Extraction, Isolation and Quantiﬁcation of Vanillin and Others Constituents in Vanilla Flavor

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A Comprehensive Review on Vanilla Flavor: Extraction, Isolation and Quantiﬁcation of Vanillin and Others Constituents

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A comprehensive review on vanilla flavor: Extraction, isolation and quantification of vanillin and others constituents

Arun K. Sinhaâ; Upendra K. Sharmaâ; Nandini Sharmaâ

aNatural Plant Products Division, Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India

First Published on: 18 September 2007

To cite this Article: Sinha, Arun K., Sharma, Upendra K. and Sharma, Nandini (2007) 'A comprehensive review on vanilla flavor: Extraction, isolation and quantification of vanillin and others constituents', International Journal of Food Sciences and Nutrition, 59:4, 299 - 326

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A comprehensive review on vanilla flavor: Extraction, isolation and quantification of vanillin and others constituents

ARUN K. SINHA, UPENDRA K. SHARMA, & NANDINI SHARMA Natural Plant Products Division, Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India

Abstract

Vanilla, being the world’s most popular flavoring materials, finds extensive applications in food, beverages, perfumery and pharmaceutical industry. With the high demand and limited supply of vanilla pods and the continuing increase in their cost, numerous efforts of blending and adulteration in natural vanilla extracts have been reported. Thus, to ensure the quality of vanilla extracts and vanilla-containing products, it is important to develop techniques to verify their authenticity. Quantitatively, vanillin is the major compound present in the vanilla pods and the determination of vanillin is a vital consideration in natural vanilla extracts. This paper provides a comprehensive account of different extraction processes and chromatographic techniques applied for the separation, identification and determination of chemical constituents of vanilla.

The review also provides an account of different methods applied for the quantification and the authentification of chemical constituents of vanilla extract. As the various properties of vanilla are attributed to its main constituent vanillin, its physico-chemical and bioactive properties have also been outlined.

Keywords: Comprehensive review, gas chromatography, high-performance liquid chromatography, vanilla flavor, Vanilla planifolia, vanillin

Introduction

In recent years, there has been growing interest in natural and healthy foods, especially with regards to the ingredients such as flavoring agents and preservatives. Amongst the variety of natural flavors in use today, vanilla occupies a prominent market place and has been in use for the preparation of ice creams, chocolates, cakes, soft drinks, pharmaceuticals, liquors, perfumery and in nutraceuticals (Ranadive 1994). Natural vanilla is a complex mixture of flavor components extracted from the cured pods of different species of plant genus Vanilla: Vanillus planifoliaand Vanillus tahitensis(Rao and Ravishankar 2000). However, V. planifolia is valued most because of its pod quality and yield. The fruity, floral fragrance of cured vanilla pods, combined with a deep, aromatic body, makes it a widely accepted flavoring agent.

The history of vanilla begins with its discovery in Mesoamerica during the 1300s.

The Aztecs (natives of Mexico) are more often cited as the first culture to use and

Correspondence: Arun K. Sinha, Natural Plant Products Division, Institute of Himalayan Bioresource Technology, IHBT Communication no.006, Post Box No. 6, Palampur 176061, Himachal Pradesh, India.

Tel: 91 1894 230426. Fax: 91 1894 230433. E-mail: [email protected] ISSN 0963-7486 print/ISSN 1465-3478 online#2008 Informa UK Ltd DOI: 10.1080/09687630701539350

June 2008; 59(4): 299326

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domesticate vanilla to flavor their drinks. Thereafter, the Spanish conquest of Aztec culture brought it to Europe. Efforts were made to cultivate vanilla outside Mexico but, owing to the absence of a natural pollinator, the crop was a failure*until the botanist Charles Morren (1836) discovered the secret of vanilla’s reluctance to bear fruit outside Mexico, which has led to the discovery of artificial pollination of vanilla flowers (Reineccius 1997). From the time of the Aztecs, vanilla has been considered an aphrodisiac, carminative and a stimulant. Venezuelans commonly use the pods for treatment of fever and spasm. It is used as an antispasmodic and aphrodisiac in Argentina. In Palau, vanilla is used for curing dysmenorrhea, fever and hysteria (Duke et al. 2003). There have been reports whereby vanilla is known to protect the skin against free radicals (Sophie and Francois 2003). Thus, owing to its medicinal properties, besides being a highly valued flavoring agent, vanilla has tremendous potential to be used as a food preservative and health food agent.

The active constituents of vanilla are responsible for its various biological and therapeutic activities. The flavor profile of vanilla contains more than 200 components, of which only 26 occur in concentrations greater than 1 mg/kg. The aroma and flavor of vanilla extract is attributed mainly due to presence of vanillin (4-hydroxy-3- methoxybenzaldehyde; Figure 1), which occurs in a concentration of 1.02.0% w/w in cured vanilla pods (Westcott et al. 1994; Bettazzi et al. 2006; Sharma et al. 2006).

True vanilla pods possess a pure delicate spicy flavor that cannot be duplicated exactly by synthetic products. For this reason, and because of limited supply, natural vanilla is able to command a premium price, leading to numerous efforts of its blending and adulteration. Also, the flavor quality of vanilla extracts vary considerably, depending upon the origin, curing technique used, storage conditions, extraction methods, and age of the vanilla extract itself. Thus, the availability of effective analytical techniques for monitoring the quality and, as far as possible, maintaining uniformity of quality over time is imperative (Poole et al. 1995). As a consequence, reliable and practical analytical methods for the identification and determination of chemical constituents in vanilla pods are of considerable interest.

Keeping the above in view, the present review is focused on various methodologies available for the extraction, separation and quantification of chemical constituents of vanilla flavor. In addition, various physico-chemical and bioactive properties of its main constituent (i.e. vanillin) and methods applied for its authentification for quality assurance have also been reviewed.

Figure 1. Structure of vanillin.

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Plant sources of vanilla

Vanilla is obtained from different species of plant genusVanilla(family: Orchidaceae), a tropical climbing orchid. Out of the total 110 species, only three species find commercial importance. These are V. planifolia Andrews or Vanillus fragrans (Salis- bury) Ames, V. tahitensis JW Moore and Vanillus pompona Scheide. However, commercial vanilla is obtained from V. planifolia. V.pompona, also called vanillon, is of lowest grade and is often used as an adulterant or by perfumers or tobacco manufacturers (Arditti 1992). Although pods obtained from V. pompona are not valued due to their low quality, the plant has some important traits such as growth under adverse conditions and resistance to root-rot disease. These traits make it an ideal candidate for use in cross-breeding programs and thus improving the commercial source of vanilla (Havkin-Frenkel and Dorn 1996). Green vanilla pods possess no flavor. The characteristic flavor and aroma of vanilla pods develops during the curing process in which enzymatic changes occur. The action of naturally induced b-glycosidases on the glycosides releases various vanilla flavor components. Curing process consists of four steps: scalding/killing, sunning/sweating, drying and con- ditioning/aging (Karas et al. 1972; Havkin-Frenkel and Dorn 1996; Dignum et al.

