FLAVOUR AND FRAGRANCE JOURNAL Flavour Fragr. J.

2008; 23: 382391 Published online 20 October 2008 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/ffj.1905

Quantitative analysis of essential oils: a complex task

John Wiley & Sons, Ltd.

Carlo Bicchi,1 Erica Liberto,1 Maura Matteodo,1 Barbara Sgorbini,1 Luigi Mondello,2,3 Barbara dAcampora Zellner,2 Rosaria Costa2 and Patrizia Rubiolo1*
Quantitative analysis of essential oils: a complex task

1 2 3

Dipartimento di Scienza e Tecnologia del Farmaco, Universit degli Studi di Torino, Via P. Giuria 9, 10125 Torino, Italy Dipartimento Farmaco-chimico, Universit degli Studi di Messina, Viale Annunziata, 98168 Messina, Italy Campus-Biomedico, Via E. Longoni 47, 00155 Roma, Italy

Received 12 May 2008; Revised 23 July 2008; Accepted 25 August 2008

ABSTRACT: This article provides a critical overview of current methods to quantify essential oil components. The fields of application and limits of the most popular approaches, in particular relative percentage abundance, normalized percentage abundance, concentration and true amount determination via calibration curves, are discussed in detail. A specific paragraph is dedicated to the correct use of the most widely used detectors and to analyte response factors. A set of applications for each approach is also included to illustrate the considerations. Copyright 2008 John Wiley & Sons, Ltd. KEY WORDS: essential oils; quantitative analysis; relative percentage abundance; normalized percentage abundance; true quantitation

Introduction
An essential oil (EO) is defined in agreement with the European Pharmacopoeia as an: . . . . Odorous product, usually of complex composition, obtained from a botanically defined plant raw material by steam distillation, dry distillation, or a suitable mechanical process without heating.1 This definition allows us to include EOs in the general framework of the volatile fraction of a plant matrix, which in turn comprises a range of other sampling approaches and/or techniques producing samples whose compositions are representative of the volatiles characterizing a plant matrix. These are almost always not comparable, and include headspace, flavours, fragrances, aromas and extracts obtained by specific techniques. The term volatile fraction in general defines a mixture of volatiles in a matrix of plant origin that can be sampled because of the volatiles ability to vaporize spontaneously and/or in suitable conditions or by appropriate techniques. These considerations appear to be obvious but, unfortunately, several articles erroneously compare the composition of EO with the different approaches of headspace sampling [not only static headspace but also headspacesolid phase micro-extraction (HSSPME) and headspace sorptive extraction (HSSE)] directly or, even worse, fail to distinguish between them. Nowadays there is also an ever-increasing demand for quantitative data in the flavour and fragrance field, in particular with regard to EOs. This is mainly because the
*Correspondence to: P. Rubiolo, Dipartimento di Scienza e Tecnologia del Farmaco, Universit degli Studi di Torino, Via Pietro Giuria 9, Torino10125, Italy. E-mail: patrizia.rubiolo@unito.it

number of controls required, in terms of quality and safety, as well as those relating to EO biological activity, is continually growing. The economic importance of such controls is also increasing, particularly in todays globalized economy. The demand for quantitation mainly derives from: (a) the pharmacopoeias, not only for EOs associated to pharmaceuticals, but also in the food and cosmetic fields; (b) international bodies and committees (e.g. EFSA, IFRA, IOFI); (c) limitations fixed by law (e.g. suspected volatile allergens, thujone, menthofurane, pulegone); (d) product characterization from/for industry; (e) in-house quality control; and, last but not least, (f) scientific implications (e.g. studies on EO or EO-component biological activity). The quantitative aspects of EO analysis are not easy to deal with, not only because quantitation has long been considered less important than the elucidation of new odorous structures, but also because several aspects of quantitation were, and still are, ambiguous. The quantitative composition of most EOs is reported in the literature in terms of relative percentage abundances although, unfortunately, this approach is of limited value, as will be discussed in detail below. However, EO complexity, the number of applications and the differing quantitative answers required in this field make the use of a unique approach to EO component quantitation difficult. Different approaches to EO quantitative analysis are therefore possible, depending on their use and destination. On the basis of a recent literature survey carried out in the authors laboratory, the most used approaches in this field are: (a) relative percentage abundance, in this article indicated simply as relative % abundance; (b) internal standard normalized percentage abundance (normalized % abundance), which also includes

Copyright 2008 John Wiley & Sons, Ltd.

