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.     

Simultaneous spectrophotometric kinetic determination of four flavor enhancers in foods with the aid of chemometrics

A sensitive kinetic spectrophotometric method was developed for the determination of four flavor enhancers--maltol, ethyl maltol, vanillin, and ethyl vanillin--in food samples. The method was based on the reduction of iron(III) by the four analytes in a sulfuric acid medium (0.012 mol/L), and the subsequent interaction of iron(II) with hexacyanoferrate(III) to form the strongly colored Prussian blue complex, which exhibited an absorption maximum at 800 nm. The optimized method had linear calibrations over the concentration ranges of 0.2-2.8 mg/L for maltol, ethyl maltol, and vanillin, as well as 0.2-1.8 mg/L for ethyl vanillin; the corresponding detection limits were 0.07, 0.07, 0.06, and 0.06 mg/L, respectively. Calibration models were constructed from the original and first-derivative spectral data with the use of partial least-squares (PLS) and principal component regression chemometrics methods. Ultimately, the proposed analytical procedure was successively applied for the determination of the four compounds in commercial food samples with the use of a PLS calibration based on the first-derivative spectral data. The results were comparable with those from a reference HPLC method.     

Food Analysis on Electrophoretic Microchips

One of the current trends of separation techniques in analytical chemistry is miniaturization. The aim of miniaturization is to attain better performance, shorter analysis time, and reduced reagent consumption. Capillary Electrophoresis (CE) microchips, the first generation of micro-total analysis systems, are the most used microsystems in food analysis. The scope of this review is to gather and discuss the different applications of such miniaturized devices in this field. Various analytes of food significance such as natural antioxidants, amino acids, proteins, dyes, vanilla flavors, DNA probes, heavy metals, toxins, allergens etc. have been successfully monitored using CE-microchips, either to assess food quality or to ensure food safety. Also, to deal with the high complexity of food matrices, the integration of sample preparation steps onto the chip and the use of new tools from nanotechnology for the detection step have been reported.     

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.     

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。     

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.     

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.     

Determination of spices in food samples by ionic liquid aqueous solution extraction and ion chromatography

In the present work, a novel method to extract three kinds of spices, namely vanillin, ethyl vanillin and ethyl maltol from food products such as biscuit, chocolate and milk powder was developed. 1-Octyl-3-methylimidazolium chloride([Omim]Cl) aqueous solution was selected as the extracting medium. A 0.5 g powder of food product was extracted by 5.0 mL of [Omim]Cl aqueous solution(0.3 mol/L, pH 6.0) under ultrasonication at 50 8C, and then the extract was centrifuged for 10 min at 4000 rpm. The extract was filtered through a syringe filter and injected into ion chromatography system for analysis. The separation of the three spices was carried out on an anion exchange column. The detection wavelength was set at 280 nm. Compared with traditional extraction solvents, [Omim]Cl aqueous solution displayed particular advantages. The applicability of the proposed method to real sample was confirmed. Under the optimal conditions, good reproducibility of extraction performance was obtained, with the relative standard deviation(RSD) values ranging from 1.9% to 6.3%. The recoveries of spiked samples were between 79.8% and 95.8%. The detection limits(LOD, S/N = 3) of vanillin, ethyl vanillin and ethyl maltol were in the range of 20–45 mg/kg. The use of ionic liquid aqueous solution as extraction solvent was operationally easy and environmental-friendly.     

CE microchips: an opened gate to food analysis

CE microchips are the first generation of micrototal analysis systems (-TAS) emerging in the miniaturization scene of food analysis. CE microchips for food analysis are fabricated in both glass and polymer materials, such as PDMS and poly(methyl methacrylate) (PMMA), and use simple layouts of simple and double T crosses. Nowadays, the detection route preferred is electrochemical in both, amperometry and conductivity modes, using end-channel and contactless configurations, respectively. Food applications using CE microchips are now emerging since food samples present complex matrices, the selectivity being a very important challenge because the total integration of analytical steps into microchip format is very difficult. As a consequence, the first contributions that have recently appeared in the relevant literature are based primarily on fast separations of analytes of high food significance. These protocols are combined with different strategies to achieve selectivity using a suitable nonextensive sample preparation and/or strategically choosing detection routes. Polyphenolic compounds, amino acids, preservatives, and organic and inorganic ions have been studied using CE microchips. Thus, new and exciting future expectations arise in the domain of food analysis. However, several drawbacks could easily be found and assumed within the miniaturization map.     

