Biosci. Biotechnol. Biochem., 77 (3), 595–600, 2013 Simultaneous Analysis of Guaiacol and Vanillin in a Vanilla Extract by Using High-Performance Liquid Chromatography with Electrochemical Detection y Makoto TAKAHASHI, Shizuka SAKAMAKI, and Akira FUJITA Technical Research Institute, R&D Center, T. Hasegawa Co., Ltd., 29-7 Kariyado, Nakahara-ku, Kawasaki, Kanagawa 211-0022, Japan Received October 30, 2012; Accepted November 28, 2012; Online Publication, March 7, 2013 [doi:10.1271/bbb.120835] We developed and validated a new high-performance vanillin.3) Hoffman et al. have statistically analyzed the liquid chromatographic analysis for electrochemically sensory evaluation results, high-performance liquid detecting guaiacol and vanillin as important compo- chromatographic (HPLC) data, and gas chromatographic nents in vanilla extract. Separation was achieved with (GC) data of vanilla extracts and determined the Capcell Pak C-18 MG, the potential of the working following factors: age-related compounds, rummy resin- electrode being set at þ1000 mV. The respective cali- ous and vanillin as positive factors, and smoky phenolic bration curves for guaiacol and vanillin were linear in as a negative factor, these factors being respectively the range of 1.60–460 g/L and 5.90–1180 g/L. The represented by hexanal diethyl acetal, 4-ethoxymethyl- respective limits for the quantities of guaiacol and phenol, vanillin, and guaiacol.4) vanillin were 1.60 g/L and 2.36 g/L. The related Guaiacol contributes to the pleasing characteristic standard deviations of the intra- and inter-day precision odor of some roasted foodstuffs, most notably of of the retention time and peak area were all less than Arabica coffee5) and barley malt.6) Yet in other analyses, 4%. The recovery of guaiacol and vanillin was both the production of guaiacol has been found to cause more than 97%, all of the validation data being within phenolic or smoky off-odors in fruit juice,7) chocolate an acceptable range. This analysis method is well suited ice cream,8) chocolate milk,9) vanilla yogurt,10) and for the simultaneous and convenient analysis of guaiacol vanilla ice cream.11) and vanillin in a vanilla extract to evaluate the quality of Grassenmeier et al. have classified the ethanol the vanilla extract. extracts of Madagascar beans into four aroma profile groups and calculated the average vanillin content of Key words: vanilla (Vanilla planiforia); high-perform- each group. The highest vanillin content was found in ance liquid chromatography; electrochemi- the group with the strongest vanillin notes. Yet intrigu- cal detection; guaiacol; vanillin ingly, another group with a strong phenolic and woody profile showed a higher vanillin content than a third Vanilla is one of the world’s most popular flavoring group with a balanced profile.12) The quality of vanilla materials and is widely applied by the food, confection- could not be evaluated only by the concentration of ery, beverage, perfume, and pharmaceutical industries. vanillin. We have shown that the concentration ratio of Commercial vanilla beans are the cured, dried, and vanillin and guaiacol in Madagascar vanilla beans could conditioned pods of the fully mature fruit of Vanilla,an be an index of the quality of vanilla beans by comparing orchid genus native to Mexico. Only 3 of 110 species of the aroma components between Madagascar standard vanilla are commercially traded today: Vanilla planifo- and substandard vanilla beans whose grades differed.13) ria Andrews, primarily cultivated in Madagascar, We therefore decided to develop an analysis method for Indonesia, Uganda, India, Comoro Islands, and Mexico; evaluating the quality of vanilla beans by simultane- Vanilla tahitensis Moore cultivated in Tahiti and Papua ously and conveniently analyzing guaiacol and vanillin New Guinea; and Vanilla pompona Shiede cultivated in in a vanilla extract made from the vanilla beans. Central America.1) The vanillin content in a vanilla extract is generally Over 200 compounds have been found to be respon- analyzed by using HPLC-ultraviolet detection sible for the flavor profile of vanilla, the most abundant (UV).14–17) Four major components in vanilla, namely, of which is vanillin.2) Perez-Silva et al. have identified p-hydroxybenzaldehyde (p-HBAld), p-hydroxybenzoic 26 odor-active compounds responsible for the overall acid (p-HB acid), vanillic acid, and vanillin, can be aroma of the Mexican vanilla beans, including guaiacol, easily measured by HPLC.14) For this reason, the a compound similar to vanillin in intensity, but present Direction Ge´ne´rale de la Concurrence, de la Consom- in vanilla beans at a concentration 1000 times lower than mation et de la Re´pression des Fraudes (DGCCRF) y To whom correspondence should be