Review of the Analytical Techniques for Sesquiterpenes And

967 (2002) 115–130Journal of Chromatography A,www.elsevier.com/ locate/chroma

Review

Review of the analytical techniques for sesquiterpenes andsesquiterpene lactones

*Irmgard MerfortInstitute of Pharmaceutical Biology, Albert-Ludwigs-University Freiburg, Stefan-Meier-Str. 19, 79104Freiburg, Germany

Abstract

In this paper the analytical techniques of about the last 2 decades for sesquiterpenes including their lactones are reviewed.13For sesquiterpenes, methods like GC, GC–EI-MS, GC–CI-MS, GC–MS–MS, GC–FT-IR, GC–UV, GC–AES, C-NMR,

PY–GC–MS, HPLC, HPLC–TSP, SFE, SFC, SFC–UV are available, GC combined with MS is the most widespread.Sesquiterpene lactones can be analysed by HPLC, HPLC–TSP, HPLC–APCI, HPLC–ESI, HPLC–PB, HPLC–NMR, SFC,MEKC, GC, GC–MS, TLC and OPLC. Here HPLC is the method of choice. The usefulness of the individual methods arebriefly discussed. 2002 Elsevier Science B.V. All rights reserved.

Keywords: Reviews; Terpenoids; Sesquiterpenes; Sesquiterpene lactones

Contents

1. Introduction ............................................................................................................................................................................ 1162. Analytical methods for sesquiterpenes (without lactones) ........................................................................................................... 116

2.1. Gas chromatography ....................................................................................................................................................... 1162.2. Gas chromatography–mass spectrometry (EI-MS, CI-MS, MS–MS) .................................................................................. 1182.3. Gas chromatography–FT-IR spectroscopy ........................................................................................................................ 1182.4. Further combined techniques: GC–UV and GC–AES ........................................................................................................ 119

132.5. Combination of C-NMR or pyrolysis with GC–MS ........................................................................................................ 1192.6. High-performance liquid chromatographic analysis ........................................................................................................... 1192.7. Combined techniques with HPLC .................................................................................................................................... 1212.8. Supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC)............................................................... 123

3. Analytical methods of sesquiterpene lactones (SLs) ................................................................................................................... 1233.1. HPLC with UV detection................................................................................................................................................. 1233.2. Combined techniques with HPLC: HPLC–MS and HPLC–NMR ....................................................................................... 1263.3. Supercritical fluid chromatography and micellar electrokinetic chromatography (MEKC) .................................................... 1273.4. Gas chromatography ....................................................................................................................................................... 1273.5. Thin-layer chromatography.............................................................................................................................................. 127

4. Nomenclature ......................................................................................................................................................................... 128Acknowledgements...................................................................................................................................................................... 128References .................................................................................................................................................................................. 129

*Tel.: 149-761-2032-804; fax:149-761-2032-803.E-mail address: [email protected] (I. Merfort).

0021-9673/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0021-9673( 01 )01560-6

967 (2002) 115–130116 I. Merfort / J. Chromatogr. A

1. Introduction 2. Analytical methods for sesquiterpenes(without lactones)

Sesquiterpenes are C-15 terpenoids which occur as Recently, Kubeczka [4] proposed that the ana-hydrocarbons or in oxygenated forms such as al- lytical methods applied to the analysis of essentialcohols, ketones, aldehydes, acids or lactones in oils can be classified into three different groups.nature. They are important constituents of essential Concentrating only on sesquiterpenes this classifica-oils, which have many applications in medical, but tion can be adopted, too:also in soap and perfume formulations. Moreover, —Chromatographic methods like gas chromatog-they are found as flavour compounds in aroma raphy (GC), high-performance liquid chromatog-mixtures. Essential oils as well as aroma mixtures raphy (HPLC) and supercritical fluid chromatog-have very complex compositions from the chemical raphy (SFC), including multidimensional and chro-standpoint. Most often the single constituents only matographic coupling techniques resulting in theoccur in traces. Therefore effective chromatographic separation to individual components.techniques are required for optimal separation and —Hyphenated techniques that means instrumentalidentification or isolation of the individual com- on-line couplings of chromatographic separationponents. devices to spectrometers like coupling of GC with

Within the group of sesquiterpenes their lactones mass spectrometry (MS), Fourier transform infrareddeserve special interest. These secondary metabo- spectrometry (FT-IR), UV or atomic emission spec-lites derive from the basic sesquiterpene carbon troscopy (AES) as well as coupling of HPLC withskeleton, in which one of the methyl groups of the MS. The advantage of those techniques is that moreisopropyl group is oxidized to the lactone group information about the structure of the separated[1]. They are mainly found in several genera of components is available and often identification isAsteraceae, but also in Umbelliferae and Mag- possible.noliaceae [2]. These compounds are of interest not —Methods like MS spectrometry and especially

13only from chemical and chemotaxonomical stand- C-NMR spectroscopy resulting in the identificationpoints, but also because many of them possess of a multi-component sample without previous sepa-biological and therapeutic activity including anti- ration.inflammatory, antitumoral, antimicrobial, anthelmin-tic and anti-feeding [3]. Because of these effects, 2.1. Gas chromatographypreparations of plants containing sesquiterpene lac-tones are often used in traditional medicine. To GC is the most efficient chromatographic tech-guarantee the same quality of the medicinal plants nique for separating volatile mixtures, because of itschromatographic techniques including qualitative high resolving power and the availability of universaland quantitative analysis are used. Moreover, these detection using flame ionization detection (FID).methods are also helpful tools in isolation and Mostly capillary columns with dimethyl polysiloxaneidentification of these compounds. (methyl silicone) non-polar and Carbowax 20M polar

In the past several papers have focused on chro- phases are used. Carbowax 20M phases include DB-matographic methods used in the analysis of ses- Wax, BP-20, PEG 20M and HP 20, while methylquiterpenes [4,5]. In the meantime some new ana- silicone phases include SE-30, SF-96, OV-1, OV-lytical techniques, especially combined ones, have 101, BP 1, CPSIL 5CB, SP 2100, DB 1, DB 5 andbeen developed. In this review all the methods of HP 1 [6]. Among these fused-silica capillary GCabout the last 2 decades successfully applied to the columns DB 1 or DB 5 and CPSil 5 are mostlyanalysis of these secondary metabolites are summa- preferred. Identification based on direct comparisonrized and discussed. Because of the importance of retention times with standards or precise knowl-

´mentioned above their lactones are discussed in a edge of retention indices, e.g., Kovats’ retention´separate chapter. indices [6–9]. Recently some 900 Kovats’ indices of

967 (2002) 115–130 117I. Merfort / J. Chromatogr. A

400 individual compounds were summarized fromthe general literature [6].