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quality characterization through statistical elaboration of the GC profile, taken as a marker; (c) true quantitation of one or more components (true quantitation ) by a validated method. In addition, since the term marker is sometimes inappropriately used, its meaning is also recalled here, assuming that a marker is a component of an EO that concurs to define its identity, specificity, organoleptic properties, origin, genuineness and quality, independently of its abundance. A quantitative EO analytical procedure consists of two main steps: (a) sample preparation and (b) the analysis itself. The present article mainly deals with analysis; however, some basic aspects about sample preparation must be followed if reliable quantitative results are to be obtained, whatever method is used to sample the volatile fraction, e.g. steam distillation, hydrodistillation, mechanical procedures or headspace (HS) techniques: (a) the variability of a plant matrix requires careful standardization, both of plant collection and of sample preparation; (b) a suitable number of samples must be analysed to obtain a representative composition of the volatile fraction of the species investigated (i.e. the average composition from a minimum of three samples from different, clearly distinct populations is necessary). This article critically discusses the current methods to quantify EO components, together with their advantages and limits, and reports basic considerations that, although they may appear obvious, are the object of serious fundamental errors that have appeared even in recently published articles.

each analyte, as it closely depends on structure, and because the number of ions produced in the MS source is often not linearly related to the amount of analyte.2 The detector that best meets the requirement of consistency of response factors for all sorts of chemical structure is the thermo-conductivity detector (TCD), which can be used successfully in combination with conventional GC for quality controls.3 Its main limitation is its sensitivity, which is more than one order of magnitude lower than that achievable with FID; it therefore cannot be used for EOs where trace components must be quantified, nor in combination with fast-GC. On the other hand, FID is the most popular GC detector: it is universal, highly sensitive and robust; however, its response factors can vary by up to 60%, depending on the chemical structure of the analyte to detect. Response factors are discussed in greater depth below. TCD should therefore be preferred in all cases where high sensitivity and fast analysis time are not required, and for laboratories with problems of security and gas supply. In addition, dedicated considerations concerning correct detection for a specific quantitation approach are given at the end of the each respective subsection.

Relative % Abundances (of All Detected Components) and Normalized % Abundances (or Relative % Abundances with Internal Standard Normalization) of all Detected Components or Groups of Selected Markers
These two approaches are discussed together, because they are closely related. Relative % abundance of all detected components is the most commonly used approach in EO analysis. Unfortunately, it is still today very often applied incorrectly, since it is used to compare compositions of EOs from the same species across a series of samples. Relative % abundance results are only correctly used to measure relative component ratios in a single sample. When the compositions of a group of EOs must be compared, this approach fails because the data are not standardized and, in consequence, are not comparable. In this situation, two strategies can be adopted, depending on the variability of the sample composition. When samples vary in qualitative and quantitative compositions within a limited range, the normalized % abundance can be applied. With this approach, the GC raw data of a selected number of markers of the EOs under investigation and, usually common to all investigated samples, are normalized vs. one or more internal standards (or an external standard, if an automatic injector is available) and from them the normalized % abundance is calculated. The internal standard allows us to contour the GC instrument performance variations. The normalized % abundance must not be confused with the internal normalization, i.e. the calculation of % after determining the response coefficients of all constituents

EO Quantitative Analysis and Detectors

GC detectors and their characteristics are discussed first because of their importance in quantitation of EO components. Various different GC detectors (universal, selective or specific detectors) can be used for quantitation purposes, depending their choice on the application; in general in the EO field, universal detectors are considered to be the most suitable for everyday work, because of the matrix chemical nature. A rapid literature survey shows that quantitative EO analysis is mainly based on flame ionization (FID) and mass spectrometry detectors (MS or MSD). The most important characteristics of a detector when used for EO quantitation purposes are high sensitivity and robustness, extension of the range of linearity, low purchase and running costs, and response factors for all analyte classes as close as possible to 1. The importance of the last condition is often underestimated: for instance, several studies use MS in full scan mode (SCAN) as detector with relative and normalized % abundance, since it provides unequivocal component identification at the same time as quantitation (in SIM mode). This is a serious mistake, both because fragment abundance is specific to

Copyright 2008 John Wiley & Sons, Ltd.

