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13.1 INTRODUCTION Bergamot peel oil is a valuable product for its olfactory properties and high commer-cial value. As unfortunately often happens, bergamot oil is subject to adulteration practices by industrial or commercial operators to increase their profi t. The fi rst information on bergamot adulteration is given by De Domenico (1854) from Reggio Calabria, who wrote a book on the medical properties of bergamot oil. De Domenico reported the addition to cold-extracted bergamot oil of a mixture obtained with "turpentine" (an essential oil extracted from the pine tree also known as turpentine oil) and sweet orange or lemon oils or oils obtained by distillation or cold extraction of unripe fruits of bergamot (called bergamottella) prematurely fallen from the trees. Similar practices were probably also applied for the adulteration of other citrus oils. Later, after Semmler and Tiemann (1892) found that linalyl acetate is the main com-ponent of bergamot oil, it became common practice to add esters obtained from sources different from bergamot, and of lower commercial value, to adulterate bergamot oil.
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349
13 Adulteration of
Bergamot Oil
Ivana Bonaccorsi, Luisa Schipilliti,
and Giovanni Dugo
13.1 INTRODUC TION
Bergamot peel oil is a valuable product for its olfactory properties and high commer-
cial value. As unfortunately often happens, bergamot oil is subject to adulteration
practices by industrial or commercial operators to increase their pro t.
The  rst information on bergamot adulteration is given by De Domenico (1854)
from Reggio Calabria, who wrote a book on the medical properties of bergamot oil. De
Domenico reported the addition to cold-extracted bergamot oil of a mixture obtained
with “turpentine” (an essential oil extracted from the pine tree also known as turpentine
oil) and sweet orange or lemon oils or oils obtained by distillation or cold extraction
of unripe fruits of bergamot (called bergamottella) prematurely fallen from the trees.
Similar practices were probably also applied for the adulteration of other citrus oils.
Later, after Semmler and Tiemann (1892) found that linalyl acetate is the main com-
po nen t of ber gam ot oil, it b eca me co mmon pra cti ce t o ad d es ter s ob taine d from sou rce s
different from bergamot, and of lower commercial value, to adulterate bergamot oil.
CONTENTS
13.1 Introduction ..................................................................................................349
13.2 Possible Adulterations of Bergamot Oil .......................................................350
13.3 Methods to Reveal Adulterations of Bergamot Oil ......................................351
13.3.1 Earlier Methods ................................................................................ 351
13.3.2 IR Spectroscopy ................................................................................ 351
13.3.3 UV Spectroscopy .............................................................................. 351
13.3.4 Paper Chromatography (PC) and ThinLayer Chromatography
(TLC) ................................................................................................ 353
13.3.5 High Performance Liquid Chromatography (HPLC) ....................... 355
13.3.6 Gas Chromatography (GC) with Conventional Stationary Phases ... 357
13.3.7 Chiral Gas Chromatography .............................................................365
13.3.8 Gas Chromatography–Isotope Ratio Mass Spectrometry
(GC-IRMS) ....................................................................................... 370
13.3.8.1 State of the Art ................................................................... 370
13.3.8.2 Application on Bergamot Oils ...........................................372
13.4 Final Remarks ............................................................................................... 377
References .............................................................................................................. 378
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350 Citrus bergamia
The  rst adulterants used to dilute bergamot oil and other citrus oils were turpen-
tine, petroleum, kerosene, and mineral oils. In addition to these products are hitherto
used by-products obtained from citrus transformation processes, such as recovered
oils obtained from the residues from the cold extraction, and terpenes, mainly from
sweet orange, obtained by fractionation of citrus oils to prepare terpene-free oils. In
this last case, the “reconstitution” of the oil is obtained by addition of substances to
increase the speci c gravity (in the past castor oil was used); addition of mixtures of
natural or synthetic substances to adjust the physicochemical properties (e.g., optical
rotation, UV absorbance, etc.); addition of the adulterated oil; or addition of synthetic
or natural ingredients of different botanical origin such as linalool or linalyl acetate
to imitate more or less the composition of the genuine oil.
When the methods for determining the physical properties of oils were not fully
developed and available, the quality assessment of the oils was limited to qualitative
assays, such as treatment with fuming sulphuric acid to reveal the presence of inert
paraf ns contained in petroleum, mineral oils, and kerosene; to sensorial methods,
which are not objective; or to simple determinations such as the nonvolatile residue
by evaporation. The development and diffusion of new methods, such as the polari-
metric, revealed the presence of compounds with optical rotation different from
the natural oil. For example, the measure of the optical rotation of the  rst 10%
distillate of the essential oil revealed addition of turpentine oil. In fact, the major
component of turpentine is α-pinene, with a boiling point lower than most of the
components present in citrus essential oils, and with an optical rotation in turpen-
tine different from the (+)-limonene that is the main monoterpene hydrocarbon in
citrus oils.
The possibility of revealing adulteration of bergamot oil, as of any other citrus
cold-pressed essential oil, is mainly linked to the development and use of spectro-
scopic and chromatographic analytical techniques. This chapter will review applica-
tions to this  eld such as ultraviolet spectroscopy (UV), thin layer chromatography
(TLC), high pressure liquid chromatography (HPLC), gas chromatography (GC)
with conventional and chiral columns, and the coupled and multidimensional chro-
matographic techniques with classic detectors and mass spectrometer (conventional
and isotopic mass spectrometers). Infrared spectrometry (IR), seldom applied to the
analysis of bergamot oil and with unsatisfactory results, and paper chromatography
will also be discussed.
13.2 POSSIBLE ADULTERATIONS OF BERGAMOT OIL
Bergamot essential oil can be adulterated in many ways. Some of these methods
were detectable by analytical methods available in the 1950s and 1960s. In the 1990s
it b ecame possible to reveal most of them by the determination of enantiomeric ratios
of volatile components and of the isotopic ratios.
Addition of turpentine, petroleum, kerosene, mineral oils, castor oil, cedar
wood stearin
Addition of natural or synthetic compounds (menthyl and n-homomenthyl
salicilate; methyl N-methyl anthranilate; 4-methoxychalcone)
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351Adulteration of Bergamot Oil
Addition of esters different from linalyl acetate
Addition of citrus oils cheaper than bergamot, or their terpenes, mainly
obtained from sweet orange
Addition of oils recovered from the residues of cold extraction
Addition of the nonvolatile residue of citrus oils, mainly lime and
grapefruit
Addition of linalool and linalyl acetate synthetic or natural (obtained from
sources different from bergamot)
Also possible, although not common, is the occasional contamination with dif ferent
citrus oils due to inadequate cleaning procedures of the extraction lines previously
used to process different citrus.
13. 3 METHODS TO REVEAL ADULTERATIONS
OF BERGAMOT OIL
13.3.1 EARLIER METHODS
The oldest methods for the analysis of citrus oil, including bergamot, developed
by pioneers of the rst half of the past century, were revised by Guenther
(1949)and by Gildermeister and Hoffman (1959). Some of these (treatment with
fuming sulphuric acid, determination of speci c gravity, optical rotation) are
mentioned in the introduction of this chapter. Photometric and chromatographic
methods will be described here. UV spectroscopy and liquid chromatography are
aimed at the analysis of the oxygen heterocyclic, present in the nonvolatile resi-
due of the oil, while gas chromatography is dedicated to the determination of the
volatiles.
13.3.2 IR SPECTROSCOPY
Presnel (1953) compared the IR spectrum obtained from a genuine bergamot oil with
that of an adulterated one. He assumed that this technique could be used to reveal
possible frauds. Theile etal. (1960) observed that the ratio between the absorbance
bands at 835 cm−1 and 801 cm−1 varied for genuine bergamot oil between 0.90 and
1.14, and could be used to reveal the addition of monoterpene hydrocarbons. Di
Giacomo (1972) reported that at the Experimental Station in Reggio Calabria the
Theile’s ratio was considered among the genuineness parameters for bergamot oil,
with a range of variability of this ratio for genuine oil between 0.830 and 1.114.
Bovalo etal. (1985), following a study on numerous genuine oils, determined a range
of Theile’s ratio between 0.75 and 1.09. These authors also related Theile’s ratio with
the optical rotation.
13.3.3 UV SPECTROSCOPY
Morton (1929) found that cold-pressed lemon oils absorb UV light at 311 nm.
Surprisingly, Morton’s studies were independently continued only after 20 years
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352 Citrus bergamia
by Cultrera etal. (1952) in Sicily and by Sale etal. (1953) in the United States.
This research led to the conclusion that the oxygen heterocyclic compounds pres-
ent in the nonvolatile residue were responsible for this absorbance, while distilled
oils did not adsorb in the UV region. Thus, it was possible to differentiate these
oils and reveal mixtures of cold-pressed oils with distilled ones even if nonvola-
tile substances, which did not absorb in the UV region, were added to mask the
dilution. Cultrera etal. (1952) reported on the same graphic the UV transmit-
tance curves ofthe maxima and minima determined for genuine oils: the result-
ing area between thetwo curves represented all the transmittance values of these
oils. This is known as the “Palermo method.” Instead of transmittance, Sale etal.
(1953) used the values of UV absorbance and expressed the results as values of a
parameter called “CD.
The studies carried out by Cultrera etal. and by Sale etal. were originally on
lemon oils, but the UV characterization was soon applied for the genuineness assess-
ment of all citrus cold-pressed oils. Van Os and Dikastra (1937) measured the UV
absorbance of bergamot oil and of other citrus to explore the potential of this tech-
nique for quality assessment of the oils. La Face (1959) determined the UV absor-
bance of numerous genuine bergamot oils in order to establish limits of variation. In
this article were reported the absorbance pro les of genuine oils, of residues from
evaporation, and of selected compounds of the volatile and nonvolatile fraction of
bergamot.
Theile etal. (1960) found that the absorbance at 312 nm of an alcoholic solu-
tion of genuine bergamot oil varied between 10 and 13. They believed that it
could be possible to reveal the addition of mineral oils by the UV pro le of
bergamot oil. Calvarano and Calvarano (1964) determined for a very large num-
ber of samples of securely genuine bergamot oils the CV values and the E1%,1c m ,
proposed by Theile etal. (1960) as parameter of genuineness of the oils, and the
transmittance values determined at 260 and 310 nm using the Palermo method
(Cultrera etal. 1952). They found for the genuine sample a range of 9.95–15.40
for E1%, 1cm, and of 0.83–1.04 for CD. In the same article they reported the results
relative to a large number of surely adulterated or altered and reconstituted ber-
gamot oils.
Calvarano and Calvarano (1964) concluded that the CD parameter, among those
investigated, was the most reliable to reveal frauds on bergamot oil. Presently the
ranges of CD values for genuine bergamot oils are  xed by the ISO 3520:1998(E)
regulation between 0.760 and 1.180. In Figure 13.1 is a typical CD graph of a genuine
bergamot oil and of a distilled oil.
Calabrò etal. (1977) determined the  uorimetric properties of some cold-pressed
citrus oils, including bergamot, furocoumarin-free, and reconstituted oils. The val-
ues of  uorescence expressed as percentage of citropten in cold-pressed oils ranged
between 0.35% and 0.45%, while they were noticeably lower in furocoumarin free
oils and were below the limit of detection in the reconstituted oils. These authors
concluded that the measure of emitted  uorescence, along with the CD, could repre-
sent a parameter for genuineness assessment, mainly useful to reveal reconstituted
oils where the CD value was arti cially brought within the limit of genuine oils by
the addition of extraneous substances.