One of the most important developments in gaschromatography has been the introduction of enan-tioselective capillary columns with a high separationefficiency in the mid-1960s by Gil-Av et al. [10].Hereby a great number of chiral substances including

Fig. 1. Structures of enantiomeric compounds separated by a 25many essential oil constituents can be separated. Form fused-silica capillary column with heptakis(6-O-tert.-butyldi-

more than 10 years, phases exclusively based onmethyl-silyl-2,3-di-O-methyl)-b-cyclodextrin (20% in OV 1701)chiral diamide structures and concomitant hydrogen [13].bonding interactions. The first optimum performancewas reached in the development of Chirasil-Val, amethyl–polysiloxane phase containing about 6% of a single column is not sufficient, especially whenbranched aliphatic side chains withL-valine in the mixture is very complex. Then two-dimensionaldiamide linkage, and similar polymeric chiral capillary gas chromatography is a valuable tool fordiamide stationary phases [11,12] which possessed reliable stereochemical assignments. At first thehigh thermal stability. Specific derivatization pro- essential oil is pre-separated on a non-chiral station-cedures, such as formation of carbamates, cyclic ary phase (e.g., a 25 m capillary with CPSil 5).carbonates or oximes, introduced sites for hydrogen Afterwards small fractions are automatically orbonding interactions and enhanced the range of manually transferred to a second column [e.g., a 25applications (for literature see Ref. [13]). The analy- m capillary with heptakis(2,6-di-O-methyl-3-O-sis of patchoulol oil used in perfume may serve as an pentyl)-b-cyclodextrin]. For this combined techniqueexample that the resolution of a sesquiterpene mix- two FIDs and a ‘‘live switching’’ coupling piece forture is sometimes better on Chirasil-Val than on peak transfer from the polysiloxane to the cyclo-methyl polysiloxane such as OV-101 [14]. dextrin column are necessary [4,13]. Enantioselective

Finally, the introduction of capillary columns capillary gas chromatography can be a valuable toolusing different hydrophobic cyclodextrin derivatives in quality control of essential oils, such as that fromwith a higher thermal stability considerably im- chamomile. It is possible to separate all four stereo-proved the separation facilities [4,13]. Cyclodextrins isomers ofa-bisabolol. Thus naturala-bisabolol canwere modified by the introduction of alkyl and acetyl be differentiated from nature-identical, racemic, ma-groups giving hydrophobic cyclodextrin derivatives, terial in pharmaceutical and cosmetic formulationswhich interact enantioselectively with a great variety [15].of chiral constituents of essential oils by forming In gas chromatography, FID is the most commondiastereomeric inclusion complexes. Thus, separation detection method used. Nitrogen-containing com-of saturated hydrocarbons is possible as well as that pounds can be identified by nitrogen–phosphorus

3of compounds with several functional groups [13]. detection (NPD) [8] and H-labeled sesquiterpenesSesquiterpene enantiomers can be preferably re- by a radioactivity monitor [16]. A serious problem insolved with 25 m capillaries with heptakis(2,6-di-O- essential oil mixtures is the complete separation ofmethyl-3-O-pentyl)-b-cyclodextrin or heptakis(6-O- the hydrocarbons from the oxygenated compounds,tert.-butyldimethyl-silyl-2,3-di-O-methyl)-b-cyclo- which sometimes possess very low polarities and aredextrin (both 50%, w/w, in OV-1701) [13] as shown obtained in the hydrocarbon fraction. This problemwith a enantiomeric mixture ofa-cubebene,b- can be solved by solid-phase extraction (SPE) orcubebene, andb-elemene (see Fig. 1). Carrying out solid-phase microextraction (SPME) followed by GCGC analysis it became evident that many of the analysis [17,18], e.g., SPE with silica gel as station-chiral constituents were present in a certain propor- ary phase and hexane and diethyl ether as mobiletion of the enantiomers. Pure chiral compounds were phases gives a good separation between sesquiter-found only in a few cases. Sometimes the selectivity penes and their oxygenated derivatives. Another


possibility is the use of oxygen flame ionization could be proven that the use of ammonia was by fardetection (O-FID) for the selective determination of superior to isobutane [29]. An additional method is

2oxygenates [4,14,19]. This application seems to be negative ion chemical ionisation (NCI) with OH asparticularly useful if very small samples have to be the reactant ion [30–32]. However, these techniquesanalyzed, e.g., from tissue cultures or oil glands. The have the disadvantage that there is no characteristicO-FID analyzer works in such a way that first a fragmentation pattern in the mass spectra. Thiscracking reaction forms carbon monoxide which is problem can be solved for sesquiterpenes with onereduced in a second reactor yielding equimolar and two non-conjugated double bonds by usingquantities of methane. The latter product can be trioxo(tert.-butylimido)osmium(VIII) [33]. The re-sensitively detected by FID. The GC analysis of agent forms cyclic osmate ester amides as inter-peppermint oil reported by Kubeczka [4] demon- mediates which gave after reduction vicinal mono-strates the usefulness of this detection method. amino alcohols, bisamino alcohols and aminotriols.

These derivatives show characteristic mass spectra2.2. Gas chromatography–mass spectrometry (EI- which can be used as a fingerprint for identificationMS, CI-MS, MS–MS) of the respective parent compound.

GC–MS is a very useful tool for the analysis ofWith the advent of high-resolution capillary GC complex mixtures, but sometimes identification is

using fused-silica columns, separation of complex limited when a single chromatographic peak containsmixtures of sesquiterpenoids was possible and the several compounds so that the recorded mass spectranumber of sesquiterpenoids isolated from essential are difficult to interpret. There are several possi-oils has increased into the hundreds in recent years. bilities to solve this problem. One is MS–MSAgainst this background identification only by GC (tandem mass spectrometry), which, when coupled

´retention data and Kovats’ retention indices alone with GC, allows separate analysis of each componenthas become uncertain. Therefore, nowadays the of such complex peaks. Moreover, the presence ofcombination of gas chromatography and mass spec- minor constituents can also be confirmed [31,34].trometry in the electron impact mode (GC–EI-MS) The analysis of vetiver oil consisting of 150 com-is a well established technique for the routine ponents shows, that the combination of GC withanalysis of essential oils. This technique offers the different MS techniques (EI-MS, CI-MS, MS–MS)possibility to gain additional information by mass gives the best results in the resolution of a complexspectra [4]. However, it has to be emphasized that mixture. Altogether, 118 compounds have beenidentification of sesquiterpene hydrocarbons based characterized [34].only on mass spectra is also virtually impossible.Molecular rearrangement and isomerization pro- 2.3. Gas chromatography–FT-IR spectroscopycesses of unsaturated hydrocarbons result in verysimilar mass spectra lacking characteristic frag- GC–MS is considered to be the method of choicementation patterns. Only combined data of retention in the identification of volatile compounds including

´times, Kovats’ or Sadler retention indices [20] and sesquiterpenoids. However, in distinguishing iso-mass spectral data offer the possibility of an un- mers, which often occur in the terpene group,ambiguous identification of sesquiterpenoid con- capillary GC–FT-IR coupling offers a useful supple-stituents. In the last decade numerous papers have mentation [35]. Herres and Kubeczka have writtenbeen published using these combined GC–MS tech- an excellent review on this method [35,36]. Advan-niques [9,21–28]. tages of FT-IR spectroscopy are high resolution and

Nevertheless, sometimes mass spectra measured in sensitivity. The more time-consuming interpretationEI mode are problematic, because they miss the and the absence of a database of reference vapourmolecular ions, specially with esters. In this case, the phase spectra may be reasons why this technique isapplication of GC–MS in the chemical ionization not generally accepted for the analysis of volatile(CI) mode using various reagent gases often yields compounds. However, a GC–FT-IR-MS instrumentvaluable additional information. For sesquiterpenes it is available, whereby simultaneously IR and mass


spectra can be obtained. Thus, the unambiguous spectrum with spectra of pure compounds. Quali-identification of critical isomeric sesquiterpenes is tative as well as quantitative analysis is possible.possible. Furthermore, PY–GC–MS can be used as a mi-

crotechnique for the simultaneous analysis of variousclasses of compounds shown withVitex agnus-castus

2.4. Further combined techniques: GC–UV andandOriganum heracleoticum [40,41]. Samples in the

GC–AESsubmilligram range are heated at typically 500–10008C in the inert atmosphere of the GC carrier gas. The

The combined techniques GC–UV or GC–AESpyrolysis products are swept directly into the gas

mentioned in the review of Kubeczka [4,37] have notchromatograph and result in a pyrogram which is

gained much importance up to now. GC–UV doescharacteristic of the original sample. On the other

not possess any significant advantage compared tohand, flash vaporization for molecules of low mass

GC–MS. UV spectra only offer low information,can be carried out by pyrolysis. The molecules are

even less than FT-IR spectra. The coupling oftightly adsorbed on the sample matrix, thereby

capillary gas chromatography with atomic emissionextraction and concentration steps are saved.

spectroscopy offers the possibility to the analyst toprovide information on the elemental composition of

2.6. High-performance liquid chromatographicthe individual components of a mixture and, more

analysisimportantly, on the percentual elemental compositionof a component [38]. However, this is also possible

The relatively good separation obtained by GC hasby precise mass measurement. Altogether, both

delayed the application of HPLC to the analysis oftechniques can give additional information being to

volatile compounds, such as sesquiterpenoids (Tablesome extend complementary to MS and/or IR data.

