FLAVOUR AND FRAGRANCE JOURNALFlavour Fragr. J. 2001; 16: 289–293DOI: 10.1002/ffj.999

Headspace–SPME analysis of volatiles of the ridge gourd(Luffa acutangula) and bitter gourd (Momordicacharantia) flowers†

L. N. Fernando and I. U. Grun∗

Department of Food Science, 256 Eckles Hall, William C. Stringer Wing, University of Missouri–Columbia, Columbia, MO65211-5160, USA

Received 29 June 2000Revised 23 February 2001Accepted 23 February 2001

ABSTRACT: The headspace (HS) volatile compounds of the flowers of ridge gourd (Luffa acutangula) andbitter gourd (Momordica charantia) were analysed by solid-phase microextraction (SPME) coupled with capillarygas chromatography/mass spectrometry (GC–MS). In the ridge gourd, 16 volatiles were positively identifiedand nine were tentatively identified, while in the bitter gourd 13 compounds were positively identified and sixwere 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 thatwith more than 90% of the headspace, the most abundant volatile compound of the flower is trans-ˇ-ocimene. Forthe 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 thatSPME 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

Ridge gourd (Luffa acutangula) and bitter gourd (Momo-rdica charantia) are best known for their food valuein Asia. The health benefits of bitter gourd have beenwell documented, especially its anti-diabetic properties.1

On the other hand, ridge gourd has not been studiedmuch for its medicinal value, although it is a well-known vegetable in Asia. The flowers of both gourdshave a pleasant odour, which has not been investigated.The volatile compounds of the fruit and vines of bittergourd have been studied using an acetone extraction withsubsequent distillation and ether extraction in search ofcompounds that are attractive to the melon fly.2 Inves-tigation of the steam volatile constituents from seeds ofbitter 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 separated150 volatile components, of which 37 were identified

ŁCorrespondence to: I. U. Grun, Department of Food Science, 256Eckles 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.

and quantified, in the flowers of L. cylindrica (spongegourd).

Usually flavours and fragrances of aromatic herbs andflowers are analysed by using the corresponding volatileoils. Various methods have been used for the extrac-tion of essential oils and fragrances from flowers forcommercial purposes as well as for research. Some ofthese methods include steam or water distillation, solventextraction with subsequent or concurrent steam or waterdistillation, and enfleurage (for flowers such as Jasmine).These methods are time-consuming, expensive and canform artifacts. Headspace analysis tends to be the pre-ferred method for analysing the volatile composition ofdelicate, odoriferous flowers, because other methods tendto significantly change the volatile profile due to thetedious and time-consuming extraction processes.

A relatively new extraction method, solid-phasemicro-extraction (SPME), was developed by Belardiand Pawliszyn5 for pollutants in environmental wateranalysis. SPME is based on the adsorption ofchemical compounds onto an extracting phase, such aspolydimethylsiloxane immobilized on a fused silica fibrevia a partitioning effect between the adsorbent and thesample matrix, such as air, water, etc.6 SPME can beused to isolate, extract and concentrate volatiles and

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290 L. N. FERNANDO AND I. U. GRUN

semi-volatile compounds in liquid or gaseous matricesand can be used with GC or GC–MS, where theadsorbed compounds are thermally desorbed in theinjection port. Although this method has been developedfor water pollutant work, SPME has found increasingapplications in flavour analysis. In one of the earlieststudies, this method was used to determine caffeinein beverages, whereas more recently it has been usedto analyse the headspace flavour compounds of orangejuice, alcohols and esters in beer, flavour additivesin tobacco products, volatile aroma compounds inwines and fruit and quantification of volatile aliphaticaldehydes in sunflower oil.7–12 A comparative study offour sampling methods for gas chromatography, namelydirect injection, dynamic headspace, gas-tight samplingand SPME, of an essential oil revealed that SPMEprovided information as to the distribution of majorand minor volatile compounds in the headspace abovethe oil sample.13 Miller and Stuart14 observed dramaticimprovements in the extraction abilities of the SPMEfibres over the traditional static headspace method.These authors used SPME for monitoring juice oxidationproducts (terpene oxidation) as well as the profilingof different juice samples. HS–SPME–GC–MS hasalso been used to study the differences in essential oilquality between flowers and leaves of various positionsof peppermint (Menthax piperita L.)15

The objective of this study was to investigate theodoriferous volatile compounds in the headspace of ridgegourd (Luffa acutangula) and bitter gourd (Momordicacharantia) flowers using HS–SPME–GC–MS.

