Rapid Sample Screening Method for Authenticity Controlling of Vanilla Flavours Using Liquid Chromatography with Electrochemical Detection Using Aluminium-Doped Zirconia Nanoparticles-Modified Electrode

A rapid and sensitive technique for frauds determination in vanilla flavors was developed. The method comprises separation by liquid chromatography followed by an electrochemical detection using a homemade screen-printed carbon electrode modified with aluminium-doped zirconia nanoparticles (Al-ZrO₂-NPs/SPCE). The prepared nanomaterials (Al-ZrO₂-NPs) were characterized by using X-ray diffraction (XRD), transmission electron microscopy (TEM) and energy dispersive X-ray (EDX). This method allows for the determination of six phenolic compounds of vanilla flavors, namely, vanillin, p-hydroxybenzoic acid, p-hydroxybenzaldehyde, vanillyl alcohol, vanillic acid and ethyl vanillin in a linear range between 0.5 and 25 µg g⁻¹, with relative standard deviation values from 2.89 to 4.76%. Meanwhile, the limits of detection and quantification were in the range of 0.10 to 0.14 µg g⁻¹ and 0.33 to 0.48 µg g⁻¹, respectively. In addition, the Al-ZrO₂-NPs/SPCE method displayed a good reproducibility, high sensitivity, and good selectivity towards the determination of the vanilla phenolic compounds, making it suitable for the determination of vanilla phenolic compounds in vanilla real extracts products.     

Detection and quantification of phenolic compounds in olive oil by high resolution H nuclear magnetic resonance spectroscopy

High resolution ¹H NMR spectroscopy has been employed as a versatile and rapid method to analyze the polar fraction of extra virgin olive oils containing various classes of phenolic compounds. The strategy for identification of phenolic compounds is based on the NMR chemical shifts of a large number of model compounds assigned by using two-dimensional (2D) NMR spectroscopy. Furthermore, 2D NMR was applied to phenolic extracts in an attempt to discover additional phenolic compounds. The ¹H NMR methodology was successful in detecting simple phenols, such as p-coumaric acid, vanillic acid, homovanillyl alcohol, vanillin, free tyrosol, and free hydroxytyrosol, the flavonols apigenin and luteolin, the lignans (+) pinoresinol, (+) 1-acetoxypinoresinol and syringaresinol, two isomers of the aldehydic form of oleuropein and ligstroside, the dialdehydic form of oleuropein and ligstroside lacking a carboxymethyl group, and finally total hydroxytyrosol and total tyrosol reflecting the total amounts of free and esterified hydroxytyrol and tyrosol, respectively. The absolute amount of each phenolic constituent was determined in the polar fraction by using anhydrous 1,3,5-triazine as an internal standard.     

RP-HPTLC densitometric determination and validation of vanillin and related phenolic compounds in accelerated solvent extract of Vanilla planifolia*

A simple, fast and sensitive RP-HPTLC method is developed for simultaneous quantitative determination of vanillin and related phenolic compounds in ethanolic extracts of Vanilla planifolia pods. In addition to this, the applicability of accelerated solvent extraction (ASE) as an alternative to microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE) and Soxhlet extraction was also explored for the rapid extraction of phenolic compounds in vanilla pods. Good separation was achieved on aluminium plates precoated with silica gel RP-18 F(254S) in the mobile phase of methanol/water/isopropanol/acetic acid (30:65:2:3, by volume). The method showed good linearity, high precision and good recovery of compounds of interest. ASE showed good extraction efficiency in less time as compared to other techniques for all the phenolic compounds. The present method would be useful for analytical research and for routine analysis of vanilla extracts for their quality control.     

