Determination of coumarin as an adulterant in vanilla flavoring products by high-performance liquid chromatography

A high-performance liquid chromatographic procedure was developed for the isolation and quantitation of coumarin from vanilla-based liquid flavorings of Mexican origin. Forty products representing fourteen different Mexican brands were assayed for coumarin, vanillin, and ethyl vanillin by the proposed method. The procedure has been adapted to the analysis of other products including domestic vanilla extracts and imitation vanilla flavorings for vanillin, ethyl vanillin, 4-hydroxybenzaldehyde and piperonal. Chromatographic retention data for thirty-seven compounds associated with vanillin and vanilla products employing two mobile phase systems are presented.     

A disposable electrochemical sensor for vanillin detection

A disposable electrochemical sensor was developed for the detection of vanillin in vanilla extracts and in commercial products. An analytical procedure based on square-wave voltammetry (SWV) was optimised and a detection limit of 0.4 μM for vanillin was found. A relative standard deviation of 2% was calculated for a vanillin concentration of 100 μM. The method was applied to the determination of vanillin in natural concentrated vanilla extracts and in final products such as yoghurt and compote. The obtained results were compared with those provided by a reference method based on HPLC. The electrochemical behaviour of other compounds (vanillic acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, etc.), generally present in natural oleoresins, were also studied, to check for interferences with respect to the vanillin voltammetric signal.     

Development and validation of an RP-HPLC method for quantitative determination of vanillin and related phenolic compounds in Vanilla planifolia

A simple and fast method was developed using RP-HPLC for separation and quantitative determination of vanillin and related phenolic compounds in ethanolic extract of pods of Vanilla planifolia. Ten phenolic compounds, namely 4-hydroxybenzyl alcohol, vanillyl alcohol, 3,4-dihydroxybenzaldehyde, 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin, p-coumaric acid, ferulic acid, and piperonal were quantitatively determined using ACN, methanol, and 0.2% acetic acid in water as a mobile phase with a gradient elution mode. The method showed good linearity, high precision, and good recovery of compounds of interest. The present method would be useful for analytical research and for routine analysis of vanilla extracts for their quality control.     

Determination of flavour components in natural vanilla extracts and synthetic flavourings by mixed micellar electrokinetic capillary chromatography

The development of a mixed micellar electrokinetic capillary chromatography (MECC) method for the qualitative and quantitative determination of key components, including vanillin, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, vanillic acid and 3-methoxybenzaldehyde, that contribute to vanilla flavour was investigated. The micellar phase consisted of sodium dodecyl sulfate (SDS) and sodium cholate (SC). The percent relative standard deviation (R.S.D.%) for migration time was <1 over six runs. The R.S.D.% for peak areas ranged between 0.85-1.96% over six runs. Peak efficiencies were excellent with theoretical plate numbers typically in the range of 130,000-200,000 per column (52 cm effective length). The limits of detection (LOD) were between 5-10 μg/ml. The quantitative data was verified by high performance liquid chromatography (HPLC) and gas chromatography (GC). The mixed MECC method was successfully applied to a number of natural vanilla extracts, nature identical extracts and synthetic flavourings.     

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.     

Rapid method for the determination of coumarin, vanillin, and ethyl vanillin in vanilla extract by reversed-phase liquid chromatography with ultraviolet detection

A method is described for determining coumarin, vanillin, and ethyl vanillin in vanilla extract products. A product is diluted one-thousand-fold and then analyzed by reversed-phase liquid chromatography using a C18 column and a mobile phase consisting of 55% acetonitrile-45% aqueous acetic acid (1%) solution at a flow rate of 1.0 mL/min. Peaks are detected with a UV detector set at 275 nm. Vanilla extracts were spiked with 250, 500, and 1000 microg/g each of coumarin, vanillin, and ethyl vanillin. Recoveries averaged 97.4, 97.8, and 99.8% for coumarin, vanillin, and ethyl vanillin, respectively, with coefficient of variation values of 1.8, 1.3, and 1.3%, respectively. No significant difference was observed among the 3 spiking levels. A survey of 23 domestic and imported vanilla extract products was conducted using the method. None of the samples contained coumarin. The surveyed samples contained between 0.4 to 13.1 and 0.4 to 2.2 mg/g vanillin and ethyl vanillin, respectively.     