2002).

Chemistry of vanilla

Since the last century, identification of the chemical components of vanilla has attracted considerable attention and more than 200 compounds have been identified (Klimes and Lamparsky 1976; Galetto and Hoffman 1978; Adedeji et al. 1993;

Ramaroson-Raonizafinimanana et al. 1997; Wekhhoff and Guntert 1997; Pe´rez-Silva et al. 2006). The characteristic aroma of the vanilla flavor is due to the presence of a large number of compounds in the vanilla extract. Various non-volatile constituents that impart the characteristic flavor to vanilla include tannins, polyphenols, free amino acids and resins (Rao and Ravishankar 2000). An extract containing large amounts of resins will retain aromatic compounds far longer than one that has smaller quantity of them (Reineccius 1997).

Volatile constituents that are responsible for the aroma and flavor of vanilla are acids, ethers, alcohols, acetals, heterocyclics, phenolics, hydrocarbons, esters and carbonyls (Klimes and Lamparsky 1976). A comprehensive account of various chemical constituents present in green and cured vanilla pods has been reviewed earlier (Dignum et al. 2001). Among the various volatile compounds reported, vanillin is the single most characteristic component of flavor. The chemical identity and physico-chemical properties of vanillin are summarized and presented in Table I.

Bioactive properties

Because of advancements in chemistry and pharmacology, most of the earlier uses of vanilla have given way to functional uses of vanillin, vanilla’s main constituent. In recent years, researchers have been exploring vanillin’s properties as an antioxidant, antimicrobial, anticarcinogenic and antisickling agent. Also, in the food industry there is a growing interest in naturally occurring flavor compounds that exhibit antioxidant and antimicrobial activity and therefore provide a potential source of novel

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preservatives. In this regard, some of the reported bioactive properties of vanillin are presented below.

Antimicrobial activity

Vanillin and its direct structural analogues exhibit varying degrees of antifungal activity against the different moulds and yeasts that cause food spoilage (Fitzgerald et al. 2005). Vanillin has been reported to inhibit the growth of moulds and yeasts in fruit purees, fruit-based agar systems (Lo´pez-Malo and Alzamora 1995; Cerrutti and Alzamora 1996; Cerrutti et al. 1997; Lo´pez-Malo et al. 1998) and in soft drinks (Fitzgerald et al. 2004a). The antifungal activity of vanillin against the medicinally important yeasts Candida albicans and Cryptococcus neoformans has also been documented (Boonchird and Flegel 1982). The aldehyde moiety of vanillin has been shown to play a key role in the antifungal activity. However, the side-group position on the benzene ring seems to be important structural feature that can contribute to these effects (Fitzgerald et al. 2005).

The role of vanillin as an antibacterial agent against Escherichia coli, Lactobacillus plantarum and Listeria innocua appears to be promising (Fitzgerald et al. 2004b).

Schiff bases derived from vanillin were evaluated for their potential as antibacterial agents against some Gram-positive and Gram-negative bacterial strains such as Pseudomonas pseudoalcaligenes, Proteus vulgaris, Citrobacter freundii, Enterobacter aero- genes,Staphylococcus subfavaandBacillus megaterium(Vaghasiya et al. 2004). However, further research needs to be conducted to determine the minimum concentration at which vanillin act as an antimicrobial agent or a natural preservative.

Antioxidant activity

Vanillin has been recognized for its antioxidant potential (Burri et al. 1989a; Teissedre and Waterhouse 2000) as well as its free radical scavenging activity (Sawa et al. 1999;

Mahal et al. 2001; Kumar et al. 2002, 2004). The presence of vanillin in micro quantity enhanced the protection of food against oxidation (Burri et al. 1989b, 1991).

The hydroalcoholic extract of vanilla also exhibited antioxidant properties (Toshio

Table I. Chemical identity and physico-chemical properties of vanillin.

Property Data

Molecular formula C8H8O3

CAS number 121-33-5

Chemical structure (CH3O)C6H3(OH) CHO

Physical state White or slightly yellow needles

Molecular weight 152.15

Melting point 80818C

Boiling point 2858C

Water solubility 1 g/100 ml

Density 1.056 g/ml

Vapor density (air1) 5.2

Vapor pressure 2.210³mmHg at 258C

Flash point 1478C

Dissociation constant pKa17.40, pKa₂11.4 (258C)

Adapted from http://cira.ornl.gov/documents/vanillin.pdf

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et al. 2000), thus acting as a food preservative (Aruoma 1999). Vanillin has also been reported to inhibit the auto-oxidation of milk fat (Mykolaiivna and Petrivna 2005).

Using rat liver mitochondria as model systems, Kamat et al. (2000) have examined the ability of vanillin to protect the cell membrane against oxidative damage induced by photosensitization.

Anticarcinogenic and antimutagenic activity

Earlier studies suggested that vanillin has antimutagenic and anticarcinogenic activity (Imanishi et al. 1990; Ferguson 1994). It has been shown to decrease the number of colon tumors induced by multiple agents in rat models (Akagi et al. 1995). Vanillin has been reported to suppress chromosomal aberrations caused by ultraviolet (UV) and X-rays (Sasaki et al. 1990; Keshava et al. 1998), to inhibit mutation at theCD59 locus on human chromosome (Gustafson et al. 2000) and also to act as a DNA-PK inhibitor, thus proving its utility in DNA strand repair (Durant and Karran 2003).

Hypolipidemic activity

Vanillin, as a food additive, is also used for preventing the development of pathological conditions like hyperlipidemia. The pharmacological action of vanillin as a hypolipidemichypotriglyceridemic agent over a wide range of concentration in the treatment of diabetes (type 2), cardiovascular disturbances and obesity has been recognized (Mokshagundam and Mokshagundam 2003).

Antisickling activity

Vanillin, in vitro, demonstrated antisickling effect through covalent bonding of the aldehyde group with the hemoglobin in the red blood cells (Abraham et al. 1991).

However, orally administered vanillin has no therapeutic effect because of its rapid decomposition in the upper digestive tract. To overcome this problem, a vanillin prodrug, MX-1520, has been synthesized, which is biotransformed to vanillinin vivo.

The bioavailability of this drug was found to be 30 times higher than that of vanillin.

At the same time, this prodrug was also found to be five times more effective in reducing sickling than natural vanilla (Zhang et al. 2004).

Other activities

Sun et al. (2001) found the alcoholic extracts of leaves ofV. fragranstoxic to mosquito larvae. The volatile oils derived from turmeric, citronella grass and hairy basil, especially with 5% vanillin, were very effective against the mosquito species (Tawatsin et al. 2001). A significant enhancement of repellence towards black fly species (Silulium venstum Say and Prosimulium hirtipes Fries) was observed when vanillin, along with toluamide, was used as a spray (Retnakaran 1984).