A rapid and simple ultra high performance liquid chromatography method for the simultaneous determination of methoxyphenol derivatives involved in the eugenol catabolic pathway

An efficient ultra high performance liquid chromatography method of separation was developed for the analysis of six important methoxyphenol derivatives involved in the eugenol catabolic pathway. In the present study, an Acquity UPLC BEH C₁₈ column was used for the chromatographic separation of the industrially important phenolic compounds such as vanillin, vanillic acid, ferulic acid, coniferyl alcohol, and coniferyl aldehyde obtained during microbial transformation of eugenol. Eluted components were identified using the dual wavelength (254 and 310 nm) UV detector. A gradient method of elution using mobile phase of aqueous 1 mM trifluoroacetic acid (Solvent A) and methanol (Solvent B) at a flow rate of 0.3 mL/min separated all the five intermediate methoxyphenol derivatives along with their precursor eugenol within 15 min with stable baseline resolution. Method validation was performed for the accurate quantification of vanillin, coniferyl aldehyde, and eugenol using the parameters of linearity, specificity, precision, limit of detection, limit of quantification, and robustness. The developed method would be helpful for clear separation and identification of the five most important intermediate metabolites of the eugenol catabolism pathway.     

Determination of vanillin and related flavor compounds in natural vanilla extracts and vanilla-flavored foods by thin layer chromatography and automated multiple development

Summary Thin layer chromatography on silica gel high performance layers and automated multiple development was used to separate the polar aromatic flavor compounds vanillin, ethyl vanillin, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, 4-hydroxybenzyl alcohol, vanillic acid, coumarin, piperonal, anisic acid, and anisaldehyde commonly found in extracts of natural and artificial vanilla flavors. The ratio of 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde and vanillic acid to vanillin in natural vanilla extracts was used to confirm the authenticity of extracts purchased in the United States of America and the United Kingdom. Natural vanilla extracts purchased in Mexico and Puerto Rico were identified as counterfeit products based on changes in the above ratio and the presence of synthetic flavor compounds such as ethyl vanillin and coumarin. It is also demonstrated that the proposed method is suitable for the determination of natural and synthetic vanilla flavors in solvent extracts from food, beverage and confectionery products. The main advantage of thin layer chromatography for the analysis of vanilla extracts and food stuffs flavored with vanilla is its high sample throughput since sample preparation requirements are minimal and multiple samples can be separated simultaneously.     

Liquid chromatographic determination of vanillin and related aromatic compounds

This paper describes a reversed-phase liquid chromatographic method for the determination of vanillin, associated natural aromatic compounds and/or synthetic precursors, ethyl vanillin, and coumarin, a commonly encountered adulterant in nonbeverage and beverage alcohol products using a ternary gradient mobile phase. The compounds were separated on a Nova-Pak C18 column by using water, methanol, and tetrahydrofuran as the mobile phase. Measurements were made by using a photodiode array detector at 275 nm. The choice of the mobile phase and the column provides baseline resolution of vanillin and the associated aromatic compounds commonly found in vanilla-flavoring material. Because this method provides low-level detection/quantitation, it is suitable for the characterization of vanilla flavoring materials that are currently added to vanilla flavored beverage alcohol products.     

Optimization of phenolic compounds analysis by capillary electrophoresis

Operational parameters like migration time, temperature, voltage, composition of background electrolyte and content of organic modifier were optimized in CZE for the determination of lignin-like phenolic compounds.The applied background electrolyte buffer consisted of a Na₂B₄O₇, KH₂PO₄ aqueous solution, pH 9.15 using acetonitrile as organic modifier with UV-detection. Compounds, such as acetosyringone, acetovanillone, syringealdehyde, p-hydroxyacetophenone, vanillin, syringic acid, ferulic acid, p-hydroxybenzaldehyde, p-coumaric acid, vanillic acid and p-hydroxybenzoic acid were applied as reference compounds.The quality and quantity of different phenolic compounds obtained upon alkaline CuO oxidation of a commercial humic acid were determined with CZE using ethylvanillin as internal standard.The optimized CZE revealed has being an appropriate method since it is quick, sensitive and quantitative and does not require a time-consuming sample preparation.     

