Simultaneous Analysis of Guaiacol and Vanillin in a Vanilla Extract by Using High-Performance Liquid Chromatography with Electrochemical Detection

Biosci. Biotechnol. Biochem., 77 (3), 595–600, 2013

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 Geńe´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 Geńe´rale de la Concurrence, de la Consommation et de la Re´pression des Fraudes; ECD, electrochemical detection; EDTA2Na, EDTA disodium salt; GC, gas chromatography; p-HBAld, p-hydroxybenzaldehyde; 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. 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 EDTA2Na 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 electrochemical 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 concentration 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 precision was determined by ten replicate injections of each moisture content of 25% were purchased from a commercial source 1) solution. (Dammann, Oakland, NJ, USA) as standard quality beans. Short split Accuracy and recovery. The accuracy of the method was evaluated Madagascar cured vanilla beans ‘‘cuts’’ (vanilla planiforia) with a by recovering standards from the samples. The working standard substandard aroma, color and a moisture content of 11% were solutions of guaiacol and vanillin were prepared in three different purchased from a commercial source (Trimeta, Antananarivo, concentrations each: 11.2, 22.5 and 44.9 mg/mL for guaiacol, and 850, Madagascar) as substandard quality beans.1) MV red whole as 1700 and 3400 mg/mL for vanillin. Each solution was added to the Madagascar cured vanilla beans ‘‘red whole’’ and MV cuts as ethanol and water (50:50 w/w) extract of MV red whole and injected Madagascar cured vanilla beans ‘‘cuts’’ were used throughout this into the HPLC system for three repeated measurements of guaiacol and study. vanillin. The recovered concentration was calculated as the remainder when subtracting the concentration of the unspiked extract from the Chemicals. The following compounds were purchased from concentration of the spiked extract. The average recovery was commercial sources: acetonitrile (Burdick & Jackson, Muskegon, calculated as the rate of the recovered concentration and the spiked MI, USA), EDTA disodium salt (EDTA2Na), guaiacol and sodium concentration. The percentage related standard deviation (RSD) of the perchlorate monohydrate (Wako Pure Chemicals, Osaka, Japan), average recovery was also calculated. ethanol (Japan Alcohol Trading, Tokyo, Japan), and vanillin (Junsei Chemicals, Tokyo, Japan). Acetonitrile was of HPLC grade. All other Sensory evaluation. reagents and solvents were of analytical grade. Samples. MV red whole and MV cuts respectively used as the standard quality and substandard quality beans,1) for the HPLC-ECD Sample preparation and quantitative analysis of the vanilla analysis were cut into 1-cm pieces, frozen with liquid nitrogen, and extracts. Vanilla beans (20 g) were cut into 1-cm pieces by knife and then ground into a fine powder in an AM-10 laboratory mill extracted with 100 g of ethanol and water (50:50 w/w) at 60 C for 4 h (Nihonseiki Kaisha, Tokyo, Japan). A 10-g amount of each bean to effectively extract the vanillin and guaiacol. The extract samples powder was directly dispensed into a closed 30-mL sensory vial. were filtered to remove the beans, diluted 2000 times with ethanol and Evaluation attributes. The phenolic attribute was used for describ- water (50:50 w/w) to minimize the influence of soluble solids in the ing and evaluating the negative phenolic aroma of the vanilla beans. vanilla extract, and subjected to a quantitative analysis by the HPLC Evaluation conditions. The evaluation panel consisted of 9 male method described next. and 4 female employees of the Technical Research Institute of T. Hasegawa Co., Ltd. (Kawasaki, Japan). Each panelist had been trained HPLC. The HPLC analysis was performed with a Prominence LC to recognize and quantify the aromas of about 100 aroma chemicals high-performance liquid chromatographic system (Shimadzu, Kyoto, and raw materials. Each was presented with two test samples, i) MV Japan) equipped with an SIL-20AC auto sampler, LC-20AD gradient red whole and ii) MV cuts (each sample was coded by a random three- pump, CTO-20A column oven, and an ED723 electrochemical detector digit number), and instructed to sniff each sample and rate the intensity (GL Science, Tokyo, Japan) operated in the DC amperometric mode of the phenolic attribute on a seven-point linear scale from 1 (none) to with a diamond working electrode and an Ag/AgCl reference 7 (very strong). The evaluation was conducted in a quiet room kept at Simultaneous Analysis of Guaiacol and Vanillin in Vanilla 597

250

200

150

100

Guaiacol

Current response (nA) 50 Vanillin

0 700 800 900 1000 1100 1200 1300

Detector cell potential (mV) vs. Ag/AgCl

Fig. 1. Hydrodynamic Voltammograms for Guaiacol and Vanillin.