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353Adulteration of Bergamot Oil
13.3.4 PAPER CHROMATOGRAPHY (PC) AND THINLAYER
CHROMATOGRAPHY (TLC)
Although Chakraborty and Bose (1956) demonstrated that partition chromatography
on paper was a suitable technique to separate and identify coumarins and psoralens,
this technique has been scantily applied to the analysis of citrus essential oils and
their residues. To our knowledge applications of this technique on bergamot oil are
limited to Calvarano (1961) and Chambon etal. (1969), who used paper chromatog-
raphy to separate and determine bergaptene and citroptene.
Stanley and Vannier (1957a) found that limettin and other similar compounds iso-
lated from the nonvolatile residue by open column chromatography and TLC were
responsible for the UV absorbance of lemon oils. The same authors (Stanley and
Vannier 1957b; Vannier and Stanley 1958) developed an analytical method for the
analysis of coumarins and psoralens in different essential oils and found that by
analyzing these compounds it could be possible to differentiate the pure oils from
mixtures of different essential oils. Stanley (1959, 1961) using similar procedures
determined some natural and synthetic compounds (methyl and n-homomethyl sali-
cilate, methyl anthranilate, and 4-methoxycalcone) which absorb in the UV region
and were used to increase the CD value of cold-pressed oils diluted with distilled
ones or terpenes.
Stanley and Jurd (1971) in their review on the distribution of coumarins in citrus
asserted that the open column chromatography, TLC, along with spectral absorbance,
uorescence emission, and chromatographic analysis provide useful information on
the quality and genuineness of essential oils. D’Amore and Calapay (1965) used ana-
lytical TLC to determine the chromatographic characters of  uorescent compounds
present in citrus essential oils, including bergamot, and preparative TLC to isolate
these components and determine their UV spectroscopic properties. Rf values deter-
mined with different eluents and the wavelengths of the maxima, minima, and  ex of
the single  uorescent components were reported, but no quantitative results. These
results are useful to detect contamination or mixtures of different oils.
Cieri (1969) determined the content of coumarins and furocoumarins in berga-
mot oil and in other natural and commercial essential oils and the ratio between
Wavelength (nm)
260
0.0
0.5
1.0
1.5
C
A
D
B
2.0
Abs
(a)
(b)
300 350 400
FIGURE 13.1 Typical CD line plots for cold-pressed (a) and distilled (b) bergamot oils.
(From Dugo G., Bonaccorsi I., Russo M., and Dugo P., Unpublished results, 2011a.)
TAF-K12886-13-0201-C013.indd 353 15/07/13 6:35 PM
354 Citrus bergamia
bergapten and the other oxygen heterocyclic compounds present in the residue of the
oils analyzed. These ratios, as stated by the authors, can provide information on the
origin of bergapten in commercial oils and in oils of unknown origin.
Madsen and Latz (1970) developed a method to determine the content of  uores-
cent compounds in different essential oils, including bergamot, by direct  uorimetric
scanning of the fraction separated by TLC; Di Giacomo and Calvarano (1974) used
Madsen and Latz’s method to determine these components in genuine bergamot oils.
They observed that the variability range of these compounds was narrow and with-
out signi cant variation during the productive season, and could be therefore used
as a valid tool for purity assessment of the oils. In Figure 13.2 is a graph obtained by
spectro uorimetric scanning of the chromatograms where the signals of bergaptol,
citroptene, and of 5-gernyloxy-7-methoxy coumarin are visible. The presence of ber-
gaptol has been reported in literature, in addition to this article, only by Späth and
Socias (1934) and Di Giacomo (1990). Rodighero and Caporale (1974) believed that
bergaptol could derive from the decomposition of bergamottin, and Guenther (1949)
observed the tendency of bergamottin to produce bergaptol under speci c analytical
conditions. Günther and Zigler (1977) reported on the possible formation of artifacts
0
0
10
20
30
40
3
2
1
I
50
24 6 8
cm
10 12 14
FIGURE 13.2 Graphic of a spectro uorimetric scan of a TLC chromatogram: (1) bergaptol;
(2) citropten; (3) 5-geranyloxy-7-methoxycoumarin. (From Di Giacomo, A., and Calvarano,
I., Essenz. Deriv. Agrum. 44, 329–339, 1974.)
TAF-K12886-13-0201-C013.indd 354 15/07/13 6:35 PM
355Adulteration of Bergamot Oil
during the TLC separation of furocoumarins in citrus essential oils. Di Giacomo
(1990) recalled that to reproduce the UV pro le in bergamot oil it was common
practice to add not only the above-mentioned compounds, but also p-aminobenzoic
acid, commonly called anticalcone, which could be detected by TLC.
13.3. 5 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
The development of HPLC allowed researchers to separate, identify, and quantita-
tively determine the oxygen heterocyclic compounds in citrus essential oil with more
reliable results than the previously applied methods. The technique is simple and
versatile; it is possible to operate in normal and reversed phase and under isocratic or
gradient elution, thus selecting the most appropriate conditions to separate the com-
ponents of interest is possible. The use of UV detectors with variable wavelengths
permits optimization of the response of single compounds. The photodiode array
(PDA) and the hyphenation to mass spectrometers (MS) allows identi cation of the
components analyzed.
McHale and Sheridan (1989) in a milestone article determined the composition of
the oxygen heterocyclic compounds in cold-pressed citrus oils and listed all the com-
ponents present in a single oil. These authors concluded with the following sentence:
“While there is not a unique oxygen heterocyclic marker for every Citrus species, the
patterns of occurrence of the various components are suf ciently diverse to permit
the detection of peel oil from one species in that of another.”
Mondello etal. (1993) determined the variability range of coumarins and pso-
ralens in 128 samples of bergamot oil produced in Calabria during an entire grow-
ing season. In the same article were analyzed some reconstituted bergamot oils
which presented an anomalous composition of the oxygen heterocyclic fraction,
which was explained by the authors as the adulteration with oil or nonvolatile resi-
due of lime oil. The chromatogram of one of these oils is compared to that of a lime
oil in Figure 13.3.
Researchers at the Experimental Station in Reggio Calabria (Calvarano etal.
1995; Gionfriddo et al. 1997) identi ed in bergamot oil oxygen heterocyclic com-
pounds not indicated by other authors, but usually detected in lemon and lime oils.
Presently, if these compounds are detected in bergamot oils, their presence is still
considered a symptom of adulteration. The presence of these components in ber-
gamot oil needs further investigation to be con rmed. Calvarano etal. (1995) con-
rmed, however, that the HPLC analysis of the oxygen heterocyclic compounds in
bergamot oil reveals adulteration with extraneous substances with UV spectra simi-
lar to the cold-pressed oils or with different citrus species.
Bonaccorsi etal. (1999, 2000) optimized a fast HPLC method to obtain a
chromatographic pro le of the oxygen heterocyclic fraction of different citrus
cold-pressed oils, including bergamot oil. This method permitted differentiation
between citrus oils, revealing possible contaminations or cross-adulteration. An
example of these chromatographic pro les of the different citrus oils is reported
in Figure 13.4.
Recently Dugo etal. (2011a) obtained the HPLC chromatograms of bergamot
oils to which had been added 5%, 10%, and 20% of different citrus oils (Persian and
TAF-K12886-13-0201-C013.indd 355 15/07/13 6:35 PM
356 Citrus bergamia
Key limes, lemon, grapefruit, bitter orange, sweet orange, and mandarin). In Figures
13.5a through 13.5g are chromatograms of a genuine bergamot oil and of the mix-
tures from which are visible the addition of oils of different citrus species.
Dugo etal. (1999), using HPLC-MS with an atmospheric pressure chemical
ionization (APCI) probe, identi ed in bergamot oil trace amounts of two polyme-
thoxylated  avones, tetra-O-methylscutellarein and sinensetin and, more recently,
Donato etal. (2013) identi ed nobiletine and tangeretine. Therefore, trace amounts
of these four compounds are not indicative of contamination or adulteration with
sweet orange oil. Dugo etal. (2012) found, in many genuine samples of bergamot oil
produced between 2008 and 2011, variable amounts of herniarin ranging between
8 and 385 ppm. The presence of herniarin at these concentrations must not be con-
sidered indicative of contamination or adulteration of bergamot oil.
3(a)
(b)
45
10
7
8
12 6 11
10
68
5
4
9
7
3
11
21
9
FIGURE 13.3 HPLC chromatogram of a reconstituted bergamot oil (a) and of a lime oil (b).
(1) bergamottin; (2) 5-geranyloxy-7-methoxycoumarin; (3) 5-geranyloxy-8-methoxypsoralen;
(4) 5-isopenthenyloxy-7-methoxycoumarin; (5) 5-isopenthenyloxy-8-methoxypsoralen; (6)
citropten; (7) 8-geranyloxypsoralen; (8) herniarin; (9) bergapten; (10) isopimpinellin; (11)
oxypeucedanin. (From Mondello, L., Stagno d’Alcontres, I., Del Duce, R., and Crispo, F.,
Flavour Fragr. J. 8, 17–24, 1993. Reproduced with permission.)
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357Adulteration of Bergamot Oil
13.3.6 GAS CHROMATOGRAPHY (GC) WITH CONVENTIONAL STATIONARY PHASES
Gas chromatography,  rst on packed columns and later on capillary columns, isthe
most appropriate analytical technique to learn the composition of the volatile fraction
of essential oils. Liberti was the  rst to speculate on the potentiality of this technique
for the analysis of essential oils in general and for citrus oils in particular. In 1956
he performed the  rst gas chromatogram of a bergamot oil (Liberti and Conte 1956).
Since then GC, working in isothermal or in temperature programmed, with universal
or selective detectors, has become the analytical technique most applied to deter-
mine the composition of the volatile fraction of all essential oils and to evaluate
contaminations and adulteration of essential oils with single components or mixtures
of other volatiles. Successively, the coupling of GC to mass spectrometry conjugated
the high separation power of one technique (GC) with the high identi cation power
of another (MS). The interactive use of the mass spectral data with chromatographic
retention parameters as the linear retention indices (LRI) rendered more reliable
the identi cation of components in complex mixtures. The use of stationary phases
capable of discriminating chiral volatile compounds, the development of multidi-
mensional chromatographic techniques, and the determination of the isotopic ratios
by mass spectrometry presently represent the most powerful tools to reveal the adul-
teration of numerous natural products, including citrus essential oils.
Theile etal. (1960) determined in bergamot oil the following variability ranges:
β-pinene 7%–10%; limonene 39%–46%; linalool 17%–24%; linalyl acetate 22%–
30%. They assumed that the addition of terpenes (mainly from sweet orange) could
2
400
600
800
ABS (mAu)
(e)
(d)
(c)
(b)
(a)
468
Time (min)
5
1
10
7
511 14 16
19
20
20
21
21
18
17
15
13
12
8
6
7
2
2
7
109
91011
11
1
4
3
10 12 14 16
FIGURE 13.4 HPLC-PDA chromatogram of mandarin (a); sweet orange (b); lemon (c); bit-
ter orange (d); bergamot (e) essential oils. (From Bonaccorsi, I., Dugo, G., McNair, H. M., and
Dugo, P., Ital. J. Food Sci. 4, 485–491, 2000. Reproduced with permission.)
