Analysis of Volatile Compoundsof Rosemary

Honey. Comparison of Different Extraction

Techniques

2003, 57, 227-233

L. Castro-Vdzquez / M. S. P6rez-Coello* / M. D. Cabezudo

Departamento de Ciencia y Tecnologia de Ahmentos. Facultad de Ciencias Quimicas. Universidad de Castilla-La Mancha.
Campus Universitario s/n, 13071 Ciudad Real.Spain; E-Maih Soledad.Perez@uclm.es

Liquid-liquid extraction using different

KeyWords organic solvents is one of the methods
most frequently used for this purpose
Gas chromatography - mass spectrometry [11 15]. Some authors have used continu-
Extraction techniques ous liquid-liquid extraction with diethyl
Volatile compounds ether and the gas-chromatographic analy-
Rosemary honey sis of the methytated extracts to character-
ise New Zealand unifloral honeys [1, 16
18]. This method was also applied by
Summary Wilkins et al. [19] to determine linalol de-
rivatives in nodding thistle honey.
The analysis of the volatile fraction from honey requires the sugar matrix to be separated prior Simultaneous distillation extraction
to the analysis by GC-MS. In this study, three extraction techniques, simultaneous extraction- (SDE) has been used under atmospheric
distillation, liquid-liquid extraction and solid-phase extraction, were compared to the extrac- conditions using the Likens and Nicker-
tion of the volatile compounds of a rosemary honey Analysis of these fractions by gas chroma- son apparatus to identify volatile com-
tography - mass spectrometry enabled the tentative identification of up to 122 volatile com- pounds from chestnut honey, some of
pounds (alcohols, ketones,aldehydes, acids, esters,terpenes, hydrocarbons, phenol, furan and them are specific of the floral source (3-
pyran compounds). SDE extracts were rich in terpenes and esters,while the other l",,votechni- aminoacetophenone) [12]. In order to
ques avoided the formation of artefacts clue to heating the sample. avoid the formation of artefacts when the
sample is subjected to distillation, some
authors propose working at reduced pres-
sure. Under these conditions hydrolysis
reactions were avoided and sugar degra-
dation products like furfural are not
Introduction posed as markers of the organoleptic qual- formed [20].
ity and authenticity of the honey [4, 5]. A preliminary extraction of volatile
Honey is a highly complex substrate to compounds from the sugar matrix with
Honey is a nutritious product with orga- analyse since it contains many volatile acetone before carrying out the extraction
noleptic importance and a characteristic components with different chemical struc- with SDE was used by Bichi et al. [21].
aroma. Many of the volatile compounds ture and low concentration in a sugar ma- The obtained extracts had a strong honey-
of honey come from the nectar of the trix where polar substances are the major like aroma and lacked furan derivatives.
flowers, and therefore monofloral honeys components. Bouseta et al. [22] optimised this method
have a distinctive pattern of volatile com- Over 300 volatile compounds have using dichloromethane in the pre-extrac-
pounds [1]. been identified in honeys: acids, alcohols, tion phase, obtaining excellent recoveries
Some volatile compounds have been ketones, aldehydes, terpenes, esters, etc. for 70 tested compounds. The optimised
described as characteristic of the floral [6 9]. The extraction of these compounds method was applied to characterise differ-
source: hexanal and heptanal in lavender prior to their analysis by gas chromato- ent unifloral honeys [23].
honey, dihydroxyketones, sulphur com- graphy has been carried out by different Solid-phase extraction using different
pounds and alkanes in eucalyptus honey methods, all of them with various advan- resins like Amberlita X A D or Porapak Q
[2, 3]. Other compounds have been pro- tages and disadvantages [10]. make it possible to obtain the volatile

Original Chromatographia 2003, 57, February (No. 3/4) 227

0009-5893/00/02 227- 07 $ 03.00/0 9 2003 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH
t,.,.h.m cl~ n o e Experimental
.9.3,:i t'l g'O r
~4
Three extraction methods were applied to
1gO 33Cr/ - 1'4
a commercial Spanish Rosemary honey
i 6g raS~20,
(Apisol) purchased in a local market. The
9D extractions were carried out in duplicate.
"-4 ~ DOOfl ..