Other important activities shown by vanilla are anti-aggregant, anti-hepatotoxic, anti-inflammatory, antiviral, analgesic, anesthetic, antiseptic, and so on (Duke et al.

2003). However, keeping the low bioavailability of vanillin in focus, additionalin vivo studies would be helpful to amplify its medicinal use and to make it an effective part of prevention diet.

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Extraction methods

Extraction or sample preparation is the most important step in the development of methods for isolation of target analytes or for the analysis of botanical and herbal preparations. It is regarded as a foremost step in the qualitative and quantitative analysis of any plant constituents. Ideally, an extraction technique should be exhaustive with respect to constituents to be analyzed, rapid, simple and inexpensive.

Since vanilla is traded in international market as an ethanolic extract, rapid, convenient and safe extraction methods will play an important role in ensuring a high-quality product for consumers worldwide.

Commercial extraction of natural vanilla flavor

In some countries, notably France and Germany, the pods themselves are frequently demanded by consumers. In America, where convenience is on a par with quality, homemakers prefer extract, which is prepared mainly by percolating or macerating chopped vanilla pods with ethyl alcohol and water, instead of distillation that destroys the gentle fragrance of aromatic compounds (Reineccius 1997). According to US FDA regulations, vanilla extract must contain at least the sapid and odorous principles extracted from one unit weight (13.35 oz pods, maximum moisture content 25% by weight per gallon of solvent) of vanilla pods by an aqueous alcohol solution of not less than 35% ethyl alcohol (Bartnick et al. 2005). The concentration of extract is noted by its ‘fold’. A single fold of vanilla extract contains the extractable material from 13.35 oz vanilla pods per gallon of solvent or 100 g extractable material per liter.

Commercially, natural vanilla extract is sold as a dilute ethanolic extract containing about 1.0 g/l vanillin. Commercial vanilla extraction may fall under two categories: the percolation method and the oleoresin method. The percolation method consists of a circulating mixture of ethanol and water containing 3550% alcohol for 4872 h, which results in a four-fold strength vanilla extract. The oleoresin method consists of pulverizing whole pods and then circulating ethanol over the pods under vacuum at about 458C. The excess ethanol is removed by evaporation. This process takes about 89 days. Using the oleoresin process, approximately 10-fold strength vanilla extract can be prepared (Rao and Ravishankar 2000). The color of vanilla extract is influenced by the quality of vanilla pods, the strength of alcoholic menstrum, the duration of extraction, and the presence of glycerin that is added to retard the evaporation and to retain the flavor in the extract (Reineccius 1997).

Other conventional methods

Other conventional methods employed for the extraction and analysis of natural vanilla include soxhlet, heat treatment, homogenization, maceration (Voisine et al.

1995) and liquidliquid and liquidsolid extraction using a special reciprocating plate extraction contactor (Zhu and Zhou 2002). Voisine et al. (1995) compared the different extraction techniques for the simultaneous extraction of vanillin and glucovanillin from Java and Bourbon vanilla pods with methanol and ethanol as the extraction solvent. The highest yield for glucovanillin was obtained with 24-h soxhlet extraction in 47.5% ethanol, and for vanillin with 24-h extraction by maceration in 47.5% ethanol or 80% methanol. In another study, customary soxhlet apparatus, the Buchi 810 soxhlet extractor and maceration/percolation has been compared for the

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extraction of vanilla pods using isopropanol, ethanol, methanol, and a mixture of ethanol/water. The results were compared on the basis of vanillin content and several ratios of other principal flavor components. The highest extraction yield for vanillin was obtained using a Buchi 810 soxhlet apparatus with methanol as the extraction solvent and an extraction time of 24 h (Ehlers et al. 1999). However, the conventional extraction procedures suffer from a number of drawbacks which include low extraction yields, large extraction time, higher solvent consumption, and so forth.

Supercritical fluid extraction

Due to increasing stringent environmental regulations, supercritical fluid extraction (SCFE) has gained wide importance as an alternative to conventional solvent extraction for the separation of organic compounds in many industrial and analytical processes. In SCFE, the solvation power of fluid can be manipulated by changing pressure and/or temperature resulting in high selectivity. Due to their low viscosity and relatively high diffusivity, supercritical fluids can penetrate through porous solid material more effectively than liquid solvents*the outcome of which is high extraction yields. The extracts are produced under gentle conditions, virtually free of solvent, and contain only the plant ingredients. Because of its low cost, little toxicity, and favorable critical parameters (Tc31.18C, Pc74.8 atm), CO2 is one of the most commonly used extracting agents. A mixture of CO₂ with modifiers (polar organic solvents) is generally used for the extraction of polar substances (Lang and Wai 2001).

SCFE has been applied successfully for the extraction of vanillin using supercritical CO₂as an extraction solvent (Ehlers and Bartholomae 1993; Fang et al. 2002; Fu et al. 2002). Extraction conditions varied from 35 MPa pressure at 458C for 150 min (Fu et al. 2002) to 350 bar at 458C for 140 min yielding 20.05 mg vanillin from 1 g vanilla pods (Fang et al. 2002). Since carbon dioxide is a lipophilic solvent, extraction is much more selective towards the flavor ingredients, leaving behind colors, sugars and other polar components of the alcoholic extraction process that do not contribute to the vanilla flavor. Purity of vanillin was found to be higher with SCFE than with conventional aqueous ethanol extraction (Nguyen et al. 1991). However, the ratio of main constituents of vanilla flavor (namely, vanillin, p-hydroxybenzaldehyde, p- hydroxybenzoic acid, vanillic acid) was found to be different from that present in conventional alcoholic extraction procedures. This could lead to erroneous results in regards to authenticity analysis, since the ratio of these compounds is used to evaluate the authenticity of vanilla extracts (Ehlers and Bartholomae 1993). Hence, serious efforts in this direction ought to be put in before ascertaining SCFE as an effective alternate to conventional methods.

Microwave-assisted extraction and ultrasound-assisted extraction

In the past 10 years, there has been increased interest in using techniques involving microwave-assisted extraction and ultrasound-assisted extraction as these are found to be simpler and more effective alternatives to conventional extraction methods. The main advantages of these techniques are a reduced extraction time and minimized solvent consumption, rendering them rapid, safe and cheap (Hroma´dkova´ et al. 2002;

Pan et al. 2002). A focused microwave has been employed for extraction of vanillin and p-hydroxybenzaldehyde from vanilla pods whereby the extraction time decreased up to 62 times with 4050% higher vanillin and p-hydroxybenzaldehyde concentrations

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when compared with the official Mexican extraction method (Longares-Patro´n and Canizares-Macı´as 2006). Recently, Sharma et al. (2006) have compared microwave- assisted extraction and ultrasound-assisted extraction methodology for the extraction of vanillin from vanilla pods, wherein microwave-assisted extraction proved to be the better of the two. The results showed that using microwave-assisted extraction and ultrasound-assisted extraction the extraction time decreased between 50 and 70 times with respect to the conventional cold percolation method, and between six and eight times with respect to Soxhlet extraction. The maximum yield of vanilla extract (29.81%) was obtained with a mixture of ethanol/water (40:60, v/v), while dehydrated ethanolic extract showed the highest amount of vanillin (1.8%). Thus these approaches provide a simple, rapid and environment friendly tool for the extraction as well as quantitative analysis of chemical constituents of vanilla flavor.

