Analytica Chimica Acta 447 (2001) 125134

Computer-aided method for identification of components in

essential oils by 13C NMR spectroscopy
Marcelo J.P. Ferreira a , Mara B. Costantin a , Patrcia Sartorelli a ,
Gilberto V. Rodrigues b , Renata Limberger c , Amlia T. Henriques c ,
Massuo J. Kato a , Vicente P. Emerenciano a,
b

a Instituto de Qumica, Universidade de So Paulo, Caixa Postal 26077, 05513-970 So Paulo, Brazil
Departamento de Qumica, ICEx, Universidade Federal de Minas Gerais, 30161-000 Belo Horizonte, Brazil
c Faculdade de Farmcia, Universidade Federal do Rio Grande do Sul, 90610-000 Porto Alegre, Brazil

Received 20 December 2000; received in revised form 6 June 2001; accepted 13 June 2001

Abstract
The aim of this paper is to present a procedure based on analyses of 13 C NMR data for identification of known and new
chemical constituents in essential oils. A novel program developed to analyze complex mixtures of terpenoid compound
was evaluated for the identification of components in the essential oils from leaves of Piper cernuum and Piper regnellii.
2001 Elsevier Science B.V. All rights reserved.
Keywords: 13 C NMR; Essential oils; Computer-aided method; Expert system; Piper sp.; Terpenoid identification

1. Introduction
Countless specialist systems have been developed
in the last decades seeking the structural determination
of novel organic substances which are not recorded
in databases yet [110]. Among these systems, the
SISTEMAT system [1122] was developed by our research group to assist the processes of structural determination of natural products. In this system and in
others, the developed programs were proved to be very
efficient to accomplish the analysis and identification
for new isolated compounds. However, such systems
do not allow the analysis of mixtures of substances,
such as in the essential oils, without previous purification, due to the large number of signals. Fig. 1 shows
Corresponding author. Fax: +55-11-38155579.
E-mail address: vdpemere@quim.iq.usp.br (V.P. Emerenciano).

an example of 13 C NMR spectra of the essential oil

where it is easy to note the difficulties faced by the
method due to the great number of signals and overlapping of these.
For such cases, we have developed a methodology specifically addressed to analyze 13 C NMR data
of constituents present in mixtures. This methodology was applied in the identification of a mixture
of triterpenes obtained from the roots of Vernonia
cognata (Asteraceae) [23]. From this mixture six
triterpenes were recognized by the SISTEMAT system. However, the analysis of volatile oils was not
possible due to large number of compounds found in
this type of mixture and also because, at that time,
SISTEMAT did not have databases of 13 C NMR
data of monoterpenes and sesquiterpenes as well.
The 13 C NMR spectroscopy has been recently introduced to analyze several classes of compounds

0003-2670/01/$ see front matter 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 0 3 - 2 6 7 0 ( 0 1 ) 0 1 2 0 4 - 1

126

M.J.P. Ferreira et al. / Analytica Chimica Acta 447 (2001) 125134

M.J.P. Ferreira et al. / Analytica Chimica Acta 447 (2001) 125134

in mixtures by the use of various specialist systems

[2432].
The aim of this paper is to introduce a new methodology to the identification of chemical constituents
present in essential oils through analyses of 13 C
NMR data. The developed method differs from others
[3133] based on signal intensity.

2. Methodology
The identification of constituents present in essential oils was based on the analysis of their 13 C NMR
spectra and comparison with these of monoterpenes
and sesquiterpenes available in the literature. These
data were encoded and stored in the SISTEMAT system [19,21].
After we have prepared the 13 C NMR databases that
contain around 1300 mono- and 2500 sesquiterpenes,
the SISCONST program [14] was developed, in order
to analyze mixtures of these compounds.