"--20DO00
Simultaneous Distillation-
'. tl fl C,~0,0 ' 53 "_ "_'..3 ~.
Extraction (SDE)
i
-',6
l~t]CC.OO A micro scale simultaneous distillation-
27 ] ,I extraction apparatus (Chrompack, Mid-
~,qCt~OO z;..J g l 36 9
delburg, The Netherlands) was used, as
4 3CI~U~ ] Zi previously described by Godefroot et al.
[30]. 15 g of honey dissolved in 40 mL of
zacoua.j: -, , ,
distillated water, and 15 ixL of 2-pentanol
:

I -,I, II, ~ i,j . ,il:',.i, I (1 g L 1) as internal standard, were ex-

0 .~ ~,a,.,.,. ~ ~ ~4,t.,.~ ...,~,14
~.rr ;. "-B.fl[; ~C.O0 ?~.CO |O.CC -~O.OC r.O.O~ 7~.05 BD.~D 90.Co I tracted using dichloromethane as solvent
during 2 hours.
Time (min)
Figure 1. Total-ion chromatographic profile of the SDE extract from Rosemary honey.
Liquid-liquid Extraction
~EE-a,~.d,a.~oe
50 g of honey and 60 mL of dichloro-
methane were shaken at 300 rpm for 60
253~30D,
-1
minutes at a temperature of 30 ~ 50 ixL
of 2-pentanol (1 g L 1) were added as in-
ternal standard). The organic phase col-
zDaODOD ~ ~B
J lected was concentrated in a Vigreux col-
umn.
3,3
153C R.~n

Solid Phase Extraction (SPE)

9~ C~

i. 0 LIO LIO D - .r-~

8]
aa27 50 g of honey dissolved in 150 mL of dis-
i lid i tillated water and 50 ixL of 2-pentanol (1 g
L 1) as internal standard were passed
500P,OB 1 through an OASIS cartridge (Waters)
I , I ' containing 1 g of divinyl benzene-N-vinyl
l/ , pirrolydone. The cartridge was previously
4:/_ conditioned with 25 mL of methanol and
~_z_i_~9- - > tO_OD ~_,3 _ 0,"I, JO.CO 43.C0 bO,O0 .... 8,:1 .,.00 qR. DO aC,,. a.":, ':If,,. ,'3.3
25 mL of distilled water at a flow of 2 mL
min 1.
Time Cmin)
After the sample had passed through
Figure 2. Total-ion chromatographic profile of the liquid-liquid extract from Rosemary honey. the cartridge the sugars and other hydro-
philic substances were eluted with 100 mL
honey fraction without the need of apply- marker described for this type of honey of distillated water. The volatile com-
ing heat [24 27]. [3]. pounds of interest were eluted with
Head-space analysis methods can be The objective of this study was to com- 100mL of dichloromethane at a flow of
used for the analysis of the more volatile pare three different fractionation meth- 2mL min 1. The organic phase colleted
honey compounds, however the extrac- ods, simultaneous extraction and distilla- was concentrated in a Vigreux column.
tion yield and the number of compounds tion (SDE), liquid-liquid extraction and In all cases, the extracts obtained were
obtained is lower than with direct extrac- solid phase extraction (SPE), for charac- concentrated to 200 ixL under a nitrogen
tion methods [2, 3, 28, 29]. terise rosemary honey by determination flow.
Rosemary honey has been studied very of its volatile composition by GC-MS
little although it is one of the most abun- analysis.
dant monofloral honeys in the Mediterra- Chromatographic Conditions
nean area due to the great number of ro-
semary bushes found there. The absence The extracts were analysed using a HP G
of acetyl furan has been the only floral 1800 B GCD System with a mass detector

228 Chromatographia 2003, 57, February (No. 3/4) Original

(Agilent Technologies, Palo Alto, CA,
USA). 2 ixL of the extracts were injected in 50300Dg

splitless mode (0.6 min) in a BP-21 capil-

4bO~O~C
lary column (50 m • 0.32 mm • 0.32 ixm
of film thickness). The temperature of the 4DCO0~O
column was 60 ~ (3 min) 2~ min
3503D00-
200 ~ 1 (30 min). Carrier gas: helium
(0.8 mL min 1). Injector temperature was /
3 0 0 C ODD "
250~ Transfer line temperature was F