Enzymatic extraction

The use of enzymes with glycosidase activity has been applied successfully for producing natural vanilla extract (Bartnick et al. 2005). The use of enzymes is quite helpful to improve the yield without affecting the flavor quality. Waliszewski et al.

(2007b) studied the effect of enzymatic pretreatment of vanilla pods using different cellulytic enzymes and found that as much as one-half of the amount of vanillin trapped in the cellulose structure of cured vanilla pods in free form or in glucovanillin form can be extracted and liberated by enzymatic pretreatment.

In another report, the amount of vanillin transformed from green vanilla pods using viscozyme and celluclast enzymes was higher than that in conventional methods. Use of these two enzymes for extraction of vanillin may increase the yield 3.13 times more than that obtained with the Soxhlet method. Thus, enzymes are useful not only in the conversion of precursor glucovanillin to vanillin, but also in extraction from the pods, avoiding the fermentationextraction process (Ruiz-Teran et al. 2001).

Solid-phase extraction

Solid phase extraction (SPE) is an effective alternative to liquidliquid extraction. SPE involves absorbing the analyte from the sample onto a modified solid support. The analyte is then desorbed either by thermal means or by using a solvent. The primary advantage of SPE is the reduced consumption of high-purity solvents, thereby reducing laboratory costs and diminishing the need for solvent disposal (Arthur and Pawliszyn 1990). The glucosides in the vanilla extract have been isolated using SPE on a 35-ml Oasis HLB cartridge, whereby these were collected by elution with methanol/

water (1:1, v/v; 60 ml) (Dignum et al. 2004).

More recent development in this field is solid phase microextraction (SPME), wherein the polymeric phase is immobilized onto a silica fiber. SPME eliminates the problems associated with SPE while retaining the advantages; solvents are completely eliminated, blanks are greatly reduced, and the extraction time can be reduced to a few minutes. In this procedure a small diameter fiber coated with a stationary phase is placed in an aqueous sample. The analytes partition into the stationary phase and are then thermally desorbed, on-column, in the injector of a gas chromatograph (Arthur and Pawliszyn 1990). Sostaric et al. (2000) have developed a SPME method for analysis of volatiles in vanilla extract using optimum conditions such as polyacrylate fiber, a 40-min sampling time at room temperature and a 2-min desorption time.

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Biphasic sonoelectroanalysis

Biphasic sonoelectroanalysis involves the use of ultrasonic emulsification and voltammetric measurements in biphasic systems. The use of ultrasound to form emulsions ensures that, regardless of the relative densities of the two liquids, both remain in constant contact with the electrode surface during voltammetric analysis.

Biphasic sonoelectroanalysis removes the need for sample degradation or a separation step, which would lengthen and complicate the analytical protocol. Thus, biphasic sonoelectroanalysis may be used as an alternative extraction technique, which demonstrates the possibilities of simultaneous sono-extraction with an organic phase sonoemulsified with the target medium (Banks and Compton 2003). Biphasic sonoelectroanalysis has been employed for the simultaneous extraction and determination of vanillin in food flavorings using ethyl acetoacetate as an electrochemical and sonoelectrochemical solvent with quantitation efficiency comparable with the high- performance liquid chromatography (HPLC)UV method (Hardcastle et al. 2001).

Qualitative and quantitative determination of chemical constituents

Owing to the limited supply and high price of vanilla pods, the creation of imitation vanilla flavors to replace the natural extracts has now reached a very high level.

Artificial vanilla flavorings usually contain synthetically produced vanillin, ethyl vanillin and coumarin (banned in several countries) in order to increase the vanilla flavor perception. In this perspective, to determine the variation in the quality of vanilla extracts as well as for the separation, identification and quantitative determination of various chemical constituents, chromatography seems to be a powerful analytical tool. Various chromatographic techniques such as thin layer chromatography (TLC), gas chromatography (GC), HPLC, capillary electrophoresis (CE) and micellar electrokinetic chromatography (MECK) offer very useful information, in terms of identification and quantitation, furnishing excellent resolution and selective retention times. Recently, techniques such as GCmass spectrometry (MS) and HPLCMS have come up for the identification of new compounds on the basis of their mass fragmentation pattern while avoiding unnecessary isolation of common compounds of minor interest. In the present review, the analytical methods currently available for the analysis of chemical constituents of vanilla flavor are summarized.

Thin layer chromatography

The separation and identification of various natural compounds including vanillin and its derivatives have been performed using paper chromatography (Anwar 1963) and TLC (Ramaroson-Raonizafinimanana et al. 1997). TLC has some advantages, such as rapidity and ease of handling, besides being economical (Stahl 1969).

Analysis of vanilla flavor metabolites obtained from biotransformation has been achieved successfully on silica gel plates with benzene as the solvent system (Rao and Ravishankar 1999). Gerasimov et al. (2003) applied TLC for the determination of vanillin and its homologue ethyl vanillin in food flavorings using hexane/ethyl acetate (9:1) as the solvent system. A mixture of heptanone, ethanol and sulfuric acid (4:5:1, v/v/v) was used as developing agent. Recently, TLC has given way to its modern counterparts: high-performance TLC (HPTLC) and automated multiple development TLC (AMD-TLC).

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HPTLC is a modern, instrumentalized, quantitative method allowing greater separation efficiency and improved detection limits as compared with conventional TLC. HPTLC using silica gel or cellulose layers combined with selective spray reagents has been used to identify the adulterated vanilla extracts (Association of Official Analytical Chemists 1990; Poole et al. 1995) and detection of principal flavor components*namely, vanillin, vanillic acid, p-hydroxybenzaldehyde and p-hydroxybenzoic acid*in vanilla extract (Lavoine et al. 1998). Kiridena et al. (1994) have applied HPTLC to quantify 5-(hydroxymethyl)-2-furfural, one of the important constituents in vanilla extract, using chloroform/ethyl acetate/1-propanol (94:2:4, v/v) as the mobile phase.

Introduction of AMD-TLC has simplified the separation of complex mixtures by TLC. The AMD-TLC technique employs incremental multiple development in combination with a stepwise solvent gradient that allows optimization of the separation selectivity through the chromatogram for mixtures of a wide polarity range. Belay and Poole (1993) applied the AMD-TLC technique for determination of vanillin and related flavor compounds in natural vanilla extract. They were able to separate nine phenolic compounds in a standard mixture with the detection of five phenolic compounds in vanilla extract. In this method, a mixture of chloroform, ethyl acetate and 1-propanol was used as the developing solvent while acetic acid was added to minimize tailing of polar compounds.