23 C. Each panelist was asked to perform the assessment alone in a 2 booth to eliminate any influence from the other panelists. guaiacol y ¼ 1160x 553 ðr ¼ 0:9999Þ Statistical analysis. The differences between the average scores of ¼ þ ð 2 ¼ Þ the evaluated samples were compared by a t-test, for which the vanillin y 629x 2320 r 0:9998 difference in average (d), standard deviation (SD), degree of freedom where y is the peak area of the analyte, x is the (df) and t-value were calculated for the phenolic attribute (p < 0:05, concentration of the analyte (mg/L), and r2 is a and 0.01). correlation coefficient. The result shows an outstanding correlation between the peak area and concentration for Results and Discussions both guaiacol and vanillin. The respective LOD values for guaiacol and vanillin were 0.480 mg/L and 0.870 Optimization of the HPLC-ECD method mg/L, and the respective LOQ values for guaiacol and When quantifying an analyte by electrochemical vanillin were 1.60 mg/L and 2.36 mg/L. LOD and LOQ detection, the signal has to be independent of the for vanillin by HPLC-ECD were approximately 30– potential applied to the working electrode. Hydrody- 1000 times lower than the values obtained by HPLC-UV namic voltammograms of guaiacol and vanillin are in previous published studies,15–17) and those for shown in Fig. 1. The detection potential versus the Ag/ guaiacol were also similar to those for vanillin. HPLC- AgCl reference electrode, namely þ1000 mV, is the ECD detected both vanillin and guaiacol with good potential at which the current response of guaiacol was specificity and high sensitivity, and quantification at highest. Isocratic elution was applied, being better suited relatively low concentrations in the range 1–1000 mg/L than gradient elution to amperometric detection at a high enabled the target compounds to be selectively quanti- operating potential. Complete resolution of guaiacol and fied without any influence from soluble solid in the vanillin was obtained by gradually adjusting the polarity vanilla extract. of the mobile phase with sodium perchlorate monohy- Precision drate and EDTA2Na. The Materials and Methods Table 3 shows the results for precision. The percent- section describes the final composition of the mobile age RSD for the intra- and inter-day precision of the phase. Figure 2 shows chromatograms of the reference retention time and peak area was generally low (less compounds and aqueous ethanol extracts of MV red than 2%), while the percentage RSD at the lowest whole and MV cuts under the optimized HPLC-ECD concentration of vanillin was somewhat higher (approx- conditions. imately 3–4%). Accuracy Method validation Table 4 shows the results for accuracy. In a compar- Specificity and sensitivity ison between the actually spiked concentration and Table 1 shows the results for specifying the system spiked concentration measured by our HPLC method, suitability. The capacity factor, resolution, theoretical the average respective recovery of guaiacol and vanillin plate, and symmetry factor of both guaiacol and vanillin was in the range of 97.1–100.9% and 98.5–100.0%. were within the values recommended by the CDER All the validation data were thus within an acceptable Reviewer Guidance: Validation of Chromatographic range. Methods.25) Table 2 shows the results for linearity, LOD, and Sensory evaluation of vanilla beans and quantitative LOQ. The linearity of the system was checked by analysis of the vanilla extracts plotting calibration curves for the five working standard The negative characteristic aromas of both MV red solutions. The respective calibration curves for guaiacol whole and MV cuts were evaluated according to the and vanillin were linear in the range of 1.60–460 mg/L phenolic attributes. The average scores are shown in and 5.90–1180 mg/L. Good linear response with respec- Table 5. MV cuts was significantly more phenolic than tive correlation coefficients of 0.9999 and 0.9998 were MV red whole. Aqueous ethanol extracts of both beans obtained. The calibration curves could be represented by were prepared and subjected to a quantitative analysis the following regression equations: by our HPLC method. The results are shown in Table 6. 598 M. TAKAHASHI et al.