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358 Citrus bergamia
TABLE 13.1
Composition of the Oxygen Heterocyclic Fraction of a Bergamot Essential Oil and a Bergamot +5% of a Key Lime, Persian
Lime, Grapefruit, and Bitter Orange Essential Oils; +10% of a Mandarin Essential Oil; and +20% of a Lemon Essential Oil
(mg/L)
No. Class Compounds Bergamot
+5%
Key lime
+5%
Persian lime
+5%
Grapefruit
+5%
Bitter orange
+10%
Mandarin
+20%
Lemon
1 CUM Meranzin hydrate 262
2 CUM Herniarin 301 517 670 2485 262 245 201
3 PSO Byakangelicin 331
4 CUM Citropten 2962 3260 3070 668 2606 2333 2224
5 CUM Isopimpinellin 225 170
6 CUM Meranzin 138 244
7 CUM Isomeranzin 1853 65
8 PSO Bergapten 2285 2243 2125 151 1950 1842 1584
9 PMF Sinensetin 72 75 59 57 62 98
10 PSO Byakangelicol 229
11 PSO Oxypeucedanin 235 168 456
12 PMF Nobiletin 78 192 403
13 PMF Tetra-O-methyl-
scutellarein
79 77 111 90 85 31
14 PMF Heptamethoxy avone 72 396
15 PMF Tangeretin 63 683
16 CUM Epoxyaurapten 301
17 PSO Phelloperin 32
18 CUM Osthol 111 75
19 PSO Epoxybergamottin 877 277
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359Adulteration of Bergamot Oil
20 CUM 5-Isopentenyloxy-7-
methoxy-coumarin
– – 159
21 PSO Cnidicin 26
22 PSO 8-Geranyloxy-
psoralen
– 167 68 224
23 CUM Aurapten 298
24 PSO 5-Geranyloxy-8-
methoxy-psoralen
– 155 64
25 PSO Bergamottin 17,606 16,473 16,927 13,986 14,712 13,660 12,413
26 CUM 5-Geranyloxy-7-
methoxy-coumarin
916 2132 2870 773 799 779 1145
Coumarins 4179 5909 6609 5036 4052 3356 3729
Psoralens 19,891 20,081 19,792 16,716 16,938 15,501 14,964
Polimethoxy avones 351 252 270 228 574 2041 129
All 22,070 24,990 25,401 21,981 20,564 23,255 18,822
TAF-K12886-13-0201-C013.indd 359 15/07/13 6:35 PM
360 Citrus bergamia
0.0
I.S.
4
8
2912
26
25315 nm, 4 nm (1.00)
mAU (×10)
0.0
2.0
4.0
6.0
8.0
(a)
5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 min
0.0
0.00
0.25
0.50
0.75
1.00
1.25
I.S.
4
8
2
3511
913 24
22
26
25315 nm, 4 nm (1.00)
mAU (×10)
1.50
(b)
5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 min
0.0
0.00
0.25
0.50
0.75
1.00
1.25
I.S.
4
8
2
511
913 24
22
2625
315 nm, 4 nm (1.00)
mAU (×10)
1.50
(c)
5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 min
FIGURE 13.5 RP-HPLC chromatograms of a cold-pressed bergamot oil (a) and of mixtures
of bergamot oil with 5% of Key lime oil (b), Persian lime oil (c), grapefruit oil (d), bitter
orange oil (e), 10% of mandarin oil (f), and 20% of lemon oil (g). For peak identi cation
see Table 13.1. (From Dugo G., Bonaccorsi I., Russo M., and Dugo P., Unpublished results,
2011a.)
TAF-K12886-13-0201-C013.indd 360 15/07/13 6:35 PM
361Adulteration of Bergamot Oil
0.0
0.00
0.25
0.50
0.75
1.00 I.S.
48
2
1
6
717
9
18
19
26
23
13
25
315 nm, 4 nm (1.00)
mAU (×10)
1.25
(d)
5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 min
0.0
0.00
0.25
0.50
1.00
1.50
0.75
1.25
I.S.
48
26
7
15
918 19
26
12 13
25
315 nm, 4 nm (1.00)
mAU (×10)
1.75
(e)
5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 min
0.0
0.00
0.25
0.75
1.25
0.50
1.00 I.S.
4
8
2
15
9
26
12 13
25
315 nm, 4 nm (1.00)
mAU (×10)
1.50
(f)
5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 min
FIGURE 13.5 (CONTINUED) RP-HPLC chromatograms of a cold-pressed bergamot oil
(a) and of mixtures of bergamot oil with 5% of Key lime oil (b), Persian lime oil (c), grapefruit
oil (d), bitter orange oil (e), 10% of mandarin oil (f), and 20% of lemon oil (g). For peak iden-
ti cation see Table 13.1. (From Dugo G., Bonaccorsi I., Russo M., and Dugo P., Unpublished
results, 2011a.)
TAF-K12886-13-0201-C013.indd 361 15/07/13 6:35 PM
362 Citrus bergamia
be detected by evaluation of the decrease in β-pinene and increase in limonene,
and by the lower percentage of linalool and linalyl acetate compared to the above-
mentioned values. The presence of other adulterants could also be found by the
detection of extraneous peaks in the chromatogram.
Calvarano and Calvarano (1964) determined in genuine and adulterated bergamot
oils the percentages of some monoterpene hydrocarbons as well as of linalool and
linalyl acetate, and the values of some physicochemical parameters. They observed
that the physicochemical indices, including the total free alcohols, determined in
the adulterated samples were compatible with those determined in genuine oils. On
the other hand, the GC analysis showed higher values of limonene and lower val-
ues of linalool, leading to the assumption that these oils were adulterated by the
addition of terpenes, probably of sweet orange, and by the addition of alcohols dif-
ferent from linalool. The same researchers (Calvarano 1965, 1968; Calvarano and
Calvarano 1968) corroborated the importance and validity of gas chromatography to
detect adulteration, perpetuated at that time on cold-pressed bergamot oil, from the
addition of recovered oils to mixtures of synthetic compounds. In particular, these
authors found that terpinen-4-ol is contained at higher percentages in recovered oils
than in cold-pressed ones; the ratio of the peak area relative to terpinen-4-ol and
that of the unidenti ed immediately following peak on a chromatogram obtained
on UCON LB 550X column (Figure 13.6) never exceeded 0.50 in cold-pressed oils,
while it increased in recovered oils.
Dugo etal. (1987, 1991), following the analysis of many hundreds of samples
of genuine bergamot oils cold-extracted during different productive seasons,
found that the quantitative composition varied during the season within such a
wide range that genuine assessment could not be based uniquely on this analyti-
cal approach. Verzera etal. (1996) calculated for 1081 samples of genuine berga-
mot oil the ranges of variability of the following ratios of components: citronellal/
0.0
0.00
0.25
0.50
0.75 I.S.
4
8
217 20
21 22
9
26
10 11
12
25
315 nm, 4 nm (1.00)
mAU (×10)
1.00
(g)
5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 min
FIGURE 13.5 (CONTINUED) RP-HPLC chromatograms of a cold-pressed bergamot oil
(a) and of mixtures of bergamot oil with 5% of Key lime oil (b), Persian lime oil (c), grapefruit
oil (d), bitter orange oil (e), 10% of mandarin oil (f), and 20% of lemon oil (g). For peak iden-
ti cation see Table 13.1. (From Dugo G., Bonaccorsi I., Russo M., and Dugo P., Unpublished
results, 2011a.)
TAF-K12886-13-0201-C013.indd 362 15/07/13 6:35 PM
363Adulteration of Bergamot Oil
24
27
28
30
34
37
36
40
38
39
35
31
29 25 16 15 12
14
97 2
32
42
43
40 30
Time in minutes
20 10 0
44
46
45 41 33
26
23
20
19
21
22
17
18
11
810 6
43 1
5
FIGURE 13.6 GC chromatogram on stainless steel capillary column coated with UCON LB 550X. 27, terpinene-4-ol; 28, unknown. (From Calvarano,
M., Essenz. Deriv. Agrum. 38, 21–30, 1968.)
TAF-K12886-13-0201-C013.indd 363 15/07/13 6:35 PM
364 Citrus bergamia
terpinene-4-ol (0.167–1.875); octyl acetate/α-terpineol (0.8424.742); γ-terpinene/
sabinene + β-pinene (0.661–1.279); and trans-sabinene hydrate acetate/α-terpineol
(0.704–3.323). These authors observed that some reconstituted bergamot oils
showed all the conventional parameters (volatile and nonvolatile composition;
enantiomeric distribution of linalool and linalyl acetate) compatible with genuine
oils. However, these samples presented values of the abovementioned ratios out-
side the limits reported for authentic oils, thus they can be considered adulterated.
Verzera etal. (1988) observed that the percentage of octanol could be used to
differentiate the cold-extracted oils, with a maximum of 0.010% of octanol, from
recovered oils (ricicli, torchi, pulizia dischi) where octanol increased from 0.023 to
0.044%. In the same article, these authors observed that the oils recovered by dis-
tillation, either at atmospheric pressure or at decreased pressure, presented lower
amounts of linalyl acetate and higher amounts of alcohols (linalool, terpinene-
4-ol, α-terpineol) and of (E)- and (Z)-β-ocimene, than cold-pressed oils. They
concluded that an increase of alcohols and of (E)- and (Z)-β-ocimene was indica-
tive of the addition of distilled oils to the cold-pressed ones.
Mondello etal. (2000) developed a method by Fast GC that allowed the elution
of some oxygen heterocyclic compounds in citrus oils in less than two minutes. This
method allowed detection of the addition of lime oil residue to bergamot oil by the
determination of the presence of herniarin and isopimpinellin (Figure 13.7).
Sweet orange oil, and therefore its terpenes, contain an average amount of 0.1%
of δ-3-carene, with a maximum of 0.3% in some cases. This component is absent,
or present at trace amounts (never exceeding 0.01%), in bergamot, lemon, manda-
rin, and bitter orange oils (Dugo etal. 2011b). The presence of sweet orange oil
or its terpenes can be revealed in bergamot oil, as well as in lemon and mandarin
(Dugo etal. 1992a) by the increased amount of δ-3-carene and of the ratios of
012
4
3
12
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
(a) (b) (c)
4
3
124
3
12
01201 2
FIGURE 13.7 Fast GC chromatogram obtained for (a) genuine bergamot oil, (b) mixtures
of bergamot and 20% of lime oil, (c) lime oil on a RTX-5 (10 m × 0.1 mm, 0.1 μm). (1) hernia-
rin; (2) citropten; (3) bergapten; (4) isopimpinellin. (From Mondello, L., Zappia, G., Errante,
G., and Dugo, G., LC-GC Europe 13, 495–502, 2000. Reproduced with permission.)
TAF-K12886-13-0201-C013.indd 364 15/07/13 6:35 PM
365Adulteration of Bergamot Oil
this component with α-terpinene and camphene. In Figure 13.8a through 13.8d
are the chromatograms obtained by Dugo etal. (2011a) for a sample of bergamot
oil, for mixtures of this oil with 5% and 10% of sweet orange oil, and of a sample
of sweet orange oil. The adulteration is visible by a simple comparison of the
chromatograms.