Rapid sample screening method for authenticity controlling vanilla flavors using a CE microchip approach with electrochemical detection

Five vanilla-related flavors of food significance, vanillic alcohol (VOH), ethyl maltol (EMA), maltol (MAL), ethyl vanillin (EVA) and vanillin (VAN), were separated using CE microchips with electrochemical detection (CE-ED microchips). A +2 kV driving voltage for both injection and separation operation steps, using a borate buffer (pH 9.5, 20 mM) and 1 M nitric acid in the detection reservoir allowed the selective and sensitive detection of the target analytes in less than 200 s with reproducible control of EOF (RSD(migration times)<3%). The analysis in selected real vanilla samples was focusing on VAN and EVA because VAN is a basic fragrance compound of the vanilla aroma, whereas EVA is an unequivocal proof of adulteration of vanilla flavors. Fast detection of all relevant flavors (200 s) with an acceptable resolution (R(s) >1.5) and a high accuracy (recoveries higher than 90%) were obtained with independence of the matrices and samples examined. These results showed the reliability of the method and the potential use of CE microchips in the food control field for fraudulent purposes.     

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.     

反相高效液相色谱法定量分析木质素的主要降解产物

建立了反相高效液相色谱定量分析玉米秸秆蒸汽爆破预处理过程中产生的主要木质素降解产物的方法。采用C18色谱柱,柱温30 ℃,乙腈-水(含1.5%的醋酸)为流动相,梯度洗脱,流速为0.8 mL/min, 254 nm和280 nm波长下紫外检测,可实现4-羟基苯甲酸、香草酸、紫丁香酸、4-羟基苯甲醛、香草醛和紫丁香醛的有效分离。6种主要木质素降解产物线性回归方程相关系数为0.9999～1.0000,加标回收率均在96%以上,相对标准偏差低于2.5%(n=6),满足定量分析要求。     

New Esters, 2-(4-Hydroxy-3-Methoxyphenyl)Ethyl Hexa- and Octacosanoates from the Leaves of Cinnamomum Reticulatum Hay

The leaves of Cinnamomum reticulatum Hay were extracted successively with hexane and acetone successively. Dipentadecyl ketone, n-dotriaconyl alcohol, 2-(4-hydroxy-3-methoxyphenyl)ethyl hexa- and octacosanoates ( two new esters from hexane extract ), β-sitosterol, fatty acid, vanillin, p-hydroxybenzaldehyde, genkwanin, vanillic acid, p-hydroxybenzoic acid, β-sitosteryl-l-β-glucopyranoside, meso-erythritol, glucose, and 4-(2-hydroxyethyl)-2-methoxyphenyl-l-β-D-glucopyranoside (from the acetone extract) were isolated. The structures of the new esters were elucidated by chemical and physical evidence.     

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.     

Determination of coumarin, vanillin, and ethyl vanillin in vanilla extract products: liquid chromatography mass spectrometry method development and validation studies

A LC-MS method was developed for the determination of coumarin, vanillin, and ethyl vanillin in vanilla products. Samples were analyzed using LC-electrospray ionization (ESI)-MS in the positive ionization mode. Limits of detection for the method ranged from 0.051 to 0.073 microg mL(-1). Using the optimized method, 24 vanilla products were analyzed. All samples tested negative for coumarin. Concentrations ranged from 0.38 to 8.59 mg mL(-1) (x =3.73) for vanillin and 0.33 to 2.27 mg mL(-1) (x =1.03) for ethyl vanillin. The measured concentrations are compared to values calculated using UV monitoring and to results reported in a similar survey in 1988. Analytical results, method precision, and accuracy data are presented.     