TLC and its modern counterparts hold ground for routine quality control applications over other techniques due to their simplicity, minimum clean-up procedures, less solvent consumption and the possibility of analyzing several samples in less time.

Gas chromatography

GC is one of the most popular chromatographic techniques for separating volatile mixtures. It has certain advantages such as high efficiency and better resolution, and can also be used for quantitative analysis. Gasliquid chromatography and gassolid chromatography have proven of value in both differentiating and quantitating vanillin and ethyl vanillin, as reported in earlier works (Martin et al. 1973). The presence of vanillin in various citrus fruits has been identified and confirmed using high-resolution GC retention index values and aroma quality (Makkar and Beeker 1994).

Identification based on GC retention data alone is not sufficient; therefore, the recent trend is to complement GC with MS in the electron impact mode for better quantitation and analysis of vanilla volatiles. This technique offers the possibility to gain additional information by mass spectra. GCMS has proven of worth for the analysis of vanilla constituents; more than 150 compounds have been identified in different extracts. Identification of some benzyl ethers in commercially prepared pentane extract of vanilla has been reported using GCMS (Galetto and Hoffman 1978). The above technique has been applied for the identification of 54 hydrocarbons from three species of Vanilla (Ramaroson-Raonizafinimanana et al. 1997).

Pe´rez-Silva et al. (2006) identified 65 volatile constituents of vanilla in pentane/ether extract by GCMS analysis, which include 25 acids, 15 phenolic compounds, 10 alcohols, four aldehydes, four heterocyclic compounds, four esters, two hydrocarbons and one ketone, of which 26 compounds were found to be odor active by GC

A. K. Sinha et al.

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olfactometry analysis. The GC conditions used for the above mentioned studies are presented in Table II.

Recently, GCMS has been used in conjugation with SPME to discriminate between different types of vanilla extracts and flavorings (Sostaric et al. 2000).

Bourbon, Tahitian and Indonesian vanilla extracts were analyzed as a part of these studies (Figure 2).

High-performance liquid chromatography

To date, HPLC is the most powerful and preferred technique for the quantification of organic molecules due to its simplicity, sensitivity, precision and selectivity. HPLC methods can be adapted according to the problem at hand using various strategies such as different types of stationary phases, mobile phases and wide range of selective detectors. This makes HPLC suitable for analysis of active components in the natural extracts.

Various HPLC methods have been reported on separation and quantitative determination of vanillin and other related phenolics in natural vanilla extract (Ranadive 1992; Lamprecht et al. 1994; Voisine et al. 1995; Negishi and Ozawa 1996; Dignum et al. 2004). For the separation of various components of vanilla flavor, the chromatographic conditions of the HPLC generally include the use of a reverse- phase C₁₈ column. Sachan et al. (2004) have developed a HPLC method for the analysis of phenolic flavor compounds using various C₁₈columns (Prodigy^TMODS2, Synergi^TM Hydro-RP, Lichrosorb^† and Columbus^TM). Six phenolics have been separated within 21 min on a reverse-phase C₁₈ column (Synergi^TM Hydro-RP).

Recently, Sinha et al. (2007) have developed a HPLC method using a Purospher^†- Star RP-18e column, resulting in separation of 21 phenolics in a standard mixture including adulterants and quantification of 10 phenolics (namely, 4-hydroxybenzyl alcohol, vanillyl alcohol, 3,4-dihydroxybenzaldehyde, 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin,p-coumaric acid, ferulic acid and piperonal) in crude extract of vanilla pods cultivated in India (Figures 3 and 4).

In addition to choice of column, the efficiency of HPLC analysis is also dependent upon the selection of mobile phase. Most of the HPLC methods (see Table III) have employed acidified water mixed with a polar organic solvent such as methanol or acetonitrile as the mobile phase. All of the reported HPLC protocols show vanillin elution time varying from 7 min (Ehlers 1999) to 36 min (Voisine et al. 1995) at detection wavelengths ranging between 254 and 340 nm (Table III), but recently a rapid HPLC technique for vanillin determination has been developed where the elution time of vanillin is as low as 2.2 min at 231 nm (Waliszewski et al. 2007b)*

however, the method does not report the quantification of other phenolic compounds.

The role of HPLC has been highlighted by various authors (Ehlers and Bartholomae 1993; Voisine et al. 1995; Ruiz-Teran et al. 2001; Sharma et al. 2006) for comparison of different vanilla extraction protocols. Besides this, HPLC has also been applied successfully for composition analysis of different species of genusVanilla (Ehlers et al. 1994; Ehlers and Pfister 1997) and to detect possible adulteration in vanilla flavorings (Ehlers 1999; Jagerdeo et al. 2000; Scharrer and Mosandi 2001).

HPLC analysis is not only confined to quantification of vanillin in vanilla extract, but has also been applied extensively for determination of vanillin and other related phenolics produced through biotransformation pathway (Rao and Ravishankar 1999;

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Table II. GC/GCMS analysis of vanilla extract/other matrices.

Analysis/matrix Sample preparation Column Conditions Reference

GC/vanilla extract Extraction of pods with pentane

Glass column packed with 5%

Carbowax 20M on Chromosorb W.A.W. 8 ft0.25 in

Helium: 60 ml/min, with column temp. 802308C at 48C/min, TCD

Galetto and Hoffman (1978)

GC/vanilla extract Soxhlet extraction with pentaneH2O; extraction with diisopropyl ether;

organic layer washed, dried and evaporated; column chromatography of unsaponifiable extracted over Al2O3gel with hexane

OV-I glass capillary column, 25 m0.31 mm i.d., 0.15mm

Hydrogen: 3 ml/min, split 60 ml/min, column temp. 702208C at 38C/min, FID

Ramaroson-Raonizafinimanana et al. (1997)

GCMS/vanilla extract OV-1701 fused capillary column,

50 m0.32 mm i.d., 0.30mm

Helium: 4 ml/min, split 80 ml/min, column temp. 1002808C; 38C/min, i.s. 2708C, i.v. 70 eV

Ramaroson-Raonizafinimanana et al. (1997)

GCMS/vanilla extract SPME DB-5 glass capillary column, 30 m0.2 mm i.d., 0.25mm

Helium: 1.0 ml/min, 2008C at 88C/

min, then 2508C at 508C/min

Sostaric et al. (2000) GC/fruit juices Extraction with 50:50 mix-

ture of pentane and diethyl ethercentrifuge

DB-5 glass capillary column, 30 m0.25 mm i.d., 0.5mm

Helium: 1.55 ml/min, 352758C at 68C/min, FID

Goodner et al. (2000)

GCMS/fruit juices RTX5-MS column, 30 m

0.25 mm, 0.25mm

352218C at 48C/min to 2758C at 108C/min, i.s. 1708C, i.v. 70 eV

Goodner et al. (2000) GC/vanilla extract Extraction with pentane/

ether

DB-Wax fused silica capillary column, 30 m0.32 mm i.d., 0.25mm

Hydrogen: 2 ml/min, column temp.

