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.     

Analysis of phenolic acids in fruits by HPLC with monolithic columns

Reversed-phase HPLC was optimised for simultaneous determination of several derivatives of benzoic and hydroxycinnamic acids (so-called phenolic acids) in plums using a commercially available monolithic column. Mobile phase pH and concentration of organic modifier (methanol and acetonitrile) were tested in order to obtain the best resolution. Satisfactory separation was achieved in gradient mode with a mobile phase consisting of 50 mM phosphate buffer at pH 2.2 (solvent A) and acetonitrile (solvent B). The limits of detection for a UV detector ranged between 0.098 and 2.04 microg/mL for vanillic acid and p-hydroxyphenylacetic acid, respectively. The developed method was used for monitoring the content of polyphenolic acids in plums during their ripening process. The presence of these constituents was confirmed by checking their MS spectra.     

Optimization and validation of a methodology based on solvent extraction and liquid chromatography for the simultaneous determination of several polyphenolic families in fruit juices

A solvent extraction procedure of freeze-dried aliquots followed by the analysis of phenolic compounds by reversed-phase high-performance liquid chromatography (RP-HPLC) with photodiode array detection (DAD) has been developed for the analysis of polyphenolic compounds in fruit juices. This methodology is focussed on the characterization of fruit juices, mainly for quality control purposes. The effects of experimental variables, such as solvent composition and volume and time and temperature on extraction, have been studied. A unique gradient program for the separation of several phenolic classes (hydroquinones, hydroxybenzoic acids, flavan-3-oles, hydroxycinnamic acids, coumarins, flavanones, flavones, dihydrochalcones and flavonols) has been optimized, using standards of 55 commercially available phenolic compounds present in fruits, as well as representative real extracts from fruit juices. All phenolic compounds showed a high repeatability within-day (n=5) and between days (n=3) in peak area (RSD<8%) and excellent stability of their retention times. High precision was also observed in calibration slopes (RSD<8%). Detection limits ranged between 0.005 and 0.03 microg/mL for the different detected polyphenols. Complete recoveries (98-100%) were obtained for the majority of the phenolic structures of all representative phenolic families present in fruits. The method was successfully employed to measure diverse phenolic families in juices from 18 different fruits and consequently could be used for evaluate the quality of fruit juices.     

Separation and identification of polyphenols in apple pomace by high-speed counter-current chromatography and high-performance liquid chromatography coupled with mass spectrometry

Apple pomace, a by-product in the processing of apple juice, was investigated as a potential source of polyphenols. Two methods of separation and purification of polyphenols from apple pomace extract were established by combination of gel chromatography with high-speed counter-current chromatography (HSCCC) and solvent extraction with HSCCC, respectively. The optimal separation was performed on a Sephadex LH-20 column using gradient aqueous ethanol as eluting solvent from 0% to 100% in increments of 10%. HPLC analysis indicated that main polyphenols existed in fractions eluted between 40% and 50% aqueous ethanol. The fractions of interest from column were separated by HSCCC with the solvent system hexane–ethyl acetate–1% aqueous acetic acid (0.5:9.5:10, v/v/v). Ethyl acetate fractionation of the apple pomace extract followed by direct HSCCC separation by the same solvent system in the volume ratio of 1:9:10 also produced a good separation of the main polyphenols of interest. Six high-purity polyphenols were achieved tentatively and identified by HPLC/MS: chlorogenic acid (1, m/z 354), quercetin-3-glucoside/quercetin-3-glacaside (2, m/z 464), quercetin-3-xyloside (3, m/z 434), phloridzin (4, m/z 436), quercetin-3-arabinoside (5, m/z 434), and quercetin-3-rhamnoside (6, m/z 448). These results provided a preliminary foundation for further development and exploration of apple pomace.     

Isolation of Anti-HIV Components from Canarium album Fruits by High-Speed Counter-Current Chromatography

The fruit of Canarium album, also called Chinese olive, is a popular food and traditional Chinese herb. The ethyl acetate extract of Canarium album fruits was found to exhibit an inhibitory effect on six-helix bundle formation of the human immune deficiency virus (HIV) glycoprotein transmembrane subunit gp41 in our previous study. In this paper, a preparative separation of anti-HIV components from an ethyl acetate extract of Canarium album fruits was performed by high-speed counter-current chromatography, using a solvent system of n-hexane–ethyl acetate–ethanol–0.5% acetic acid aqueous (0.5:9.5:2:8, v/v/v/v). Among the six fractions obtained from a single separation process, three exhibited over 90% purity as determined by HPLC, two compounds were identified as 5-hydroxymethylfurfural and 3,4,5-trihydroxybenzoic acid, and four fractions displayed significant inhibitory effects on HIV-1 IIIB infection. These findings suggest that the high-speed counter-current chromatography is an effective and reliable technique for separating bioactive components from medicinal herbs. Further identification of the active compounds in Canarium album fruits and studying their mechanism of action are warranted.     