Biotransformation of Rice Bran to Ferulic Acid by Pediococcal Isolates

Ferulic acid (FA) is widely used in foods, in beverages, and in various pharmaceutical industries as a precursor of vanillin. FA biotransformation can occur during the growth of lactic acid bacteria (LAB), and its conversion to other phenolic derivatives is observed by many scientists, where ferulic acid esterase (FAE) and ferulic acid decarboxylase (FDC) play significant roles. The present study aimed at screening a panel of LAB for their ability to release FA from rice bran, an agro waste material. FAE and FDC activities were analyzed for the preliminary screening of various dairy isolates. Two Pediococcus acidilactici isolates were selected for studying further the hydrolysis of FA from rice bran and its bioconversion into phenolic derivatives like 4-ethylphenol, vanillin, vanillic acid, and vanillyl alcohol. P. acidilactici M16, a probiotic isolate, has great potential for the production of FA from rice bran and could be exploited as starter culture in the food industry for the production of biovanillin.     

Fractionation of red wine polyphenols by solid-phase extraction and liquid chromatography

A systematic method for separation of aged red wine polyphenols into various distinct fractions using combined techniques of solid-phase extraction and liquid chromatography was proposed. The aged red wine polyphenols were separated into various distinct fractions including phenolic acid fraction, monomer flavanol fraction, oligomer procyanidin fraction, anthocyanin and its pyruvic acid derivative fraction, free or non-colored proanthocyanidin fraction, fraction of direct condensation products between anthocyanins and proanthocyanidins and fraction of other pigmented complexes. The phenolic composition of each fraction was verified by HPLC with diode array detection (HPLC-DAD), thiolysis, vanillin assay, HPLC coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS) and multi-stage MS fragment analysis. For the first time, anthocyanins and their pyruvic derivatives were separated from other phenolic compounds, while free or non-pigmented polymer proanthocyanidins from other pigmented complexes. The fractionation method would be of particular interest in further studying the detailed composition of polymeric polyphenols in red wine.     

Probing occurrence of phenylpropanoids in Morinda citrifolia in relation to foliar diseases

Accumulation of phenolic compounds in cell walls of different plant organs leading to increased lignification is an early defence response of plants against biotic stress. The aim of this work was to delineate occurrence of cell wall-bound (CWB) phenolic compounds in Morinda citrifolia leaves. Alkaline hydrolysis of the cell wall material of leaf tissues yielded 4-coumaric acid (4-CA) as the major bulk of the phenolic compounds in all Morinda germplasms. Next in line was 4-hydroxybenzoic acid. Other phenolics identified were vanillic acid, 4-hydroxybenzaldehyde, vanillin and ferulic acid. Concentrations of all the CWB phenolics were highest in the germplasm CHN-5, followed by the germplasm CHN-1. Incidentally, these two Morinda germplasms recorded lowest incidence of foliar diseases. Significantly higher amounts of 4-CA in combination with other phenolics may be the reasons for lowest incidence of foliar diseases in CHN-5 and CHN-1 germplasms of M. citrifolia.     

Propolis oil extract: quality analysis and evaluation of its antimicrobial activity

Designing propolis products for external use involves determining the optimal form of propolis for the introduction into dermatological pharmaceuticals and cosmetic preparations. As a potent ingredient, propolis oil extract from raw material harvested in Lithuania was analysed. The rheological characteristics, content of phenolic compounds, major compounds and antimicrobial activity of the propolis oil extract are investigated here for the first time. The propolis oil extract was produced by maceration using different solvents, raw material was collected in Lithuania. Solvent mixture with 96% ethanol increased the rheological stability and extracted amount of phenolic compound. High-performance liquid chromatography identified the potent quality markers for Lithuanian propolis, phenylpropanoid vanillin, coumaric acid and ferulic acid. Antimicrobial activity of propolis oil extract was evaluated in experimental studies in vitro, and the minimal concentration of phenolic compounds that inhibited respective microorganisms was determined. The results demonstrate that phenolic compounds have effective antimicrobial activity in propolis oil extract; thus, it can be compatible with the semisolid preparation.     