402458C at 38C/min, FID

Pe´rez-Silva et al. (2006)

GCMS/vanilla extract DB-Wax fused silica capillary column, 30 m0.32 mm i.d., 0.25mm

Helium: 1.1 ml/min, column temp.

402458C at 38C/min, i.s. 2308C, i.v.

70 eV

Pe´rez-Silva et al. (2006)

A.K.Sinhaetal.

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Tripathi et al. 2002; Suresh et al. 2003; Li et al. 2004; Velanker and Heble 2004) as well as to quantify vanillin in several other matrices (Anklam et al. 1997; Farthing et al. 1999; Sobolev 2001). The HPLC conditions used for the above-mentioned studies are presented in Table III.

More recently, hyphenated techniques coupling HPLC with MS detection have been developed, allowing the online detection and identification of chemical

Figure 2. GC profile of headspace volatile components sampled by solid-phase microextraction at room temperature from (a) Tahitian natural vanilla extract, (b) Indonesian natural vanilla extract and (c) Bourbon natural vanilla extract (for GC conditions see Table II). Peak identification: (1) ethyl hexanoate, (2) p-methoxybenzaldehyde, (3) 5-propenyl-1,3-benzodioxole, (4) ethyl nonanoate, (5) unidentified component, (6) p-methoxybenzoic acid methyl ester, (7) 3-phenyl-2-propenoic acid methyl ester, (8) ethyl decanoate, and (9) vanillin. Adapted from Sostaric et al. (2000).#2000 American Chemical Society.

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AU

0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16

Minutes

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 1

2 3

4 5

6

11 7

8 12

13 9 14

15, 16

10

18, 19 20

21 17

Figure 3. HPLC chromatogram of a standard mixture of phenolic compounds (121) (for HPLC conditions see Table III). Peak identification: (1) 4-Hydroxy benzyl alcohol, (2) Vanillyl alcohol, (3) 3, 4-Dihydroxybenzaldehyde, (4) 4-hydroxybenzoic acid, (5) vanillic acid, (6) 4-hydroxybenzaldehyde, (7) vanillin, (8)p-coumaric acid, (9) ferulic acid, (10) piperonal, (11) isovanillin, (12) syringaldehyde, (13) acetovanillone, (14)m-coumaric acid, (15) vanillin methyl ester, (16) ethyl vanillin, (17) 2,4-dihydroxy acetophenone, (18)o-vanillin, (19) coumarin, (20)p-anisaldehyde, and (21) eugenol. Adapted from Sinha et al. (2007).#2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

UA

-0.05 0.00 0.05 0.10 0.15 0.20 0.25

Minutes

5.00 10.00 15.00 20.00 25.00 30.00

1 2

3 4 5

6 7

8

9 10

Figure 4. HPLC chromatogram of ethanolic extract of pods ofV. planifolia(for HPLC conditions see Table III). Peak identification: (1) 4-hydroxy benzyl alcohol, (2) vanillyl alcohol, (3) 3,4-dihydroxybenzaldehyde, (4) 4-hydroxybenzoic acid, (5) vanillic acid, (6) 4-hydroxybenzaldehyde, (7) vanillin, (8)p-coumaric acid, (9) ferulic acid, and (10) piperonal. Adapted from Sinha et al. (2007).#2007 WILEY-VCH Verlag GmbH

& Co. KGaA, Weinheim.

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Analysis/matrix Sample preparation Stationary phase (column) Mobile phase Detector Reference

MPLC/vanilla extract Extraction with 44% EtOH, filtered

MPLC RP-8 spheri-5 column, 1004.6 mm, 5mm

Methanol/acidified water (10:90), 1.5 ml/min

UV at 254 nm Ranadive (1992) Prep. HPLC/vanilla extract Crude vanilla extract diluted with

water/methanol 1:1, extract with ether, added ethanol30%, concentration

Lichrospher 100 RP-18, 251 cm i.d., 5mm

(A) H2O acidified with HCl, pH 2.8; (B) methanol.

Isocratic elution at 0% 7A and 30% B, 2.7 ml/min.

DAD at 340 nm Lamprecht et al.

(1994)

HPLC/vanilla extract Vanilla extract diluted with methanol/water 1:1

LiChroCart Superspher 100 RP-18, 504 mm i.d., 4mm, guard: RP-18, 354 mm, 5mm

Aqueous sol. 0.01 M CH3COONa, pH 4.0 with (A) HCl and (B) methanol;

gradient: 15% B85% B, 025 min; 100% B, 2530 min; 0.8 ml/min

DAD at 340 nm Lamprecht et al.

(1994)

Prep. HPLC/ vanilla extract Extraction and filtration mBondapak RP C18, 25 100 mm i.d., 10mm, guard:

2510 mm

Water acidified with 1.25%

CH3COOH and methanol in 90:10, isocratic elution, 8 ml/min

UV at 270 nm Voisine et al.

(1995)

HPLC/gluco-vanillin and vanillin

_ ODS 2 Spherisorb, RP

C18, 250.46 cm, 10mm

Solvent A, water acidified with 1.25% CH3COOH;

Solvent B, methanol;

gradient: 9590%, 0.5 min, hold for 5 min; 65% A, 535 min, hold for 10 min, 1 ml/

min

DAD at 270 nm Voisine et al.

(1995)

HPLC/vanilla extract Vanilla beans extracted with MeOH20 ml H2O, extracted with pentane then ether, aqueous passed through XAD-2 and eluted by H2O and MeOH

C18 TSK-gel, ODS 80Ts, 1504.6 mm i.d., 5mm

Solvent system I, H2O:MeOH:AcOH:Et3N, A97:3:0.3:0.3. pH 4.6;

B20:80:0.3:0.3. Solvent system II, 1 mM H3PO4, pH3.1:MeOH, A97:3, B20:80, 0.8 ml/min

UV at 280 nm Negishi and Ozawa (1996)

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Downloaded By: [CSIR eJournals Consortium] At: 13:10 28 April 2008 Table III (Continued)

HPLC/plasma and red blood cell (RBC) analysis for vanillin

150ml plasma150ml ACN, vortex 15 sec, 13,000g, 10 min, 100ml RBCs100ml deionized H2O, vortex 15 sec200ml ACN, 13,000g, 10 min, 40ml urine460 ml deionized H2O, vortex 10 sec

ODS, 150 mm3.2 mm i.d., 3mm guard: C18, 30 mm4.6 mm i.d., 4050 mm

For plasma and RBC analysis: aq. CH3COOH (1%)/ACN (85:15), pH 2.9, for urine analysis: aq.

CH3COOH (1%)/ACN;

gradient: 90:10 for 10 min;

70:30 at 10 min and hold 5 min, 0.5 ml/min for plasma, 0.6 ml/min for urine and RBCs

UV at 280 nm Farthing et al.

