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.     

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.     

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.     

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 of a LC-ESI/MS/MS assay for the quantification of vanillin using a simple off-line dansyl chloride derivatization reaction to enhance signal intensity

Vanillin is responsible for producing the familiar smell of vanilla. Vanillin has many similarities with other flavor phenolic compounds and could potentially show similar pharmacological activity. A previously published analytical method was adapted, developed and tested. Vanillin was extracted from rat plasma using protein precipitation with acetone. Prior to LC-ESI/MS/MS analysis, an aliquot of the supernatant was used to proceed to the derivatization of vanillin and the internal standard with dansyl chloride to enhance signal intensity in positive electrospray mode. The chromatography was performed on a 100 x 2.1 mm C8 column and an isocratic mobile phase composed of 75:25 acetonitrile:0.5% formic acid in water with a flow rate fixed at 500 microL/min. A linear (weighted 1/concentration) relationship was used to perform the calibration over an analytical range of 10-10,000 ng/mL. The intra-batch precision and accuracy at the limit of quantitation (10 ng/mL), medium (500 ng/mL) and high (10,000 ng/mL) concentrations were 10.7, 7.0 and 7.2% and 103.5, 108.0 and 100.1%, respectively. The observed recovery was greater than 87% and no significant ionization suppression or matrix effect was observed. This LC-ESI/MS/MS method for the determination of vanillin in rat plasma provided results within generally accepted criteria used for bioanalytical assay.     

固相萃取-气相色谱/质谱联用法测定卷烟主流烟气中的香兰素和乙基香兰素

采用固相萃取-气相色谱/质谱联用技术,建立了一种同时测定卷烟主流烟气中香兰素和乙基香兰素的方法。方法优化了萃取条件,考察了萃取溶剂、萃取时间对检测结果的影响。通过硅藻土固相萃取柱净化并浓缩后,选取DB-HeavyWAX毛细管柱进行目标物分离,以香兰素-d3作为内标,对香兰素和乙基香兰素进行定量分析。结果表明,香兰素和乙基香兰素在浓度0.2~20.0 mg/L范围内具有良好的线性关系,其检出限分别为9.1 ng/支和6.5 ng/支,回收率均在96.3%~107.7%之间。     

Determination of site-specific (deuterium/hydrogen) ratios in vanillin by 2H-nuclear magnetic resonance spectrometry: collaborative study

The results of collaborative study are reported for a method that determines the site-specific isotope ratios of deuterium/hydrogen (D/H)i in vanillin by deuterium-nuclear magnetic resonance (2H-NMR) spectrometry. This method allows characterization of all the main commercial sources of commercial vanillin and detection of undeclared mixtures. It is based on the fact that the amounts of deuterium at various positions in the vanillin molecule are significantly different from one source to another. Vanillin is dissolved in acetonitrile and analyzed with a high-field NMR spectrometer fitted with a deuterium probe and a fluorine lock. The proportions of isotopomers monodeuterated at each hydrogen position of the molecule are recorded, and the corresponding (D/H) ratios are determined by using a calibrated reference. Nine laboratories analyzed 5 materials supplied as blind duplicates (1 natural vanillin from vanilla beans, 2 synthetic vanillins from guaiacol, 1 semisynthetic vanillin from lignin, and a mixture of natural and synthetic vanillins). The precision of the method for measuring site-specific ratios was as follows: for (D/H)1 the within-laboratory standard deviation (Sr) values ranged from 2.2 to 5.8 ppm, and the among-laboratories standard deviation (sR) values ranged from 3.6 to 5.1 ppm; for (D/H)3 the Sr values ranged from 1.7 to 3.2 ppm, and the SR values ranged from 2.4 to 3.7 ppm; for (D/H)4 the Sr values ranged from 2.3 to 6.2 ppm, and the SR values ranged from 2.4 to 6.4 ppm; for (D/H)5 the Sr values ranged from 0.8 to 2.7 ppm, and the SR values ranged from 0.9 to 2.3 ppm. It was shown that these values allow a satisfactory discrimination between vanillin sources. Therefore, the Study Director recommends the method for adoption as a First Action Official Method by AOAC INTERNATIONAL.     

Optimization of enzymatic process for vanillin extraction using response surface methodology

Vanillin was extracted from vanilla beans using pretreatment with cellulase to produce enzymatic hydrolysis, and response surface methodology (RSM) was applied to optimize the processing parameters of this extraction. The effects of heating time, enzyme quantity and temperature on enzymatic extraction of vanillin were evaluated. Extraction yield (mg/g) was used as the response value. The results revealed that the increase in heating time and the increase in enzyme quantity (within certain ranges) were associated with an enhancement of extraction yield, and that the optimal conditions for vanillin extraction were: Heating time 6 h, temperature 60 °C and enzyme quantity 33.5 mL. Calculated from the final polynomial functions, the optimal response of vanillin extraction yield was 7.62 mg/g. The predicted results for optimal reaction conditions were in good agreement with experimental values.     

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.     