Lakszener and Szepsy (1988) used a selective detector for oxygenated compounds
(O-FID) to differentiate natural essential oils from sophisticated ones. Figure 13.9
compares the chromatograms obtained by these authors for a genuine and a sophis-
ticated oil. The two chromatograms are evidently different, but the identi cation of
the components is not reported.
13.3.7 CHIRAL GAS CHROMATOGRAPHY
The study of the enantiomeric distribution of volatile components in citrus oils
like bergamot by gas chromatography on columns with cyclodextrine as the
9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 min
9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 min
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
uV (×10,000)
Chromatogram
9.494
9.494
10.816
10.815
11.574
11.709
11.831
12.166
12.504
12.810
10.964
10.584
10.355
12.928
11.576
11.712
11.844
11.935
12.167
12.506
12.929
10.356
10.585
10.965
12.809
1
2
3
12 3
(a)
uV (×10,000)
Chromatogram
(b)
FIGURE 13.8 GC chromatogram of monoterpene zone of a bergamot oil (a), of a mixture
of bergamot oil with 5% (b), or 10% (c) of sweet orange oil, and of a sweet orange oil (d).
(1) camphene; (2) δ-3-carene; (3) α-terpinene. (From Dugo G., Bonaccorsi I., Russo M., and
Dugo P., Unpublished results, 2011a.)
TAF-K12886-13-0201-C013.indd 365 15/07/13 6:35 PM
366 Citrus bergamia
stationary phase started in the 1990s. It immediately showed its power to investi-
gate the genuineness of the oils and to reveal adulterations until then undetectable
by the commonly used analytical approaches. In fact, almost all the commercial
samples analyzed during the  rst half of the 1990s showed for limonene (Mosandl
etal. 1990; Hener et al. 1990; Weinreich and Nitz 1992; Mosandl 1995) and
for linalool and linalyl acetate (Hener etal. 1990; Schubert and Mosandl 1991;
Bernreuther and Schreier 1991; Weinreich and Nitz 1992; Neukom etal. 1993;
Casabianca and Graff 1996) enantiomeric distributions incompatible with those
later determined for genuine bergamot oils. Values of the enantiomeric distribu-
tion of linalool and linalyl acetate compatible with natural oils were reported
only for few commercial samples (Bernreuther and Schreier 1991; Neukom etal.
1993; Ravid etal. 1994) and laboratory extracted oils (Schubert and Mosandl
1991; Weinreich and Nitz 1992). The enantiomeric distribution of linalool in tea
9.319
9.490
1
2
3
1
2
3
9.0 9.5
9.491
10.574
11.716
11.839
11.571
10.970
10.812
10.577
10.961
12.808
10.350
11.568
11.706
11.829
12.163
12.502
12.925
12.888
10.354
12.174
12.523
10.0 10.5 11.0 11.5 12.0 12.5 min
9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
uV (×10,000)
Chromatogram
uV (×10,000)
Chromatogram
(c)
(d)
FIGURE 13.8 (CONTINUED) GC chromatogram of monoterpene zone of a bergamot
oil (a), of a mixture of bergamot oil with 5% (b), or 10% (c) of sweet orange oil, and of a sweet
orange oil (d). (1) camphene; (2) δ-3-carene; (3) α-terpinene. (From Dugo G., Bonaccorsi I.,
Russo M., and Dugo P., Unpublished results, 2011a.)
TAF-K12886-13-0201-C013.indd 366 15/07/13 6:35 PM
367Adulteration of Bergamot Oil
extracts was racemic or close to racemic in the samples analyzed by Neukom
etal. (1993) and Casabianca etal. (1995). Similarly, the enantiomeric distribu-
tion of linalool and linalyl acetate determined by Becker (1995) in 37 out of 53
samples of perfumes was not compatible with the values obtained for these com-
ponents in natural bergamot oils.
The analytical approaches used to determine the enantiomeric distribution of
volatiles in bergamot were direct GC with chiral columns (Cotroneo etal. 1992;
Ravid etal. 1994; Dellacassa etal. 1997; Costa etal. 2010); multidimensional GC
(MDGC with a conventional column in the  rst dimension and in second dimen-
sion a column with stationary phase capable of discriminating chiral compounds,
connected through an interface based on pressure balancing [line-T-piece])
(Mosandl etal. 1990; Hener etal. 1990; Casabianca etal. 1995; Casabianca and
Graff 1996; Juchelka and Mosandl 1996; Mosandl and Juchelka 1997a, 1997b);
MDGC with mechanical valve as interface (Mondello etal. 1997, 1998) or with
an innovative system based on pressure balancing (Dugo etal. 2012) developed by
Mondello et al. (2006); pre-separation by  ash chromatography (Casabianca and
Graff 1994) or by HPTLC (Mosandl and Juchelka 1997b) followed by multidi-
mensional GC separation of the fractions. For the analysis of alcohols in essential
oils of bitter and sweet orange, mandarin, and lemon, an HPLC-HRGC system
was also used (Dugo etal. 1994a, 1994b; Mondello etal. 1996). For the analysis
of limonene in lemon and mandarin oils (Dugo etal. 1992a, 1992b, 1993) two col-
umns were used—one conventional, the second chiral, installed in series. The last
method can also be applied to bergamot oils. It must be emphasized, however, that
some of the earliest chiral separations of linalyl acetate were carried out on chiral
columns of Ni(HFC)2 (Hener etal. 1990) or by carrying out the analysis after
(a)
(b)
FIGURE 13.9 O-FID chromatogram of a synthetic (a) and a natural (b) bergamot oil.
(From Lakszener, K., and Szepesy, L. Chromatographia 26, 91–96, 1988. Reproduced with
permission.)
TAF-K12886-13-0201-C013.indd 367 15/07/13 6:35 PM
368 Citrus bergamia
hydrogenation of the compound separated by  ash chromatography (Casabianca
and Graff 1994).
Dugo etal. (1992a) and Cotroneo etal. (1992) analyzed 150 samples of gen-
uine bergamot oils produced in Calabria, 8 samples of commercial linalool, and
20 samples of reconstituted oils obtained by addition to genuine oils of variable
amounts of commercial linalool, and determined that in genuine samples of berga-
mot oil the S-(+)-enantiomer of linalool never exceeded 0.5% of the total amount
of linalool. Values above this limit indicated adulteration by addition of synthetic or
natural linalool of different origin than bergamot with high percentage of the S-(+)-
enantiomer. These results were substantially con rmed by all successive studies,
although a few authors found S-(+)-enantiomer values that were slightly higher (up
to 1%) (Juchelka and Mosandl 1996). The same authors asserted, however, that the
R-()-enantiomer of linalool in bergamot oil must be higher than 99% (Mosandl and
Juchelka 1997a, 1997b).
In all the studies the enantiomeric distribution of linalyl acetate was determined
in proven genuine bergamot oils (see Chapter 11, Table 11.1, this volume) with the
S-(+)enantiomer never exceeding 0.4%. The only exception was in a sample from the
Ivory Coast (Sciarrone 2009) where the S-(+)-linalyl acetate was 1%. Mosandl and
Juchelka (1997a, 1997b) considered for the R-()-enantiomer of linalyl acetate the
same limits established for linalool for bergamot oils, with this enantiomer always
above 99% in genuine samples.
In oils recovered by distillation, the total amount of linalyl acetate decreases
(König etal. 1997) while its enantiomeric distribution does not change unless in
oils recovered in the past by inappropriate techniques (Mondello etal. 1998). On
the contrary, linalool shows a tendency to racemization, more or less evidently,
in recovered oils (Juchelka and Mosandl 1996; Mondello etal. 1996; König etal.
1997; Dugo etal. 2001, 2012; Sciarrone 2009); therefore its enantiomeric distribu-
tion results in a useful parameter to differentiate recovered oils from cold-extracted
ones. In Figures 13.10a through 13.10c are the chromatograms relative to the enan-
tiomeric separation of linalool and linalyl acetate in a genuine bergamot oil, in an
adulterated sample, and of a mix of synthetic linalool and linalyl acetate (Dugo
1997, unpublished results).
The enantiomeric distribution of linalool and linalyl acetate represents a funda-
mental parameter of genuineness and quality of bergamot oil. However, this param-
eter alone is not suf cient to evaluate the adulteration of bergamot oils with linalool
and linalyl acetate of natural origin, with enantiomeric distributions identical or
similar to that of natural bergamot. Small dilutions with terpenes, without the addi-
tion of extraneous linalool and linalyl acetate, would not change their enantiomeric
distribution.
The enantiomeric distribution of other volatiles in genuine bergamot oils was
determined by numerous authors (see Chapter 11, Table 11.1, this volume). For
some components the results are limited or the ranges of variations determined
are too wide and therefore not characteristic and cannot be used for quality assess-
ment; for other components the ranges of variation are narrow, so their enantio-
meric distribution can be used for quality assessment. These are reported in the
table below.
TAF-K12886-13-0201-C013.indd 368 15/07/13 6:35 PM
369Adulteration of Bergamot Oil
4
(a)
8
12
16
20
24
28
32
36
(+)-linalile acetato 0.2
()-linalile acetato 99.8
()-linalolo 99.4
(+)-linalolo 0.6
4
(b)
8
12
16
20
24
28
32
36
(+)-linalile acetato 19.2
()-linalile acetato 80.8
(+)-linalolo 76.0
(+)-linalolo 24.0
4
(c)
8
12
16
20
24
28
32
36
(+)-linalile acetato 35
()-linalile acetato 65
()-linalolo 50
(+)-linalolo 50
FIGURE 13.10 Direct enantio GC of a cold-pressed bergamot oil (a), an adulterated ber-
gamot oil (b), and a mixture of synthetic linalool and linalyl acetate (c). (From Dugo, G.,
Unpublished results, 1997.)
TAF-K12886-13-0201-C013.indd 369 15/07/13 6:35 PM
370 Citrus bergamia
Enantiomeric Excesses of Characteristic Volatiles in Bergamot Oil
a Value reported uniquely by Mosandl and Juchelka (1997b)
The dilution of the most valuable citrus oils with cheap sweet orange terpenes
leads to the increase in optical rotation. To correct this value, the addition of sweet
orange terpenes is always followed by the addition of a commercial product, ()-lim-
onene. This addition brings the optical rotation of the adulterated oil back to the
values expected for genuine bergamot, but the resulting enantiomeric distribution of
limonene is not compatible with any of the natural citrus oils. Dugo etal. (1992a,
1992b, 1993) revealed this adulteration in lemon and mandarin oils. Similarly, this
fraud can be revealed in bergamot. The enantiomeric excess of R-(+)-limonene in
bergamot oil is never less than 94 and never more than 96. In sweet orange this
excess is about 99. Value of enantiomeric excess of limonene in bergamot oils above
96 could be indicative of the addition of sweet orange terpenes without the addition
of ()-limonene.
Additional information on the enantiomeric distribution of volatile components in
bergamot oil is reported in Chapter 11, this volume.