(1999)

HPLC/vanilla flavorings Lichrospher RP H3PO4in H2O (1:10,000)/

ACN (14:86), 1 ml/min

UV at 278 nm Ehlers (1999)

HPLC/vanilla flavorings Nova-Pak C18 Aqueous AcOH (0.05%)/

MeOH/THF (70:30:0.2), 1 ml/min

UV at 275 nm Jagerdeo et al.

(2000) HPLC/green vanilla beans Enzymatic extraction

(viscoenzyme and celluclast) with 47.5% aq. EtOH

Beckman C18 column 4.66 mm25 cm i.d.

MeOH/H2O acidified with AcOH, pH 4; gradient:

acidified H2O/MeOH, 6040% for 3 min. at 0.8 ml/

min, 6535% for 9 min at 1.0 ml/min, 6040% at 0.8 ml/min

UV at 278 nm Ruiz-Teran et al. (2001)

HPLC/boiled peanuts Extraction with ACN, purified over Al2O3

Zorbax Rx-SIL column, 250 mm4.6 mm i.d., 5mm

Isocratic elution with n-hexane/2-propanol/H2O/

AcOH (2,100/540/37/2, v/

v), 1.5 ml/min

DAD at 220450 nm Sobolev (2001)

HPLC/vanilla beans Lichrospher 60 Aqueous H3PO4(1%)/

ACN/MeOH (95:2:3), 1 ml/

min

UV at 275 nm Scharrer and Mosandi (2001)

A.K.Sinhaetal.

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HPLC/biotransformation (vanilla flavor metabolites) Capsicum frutescenscell culture

Medium was extracted with EtOAc (340 ml), dried and concentrated

Shim-pack ODS column, 4.6 mm150 mm i.d., 5 mm

Solvent A, 0.1% HCOOH aq. sol. Solvent B, ACN.

Solvent B started at 20%, hold 5 min, 40% at 5 min, 80% at 6.5 min, hold 1.5 min, return to 20% at 10 min, re-equilibrated for 5 min, 1.0 ml/min

UV at 280 nm Tripathi et al.

(2002)

HPLC/vanilla extract Commercial extract diluted with H2O, filtered; for reference extract, beans extracted with H2O/

MeOH by maceration and filtered

RP-18-5, 2504 mm Aqueous CH3COONa (10 mM/l) pH 4.0, with conc.

HCl (compound A) and methanol (compound B);

gradient: A/B85:15, in 35 min to A/B15:85, in 5 min to A/B85:15, 0.8 ml/

min

DAD at 210360 nm Pyell et al.

(2002)

HPLC/biotransformation (vanilla flavor metabolites) Capsicum frutescensroot culture

Roots were extracted with EtOAc (three times), dried and

concentrated

Shim-pack ODS column, 4.6 mm150 mm i.d., 5mm

Isocratic elution with MeOH/AcOH/H2O (20:5:75) 1.0 ml/min

UV at 280 nm Suresh et al.

(2003)

HPLC/vanilla glucosides Extraction with 0.1 M acetate buffer, pH 5, then SPE

Phenomenex ODS-Hypersil C18, 254.6 mm, 5mm, guard: C18 as precolumn

(A) H2O/ACN/

CH3COOH95:5:0.75, (B) H2O/ACN/

CH3COOH5:95:0.75;

gradient: 0% B20% B, 030min; 80% B, 3037.5 min, 2.5 ml/min

DAD at 254275 nm Dignum et al.

(2004)

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Downloaded By: [CSIR eJournals Consortium] At: 13:10 28 April 2008 Table III (Continued)

Semi Prep HPLC/glucosides Bondapak C18, 3007.8

mm i.d.

Solvent A, H2O/ACN/

CH3COOH95:5:0.75.

Solvent B, H2O/ACN/

CH3COOH5:95:0.75;

gradient: 010% B in 30 min, 80% B in 7.5 min, 100% B for 1.5 min, 2.5ml/

min

DAD at 254275 nm Dignum et al.

(2004)

HPLC/bioconversion broth 20 g/l isoeugenol as substrate, bioconversion with enzyme at 288C, 180 rpm, pH 9, for 3 days

Lichrospher 100 RP-18-5, 2504 mm i.d., 5mm

Solvent A, 0.01% AcOH aq.

sol. Solvent B, methanol;

gradient: 60% B for 5 min, 50% B for 7 min, 100% B for 18 min, 60% B for 5 min, 1 ml/min

UV at 270 nm Li et al. (2004)

HPLC/microbial plant sources

Synergi^TMHydro-RP C18 Isocratic elution with aq.

CF3COOH (1 mM)/

methanol (17:8), 1.0 ml/min

Sachan et al.

(2004) HPLC/vanilla extract Extraction with EtOH RP-C18 ODS column, 4.6

mm i.d.250 mm, 5mm

Solvent A, ACN. Solvent B, H2O/H3PO4, 99.999:0.001, pH 6.0; gradient: 010 min, 1040% A, 1020 min, 4080% A, 2025 min, 80100% A, 1.4 ml/min

PDA at 254 nm Sharma et al.

(2006)

HPLC/vanilla extract Extraction with EtOH Nucleosil C18 H2O/MeOH (40:60), 05 min. H2O/MeOH (40:60), 510 min. MeOH (100), 10 15 min. H2O/MeOH (40:60), 1 ml/min

UV at 231 nm Waliszewski et al. (2007)

Note: AcOH, acetic acid; ACN, acetonitrile; MeOH, methanol; CF3COOH, trifluoroacetic acid; EtOH, ethanol; THF, tetrahydrofuran.

A.K.Sinhaetal.

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constituents. de Jager et al. (2007) have applied liquid chromatographyelectrospray ionizationmass spectrometry for the determination of vanillin, coumarin, and ethyl vanillin in vanilla products. All MS data were acquired in the positive ionization mode.

Also, comparison made between liquid chromatographyMS and liquid chromatographyUV in terms of precision and accuracy found liquid chromatographyMS to be more accurate due to its higher level of specificity.

Capillary electrophoresis

Although HPLC remains the most dominating technique for the analysis of chemical constituents of vanilla, CE is gaining popularity as it has several unique advantages over HPLC because of its speed, efficiency, reproducibility, small sample volume, low consumption of solvent and ease of removal of contaminants. A high-performance CE method has been developed for analysis of vanillin, syringaldehyde, coniferaldehyde and sinapaldehyde in brandy and wine. The optimized conditions selected for the purpose of monitoring were a capillary of length 53.5 cm, borate buffer of pH 9.3 (50 mM) as a mobile phase at temperature 208C, voltage at 30 kV and UV absorptions at 348, 362, 404, and 422 nm. Recoveries ranged between 99.9% and 107.7% for all of the compounds tested. The repeatability of the method was found to be high (Panossian et al. 2001). In addition to CE, more recent capillary techniques such as micellar electrokinetic capillary chromatography (MEKC) and mixed micellar electrokinetic capillary chromatography are also being investigated for component analysis.