addressed. Tel: +81-44-411-0134; Fax: +81-44-411-8353; E-mail: makoto [email protected] Abbreviations: d, difference of average; df, degree of freedom; DGCCRF, Direction Ge´ne´rale de la Concurrence, de la Consommation et de la Re´pression des Fraudes; ECD, electrochemical detection; EDTAÁ2Na, EDTA disodium salt; GC, gas chromatography; p-HBAld, p-hydroxyben- zaldehyde; p-HB acid, p-hydroxybenzoic acid; ICH, International Conference on Harmonization; HPLC, high-performance liquid chromatography; LOD, limit of detection; LOQ, limit of quantification; MV, Madagascar cured vanilla beans; RSD, related standard deviation; SD, standard deviation; S/N, signal/noise; UV, ultraviolet detection 596 M. TAKAHASHI et al. guidelines fix the content ratio between these compo- electrode. The potential of the working electrode was set at nents within a certain range for assessing the quality and +1000 mV. A C-18 MG Capcell Pak column (3:0 mm I.D. Â authenticity of a vanilla product. Yet according to 150 mm,3mm; Shiseido, Tokyo, Japan) was used for separation. The mobile phase consisted of 100 mM sodium perchlorate monohydrate in several analyses, the ratios of vanillin/p-HBAld and water, 0.2 mM EDTAÁ2Na in water, and acetonitrile (415:415:170 v/v) vanillic acid/p-HBAld for Indian vanilla beans are for isocratic elution at a flow rate of 0.3 mL/min and 40 C. The 1,12,18) higher than this DGCCRF guideline. injection volume for each sample was 10 mL. Data was acquired by The guaiacol content of a vanilla extract is officially using Labsolution software (Shimadzu, Kyoto, Japan). analyzed by using GC.19) A GC analysis is complicated and unsuitable for rapidly analyzing abundant samples Optimization of the potential of the working electrode. The working because of the extra steps required for solvent-extracting potential for amperometric detection was optimized by constructing the aroma components in the vanilla extract and then hydrodynamic voltammograms of 0.460 mg/mL of guaiacol and 0.590 mg/mL of vanillin in ethanol and water (50:50 w/w) by repeated evaporating the solvent from the extract. We assumed injection, with the detector set at a progressively higher electrode that the guaiacol concentration in vanilla beans would be potential over the range of þ700 mV to þ1300 mV. too low3) to measure by HPLC-UV. Although electro- chemical detection (ECD) is more sensitive than UV Validation method. The suitability, sensitivity, precision, and and can selectively distinguish electrochemically active accuracy of the HPLC method were validated according to Center compounds, no previous reports have been published on for Drug Evaluation and Research (CDER) Reviewer Guidance: Validation of Chromatographic Methods25) and International Confer- an HPLC-ECD analysis of guaiacol and vanillin in the 26) vanilla extract. We therefore decided to perform an ence on Harmonization (ICH) guidelines. System suitability and sensitivity. Standard solutions of guaiacol and HPLC analysis for guaiacol and vanillin in the vanilla vanillin were prepared by respectively dissolving 9.20 mg and 11.8 mg extract by using ECD, a detector widely used for the in 10 mL of acetonitrile. Working standard solutions of guaiacol and HPLC analysis of trace phenolic compounds (e.g., vanillin were prepared in respective concentration ranges of 0.184– catecholamine20) and catechol estrogen21) in urine, 92000 mg/L and 0.590–118000 mg/L by serially diluting the standard phenol, guaiacol and eugenol in dental drugs,22) and stock solutions. The working standard solutions were respectively the phenolic compounds in beer and wort23,24)). This injected into the HPLC system to obtain chromatograms and paper reports the development and validation of a calibration curves for guaiacol and vanillin by plotting the concen- tration of each standard solution versus the peak area. The following simultaneous HPLC-ECD analysis of guaiacol as a values were calculated: the capacity factor for localizing the peak of negative component and of vanillin as a positive interest, the resolution as a measure of separation of the two peaks, the component in a vanilla extract to evaluate the quality theoretical plate as a measure of column efficiency, and the symmetry of the vanilla extract. factor as a measure of peak tailing.25) The limit of detection (LOD) and limit of quantification (LOQ) were determined at respective signal/ Materials and Methods noise (S/N) ratios of 3 and 10. Precision. Working standard solutions of guaiacol and vanillin were prepared at three different concentrations each: 9.20, 46.0 and 460 mg/ Plant materials. Madagascar cured vanilla beans ‘‘red whole’’ mL for guaiacol, and 5.90, 59.0 and 590 mg/mL for vanillin. Intra- and (vanilla planiforia) with a reddish chocolate brown color and a inter-day