Flavour Fragr. J. 2008; 23: 382391 DOI: 10.1002/ffj

384 C. BICCHI ET AL.

Table 1. Absolute areas, normalized % abundance, and true concentration of menthone and linalool in peppermint samples A and B Sample Peppermint EO Sample A Menthone
Absolute areas (counts) Normalized % abundance True quantitation (mg/l) 2 566 296 11.20 2233

Peppermint EO Sample B Menthone

2 456 887 20.70 2138

Linalool
48 551 0.39 33

Linalool
77 040 0.35 53

Figure 1. GC profiles of two peppermint EOs (samples A and B) of different origins and compositions. Analysis conditions: column, Megawax (Mega, Legnano, Italy), 60 m 0.25 mm i.d., 0.25 m film thickness; temperature rate, 75C for 8 min, rising at 4C/min to 220C, then held for 5 min; injection mode, split (ratio 1:100); injector temperature, 230C; detector (FID) temperature, 250C; carrier, H2; flow rate, 1.5 ml/min in constant flow mode

is about 35% larger in sample B. On the basis of the absolute areas, the comparison of data resulting from normalized % abundance are contradictory and incorrect, because the menthone percentage in sample B is much higher than in A (about double, 20.7% vs. 11.2%), although they show similar areas, while the linalool percentages are almost the same (0.39% vs. 0.35%), despite their different absolute areas. Similar results are obtained with relative % abundance. On the other hand, if the normalized absolute areas or a true quantitation through calibration curves are applied, the results are fully consistent with the absolute peak areas. Normalized % abundance can therefore be used in quality control, but if the number of samples to be periodically analysed is a sufficiently high, several approaches can be applied to exploit the results, so as to improve the effectiveness and reliability of quality control.

Statistical Elaboration of the GC Profile If the samples of a group must only be classified and/or discriminated, e.g. in terms of chemical composition or geographical origin, and if comparison between specific marker abundances is not necessary, the GC profile itself (i.e. the normalized data of a selected number of markers in toto) can be taken as a parameter peculiar to the EO investigated, and can be elaborated statistically. This approach is not a true quantitative method, but it requires the use of correct and appropriate quantitative data that, obviously, can only derive from normalized areas or normalized % abundance data. The most widely used statistical tool for GC profile elaboration is multivariate analysis (MVA), and in particular principal component analysis (PCA), a method that can highlight differences among the samples of a set characterized by a suitable number of components (variables) through the linear combination of those explaining most of the variability.4,5 In this case, the more samples are analysed, the better is the reliability of the statistical results obtained. Figure 2 reports the PCA discrimination of 92 chamomile (Matricaria recutita L.) EOs by chemotype, using normalized % abundances of seven characterizing sesquiterpenoid components after normalization to a single internal standard (n-tridecane) and standardization of variables. Four

(see below). On the other hand, when the qualitative quantitative compositions of the samples vary significantly, a comparison can only be made either through the component normalized area approach or by true quantitation via the calibration curve (the latter is described in detail in the next section). The normalized area approach is based on the normalization of the raw areas of a part or all of the sample components vs. one or more internal standards, thus enabling the analyst to compare the intensity of each individual component across the group of samples to be analysed. This approach is very seldom used, because its results are specific to each EO component, whereas normalized % abundance correlates the components of each EO through their percentages; however, it can give erroneous results, as will be clear from the following example. Figure 1 reports the GC profiles of two peppermint EOs (samples A and B) of different origins. Table 1 shows that the menthone absolute area in sample A is about 5% larger than in sample B, while the linalool area

Copyright 2008 John Wiley & Sons, Ltd.

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Table 2. List of Lawrences indices7 calculated on 142 peppermint EOs from Piedmont Lawrences indices
A B C D E F G H I L M N 1,8-Cineole/limonene 1,8-Cineole/menthofuran 1/2 1,8-Cineole/menthyl acetate 1,8-Cineole/menthol 50 1,8-Cineole/menthone 25 Menthofuran/menthone 100/6 Menthofuran/menthol 50 Mentofhuran/menthyl acetate 1/2 Menthofuran/limonene Menthone/menthyl acetate 1/2 Menthol/menthone Menthol/menthyl acetate 1/2

Min
1.71 0.28 0.59 3.20 4.46 0.75 1.18 0.99 0.38 0.73 1.29 2.99

Max
3.46 3.97 2.33 9.38 13.19 7.82 11.87 10.92 4.53 5.83 4.45 7.94

Figure 2. PCA chemotype discrimination of 92 chamomile (Matricaria recutita L.) EOs chemotypes following the Schilcher classification:6 (A) bisabolol oxide A; (B) bisabolol oxide B; (C) -bisabolol; and (D) -bisabolol and bisabolol oxides A and B