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

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

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 the active ingredients in Gastrodia rhizoma by capillary electrophoresis with electrochemical detection

A simple, reliable and reproducible method, based on capillary electrophoresis (CE) with electrochemical detection (ED), for the determination of five active ingredients and three carbohydrates in extracts of Gastrodia rhizoma is described in this work. The main active ingredients are gastrodin, 4-hydroxybenzyl alcohol, vanillyl alcohol, 4-hydroxybenzylaldehyde and vanillin. Operated in a wall-jet configuration, a 300 microm diameter carbon disc electrode was used as a working electrode, with a good response at +1000 mV (vs. SCE) for 4-hydroxybenzyl alcohol, vanillyl alcohol, 4-hydroxybenzylaldehyde and vanillin; a 300 microm diameter copper disc electrode exhibits a good response at +650 mV (vs. SCE) for gastrodin, sucrose, glucose and fructose. Under optimum conditions, 4-hydroxybenzyl alcohol, vanillyl alcohol, 4-hydroxybenzylaldehyde and vanillin in 100 mmol l(-1) borate buffer (pH 9.2) and gastrodin, sucrose, glucose and fructose in 50 mmol l(-1) sodium hydroxide buffer were baseline separated within 18 min. The response was linear over two orders of magnitude with a detection limit (S/N = 3) in the range 3 x 10(-7)-1.8 x 10(-6) mol l(-1) for all eight analytes. This method was successfully used in the analysis of traditional Chinese medicine, and the assay results were satisfactory.     

Cobalt‐catalyzed Aerobic Oxidation of Eugenol to Vanillin and Vanillic Acid

A novel, simple, one‐step method of synthesizing vanillin and vanillic acid from eugenol has been developed. The method uses ligand‐ and additive‐free Co(OAc)₂ ? 4H₂O as catalyst and molecular oxygen as oxidant to achieve catalytic process without isomerizing eugenol. Extensive screening efforts were used to optimize eugenol to obtain vanillin and vanillic acid. Under optimal conditions, the reaction provided vanillin and vanillic acid with 68.5 % and 15.2 % yields, respectively. Apart from the desired products, coniferyl alcohol 9‐methyl ether and 4‐hydroxy‐3‐methoxycinnamaldehyde as two intermediates were also detected in the reaction process. Level changes of all compounds over time were presented in the reaction. The reaction pathway from eugenol to vanillic acid was validated by conducting several control experiments. Furthermore, a possible reaction mechanism dominated by a circular redox reaction from Co(III) and Co(II) species was proposed. This method offers a potentially practical alternative for manufacturing vanillin and vanillic acid efficiently.     

Salt addition effect on partition coefficient of some phenolic compounds constituents of olive mill wastewater in 1-octanol-water system at 298.15 K

The 1-octanol-water partition coefficient is an important property to measure the hydrophobicity of organic compounds, which has been demonstrated to be a parameter in studying the conformation of biomolecules in aqueous solutions. For biological systems, electrolytes play an important role in thermodynamic properties. The salt addition effect on the distribution of phenolic compounds between water and 1-octanol at 298.15 K has been studied. The phenolic compounds used were vanillic acid, protocatechuic acid, vanillin, tyrosol, cathecol, caffeic acid and syringic acid, and the considered salts were potassium chloride, sodium chloride and lithium chloride. The influence of both the concentration and size of the added salt on the partition coefficient (K _ow) have been considered. This study shows a salting in with the following decreasing order: LiCl > NaCl > KCl. The Gibbs energies of transfer of phenolic compounds (168–1) form chloride solutions to organic phase have been calculated using experimental 1-octanol-water partition coefficients.     