127

The sequence of information to be supplied to the

SISCONST program is the following:

13 C chemical shifts and their multiplicities obtained

experimentally from the DEPT spectra;

the minimum number of carbon atoms for the substructure search, i.e. this number is demanded by
the user, and generally it is the half of the total carbon number of the chemical class studied;
the error range admitted by the program, being generally 1.0;
the gradient, that allows the automatic increase of
the error range;
specify the possible classes of compounds to be
searched.
The search process for a probable skeleton is limited to compounds having a substructure containing
at least half of the total carbons number. So, from
each chosen compound and for its skeleton, the program associates a statistical weight (Ws ) calculated
by: W s = (N C TNC)2 (ER/ERM), where NC is

2.1. The SISCONST program

The SISCONST program [14] was developed to aid
the process of structural determination of natural products by means of analysis of chemical shifts and multiplicities obtained from the 13 C NMR DEPT spectra.
The program may predict the most probable skeleton type for a compound under analysis and suggest
several substructures with the assigned signals of 13 C
NMR.
The program matchs the signals of the spectrum
obtained with all spectra stored in the database. If a
spectrum signal and the its respective multiplicity are
present in a determined carbon atom, the signals of the
interlinked carbons are matched with the spectrum in
question. This searching process is repeated so that the
largest fragments of the substructure bearing compatible chemical shifts with the 13 C NMR data from the
spectrum are obtained. Except for the reduction process of associative groups [13], the system basically
follows the Munks algorithm [2].
Earlier works using a similarity searching method
are reported in the literature concerning the identification of known and new substances through infrared
spectra, mass spectrometry, 1 H NMR and 13 C NMR
spectroscopic databases [3446].

Fig. 2. Performance flow chart of the SISCONST program.

128

M.J.P. Ferreira et al. / Analytica Chimica Acta 447 (2001) 125134

the number of carbons found in the substructure, TNC

the total number of carbons, ERM the absolute value
of the largest difference between the substructure signals and the corresponding signal of the spectrum in
question, and ER is the error range. Thus, the skeleton
probability of the substance in question is computed.
Table 1
Chemical constituents of the P. cernuum volatile oil with their
respective Kovats indexes and relative concentrations analyzed by
GCMS
KI-DB5a

Chemical
constituents

Concentration
(%)

924
961
966
978
1006
1013
1017
1034
1045
1074
1357
1366
1371
1374
1404
1421
1437
1444
1467
1472
1487
1489
1495
1509
1548
1551
1562
1566
1569
1576
1585
1610
1621
1626
1630
1636
1640

-Pinene
Sabinene
-Pinene
Myrcene
-Terpinene
p-Cymene
Limonene
(Z)--ocimene
(E)--ocimene
Terpinolene
-Copaene
-Bourbonene
-Cubebene
-Elemene
-Caryophyllene
Aromadendrene
-Humulene
Alo-aromadendrene
Germacrene-D
Viridiflorene
Bicyclogermacrene
-Muurolene
Germacrene-A
-Cadinene
(E)-nerolidol
Ledol
Spathulenol
Caryophyllene oxide
Globulol
Epi-globulol
Eudesmol (isomer not identified)
1-10-Di-epi-cubenol
Iso-spathulenol
-Cadinol + -muurolol
Cubenol
Not identified
-Cadinol

7.16
0.27
6.15
0.50
0.97
0.61
0.52
0.14
1.88
0.68
1.48
0.20
0.45
3.02
20.69
0.36
1.74
0.23
6.68
0.62
21.88
0.31
4.15
1.54
1.28
0.67
2.26
0.74
3.08
2.21
0.82
0.20
0.26
1.72
0.60
0.49
2.84

Total
Non-identified
Identified

99.40
0.49
98.91

Kovats index on DB-5 column [54].

For this search process an error range of the 13 C

NMR chemical shift is fixed at 1.0 for the substructure and 5.0 for skeleton. If this error range is too
narrow and does not allow a successful matching, the
ranges are automatically increased with a gradient of
0.5 and 1.0 for substructures and skeletons, respectively. If the error range of the search reaches the upper
limit of 3.0 for substructures and 10.0 for skeletons,
the program automatically will stop the search, and no
substructures and/or skeletons will be presented.
Since the system is supposed to create constraints
in structural determination, the assignments of the carbon belonging to the substructures are important data
as well as their respective chemical shifts. Due to this
fact, we introduced the Kalchausers algorithm [47]
which we modified slightly, because the only data

Table 2
Chemical constituents of the P. regnellii essential oil analyzed by
GCMS
KI-DB5a

Chemical constituents

917
924
962
966
984
996
1007
1014
1018
1045
1088
1161
1174
1358
1402
1422
1437
1477
1483
1509
1542
1548
1562
1626
1630