20 a 1

mV 10

2 5

0 0 5 10 15 2025 30 Rt

20 b 1

mV 10

2 5

0 0 5 10 15 2025 30 Rt

20 c

15 1

mV 10

2 5

0 0 5 10 15 2025 30 Rt

Fig. 2. HPLC-ECD Chromatograms of (a) Standards, Vanillin (1) and Guaiacol (2), (b) the Aqueous Ethanol Extract of MV Red Whole, and (c) the Aqueous Ethanol Extract of MV Cuts.

Table 1. Speciﬁcity Data for the System Suitability for Guaiacol and Vanillin by the HPLC-ECD Analysis

Analyte Capacity factor Resolution Theoretical plate Symmetry factor Guaiacol 5.62 9.98 9510 1.06 Vanillin 3.20 17.8 6290 1.08 Recommened valuea >2 >2 >2000 52

aThe value recommened in CDER Reviewer Guidance: Validation of Chromatographic Methods.24)

Table 2. Calibration Parameters and Sensitivity Data for Guiacol and Vanillin by the HPLC-ECD Analysis

Linearity range LOD LOQ Analyte Slope (a) Intercept (b) r2 (mg/L) (mg/L) (mg/L) Guaiacol 460–1.60 1160 553 0.9999 0.48 1.60 Vanillin 1180–5.90 629 2320 0.9998 0.87 2.36

For each curve, the regression equation is y ¼ ax þ b, where y is the peak area, x is the concentration of the analyte (mg/L) and r2 is the correlation coeﬃcient. Simultaneous Analysis of Guaiacol and Vanillin in Vanilla 599 Table 3. Intra-Day and Inter-Day Precision Data for Guaiacol and Vanillin by the HPLC-ECD Analysis

Intra-day precision Inter-day Concentration (n ¼ 10, mean) RSD% precision Analyte of standard Day 1 Day 2 Day 3 (n ¼ 30, mean) RSD% solution (mg/L) a b a b a b a b tR Pa tR Pa tR Pa tR Pa Guaiacol 9.20 0.07 1.14 0.05 1.70 0.07 1.36 0.14 2.13 46.0 0.21 0.48 0.05 0.35 0.04 1.40 0.78 1.95 460 0.21 0.16 0.12 0.09 0.04 0.35 0.76 0.41 Vanillin 5.90 0.11 2.92 0.14 3.86 0.12 1.15 0.18 2.86 59.0 0.15 0.46 0.06 0.35 0.08 0.40 0.18 1.35 590 0.02 0.23 0.02 0.18 0.16 0.46 0.16 0.67

aRSD% of retention time bRSD% of peak area

Table 4. Accuracy Data for the HPLC-ECD Analysis of Guaiacol and Vanillin in the Aqueous Ethanol Extract of Vanilla Beans

Spiked Recovered Average RSD% of average Concentration Analytes concentration concentrationa recoveryb recovery (mg/mL) (mg/mL) (mg/mL) (%) n ¼ 3 Guaiacol 0 17.8 — — — 11.2 29.1 11.3 100.9 1.15 22.5 40.1 22.3 99.1 0.58 44.9 61.4 43.6 97.1 0.74 Vanillin 0 2480 — — — 850 3330 850 100.0 1.37 1700 4170 1690 99.4 0.12 3400 5830 3350 98.5 0.96

a(Concentration of spiked extract) (Concentration of unspiked extract) b100 (Recovered concentration)/(spiked concentration)

Table 5. Sensory Evaluation of Vanilla Beans Table 6. Quantitative Analysis of the Vanilla Extracts

Average score for phenolic Concentration (mg/mL) Vanilla beans attributea Analyte MV red wholea MV cutsb MV red wholeb 4.0 Guaiacol 17:8 0:0837c 88:7 0:612c MV cutsc 5.5 Vanillin 2480 14:8c 1540 25:8c an ¼ 13. The scores were signiﬁcantly diﬀerent by t-test (p < 0:01). aMadagascar vanilla beans ‘‘red whole’’ with standard quality, reddish bMadagascar vanilla beans ‘‘red whole’’ with standard quality, reddish chocolate brown color and 25% moisture1) chocolate brown color and 25% moisture1) bMadagascar vanilla beans ‘‘cuts’’ with substandard aroma, color and 11% cMadagascar vanilla beans ‘‘cuts’’ with substandard aroma, color and 11% moisture1) moisture1) cstandard deviation

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