FLAVOUR AND FRAGRANCE JOURNAL, VOL.

7, 187-193 (1992)

Chiral Compounds of Essential Oils. Part XI. Simultaneous

Stereoanalysis of Lauandula Oil Constituents
Peter Kreis and Armin Mosandl
Imtitut fur Lebensmiltekhemie, Johann Wolfgang Goethe- Universitat, Robert-Mayer-Strasse 7-9, 0-6000 FrankjurtlMain I I,
Germany

Enantioselective multidimensional gas chromatography using the column combination Carbowax 20 M/heptakis-
(2,3-di-O-acetyl-6-0-tert-butyldimethylsilyl~~-cyclodextrin in OV 1701-vinyl allows the simultaneous stereodifferen-
tiation of trans- and cis-linalol oxides (furanoid), camphor, octan-3-01, oct-1-en-3-01, linalyl acetate, lavandulol,
terpinen-4-01 and linalol. The method is applied to commercially available as well as self-prepared essential oils of
Lauandula species. Chirality evaluation is discussed in view of quality assessment of these essential oils.

KEY WORDS Enantiomeric separation of essential oil compounds

Enantioselective multidimensional gas chromatography trans/&-Linalol oxides (furanoid)
Camphor Octan-3-01 Oct-1-en-3-01 Linalyl acetate Lavandulol Terpinen-4-01 Linalol
Direct and simultaneous stereoanalysis Genuineness of essential oils Quality assessment
Lauandula oils Perfume oils

INTRODUCTION and linalol (17,18), the characteristic chiral com-

Since early antiquity, spices and resins from natural
sources have been used extensively for perfumery
and flavour purposes.' As pleasant odours enhance
the enjoyment of well-being, nowadays, the cus-
tomers pay close attention to the naturalness of
aroma in foods and beverages.
The demand for genuine flavourings and essen-
tial oils is continuously increasing worldwide, but
natural resources are rather limited. Against this
background legal regulations order the differentia-
tion of natural and non-natural substances and
efficient tools in the authenticity control of flavours
and fragrances are needed.* Chirality evaluation of
essential oil compounds has been introduced as
new and substantial indicators of
and have been adopted into quality assurance by
the flavour and fragrance industry.'
This paper presents new and powerful tools,
which allow the simultaneous determination of the
enantiomeric distribution of trans (1,2), and cis
(3,4)-linalol oxide (furanoid), camphor (5,8), octan-
3-01 (6,7), oct- 1-en-3-ol (9,10), linalyl acetate
(1 1,12), lavandulol (13,14), terpinen-4-01 (15,16)
Dedicated to Drs W. Jennings and R. Teranishi on the occasion of their 70th birthdays.
pounds of the essential oils from Lauandula species.
EXPERIMENTAL
Column Preparation and Coating
Column preparation and coating was according
to Reference 8.
Instrumental ion
1. Capillary Gas Chromatography (One-dimen-
sional). Perkin-Elmer G C F 22 with a Gerstel PAS
1 injector system (Bodenseewerk Perkin-Elmer &
Co GmbH, D-7770 uberlingen, FRG; Gerstel, D-
4330 Miilheim a.d. Ruhr, FRG).
Injector temperature: 180C; detector: FID,
190C; injection mode: split 1 :50; injection vol-
ume: 1.0 p1 essential oil sample (0.5% in n-pen-
tane); column: Duran glass capillary, 2 6 m x
0.23 mm id., 0.25 p n film thickness; coated with a