Tricyclene
-Pinene
Sabinene
-Pinene
Myrcene
-Phellandrene
D3-carene
p-Cymene
Limonene+-phellandrene
-Terpinene
Linalool
Terpinen-4-ol
-Terpineol
-Copaene
-Caryophyllene
Aromadendrene
-Humulene
Germacrene-D
Bicyclogermacrene
-Cadinene
Germacrene-B
(E)-nerolidol
Spathulenol
-Muurolol
-Muurolol
Total
Non-identified
Identified

Kovats index on DB-5 column [55].

Concentration
(%)
0.30
0.50
1.30
0.20
52.6
0.20
0.20
1.50
4.10
0.20
15.9
0.70
1.20
0.30
8.50
0.40
0.40
0.30
2.90
0.50
0.50
4.20
0.90
0.30
0.70
100.0
1.30
98.70

M.J.P. Ferreira et al. / Analytica Chimica Acta 447 (2001) 125134

129

Table 3
13 C NMR DEPT spectral data obtained for crude essential oil from leaves of P. cernuum
s
154.9
153.7
152.4
151.1
149.1
147.9
144.7
142.6
139.6
135.3
135.1
134.1
133.2
131.5
127.7
126.5
116.4
81.1

d
75.4
73.6
72.5
54.2
40.8
36.5
36.4
28.1
26.0
24.9
20.5
19.4

150.5
145.4
140.3
135.8
133.7
132.0
129.2
128.2
126.7
126.1
125.2
124.8
124.6
123.0
121.2
119.8
116.7
116.3

t
77.5
58.5
57.3
54.6
54.5
53.9
53.7
53.2
53.0
52.1
50.3
49.6
48.7
47.3
47.1
45.7
44.6
41.4

41.0
40.7
40.1
39.7
38.7
37.2
36.6
34.8
33.3
32.5
31.6
30.2
28.6
27.8
27.2
27.0
26.2
22.6

which are necessary are the signal values and their

multiplicities. Therefore, from this structure search
process, the chemical shift values are attributed to the
respective carbons during the similarity searching process. Therefore, the reliability of each assignment of
the experimental spectrum is achieved by the program
with the stored data obtained from the literature.
However, so far, this program had only been previously tested for the individual identification, i.e. previously separated and purified compounds. In order to
allow the analysis of mixtures, we have increased the
data storage capacity of the program to a largest number of chemical shifts and multiplicities into the system during the analysis. The analyses with a minimum
of nine coincident signals could be successfully carried
out in the analysis of mono- and sesquiterpenes, once
this value embodies structures of both compounds.
The SISCONST program is able to recognize structures for which the 13 C NMR data are present in the
database and structures whose 13 C NMR data are absent in the database. In the last case, the program
searches for substructures, parts of a structure compatible with the experimental data that, in many cases,
when overlapped, they furnish the complete structure. Various examples of new substances which were
tested by the program are shown with excellent results
[11,14,16,19,22]. The self-training is the main charac-

115.9
113.2
112.3
111.9
111.1
110.1
109.3
108.5
107.6
106.2
44.9
43.7
42.5
42.3
42.0
41.0
40.6
40.5

q
40.0
39.8
38.2
37.5
36.5
34.9
34.8
33.2
32.6
31.7
31.5
31.2
31.1
30.7
30.0
29.6
29.5
29.2

28.2
27.2
27.1
27.0
26.8
26.4
26.3
25.1
23.8
23.2
22.9
22.2
21.5
20.4

32.4
30.3
30.1
29.5
28.9
28.1
27.2
26.6
26.4
25.0
24.1
23.8
23.2
22.9
22.1
21.8
21.5
21.4

21.1
21.0
21.0
20.5
20.0
19.2
18.2
16.9
16.8
16.5
16.3
16.1
15.9
15.7
15.4

theristic of this program, since it identifies known and

new substances present in the sample. Fig. 2 shows the
performance flow chart of the SISCONST program.
3. Results
The identification capacity of the SISCONST program was evaluated in the analysis of terpene mixtures
Table 4
13 C NMR DEPT spectral data obtained for crude essential oil
from leaves of P. regnellii
s