chemotypes can clearly be distinguished, with a total variability explained by the two principal components of about 80%. The markers characteristic of the chemotypes, following the Schilcher classification5,6 are: (A) bisabolol oxide A; (B) bisabolol oxide B; (C) -bisabolol; and (D) -bisabolol and bisabolol oxides A and B. Definition of the Reference Profile When the percentage of one or more markers is specifically required to characterize an EO (e.g. European Pharmacopoeia) or is needed for quality control, and if a rather large number of samples must periodically be analysed, a reference profile of the investigated EO can be built up. This profile in general includes the average, minimum and maximum percentage values of each marker, calculated from a set of reference and/or accepted samples. As already mentioned above, in this case normalized % abundance can correctly be applied, because the reference profile involves well-defined component ratios, which in consequence vary within a relatively narrow percentage range. Each markers acceptance range can be continually updated and refined with the data relating to accepted samples from each analysis campaign. This approach enables (a) to establish the acceptability of the investigated EO sample, by comparing the normalized % abundances of its markers to those fixed from the reference profile, so that those components that are outside the percentage window are immediately indicated; and (b) to improve the flexibility of the acceptance criteria, taking into account seasonal or climatic variations in EO composition.

When a large number of markers must be quoted, this approach can be time-consuming; matching the markers of a sample to reference profiles can better be carried out automatically and/or graphically through statistical computer software. For EOs having a widespread world market, and/or originating from several countries, such as peppermint, citrus oils, etc., an additional approach to characterizing their quality is to determine the ratio between the normalized % abundances of some characteristic or biosyntheticallyrelated components that must simultaneously be met to define EO quality. For peppermint EO, these values are known as Lawrences indices;7 Table 2 reports the values from a set of peppermint oils from Piedmont. In this case the ratios between components of this EO were chosen because of: (a) their biosynthetic sequence (e.g. limonene/ 1,8-cineole, menthol/menthone or menthol/menthyl acetate); (b) their common biosynthetic precursors, e.g. pulegone for menthofuran/menthone; and (c) their influence on the EO odour (e.g. 1,8-cineole/menthofuran). Detection for the purpose of calculating relative % abundance and/or normalized % abundance can correctly be either by TCD or by FID (provided that at least class response factors are available; see below) but not by MS in either SIM or SCAN modes. The reasons why SCAN MS is inapplicable to these approaches have already been discussed. SIMMS obviously gives a signal only for those components producing the selected ion(s) in their fragmentation pattern, and thus induces discriminative detection; it may therefore only be used to establish ratios between the members of homologous series showing the same diagnostic ions of similar intensity. Provided that the correct enantiomer elution order is known, this characteristic can be exploited to measure the enantiomeric excess (EE) or ratio (ER) of optically active EO markers directly, since standard MS is known to be unable to discriminate between optical isomers, not being a selective chiral probe in this sense and therefore giving indistinguishable spectra.8 For example, the genuineness of bergamot EO can be detected through the enantiomer ratios

Copyright 2008 John Wiley & Sons, Ltd.

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Figure 3. ESGCTICMS profile of bergamot EO. (A) Peak identification: 1, -pinene; 2, -pinene; 3, sabinene; 4, limonene; 5, linalool; 6, linalyl acetate; 7, -terpineol; a, (R) enantiomer; b, (S) enantiomer. ESGCSIMMS profiles and enantiomeric ratios of (B) linalool and linalyl acetate (80, 93, 121 m/z) and (C) -terpineol (59, 93, 136 m/z). Analysis conditions: column, heptakis-2,3-di-O-ethyl-6-O-t-butyldimethylsilyl--CD, 30% in PS086 (Megadex, Mega), 25 m 0.25 mm i.d., 0.25 m film thickness; temperature programme, 50C, rising at 2C/min to 200C, then held for 5 min; injection mode, split (ratio 1:50); injector temperature, 220C; EI mode, 70 eV; ion source temperature, 230C; carrier, helium; flow rate, 1.0 ml/min in constant flow mode. Full scan mode: scan range, 35 350 m/z; SIM mode: dwell time, 0.2 s

of linalool, linalyl acetate, -pinene, sabinene, limonene and -terpineol.9 Figure 3 reports the enantioselectivegas chromatographytotal ion mass spectrometry (ESGC SCANMS) profile of an adulterated bergamot EO together with those of linalool and linalyl acetate and -terpineol in enantioselectivegas chromatography selected ion monitoring mass spectrometry (ESGC SIMMS) and their enantiomeric ratios.