13.3.8 GAS CHROMATOGRAPHY–ISOTOPE RATIO
MASS SPECTROMETRY (GC-IRMS)
Mass discrimination (kinetic isotope effect) and enantioselectivity are related to
the biosynthetic pathway of the plant. Both phenomena can represent characteristic
parameters of a single component to assess the origin and authenticity of natural
avors and fragrances.
13.3.8.1 State of the Art
One of the  rst systems for the determination of the isotope ratios 13C/12C and
15N/14N was developed by Matthews and Hayes (1978). This system was obtained
by the online coupling of a gas chromatograph with a combustion chamber
(CG-Combustion). Isotope ratio analysis of CO2 and N2 produced by combustion of
GC-separated compounds was performed by a single collector mass spectrometer.
Later, Barrie etal. (1984) improved the system by coupling to the GC-Combustion a
double mass collector capable of registering simultaneously two successive masses
and of continuous recording of their ratio (m + 1/m).
Isotope ratio mass spectrometry (IRMS) coupled with gas chromatography (GC)
is a useful analytical tool to evaluate origin and authenticity of natural citrus oils
R-()-α-Thujiene >97.0
S-()-α-Pinene >30.0
S-()-β-Pinene >80.0
S-()-Sabinene >60.0
R-(+)-Limonene >94.0
R-()-Linalool >98.0
R-()-Linalyl acetate >99.0
S-(+)- (E)-Nerolidola>60.0
TAF-K12886-13-0201-C013.indd 370 15/07/13 6:35 PM
371Adulteration of Bergamot Oil
as well of other natural  avors and fragrances as well as food. IRMS enables the
measurements of deviation of isotope abundance ratios, from an agreed standard,
by only a few parts per thousand for C, as well for other elements such as H, N, O,
S. Prerequisite for GC-IRMS analysis is high GC resolution (baseline: R2 1.5),
enabling the quantitative transfer of high-purity compounds to the mass spectrom-
eter, thus avoiding GC isotopic discrimination. Each compound must be converted
in a gaseous species before the transfer into the ion source (i.e., determination of the
13C/12C ratio via IRMS is obtained by converting the C atoms of the analytes into
CO2 by using a combustion chamber and then by comparing the C isotope ratio of
each analyte to that of a known reference). An adimensional quantity (δ) is used to
express the isotope ratio value of a speci c analyte in relation to the reference and
is expressed in ‰ (Brenna etal. 1997). Standard deviation is generally between the
fourth and sixth  gures.
Most of the plants used for human consumption belong to the C3 group. In these
plants the pathway of CO2 xation, known as Calvin cycle, results in the formation
of a C3 body, 3-phosphoglycerate [3(PGA)]. When the IRMS analysis is performed
on the metabolites of these plants the evaluation is critical. In fact, the values of δ13C
can overlap with those of synthetic compounds derived from fossil sources or CAM
plants. To avoid this inconvenience the concept of an internal standard, i-STD, was
introduced (Braunsdorf etal. 1993a; Mosandl 1995) based on the following criteria:
The component selected as the internal standard should be characteristic of
the matrix, but of low sensorial importance.
The component must be present in a signi cant amount in the sample and
must not undergo mass discrimination during sample preparation.
It must be biogenetically related to the components investigated.
It must be inert during storage and during the preparative processes applied
to the matrix.
The component selected as standard must not be a legally authorized additive.
This approach eliminates the in uences, due to the geographic origin and climate
conditions on δ13C values, which occur during the CO2 xation step in photosynthe-
sis. Thus it is possible to evaluate only the contribution of mass discrimination, which
occurs in the enzymatic reaction of the secondary biogenetic pathway. Figure13.11
provides an example of the reliability of this approach: application on a lemon essen-
tial oil (Mosandl 1995).
Enantio GC-IRMS also represents a unique method to reveal the addition of mix-
tures of natural chiral compounds with synthetic ones not detectable by enantio-GC
or by IRMS measurements (Mosandl 1995).
Using the guidelines suggested by Braunsdorf et al. (1993a) and Mosandl
(1995), the carbon isotope ratio has largely been applied to evaluate  avor and fra-
grance genuineness. The matrices studied, not including bergamot, were balm oils
(Hener etal. 1995), coriander oils (Frank etal. 1995), orange oils (Braunsdorf etal.
1993b), mandarin oils (Faulhaber etal. 1997a, 1997b; Schipilliti etal. 2010), lemon
(Schipilliti etal. 2011a), mentha piperita oils (Faber etal. 1995), volatile components
from strawberry (Schumacher etal. 1995; Schipilliti etal. 2011b) and apple (Karl
TAF-K12886-13-0201-C013.indd 371 15/07/13 6:35 PM
372 Citrus bergamia
etal. 1994), and nerolì and lime oils (Bonaccorsi etal. 2011a, 2011b). Applications
of IRMS to avor and fragrances were revised by Meier-Augenstein (1999) and by
Mosandl (2007).
13.3.8.2 Application on Bergamot Oils
Weinreich and Nitz (1992) studied the enantiomeric distribution and the carbon iso-
topic ratio by MDGC-IRMS of linalool and linalyl acetate in different essential oils
to establish a method to determine their origin (to differentiate them). For linalool
in bergamot oil, they reported values of δ13CPDB ‰ (29.77 ± 0.1) lower than those
obtained in lavender oil (27.93 ± 0.1) and in coriander (26.90 ± 0.1), and very
similar to that obtained in geranium oil (29.82 ± 0.1). Also for linalyl acetate, the
value of δ13CPDB ‰ (29.82 ± 0.1) obtained in bergamot oil was lower than that
obtained in lavender oil (28.41 ± 0.1).
1
–31
–30
–29
–28
–27
–26
–25
0
1
2
3
4
5
–1
23456789
1234
Italy
δ13C
δ13C
Argentina
56789
FIGURE 13.11 δ13C  ngerprint of biogenetically related compounds in lemon oils of differ-
ent geographical origin (top graph). The graph at the bottom shows the δ13C  ngerprint of the
samples obtained when using neryl acetate as internal standard. (1) β-pinene; (2) limonene;
(3) γ-terpinene; (4) nerol; (5) geraniol; (6) neral; (7) geranial; (8) neryl acetate; (9) geranyl
acetate. (From Mosandl, A., Food Rev. Int. 11, 597–664, 1995. Reproduced with permission.)
TAF-K12886-13-0201-C013.indd 372 15/07/13 6:35 PM
373Adulteration of Bergamot Oil
Casabianca etal. (1995), in a study of the authentication of  avors including lin-
alyl acetate from lavender and bergamot oils, asserted that the isotope ratio 13C/12C
was not useful due to the great similarity with synthetic products, and found more
reliable the ratio D/H determined by NMR.
Mosandl and Juchelka (1996, 1997a) used enantio-MDGC (see Section 13.3.7 of
this chapter) and GC-C-IRMS to establish the genuineness of bergamot oils and
unveil possible adulterations in commercial samples. They determined the ranges
of authenticity of the isotope ratios determined in genuine samples for α-pinene,
β-pinene+sabinene, γ-terpinene, limonene, myrcene, linalool, linalyl acetate, neryl
acetate, and caryophyllene and the values of the same components in seven com-
mercial oils. The adulteration of two commercial oils (samples 12 and 15) was
highlighted based on the enantiomeric distribution, and/or on the values of isotopic
ratios; the adulteration of sample 16 could not be unveiled based on enantiomeric
distribution but was clearly evident based on isotopic ratios (Figure 13.12).
Recently, a new study on the stable isotope ratio of carbon determined on volatiles
of bergamot oil was published by Schipilliti etal. (2011c). The genuineness assess-
ment was established based on the values of δ13CVPDB determined on numerous
Italian cold-pressed bergamot oils. These values were compared to those obtained
from oils of different geographic origin, from commercial samples, from surely
adulterated oils, and from distilled oils. This analytical approach was useful to dif-
ferentiate bergamot oils of different geographic origin, and by the use of internal
standard it was possible to con rm the authenticity of bergamot oils obtained from
fruits cultivated in the Ivory Coast. In addition, this technique proved the results
Limonene
12 15
16
Myrcene
Linalol
Linalyl acetate
Neryl acetate
Caryophyllene
γ-terpinene
α-pinene
–34
–32
–30
–28
–26
–24
–22
[‰]
CPDB
β-pinene/
sabinene
δ13
FIGURE 13.12 Commercial bergamot oils compared to the authenticity isotopic range
obtained for genuine bergamot oils. (From Juchelka, D., and Mosandl, A., Pharmazie 51,
417–222, 1996. Reproduced with permission.)
TAF-K12886-13-0201-C013.indd 373 15/07/13 6:35 PM
374 Citrus bergamia
–9
–7
–5
–3
–1
1
3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
1234567891011121314151617
18
1 2 3 4 5 6 7 8 9 101112131415161718
Min c-p i-std
Max c-p i-std
Ivory Coast 1 i-std
Ivory Coast 2 i-std
–10
–8
–6
–4
–2
0
2Min c-p i-std
Max c-p i-std
Co1 i-std
Co2 i-std
Co3 i-std
–9
–7
–5
–3
–1
1
3Min c-p i-std
Max c-p i-std
Co1 i-std
A50%Co1 i-std
A30%Co1 i-std
A20%Co1 i-std
(a)
(b)
(c)
δ13CMyrcene δ13CMyrcene
δ13CMyrcene
FIGURE 13.13 δ13CMyrcene values of Ivory Coast bergamot oils (a) of commercial berga-
mot oils (b) and of self-adulterated bergamot oils (c) compared to the authenticity range
determined for cold-pressed (c-p) bergamot oils. Compounds: (1) α-thujene; (2) α-pinene;
(3) β-pinene; (4) myrcene; (5) limonene; (6) γ-terpinene; (7) linalool; (8) linalyl acetate;
(9) α-terpinyl acetate; (10) neryl acetate; (11) geranyl acetate; (12) (E)-caryophyllene; (13)
trans-α-bergamotene; (14) β-bisabolene; (15) 2,3-dimethyl-3-(4-methyl-3-pentenyl)-2-nor-
bornanol; (16) campherenol; (17) α-bisabolol; (18) nootkatone. (From Schipilliti, L., Dugo,
P., Mondello, L., Santi, L., and Dugo, G., J. Essent. Oil Res. 23(2), 60–70, 2011. Reproduced
with permission.)
TAF-K12886-13-0201-C013.indd 374 15/07/13 6:35 PM
375Adulteration of Bergamot Oil
obtained by es-GC, unveiling the presence of adulteration in commercial samples and
in samples prepared in laboratory. The values of δ 13CVPDB determined in distilled sam-
ples matched, as predicted, the authenticity range determined for genuine cold-pressed
ones. These results are illustrated in Figure 13.13a through 13.13c and in Figure 13.14.
In another article from the same research group (Dugo etal. 2012), cold-pressed
Italian bergamot oils produced in the seasons 2008–2009, 2009–2010, and 2010–
2011 were analyzed by GC-C-IRMS. Dugo etal. (2012) also analyzed by GC-C-
IRMS different processed samples of bergamot oils (bergapten-free obtained by
treatment with alkali and by distillation), recovered (Peratoner and “fecce” oils), and
concentrated (crude and wax free) oils. The δ13CVPDB values determined for these
samples matched the range of authenticity determined for cold-pressed oils. These
results are illustrated in Figure 13.15a through 13.15c.