154.7
146.1
134.2
131.9
129.6
129.0
126.3
110.0
108.4
73.5
40.0
19.7

146.2
145.1
139.0
131.8
126.3
124.4
124.3
124.2
53.6
48.5
40.4
30.1
26.8

115.7
113.0
111.7
42.1
40.0
39.7
34.8
31.5
31.4
29.4
28.4
27.9
26.0
24.1
22.6

30.2
27.5
26.8
26.7
25.7
22.8
20.8
19.5
17.7
16.3
16.1

130

M.J.P. Ferreira et al. / Analytica Chimica Acta 447 (2001) 125134

Fig. 3. Structures of compounds identified by the SISCONST program.

in volatile oil samples obtained from leaves of Piper

cernuum and Piper regnellii (Piperaceae family). The
system efficiency was based on the comparison of results obtained in the identification of compounds by
analysis of 13 C NMR spectra with those obtained by
GCMS (gas cromatographymass spectrometry) and
by GCKI (gas chromatographyKovats indexes). The
data obtained through GCMS and GCKI for P. cer-

nuum and P. regnellii are listed in Tables 1 and 2, respectively.

Both samples were also submitted to the aquisition
of 13 C NMR data in which 167 chemical shifts and
their respective multiplicities were obtained by 45, 90
and 135 DEPT for P. cernuum (Table 3) and 49 chemical shifts for P. regnellii (Table 4). All chemical shifts
obtained from the list of the peaks in the 13 C NMR

M.J.P. Ferreira et al. / Analytica Chimica Acta 447 (2001) 125134

131

132

M.J.P. Ferreira et al. / Analytica Chimica Acta 447 (2001) 125134

Table 6
Experimental

13 C

NMR data from crude essential oil of P. regnellii assigned by SISCONST program

Linalool

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

Myrcene

-Caryophyllene

Bicyclogermacrene

Nerolidol

Exp.

Lit. [19]

Exp.

Lit. [19]

Exp.

Lit. [50]

Exp.

Lit. [50]

Exp.

Lit. [57]

25.7
129.6
124.4
22.6
42.1
73.5
145.1
111.7
17.7
27.5

25.2
130.3
124.5
22.6
41.2
72.6
145.0
111.3
17.5
27.2

26.8
126.3
131.8
27.9
31.5
146.1
139.0
115.7
16.3
113.0

27.7
124.4
131.5
27.0
31.7
146.3
139.1
115.5
17.7
112.9

53.6
40.0
29.4
48.5
154.7
40.0
29.4
124.3
134.2
34.8
28.4
30.2
22.8
111.7
16.1

53.5
40.0
30.1
48.2
154.3
40.4
29.3
124.5
135.0
34.7
28.3
30.1
22.6
111.8
16.2

124.2
26.0
42.1
129.0
126.3
26.8
30.1
26.0
39.7

19.7
30.2
16.1
16.3
20.8

124.8
26.0
41.0
128.0
126.5
26.8
30.0
26.7
37.2
140.6
19.7
29.2
15.3
16.5
20.7

25.7
131.9
124.2
24.1
39.7
134.2
124.4
24.1
42.1
73.5
145.1
111.7
17.7
16.1
25.7

25.1
130.5
124.2
24.3
39.2
134.3
124.7
24.2
42.0
72.2
145.3
110.8
17.2
15.4
25.0

spectra were entered in the program for analysis. The

additional parameters of the obtained spectra are described in the Section 4.1.
The data of P. cernuum were loaded into the SISCONST program which, at the first stage of run,
identified the sesquiterpene germacrene-D. The assignment of its 13 C NMR signal was in full agreement
with the data previously published in [48]. Then, in
the second step, the program continued the analysis and identified additionally three sesquiterpenes:
-caryophyllene, germacrene-A and bicyclogermacrene. Thus, successively, the program identified
the following compounds: -pinene, spathulenol,
-elemene, -pinene and globulol (Fig. 3). The experimental 13 C NMR data for identified compounds
was compared to those available in the literature, and
they are listed in Table 5.
The analysis of the components in the essential oil
of P. regnellii was also carried out (Table 6) and the
SISCONST program was able to identify sequentially
linalool, myrcene, nerolidol, -caryophyllene and bicyclogermacrene. The reliability in each assignment
of the compound was obtained in both samples by
GCMS, GCKI and 13 C NMR data previously published in [4853].
In summary, the SISCONST program was able to
identify two monoterpenes (- and -pinene) and
eight sesquiterpenes (germacrene-D, germacrene-A,