超高效液相色谱-串联质谱法同时测定食品中4种常用香精

建立了超高效液相色谱-串联质谱法同时测定食品中香兰素、乙基香兰素、麦芽酚和乙基麦芽酚4种香精的方法。样品用水提取,固相萃取小柱净化,目标化合物采用UPLC^TMBEH C18色谱柱(50 mm×2.1 mm, 1.7 μm)分离,以甲醇和含0.002 mol/L乙酸铵及0.1%(v/v)甲酸的水溶液为流动相进行梯度洗脱,采用电喷雾离子源电离、正离子多反应监测模式质谱检测。4种香精在5~500 μg/L或10~1000 μg/L质量浓度范围内线性良好,相关系数均在0.9995~0.9998之间;回收率为75.8%~116%,相对标准偏差(RSD, n=6)为1.58%~4.01%。该方法灵敏、准确,检测范围广,分析速度快,适合食品中香兰素、乙基香兰素、麦芽酚和乙基麦芽酚4种香精的检测。     

HPLC characterization of phenolics in lignocellulosic materials

Summary The application of HPLC with an electrochemical detector for the determination of phenolics in lignocellulosic materials is reported. The separation of phenolic acids and aldehydes (gallic acid, p-hydroxybenzoic acid, vanillic acid, p-coumaric acid, syringic acid, ferulic acid, vanillin, syringaldehyde and p-hydroxybenzaldehyde) on two different columns (reversed phase C6 and styrene-divinylbenzene PLRP-S) is shown. Chromatograms of phenolic compounds in neutral, basic and oxidative extracts of wheat straw treated with NaOH and white rot fungusStropharia rugosoannulata are reported along with quantitative results.     

Comparison of the simple cyclic voltammetry (CV) and DPPH assays for the determination of antioxidant capacity of active principles

Antioxidant activity of a number of small (low molecular weight) natural compounds found in spices, condiments or drugs (gallic acid, sesamol, eugenol, thymol, carvacrol, vanillin, salicylaldehyde, limonene, geraniol, 4-hexylresorcinol, etc.) has been evaluated using electrochemical and DPPH? radical scavenging measurements. Structural analysis of the tested compound suggest a remarkable activity for phenol derivatives and the importance of the -R groups located on the phenolic ring in the molecule's ability to act as free radical scavenging as well as their influence in the electrochemical behavior. The voltammetric method can be used for the determination of the antioxidant capability in the same manner as the DPPH? radical scavenging because of the correlation found between oxidation potentials and anti-radical power (ARP = 1/EC??). Such electrochemical determination is fast and cheap and allows making measurements under a variety of experimental conditions. The accuracy of the electrochemical measurements is the same for all the compounds, irrespective of their scavenging activity, the opposite of what occurs in the DPPH? test.     

Bioprocess Design for the Microbial Production of Natural Phenolic Compounds by Debaryomyces hansenii

Debaryomyces hansenii NRRL Y-7426 metabolised ferulic acid into different phenolic compounds using a factorial design where glucose concentration (in the range of 1?C20?g/L), peptone concentration (2?C20?g/L) and yeast extract concentration (0.2?C10?g/L) were the independent variables. The interrelationship between dependent and operational variables was well fitted (R ²?>?0.95) to models including linear, interaction and quadratic terms. Depending on the glucose and nitrogen concentrations, which redirected the metabolism, the major degradation products were 1,226.2?mg 4-vinyl guaiacol/L after 72?h (molar yield of 86.0?%), 1,077.8?mg vanillic acid/L after 360?h (molar yield of 91.1?%) or 1,682.6?mg acetovanillone/L after 408?h (molar yield of 98.8?%) in fermentations carried out with 2,000?mg ferulic acid/L. Other metabolites such as vanillin, vanillyl alcohol or 4-ethylguaiacol were present in lower amounts.