Hör etal. (2001) determined the hydrogen isotope ratio δ2HSMOW of linalool and
linalyl acetate, synthetic and natural of different botanical origins, including ber-
gamot, by gas chromatography-pyrolysis-isotope ratio mass spectrometry (GC-P-
IRMS). The range of δ2HSMOW (isotope ratio 2H/1H expressed in ‰ deviation relative
to the standard mean ocean water) determined for linalool isolated from bergamot
(273 to 294) was different from the values obtained for linalool isolated from
coriander, ho-oil, laurel leaf, neroli, and orange oils and partially or totally over-
lapped with the ranges determined in lavender, lavandin, petitgrain, rose wood, and
tea tree oils. The range determined for synthetic linalool (207 to 301), however,
overlapped, totally or partially, with all those of natural sources; thus based on this
–34
–32
–30
–28
–26
–24
–22
Min c-p
Max c-p
P1
P2
P3
123456789101112131415161718
δ13CVPDB
FIGURE 13.14 δ13CVPDB values of bergamot Peratoner oils compared to the authenticity
range determined for cold-pressed bergamot oils. Compounds: (1) α-thujene; (2) α-pinene;
(3) β-pinene; (4) myrcene; (5) limonene; (6) γ-terpinene; (7) linalool; (8) linalyl acetate; (9)
α-terpinyl acetate; (10) neryl acetate; (11) geranyl acetate; (12) (E)-caryophyllene; (13) trans-
α-bergamotene; (14) β-bisabolene; (15) 2,3-dimethyl-3-(4-methyl-3-pentenyl)-2-norbor-
nanol; (16) campherenol; (17) α-bisabolol; (18) nootkatone. (From Schipilliti, L., Dugo, P.,
Mondello, L., Santi. L., and Dugo, G., J. Essent. Oil Res. 23(2), 60–70, 2011. Reproduced
with permission.)
TAF-K12886-13-0201-C013.indd 375 15/07/13 6:35 PM
376 Citrus bergamia
parameter it was not possible to differentiate the natural and synthetic samples. The
range of δ2HSMOW linalyl acetate isolated from bergamot (252 to 280) was different
from those determined in synthetic linalyl acetate (199 to 239) and from nerolì oil
(213 to 232) but totally or partially overlapped with those determined in lavender,
lavandin, and petitgrain oils. Based on these results researchers could differentiate
Mean value bergamot oil
bergapten free distilled
Mean value bergamot oil
bergapten free alkali
treated
Min c-p 2008–2011
Max c-p 2008–2011
–31
–30
–29
–28
–27
–26
–25
–24
–33
–32
–31
–30
–29
–28
–27
–26
–25
–23(a)
(b)
–24
7 8 9 1011121314
Mean value bergamot oil
deterpenated and colorless
Mean value bergamot oil
deterpenated crude
Min c-p 2008–2011
Max c-p 2008–2011
12 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
δ13CVPDB
δ13CVPDB
FIGURE 13.15 Carbon isotope ratio values relative to volatile components of bergap-
ten-free distilled and alkali treated (a), deterpenated colorless and deterpenated crude (b),
recovered (c) bergamot oils compared to the authenticity range from cold-pressed bergamot
oils. Compounds: (1) α-thujene; (2) α-pinene; (3) β-pinene; (4) myrcene; (5) limonene; (6)
γ-terpinene; (7) linalool; (8) linalyl acetate; (9) α-terpinyl acetate; (10) neryl acetate; (11)
geranyl acetate; (12) (E)-caryophyllene; (13) trans-α-bergamotene; (14) β-bisabolene; (15)
2,3-dimethyl-3-(4-methyl-3-pentenyl)-2- norbornanol; (16) campherenol; (17) α-bisabolol;
(18) nootkatone. (From Dugo, G., Bonaccorsi I., Sciarrone D., etal., J. Essent. Oil Res. 24:
93–117, 2012. Reproduced with permission.)
TAF-K12886-13-0201-C013.indd 376 15/07/13 6:35 PM
377Adulteration of Bergamot Oil
synthetic linalool from the natural one isolated from lavender and lavandin but not
from those isolated from nerolì and petitgrain. Making a comparison between the
δ2HSMOW values of linalool and linalyl acetate of different natural origins, the authors
observed a depletion of linalool between 7% and 30‰.
In addition to the studies carried out by GC-C-IRMS, Hanneguelle etal. (1992)
used SNIF NMR and IRMS to characterize samples of linalool and linalyl acetate of
natural and synthetic origin. They determined the isotope ratios 13C/12C, expressed
as δ, and the total ratio D/H determined by IRMS and the site-speci c isotope ratio
(D/H)i obtained by NMR and IRMS data. These authors did not observe signi cant
differences for the values of δ13C determined for synthetic and natural linalool and
linalyl acetate. However, for the D/H values noticeable differences between syn-
thetic and natural samples were seen. Values of site-speci c isotopic ratios (D/H)i
and the statistical results based on principal component analysis (10 deuterium site-
speci c isotope ratios and δ13C) were useful to differentiate natural from synthetic
samples but could not discriminate between bergamot and other natural sources
investigated in this study.
13.4 FINAL REMARKS
The analytical tools available at this time, if applied properly, can successfully reveal
almost all the economically signi cant adulteration practices used in bergamot oil.
Mean value bergamot
oil recovered
Mean value bergamot
oil recovered Peratoner
Min c-p 2008–2011
Max c-p 2008–2011
1 2 3 4 5 6 7 8 9 10 11 12 13 14
–33
–32
–31
–30
–29
–28
–27
–26
–25
–23(c)
–24
δ13CVPDB
FIGURE 13.15 (CONTINUED) Carbon isotope ratio values relative to volatile components of
bergapten-free distilled and alkali treated (a), deterpenated colorless and deterpenated crude (b),
recovered (c) bergamot oils compared to the authenticity range from cold-pressed bergamot oils.
Compounds: (1) α-thujene; (2) α-pinene; (3) β-pinene; (4) myrcene; (5) limonene; (6) γ-terpinene;
(7) linalol; (8) linalyl acetate; (9) α-terpinyl acetate; (10) neryl acetate; (11) geranyl acetate; (12)
(E)-caryophyllene, (13) trans-α-bergamotene; (14) β-bisabolene; (15) 2,3-dimethyl-3-(4-methyl-
3-pentenyl)-2- norbornanol; (16) campherenol; (17) α-bisabolol; (18) nootkatone. (From Dugo,
G., Bonaccorsi I., Sciarrone D., et al., J. Essent. Oil Res. 24, 93–117, 2012. Reproduced with
permission.)
TAF-K12886-13-0201-C013.indd 377 15/07/13 6:35 PM
378 Citrus bergamia
However, it will always be necessary to improve the methodologies and develop new
dedicated methods to reveal more subtle adulterations, which evolve in parallel to
the depth of the analytical investigations. In these cases chiral and isotopic analyses
performed by heart-cutting multidimensional chromatography appear to be the most
promising techniques and, although not yet fully demonstrated, comprehensive chro-
matography techniques such as LC × LC could represent highly powerful tools still
to be exploited for deep analyses of nonvolatiles in bergamot oil.
REFERENCES
Barrie, A., Bricout, J., and Koziet, J. 1984. Gas chromatography-stable isotope ratio analysis
at natural abundance level. Biomed. Mass Spectrom. 11:583–588.
Becker etal. 1995. Ätherische öle-gepanschte seelen. Öko-Test 10:41–49.
Bernreuther, A., and Schreier, P. 1991. Multidimensional gas chromatography/mass spectrom-
etry: A powerful tool for the direct chiral evaluation of aroma compounds in plant tis-
sues. II. Linalol in essential oils and fruits. Phytochem. Anal. 2:167–170.
Bonaccorsi, I., McNair, H. M., Brunner, L. A., Dugo, P., and Dugo, G. 1999. Fast HPLC for
the analysis of oxygen heterocyclic compounds of citrus essential oils. J. Agric. Food
Chem. 47:4237–4239.
Bonaccorsi, I., Dugo, G., McNair, H. M., and Dugo, P. 2000. Rapid HPLC methods for the
analysis of the oxygen heterocyclic fraction in citrus essential oils. Ital. J. Food Sci.
4:485–491.
Bonaccorsi, I., Sciarrone, D., Schipilliti, L., etal. 2011a. Composition of Egyptian nerolì oil.
Nat. Product. Commun. 6:1009–1014.
Bonaccorsi, I., Sciarrone, D., Schipilliti, L., et al. 2011b. Multidimensional enantio gas
chromatography/mass spectrometry and gas chromatography-combustion- isotopic
ratio mass spectrometry for the authenticity assessment of lime essential oils
(C. aurantifolia Swingle and C. latifolia Tanaka). J. Chromatogr. A. In press. http://
dx.doi.org/10.1016/j.chroma.2011.10.038.
Bovalo, F., Cappello, C., Sardo, C., and Di Giacomo, A. 1985. A proposito dello spettro IR
dell’essenza di bergamotto. Essenz. Deriv. Agrum. 55:36–47.
Braunsdorf, R., Hener, U., Stein, S., and Mosandl, A. 1993a. Comprehensive cGC-IRMS
analysis in the authenticity control of  avours and essential oils. Part I: Lemon oil. Z.
Lebensm Unters. Forsch. 197:137–141.
Braunsdorf, R., Hener, U., Przibilla, G., Piecha, S., and Mosandl, A. 1993b. Analytische
und technologische Ein üsse auf das13C/12C-Isotopenverhältnis von Orangenöl-
Komponenten. Z. Lebensm Unters. Forsch. 197:24–28.
Brenna J. T., Corso, T. N., Tobias, H. J., and Caimi, R. J. 1997. High precision continuous- ow
isotope ratio mass spectrometry. Mass Spectrometry Rev. 16:227–258.
Calabrò, G., Currò, P., and Lo Coco, F. 1977. Studio spettro uorimetrico delle essenze agru-
marie. Essenz. Deriv. Agrum. 47:286–304.
Calvarano, M. 1961. Sulle cumarine dell’essenza di bergamotto. Essenz. Deriv. Agrum. 31:167.
Calvarano, M., and Calvarano, I. 1964. La composizione delle essenze di bergamotto. II.
Contributo all’indagine analitica mediante spettrofotometria nell’UV e gascromatogra-
a. Essenz. Deriv. Agrum. 34:71–92.
Calvarano, M. 1965. La composizione delle essenze di bergamotto. Nota III. Essenz. Deriv.
Agrum. 47:473–484.
Calvarano, M. 1968. Variazioni nella composizione dell’essenza di bergamotto durante la
maturazione del frutto. Essenz. Deriv. Agrum. 38:3–20.
Calvarano, M., and Calvarano, I. 1968. Applicazione della gascromatogra a all’analisi delle
essenze di bergamotto. Essenz. Deriv. Agrum. 38:21–30.
TAF-K12886-13-0201-C013.indd 378 15/07/13 6:35 PM
379Adulteration of Bergamot Oil
Calvarano, I., Calvarano, M., Gionfriddo, F., Bovalo, F., and Postorino, E. 1995. HPLC pro le of
citrus essential oils from different geographic origin. Essenz. Deriv. Agrum. 65:488–502.
Casabianca, H., and Graff, J.-B. 1994. Separation of linalyl acetate enantiomers: Application to
the authentication of bergamot food products. J. High Resolut. Chromatogr. 17:184–186.