-elemene, -caryophyllene, spathulenol, globulol,

bicyclogermacrene and -cadinol) in the essential
oil from P. cernuum. In P. regnelliis essential oil,
two monoterpenes (myrcene and linalool) and three
sesquiterpenes (-caryophyllene, nerolidol and bicyclogermacrene) were identified.

4. Discussion and conclusions

The SISCONST program was capable to identifying
efficiently all the constituents present in the volatile
oils at the same or superior concentration of 2.26%.
The non-identification of minor compounds (<2.26%)
is caused by interrelated factors: the small concentration of the constituent in our sample of the volatile oil
which did not allow the achievement of the chemical
shifts of the compound. This limitation is inherent to
the own analysis type. However, if a larger amount of
oil were obtained, starting, for example, from a larger
biomass, and we had haven more time of accumulation
to run the spectrum, we probably could have identified, through 13 C NMR, the other constituents present
in smaller amounts.
In summary, the method here described allows the
correct identification of major compounds present in
volatile oils. The method can be suggested as a complementary tool for analysis of essential oils based on

M.J.P. Ferreira et al. / Analytica Chimica Acta 447 (2001) 125134

GCMS data. One of the advantage of this method is

the correct identification of isomers of a certain compound present in the sample or to detect the presence
of new compounds in essential oils.

133

series of n-alkanes) and mass spectra with that available in the system [55,56].

Acknowledgements
4.1. Experimental
Plant material and extraction: leaves of Piper cernuum Vell. and Piper regnellii (Miq.) C.DC. were
collected at the Campus of So Paulo University in
So Paulo city, Brazil, in June 2000. Voucher specimens (Kato-0137 and E. Guimares-1961, respectively) were deposited at Herbarium of Instituto de
Botnica, So Paulo, Brazil. The essential oils were
obtained from steam distillation of 570 and 400 g of
fresh leaves of P. cernuum and P. regnellii, respectively, in agreement with the literature [54], yielding
102 mg of the crude oil for P. cernuum and 400 mg for
P. regnellii.
4.1.1. 13 C NMR analysis
The 13 C NMR spectra were obtained through a Varian 300 MHz, using CDCl3 as solvent and TMS as internal standard. The 13 C NMR spectra were measured
using 45 pulse (13.4 ms) and 3600 pulses; repetition
time, 2 s; threshold, 15; signal to noise, 80:1; spectral
width, 18.1 kHz; data set, 65.5 kilo-words zero-filled;
temperature, 25 C.
4.1.2. GC and GCMS analysis
Gas chromatography analysis was performed in a
chromatograph (Shimadzu GC-17A) equipped with a
Shimadzu GC 10 software, using a fused silica capillary column, DB-5 (30 m 0.25 mm 0.25 m), and
a flame ionization detector. Injector and detector temperatures were set at 220 C. The oven temperature was
programmed from 60 to 300 C at 3 C/min and helium was employed as carrier gas (1 ml/min) for both
analyses. The percentage compositions were obtained
from electronic integration measurements, using flame
ionization detection without taking into account relative response factors. Gas chromatographymass spectrometry: the sample was analyzed by GCMS, using a Shimadzu capillary GC-quadrupole MS system
(QP 5000) operating at 70 eV at the same conditions
as described above. The identification of the compounds was performed by comparison of retention indexes (determined relatively to the retention times of a