MEKC, a separation mode of CE, was introduced by Terabe et al. (2001). It is one of the latest chromatographic techniques incorporating many unique features and is highly competitive with HPLC in the field of food analysis. Because of the absence of a stationary phase and solubilization of the sample by the micellar phase, direct sample injection is possible in many cases. Other advantages are the high peak capacity and short run times. MEKC has been used as a rapid screening method for the analysis of vanilla flavorings and vanilla extracts. Under optimized conditions, the separation of nine vanilla constituents and three probable adulterants was possible within 9 min (Bu¨ tehorn and Pyell 1996).

There have been attempts to study the comparative advantages of MEKC vis-a-vis HPLC for the component analysis of vanilla extracts and vanilla-containing commercial preparations (Pyell et al. 2002). The results showed that MEKC could be regarded as a competitive alternative to HPLC, since investigation can be concluded in much shorter analysis time with high resolution and efficiency (Pyell et al. 2002) (Figure 5). This study used a capillary electrophoresis system equipped with an UV absorbance detector and fused-silica capillaries (75mm i.d., 375mm o.d.).

The detection was performed at 230 nm. The separation buffer was an aqueous solution of sodium dodecyl sulfate (80 mmol/l), disodium tetraborate (10 mmol/l), boric acid (10 mmol/l) and urea (1.5 mol/l) having pH 9.14.

Recently, a mixed micellar electrokinetic capillary chromatography method for the qualitative and quantitative determination of key components of vanilla flavor, including vanillin, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, vanillic acid and 3-methoxybenzaldehyde, has been developed (Boyce et al. 2003). The separations were performed using a fused silica capillary (52 cm effective length75mm i.d.) at 258C with an applied voltage of 18 kV. The detection wavelength was 214 nm. Buffer

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consisted of 10 mM sodium tetraborate, 10 mM boric acid, 100 mM sodium dodecyl sulphate and 40 mM sodium cholate at pH 7.0.

Production of vanillin

Vanillin, being the most characteristic component of vanilla flavor, has an annual consumption in the world flavor market touching 12,000 t per annum. The limited supply and consequently high price of natural vanillin (i.e. US$4,000/kg) (Rao and Ravishankar 2000) has led to the usage of a large amount of synthetic equivalents. The production of vanillin, from its earliest days to the present, is a fascinating story of the progress made in chemistry and chemical engineering, which is reflected by the gradual lowering of price up to $12/kg. At present about 97% of vanillin sold in the market comes mainly from the synthetic sources using coniferin, eugenol, safrole, guaiacol (Bedoukian 1986) and lignin (Hearon and Lo 1980; Wu et al. 1994; Sande and Sears 1996; Hocking 1997; Bjørsvik 1999; Qiang and Zhonghao 2001; Kozlov and Gogotov 2001). Although vanillin produced by these means is able to meet the global annual demand, it suffers from serious drawbacks. For one, the aroma of synthetically produced vanillin is not comparable with that of natural vanillin.

Secondly, chemical synthesis involves use of hazardous chemicals (and hence under current US and European legislations cannot be used in natural flavors), resulting in decreased consumer appeal the world over.

However, the production and isolation of vanillin from natural sources present an altogether different scenario. The reason behind it is the huge disparity in efforts put in and the yield per hectare. The cultivation of vanilla is a time-consuming and labor- intensive process, yet the yield is not very high (Rao and Ravishankar 2000). Very few attempts have been reported for the isolation of natural vanillin from vanilla extract. In one process, extract containing about 0.10.2 g vanillin was extracted three times with ether. The combined ether layers were concentrated and the residue was taken up in

Figure 5. Electropherogram (MEKC) of commercial vanilla extract. Peak identification: (2) 4-hydroxybenzyl alcohol, (3) vanillyl alcohol, (4) 4-hydroxybenzoic acid, (5) vanillic acid, (6) 4-hydroxybenzaldehyde, (7) vanillin, and (8) syringaldehyde. Adapted from Pyell et al. (2002).#2000 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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methylene chloride, removing any water, and dried (Na₂SO₄). This solution was concentrated to solid, from which approximately a 10% solution in ether was prepared. Pure vanillin was then isolated by preparative GC of the solution (Hoffman and Salb 1979). In another report, Krueger and Krueger (1985) adopted the procedure suggested by Dr Warren Wong of Givaudan Cooperation for the isolation of natural vanillin. To a 100 ml portion of one-fold extract (or an equivalent amount of multi-fold extract diluted to 100 ml with 35% alcohol) taken in a beaker, 200 ml methylene chloride was added and the solution was stirred magnetically overnight (it was found that extraction in a separatory funnel yielded intractable emulsions). The methylene chloride layer was separated, dried over Na₂SO₄and evaporatedin vacuoto dryness. The residue in flask was extracted with 50 ml boiling pentane whereby vanillin crystallized out on cooling in an ice bath.

Recently, there has been a huge upsurge in the exploration of more eco-friendly biosynthetic procedures for the production of natural flavors as the products derived from these have already been identified in plants or other natural sources, and hence in principle defined as ‘natural’. Biotechnological approaches for the production of vanillin have been based on bioconversion of certain natural precursors such as lignin, eugenol, isoeugenol, ferulic acid and phenolic stilbenes and on de novobiosynthesis applying fungi, bacteria, plant cells, or genetically engineered microorganisms (Priefert et al. 2001; Rao and Ravishankar 2000). However, the yields from most of the precursors are very low since the toxicity of both the precursor and the vanillin formed presents a major obstacle. Ferulic acid, being least toxic of all the above, is a suitable candidate for these microbial conversions (Benz and Muheim 1996), thereby providing a unique biocatalytic pathway for commercial production. Another notable feature about ferulic acid is that it is a cheap raw material and can be easily obtained from agricultural waste residues such as cereal bran and sugar beet pulp (Zheng et al.

2007). Several reviews are already documented on the biotechnological production of vanillin (Rao and Ravishankar 2000; Priefert et al. 2001; Walton et al. 2003).

Although the consumers’ preference for ‘natural’, ‘bio’ or ‘organic’ products is undoubtedly an important market pull in the food sector (Cheetham 1997), the industrial interest lies in the cost-effective production of flavor molecules, which are otherwise difficult to obtain in the proportions present in natural extracts. In this context, research exploiting the advantages of biocatalysis is certainly to overplay chemical approaches in the near future.

Authenticity of vanilla extract for quality assurance

A huge market demand together with a high price of authentic vanilla extract has provided an economic incentive to pretend inferior and synthetic products as natural vanilla. Often adulterants like vanillin isolated from lignin, ethyl vanillin, coumarin or piperonal are heavily used to mimic the flavor. Also, the flavor of extracts varies significantly with environmental, geographical and storage conditions. Thus, determination of the origin of vanilla is a significant problem nowadays. Several methods have been developed in an attempt to identify the primary components responsible for the delicate and unique flavor and to verify the authenticity of vanilla extract, including AOAC methods. Adulteration can be detected chromatographically by an abnormal excess of vanillin relative to the profile of minor components in a vanilla preparation, but again the possibility is there to manipulate this profile artificially