Rapid high-performance liquid chromatography-electrospray ionization tandem mass spectrometry method for qualitative and quantitative analysis of virgin olive oil phenolic metabolites in human low-density lipoproteins

A rapid method for detection and quantification of metabolites of specific olive oil phenolic compounds (hydroxytyrosol monoglucuronide, hydroxytyrosol monosulfate, tyrosol glucuronide, tyrosol sulfate and homovanillic acid sulfate) in low-density lipoprotein (LDL) fractions by solid-phase extraction (SPE) and high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) is described. A 3 microm particle size fast C18 Luna column, 5 cm x 2.0 mm I.D., was used at a flow rate of 0.6 mL/min with a mobile phase consisting of 0.1% (v/v) formic acid (A) and acetonitrile (B). A linear gradient profile was used for separation at column temperature 40 degrees C. The proposed chromatographic procedure is rapid without loosing its separation efficiency and sensitivity. Validation proofs were carried out for the method described, showing a linear system (r>0.99) and a recovery of 81.9 and 101.3% for hydroxytyrosol and homovanillic acid, respectively. The results show that this method is effective and can be used in routine analysis.     

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

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

A new and highly sensitive TLC method to measure hypericin using chemiluminescence

ABSTRACT

Hypericin is a polyphenolic compound belonging to the group of polyphenols and is the active constituents of Hypericum perforatum (Saint John’s wort). We present a new high-performance thin-layer chromatography (HPTLC) method to measure a large number of hypericin extracts using chemiluminescence. On a 10?×?10?cm HPTLC plate (LiChrospher® Merck, 1.05586), more than 40 tracks can be simultaneously quantified using a piezoelectric application system (pipeJet) which can apply 56?nL of a methanolic hypericin extract contactless with high precision. For separation, a solvent mixture of ethyl acetate, water, formic acid, methyl tert-butyl ether, and cyclohexane (180?+?14?+?14?+?80?+?30, v/v) was used. The R_f-value of hypericin is 0.27. The method presented is specific for hypericin and offers a limit of quantification of 690 pg hypericin per band.     

Combined application of chromatographic techniques for the separation of phenolic compounds from Stenoloma chusanum Ching

High‐speed countercurrent chromatography combined with preparative high‐performance liquid chromatography was successfully used to separate seven phenolic compounds from Stenoloma chusanum Ching. A biphasic solvent system composed of hexane/ethyl acetate/methanol/water (1:2:1:2, v/v) was used for the first step high‐speed countercurrent chromatography separation in elution–extrusion mode. A mobile phase composed of acetonitrile (18%) and pure water (82%) was used for further preparative high‐performance liquid chromatography purification. In total, the combined separation yielded seven compounds, including 3,4‐dihydroxy benzoic acid, 3,4‐dihydroxy benzaldehyde, esculetin, caffeic acid, syringic acid, luteolin, and apigenin, at a purity of over 90%. Esculetin was separated from Stenoloma chusanum Ching for the first time. The results suggest that the proposed combination method is a useful strategy for separating compounds from complex samples.     

Micellar electrokinetic capillary chromatography as an alternative method for determination of hydrocortisone and its most important associated compounds in local pharmaceutical preparations

Summary A new, simple, and accurate micellar electrokinetic chromatographic (MEKC) method is described for quantification of hydrocortisone, hydrocortisone hemisuccinate, hydrocortisone acetate, mystatin, oxytetracycline, Zn-bacitracin, polymyxin B, and lidocaine in ocular and cutaneous pharmaceutical products. The separation was performed at 25°C and 25 kV, with 15mm phosphate +15mm borate buffer, pH 8.2, and 60mm sodium dodecylsulfate (SDS) in 10∶1 (%,v/v) methanol-water as background electrolyte. Under these conditions the analysis time is approximately 23 min. The method has been used for quantification of these compounds in different commercial pharmaceutical products and gave good results when compared with reference spectrophotometric and HPLC methods.     