This work was supported by grants from the Fundao de Amparo Pesquisa do Estado de So Paulo
(FAPESP), the Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico (CNPq) and by the
Fundao Coordenao de Aperfeioamento de Pessoal de Nvel Superior (CAPES). The authors thank
Antnio J.C. Brant for helpful discussion during the
preparation of the manuscript.
References
[1] R.K. Lindsay, B.G. Buchanan, E.A. Fergenbaum, J.
Lederberg, Applications of Artificial Intelligence for Organic
Chemistry: the DENDRAL Project, McGraw-Hill, New York,
1980.
[2] C. Shelley, M.E. Munk, Anal. Chem. 54 (1982) 516.
[3] N.A.B. Gray, in: Computer-Assisted Structure Elucidation,
Willey, New York, 1986.
[4] B.D. Christie, M.E. Munk, Anal. Chim. Acta 200 (1987) 347.
[5] K. Funatsu, N. Miyabashi, S. Sasaki, J. Chem. Inform.
Comput. Sci. 28 (1988) 18.
[6] M. Carabedian, I. Dagane, J.-E. Dubois, Anal. Chem. 60
(1988) 2186.
[7] M. Will, W. Fachinger, J.R. Richert, J. Chem. Inform.
Comput. Sci. 36 (1996) 221.
[8] M.E. Munk, J. Chem. Inform. Comput. Sci. 38 (1998) 997.
[9] M. Jaspars, Nat. Prod. Rep. 16 (1999) 241.
[10] M. Badertscher, A. Korytko, K.P. Schulz, M. Madison, M.E.
Munk, P. Portmann, M. Junghans, P. Fontana, E. Pretsch,
Chemometrics Intell. Lab. Syst. 51 (2000) 73.
[11] J.P. Gastmans, M. Furlan, M.N. Lopes, J.H.G. Borges, V.P.
Emerenciano, Qumica Nova 13 (1990) 10.
[12] V.P. Emerenciano, G.V. Rodrigues, P.A.T. Macari, S.A.
Vestri, J.H.G. Borges, J.P. Gastmans, D.L.G. Fromanteau,
Spectroscopy 12 (1994) 91.
[13] J.P. Gastmans, M. Furlan, M.N. Lopes, J.H.G. Borges, V.P.
Emerenciano, Qumica Nova 13 (1990) 75.
[14] D.L.G. Fromanteau, J.P. Gastmans, S.A. Vestri, V.P.
Emerenciano, J.H.G. Borges, Comput. Chem. 17 (1993) 369.
[15] P.A.T. Macari, J.P. Gastmans, G.V. Rodrigues, V.P.
Emerenciano, Spectroscopy 12 (1994/1995) 139.
[16] G.V. Rodrigues, I.P.A. Campos, V.P. Emerenciano,
Spectroscopy 13 (1997) 191.
[17] M.J.P. Ferreira, Llm sistema especialista em determinacao
estructural de monoterpenos e indides; M. Sc. Dissertation,
IQ-USP (1999).
[18] M.J.P. Ferreira, A.J.C. Brant, G.V. Rodrigues, V.P.
Emerenciano, Anal. Chim. Acta 429 (2001) 151.