1). However, HPLC analysis, which is has been wellreviewed previously [5,42], offers some advantages

132.5. Combination of C-NMR or pyrolysis with compared to GC. HPLC can be carried out when GCGC–MS analysis of thermolabile and/or polar compounds is

difficult to achieve, involatile materials has to beSometimes it may be useful to confirm by an handled or preparative isolation of volatile com-

additional analytical method the results of capillary pounds is required. The coupling with diode arrayGC and GC–MS, as it was shown with the rhizome detection (DAD) can be useful. Restricting factors

13oil of Piper betle [39]. Here C-NMR followed GC for application of HPLC in sesquiterpene analysis areand GC–MS analysis to confirm the structure assign- the limitations inherent in the commonly availablements proposed by retention data and mass spectra. detectors for HPLC. Many volatile sesquiterpenoidStructurally, closely related molecules such as components lack chromophoric groups and cannot bestereoisomers, which exhibit insufficiently resolved analysed by HPLC with UV detection at 254 nm. Inmass spectral patterns, and compounds inseparable these cases refractive index or low UV monitoringby GC, such as T-muurolol and T-cadinol can be are necessary. Often low UV monitoring is preferred,identified in this way. However, this analysis has because refractive index (RI) detection is not sensi-sometimes limitations, too. Spectra containing an tive enough and low concentrations are not detect-immense density of individual lines, especially in the able. These problems limit the applicability of sever-aliphatic region, are difficult to elucidate. Here the al solvents, because considerable background absorp-ambiguous assignment of signals may be very dif- tion at low wavelengths has to be avoided. Anotherficult. To make specific assignments more certain, problem in HPLC is the restricted peak capacity anddistorsionsless enhancement by polarization transfer the relatively small range ofk9 values of a liquid(DEPT) experiments can be carried out [4]. By this chromatographic system, by which effective sepa-method, primary, secondary, tertiary or quarternary ration of multicomponent mixtures in one operationcarbon atoms can be distinguished. In both cases is often not possible. Temperature is an importantidentification is based on comparison of the sample factor which controlsk9 values. Therefore analysis at


Table 1Analytical separations of sesquiterpenes by HPLC

Sample Column Detection Ref.

Solanum tuberosum mBondapak C (10mm) 255 nm [47]18

(sesquiterpenoid stress metabolites) (30 cm33.9 mm)

Valeriana officinalis RP 18 (Knauer) 225 nm [52](25 cm34.6 mm)Superspher (5mm) 254 nm [53](25 cm34 mm)

Lime oil Whatman Partisil-PXS (5 or 10mm) Refractometer [45](mono- and sequiterpenes) (25 cm34.6 mm)

Sesquiterpene hydrocarbons, Nucleosil C (7mm) 220 nm [44]18

sesquiterpene alcohols LiChrosorb RP 18 (10mm)Zorbax C (7–8mm)18

(25 cm34.6 mm)

Echinacea species Hibar 125-4 with LiChrospher 100 280 nm [54](sesquiterpene ester)

Cotton stele Radial PAK C (10mm) 254 nm [46]8

(sesquiterpenoid stress metabolites) (10 cm38 mm)

Sesquiterpene Spherisorb CN (5mm) 205 nm [48](phytoalexins) (10 cm33 mm)

Polygonum hydropiper Microsorb C (5mm) 116 nm [50]18

(polygodial) (15 cm34.6 mm)

Petasites hybridus Nucleosil-100 (3mm) 254 nm [49](sesquiterpenes) (2534 mm)

Ylang ylang oil Chiralcel OB 255 nm [51]Solidago altissima (25 cm34.6 mm)(germacrene-D derivative)

low temperatures, e.g.,215 8C afforded better —Qualitative and quantitative detection of specificseparations [5]. This could be proven on a LiCh- volatile constituents.rosorb Si 60 (7mm) column with n-pentane as —Semipreparative or preparative isolation of vola-solvent [42]. By these conditions a mixture of the tile compounds.sesquiterpene hydrocarbons, containinga-copaene, HPLC is often used as prefractionation of complexd-elemene,b-elemene,b-caryophyllene,a-bergam- mixtures into groups of components in order tootene,b-bisabolene,a-humulene andd-cadinene was simplify GC and GC–MS analysis. Several examplesseparated. Silica gel was at first deactivated by are given in Ref. [42,43]. Using reversed-phaseadding 4.8% water, because otherwise dry or fully materials, e.g., RP18, fractionation is carried outactivated adsorbents would possess undesirable prop- according to the polarity and the chain length oferties such as sample alteration and irreversible essential oil constituents [44]. Most often threeadsorption [37,38]. groups containing either oxygenated terpenes, mono-

The applications of HPLC in the analysis of terpene hydrocarbons or sesquiterpene hydrocarbonsvolatile compounds can be placed in three major are obtained. The use of a reversed-phase columngroups: involves aqueous solvents, isocratic or gradient

—Prefractionation of complex volatile mixtures elution and UV detection. The pattern of elution isprior to GC or GC–MS analysis. reversed on silica gel, that means starting with


hydrocarbons, etc. [45]. In this approach isocratic Previously it was shown that enantiomeric resolutionnon-aqueous solvent mixtures and, in most cases, RI can also be achieved by HPLC analysis. This wasdetection is necessary [45]. The obtained fractions demonstrated with germacrene-D, which was sepa-can be subsequently separated by GC analysis. In rated in its enantiomers by a Chiralcel OD columnRef. [5] numerous examples are given, e.g., prefrac- [51]. HPLC technique of sesquiterpenes can be usedtionation of Citrus, or Valeriana essential oil. in quality control of medicinal plants, when these

Another field of HPLC application is the quali- compounds can serve as lead compounds. Valerenictative or quantitative determination of specific con- acid and valerenal (for structures see Fig. 3) arestituents in complex mixtures. In Ref. [46] a HPLC sesquiterpenes which only occur in roots fromprocedure is described for the determination of four Valeriana officinalis [52,53]. They can be used toantifungal sesquiterpenoid stress metabolites, exclude the occurrence of otherValeriana species.hemigossypol, 6-methoxyhemigossypol, desoxyhem- This is the same withEchinacea species which canigossypol and 6-methoxydesoxyhemigossypol (for also be differentiated by sesquiterpenes [54].structures see Fig. 2), in cotton stele infected with A further application of HPLC which has to beVerticillium dahliae. Analysis was carried out on a mentioned is its use in the isolation of pure com-Radial-PAK reversed-phase C column (10mm), pounds. Here often a combination of reversed-phase8

eluted with a 0.1% aqueous phosphoric acid–metha- HPLC pre-fractionation followed by liquid–solidnol gradient, and monitored at 254 nm. Stress-in- chromatographic HPLC separation can afford pureduced sesquiterpenes of the potato can be determined compounds. Another successful way for difficulton a reversed-phasemBondapak C column using separations is modifying commercial prepacked pre-18

methanol–water (7:3) and a detection wavelength of parative silica gel columns by treatment with silver200 nm [47]. Other systems used for sesquiterpene ions [55–57]. To achieve quantitative coating of theanalysis are: a Spherisorb CN column (5mm) and silver salt on the support it seems the best to dissolvehexane–isopropanol as solvent [48] or a Nucleosil- silver perchlorate in a non-polar solvent such as100 column (3mm) and a hexane–diisopropylether– toluene. Thus, a couple of isomeric non-polar ses-acetonitrile gradient [49]. In Ref. [50] the quantita- quiterpene olefins from tolu balsam could be isolatedtive analysis of polygodial inPolygonum hydropiper (see Fig. 4).leaves is described using a Microsorb 5mm C18

stationary phase and acetonitrile–water as isocraticsolvent. Additional examples are given in Ref. [5]. 2.7. Combined techniques with HPLC