280 ~ The mass detector was operated in 25~c0~0 2: I , i",

:Ig
~ !"i
EI mode; the electron energy was 70 eV.
Mass acquisition range: 40 450 amu.
Peak identifications were based on
comparison with spectral data from the
Wiley G 1035 library, retention data was lco0~c0 i gJ s~o I':bs ~ r,, ' I 44
used when standard compounds were
5ou~cu:
I :
; ~
0
2~, ~ ~IJl
',,1"
I[
1 t '1-"'11 :,~ , l
IIII Jr ,
1
"
available. Semi-quantitative analysis of , 2: ,1,; j' r ~ ~ '
positively identified compounds was per- r, I. ' L --.'S...
~ l.__a~.--- _~_.} e . ~_9_ . z u . ~ u ~.ue 4c.3o sc.~: ~c.a~ ;c,o~ ~.ce 1_~.._s ..
formed assuming that compounds have
the same response factor to that the inter-
nal standard. Time (ram)
Figure 3. Total-ion chromatographic profile of the SPE extract from Rosemary honey9 (*) resin im-
purity9
Results and Discussion
Gas-chromatographic analysis of the ex- 3-hydroxy-2-butanone (acetoin) is a Some benzene derivatives (benzalde-
tracts obtained by means of SDE, liquid- characteristic honey compound [15, 31]. hyde, 2-phenylethanol, and benzylic alco-
liquid extraction and solid phase extrac- This compound is extracted in high con- hol) are characteristic compounds in hon-
tion (SPE) made it possible to identify and centration by liquid-liquid extraction. ey. Extracts obtained by means of liquid-
quantify a total of 122 volatile compounds Other ketones were extracted and identi- liquid extraction and solid-phase extrac-
of rosemary honey. The chromatograms fied. SDE showed low recovery percentage tion (SPE) contained higher concentra-
of the extracts are shown in Figures 1, 2 for the 3-hydroxy-2-butanone in a study tions of these compounds that SDE ex-
and 3. carried out with model solutions [32]. tracts, except in the case of the bencenea-
Table I shows the concentrations of the Acetic acid was obtained by liquid-li- cetaldehyde, which can be formed by
identified compounds arranged according quid extraction (2.16 mg kg 1), while Maillard reaction in the SDE tecnique
to their functional group. smaller amounts of all acids in general are [32].

13 linear alcohols were identified extracted with SDE. In this technique Furan and pyran compounds have
among which the levo- and meso- isomers losses of compounds with low boiling been associated with honey storage and
of 2,3-butanediol are notably due to their point could take place due to re-distilla- processing conditions. Furfural and hy-
high concentration (more than 3 mg kg 1). tions in the solvent flask. On the other droxymethylfurfural (HMF) are well
Despite their high concentration, they are hand, high polar compounds like acids, known quality markers in honey, exces-
only obtained by means of liquid-liquid ex- alcohols and ketones, could not have been sive amounts of these compounds indicate
traction and do not appear in the other two totally extracted by SDE because of their the possible loss of freshness because of a
extracts, probably because their volatilities high affinity for the aqueous phase and prolonged storage period or for exposure
are too low to be distillated in the SDE, consequently their low water/solvent par- to high temperatures [4, 11].
and they have too little affinity for the resin tition coefficients [32]. In order to quantify these compounds,
used in the SPE method. These compounds The esters of medium- and long-chain it is preferable to use a method where the
have been found in eucalyptus, and nod- fatty acids were obtained in higher con- sample is not heated. SDE is the least pre-
ding thistle honey, by other authors also centrations in both SDE and liquid-liquid ferred technique for this determination.
applying liquid-liquid extraction with extraction than by solid phase extraction. The amount of furfural is higher in the
ethyl acetate and diethyl ether, respectively Terpenic compounds (linalool, linalool SDE extract due to its formation during
[15,19]. oxides, hotrienol, nerol, geraniol ...) are the distillation of the sample. Other furan
Methyl alcohols of C4 alcohols (3- found in monofloral honeys coming from and pyran compounds were not comple-
methyl-i- butanol; 3-methyl-3-buten-l-ol; the plant pollen and have pleasant flower tely extracted with this method [32, 33],
2-methyl-2-buten-l-ol) and C6 alcohols aromas that are reminiscent of the plant but they were present in the extracts ob-
(1-hexanol; 2-hexanol; cis-3-hexen-l-ol),- source [22]. Due to their low odour thresh- tained without heating the sample in sig-
characterised by having fresh herb aro- olds they can be of organoleptic signifi- nificant concentrations (like furfuryl alco-
mas, were found in low concentration in cance despite their low concentrations. hol, methyl furoate, hydroxymethylfur-
the three extracts. These compounds also SDE has been frequently applied for the fural and the two pyranones).
appear in higher concentrations in SDE extraction of terpenes in plants and gives Some lactones such as y-butyrolactone
extracts from lavender and eucalyptus good recoveries in the analysis of these and pantolactone are characterised by
honeys [31]. compounds in honey [23]. their sweet odour. These compounds were

Original Chromatographia 2003, 57, February (No. 3/4) 229

Table I. Volatile compound concentrations (mg kg 1) in the three extracts of Rosemary honey.