134

M.J.P. Ferreira et al. / Analytica Chimica Acta 447 (2001) 125134

[19] M.J.P. Ferreira, V.P. Emerenciano, G.A.R. Linia, P. Romoff,

P.A.T. Macari, G.V. Rodrigues, Prog. Nucl. Magn. Reson.
Spectros. 33 (1998) 153.
[20] V.P. Emerenciano, A.C. Bussolini, M. Furlan, G.V. Rodrigues,
D.L.G. Fromanteau, Spectroscopy 11 (1993) 95.
[21] F.C. Oliveira, M.J.P. Ferreira, C.V. Nez, G.V. Rodriguez,
V.P. Emerenciano, Prog. Nucl. Magn. Reson. Spectros. 37
(2000) 1.
[22] S.A.V. Alvarenga, J.P. Gastmans, G.V. Rodrigues, V.P.
Emerenciano, Spectroscopy 13 (1997) 227.
[23] V.P. Emerenciano, N.F. Roque, M. Furlan, L.M.B. Torres,
Anal. Chim. Acta 236 (1990) 501.
[24] D.A. Laude, C.L. Wilkins, Anal. Chem. 58 (1986) 2820.
[25] A. Bighelli, F. Tomi, J. Casanova, Biomass Bioenergy 6
(1994) 461.
[26] K.F. Wollenberg, J. Am. Chem. Soc. 67 (1990) 487.
[27] F.D. Gunstone, Chem. Phys. Lipids 58 (1992) 239.
[28] N. Low, T. Brisbane, G. Bigam, P. Sporns, J. Agric. Food
Chem. 36 (1988) 953.
[29] T. Brekke, D.W. Aksnes, E. Sletten, M. Stcker, Anal. Chem.
60 (1988) 591.
[30] M. Matlengiewicz, N. Henzel, J.C. Lauer, J.C. Laurens, D.
Nicole, P. Rubini, Analyst 117 (1992) 387.
[31] M. Corticchiato, J. Casanova, Analysis 20 (1992) M51.
[32] F. Tomi, P. Bradesi, A. Bighelli, J. Casanova, J. Magn. Reson.
Anal. 1 (1995) 25.
[33] V. Formcek, K.H. Kubeczka, Essential Oils Analysis
by Capillary Gas Chromatography and Carbon-13 NMR
Spectroscopy, Willey, Chichester, 1982.
[34] P. Willett, J.M. Barnard, G.M. Downs, J. Chem. Inform.
Comput. Sci. 38 (1998) 983.
[35] K. Baumann, J.T. Clerc, Anal. Chim. Acta 348 (1997) 327.
[36] C. Affolter, K. Baumann, J.T. Clerc, H. Schriber, E. Pretsch,
Mikrochim. Acta Suppl. 14 (1997) 143.
[37] K. Baumann, C. Affolter, E. Pretsch, J.T. Clerc, Mikrochim.
Acta Suppl. 14 (1997) 275.
[38] F. Gong, Y.Z. Liang, Q.S. Xu, F.T. Chau, J. Chromatogr. A
905 (2001) 193.

[39] M. Farkas, J. Bendl, D.H. Welti, E. Pretsch, S. Dutsch, P.

Portmann, M. Zurcher, J.T. Clerc, Anal. Chim. Acta 206
(1988) 173.
[40] A. Tsipouras, J. Ondeyka, C. Dufresne, S. Lee, G. Salituro,
N. Tsou, M. Goetz, S.B. Singh, S.K. Kearsley, Anal. Chim.
Acta 316 (1995) 161.
[41] W. Bremser, M. Klier, E. Meyer, Org. Magn. Reson. 7 (1975)
97.
[42] W. Bremser, Z. Fresenius, Anal. Chem. 286 (1977) 1.
[43] W. Mlynarik, M. Vida, V. Kello, Anal. Chim. Acta 122 (1980)
47.
[44] B.D. Christie, M.E. Munk, Anal. Chim. Acta 200 (1988) 346.
[45] M.E. Munk, B.D. Christie, Anal. Chim. Acta 216 (1989) 57.
[46] B.D. Christie, M.E. Munk, J. Am. Chem. Soc. 113 (1991)
3750.
[47] H. Kalchauser, R. Wolfgang, J. Chem. Inform. Comput. Sci.
25 (1985) 103.
[48] R. Randriamiharisoa, E.M. Gaydou, R. Faure, Magn. Reson.
Chem. 24 (1986) 275.
[49] R. Brauchli, A.F. Thomas, J. Agric. Food Chem. 39 (1991)
431.
[50] F. Bohlmann, C. Zdero, H. Robinson, R.M. King,
Phytochemistry 19 (1980) 2381.
[51] A. Ulubelen, G. Topcu, C. Eris, U. Sonmez, M. Kartal, S.
Kurucu, C. Bozokjohansson, Phytochemistry 36 (1994) 971.
[52] H. Weenen, M.H.H. Nkunya, Q.A. Mgani, M.A. Posthumus,
R. Waibel, H. Achenbach, J. Org. Chem. 56 (1991) 5865.
[53] J.C. Chalchat, B.P. Garry, A. Michet, Planta Med. 51 (1985)
285.
[54] Farmacopia Brasileira, Ateneu, So Paulo, 4th Edition, Part
I, 1988.
[55] R.P. Adams, Identification of Essential Oils by Ion Trap Mass
Spectrometry, Academic Press, New York, 1995.
[56] W. Jennings, T. Shibamoto, Qualitative Analysis of Flavor and
Fragrance Volatiles by Glass Capillary Gas Chromatography,
Academic Press, New York, 1980.
[57] Software ACD/Labs, Advanced Chemistry Development Inc.,
CNMR Predictor 4.5 Stardalon.