FLAVOUR AND FRAGRANCE JOURNAL

Flavour Fragr. J. 2001; 16: 289–293

DOI: 10.1002/ffj.999

Headspace–SPME analysis of volatiles of the ridge gourd

(Luffa acutangula) and bitter gourd (Momordica
charantia) flowers†
L. N. Fernando and I. U. Grün∗
Department of Food Science, 256 Eckles Hall, William C. Stringer Wing, University of Missouri–Columbia, Columbia, MO
65211-5160, USA
Received 29 June 2000
Revised 23 February 2001
Accepted 23 February 2001

ABSTRACT: The headspace (HS) volatile compounds of the flowers of ridge gourd (Luffa acutangula) and
bitter gourd (Momordica charantia) were analysed by solid-phase microextraction (SPME) coupled with capillary
gas chromatography/mass spectrometry (GC–MS). In the ridge gourd, 16 volatiles were positively identified
and nine were tentatively identified, while in the bitter gourd 13 compounds were positively identified and six
were tentatively identified. Typical compounds that are found in essential oils and fragrances such as terpenes,
hydrocarbons and oxygenated terpenes were successfully identified. For the ridge gourd, the results showed that
with more than 90% of the headspace, the most abundant volatile compound of the flower is trans-ˇ-ocimene. For
the bitter gourd flower, the four most abundant compounds were identified as linalool (5% of total headspace),
2-aminobenzaldehyde (27% of total headspace), 1H-indole (33% of total headspace) and methyl anthranilate (32%
of total headspace), accounting for over 95% of the headspace volatiles of the flower. These results indicate that
SPME coupled with GC–MS is a potential alternative for the effective extraction and analysis of odours of rare,
exotic, delicate flowers. Copyright  2001 John Wiley & Sons, Ltd.
KEY WORDS: SPME; GC–MS; Luffa acutangula; Momordica charantia; gourd; headspace volatiles

Introduction and quantified, in the flowers of L. cylindrica (sponge

Ridge gourd (Luffa acutangula) and bitter gourd (Momo-
rdica charantia) are best known for their food value
in Asia. The health benefits of bitter gourd have been
well documented, especially its anti-diabetic properties.1
On the other hand, ridge gourd has not been studied
much for its medicinal value, although it is a well-
known vegetable in Asia. The flowers of both gourds
have a pleasant odour, which has not been investigated.
The volatile compounds of the fruit and vines of bitter
gourd have been studied using an acetone extraction with
subsequent distillation and ether extraction in search of
compounds that are attractive to the melon fly.2 Inves-
tigation of the steam volatile constituents from seeds of
bitter gourd has revealed the presence of a compound
(MC 152) with a unique fragrance, in addition to vari-
ous other volatile compounds.3 Chang et al.4 separated
150 volatile components, of which 37 were identified
Ł Correspondence to: I. U. Grün, Department of Food Science, 256
Eckles Hall, William C. Stringer Wing, University of Missouri–
Columbia, Columbia, MO 65211-5160, USA.
E-mail: grueni@missouri.edu
† Contribution from the Missouri Agricultural Experiment Station.
Journal Series Number 13 048.
gourd).
Usually flavours and fragrances of aromatic herbs and
flowers are analysed by using the corresponding volatile
oils. Various methods have been used for the extrac-
tion of essential oils and fragrances from flowers for
commercial purposes as well as for research. Some of
these methods include steam or water distillation, solvent
extraction with subsequent or concurrent steam or water
distillation, and enfleurage (for flowers such as Jasmine).
These methods are time-consuming, expensive and can
form artifacts. Headspace analysis tends to be the pre-
ferred method for analysing the volatile composition of
delicate, odoriferous flowers, because other methods tend
to significantly change the volatile profile due to the
tedious and time-consuming extraction processes.
A relatively new extraction method, solid-phase
micro-extraction (SPME), was developed by Belardi
and Pawliszyn5 for pollutants in environmental water
analysis. SPME is based on the adsorption of
chemical compounds onto an extracting phase, such as
polydimethylsiloxane immobilized on a fused silica fibre
via a partitioning effect between the adsorbent and the
sample matrix, such as air, water, etc.6 SPME can be
used to isolate, extract and concentrate volatiles and

Copyright  2001 John Wiley & Sons, Ltd.