Determination of gambogic acid in dog plasma by high-performance liquid chromatography for a pharmacokinetic study

A rapid, simple and sensitive high-performance liquid chromatography method for the quantification of gambogic acid in dog plasma was developed and validated. After acidification with hydrochloric acid, dog plasma was extracted with ethyl acetate and determined by HPLC. The analysis was carried out on a reversed-phase C(18) analytical column. The mobile phase consisted of a mixture of methanol-0.05% phosphoric acid (94:6, v/v), and the column temperature was maintained at 35 degrees C. A constant mobile phase flow rate of 1.0 mL/min was employed throughout the analyses. The ultraviolet detector was set at 360 nm. Chromatographic separation was achieved in less than 10 min and the calibration curve was linear over a concentration range of 0.156-20 microg/mL. The intra-assay and inter-assay variability values were less than 10.0%. The accuracy ranged from 93.0 to 104.2%. The established method has been successfully applied to a pharmacokinetic study of gambogic acid in dogs.     

Binary–solvent–based ionic–liquid–assisted surfactant‐enhanced emulsification microextraction for the determination of four fungicides in apple juice and apple vinegar

A binary–solvent–based ionic–liquid–assisted surfactant‐enhanced emulsification microextraction method was developed for the separation/preconcentration and determination of four fungicides (pyrimethanil, fludioxonil, cyprodynil, pyraclostrobin) in apple juice and apple vinegar. A nonchlorinated solvent amyl acetate, which has a lower density than water, was used as the extraction solvent, and an ionic liquid 1‐hexyl‐3‐methylimidazolium hexafluorophosphate, which has a high density and low toxicity, was used as a secondary solvent mixed with the extraction solvent. After centrifugation, the binary solvent drop with a relatively high density was deposited on the bottom of the tube. Some parameters influencing the extraction efficiency of analytes such as type of extraction solvent, ratio of ionic liquid, volume of mixed solvent, type and concentration of surfactant, sample pH, NaCl concentration, and vortex time were investigated and optimized. Under the optimized conditions, the proposed method provided a good linearity in the range of 5–200 μg/L. The limits of quantification of the method were in the range of 2–5 μg/L. The relative standard deviations for interday assays were 1.7–11.9%. The method was applied to the determination of pyrimethanil, fludioxonil, cyprodynil, and pyraclostrobin in apple juice and apple vinegar samples, and the accuracy was evaluated through recovery experiments.     

Development and validation of a HPLC–UV based method for the extraction and quantification of methotrexate in the skin

An innovative and sensitive HPLC–UV method for the extraction and quantification of methotrexate (MTX) in skin layers was developed and validated. Owing to the physico-chemical characteristics of the drug and the nature of the tissue, it was necessary to use folic acid (FA) as an internal standard for MTX quantification in the dermis. MTX (and FA) analysis was performed on a Phenomenex Jupiter C₁₈ column, using a 50 mm sodium acetate buffer (pH 3.6) and methanol mixture (87:13, v/v) as mobile phase, pumped at 1 ml/min. The absorbance was monitored at 290 nm. The method was selective, linear in the range 0.11–8.49 μg/ml for extraction solvent and 0.05–8.94 μg/ml for pH 7.4 phosphate-buffered saline, precise and accurate, with lower limits of quantitation of 0.11 μg/ml (extraction solvent) and 0.05 μg/ml (pH 7.4 phosphate-buffered saline). The method developed is suitable for the quantification of MTX in skin layers at the end of in vitro permeation experiments; the overall mass balance was 96.5 ± 1.4%, in line with the requirements of the Organisation for Economic Co-operation and Development guideline for the testing of the chemicals (Skin absorption: in vitro method).     

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

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

Determination of some phenolic compounds in red wine by RP-HPLC: method development and validation

A methodology employing reversed-phase high-performance liquid chromatography (RP-HPLC) was developed and validated for simultaneous determination of five phenolic compounds in red wine. The chromatographic separation was carried out in a C(18) column with water acidify with acetic acid (pH 2.6) (solvent A) and 20% solvent A and 80% acetonitrile (solvent B) as the mobile phase. The validation parameters included: selectivity, linearity, range, limits of detection and quantitation, precision and accuracy, using an internal standard. All calibration curves were linear (R(2) > 0.999) within the range, and good precision (RSD < 2.6%) and recovery (80-120%) was obtained for all compounds. This method was applied to quantify phenolics in red wine samples from Santa Catarina State, Brazil, and good separation peaks for phenolic compounds in these wines were observed.     