Determination of Vanillin and Ethylvanillin in Vanilla Flavorings by Planar (Thin-Layer) Chromatography

Results of a determination of vanillin and its homologue, ethylvanillin (vanillal), in food flavorings by thin-layer chromatography are presented. A mixture of hexane and ethyl acetate with a volume ratio 9 : 1–8 : 2 is preferable as an eluent for the separation of the flavorings. A mixture of heptanone, ethanol, and sulfuric acid (a volume ratio 4 : 5 : 1) was proposed as a developing agent. Different eluents and developing agents used for the separation of vanillin and ethylvanillin were compared; the results are presented. Solutions of vanillin and ethylvanillin and commercially produced vanilla flavorings were analyzed. The results can be used in the monitoring of the composition of vanilla flavorings, as well as for the authentication of artificial or identical-to-natural flavorings.     

Development of an Aluminium Doped TiO2 Nanoparticles‐modified Screen Printed Carbon Electrode for Electrochemical Sensing of Vanillin in Food Samples

A new chemically modified electrode based on titanium dioxide nanoparticles (TiO₂‐NPs) has been developed. Aluminium was incorporated into the TiO₂‐NPs to prepare aluminium doped TiO₂ nanoparticles (Al‐TiO₂‐NPs). Aluminium doped TiO₂ nanoparticles‐modified screen printed carbon electrode (Al‐TiO₂‐NPs/SPCE) was employed as easy, efficient and rapid sensor for electrochemical detection of vanillin in various types of food samples. Al‐TiO₂‐NPs were characterized by energy‐dispersive X‐ray (EDX), transmission electron microscopy (TEM), and X‐ray diffraction (XRD) and analyses showing that the average particle sizes varied for the Al‐NPs (7.63 nm) and Al‐TiO₂‐NPs (7.47 nm) with spherical crystal. Cyclic voltammetry (CV) and linear sweep voltammetry (LSV) were used to optimize the analytical procedure. A detection limit of vanillin was 0.02 μM, and the relative standard deviation (RSD) was 3.50 %, obtained for a 5.0 μM concentration of vanillin. The electrochemical behaviour of several compounds, such as vanillic acid, vanillic alcohol, p‐hydroxybenzaldehyde and p‐hydroxybenzoic, etc., generally present in natural vanilla samples, were also studied to check the interferences with respect to vanillin voltammetric signal. The applicability was demonstrated by analysing food samples. The obtained results were compared with those provided by a previous method based on liquid chromatography for determination of vanillin.     

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.     

Determination of available phenolic compounds in soils by liquid chromatography with solid-phase extraction

A fast, selective, and sensitive liquid chromatographic (LC) method was developed for determination of derivatives of benzoic and cinnamic acids (gallic, protocatechuic, 2,3,4-trihydroxybenzoic, 4-hydroxybenzoic, vanillic, caffeic, syringic, 4-coumaric, ferulic, sinapic, benzoic, 2-coumaric, cinnamic acids, and 4-hydroxybenzaldehyde and vanillin) in soil samples. The method for sample pretreatment is based on temperature-controlled extraction with water (pH 5.6) for 60 min. Extracts were preconcentrated and purified by solid-phase extraction on OASIS HLB sorbent, with subsequent separation and quantification of individual substances by LC with UV diode-array detection. Limits of detection (3 signal-to-noise LODs) better than 65 ng/g (dry weight) and recoveries from 88 to 99% were found for each compound at absorbance 280 nm. The method was used for determination of bioavailable phenolic compounds in different soil samples.     

气相色谱-三重四极杆质谱法同时测定巴氏杀菌乳中9种香精成分

建立了气相色谱-三重四极杆质谱（GC-MS/MS）同时检测巴氏杀菌乳中9种香精成分（二氢香豆素、香兰素、香豆素、乙基香兰素、甲基香兰素、7-甲基香豆素、7-甲氧基香豆素、7-乙氧基-4甲基香豆素和环香豆素）的分析方法。巴氏杀菌乳样品用乙醇溶液萃取,低温涡旋离心,取上清液过0.22 μm滤膜,以DB-5MS色谱柱分离,在MRM模式下测定,基质曲线外标法定量。实验结果表明,9种香精成分在1~200 μg/L范围内呈良好线性,线性相关系数（R²）均大于0.997,方法的检出限为0.002~0.1 μg/kg,定量限为0.001~2 μg/kg,平均回收率为90.3%~110.6%,日内、日间精密度均小于10%。该方法前处理简单快速,检测准确度和灵敏度高,可用于巴氏杀菌乳中9种香精成分的同时检测。     