As mentioned in Section 2.6. HPLC can be usedas pre-fractionation followed by further chromato-graphic analysis, such as GC–MS. This procedurecan be recommended when the essential oil is a verycomplex mixture, so that overlap of some peaksoccur and minor compounds are not detected. Thisanalytical performance is described in Ref. [44] forVervain essential oil or in Ref. [45] for lime oils.

In Ref. [58] this technique is more developed. Anautomated HPLC–high resolution GC (HRGC) anal-ysis is described for the volatile fraction of citrus oil.HPLC was used to separate the oil in three fractions,which were automatically transferred to the GCwithout interferences. Furthermore, it is possible to

Fig. 2. Structures of sesquiterpenoid stress metabolites fromcouple GC and MS so that the components can becotton stele, hemigossypol (1a), 6-methoxyhemigossypol (1b),identified by their retention time as well as by theirdesoxyhemigossypol (2a) and 6-methoxyhemigossypol (2b), sepa-

rated by HPLC analysis [46]. mass spectrum. The results of this work show that


Fig. 3. Sesquiterpenes from the medicinal plantsValeriana officinalis andEchinacea purpurea—HPLC analysis for quality control [52–54].

combined HPLC–HRGC has a great potential for the Mondello et al. [58] who separated aliphatic, mono-analysis of complex mixtures, specially valuable for terpene and sesquiterpene aldehydes from sweetquality control of essential oils in industrial lab- orange oil by this technique.oratories. The technique enables identification and For the solution of special problems, e.g., whenseparation of compounds of the same polarity from a thermosensitive sesquiterpenes have to be separatedmixture of components of different polarity, even if and identified, HPLC coupled with mass spec-the relative concentrations of the various classes of trometry can be used. This is demonstrated withcomponents vary considerably. This was shown by prehelminthosporol, a phytotoxin formed by the

plant parasitic fungusBipolaris sorokiniana. Quanti-fication was possible using reversed-phase on-lineHPLC–MS and plasma spray ionization [59]. Adetection limit of¯5 ng was obtained using selectedion monitoring atm /z 219. HPLC combined withthermospray mass spectrometry is a further couplingtechnique often used in phytochemical analysis forscreening of extracts. The usefulness was shownwith crude extracts of the pathogenic basidiomyceteArmillaria which were screened for sesquiterpenearyl esters [60]. However, it has to be mentioned thatmass spectra obtained by ionisation methods lackcharacteristic fragmentation pattern. Therefore, un-ambiguous identification of unknown compounds isnot possible using only retention time, UV spectraFig. 4. Structures of aromadendrene (1, 4, 5) and guaiane (2, 3)and molecular mass, which is obtained from thederivatives from tolu balsam separated by HPLC on silver-loaded

LiChrosorb Si 60 [55]. mass spectra. Liquid chromatography–mass spec-


trometry has to be considered only as a suitable HPLC techniques [63]. By modifying carbon dioxidemethod for screening of crude fractions or for with 0.5% of methanol sesquiterpene alcohols, suchcomparative analysis. as nerolidol,a-bisabolol and (2E,6E)-farnesol were

separately eluted in a rather short time (10 min).2.8. Supercritical fluid extraction (SFE) and Addition of methanol is essential, otherwise thesupercritical fluid chromatography (SFC) sesquiterpene alcohols would not be eluted from the

silica-packed column. This is due in part to theAnalysis of sesquiterpenes may often cause prob- carbon dioxide, which is not a good enough solvent

lems because of their thermosensitivity. This proper- for sesquiterpene alcohols, but also to the absorptionty may have a negative effect in the isolation of hydroxyl groups of sesquiterpene alcohols onprocedure as well in analysis carried out for identifi- residual silanol sites of the stationary phase.cation, e.g., GC or GC–MS. Liquid phase solventextraction, steam distillation or headspace are fre-quently used, even though they are time consuming, 3. Analytical methods of sesquiterpene lactonesmay yield low extraction efficiencies, and may result (SLs)in loss or degradation of the components. Theseproblems can be solved by using SFE. It has been Following the classification of the sesquiterpenesreported that this technique is selective and highly in different groups analytical methods of SLs can beefficient in the isolation of sesquiterpenes [61,62] divided into two groups:and even superior to distillation–solvent extraction —Chromatographic methods like HPLC; SFC,[62]. Many supercritical fluids, such as carbon GC, TLC, OPLC and MEKC for the separation indioxide, have low critical temperatures which en- individual components.ables extractions to be performed at relatively low —Hyphenated techniques such as GC–MS,temperatures, thus possible degradation of thermally HPLC–UV, HPLC–NMR or SFC–UV by whichlabile compounds is reduced. Concentration steps are characterization and often identification of the sepa-simplified, because carbon dioxide is a gas at room rated compound is possible.temperature. Therefore it is possible to couple theextraction step directly with chromatographic analy- 3.1. HPLC with UV detectionsis, e.g., with capillary gas chromatography [61].Nevertheless, by using SFE and on-line GC degra- SLs are compounds with low volatility and manydation may occur during GC analysis as already of them are thermolabile. Because of these propertiesmentioned above. HPLC, mainly on reversed phase, is the analytical

An analytical method which also uses supercritical method of choice for SL analysis in crude plantcarbon dioxide and bare silica as stationary phase is extracts (see Table 2 and Fig. 5). However, normal-SFC. Detection can follow by on-line coupling with phase gradient run HPLC byn-hexane–acetonitrile–UV spectroscopy [63] or FT-IR [64–66]. These isopropanol may be sometimes preferable [67,68].attractive techniques at low temperatures are suitable HPLC has been applied successfully to the analysisfor the analysis of extreme thermally labile com- of 33 pseudoguaianolides and xanthanolides of thepounds such as sesquiterpene hydrocarbons. The genusParthenium [69] as well as 21 pseudo-advantages as well as disadvantages of on-line guaianolides ofArnica chamissonis and 15 of A.coupling with FT-IR, have already been discussed in montana [70,71]. Acetonitrile–water or methanol–Section 2.3 and are excellently summarized in Ref. water gradients are used, respectively. In a com-[64]. Carbon dioxide offers large transparency re- prehensive study 37 SLs of theMelampodiinae havegions in infrared, so that many functional groups can been investigated [72]. Based on this method andbe detected. Chromatograms are reconstructed either combined with GC (see Section 3.3) a crude extractfrom interferograms by Gram-Schmidt algorithm or ofMelampodium cinereum was analysed. Furtherthrough an infrared absorbance window [65]. Com- studies revealed the usefulness of qualitative HPLCbined SFC–UV offers an alternative to the existing analysis for the detection of Sls in plant extracts,


Table 2Analytical separations of sesquiterpenes lactones by HPLC

Sample Skeleton* Column Solvent Detection Ref.