Optimization and Validation of a Fast Liquid Gradient for Determination of Prominent Flavan‐3‐ols and Flavonols in Fresh Vegetables

A fast high‐performance liquid chromatography method was used for analysis of prominent flavan‐3‐ols and flavonols in vegetables. Gradient elution with phosphoric acid‐acetonitrile mixtures and phosphoric acid‐methanol mixtures allowed fast and complete separation of the studied phenolic compounds within analysis times less than 10 min. The development of two elution gradients using methanol and acetonitrile as modifiers proved to be an excellent approach for the verification of the real polyphenolic composition in vegetables samples because the two optimized methods allowed the separation of the same number of compounds in the same elution order. Diode‐array detection was employed for the provisional identification of phenolic compounds that were not available as standards. We preferred methanol as a modifier because it was less toxic and cheaper than acetonitrile. Detection limits ranged between 0.12 and 0.59 μg mL^–1. High recoveries of phenolics from fresh vegetables were measured in all studied cases, independent of the phenolic structure, matrix, and vegetable in question. High levels of procyanidins between 150 and 450 mg kg^–1 were found in all studied vegetables. Quantification of quercetin and kaempferol glycosides was only possible in marrow and onion, respectively.     

Comparison of high performance TLC and HPLC for separation and quantification of chlorogenic acid in green coffee bean extracts

Two chromatographic methods, high-performance TLC (HPTLC) and HPLC, were developed and used for separation and quantitative determination of chlorogenic acid in green coffee bean extracts. For HPTLC silica gel Kieselgel 60 F 254 plates with ethyl acetate/dichlormethane/formic acid/acetic acid/water (100:25:10:10:11, v/v/v/v/v) as mobile phase were used. Densitometric determination of chlorogenic acid by HPTLC was performed at 330 nm. A gradient RP HPLC method was carried out at 330 nm. All necessary validation tests for both methods were developed for their comparison. There were no statistically significant differences between HPLC and HPTLC for quantitative determination of chlorogenic acid according to the test of equality of the means.     

Development and validation of a HPLC method for quantification of rivastigmine in rat urine and identification of a novel metabolite in urine by LC‐MS/MS

A sensitive, specific and accurate HPLC method for the quantification of rivastigmine (RSM) in rat urine was developed and validated. The method involves the simple liquid–liquid extraction of RSM and pyridostigmine as an internal standard (IS) from rat urine with tertiary methyl butyl ether. The chromatographic separation of RSM and IS was achieved with 20 mm ammonium acetate buffer (pH 6.5) and acetonitrile (65:35, v/v) delivered at flow‐rate of 1 mL/min on a Kromasil KR‐100. The method was in linear range from 50 to 5000 ng/mL. The validation was done as per FDA guidelines and the results met the acceptance criteria. The method was successfully applied for the quantification of RSM in rat urine. Besides method validation, we have identified two metabolites of RSM in urine. Both the metabolites were characterized by HPLC‐PDA and LC‐MS/MS and it was found that one metabolite is novel. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

High-performance liquid chromatographic determination of doxazosin in human plasma for bioequivalence study of controlled release doxazosin tablets

A highly sensitive high-performance liquid chromatographic quantification method with fluorescence detection was developed and validated for the determination of doxazosin in human plasma. The developed method employed one-step extraction of doxazosin from plasma matrix with ethyl acetate using propranolol as an internal standard. Chromatographic separation was obtained within 8.0 min using a reverse-phase Capcell-Pak C(18) column (150 x 4.6 mm i.d., 5 microm) and the mobile phase consisted of methanol-water containing 10 mM perchloric acid and 1.8 mM sodium heptane sulfonic acid (50:50, v/v) and was set at a flow rate of 1.5 mL/min. The calibration curve constructed was linear in the range of 0.3-50.0 ng/mL. The proposed method achieved a lower limit of quantification of 0.3 ng/mL, better than the reported HPLC methods. Average recoveries of doxazosin and the internal standard from human plasma matrix were 87.0 and 85.9%, respectively. The present method was validated by evaluating the precision and accuracy for inter- and intraday variation in the concentration range 0.3-50 ng/mL. The precision values expressed as relative standard deviations in the inter- and intraday validation were 1.17-6.29 and 0.84-5.94%, respectively. This method was successfully applied to the bioequivalence study of two doxazosin controlled release tablets in healthy, male human subjects.