Experimental

Sample Preparation

Bitter gourd and ridge gourd flowers were obtained froma local garden, in Columbia, Missouri, over a 2 yearperiod. Bitter gourd flowers, blooming in the early morn-ings, and ridge gourd flowers, blooming in the evenings,were picked and immediately placed into screw-cap5 ml microreaction vessels (Supelco, Bellefonte, PA,USA) and tightly capped with caps containing 20 mmPTFE/silicone septa (Supelco). Flowers were sampled,both as the complete flower or as just one petal, in thevessels in order to facilitate the measurement of minorand major components in the headspace, respectively.The time between picking the flowers and extraction ofheadspace 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,

was inserted through the septum of the microreactionvessel cap into the headspace of the sample. The fibre(1 cm) was exposed to the headspace for 30 min at roomtemperature after an equilibration time of 2 h. The SPMEfibre was then retracted, the syringe was removed and thevolatiles were thermally desorbed into the GC columnby placing the SPME injection unit in the injector of theGC–MS system. Trials were carried out to establish theoptimum times required for equilibration and absorptionof the headspace volatiles. Absorption times of 0.5,10, 15 and 30 min were evaluated, and the effect ofmultiple extractions from the same extraction vessel wasdetermined. Equilibration times of 2, 6 and 18 h werealso investigated. Approximately 2 h of equilibrationtime and 30 min of absorption time were sufficientfor identification of most of the compounds. However,shorter absorption times were used to identify the majorHS volatile compounds. These trials were necessarymainly because of the inability of the ion trap of theMS system to handle concentrations of molecules in thetrap too high for proper ionization, which in turn couldgive erroneous results.

Gas Chromatography–Mass SpectrometryConditions

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 chromatographiccolumn. Carrier gas (ultra-pure helium) flow rate was0.63 ml/min and the injector, transfer line and ion traptemperatures were maintained at 250, 250 and 150 °C,respectively. The MS detector was used in the EI modewith an ionization voltage of 70 eV. The column washeld at 35 °C for 5 min and then programmed at 3 °C/minto 250 °C. The volatiles were desorbed from the SPMEfibre in the splitless mode for 4 min before operating thesystem 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 indicesof authentic standards were used to identify the chemicalcompounds.

Results and Discussion

Twenty-five and 19 compounds from the headspace ofridge 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

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HEADSPACE–SPME OF GOURD FLOWER VOLATILES 291

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

Retention Retention Method ofName of compound time index identification

3-Methyl-1-butanol 9.4 MS4,5-Dimethyl-1-hexene 18.84 907 MS˛-Thujene 20.09 930 MS,RI˛-Pinene 20.54 938 MS,RI,STDSabinene 22.98 978 MS,RIˇ-Pinene 23.25 982 MS,RI,STDˇ-Myrcene 23.94 992 MS,RI,STDD,L-Limonene 26.299 1034 MS,RI,STD1,8-Cineole 26.525 1038 MS,RI,STDˇ-Ocimene(Z) 26.567 1039 MS,RI,STDˇ-Ocimene(E) 27.208 1050 MS,RI,STDˇ-Terpinene 28.424 1071 MS�-Terpinene 28.557 1073 MS,RIMethyl, methyl ethyl substituted benzeneŁ 29.3 1085 MStrans-Linalool oxide 29.759 1093 MStrans-Dihydrocarvone 30.092 1098 MSLinalool 30.36 1103 MS,RI,STDcis-Sabinene hydrate 30.53 1106 MS,RI˛-Thujone 30.701 1110 MS,RI1,7-Octadien-3-one, 2-methyl-6-methylene 31.066 1117 MS2,4,6-Octatriene 3,4-dimethyl 31.859 1132 MSEpoxylinelol 34.16 1175 MS˛-Terpineol 35.56 1199 MS,RI,STD1H-Indole 40.391 1296 MS,RI,STDNeryl 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 ofName of compound time index identification