Parthenium species PGU, XA Ultrasphere-ODS CH CN–water 215 nm [69]3

(1534.6 mm)

Laurus nobilis GE, GU LiChrosorb RP 18 Water–CH CN 210 nm [78]3

(5 mm, 2534 mm)LiChrosorb RP 8(5 mm, 2534 mm)LiChrosorb RP 8(5 mm, 12.534 mm)

Cichorium intybus GU Radial-PAK C MeOH–water 258 nm [74]18

(1038 mm)

Arnica chamissonis PGU Shandon Hypersil ODS MeOH–water 225 nm [70](5 mm, 2534.6 mm)

Arnica montana PGU Shandon Hypersil ODS MeOH–water 225 nm [71](5 mm, 2534.6 mm)

Ambrosia maritima PGU Nucleosil C Water–CH CN 220 nm [73]8 3

(5 mm, 12.534 mm)

Helianthus species GE, GU Shandon Hypersil ODS MeOH–water 225 nm [80](5 mm, 2534 mm) 265 nm

Cichorium intybus GU Spherisorb C MeOH–water, 258 nm [75]18

(5 mm, 2034.6 mm)/ Water–CH CN,3

THF–water /LiChrosorb Si CH Cl –MeOH,2 2

(10 mm, 2534.6 mm) EtOAc–hexane,tert.-butyl-methyl ether–MeOH

Melampodium cinereum GE Shandon Hypersil ODS MeOH–water 210–254 nm [72]species of subtribeMelampodiinae (5 mm, 1534.6 mm)

SLs from different plants GE RP 8 MeOH–water 230 nm [67,68]EU (10 mm, 2034.6 mm)

Si-100 n-Hexane–CH CN–isopropanol3

(10 mm, 2034.6 mm)

Artemisia annua CLC-ODS Water–CH CN (1% TFA) 220 nm [84]3

(2534.6 mm) andBondclone 10 CN(3033.9 mm)

Artemisia leucodes GU Microcolumn of KAKh-2 MeOH–0.5 acetate buffer or 254 nm [79](2362 mm) THF–0.01M phosphate buffer

SLs from different plants GE, EU, GU LiChrosorb RP 18 MeOH–water 254 nm [87]PGU (7mm, 2534.6 mm)

Tanacetum species GE, EU, GU LiChrosorb RP 18 MeOH–water 250 nm [77](5 mm, 6232 mm)

Artemisia caerulescens EU LiChrospher 100-RP Water–CH CN 236 nm [82]3

(2434 mm)

Parthenium species PGU Nucleosil C MeOH–water 210 nm [76]18

(5 mm, 2534 mm)

*EU: Eudesmanolide; GE: germacranolide; GU: guaianolide, PGU: pseudoguaianolide; XA: xanthanolide.


Fig. 5. Structures of some sesquiterpene lactones analysed by HPLC.

e.g., pseudoguaianolides from the ambrosanolide- separation quality of some structural different SLstype from Ambrosia maritima [73], bitter SLs with a using either an RP 8 column of 12534 mm orguaianolide skeleton from chicory roots [74,75], 25034 mm. However, because of lower back pres-pseudoguaianolides (ambrosanolides and parthen- sure and shorter time of analysis the shorter columnolides) from Parthenium species [76] or germac- is preferred. For special applications a microcolumnranolides and eudesmanolides fromTanacetum may be useful [79].species [77]. In Ref. [78] some hints for application For a better sample preparation avoiding undesir-are given. To avoid severe peak leading, especially able compounds Spring developed a micro-samplingin early peaks, SLs should be solved in the same technique [80]. It was demonstrated that it is possibleratio of the solvent mixture rather than in only one to directly sample 20 glandular trichomes of leavessolvent. It was shown that there is no difference in and flower heads, where SLs are sequestered, under a


dissection microscope and prepare a water /water 3.2. Combined techniques with HPLC: HPLC–MSextract for analysis. This method is suitable for and HPLC–NMRchemotaxonomical studies as shown with the genusHelianthus [80]. As mentioned above, UV detection is sometimes

HPLC is a valuable tool for quantitative analysis, unsuccessful for SL analysis, because several SLstoo. The content of single SLs as well as the total show weakly absorbing chromatophoric groups or noamount can be determined shown with the complex chromophores at all. Therefore, other detection meth-mixture of SLs in flowers ofArnica chamissonis and ods, such as RI or evaporative light scatteringA. montana [70,71] which are used for Arnicae flos detection (ELSD) can be used to overcome thisin the German pharmacopoea. Quality control of the limitation [87,88]. Whereas these detection tech-flower drug as well as standardization of preparations niques are not yet widespread in HPLC analysis ofused in traditional medicine are possible by this SLs the combination with mass spectrometry hasmethod. Further reports on the same topic, but with become extremely helpful in the detection andless complex mixtures are the quantitation of hy- identification of SLs in raw plant extracts. Themenoxon inHymenoxys odorata and of helenalin, thermospray (TSP) interface was one of the firstmexicanin E and helenalin inHelenium species [81] which was developed in this field [89]. The massas well as santonin inArtemisia caerulescens [82]. spectra obtained by HPLC–TSP yield different

1 1In Ref. [83] it is reported that the combination of quasi-molecular peaks, e.g., [M1H] , [M1NH ] ,4

ODS and CN columns results in a good resolution or ions formed from solvent adducts. Further frag-with maximum recovery of the three SLs arte- ment ions resulting from the loss of water or acidmisinin, arteannuin-B and artemisinic acid in the residues may be obtained. The mass spectra arechinese medicinal plantArtemisia annua (‘‘qin- comparable with those from the direct chemicalghao’’). Because of the importance of artemisinin as ionization (DCI) technique in the positive ion modea novel antimalarial agent, several reports on quanti- [90].This method was successfully applied to thetation can be found in literature (see Ref. [84]). One identification of artemisinin inArtemisia annua [90]problem in HPLC analysis of artemisinin, specially or of chlorinated SLs being formed in the isolationin plant extracts, is its absorption at the low end of procedure in the neurotoxic thistleCentaurea sol-the UV spectrum, between 210 and 220 nm. There- stitialis [91]. Further techniques which give similarfore, ElSohly et al. [84] published a HPLC method results as with TSP are atmospheric pressure chemi-with postcolumn derivatization. Artemisinin is modi- cal ionization (APCI) or electrospray (ESP) coupledfied by 0.2% NaOH resulting in an UV absorption at with HPLC. Stuppner et al. showed that APCI292 nm. The mentioned problem of low absorption technique has to be considered more suitable for themaximum in UV can be applied to most sesquiter- detection of pseudoguaianolides in extracts frompene lactones. This property is often a limited factor Arnica montana [92]. Minor constituents could onlyin carrying out successful HPLC analysis of SLs in be detected by APCI. Nevertheless, HPLC–ESIcrude plant extracts especially of minor components gains more and more importance in the structure[85,86]. A solution of this problem, but only for SLs elucidation of isolated SLs for information on molec-containing a-methylenebutyrolactone functions, is ular mass [93], because the molecular ion is oftenderivatization by 9-thiomethylanthracene [85]. The missed in EI mass spectra. A further coupled tech-thiol containing reagent reacts in a Michael type nique worth to be mentioned is HPLC combinedaddition with thea-methylenebutyrolactone of the with particle beam (PB) mass spectrometry [87].SL and increases the sensitivity, so that routinely One of the main advantages of a PB interface is thenanogram quantities of the lactones can be detected possibility of recording mass spectra in both EI andby HPLC and monitoring at 369 nm. Parthenolide, CI modes, through a single analyser mass spectrome-the effective compound inTanacetum parthenium ter with medium-high sensitivity. In Ref. [87] 22used for the treatment of migraine and arthritis has structurally different SLs were separated and massbeen determined in such a way [85]. spectra in the EI as well as in CI mode, positive-ion