290 L. N. FERNANDO AND I. U. GRÜN
semi-volatile compounds in liquid or gaseous matrices
and can be used with GC or GC–MS, where the
adsorbed compounds are thermally desorbed in the
injection port. Although this method has been developed
for water pollutant work, SPME has found increasing
applications in flavour analysis. In one of the earliest
studies, this method was used to determine caffeine
in beverages, whereas more recently it has been used
to analyse the headspace flavour compounds of orange
juice, alcohols and esters in beer, flavour additives
in tobacco products, volatile aroma compounds in
wines and fruit and quantification of volatile aliphatic
aldehydes in sunflower oil.7 – 12 A comparative study of
four sampling methods for gas chromatography, namely
direct injection, dynamic headspace, gas-tight sampling
and SPME, of an essential oil revealed that SPME
provided information as to the distribution of major
and minor volatile compounds in the headspace above
the oil sample.13 Miller and Stuart14 observed dramatic
improvements in the extraction abilities of the SPME
fibres over the traditional static headspace method.
These authors used SPME for monitoring juice oxidation
products (terpene oxidation) as well as the profiling
of different juice samples. HS–SPME–GC–MS has
also been used to study the differences in essential oil
quality between flowers and leaves of various positions
of peppermint (Menthax piperita L.)15
The objective of this study was to investigate the
odoriferous volatile compounds in the headspace of ridge
gourd (Luffa acutangula) and bitter gourd (Momordica
charantia) flowers using HS–SPME–GC–MS.
Experimental
Sample Preparation
Bitter gourd and ridge gourd flowers were obtained from
a local garden, in Columbia, Missouri, over a 2 year
period. Bitter gourd flowers, blooming in the early morn-
ings, and ridge gourd flowers, blooming in the evenings,
were picked and immediately placed into screw-cap
5 ml microreaction vessels (Supelco, Bellefonte, PA,
USA) and tightly capped with caps containing 20 mm
PTFE/silicone septa (Supelco). Flowers were sampled,
both as the complete flower or as just one petal, in the
vessels in order to facilitate the measurement of minor
and major components in the headspace, respectively.
The time between picking the flowers and extraction of
headspace volatiles was approximately 2 h.
Headspace–Solid Phase–Microextraction
The syringe injector of the SPME unit (Supelco), equi-
pped with a 100 µm polydimethylsiloxane (PDMS) fibre,
Copyright  2001 John Wiley & Sons, Ltd.
was inserted through the septum of the microreaction
vessel cap into the headspace of the sample. The fibre
(1 cm) was exposed to the headspace for 30 min at room
temperature after an equilibration time of 2 h. The SPME
fibre was then retracted, the syringe was removed and the
volatiles were thermally desorbed into the GC column
by placing the SPME injection unit in the injector of the
GC–MS system. Trials were carried out to establish the
optimum times required for equilibration and absorption
of the headspace volatiles. Absorption times of 0.5,
10, 15 and 30 min were evaluated, and the effect of
multiple extractions from the same extraction vessel was
determined. Equilibration times of 2, 6 and 18 h were
also investigated. Approximately 2 h of equilibration
time and 30 min of absorption time were sufficient
for identification of most of the compounds. However,
shorter absorption times were used to identify the major
HS volatile compounds. These trials were necessary
mainly because of the inability of the ion trap of the
MS system to handle concentrations of molecules in the
trap too high for proper ionization, which in turn could
give erroneous results.
Gas Chromatography–Mass Spectrometry
Conditions
A Varian GC 3400CX (Varian, Walnut Creek, CA,
USA) equipped with a 1078 programmable injector con-
nected to a Varian Saturn 2000 ion trap mass spec-
trometric detector was used for the GC–MS analysis.
Compound separation was achieved on a 60 m, DB-
5MS with 0.25 mm i.d. and 0.25 µm film thickness (J&
W Scientific, Folsom, CA, USA) gas chromatographic
column. Carrier gas (ultra-pure helium) flow rate was
0.63 ml/min and the injector, transfer line and ion trap
temperatures were maintained at 250, 250 and 150 ° C,
respectively. The MS detector was used in the EI mode
with an ionization voltage of 70 eV. The column was
held at 35 ° C for 5 min and then programmed at 3 ° C/min
to 250 ° C. The volatiles were desorbed from the SPME
fibre in the splitless mode for 4 min before operating the
system in the split mode with a split flow of 100 ml/min.
The NIST 1992 and Wiley5 Mass Spectral libraries,
retention indices from the literature and retention indices
of authentic standards were used to identify the chemical
compounds.
Results and Discussion
Twenty-five and 19 compounds from the headspace of
ridge gourd and bitter gourd flowers (Tables 1 and 2),
respectively, were either positively or tentatively iden-
tified by SPME–GC–MS. In ridge gourd, 16 volatiles
Flavour Fragr. J. 2001; 16: 289–293
 HEADSPACE–SPME OF GOURD FLOWER VOLATILES 291

