3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P31 ANALySIS OF FLAVOR COMPOuNDS IN
FRuITS by GC wITh TwO DIMENSIONAL
DETECTION by FID AND MS
R. JARA BIEDMA, T. GARCíA-BARRERA and
J. L. GóMEZ-ARIZA
Dpto. de Química y CC.MM. Facultad de Ciencias Experimentales.
Universidad de Huelva. Campus de El Carmen,
21007 Huelva, Spain,
tamara@dqcm.uhu.es
Introduction
The fruit juice industry has become one of the world´s
major agricultural businesses with world trade in fruit juices
annually exceeding 10 billion $ 1 . A key characteristic of
this product is the flavour. 2–7 Sweet orange (Citrus sinensis)
typical aroma is attributed to alcohols, hydrocarbons, esters
and aldehydes considering solely the number of compounds
involved. Among these compounds, citral, limonene, linalool,
α-pinene, ethyl butanoate, acetaldehyde and octanal have
been identified as most contributing to orange flavour, and
can be used in orange juice authentication. In addition, some
orange juice off-flavours can be developed such as α-terpineol
that is a well known off-flavour compound present in
stored citrus products formed from d-limonene or linalool 8 .
In this work, a method based on liquid-liquid extraction
with ethyl acetate and later preconcentration has been optimised
for the extraction of about twenty flavour compounds
in orange juice. The method is complemented by a chromatographic
separation (GC) for the identification of different
compounds based on the retention times obtained by gas
chromatography. 4–9 with flame ionization detector (GC-FID)
and the mass spectra of each analyte (GC-MS). Quantification
data was obtained with both detectors. The levels of the
flavour compounds have been used to enhance the quality
of oranges for industrial juice production and authentication
purposes.
Experimental
M a t e r i a l s
The gas chromatograph used for this study is an Agilent
6890n.
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•
•
•
Chromatographic parameters:
Column: HP-5MS. 30 m in length 0.25 mm ID. 0.25 μm
film.
Stationary phase (5% phenyl)-methylpolysiloxane
Injector Temperature: 250 °C
Oven Programme (Table I):
Table I
Oven program for the chromatographic separation
Temperature [°C] Rate [°C min –1 ] Hold [min] Total [min]
50 5 5
225 3 10 73.33
s641
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•
•
•
Detector temperature: 300 °C.
Column flow rate: 1 ml min –1
Carrier gas: Helium
Injection volume: 5 μl
A n a l y t i c a l M e t h o d s
Sample preparation
An aliquot of 30 ml of juice, previously tempered and
homogenized by manual or mechanical shaking, was measured
using a test tube of 50 ml Class A and transferred using a
funnel tapered to a 100 ml separating funnel, provided with
a Teflon key.
After that, 20 ml of juice was extracted with a mixture
of ethyl ether: methanol: ethyl acetate (18.5 : 1 : 0.5) and centrifuged
to 5 °C and 4000 rpm for 5 minutes. Supernatant was
withdrawn and passed to a 100 ml bottle with screw cap. The
juice was extracted again with 20 ml of pentane and centrifuge
to 5 °C and 4,000 rpm for 5 minutes. Supernatant was
withdrawn and passed to 100 ml container together with the
previous organic phase.
The third extraction was carried out with 20 ml of dichloromethane:
methanol (19 : 1) mixture and centrifuged to 5 °C
and 4,000 rpm for 5 minutes. The contents of the centrifuge
tube were transferred carefully to a 100 ml separating conical
funnel. After few minutes the organic phase (bottom) was
separated in a vial. Anhidrous sodium sulphate was added
and the content was stirred and centrifuged under the same
conditions above described.
The total of all three phases was passed to a functional
round-bottoned flask for evaporation with a rotary evaporator,
keeping the bath temperature at 70 °C to eliminate the
most volatile solvents (all of them with boiling point lower
than 70 °C except ethyl acetate). The extract was transferred
to an eppendorf tube and the internal standard (α-ionone)
was added to achieve a final concentration 50 mg dm –3 . The
extract was concentrated to a final volume of 1 ml.
Chromatographic analysis
5 μl of the extract with the internal standard were injected
in the gas chromatograph with a FID detector. For the inequi-
Fig. 1. Chromatogram of a sample of orange juice using GC-
FID: 1. hexanal + ethyl butyrate, 2. Cis-3-hexen-1-ol, 3. hexanol,
4. α-Pinene, 5. (-) β-Pinene, 6. Ehyll hexanoate, 7. Limonene, 8.
γ-Terpinene, 9. L-linalool, 10. (+)Terpinen-4-ol, 11. S-α-terpineol,
12. Decanal, 13. Neral, 14. Carvone, 15. Geranial, 16. Dodecanal,
17. α-ionone (internal standard), 18. β-ionone, 19. Valencene