CI (PICI) and electron-capture negative ionization LiChrospher 100 RP 18, LiChrosorb RP 8, LiChro-(ECNI) with methane as reagent gas, recorded. The sorb Diol, S3W Silica Spherisorb, S3-nitrileHPLC–PB-EI mass spectra of the investigated SLs Spherisorb (CN). A CN separation phase withshowed exactly the same patterns as those obtained MeOH–water (95:5) as modifier for supercriticalby direct introduction. HPLC–PB-CI mass spectra CO was the most successful combination in the SLs2

often gave intense SL molecular or quasi-molecular investigated.ions, together with some other diagnostic ions. Based A further analytical method which gives similaron these data fractions fromArtemisia umbelliformis results as proven for the qualitative analysis of SLswere analysed. ofArnica flowers is MEKC [97]. MEKC is a rapid

LC–NMR is a further technique which combines analytical method which saves both time and solventhigh-performance separation techniques with a struc- compared with HPLC. However, retention times,turally informative spectroscopic method (for litera- especially the longer ones, are somewhat difficult toture see Ref. [94]). It allows extracts to be screened reproduce by MEKC.not only for structural classes but also for com-pounds without isolation of individual compounds.

3.4. Gas chromatographyIn the beginning this method has achieved limitedsuccess due to the lack of sensitivity. Recently, this

GC analysis are often used in the routine analysissituation has changed as new solvent suppression

of plant extracts which are screened for SLs. How-techniques have been introduced. The successful

ever, thermal degradation has to be considered.application of LC–NMR is demonstrated withVer-

Owing to this property, many heteroatom-substitutednonia fastigiata extracts, in which nine SLs includ-

sesquiterpene lactones cannot be analysed by GCing minor constituents could be identified [94].

without derivatization and trimethylsilyl ethers haveto be prepared [98]. Nevertheless, successful applica-

3.3. Supercritical fluid chromatography andtion of GC and GC–MS to underivatized SLs not

micellar electrokinetic chromatography (MEKC)being thermolabile have been reported in the litera-ture [70–72]. Qualitative and quantitative analysis of

Sesquiterpene lactones can be separated by SFC asextracts fromArnica flowers were carried out on a

already shown with sesquiterpenes in Section 2.825 m fused-silica capillary column coated with OV-

[86,95]. This method is especially suitable to ther-01-CB-0.25. Capillary GC analysis on a bonded

molabile SLs. Packed column SFC has a samplepolymethylsilicone stationary phase (RSL-150) of 37

capacity similar to HPLC, operates with the sameSLs of the Melampodiinae revealed good separa-

columns, but requires shorter analysis times andtions, but several of the compounds under study

allows more consistent column conditions duringdecomposed, either in the injector port or in the

repeated analysis. SFC has the disadvantage that thecolumn itself [72]. This was evidenced by multiple,

polar stationary phase can retain polar componentspoorly shaped peaks. Comparing separation power of

[86]. In Ref. [96] a method is described how aGC and HPLC analysis it was concluded that the use

synthetic mixture of three difficult-to-separate SLsof both systems in conjunction with one another is a

was separated by optimization on a short columnpowerful technique for the rapid analysis of plant

using the three-parameter simplex and then transfer-samples for these SLs.

ring the method to a longer column. Detection can becarried either by UV spectroscopy, demonstratedwith Artemisia umbelliformis and Carduus bene- 3.5. Thin-layer chromatographydictus [86] extract or by ELSD as proven withbilobalides in Ginkgo biloba and Artemisia annua TLC was one of the first chromatographic methods[88,95]. The latter paper also describes detection of and was also applied to analysis of SLs. Its impor-artemisinin and artemisinic acid by SFC–FID. In tance has decreased nowadays. However, in combi-Ref. [86] a couple of stationary columns were tested: nation with special spray reagents TLC can provide


valuable information about the type of SL.Visualiza- 4. Nomenclaturetion reagents are numerous, e.g., vanillin /o-phos-phoric acid, anisaldehyde orp-dimethylaminobenzal- AES Atomic emission spectroscopydehyde-sulfuric acid, sulfuric acid, resorcin-sulfuric APCI Atmospheric pressure chemical ioniza-or phosphoric acid, aluminium chloride or hydroxyl- tionamine [67,68,99–104]. SLs lacking an exocyclic-a- CC Column chromatographymethylene group can be visualized by dimethylamine CI Chemical ionizationfollowed by Dragendorff reagent [68]. In general, it DAD Diode array detectiondepends on the skeleton which of the reagents is DCI Direct chemical ionizationmore favourable. TLC can be used for isolation ECNI Electron-capture negative ionizationcontrol in column chromatography, e.g., open col- EI Electron impactumn chromatography (CC) or middle-pressure liquid ELSD Evaporative light scattering detectionchromatography (MPLC) and identity control of ESI Electrospray ionizationmedicinal plants as published forArnica flowers DEPT Distortionless enhancement by polariza-[105]. TLC is here advantageous, because of its tion transferrapid, easy and cheap performance without the need FID Flame ionization detectionof a large instrumental equipment. However, com- FT-IR Fourier transform infrared spectrometrypared with HPLC– or GC–MS this chromatographic GC Gas chromatographymethod provides less information. Moreover, TLC HPLC High-performance liquid chromatographycan be used to separate diastereoisomers, such as HRGC High-resolution gas chromatographyparthenin and hymenin by multiple development of LC Liquid chromatographyTLC plates in the same solvent system [106]. These MEKC Micellar electrokinetic chromatographytwo SLs only differ in the configuration of the MPLC Middle-pressure liquid chromatographycylopentenone ring. In contrast, screening of com- MS Mass spectrometryplex plant extracts by TLC often causes problems MS–MS Tandem mass spectrometry[67,68], because the wide range of colors produced NICI Negative chemical ionizationwith all types of SLs makes its interpretation dif- NPD Nitrogen–phosphorus detectionficult. O-FID Oxygen flame ionization detection

A special TLC method, worth mentioning is OPLC Overlay pressure liquid chromatographyoverpressured layer chromatography (OPLC) PB Particle beam[67,68]. As the separation in OPLC chamber is much PICI Positive ion chemical ionizationfaster than in normal TLC, the separation time is PY Pyrolysisrelatively short and in the closed system there is no RI Refractive indexor limited chance providing artefacts. Therefore, SFC Supercritical fluid chromatographyOPLC is especially suitable for studying big number SFE Supercritical fluid extractionof samples. The two directional development allows SL Sesquiterpene lactonethe separation of up to 34 samples in a single run, SPE Solid-phase extractionwhile HPLC using RP 8 or Si 100 phases by SPME Solid-phase microextractionisocratic and gradient elutions, respectively, may be TLC Thin-layer chromatographythe preferred technique when rapid qualitative and TSP Thermosprayquantitative informations are needed. However, itwas demonstrated that OPLC combined with directdensiometry also allows rapid quantitative analysisof SLs, e.g., parthenolide derivatives in different Acknowledgementsplants of Zoegea leptaurea. Parthenolide can also

¨quantitatively determined inChrysanthmum parth- The author is grateful to Mrs. I. Hirschmuller-enium by using TLC–FID [107]. Ohmes for the help in collecting the literature.


[27] R.P. Adams, Identification of Essential Oil Components byReferencesGas Chromatography/Mass Spectrometry, Allured Publish-ing Corporation, Carol Stream, IL, 1995.