˛-Pinene 20.533 938 MS,RI,STDSabinene 22.925 977 MS,RIˇ-Myrcene 23.892 991 MS,RI,STDTrimethylbenzene 24.342 998 MSDichlorobenzene 25.62 1021 MSD,L-Limonene 26.265 1033 MS,RI,STD1,8-Cineole 26.474 1037 MS,RI,STDˇ-Ocimene(Z) 26.616 1040 MS,RI,STDHexanoic acid,2-ethyl-methyl ester 26.815 1043 MS2-Hydroxybenzaldehyde 27.157 1049 MSˇ-Ocimene(E) 27.223 1051 MS,RI,STDLinalool 30.241 1100 MS,RI,STDNonanal 30.525 1106 MS,RI,STD4-Octenoic acid, methyl ester 31.2 1120 MSUnsaturated hydrocarbon 35.164 1193 MS2-Aminobenzaldehyde 36.642 1222 MS,STD1H-Indole 40.367 1296 MS,RI,STDMethylanthranilate 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. Thetotal ion chromatograms (TIC) of headspace volatilesof ridge gourd and bitter gourd flowers are shown inFigure 1A, B, respectively. Typical compounds such as

those found in essential oils and fragrances, namelyterpenes, hydrocarbons and oxygenated terpenes, weresuccessfully identified using mass spectra of publisheddata, retention indices as well as authentic standards. Forridge gourd the results showed that the most abundant(> 90% relative abundance) volatile compound in the

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292 L. N. FERNANDO AND I. U. GRUN

MCounts

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B

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75

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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 wereable to separate and identify both the cis and trans iso-mers of ocimene. This acyclic terpene hydrocarbon ispresent in many fruits and essential oils and gives asweet, green terpene-like tropical odour. Although trans-ˇ-ocimene was the most abundant compound in theheadspace of the flower, the odour of the flower is muchmore complex, with a strong influence from minor com-ponents. For bitter gourd flower, four key compounds,three of which are nitrogen-containing, constituted themajor part (> 95% relative abundance) of the headspacevolatiles of the flower. These were linalool (5%), indole(33%), 2-amino benzaldehyde (27%) and methyl anthr-anilate (32%). Indole has been identified in the flo-ral odour of many Cucurbitaceae species and has beenreported as an attractant for western corn rootworm (Dia-brotica virgifera virgifera) and the striped cucumber bee-tle (Acalymma vittatum).16,17 Linalool is present in manyessential oils, is reminiscent of lily of the valley and isone of the most frequently used fragrance substances.18

Joulain19 reported the presence of 2-aminobenzaldehyde(31.5%) and linalool (14.6 %) in the headspace of theflowers of false acacia (Robina pseudoacacia L.). He

also cautioned about the close resemblance of the massspectra of 2-aminobenzaldehyde and formanilide (n-phenyl formamide) in most of the commercial massspectral libraries. We can confirm his findings, sincewe came across the same results until we obtained thestandards of 2-aminobenzaldehyde and formanilide andcompared the retention indices. This study was repeatedduring two consecutive summers and we obtained simi-lar results each time.

This study shows that although the major chemicalcompounds in extracts and essential oils of exotic odor-iferous flowers give the basic odour of the flower, theminor components, which reveal the specific differencesbetween flowers, can be studied using HS–SPME cou-pled with GC–MS. In fact, SPME allows one to studythe volatile compounds of flowers emitted on differentdays and times from the same flower in a non-destructiveway while the flower is intact in the plant. As Joulain19

suggested, the nitrogen-containing compounds that arepresent in the headspace of odoriferous flowers may notbe even detectable by conventional methods such as sol-vent extractions and hydrodistillations, while they canbe studied using HS–SPME. This method also avoids

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HEADSPACE–SPME OF GOURD FLOWER VOLATILES 293

the chemical modification and artifact formation that canoccur in conventional methods. However, SPME anal-ysis still has considerable shortcomings when absolutequantitation of compounds is of importance, becauseday-to-day variations in the analytical methodology, aswell 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 expertguidance and assistance in the interpretation of mass spectral data.

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