Table 1. Chemical compounds identified in the headspace of ridge gourd flower

Retention Retention Method of

Name of compound time index identification
3-Methyl-1-butanol 9.4 MS
4,5-Dimethyl-1-hexene 18.84 907 MS
˛-Thujene 20.09 930 MS,RI
˛-Pinene 20.54 938 MS,RI,STD
Sabinene 22.98 978 MS,RI
ˇ-Pinene 23.25 982 MS,RI,STD
ˇ-Myrcene 23.94 992 MS,RI,STD
D,L-Limonene 26.299 1034 MS,RI,STD
1,8-Cineole 26.525 1038 MS,RI,STD
ˇ-Ocimene(Z) 26.567 1039 MS,RI,STD
ˇ-Ocimene(E) 27.208 1050 MS,RI,STD
ˇ-Terpinene 28.424 1071 MS
-Terpinene 28.557 1073 MS,RI
Methyl, methyl ethyl substituted benzeneŁ 29.3 1085 MS
trans-Linalool oxide 29.759 1093 MS
trans-Dihydrocarvone 30.092 1098 MS
Linalool 30.36 1103 MS,RI,STD
cis-Sabinene hydrate 30.53 1106 MS,RI
˛-Thujone 30.701 1110 MS,RI
1,7-Octadien-3-one, 2-methyl-6-methylene 31.066 1117 MS
2,4,6-Octatriene 3,4-dimethyl 31.859 1132 MS
Epoxylinelol 34.16 1175 MS
˛-Terpineol 35.56 1199 MS,RI,STD
1H-Indole 40.391 1296 MS,RI,STD
Neryl acetate 43.35 1360 MS, RI
MS D mass spectra.
RI D published retention index.
STD D authentic standards.
Ł Methyl(1-methylethyl)-benzene; or 1-methyl-4-(1-methylethyl)-benzene; or 1-methyl-2-(1-methylethyl)-benzene; or 1-

methyl-3-(1-methylethyl)-benzene.

Table 2. Chemical compounds identified in the headspace of bitter gourd flower

Retention Retention Method of

Name of compound time index identification
˛-Pinene 20.533 938 MS,RI,STD
Sabinene 22.925 977 MS,RI
ˇ-Myrcene 23.892 991 MS,RI,STD
Trimethylbenzene 24.342 998 MS
Dichlorobenzene 25.62 1021 MS
D,L-Limonene 26.265 1033 MS,RI,STD
1,8-Cineole 26.474 1037 MS,RI,STD
ˇ-Ocimene(Z) 26.616 1040 MS,RI,STD
Hexanoic acid,2-ethyl-methyl ester 26.815 1043 MS
2-Hydroxybenzaldehyde 27.157 1049 MS
ˇ-Ocimene(E) 27.223 1051 MS,RI,STD
Linalool 30.241 1100 MS,RI,STD
Nonanal 30.525 1106 MS,RI,STD
4-Octenoic acid, methyl ester 31.2 1120 MS
Unsaturated hydrocarbon 35.164 1193 MS
2-Aminobenzaldehyde 36.642 1222 MS,STD
1H-Indole 40.367 1296 MS,RI,STD
Methylanthranilate 43.11 1346 MS,STD
˛-Humulene 46.607 1430 MS,RI
MS D mass spectra.
RI D published retention index.
STD D authentic standards.