[1] W. Herz, in: V.H. Heywood, J.B. Harborne, B.L. Turner ¨[28] D. Joulain, W.A. Konig, The Atlas of Spectral Data of(Eds.), The Biology and Chemistry of the Compositae, Sesquiterpene Hydrocarbons, E.B.-Verlag, Hamburg, 1998.Academic Press, London, 1977, p. 337, Chapter 11. [29] G. Lange, W. Schultze, in: P. Schreier (Ed.), Proceedings of

[2] F.C. Seaman, Bot. Rev. 48 (1982) 121. the International Conference, Bioflavour 87, de Gruyter,[3] A.K. Picman, Biochem. Syst. Ecol. 14 (1986) 255. Berlin, 1988, p. 104.[4] K.H. Kubeczka, in: A. San Fleiciano, M. Grande, J. Casado [30] H. Hendriks, R. Bos, A.P. Bruins, Planta Med. 51 (1985)

´ 541.(Eds.), Conferencias Plenarias de la XXIII Reunion Bienal[31] A. Cazaussus, R. Pes, N. Sellier, J.-C. Tabet, Chromato-´ ´de Quımica, Universidad de Salamanca, Seccion de la

graphia 25 (1988) 865.R.S.E.Q, 1991, p. 169.[32] R. Bos, A.P. Bruins, H. Hendriks, in: K.-H. Kubeczka (Ed.),[5] D. Barron, A. Pabst, in: N.H. Fischer (Ed.), Modern Phyto-

¨ ¨Atherische Ole, Analytik, Physiologie, Zusammensetzung,chemical Methods, Plenum Press, New York, 1991, p. 33.G. Thieme, Stuttgart, New York, 1982, p. 25.[6] N.W. Davies, J. Chromatogr. 503 (1990) 1.

¨[33] G. Rucker, M. Neugebauer, B. Daldrup, Flav. Fragr. J 5[7] H. Rotzsche, in: E. Leibnitz, G. Struppe (Eds.), Handbuch(1990) 1.der Gaschromatographie, Akadem. Verlagsgesellschaft, Leip-

[34] N. Sellier, A. Cazaussus, H. Budzinski, M. Lebon, J.zig, 1984, p. 528.Chromatogr. 557 (1991) 451.[8] E.E. Stashenko, M.A. Puertas, M.Y. Combariza, J. Chroma-

[35] W. Herres, HRGC–FTIR: Capillary Gas Chromatography–togr. A 752 (1996) 223.Fourier Transform Infrared Spectroscopy. Theory and Appli-

[9] E.E. Stashenko, J.R. Martinez, C. Macku, T. Shibamoto, J.¨cations, Huthig, Heidelberg, Basel, New York, 1987.

High Resolut. Chromatogr. 16 (1993) 441.[36] K.-H. Kubeczka, W. Herres, GIT Suppl. 4 (1984) 33.

[10] E. Gil-Av, B. Feibush, R. Charles-Siegler, Tetrahedron Lett. [37] K.-H. Kubeczka, W. Schultze, S. Ebel, M. Weyandt-Spangen-10 (1966) 1009. ¨berg, in: W. Gunther, J.P. Matthes (Eds.), Instrumentalized

[11] H. Frank, G.J. Nicholson, E. Bayer, J. Chromatogr. Sci. 15 Analytical Chemistry and Computer Technology, GIT Ver-(1977) 174. lag, 1989, p. 131.

¨[12] W.A. Konig, The Practise of Enantiomer Separation By ¨[38] T. Brandemer, in: W. Gunther, J.P. Matthes (Eds.), In-¨Capillary Gas Chromatography, Huthig-Verlag, Heidelberg, strumentalized Analytical Chemistry and Computer Technol-

1987. ogy, GIT Verlag, 1989, p. 348.¨[13] W.A. Konig, Chirality 10 (1998) 499. [39] L. Thanh, N.X. Dung, A. Bighelli, J. Casanova, P.A. Leclerq,

[14] T.J. Betts, J. Chromatogr. A 664 (1994) 295. Spectroscopy 13 (1996-1997) 131.¨ ¨[15] W.A. Konig, B. Gehrcke, D. Icheln, P. Evers, J. Donnecke, [40] G.C. Galletti, M.R. Russo, P. Bocchini, Rapid Commun.

W. Wang, J. High Resolut. Chromatogr. 17 (1992) 367. Mass Spectrom. 9 (1995) 1252.[16] R.B. Croteau, D.M. Satterwhite, J. Chromatogr. 500 (1990) [41] P. Bocchini, M. Russo, G.C. Galletti, Rapid Commun. Mass

349. Spectrom. 12 (1998) 1555.[17] J. Faldt, M. Jonsell, G. Nordlander, A.-K. Borg-Karlson, J. [42] K.-H. Kubeczka, in: P. Schreier (Ed.), Flavour 81, de

Chem. Ecol. 25 (1999) 567. Gruyter, Berlin, 1981, p. 345.[18] A. Rustaiyan, S. Masoudi, A. Monfared, H. Komeilizadeh, [43] J. Schwanbeck, K.-H. Kubeczka, in: K.-H. Kubeczka (Ed.),

Flav. Fragr. J. 14 (1999) 276. ¨ ¨Vorkommen und Analytik Atherischer Ole, G. Thieme[19] W. Schneider, J.C. Frohne, H. Bruderreck, J. Chromatogr. Verlag, Stuttgart, 1979, p. 72.

245 (1982) 71. [44] P. Morin, M. Caude, H. Richard, R. Rosset, J. Chromatogr.[20] R. Richmond, E. Pombo-Villar, J. Chromatogr. A 760 (1997) 363 (1986) 57.

303. [45] T.S. Chamblee, B.C. Clark, T. Radford, G.A. Iacobucci, J.[21] S.K. Ramaswami, P. Briscese, R.J. Gargiullo, T. von Gel- Chromatogr. 330 (1985) 141.

dern, in: B.M. Lawrence, B.D. Mookherjee, B.J. Willis [46] S.M. Lee, N.A. Garas, A.C. Waiss, J. Agric. Food Chem. 34(Eds.), Proceedings of the 10th International Congress of (1986) 490.Essential Oils, Fragances and Flavors, Washington, DC, [47] E.G. Heisler, J. Siciliano, E.B. Kalan, S.F. Osman, J.Flavors and Fragrances: A World Perspective, 1986, p. 951. Chromatogr. 210 (1981) 365.

[22] V.O. Elias, B.R.T. Simoneit, J.N. Cardoso, J. High Resolut. [48] R.A. Moreau, C.L. Preisig, S.F. Osman, Phytochem. Anal. 3Chromatogr. 20 (1997) 305. (1992) 125.

[23] A. Baaliouamer, B.Y. Meklati, J. Soc. Alger. Chim. 1 (1991) [49] B. Debrunner, M. Neuenschwander, R. Brenneisen, Pharm.51. Acta Helv. 70 (1995) 167.

[24] G. MacLeod, J.M. Ames, Phytochemistry 30 (1991) 883. [50] T.A. van Beek, N. van Dam, A. de Groot, T.A.M. Geelen,[25] A. Kamel, P. Sandra, Biochem. Syst. Ecol. 22 (1994) 529. L.H.W. van der Plas, Phytochem. Anal. 5 (1994) 19.[26] V.A. Zamureenko, N.A. Kluyev, L.B. Dmitriev, I.I. Gran- [51] Y. Nishii, T. Yoshida, Y. Tanabe, Biosci. Biotech. Biochem.

dberg, J. Chromatogr. 303 (1984) 109. 61 (1997) 547.