True Quantitation of EO Marker Components and Components Restricted by Legislation Using Internal and/or External Standards
Normalized % abundance can be assumed to be a quantitative comparison of GC profiles, but for some applications it is not sufficient to satisfy all quantitation

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requirements of an EO. An EO or products containing it, such as cosmetics, food or pharmaceuticals, must often be characterized through the concentration (p.p.m. or mg/l) or, less frequently, through the absolute amount of one or more of its quality markers or components restricted by legislation. This approach involves the true quantitation of a marker, in general by official methods or those described in the literature, as for instance is the case of the determination of - and -thujone in bitters,10 or of suspected allergens in cosmetics.11 Marker concentration or absolute amount in a real-world sample is determined from its chromatographic area normalized vs. an internal (or external) standard and calculated by a calibration curve constructed from amounts of marker pure standard in the operative concentration range. Since pure standards are not always commercially available, or are difficult and time-consuming to isolate, an accepted compromise is to adopt compounds belonging to the same class (hydrocarbons, aldehydes, alcohols, esters, etc.) and with structures as similar as possible to the investigated analyte (this approach is discussed in detail in the next paragraph). Moreover, for analysis in GCMS, specific labelled internal standards (in general 2D or 13C marked compounds) can also be used: although expensive, a labelled standard assures a response factor equal to or very close to that of the corresponding marker, and it is easy to detect because it gives the same fragmentation pattern, because it has the same structure but with a known increase in molecular weight, easily detectable by MS. It must be stressed that a true quantitation of all components in an EO is, in general, unrealistic for several reasons, including: (a) the unacceptably long analysis time, in particular for complex EOs; (b) standards of most components are unavailable commercially; (c) sufficient EO must be available to construct reliable calibration curves. Validated quantitative methods for biologically-active EO components are increasingly required, in particular for those used in the pharmaceutical industry. The development of a method meeting all the conditions and parameters required for a pharmaceutical validation is time-consuming (up to 3 months) and expensive, because of the large number of experiments and data to be collected. Full validation of a quantitation method for all EO components is even more unrealistic, and must therefore be limited to those applications where it is expressly required and, above all, to a small number of selected representative markers. A further limit is that validation requires a relatively large amount of EO and, in consequence, an abundance of plant material, which is not always available for rare species and/or those with low EO yield. Dedicated guidelines established by the international regulatory bodies and committees (Eurachem CITAC, IUPAC, etc.1214) must be followed to validate a method and to evaluate its performance through parameters such as selectivity, specificity, linearity over the working range, repeatability, precision, intermediate precision,

reproducibility, limit of detection (LOD), limit of quantitation (LOQ), accuracy and assessment of uncertainty. A reliable analytical approach must be applied before submitting it to validation and the choice of validation parameters must therefore be rational. Repeatability, intermediate precision and linearity in the working range are indispensable for all quantitation methods; LOD and LOQ are mainly important for trace analysis; reproducibility is necessary when different laboratories are involved in control procedures, and so on. Detection for true quantitation can correctly be carried out by TCD or FID (provided that class response factors are available, see below) or by MSD in SIM mode, but not by MS in SCAN mode (see above). SIM detection is not only effective for quantitation, but also increases the reliability of identification, because it combines diagnostic ions with chromatographic identification tools (i.e. linear retention indices, retention time locking and relative retention time). Some examples of true quantitation follow. The first concerns a group of 10 markers of peppermint EO, six of them indicated by the European Pharmacopoeia. A calibration curve has been constructed for each marker, using six different calibration levels in the range 2100 mg/l (i.e. 2100 p.p.m.); three more calibration levels had to be included for menthone and menthol because of their EO abundances. Tridecane (ISTD1) and octadecane (ISTD2) were used as internal standards. The analyses were carried out by GCFID and GCMS in SIM and SCAN modes. Table 3 lists the marker diagnostic ions used for SIMMS quantitation, together with the calibration range, calibration curve equation, correlation values (R2) and regression standard error of each, when analysed by GCSIMMS and GCFID. Table 4 gives the average marker areas normalized vs. the internal standards and their concentrations in the peppermint EO investigated, when analysed by GCFID, together with the GCSIM MS and GCSCANMS standard error calculated vs. GCFID. These results show that the linearity of each marker is very good, as is the regression standard error with both GC FID and GCSIMMS, and that the quantitative results are highly comparable. As expected, GCSCANMS gives higher relative standard errors, confirming that the SCAN mode cannot reliably be applied for true quantitation. The quantitation of isopulegol in mint oils is given here as an example of a marker analysis to evaluate the genuineness of an EO. The chief goal of this analysis was to detect whether Mentha piperita (peppermint) EO has been adulterated with that of the cheaper Mentha arvensis (cornmint). Isopulegol is reported in the literature to be present in peppermint EO in amounts never exceeding 0.20.3%, while in cornmint it is present in the range 0.82.0%.15 A normalized % abundance quantitation procedure of isopulegol is sufficient to detect peppermint EO adulteration, but a true quantitation is necessary to

Copyright 2008 John Wiley & Sons, Ltd.

