiazoline‐6‐sulfonic acid assay, free radical 2,2‐diphenyl‐1‐picrylhydrazyl assay, electron paramagnetic resonance spectroscopy and the measurement of the total polyphenol content. The contents of total polyphenols in the tested extracts were ranging between 5.4 and 21.1% m/m and ranked as follows: Chinese seeds ? Belgian seeds ? Flowers ? Cake ? Leaves.     

Associations de chromatographie et d'électrophorèse sur couche mince de celllose amorphe pour la séparation des aminoacides. Influence des équilibires ioniques et des constituants des solvants sur les séparations chromatographiques dans le cas des solvants du type alcool/acide/eau

Résumé Des associations d'électrophorèse et de chromatographie dans des milieux acides sur couche mince de cellulose à fibres courtes sont étudiées: acide formique 1 M.—tertbutanol ou tert-pentanol/éthanol/acide formique/eau (40:38:2:20, v/v) ou propanol/éthianol/acid formique/eau (35:35:5:20). Les relations existant entre le pouvoir séparateur et les constituants (alcools) des solvants ou les équilibres ioniques en chromatographie sont examinés. On discute des problèmes relatifs à l'optimisation des méthodes et des différences de pouvoir séparateur existant entre les nouveau procédés et les procédés antérieurement décrits.

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Coupling of electrophoresis and chromatography on amorphous cellulose thin layers for the separation of amino acids. Influence of ionic equilibria and solvent components on chromatographic separations with alcohols/acid/water solvents

Summary Associations of electrophoresis and chromatography in acidic media on thin layers of cellulose with short fibres, are studied: 1 mol. dm^–3 formic acid-tert. butanol or tert, pentanol/ethiano/formic acid/water (40:38:2:20, v/v) or propanol/ethano/formic acid/water 935:35:5: 20). The relationship between separation power and solvent components (alcohols) or ionic equilibria in chromatography is examined. Optimization of emthods and origins of differences in separation power between niew and previously described procedures are discussed

     

Identification of phenolic compounds in aqueous and ethanolic rooibos extracts (<Emphasis Type="Italic">Aspalathus linearis</Emphasis>) by HPLC-ESI-MS (TOF/IT)

Rooibos (Aspalathus linearis) is a rich source of polyphenols and used to make a mild-tasting tea containing no caffeine, is low in tannins compared to green or black teas, and has antioxidant and antimutagenic/antitumoral properties. In vivo results show that rooibos has beneficial effects upon the lipid profile by decreasing serum triglycerides and cholesterol. In this sense, we have developed a simple and rapid method to separate and characterize simultaneously the polyphenolic compounds in aqueous and ethanolic rooibos extracts using high-performance liquid chromatography coupled to electrospray ionization time-of-flight mass spectrometry (HPLC-ESI-TOF-MS) and ion trap multiple mass spectrometry (HPLC-ESI-IT-MS²). The phenolic compounds were separated on a C₁₈ column (4.6 × 150 mm, 1.8 μm) with 1% formic acid in water/acetonitrile 90:10 v/v and acetonitrile as mobile phases. The accuracy mass data generated by TOF-MS together with the fragmentation pattern obtained by IT-MS² experiments confirmed the presence of 25 and 30 phenolic compounds in the aqueous and ethanolic extracts, respectively.