¨[52] R. Hansel, J. Schulz, Dtsch. Apoth. Zgt. 122 (1982) 215. [82] E. Miraldi, S. Ferri, G.G. Franchi, Phytochem. Anal. 9[53] R. Bos, H.J. Woerdenbag, H. Hendriks, J.H. Zwaving, (1998) 296.

¨P.A.G.M. de Smet, G. Tittel, H.V. Wikstrom, J.J.C. Scheffer, [83] M.M. Gupta, R.K. Verma, A.P. Gupta, K.G. Bhartariya, S.Phytochem. Anal. 7 (1996) 143. Kumar, J. Med. Aromat. Plant Sci. 19 (1997) 968.

[54] R. Bauer, I. Khan, H. Wagner, Dtsch. Apoth. Ztg. 126 (1986) [84] H.N. ElSohly, E.M. Croom, M.A. ElSohly, Pharm. Res. 41065. (1987) 258.

[55] H.D. Friedel, R. Matusch, J. Chromatogr. 407 (1987) 343. [85] D.M. Dolman, D.W. Knight, U. Salan, D. Toplis, Phytochem.[56] M.J. Pettei, F.G. Pilkiewicz, K. Nakanishi, Tetrahedron Lett. Anal. 3 (1992) 26.

24 (1977) 2083. [86] C. Bicchi, C. Balbo, P. Rubiolo, J. Chromatogr. A 779[57] M. Morita, S. Mihashi, H. Itokawa, S. Hara, Anal. Chem. 55 (1997) 315.

(1983) 412. [87] C. Bicchi, P. Rubiolo, J. Chromatogr. A 727 (1996) 211.[58] L. Mondello, K.D. Bartle, G. Dugo, P. Dugo, J. High [88] J.T.B. Strode III, L.T. Taylor, T. van Beek, J. Chromatogr. A

Resolut. Chromatogr. 17 (1994) 312. 738 (1996) 115.[59] H. Carlson, P. Nilsson, H.-B. Jansson, G. Odham, J. Mi- [89] A.P. Bruins, Methodol. Surv. Biochem. Anal. 18 (1988) 329.

crobiol. Methods 13 (1991) 259. [90] M.P. Maillard, J.-L. Wolfender, K. Hostettmann, J. Chroma-[60] P. Cremin, D.M.X. Donnelly, J.-L. Wolfender, K. Hostet- togr. 647 (1993) 147.

tmann, J. Chromatogr. A 710 (1995) 273. [91] M. Hamburger, J.-L. Wolfender, K. Hostettmann, Nat. Tox-[61] C.R. Blatt, R. Ciola, J. High Resolut. Chromatogr. 14 (1991) ins 1 (1993) 315.

775. [92] H. Stuppner, H. Seeber, H. Santer, in: 44th, Annual Congress[62] E.E. Stashenko, M.A. Puertas, M.Y. Combariza, J. Chroma- of the Society for Medicinal Plant Research and a Joint

togr. A 752 (1996) 223. Meeting with the Czech Biotechnology Society, Prague,[63] P. Morin, Fresenius J. Anal. Chem. 348 (1994) 327. 1996, p. 135.[64] P. Morin, H. Pichard, H. Richard, M. Caude, R. Rosset, J. ¨[93] V. Castro, P. Rungeler, R. Murillo, E. Hernandez, G. Mora,

Chromatogr. 464 (1989) 125. H.L. Pahl, I. Merfort, Phytochemistry 53 (2000) 257.[65] P. Morin, M. Caude, H. Richard, R. Rosset, Analusis 15 [94] B. Vogler, I. Klaiber, G. Roos, C.U. Walter, W. Hiller, P.

(1987) 117. Sandor, W. Kraus, J. Nat. Prod. 61 (1998) 175.[66] H. Pichard, M. Caude, P. Morin, H. Richard, R. Rosset, [95] M. Kohler, P. Christen, J.-L. Veuthey, in: 44th, Annual

Analusis 18 (1990) 167. Congress of the Society for Medicinal Plant Research and a´ ´ ´ ´ ´ ´[67] A. Kery, G.Y. Turiak, I. Zambo, P. Tetenyi, Acta Pharm. Joint Meeting with the Czech Biotechnolgy Society, Prague,

Hung. 57 (1987) 228. 1996, p. 135.´[68] A. Kery, G. Petri, Herb. Hung. 26 (1987) 159. [96] J.A. Crow, J.P. Foley, Anal. Chem. 62 (1990) 378.

[69] B. Marchand, H. Mohan Behl, E. Rodriguez, J. Chromatogr. [97] I. Merfort, P.G. Pietta, P.L. Mauri, L. Zini, G. Catalano, G.265 (1983) 97. Willuhn, Phytochem. Anal. 8 (1997) 5.

[70] W. Leven, G. Willuhn, J. Chromatogr. 410 (1987) 329. ¨ ´[98] H. Pyysaldo, E.-L. Seppa, K.-G. Widen, J. Chromatogr. 190[71] G. Willuhn, W. Leven, Pharm. Ztg. Wiss. 136 (1991) 32. (1980) 466.[72] J.D. Weidenhamer, E.D. Jordan, N. Fischer, J. Chromatogr. ¨[99] N.L.T.R.M. von Jeney de Boresjeno, D.J.J. Potgieter, N.M.J.

504 (1990) 151. Vermeulen, J. Chromatogr. 94 (1974) 255.[73] I. Slacanin, D. Vargas, A. Marston, K. Hostettmann, J.

[100] R.G. Kelsey, M.S. Morris, N.R. Bhadane, F. Shafizadeh,Chromatogr. 457 (1988) 325.

Phytochemistry 12 (1973) 1345.[74] E. Leclercq, J. Chromatogr. 283 (1984) 441.

[101] B. Drozdz, E. Bloszyk, Planta Med. 33 (1978) 379.[75] T.A. van Beek, P. Maas, B.M. King, E. Leclercq, A.G.J.

[102] A.K. Picman, R.L. Ranieri, G.H.N. Towers, J. Lam, J.Voragen, A. de Groot, J. Agric. Food Chem. 38 (1990) 1035.

Chromatogr. 189 (1980) 187.[76] I.M. Lomniczi de Upton, J.R. de la Fuente, J.S. esteve-

´[103] A. Villar, J.L. Rios, S. Simeon, M.C. Zafra-Polo, J. Chro-Romero, M.C. Garcia-Alvarez-Coque, S. Carda-Broch, J.

matogr. 303 (1984) 306.Liq. Chromatogr. Rel. Technol. 22 (1999) 909.

[104] G. Nowak, Chromatographia 35 (1993) 325.[77] N.F. Belyaev, G.M. Zapol’skaya-Dovnar, S.M. Adekenov,

[105] G. Willuhn, J. Kresken, I. Merfort, Dtsch. Apoth. Ztg. 123Chem. Nat. Comp. 32 (1996) 866.(1983) 2431.¨[78] D. Strack, P. Proksch, P.-G. Gulz, Z. Naturforsch. C 35

[106] A.K. Picman, I. Panfil, G.H. Towers, J. Chromatogr. 212(1980) 915.(1981) 379.[79] M.R. Nuriddinova, K.R. Nuriddinov, I.D. Sham’yanov, V.M.

¨[107] D. Gromek, W. Kisiel, A. Stojakowska, S. Kohlmunzer, Pol.Malikov, Chem. Nat. Comp. 29 (1993) 458.J. Pharmacol. Pharm. 43 (1991) 213.[80] O. Spring, Biochem. Syst. Ecol. 17 (1989) 509.

[81] H.L. Kim, S.H. Safe, M.C. Calhoun, J. Agric. Food Chem.35 (1987) 891.

Documents

Review of the Analytical Techniques for Sesquiterpenes And