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Table 3. Marker diagnostic ions used for SIMMS quantitation, calibration range, calibration curve equation, correlation values (R2) and regression standard error of each when analysed by GCFID and GCSIMMS Compounds Calibration level (mg/l) SIM (m/z ions) GCFID Regression equation
-Pinene
Limonene 1,8-Cineole -Terpinene 3-Octanol Menthone Menthofuran Linalool i-Pulegol Menthol ISTD1 ISTD2 2-100 2-600 2-600 2-100 2-100 2-4500 2-600 2-100 2-100 2-4500 93, 69, 91 68, 93, 136 81, 43, 108 119, 91,93 59, 55, 83 112, 69, 139 108, 79, 150 93, 71, 121 81, 71, 121 95, 71, 81 57, 43, 71 57, 71, 85 y = 0.0038x + 0.0005 y = 0.0041x 0.0003 y = 0.0034x + 0.0002 y = 0.004x 0.0007 y = 0.003x + 0.0018 y = 0.0028x + 0.0029 y = 0.0033x + 0.0013 y = 0.0035x + 0.0012 y = 0.0033x + 0.0014 y = 0.0035x + 0.00245

GCMS Correlation coefficient (R2)

0.9999 0.9999 0.9999 0.9999 0.9993 0.9999 0.9994 0.9991 0.9994 0.9999

Regression equation
y = 0.0045x + 0.00037 y = 0.0022x 0.0036 y = 0.0010x 0.00054 y = 0.00415x 0.0024 y = 0.0028x + 0.0007 y = 0.0013x 0.0060 y = 0.0057x + 0.0221 y = 0.0017x + 0.0012 y = 0.00063x + 0.00027 y = 0.0019x + 0.0223

Correlation coefficient (R2)

0.9999 0.9999 0.9999 0.9999 0.9997 0.9992 0.9997 0.9996 0.9979 0.9991

Table 4. Average marker areas normalized vs. the internal standards and their concentrations in the peppermint EO investigated, when analysed by GCFID, together with the GCSIMMS and GCSCANMS standard error (SE) calculated vs. GCFID Compounds Av. norm. area EO concntration (mg/l) FID
-Pinene
Limonene 1,8-Cineole -Terpinene 3-Octanol Menthone Menthofuran Linalool i-Pulegol Menthol 0.3 0.6 1.8 0.1 0.1 4.9 0.8 0.1 0.05 14.4 88.2 136.4 517.3 27.0 26.0 1756.2 239.9 26.8 14.1 4109.0

FID relative SE SIMMS

1.2 1.1 1.8 1.1 5.4 1.2 6.2 3.7 7.1 1.6

TICMS
10.5 11.7 8.5 4.5 6.5 4.5 21.5 3.2 1.3 9.0

determine the amount of added cornmint EO. The quantitative determination of isopulegol in a peppermint EO whose chromatogram is reported in Figure 4 was carried out through the calibration curve obtained with the above experiment. This sample showed an amount of isopulegol of 1 g/100 g EO, corresponding to 1.01% isopulegol. An approximate adulteration of 30% of cornmint can be calculated, assuming an average isopulegol content of 1.6% from a GCFID reference profile built up with 42 cornmint EO samples (data not reported). In general, legislation in the cosmetic, food and pharmaceutical fields limits the amount of specific EO components in finished products. However, it is often easier to run/perform the direct quantitation of legallyrestricted components in the EO than in the final formulation, in particular when it is possible to correlate EO and finished product. In contrast, routine control of final products should be adopted with rare and expensive plants, characterized by small amounts of EO, provided that a sufficiently simple and reliable method is available. This is not least because hydrodistillation is not an easy

Figure 4. GC profile of peppermint EO. Analysis conditions: Megawax (60 m 0.25 mm i.d., 0.25 m film thickness); temperature programme, 75C for 8 min, rising at 4C/min to 220C, then held for 5 min; injection mode, split (ratio 1:100); injector temperature, 230C; detector (FID) temperature, 250C; carrier, H2; flow rate, 1.5 ml/min in constant flow mode

Copyright 2008 John Wiley & Sons, Ltd.