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.     

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.     

Focused microwaves-assisted extraction and simultaneous spectrophotometric determination of vanillin and p-hydroxybenzaldehyde from vanilla fragans

A new method to quick extraction of vanillin and p-hydroxybenzaldheyde (PHB) of vanilla beans from vanilla fragans is proposed. Samples were irradiated with microwaves energy to accelerate the extraction process and photometric monitoring was performed at 348 and 329 nm (vanillin and PHB, respectively). The simultaneous determination of vanillin and PHB from extracts was performed using the Vierordt's method, which showed a precision, expressed as relative standard deviation, smaller 2.5% for both analytes. Conditions such as microwaves irradiation power, number of irradiation and non-irradiation cycles, irradiation time and ethanol concentration were optimized by means of multivariate screening that showed that irradiation power and number of irradiation cycles are the most significant condition in the vanilla extraction process. The focused microwave-assisted extraction (FMAE) was applied to commercial (dried vanilla beans from fresh green vanilla beans), lyophilised and dried (commercial vanilla dried at 135 °C in oven) vanilla beans samples. The results showed that the extraction of vanillin and PHB in the commercial vanilla samples were higher than in dried and lyophilised samples. With the proposed FMAE a decrease in the extraction time of 62 times and an increase in the vanillin and PHB concentrations between 40 and 50% with respect to the official Mexican extraction method, were obtained.     

Determination of flavor enhancers in milk powder by one‐step sample preparation and two‐dimensional liquid chromatography

Maltol, ethyl maltol, vanillin, and ethyl vanillin are important food additives as flavor enhancers. To quantify the four additives in milk powder, a novel 2D liquid chromatographic (2DLC) method was developed in this article. In such a 2DLC system, the target fractions eluted from the first dimensional column (C₄) are stored onto the trapping column (C₈) for subsequent analysis; after that, they were switched into the second dimensional column (C₁₈) by a two‐position six‐port switching valve. A one‐step sample preparation method was used prior to 2DLC chromatographic analysis, which was easy and convenient. After optimization of all experimental parameters, the new method was validated in terms of linearity, LODs, and LOQs, intra‐ and interday precision, and accuracy. A conventional single‐dimensional liquid chromatographic method was also proposed in this work for comparison. In order to evaluate the applicability of the new 2DLC method, five brands of commercial milk powder samples (n = 8) were analyzed. Vanillin and ethyl vanillin were detected in two samples, respectively. It is showed that the 2DLC method is effective in quality control programs of milk powder products.     

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.     

Fe3O4 nanoparticles as the adsorbent of magnetic solid-phase extraction for clean and preconcentration of maltol and ethyl maltol in food samples followed by HPLC analysis

In this work, a magnetic solid-phase extraction protocol followed with high-performance liquid chromatography analysis was developed for the determination of maltol and ethyl maltol in food samples. The Fe₃O₄ nanoparticles were prepared by one-step hydrothermal method and was used as adsorbent for clean and preconcentrated maltol and ethyl maltol in food samples. The as-prepared Fe₃O₄ nanoparticle was characterized by transmission electron microscope, Fourier transform infrared spectroscopy, X-ray diffraction, and vibrating sample magnetometer. The extraction conditions including the amount of material, extraction time, pH, temperature, and desorption solvents were investigated, respectively. Under the optimized conditions, the detection limits of 0.04 and 0.05?µg?mL^?1 could be achieved for maltol and ethyl maltol. The recoveries between 84.2 and 103.4% with RSDs lower than 2.30% were obtained for the analysis of spiked plum candy sample. The developed method was successfully applied to the analysis of real samples, such as juice, chocolate, and plum candy.     