were positively identified and nine were tentatively iden-
tified, while in bitter gourd 13 compounds were posi-
tively identified and six were tentatively identified. The
total ion chromatograms (TIC) of headspace volatiles
of ridge gourd and bitter gourd flowers are shown in
Figure 1A, B, respectively. Typical compounds such as
those found in essential oils and fragrances, namely
terpenes, hydrocarbons and oxygenated terpenes, were
successfully identified using mass spectra of published
data, retention indices as well as authentic standards. For
ridge gourd the results showed that the most abundant
(> 90% relative abundance) volatile compound in the

Copyright  2001 John Wiley & Sons, Ltd. Flavour Fragr. J. 2001; 16: 289–293
292 L. N. FERNANDO AND I. U. GRÜN

MCounts
17.5 A

Sabinene
15.0

β -Ocimene( E )
β -Ocimene( Z )
12.5

β -Myrcene
10.0

α - Terpineol
7.5
α - Pinene

Thujone
5.0
α - Thujene

2.5

0.0
KCounts
125 B

100
Linalool

1H-Indole

Methyl anthranilate
75
2 - Aminobenzaldehyde
D - Limonene
α - Pinene

50
β - Myrcene

25

0
20 30 40 50 60 minutes

Figure 1. TIC of the headspace volatile compounds of ridge gourd (A) and bitter gourd flower (B)

headspace of the flower is trans-ˇ-ocimene. We were also cautioned about the close resemblance of the mass
able to separate and identify both the cis and trans iso- spectra of 2-aminobenzaldehyde and formanilide (n-
mers of ocimene. This acyclic terpene hydrocarbon is phenyl formamide) in most of the commercial mass
present in many fruits and essential oils and gives a spectral libraries. We can confirm his findings, since
sweet, green terpene-like tropical odour. Although trans- we came across the same results until we obtained the
ˇ-ocimene was the most abundant compound in the standards of 2-aminobenzaldehyde and formanilide and
headspace of the flower, the odour of the flower is much compared the retention indices. This study was repeated
more complex, with a strong influence from minor com- during two consecutive summers and we obtained simi-
ponents. For bitter gourd flower, four key compounds, lar results each time.
three of which are nitrogen-containing, constituted the This study shows that although the major chemical
major part (> 95% relative abundance) of the headspace compounds in extracts and essential oils of exotic odor-
volatiles of the flower. These were linalool (5%), indole iferous flowers give the basic odour of the flower, the
(33%), 2-amino benzaldehyde (27%) and methyl anthr- minor components, which reveal the specific differences
anilate (32%). Indole has been identified in the flo- between flowers, can be studied using HS–SPME cou-
ral odour of many Cucurbitaceae species and has been pled with GC–MS. In fact, SPME allows one to study
reported as an attractant for western corn rootworm (Dia- the volatile compounds of flowers emitted on different
brotica virgifera virgifera) and the striped cucumber bee- days and times from the same flower in a non-destructive
tle (Acalymma vittatum).16,17 Linalool is present in many way while the flower is intact in the plant. As Joulain19
essential oils, is reminiscent of lily of the valley and is suggested, the nitrogen-containing compounds that are
one of the most frequently used fragrance substances.18 present in the headspace of odoriferous flowers may not
Joulain19 reported the presence of 2-aminobenzaldehyde be even detectable by conventional methods such as sol-
(31.5%) and linalool (14.6 %) in the headspace of the vent extractions and hydrodistillations, while they can
flowers of false acacia (Robina pseudoacacia L.). He be studied using HS–SPME. This method also avoids

Copyright  2001 John Wiley & Sons, Ltd. Flavour Fragr. J. 2001; 16: 289–293

the chemical modification and artifact formation that can
occur in conventional methods. However, SPME anal-
ysis still has considerable shortcomings when absolute
quantitation of compounds is of importance, because
day-to-day variations in the analytical methodology, as
well as fibre-to-fibre variations within and between pro-
duction lots, can cause low precision of the method.20
Acknowledgements—We thank Dr Klause O. Gerhardt for his expert
guidance and assistance in the interpretation of mass spectral data.
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