0882 - S734/92/O40187-07$08.50 Received 2 April 1992

c)1992 by John Wiley & Sons, Ltd. Accepied I M a y 1992
188 P.KREIS AND A. MOSANDL

solution (0.4%) of heptakis(2,3-di-O-acetyl-6-O- ides (furanoid) (Roth, Karlsruhe); octan-3-01 (Al-

rerf-butyldimethylsily1)-fl-cyclodextrin(50%) and drich, Steinheim); oct- l-en-3-01 (Aldrich,
OV- 1701-vinyl (50 ?A), in n-pentane/dichlorometh- Steinheim); lavandulol (Roth, Karlsruhe).
ane ( 1 : I , v/v), according to Reference 22; carrier
gas: H 2 95 kPa; temperature programme: 60C +
Optically Active References. (4S)( )-Terpinen-4-
(18 rnin isothermal) lC/min + 120C; 10C/ 01 (Aldrich, Steinheim); (IS)( -)-camphor (Aldrich,
min + 200C. Steinheim); ( I R)( +)-camphor (Aldrich, Stein-
heim).
2. Multidiniensional Gas Chromatography
( MDGC). Siemens Sichromat 2 double-oven sys- (3R) ( - )-Linalol, (3R) ( - )-Linalyl acetate.
tem with two independent temperature controls, Isolated from authentic lavender oils using TLC:
two flame ionization detectors and a 'live switch- plates: silica gel 60, 0.25 mm, 20 x 20 cm (Merck
ing' coupling piece ('live-T-piece'; Siemens AG, D-5721); eluent: toluene/ethyl acetate (97:3, v/v);
7500 Karlsruhe 21, FRG). Injector temperature: detection : vanillin/phosphoric acid or anise alde-
I80 'C: detector temperature: 190C each; injectionhyde/sulphuric acid.
mode: split; injection volume: 0.2 p1 neat essential 5 p1 of authentic lavender oils giving 50 p1 sol-
oil: sample back flush after 20 min. ution (10% in dichloromethane) are developed by
TLC. The fractions of interest are isolated by
scratching out the corresponding zones (linalol,
Prrcolimzn Rf = 0.28-036; linalyl acetate, Rf: 0.63-0.70) and
SupelcowaxTM10 (Carbowax 20 M, chemically eluating the silica gel with 200-500 p1 n-pentane.
bonded) 30 m x 0.32 mm i.d., 0.25 pm film thick-
ness. Carrier gas: H, 135 kPa; temperature pro- ( R ) ( -)-Lauandulol. Isolated from authentic la-
gramme: 8 0 T (2 min isothermal) 3"C/min + vandin oil, according to Reference 10; the fraction
140 C. of primary alcohols received, also contains high
amounts of geraniol and nerol besides (R)(-)-
Main Column lavandulol (13).13 is isolated by enantioselective
MDGC and structurally assigned by MS-detection.
See 'Capillary gas chromatography (one-dimen- trans/cis-Linalol oxides (furanoid). Structure
sional)'. Carrier gas: H, 95 kPa; temperature pro- elucidation of the stereoisomers according to Refer-
gramme: 60'C (32 rnin isothermal) l"C/min + ence 1 1. 3-Octanol and 1-octen-3-01 enantiomers,
100C. referring to the literature.'2*'3Reference mass spec-
tral data for all compounds investigated taken from
H e m Cutting Reference 14.
The chronological order of the 'heart cuts' was
as follows, with the cut times (min) adapted to the RESULTS AND DISCUSSION
amounts of the substances: octan-3-01 (6,7):
7.50-7.60; trans-linalol oxide (1,2):8.93-9.03; oct- The present situation in the field of flavours and
I-en-3-01 (9.10): 9.10-9.20; cis-linalol oxide (3,4): fragrances is characterized by (i) continuously in-
9.75-9.85; camphor (5,8): 11.10-1 1.15; linalol creasing demands for high quality and genuine
(17,18): 12.615-12.62; linalyl acetate (11,12): materials, (ii) rather limited natural resources, and
13.10-13.1 1; terpinen-4-01 (15,16): 14.18-14.25; la- (iii) large-scale processes for many important essen-
vandulol (13J4):16.98-17.08. tial oil constituents.
So, highly selective analytical methods exposing
the fraudulent addition of synthetic chemicals to
Reference Compounds
essential oils are needed.
The order of elution was assigned by using For many years the adulteration of lavender
racemates, including enantiomerically pure or en- oils by means of synthetic linalol and/or linalyl
riched references. acetate has been easily revealed, by detecting the
synthetic by-products dihydrolinalol and/or
Racx~matps.Linalol (Aldrich, Steinheim); linalyl dehydrolinalol.' '*16 But this detection fails, if un-
acetate (Merck, Darmstadt); trans-/cis-linalol ox- natural by-products are not present.
 STEREOANALYSISOF LAVANDULA OILS 189