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Table 5. Calibration curves and quantitative results of - and -thujones in a set of bitters prepared with different amounts of the two A. umbelliformis chemotypes and grown under different conditions
Calibration curves -Thujone: y = 0.3105x + 3.311 R2 = 0.9995

-Thujone: y = 0.2896x + 0.1380

R 2 = 0.9994 (mg/l) 55.8 71.3 37.5 45.4 25.7 Bitters 6 7 8 9 10 Chemotype B B B B B (mg/l) 2.1 0.2 0.2 1.7 2.5

- and -Thujone (mg/l) in bitters

Bitters 1 2 3 4 5 Chemotype A A A A A

is also available.18 The European Community has limited the amount of - and -thujone in marketed bitters to 35 p.p.m. because of their toxicity.10 Thujones determination in the finished bitter by GCFID after iso-octane/ dichloromethane extraction, using methyl undecanoate as internal standard, is here to be preferred over EO analysis from plant material. Table 5 reports the calibration curves of both thujones, together with the results of their quantitation, in a set of bitters prepared with different amounts of the two A. umbelliformis chemotypes and grown in different conditions. Experiments are still under way to try to correlate their amounts in EO and bitters, in order to enable manufacturers operating with a standardized procedure to predict - and -thujone amounts in the bitter prepared by suitably mixing the two chemotypes directly from their percentages in the EO.
Figure 5. GC profiles of the two chemotypes of A. umbelliformis EOs. Analysis conditions: column, Mega5 (Mega), 25m 0.25 mm i.d., 0.25 m film thickness; temperature programme, 50C for 1 min, rising at 3C/min to 250C, then held for 5 min; injection mode, split (ratio: 1:20); injection temperature, 230C; detector (FID) temperature, 250C; carrier, H2; flow rate, 1 ml/min in constant flow mode

Response Factors and Accurate Quantitative Analysis

As already mentioned, the signals generated by GC detectors depend not only on concentrations but also on chemical structures and the elementary composition of organic compounds. As a consequence, in cases where detector responses to analytes with different structures are different, a response factor (RF) must be applied to normalize the results. Peak area normalization with RFs may be applied when: (a) all components of a sample are separated and detected; (b) the detector responds linearly to all components; and (c) no loss of sample occurs because of injector discrimination, column absorption or chemical interaction.19 The following considerations concern only FID; TCD performances are at present under study in the authors laboratories and will be the focus of a forthcoming article. A further advantage for quantitation is the nearly linear dependence of its response to the carbon number of an analyte, i.e. the relationship between absolute concentration and peak area of the corresponding detector signal. In a true quantitation, FID responses to individual EO components must be verified through calibration functions and RFs,

procedure to standardize and is time-consuming. - and -thujone characterize the EOs of several Artemisia species, some of them used in the preparation of several bitters (e.g. absinth). A typical alpine bitter called genep is prepared from an infusion of a mixture of A. umbelliformis Lam., A. genipi Weber and A. glacialis L. This bitter is characterized by its flavour, which is closely related to the plants volatile fractions and by its bitter taste, due to germacrene-derived sesquiterpene lactones.16 A. umbelliformis is a relatively rare and expensive plant (about 80 $/kg) growing wild at high altitudes; it is difficult to cultivate because it requires a specific habitat. Its EO is characterized by a very low yield (0.1% on dry weight) with percentages of - and -thujone in the range 4575%.17 Figure 5 shows the GC profiles of the two chemotypes of A. umbelliformis EOs. A cultivated thujone-free chemotype (1,8-cineole and borneol) with a different smell and taste

Copyright 2008 John Wiley & Sons, Ltd.

Flavour Fragr. J. 2008; 23: 382391 DOI: 10.1002/ffj

390 C. BICCHI ET AL.

Table 6. Measurements of response factors (RF) for the different chemical groups Compounds
Aldehydes Citronellal Decanal Octanal Aromatic aldehydes Benzaldehyde Alcohols Citronellol Linalool Nerol Terpinen-4-ol -Terpineol Esters Citronellyl acetate Geranyl acetate Linalyl acetate Neryl acetate Nonyl acetate

Mean STD
1.31 0.04 1.29 0.01 1.32 0.04 1.27 0.01 1.30 0.02 1.31 0.03 1.32 0.02 1.30 0.03 1.26 0.02 1.61 0.03 1.62 0.01 1.56 0.01 1.59 0.02 1.59 0.01

RF

Compounds
Monoterpene hydrocarbons -Pinene -Pinene -Terpinene Limonene Myrcene Sesquiterpene hydrocarbons -Humulene (E)-Caryophyllene Aromatic hydrocarbons p-Cymene Ketones 6-Methyl-5-hepten-2-one Camphor Ethers 1,8-Cineole Oxides Caryophyllene oxide

Mean STD
1.02 0.02 1.04 0.02 1.02 0.02 1.03 0.02 1.05 0.02 0.96 0.01 1.00 0.03 0.99 0.03 1.31 0.01 1.29 0.03 1.28 0.01 1.53 0.02