Thermal,morphological, and mechanical properties of ethyl vanillin immobilized in polyvinyl alcohol by electrospinning process

In this study, polyvinyl alcohol (PVA) nanofibers with ethyl vanillin as an active compound were prepared using electrospinning technique. The final products of electrospinning process were in the form of nanofibers films. PVA/ethyl vanillin nanofibers, having fibers diameters in the range 100–1700 nm, were successfully electrospun from ethanol/water mixture of PVA and ethyl vanillin. The effects of immobilization process on ethyl vanillin thermal properties were investigated by differential scanning calorimetry (DSC). The results of DSC showed significant influence of immobilization process on thermal properties of ethyl vanillin. It was noticed that melting point of immobilized ethyl vanillin was lower (~55 °C) compared to free flavor (~77 °C). Our results showed that films based on PVA/ethyl vanillin nanofibers are mechanically stable.     

Microwave- and ultrasound-assisted extraction of vanillin and its quantification by high-performance liquid chromatography in Vanilla planifolia

Microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE) and conventional extraction of vanillin and its quantification by HPLC in pods of Vanilla planifolia is described. A range of nonpolar to polar solvents were used for the extraction of vanillin employing MAE, UAE and conventional methods. Various extraction parameters such as nature of the solvent, solvent volume, time of irradiation, microwave and ultrasound energy inputs were optimized. HPLC was performed on RP ODS column (4.6 mm ID x 250 mm, 5 microm, Waters), a photodiode array detector (Waters 2996) using gradient solvent system of ACN and ortho-phosphoric acid in water (0.001:99.999 v/v) at 25 degrees C. Regression equation revealed a linear relationship (r2 > 0.9998) between the mass of vanillin injected and the peak areas. The detection limit (S/N = 3) and limit of quantification (S/N = 10) were 0.65 and 1.2 microg/g, respectively. Recovery was achieved in the range 98.5-99.6% for vanillin. Maximum yield of vanilla extract (29.81, 29.068 and 14.31% by conventional extraction, MAE and UAE, respectively) was found in a mixture of ethanol/water (40:60 v/v). Dehydrated ethanolic extract showed the highest amount of vanillin (1.8, 1.25 and 0.99% by MAE, conventional extraction and UAE, respectively).     

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。     

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 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.     

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.     

Chemiluminometric determination of vanillin in commercial vanillin products

Vanillin, ethylvanillin and 4-hydroxy-3-methoxy-benzylalcohol have been found to chemiluminesce by the action of potassium permanganate in sulphuric or polyphosphoric acid media. Both acid media have been compared and sulphuric acid allows the sensitive determination of 0.15-10.0, 0.010-1.0 and 0.0030-0.30 μg mL⁻¹ of vanillin, ethylvanillin and 4-hydroxy-3-methoxy-benzylalcohol with limits of detection equal to 0.045, 0.0030 and 0.00090 μg mL⁻¹, respectively. Recoveries of vanillin from commercial vanillin products are within the range of 95-109%. Comparison with results from the official method shows differences within the range of 0.5-3.0%. The chemiluminogenic reaction mechanism is also discussed.     

Determination of phenolic disinfectant agents in commercial formulations by liquid chromatography.

Using liquid chromatography (LC), a convenient and versatile method was developed for the assay of disinfectant products containing the commonly used phenolic agents, alone or in combination, including phenol, 2-phenylphenol, 4-t-amylphenol, 4-chloro-3,5-dimethylphenol (PCMX), 2-benzyl-4-chlorophenol, and triclosan. The procedure involves a simple dilution or dissolution of the product aliquot or portion followed by membrane filtration and LC. The method was applied to 19 different products representing 17 manufacturers and containing from one to 4 phenolic agents. Chromatography was performed using a Zorbax SB-C18 column, acetonitrile-0.05M KH2PO4 (55 + 45, v/v) pH 3.00, as mobile phase and UV detection at 280 nm. The intralaboratory precision of the product assays ranged form 0.34-5.35% (n = 5) and recoveries via fortification varied from 96.1-103.8%. The limits of quantitation (LOQ) and detection (LOD) ranged from 5.58 x 10(-5) to 2.50 x 10(-4) mg/mL and from 1.76 x 10(-5) to 0.67 x 10(-4) mg/mL, respectively, for the 6 analytes. The response for all analytes was observed to be linear for at least a 50-fold range in concentration (0.001-0.05 mg/mL). The proposed method provides an efficient means for the isolation and quantitation of these phenolic compounds.     

Determination of 2-methoxyestradiol in human plasma, using liquid chromatography/tandem mass spectrometry

A liquid chromatography/tandem mass spectrometric (LC/MS/MS) assay was developed for the quantitative determination of 2-methoxyestradiol (2ME2) in human plasma. Sample pretreatment involved liquid-liquid extraction with ethyl acetate of 0.3-mL aliquots of plasma spiked with the internal standard, deuterated 2ME2 (2ME2-d5). Separation was achieved on a Zorbax Eclipse C18 column (2.1 x 50 mm, i.d., 5 microm) at room temperature using a gradient elution with methanol and water at a flow rate of 0.25 mL/min. Detection was performed using atmospheric pressure chemical ionization MS/MS by monitoring the ion transitions from m/z 303.1 --> 136.8 (2ME2) and m/z 308.1 --> 138.8 (2ME2-d5). Calibration curves were linear in the concentration range of 1-100 ng/mL. The accuracy and precision values, obtained from three different sets of quality control samples analyzed in quintuplicate on four separate occasions, ranged from 105-108% and from 3.62-5.68%, respectively. This assay was subsequently used for the determination of 2ME2 concentration in plasma of a patient with cancer after a single oral administration of 2ME2 at a dose of 2200 mg.     

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.