Casabianca, H., Graff, J.-B., Jame, P., Perrucchietti, C., and Chastrette, M. 1995. Application
of hyphenated techniques to the chromatographic authentication of  avors in food prod-
ucts and perfumes. J. High Resolut. Chromatogr. 18:279–285.
Casabianca, H., and Graff, J.-B. 1996. Chiral analysis of linalol and linalyl acetate in various
plants. Riv. Ital. EPPOS 7:227–243.
Chakraborty, D. P., and Bose, P. K. 1956. Paper chromatographic studies of some natural cou-
marins. J. Ind. Chem. Soc. 23:905–910.
Chambon, P., Huit, B., and Chambon-Mougenot R. 1969. Dosage spectro uorimétrique
du bergaptène et du citroptène dans les essence de bergamote. Ann. Pharm. Franc.
27:635–638.
Cieri, U. R. 1969. Characterization of the steam non-volatile residue of bergamot oil and some
other essential oils. J. Assoc. Off. Anal. Chem. 52:719–728.
Costa, R., Dugo, P., Navarra, M., etal. 2010. Study on the chemical variability of some pro-
cessed bergamot (Citrus bergamia) essential oils. Flavour Fragr. J. 25:4–12.
Cotroneo, A., Stagno d’Alcontres, I., and Trozzi, A. 1992. On the genuineness of citrus essen-
tial oils. Part XXXIV. Detection of added reconstituted bergamot oil in genuine berga-
mot essential oil by high resolution gas chromatography with chiral capillary columns.
Flavour Fragr. J. 7:15–17.
Cultrera, R., Buffa, A., and Tri ro, E. 1952. Spectrophotometric analysis in the evaluation of
lemon oils. Conserve Deriv. Agrum. (Palermo) 1(2): 18–20.
D’Amore, G., and Calapay, R. 1965. Le sostanze  uorescenti contenute nelle essenze di
limone, begamotto, mandarino, arancio dolce, arancio amaro. Rass. Chim. 17: 264–269.
De Domenico, V. 1854. Sulla virtù medicamentosa dell’essenza di bergamotto. From a reprint
held by Stazione Sperimentale per l’Industria delle Essenze, Reggio Calabria.
Dellacassa, E., Lorenzo, D., Moyna, P., Verzera, A., and Cavazza, A. 1997. Uruguayan essen-
tial oils. Part V. Composition of bergamot oil. J. Essent. Oil Res. 9:419–426.
Di Giacomo, A. 1972. I criteri seguiti dalla Stazione Sperimentale di Reggio Calabria per
l’accertamnto della qualità degli oli essenziali agrumari estratti a freddo. Essenz. Deriv.
Agrum. 42:232–242.
Di Giacomo, A., and Calvarano, I. 1974. Estudio del aceite de bergamotta por cromatogra a
en lamina delgada y espectro uorimetria. Essenz. Deriv. Agrum. 44:329–339.
Di Giacomo, A. 1990. Valutazione della qualità delle essenze agrumarie “cold-pressed” in
relazione al contenuto in composti cumarinici e psoralenici. Essenz. Deriv. Agrum.
60:313–334.
Donato, P., Russo, M., Bonaccorsi, I., and Dugo, P. 2013. Determination of bio avonoids in
bergamot (C. bergamia) peel oils by liquid chromatography coupled to tandem ion-trap
time of  ight mass spectrometry. Flavour Fragr. J. submitted.
Dugo, G., Lamonica, G., Cotroneo, A., etal. 1987. Sulla genuinità delle essenze agruma-
rie. Nota XVII. La composizione della frazione volatile dell’essenza di bergamotto
Calabrese. Essenz. Deriv. Agrum. 57:456–534.
Dugo, G., Cotroneo, A., Verzera, A., etal. 1991. Genuineness characters of Calabrian berga-
mot essential oil. Flavour Fragr. J. 6:39–56.
Dugo, G., Lamonica, G., Cotroneo, A., etal. 1992a. High resolution gas chromatography for
detection of adulterations of citrus cold-pressed essential oils. Perfum. Flav. 17(5): 57–74.
Dugo, G., Stagno d’Alcontres, I., Cotroneo, A., and Dugo, P. 1992b. On the genuineness of cit-
rus essential oils. Part XXXV. Detection of added reconstituted mandarin oil in genuine
cold-pressed mandarin essential oil by high resolution gas chromatography with chiral
capillary columns. J. Essent. Oil Res. 4:589–594.
TAF-K12886-13-0201-C013.indd 379 15/07/13 6:35 PM
380 Citrus bergamia
Dugo, G., Stagno d’Alcontres, I., Donato, M. G., and Dugo, P. 1993. On the genuineness of
citrus essential oils. Part XXXVI. Detection of added reconstituted lemon oil in genuine
cold-pressed lemon essential oil by high resolution gas chromatography with chiral cap-
illary columns. J. Essent. Oil Res. 5:21–26.
Dugo, G., Verzera, A., Trozzi, A., etal. 1994a. Automated HPLC-HRGC: A powerful method
for essential oils analysis. Part I. Investigation on enantiomeric distribution of monoter-
pene alcohols of lemon and mandarin essential oils. Essenz. Deriv. Agrum. 64:35–44.
Dugo, G., Verzera, A., Cotroneo, A., etal. 1994b. Automated HPLC-HRGC: A powerful method
for essential oil analysis. Part II. Determination of the enantiomeric distribution of linalol
in sweet orange, bitter orange and mandarin essential oils. Flavour Fragr. J. 9:99–104.
Dugo, G., 1997. Unpublished results.
Dugo, P., Mondello, L., Sebastiani E., etal. 1999. Identi cation of minor oxygen heterocy-
clic compounds of citrus essential oils by liquid chromatography-atmospheric pressure
chemical ionization mass spectrometry. J. Liq. Chrom.& Rel. Technol. 22:2991–3005.
Dugo, G., Mondello, L., Cotroneo, A., Bonaccorsi, I., and Lamonica, G. 2001. Study on the
enantiomeric distribution of volatile components of citrus essential oils by multidimen-
sional gas chromatography (MDGC). Perfum. Flav. 27(1): 20–35.
Dugo, G., Bonaccorsi I., Russo M., and Dugo P. 2011a. Unpublished results.
Dugo, G., Cotroneo, A., Bonaccorsi, I., and Trozzi, A. 2011b. Composition of the volatile frac-
tion of citrus peel oils. In: Citrus Oils: Composition, Advanced Analytical Techniques,
Contaminants, and Biological Activity, eds. G. Dugo and L. Mondello, 1–162. London
and New York: Taylor& Francis.
Dugo, G., Bonaccorsi, I., Sciarrone, D., etal. 2012. Characterization of cold-pressed and pro-
cessed bergamot oils by using GC-FID, GC-MS, GC-C-IRMS, enantio-GC, MDGC,
HPLC, and HPLC-MS-IT-TOF. J. Essent. Oil Res. 24:93–117.
Faber, B., Krause, B., and Mosandl, A. 1995. Gas chromatography-isotope ratio mass spec-
trometry in the analysis of peppermint oil and its importance in the authenticity control.
J. Essent. Oil Res. 7:123–131.
Faulhaber, S., Hener, U., and Mosandl, A. 1997a. GC/IRMS analysis of mandarin essential
oils. 1. δ13CPDB and δ15NAIR values of methyl N-methylanthranilate. J. Agric. Food Chem.
45:2579–2583.
Faulhaber, S., Hener, U., and Mosandl, A. 1997b. GC/IRMS analysis of mandarin essen-
tial oils. 2. δ13CPDB values of characteristic  avour components. J. Agric. Food Chem.
45:4719–4725.
Frank, C., Dietrich, A., Kremer, U., and Mosandl, A., 1995. GC-IRMS in the authenticity
control of the essential of coriandrum-sativum L. J. Agric. Food Chem. 43:1634–1637.
Gildermeister, E., and Hoffmann, F. 1959. Die Aetherischen Oele, Vol. 5. Berlin:
Akademie-Verlag.
Gionfriddo, F., Postorino, E., and Bovalo, F. 1997. On the authenticity of bergamot oil: HPLC
pro le of heterocyclic components. Essenz. Deriv. Agrum. 67:342–352.
Guenther, E. 1949. The essential oils, vol. 3. New York: D. Van Nostrand.
Günther, H. O., and Zigler, E. 1977. Formation of artefacts during thin layer chromatography
of furanocoumarins of citrus oils. Essenz. Deriv. Agrum. 47:473–484.
Hanneguelle, S., Thibault, J. N., Naulet, N., and Martin G. J. 1992. Authentication of essential
oils containing linalool and linalyl acetate by isotopic methods J. Agric. Food Chem.
40:81–87.
Hener, U., Hollnagel, A., Kreis, P., etal. 1990. Direct enantiomer separation of chiral volatiles
from complex matrices by multidimensional gas chromatography. In Flavour Science
and Technology, eds. Y. Bessiere and A. F. Thomas. Chichester, West Sussex, England:
John Wiley& Sons.
Hener, U., Faullhaber, S., Kreis, P., and Mosandl, A., 1995. On the authenticity evaluation of
balm oil. (Melissa of cinalis L.). Pharmazie. 50:60–62.
TAF-K12886-13-0201-C013.indd 380 15/07/13 6:35 PM
381Adulteration of Bergamot Oil
Hör, K., Ruff, C., Weckerle, B., König, T., and Schreier, P. 2001. Flavor authenticity studies
by 2H/1H ratio determination using on-line gas chromatography pyrolysis isotope ratio
mass spectrometry. J. Agric. Food Chem. 49:21–25.
Juchelka, D., and Mosandl, A. 1996. Authenticity pro les of bergamot oil. Pharmazie
51:417–422.
Karl, V., Dietrich, A., and Mosandl, A. 1994. Gas chromatographyisotope ratio mass
spectrometry measurements of some carboxylic esters from different apple varieties.
Phytochem. Anal. 5:32–37.
König, W. A., Fricke, C., Saritas, Y., Momeni, B., and Hohenfeld, G. 1997. Adulteration or
natural variability? Enantioselective gas chromatography in purity control of essential
oils. J. High Resolut. Chromatogr. 20:55–61.
La Face, D. 1959. Ricerche sull’essenza di bergamotto. I. Comportamento spettrofotometrico
delle essenze pure nell’ultravioletto e nell’infrarosso. Essenz. Deriv. Agrum. 29:45–56.
Lakszener, K., and Szepesy L. 1988. Applications of the O-FID oxygenes analyzer in the cos-
metic industry. Chromatographia 26:91–96.
Liberti, A., and Conte, G. 1956. Possibilità di applicazione della cromatogra a in fase gas-
sosa allo studio delle essenze. Atti I Congresso Internazionale di Studi e Ricerche sulle
Essenze, Reggio Calabria, Italy, March.
Madsen, B. C., and Latz, H. L. 1970. Qualitative and quantitative in situ  uorimetry of citrus
oil thin-layer chromatograms. J. Chromatogr. 50:288–303.
Matthews, D. E., and Hayes, J., M. 1978. Isotope-ratio-monitoring gas-chromatography-mass
spectrometry. Analytical Chem. 50:1465–1473.