Recently, chirality evaluation of linalyl acetate 11 4

and linalol have been introduced as new and sub-
stantial criteria in control of the authenticity of
lavender 7--20 In particular, genuine linalyl
acetate from such oils has high enantiomeric purity
favouring the (R)-configuration, irrespective of the
Lmandula species, and storage or work-up
condition^.^
Developing 2,3-di-O-acylated-6-O-silylated
cyclodextrins2'*22as a new generation of chiral
stationary phases in enantioselectiveCGC, most of
chiral impact compounds of rose oil, geranium or
Laoandufa oils are now stereoanalysed simul-
taneously.2 z I
Already one-dimensionalchirospecificCGC may
serve as an orientating aid in the direct stereoanaly- I
sis of the main constituents of lavender oils (Figure
l), if structurally selective MS-detection is avail- 10 20 30 40 50 60 min-
able. As outlined in Figure 2 the falsification of a
commercially available sample is easily detected Fig. 2. Commercially available sample 'Oleum Lavandulae
even by simple injection of the diluted oil, indicat- DAB 9, Mont Blanc 38-40%', adulterated by the addition of
racemic linalyl acetate (11,12) and racemic linalol (17J8); for
ing racemates of linalyl acetate (1132) and linalol conditions, see Figure 1
(17,lS). But with respect to the authenticity control
of Luoandula oils the simultaneous stereoanalysis of
1-18 is of considerable interest. Using enantioselective MDGC and the column
combination Carbowax 20 M/heptakis(2,3-di-O-
acetyl-6-0-tert-butyldimethylsilyl)-~-cyclodextrin~
the enantiomeric pairs of trans (1,2) and cis (3,4)-
linalol oxides (furanoid), camphor (5,8), octan-3-01
(6,7), oct-l-en-3-01 (9,10), linalyl acetate (11,12),
lavandulol(13,14), terpinen-4-01(15,16) and linalol

10 20 30 40 50 60 min'

Fig. 1. One-dimensional stereodifferentiationof linalyl acetate

(11,12) and linalol(17,18) from an authentic lavender oil sample
(Provence, France). (RX->linalyl acetate (II), (R)(->linalol
(17) of high enantiomeric purity (>96%); for conditions, see 13,14 15,16 17,18
'Experimental' section; sample kindly provided by ASTA Me-
dica, Frankfurt/Main Scheme 1.
 P.KRElS AND A. MOSANDL

6
7
- 16

9
10-

-15

b 30 40 50 60 min
0 5 10 15

(a) (b)

Fig. 3. Simultaneousstereoanalysis of Lovundulu oil constituents, using enantioselective MDGC (standard mixture). (a) Presepara-
tion of racemic compounds; unresolved enantiomericpairs of octan-3-01(6,7), trans-linalol oxide (1,2), oct-len-3-01(9,10), cis-linalol
oxide (3.4). camphor (5,8), linalol (17.18). linalyl acetate (11,12), terpinen-4-01 (15,16), lavandulol (13.14); for conditions, see
Experimentalsection, MDGC. (b) Chiral resolution of enantiomericpairs, transferred from the precolumn:trans-linalol oxide: 1
(2S, SS), 2 (ZR, SR); cis-linalol oxide: 3 (ZR, 5S), 4 (2S, 5R); camphor: 5 (lS), 8 (1R); octan-3-01:6 (R), 7 (S); oct-l-en-3-01:9 (S), 10
( R ) ; linalyl acetate: 11 ( R ) , 12 (S); lavandulol: 13 ( R ) , 14 (S); terpinen-4-01: 15 (R), 16 (S); linalol: 17 ( R ) , 18 (9;
for conditions, see
Experimentalsection, MDGC