RF

1.30 0.02 1.27 0.01

1.03 0.01

0.98 0.03 0.99 0.03 1.30 0.01 1.28 0.01 1.53 0.02

1.30 0.02

1.59 0.02

the latter being useful to correct possible shift of FID responses (absolute area) with different chemical groups of analytes. As already observed for true quantitation, the complexity of an EO, together with the lack of standards, makes RF determination of all its components unrealistic. Recently, Costa et al.20 introduced a possible solution based on grouping the EOs by their functional groups, such as aldehydes and alcohols, and then by their chemical classes, such as mono- and sesquiterpenic components, etc. Calibration curves were then constructed with commercially available standards representative of each group; moreover, the reliability of the results is increased if more than one standard per group, if available, is used. RFs were calculated through equation (1), adopting nonane as internal standard20 and assuming that strict linearity exists between the GCFID response and the carbon number of each hydrocarbon: RF = Canalyte/[(Aabs analyte/Aabs i.s.)] Ci.s. (1)

CVOC = {[(AabsVOC / Aabs i.s.) Ci.s. RF ]/Woil} 100 (2) where CVOC is the concentration, expressed as g/100 g EO, of the target volatile compound, AabsVOC is its absolute peak area, Woil is the weight of the oil expressed in grams, and the other terms are as stated above. It must clearly be stated that a reference standard with similar structure and carbon atom number must carefully be chosen to calculate a reliable RF for each group of compounds. This approach was successfully applied to the quantitation of Tarchonanthus camphoratus L. and Pistacia lenstiscus L. EOs. In both studies, RFs and calibration curves were determined by diluting each standard in hexane, at five different concentrations, each specimen containing an internal standard with known absolute concentration; analyses were performed in quintuplicate. From the chromatographic peak areas, the total identified fraction of the T. camphoratus oil accounts for 85.66%, mainly consisting of monoterpene hydrocarbons (38.71%) and oxygen-containing components (37.94%; oxygenated monoterpenoids, alcohols, aldehydes, ketones, esters and oxides). On the other hand, true quantitative GCFID analyses using correction factors revealed an amount of identified volatile fraction of the T. camphoratus oil equivalent to 84.71 g/100 g, chiefly consisting of 33.73 g/ 100 g monoterpene hydrocarbons and 43.09 g/100 g oxygencontaining components. The sesquiterpene hydrocarbon fraction is also noteworthy: it accounted for 6.54% and 5.70 g/100 g, respectively, of the identified compounds in the relative and true quantitation analysis. True quantitative (g/100 g) data showed a decrease in total hydrocarbons compared to percentage peak areas, while at the same time the oxygenated fraction increased. This variation may be due to the FID response that, for hydrocarbons, is equal to 1.0, while of the oxygenated compounds it is 1.3 for alcohols and 1.4 for aldehydes. A similar trend was observed with the EOs of Pistacia lenstiscus twigs and

where Canalyte is the concentration of the standard compound representing a chemical group (e.g. limonene for monoterpene hydrocarbons), Aabs analyte is its absolute peak area, Aabs i.s. is the nonane absolute peak area, and Ci.s. is its concentration (0.1 g/100 g). The nonane RF is taken here as a unitary reference, and the average RFs obtained for each standard compound within a chemical class are then used as correction factors specific for each chemical class. This procedure gave RF values, relative to nonane, of 1.0 for monoterpene and sesquiterpene hydrocarbons, 1.3 for compounds with one oxygen atom, such as aldehydes, alcohols, ethers and ketones, and 1.6 for those with two oxygen atoms, such as the esters. Table 6 reports the experimental RF values relative to nonane.20 With these response factors, or correction factors, the components of an EO can be quantified through the following equation:

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Flavour Fragr. J. 2008; 23: 382391 DOI: 10.1002/ffj

QUANTITATIVE ANALYSIS OF ESSENTIAL OILS: A COMPLEX TASK

391

leaves obtained by hydrodistillation (HD) and simultaneous distillationextraction (SDE), and analysed by GCFID, using the calibration curves and RF values as determined for the T. camphoratus EO analysis.21
AcknowledgementsThis research was carried out within the project entitled Sviluppo di metodologie innovative per lanalisi di prodotti agroalimentari (FIRB Cod.: RBIP06SXMR_002) of the Ministero dell Istruzione, dell Universit e della Ricerca (MIUR), Italy.

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Flavour Fragr. J. 2008; 23: 382391 DOI: 10.1002/ffj