SDE Liquid-liquid SPE

Peak Compounds
Mean RSD(%) Mean RSD(%) Mean RSD(%)
Alcohols
1 2-Hexanol 0.01 4 0.01 5 0.02 4
2 2-Methyl-l-butanol 0.13 13 0.14 1 0.16 15
3 3-Methyl-3-buten-l-ol 0.09 6 0.12 3 O.O9 3
4 2-Heptanol 0.01 1 0.01 2
5 2-Methyl-2-buten-l-ol 0.11 8 0.11 1 0.15 8
6 1-Hexanol 0.03 6 0.03 3 0.07 4
7 (Z) 3-Hexen-1- ol traces traces 0.01 2
8 2-Butoxyethanol 0.04 3 0.04 7 0.04 4
9 2-Ethyl-l-hexanol 0.01 2 0.02 6
10 2,3-Butanediol (levo) 3.24 6
11 2,3-Butenediol (meso) 11.20 6
12 4-Methyl-3-cyclohexen-l-ol traces
13 1-Nonenol 0.010 5 0.08 1 0.10 8
Ketones
14 3-Hydroxy-2-butanone 0.05 10 0.24 3 0.02 10
15 6-Methyl-5-hepten-2-one 0.02 3
16 1-Hydroxy-2-butanone 0.01 12 0.06 2
17 3,5,5--Trimethyl-2-cyclohexen-1,4-dione 0.02 2 0.02 3 0.02 5
18 6-Methyl-3,5-heptadien-2-one 0.01 6
19 3-Methyl-2-hydroxy-l,2-cyclopentanone 0.03 11 traces
20 8-Nonen-2-one 0.16 12
Aldehydes
21 Nonanal 0.11 22 0.04 47 0.05 8
22 Decanal 0.04 10 0.03 4 0.04 10
23 3-Phenyl-2-propenal 0.01 4
Acids
24 Acetic acid 0.03 8 2.16 3 0.12 12
25 2-Methyl propanoic acid 0.02 7 0.10 8 0.11 13
26 Butanoic acid 0.07 14 0.39 6 0.37 12
27 2-Methyl butanoic acid 0.20 6 0.43 6 0.37 17
28 3~ 2-butenoic acid traces 0.03 2 0.03 8
29 Hexanoic acid 0.11 15 0.17 12 0.19 15
30 2-Methyl 2-butenoic acid 0.01 2 0.03 13 0.03 6
31 2-Ethyl hexanoic acid 0.03 8 0.05 1
32 Heptanoic acid 0.02 6 0.04 2 traces
33 3-Hexenoic acid 0.11 1 0.68 5 0.70 16
34 3,5,5-Trimethyl hexanoic acid 0.03 8 0.10 1
35 Octanoic acid 0.11 7 0.24 5 0.24 7
36 Nonanoic acid 0.14 11 0.14 7 0.17 13
37 3-Phenyl 2-propenoic acid 0.04 16
38 Decanoic acid 0.07 2 0.07 7 0.08 4
39 4-Amino-l,5- pentanoic acid 0.16 2 0.13 5
40 Benzoic acid 1.53 1 1.13 14
41 Hexadecanoic acid 1.23 8 1.32 5 0.50 16
42 Dodecanoic acid 0.12 9 0.14 6 0.15 14
43 Benzenacetic acid 0.83 11 0.60 17
44 Tetradecanoic acid 0.38 12 0.77 8 0.53 21