(17,18) are analysed by a single chromatographic
run without any preseparation (Figure 3).
Referring to the extremely different concentra-
tions of these constituents in genuine essential oils
the chromatographic selectivity is optimized by
adjusting the cut-intervals with the amount of the
chiial compound to be analysed (cut-intervals, Ex-
perimental section).
It remains essential to put a lot of care into
optimum chromatographic conditions, to avoid
unsuccessful cuts and misinterpretations of coelut-
ing compounds respectively.
( R ) ( - )-Linalyl acetate (11) has already been
described as the genuine main component of la-
vander oils (Figures 4 and 5). Even if 11 is hydro-
lysed in considerable amounts during prolonged
procedures of hydrodistillation, the absolute con-
figuration of the ester remains unchanged to be a
reliable indicator of genuineness. Besides (RX -)-
linalyl acetate (11) its corresponding alcohol linalol
has been identified from self-prepared diethyl ether
extracts and steam distillation of Lauandulu species
and spices, also preferring the (R)-configuration
(17; > 95 %) (Figures 4 and 5). Although the chiral
stability of linalol may be interfered, (S)( +) linalol
in amounts up to 15% may be formed during
unusual and extremely prolonged times of hydro-
distillation (> 1.5 h),23-24 higher amounts of
(SX + >linalol(l8) ( > 15%) have to be interpreted
as a blend with synthetic racemate (Figure 6). The
sample investigated in Figure 6 also contains high
amounts of racemic linalyl acetate (1132). In com-
parison to the genuine samples in Figures 4 and 5
the adulterated oil of Figure 6 shows a significant
decrease in the content of linalol oxides (1-4) and in
the amount of the characteristic fingerprint of
hydrocarbons (see Precolumn chromatogram,
2-5 min).
Owing to its musty odour ( + )-camphor (8), a
genuine minor compound of lavender oils has to be
interpreted as a quality-limiting factor. So, a fraud-
ulent blend of lavender oils by means of racemic
camphor seems to be irrelevant. Higher amounts of
camphor (and of the non-chiral compound 1,8-
cineole) are characteristic of lavandin oils (Figure
5)
As (+)-camphor (8) coelutes with (S)-oct-1-en-3-
01 (9) Figures 4(C)-6(C) show enantioselective
 STEREOANALYSIS O F LA VANDCILA OILS 191

16
1.8 -Cinwla B

L
\

8
4
I

0 5 10 15
. 30 40 50
L
60 mi; 2O 30 40 SO 80 min-

Fig. 4. Enantioselective MDGC of an authentical lavender oil sample (Provence, France; see Figure l), indicating genuine chiral
L

compounds of high ee-values. A: precolumn, B: main column separation, including stereoanalysis of camphor (5,8), C: stereoanalysis
without camphor transfer, indicating the absence of (a + )-oct-l-en-3-01(9); for conditions, see Figure 3

i:
17

[ 3 .

0 I 10 15
. 30 40 50 wmin
.-
20 30 40 50 80 min .
Fig. 5. Enantioselective MDGC of Lovandula mgusr. x lauandula IariJ; high amounts of 1.8-cineole and camphor (8); further
conditions, see Figures 3 and 4. Sample provided by courtesy of ASTA Medica, Frankfurt/Main