McHale, D., and Sheridan, J. B. 1989. The oxygen heterocyclic compounds of citrus peel oils.
J. Ess. Oil Res. 1:139–149.
McHale, D. 2002. Adulteration of citrus oils. In: Citrus. The Genus Citrus, eds. G. Dugo and
A. Di Giacomo, 496–517. London and New York: Taylor& Francis.
Meier-Augenstein, W. 1999. Applied gas chromatography coupled to isotope ratio mass spec-
trometry. J. Chromatogr. A 842:351–371.
Mondello, L., Stagno d’Alcontres, I., Del Duce, R., and Crispo, F., 1993. On the genu-
ineness of citrus essential oils. Part 40. Composition of the coumarins and pso-
ralens of Calabrian bergamot essential oil (Citrus bergamia Risso). Flavour Fragr.
J. 8:17–24.
Mondello, L., Dugo, G., Dugo, P., and Bartle, K. D. 1996. On-line HPLC-HRGC in the ana-
lytical chemistry of citrus essential oils. Perfum. Flav. 21(4): 25–49.
Mondello, L., Catalfamo, M., Dugo, P., Proteggente, A. R., and Dugo, G. 1997. La gascro-
matogra a multidimensionale per l’analisi di miscele complesse. Nota preliminare.
Determinazione della distribuzione enantiomerica di componenti degli olii essenziali
agrumari. Essenz. Deriv. Agrum. 67:62–85.
Mondello, L., Verzera, A., Previti, P., Crispo, F., and Dugo, G. 1998. Multidimensional capil-
lary GC-GC for the analysis of complex samples. 5. Enantiomeric distribution of mono-
terpene hydrocarbons, monoterpene alcohols, and linalyl acetate of bergamot (Citrus
bergamia Risso et Poiteau) oils. J. Agric. Food Chemists 46:4275–4282.
Mondello, L., Zappia, G., Errante, G., and Dugo, G. 2000. Fast GC and fast GC-MS for the
analysis of natural complex matrices. LC-GC Europe 13:495–502.
Mondello, L., Casilli, A., Tranchida, P. Q., et al. 2006. Fast enantiomeric analysis of a com-
plex essential oil with an innovative multidimensional chromatographic system. J.
Chromatog. A 1105:11–16.
Morton, R. A. 1929. Radiation in connection with essential oils and perfumery chemicals.
Perfumery and Essential Oil Record. 20:258–267.
Mosandl, A., Hener, U., Kreis P., and Schmarr, H-G. 1990. Enantiomeric distribution of
α-pinene, β-pinene and limonene in essential oils and extracts. Part 1. Rutaceae and
Gramineae. Flavour Fragr. J. 5:193–199.
TAF-K12886-13-0201-C013.indd 381 15/07/13 6:35 PM
382 Citrus bergamia
Mosandl, A. 1995. Enantioselective capillary gas chromatography and stable isotope ratio
mass spectrometry in the authenticity control of  avors and essential oils. Food Rev. Int.
11:597–664.
Mosandl, A., and Juchelka, D. 1997a. Advances in the authenticity assessment of citrus oils.
J. Essent. Oil Res. 9:5–12.
Mosandl, A., and Juchelka, D. 1997b. The bitter orange tree – a source of different essen-
tial oils. In Flavour Perception, eds. H-P. Kruse and M. Rothe, 321–331. Eigenverlag
Deutsch Inst. F. Ernahrungsforsch.
Mosandl, A. 2007. Enantiomeric and isotope analysis. Key steps to  avour authentication.
In: Flavours and Fragrances: Chemistry, Bioprocessing and Sustainability, ed. R. G.
Berger. Berlin and Heidelberg: Springer-Verlag.
Neukom, H-P., Meier D. J., and Blum D. 1993. Nachweis von natürlichem oder rekonstitu-
iertem bergamottöl in earl gray tees anhand der enantiomerentrennung von linalool und
dihydrolinalool. Mitt. Gebiete Lebensm. Hyg. 84:537–544.
Presnel, A. K., 1953. Infrared spectroscopy of essential oils. J. Soc. Cosmetic Chemists.
4:101–109.
Ravid, U., Putievsky, E., and Katzir, I. 1994. Chiral GC analysis of enantiomerically pure (R)
()-linalyl acetate in some Lamiaceae, myrtle and petitgrain essential oils. Flav. Fragr.
J. 9:275–276.
Rodighero, G., and Caporale, G. 1954. The coumarins obtained from extracts of Citrus ber-
gamia. Atti Ist. Veneto Sci. Nat. 112:97–102.
Sale, J. W., Winkler, W. O., Gnagy, M. J., etal. 1953. Analysis of lemon oils. J. Assoc. Of c.
Agr. Chem. 36:112–119.
Schipilliti, L., Tranchida, P. Q., Sciarrone, D., etal. 2010. Genuineness assessment of manda-
rin essential oils employing gas chromatography-combustion-isotope ratio MS (GC-C-
IRMS). J. Sep. Sci. 33:617–625.
Schipilliti, L., Dugo, P., Bonaccorsi, I., and Mondello, L. 2011a. Authenticity control on lemon
essential oils employing gas chromatography-combustion-isotope ratio mass spectrom-
etry (GC-C-IRMS). Food Chem. doi: 10.1016/j.foodchem.2011.09.119.
Schipilliti, L., Dugo, P., Bonaccorsi, I., and Mondello, L. 2011b. Headspace-solid phase micro-
extraction coupled to gas chromatography-combustion-isotope ratio mass spectrometer
and to enantioselective gas chromatography for strawberry  avoured food quality con-
trol. J. Chomatogr. A 1218:7481–7486.
Schipilliti, L., Dugo, P., Mondello, L., Santi. L., and Dugo, G. 2011c. Authentication of ber-
gamot essential oil by gas-chromatography-combustion-isotope ratio mass spectrometer
(GC-C-IRMS). J. Essent. Oil Res. 23(2): 60–70.
Schubert, V., and Mosandl, A. 1991. Chiral compounds of essential oils. VIII:
Stereodifferentiation of linalol using multidimensional gas chromatography.
Phytochem. Anal. 2:171–174.
Schumacher, K., Turgeon, H., and Mosandl, A. 1995. Sample preparation for gas-chromatog-
raphy isotope ratio mass-spectrometry—an investigation with volatile components from
strawberries. Phytochem. Anal. 6:258–261.
Sciarrone, D. 2009. Personal communication.
Semmler, F. W., and Tiemann, F. 1892. Ueber sauerstoffhaltige bestandtheile einiger aetheri-
scher oele. Ber. 25:1180–1188.
Späth, E., and Socias, L., 1934. Über Bergaptol, einen neuen Inhaltsstoff des Calabrischen
Bergamottöles (VIII. Mitteil. über natürliche Cumarine). Ber. 67B:59–61.
Stanley, W. L. 1959. Determination of menthyl salicylates in lemon oil. J. Assoc. Of c. Agr.
Chemists 42:643–646.
Stanley, W. L. 1961. A test for chalcones in lemon oil. J. Assoc. Of c. Agr. Chemists 44:546–548.
Stanley, W. L., and Vannier, S. H. 1957a. Chemical composition of lemon oil. I. Isolation of a
series of substituted coumarins. J. Amer. Chem. Soc. 79:3488–3491.
TAF-K12886-13-0201-C013.indd 382 15/07/13 6:35 PM
383Adulteration of Bergamot Oil
Stanley, W. L., and Vannier, S. H. 1957b. Analysis of coumarin compounds in citrus oils by
liquid solid partition. J. Assoc. Of c. Agr. Chemists 40:582–588.
Stanley, W. L., and Jurd, L. 1971. Citrus coumarins. J. Agric. Food Chem. 19:1106–1110.
Theile, F. C., Dean, D. E., and Suf s, R. 1960. The evaluation of bergamot oil. Drug and
Cosmetic Ind. 86:758–759, 837–840.
Van Os, D., and Dykstra, K. 1937. Examination of essential oils by measurement of absorption
in the ultraviolet. J. Parm. Chim. 25:437–454, 485–501.
Vannier, S. H., and Stanley, W. L. 1958 Fluorometric determination of 7-geranoxycoumarin
in lemon oil: Analysis of mixtures of grapefruit oil in lemon oil. J. Assoc. Of c. Agric.
Chem. 41:432–435.
Verzera, A., Lamonica, G., Mondello, L., Trozzi, A., and Dugo, G. 1996. The composition of
bergamot oil. Perfum. Flav. 21(6): 19–34.
Verzera, A., Trozzi, A., Stagno d’Alcontres, I., etal. 1998. The composition of the volatile
fraction of Calabrian bergamot essential oil. Riv. Ital. EPPOS 25:17–38.
Weinreich, B., and Nitz, S. 1992. In uences of processing on the enantiomeric distribu-
tion of chiral  avour compounds. Part A: Linalyl acetate and terpene alcohols. Chem.
Microbiol. Technol. Lebensm. 14:117–124.
TAF-K12886-13-0201-C013.indd 383 15/07/13 6:35 PM
... All these variables combined with the limited production, availability, and high market value of the BEO make it a favorite target of fraudulent manipulation [14]. Dilution by adding less valuable oils, addition of chemical additives, and preparation of the oil by mixing pure substances are some of the most frequent frauds in the essential oil sector that represent a serious problem for regulatory agencies [36]. For all these reasons, the use of advanced methodologies, able to identify and quantify the compounds present in BEO, is essential to ensure its quality and genuineness. ...
... For all these reasons, the use of advanced methodologies, able to identify and quantify the compounds present in BEO, is essential to ensure its quality and genuineness. The analytical techniques commonly used to perform compositional studies and currently accepted for the release of the "Protected Designation of Origin (PDO)" of "Bergamotto di Reggio Calabria-Essential oil," are gas chromatography (GC) and high-performance liquid chromatography (HPLC) [18,36]. A valid alternative to traditional analytical methods is nuclear magnetic resonance (NMR) spectroscopy. ...
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In this work, a sample of pure and certified bergamot essential oil (BEO) was extensively studied for the first time directly by NMR spectroscopy with the aim of investigating its metabolic composition, quantifying the main components of this complex natural matrix and simultaneously assessing whether the NMR technique is able to highlight possible frauds to which this high-cost product may be subjected. Eleven low molecular weight compounds have been identified by using 1D 1H and 13C-{1H} NMR experiments, 2D homo- and heteronuclear correlation NMR spectra, and 2D 1H DOSY experiments; the most abundant of them, i.e., about 90% of the sample analyzed, has been quantified by employing benzoic acid as an internal standard by 1H NMR spectrum. Moreover, since the commercial fraud of this precious oil is often due to the addition of less expensive oils, we have simulated a possible adulteration through the preparation of BEO samples to which different percentages of orange essential oil (OEO) were added. The results, obtained by combining the 1H NMR spectra collected on the adulterated samples and on pure BEO, with chemometric analysis, principal component analysis (PCA), indicate that it is possible to distinguish the sample of pure BEO from the adulterated ones and also, among them, to differentiate between the degrees of adulteration.
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Enantioselective capillary gas chromatography, as well as isotope ratio mass spectrometry (IRMS), online coupled with capillary gas chromatography are used in the origin specific analysis and authenticity control of balm oil compounds. Scope and limitations of the methods are discussed.