Simultaneous quantification of five proteins and seven additives in dairy products with a heart‐cutting two‐dimensional liquid chromatography method

A heart‐cutting two‐dimensional high‐performance liquid chromatography method was developed to simultaneously quantify five major proteins and seven food additives (maltol, ethyl maltol, vanillin, ethyl vanillin, benzoic acid, sorbic acid, and saccharin sodium) in milk and milk powders. In this two‐dimensional system, a Venusil XBP‐C4 column was selected in the first dimension for protein separation, and a Hypersil ODS‐2 C18 column was employed in the second dimension for additive separation; a two‐position, six‐port switching valve was used to transfer the targets (additives) from the first dimension to the second dimension. Method validation consisted of selectivity, response function, linearity, precision, sensitivity, and recovery. In addition, a conventional one‐dimensional high‐performance liquid chromatography method was also tested for comparison. The two‐dimensional method resulted in significantly improved recovery of the food additives compared to the conventional method (90.6–105.4% and 65.5–86.5%, respectively). Furthermore, this novel method has a simple one‐step sample preparation procedure, which shortens the analysis time, resulting in more efficient analysis and less solvent usage.     

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.     

A Graphene‐based Electrochemical Sensor for the Individual,Selective and Simultaneous Determination of Total Chlorogenic Acids,Vanillin and Caffeine in Food and Beverage Samples

An electrochemical sensor based on the electrocatalytic activity of graphene (GR) was prepared, and used for the individual, selective and simultaneous determination of 5‐O‐Caffeoylquinic acid (5‐CQA) that is major compound of chlorogenic acids in coffee, vanillin (VAN) and caffeine (CAF). The electrochemical behaviors of these compounds on GR modified glassy carbon electrode (GR/GCE) were investigated by cyclic voltammetry and square‐wave adsorptive stripping voltammetry. By using stripping conditions after 30 s accumulation under open‐circuit voltage, the electrochemical oxidation peaks appeared at +0.53, 0.83 and 1.39 V in phosphate buffer pH 2.5, and good linear current responses were obtained with detection limits of 4.4×10⁻⁹, 5.0×10⁻⁷, and 3.0×10⁻⁷ M for 5‐CQA, VAN and CAF, respectively. The potential applicability of the proposed method was illustrated in commercial food and beverage samples.     

Rapid Electrochemical Detection of Vanillin in Natural Vanilla

A commercially available and disposable multiwalled carbon nanotube screen‐printed electrode (CNT‐SPE) was employed to detect and determine vanillin compounds in natural vanilla. The voltammetric behaviour of vanillin at the CNT‐SPE is examined and shown to be a sensitive method for quantifying vanillin. Linear calibration for vanillin in the range of 2.5–750 μM was obtained with a detection limit of 1.03 μM and a quantification limit of 3.44 μM. The developed method comprises a simple sample preparation method and a sensitive electrochemical detection for the quantification of vanillin in vanilla pods and is an easy and simple procedure for manufacturers and consumers.     

A Novel Sensitive and Selective Amperometric Detection Platform for the Vanillin Content in Real Samples

Accurate and sensitive determination of vanillin in commercial samples is significant for food safety & quality. In the proposed study, copper particles were coated on an indium tin oxide electrode (ITO) by cyclic voltammetry (CV) for the determination of vanillin in food samples. CV studies indicated that the electrodeposition of Cu particles provides a good electrocatalytic effect towards the oxidation of vanillin. The fabricated sensor determines vanillin linearly between 0.50 μM–2.0 mM (Limit of detection: 0.15 μM). The Cu/ITO was successfully tested on vanillin samples and the recommended method provides accurate and selective determination of vanillin in daily samples.