analyses, camphor excluded; compared with the
stereoanalyses of Figures 5(B) and 6(B), (1R)-( +)-
camphor (8) is detected with high enantiomeric
purity. But in the case of an authentic lavender oil
higher amounts of (1s)-camphor (5) are detected
(Figure 4). At the present time, a final explanation
of this fact cannot be given and systematic investi-
gations on the stereoanalysisof camphor (5,8) from
authentic Luvandulu oils are in hand. Furthermore,
(SX +)-octan-3-01 (7 (>95 %), (RX - )-oct-l-en-3-
01 (10) (>99%), (RX->lavandulol (13)25*26as
well as (SX+)-terpinen4ol (16) may be inter-
preted as characteristic and genuine chiral constitu-
ents of Luvandula oils with high enantiomeric
purities. Further enantiomeric distributions are
outlined in Table 1.
Finally, it is interesting to note that the enantio-
meric distributions of linalol oxides (furanoid) do
not strictly correlate with the enantiomeric ratios of
linalol which should be considered as their genuine
precursor. At the present state of knowledge a
definitive explanation for this phenomenon cannot
be given.
192 P. KREIS AND A. MOSANDL

8 1' I2 11 1
(91

Il6
11
-12
16

1111 n f3 II
J.
Fig. 6. Simultaneous stereoanalysis of a commercially available sample of 'Oleum Lavandulae DAB 9, Bareme 50-52%',
adulterated by the addition of racemic linalyl acetate (11,12) and racemic linalol (17.18); content of linalol oxides (1-4) decreased;
amount of genuine (lavandin) oil: I5 % (approximately)

Table I. Enantiomeric distributions of stereoisomers, investigated from authentic samples of Laoandub oils; for conditions, see
'Experimental' section. Samples kindly provided by Ministere de I'Agriculture, Nyons, France (samples 1-11) and by ASTA
Media AG, Frankfurt/Main (samples 12 and 13)

trans-Linalol cis-Linalol Linalyl Lavandulol Terpinen-4-01 Linalol

oxide oxide acetate
Sample (2s.5s) (2R.5R) (2R.5s) (2s.5R) (R) (S) (R) (S) (R) (S) (R) (S)
no. 1 2 3 4 11 12 13 14 15 16 17 I8

I 13.6 86.4 88.5 11.5 >99 93.2 6.8 - - 97.4 2.6

2 - - - - >99 98.5 1.5 2.2 97.8 94.5 5.5
3 11.4 88.6 86.0 14.0 >99 98.0 2.0 - - 96.6 3.4
4 15.4 84.6 86.7 13.3 >99 89.8 10.2 2.0 98.0 95.1 4.9
5 9.8 90.2 91.5 8.5 >99 >99 <I 5.8 94.2 91.5 2.5
6 3.9 96.I 91.5 8.5 >99 <I >99 <I 1.8 98.2 97.3 2.7
1 4.2 95.8 92.9 7.1 >99 98.3 1.7 2.0 98.0 96.9 3.1
8 <5 > 95 > 95 <5 199 <I 98.1 1.3 1.7 98.3 98.2 1.8
9 14.6 85.4 90.0 10.0 >99 <1 > 99 <1 1.9 98.1 96.1 3.9
10 12.5 87.5 95.8 4.2 >99 <1 >99 <1 1.6 98.4 97.1 2.9
I1 - - - - 299 <1 > 99 <I 1.9 98.1 95.2 4.8
12 23.3 76.1 82.9 17.1 98.8 1.2 >99 <1 1.9 98.1 97.2 2.8
13 13.8 86.2 89.3 10.7 >99 <I 96.2 3.8 10.9 89.1 95.1 4.9

CONCLUSIONS acetyl-6-0-tert- but yldimethylsil y1)-P-cyclodextrin

now has proved to be a powerful tool, which allows
While the direct analysis of single compounds has the simultaneous stereoanalysis of almost all the
been reported in the first stage of stereoanalysis of characteristic compounds of the essential oils from
chiral essential oils corn pound^,^'^ the simulta- Lauandula species.
neous stereodifferentiation of chiral compounds
with different functionalities has been
achieved.4*27'28Enantio-MDGC, using the column Acknowledgements-The authors are indebted to F. Dettmar for
combination Carbowax 20 M/heptakis(2,3-di-O- reliable technical assistance.
 STEREOANALYSIS O F LA VANDULA OILS
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