230 Chromatographia 2003, 57, February (No. 3/4) Original

Table I. (Continued).
SDE Liquid-Liquid SPE
Peak Compounds
Mean RSD(%) Mean RSD(%) Mean RSD(%
Esters
45 Ethyl3-hexenoate 0.01 4 0.01 3
46 Ethyl2-hydroxypropanoate 0.02 7 0.04 8 0.05 10
47 Ethyloctanoate 0.01 4 0.01 4
48 Ethylnonanoate 0.02 6 0.02 5
49 Diethyl malonate 0.01 4
50 Ethyldecanoate 0.01 3
51 Methyl2-hydroxybenzoate 0.03 8 0.05 9 0.06 15
52 Ethyltetradecanoate 0.07 14 0.17 4
53 Ethylhexadecanoate 0.40 11 0.34 11 0.11 14
54 Ethyloleate 1.26 19 0.90 9 0.11 16
55 Methyl 3-hydroxybenzoate 0.07 13
56 Methyl9,12,15-octadecatrienoate 0.52 14 0.51 4
57 Dibutyl 1,2-benzenedicarboxylate 0.29 19 0.81 16 0.81 21
Terpenes Derivatives
58 (Z) Linalool oxide 0.07 15 0.03 13 0.04 3
59 (E) Linalool oxide 0.03 1 0.02 5 0.03 2
60 Camphor 0.01 3 0.01 3 0.01 9
61 Linalool 0.04 14
62 Hotrienol 0.11 16 0.08 4 0.06 9
63 Borneol 0.01 13 0.03 13 0.01 13
64 2-Car-en-4-one 0.08 15 0.07 4 0.13 17
65 Nerol 0.08 9
66 13-Damascenone 0.01 12 0.01 8
67 Geraniol 0.18 10
68 Hydroxycineole 0.01 2 0.02 2
69 2,6-Dimetil 2,6 diol 3,7-octadiene 0.18 15 0.18 8
70 13-Maliene 0.06 4
71 I~-Eudesmol 0.03 14
72 Hydroxylinalool 0.11 7
Bencene Compounds
73 1,3-Dimethylbenzene 0.01 3 0.01 7 0.01 4
74 2-Methyl- 1-ethylbenzene t races t races t races
75 1,3,5-Trimethyl-benzene 0.01 3 traces
76 Benzaldehyde 0.02 1 0.02 9 0.19 15
77 Phenylacetaldehyde 1.62 15 0.29 7 0.37 15
78 Guaiacol 0.01 5 0.01 5
79 Eugenol 0.02 5 0.03 11
80 Benzylalcohol 0.10 15 0.28 6 0.20 1
81 2-Phenylethanol 0.19 14 0.51 11 1.49 4
82 o-Cresol 0.01 4
83 2-Phenoxyethanol 0.03 11 0.06 16
84 2,3,5,6-Tetram ethylphenol 0.05 10 0.16 3 0.10 14
85 2,4,6-Trimethylphenol 0.05 13 0.07 15
86 3-Phenyl-2-propen-l-ol 0.04 11 0.17 4 0.12 20
87 3,4,5-Trim ethyl-phenol 0.11 19 0.30 7 0.21 16

Original Chromatographia 2003, 57, February (No. 3/4) 231

Table I. (Continued).

SDE Liquid-Liquid SPE

Peak Compounds
Mean RSD(%) Mean RSD(%) Mean RSD(%'
Furan Compounds
88 2-Methylfuran traces
89 Dihydro-2-methyl-3(2H)-furanone, traces 0.24 3
90 Furfural 0.53 9 0.08 8 0.10 8
91 l(2-Furanyl)-ethanone 0.04 9 0.03 9 0.06 13
92 5-Methyl-2-furfural 0.05 9 0.06 4
93 Furfuryl alcohol 0.09 12 0.31 0 0.24 7
94 Methylfuroate 0.02 5 0.73 1 0.83 13
95 Hydroxymethylfurfural 7.58 4 5.41 14
Lactones
96 "/-Valerolactone 0.02 1 0.09 16 0.06 12
97 "pButyrolactone 0.01 15 0.16 13
98 "pHexalactone 0.02 6 0.02 9 0.03 17
99 Pantolactone 0.40 12 0.34 20
Pyran Compounds
100 3,4-Dihydro-2H-Pyran 0.02 8
101 6-Methyl- 2-methoxypyrazine 0.02 7 0.24 13
102 Maltol 0.01 9 0.13 14
103 5-Hydroxy-2-methyl-4H-pyran-4-one 0.09 11 0.12 17
104 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H- 0.70 1 0.13 9
pyran-4-one
105 3,5-Dihydroxy-2-methyl-4H-pyran-4-one 0.20 5 0.21 12
Hydrocarbons
106 Undecane 0.02 5 0.02 4 0.04
107 Tetradecane 0.10 16
108 2,6,10-Trimethyldodecane 0.02 9 0.01 4 traces
109 Pentadecane 0.03 5 0.05 10 0.05 13
110 1-Acetaldehyde, ~-4-dimethyl-3- 0.01 5
cyclohexene
111 Hexadecane 0.26 21
112 1-Hexadecene 0.17 9
113 Tetratiacontane 0.13 21
114 Docosane 0.06 15
115 3,3-Dimethyl-l,5-heptadiene 0.01 3
116 5-Octadecene 0.19 13
117 Nonadecene 0.17 1 0.16 16 0.04 4
118 Heneicosane 0.26 18 0.21 15 0.09 8
119 Tricosane 1.11 9 1.13 8 0.25 18
120 7-Hexadecene 0.51 15 0.15 11
Others
121 Dimethyl sulfoxide 0.21
122 N-(3-Methylbutyl) acetamide 0.03