Solid-phase/supercritical-fluid extraction for liquid chromatography of phenolic compounds in freshwater microalgae and selected cyanobacterial species

In the present paper a new extraction technique based on the combination of solid-phase/supercritical-fluid extraction (SPE/SFE) with subsequent reversed-phase HPLC is described. The SPE/SFE extractor was originally constructed from SPE-cartridge incorporated into the SFE extraction cell. Selected groups of benzoic acid derivatives (p-hydroxybenzoic, protocatechuic, gallic, vanillic and syringic acid), hydroxybenzaldehydes (4-hydroxybenzaldehyde and 3,4-dihydroxybenzaldehyde) and cinnamic acid derivatives (o-coumaric, p-coumaric, caffeic, ferulic, sinapic and chlorogenic acid) were extracted. Cyclic addition of binary extraction solvent system based on methanol:water (1:1, v/v) and methanol/ammonia aqueous solution was used for extraction at 40 MPa and 80 °C. The p-hydroxybenzoic, protocatechuic, vanillic, syringic, caffeic and chlorogenic acid; 4-hydroxybenzaldehyde and 3,4-dihydroxybenzaldehyde were identified by HPLC-electrospray mass spectrometry in SPE/SFE extracts of acid hydrolyzates of microalga (Spongiochloris spongiosa) and cyanobacterial strains (Spirulina platensis, Anabaena doliolum, Nostoc sp., and Cylindrospermum sp.). For the identification and quantification of the compounds the quasi-molecular ions [M−H]⁻ and specific fragments were analysed by quadrupole mass spectrometry analyzer. Our analysis showed that the microalgae and cyanobacteria usually contained phenolic acids or aldehydes at μg levels per gram of lyophilized sample. The proposed SPE/SFE extraction method would be useful for the analysis of different plant species containing trace amount of polar fraction of phenols.     

Chemical characterization of ozonated lignin solutions from corn (Zea mays) stalk and poplar (Populus deltoides) wood by capillary gas chromatography

Corn (Zea mays) stalk and poplar (Populus deltoides) wood lignin was converted into monomeric aromatic compounds and short chain aliphatic carboxylic acids. The main reaction products were separated and identified using capillary gas chromatography-mass spectrometry on an HP-5 column. The compounds were considered as positively identified when their mass spectra and GC retention times agreed with those of authentic standard samples. The quantitative estimation of the identified reaction products was accomplished on an OV-101 capillary column by gas chromatography-FID using the internal standard method. Among the aromatic compounds, aldehydes (p-hydroxybenzaldehyde, vanillin, and syringaldehyde), acids (p-hydroxybenzoic, vanillic, 3,4-dihydroxybenzoic, and syringic), and one phenol (hydroquinone) were determined. In addition, the aliphatic carboxylic acids: glycolic, oxalic, malonic, glyoxylic, butanedioic, glyceric, and malic acid were identified. All the calibration curves of the quantified compounds approximated to a straight line. For both corn stalk and poplar wood lignins, the major components were the aromatic aldehydes (71 and 64% of the characterized fraction, respectively), followed by the aliphatic carboxylic acids (20 and 21% of the characterized fraction, respectively).     

Studies on some microbiological and chemical aspects of irradiated vanilla beans

Vanilla bean is a valuable food additive used in many branches of food industry as a source of natural vanillin. The influence of⁶⁰Co -radiation in the dose range of 5–50 kGy has been investigated on: (1) the survival of contaminating microflora, and (2) more important vanilla constituents like vanillin and sugars. It has been found that the main vanilla contamination is connected with mesophylic microorganisms in the amount of 8.4·10⁴/g. The dose of 15 kGy is sufficient for decontamination of commercial raw material. The samples under investigation on account of sensory properties and chemical composition were in agreement with country regulations. Doses up to 50 kGy do not cause undesirable changes in the content of sugars and vanillin, which has been confirmed by studies on model compounds.