present in the liquid-liquid extract in sig- have been described in Eucalyptus honey this technique presented poor recoveries
nificant concentrations when compared [2]. We found dimethylsulfoxide in the li- of compounds with high polarity and low
with lactones contents in other kind of quid-liquid extracts that can be formed by volatility like acids, 2,3 butanediol and
honeys. oxidation from the DMS. benzene derivatives.
Linear hydrocarbons are found in all SDE is a simple and fast technique use-
honeys coming from the bee wax and they ful when terpene derivatives are used to
were mostly extracted by solid phase ex- Conclusions differentiate monofloral honey. For the
traction. extraction of furan and pyran com-
Sulphur compounds have sensorial im- It can be concluded that sufficient volatile pounds, it would be better to use liquid-li-
portance because of their special odour compounds were obtained with all of the quid extraction to avoid the appearance
and low odour threshold. Dimethylsul- three extraction methods to get good in- of artefacts when the sample is heating.
phide (DMS) is present in many mono- formation about the sample. SDE extracts Liquid-liquid extraction is a recom-
floral honeys and dimethyldisulphide were rich in terpenes, and esters, although mended method for the quantitative ana-

232 Chromatographia 2003, 57, February (No. 3/4) Original

lysis of volatile compounds from honey, [5] Singh, N.; Bath, P.K. Food Chem. 1997, [21] Bicchi, C.; Belliardo, F.; Frattini, C. J.
since it provided a good yield and low 58, 129 133. Apic. Res. 1983,22,130 136.
[6] White, J. Honey, a Comprehensive Survey; [22] Bouseta, A.; Collin, S. J. Agric. Food
standard deviations for most of the ana- Grane, Ed.; Heinemann: London. 1975. Chem. 1995, 43, 1890 1897.
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Similar yields were obtained by solid [7] Graddon, A.D.; Morrison, J.D.; Smith, Collin, S. J. Agric. Food. Chem. 1998, 46,
J.F.J. Agric. Food. Chem. 1979, 27, 832 625 633.
phase extraction (SPE) for all compounds
837. [24] Shimoda, M.; Wu, Y.; Osajima, Y. J. Ag-
except for esters, that were poorly ex- [8] Hfiusler, M.; Montag, A. Z. Lebensm. Un- ric. FoodChem. 1996,44, 3913 3918.
tracted with this technique. Standard de- ters. Forsch. 1989,89, 113 115. [25] Dalla Serra, A.; Franco, M.A.; Mattivi,
viations were higher than when using li- [9] Hfiusler, M.; Montag, A. Deutsch. Le- F.; Ramponi, M.; Vacca, V.; Versini, G.
bensm.-Rdsch. 1990,86, 171 174. ital. J. FoodSci. 1999, 1 (11), 47 56.
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[12] Bonaga, G.; Giumanini, A . G . J . Apic.
[27] Guyot-Declerck, C.; Chevance, F.; Ler-
musieau, G.; Collin, S. J. Agric. Food
Res. 1986,25, 113 120. Chem. 2000,48(12), 5850 5855.
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cultura Pesca y Alimentacidn for the fi- Forsch. 1987,184, 17 19. boratory. 1994, 26 (6) 45, 47 53.
[14] Berahia, T.; Cerrati, C.; Sabatier, S.; [29] Campos, G.; Nappi, G.U.; Raslan, D.S.;
nancial support (API99-004-C2), in parti-
Amiot, M.J. Sci. Aliments. 1993,13, 15 Augusti, R. Cidnc. Tecnol. Aliment. Cam-
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