molecules
Communication
The First Application of 1H NMR Spectroscopy for the
Assessment of the Authenticity of Perfumes
Barbara Pacholczyk-Sienicka * , Grzegorz Ciepielowski and Łukasz Albrecht *






Citation: Pacholczyk-Sienicka, B.;
Ciepielowski, G.; Albrecht, Ł. The
First Application of 1 H NMR
Spectroscopy for the Assessment of
the Authenticity of Perfumes.
Molecules 2021, 26, 3098. https://
doi.org/10.3390/molecules26113098
Academic Editor: Luca Forti
Received: 5 May 2021
Accepted: 20 May 2021
Published: 22 May 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Molecules 2021, 26, 3098. https://doi.org/10.3390/molecules26113098
Institute of Organic Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego 116,
90-924 Lodz, Poland; grzegorz.ciepielowski@p.lodz.pl
* Correspondence: barbara.pacholczyk@p.lodz.pl (B.P.-S.); lukasz.albrecht@p.lodz.pl (Ł.A.)
Abstract: The manufacture of counterfeit goods is one of the world’s largest underground businesses
and is rapidly growing. Counterfeits can lead not only to the loss of profit for honest producers
but also have a negative impact on consumers who pay excessive prices for poor quality goods
that may result in health or safety problems. The perfume industry is constantly vulnerable to
counterfeits, particularly in the fast developing market of “smell-alike” designer-inspired perfumes
because these prompt the identification of the methods that classify their quality. In this paper,
the application of proton nuclear magnetic resonance (1 H NMR) spectroscopy is employed for
the first time to authenticate perfumery products. The molecular composition of several types of
authentic brand fragrances for women was compared with cheap inspired equivalents and fakes.
Our approach offers the prospect of a fast and simple method for detecting counterfeit perfumes
using 1 H NMR spectroscopy.
Keywords: authentication of perfumes; flavors; nuclear magnetic resonance; counterfeits
1. Introduction
The globalization of products with recognized market position such as beverages,
medicines, food and perfumes has led to the expansion of counterfeiting. These fake goods
purchased through Internet shops and websites may cost less than the originals, but they
are of inferior quality [1,2]. Moreover, these counterfeit goods may be dangerous because
they do not undergo the same rigorous safety testing conducted by honest manufacturers.
These inferior imitations affect not only companies by damaging the brand’s reputation
but also consumers, who may be unaware that they do not comply with safety standards
and could be dangerous. For the most part, these products do not undergo dermatological
testing and may contain chemicals that can cause skin reactions such as contact allergies or
dermatitis [3,4].
Nowadays, fragrances are found in almost all cosmetics and personal care products
(e.g., perfumes, deodorants, aftershave and skin lotions), hair products, facial cleansers and
sunscreen as well as cleaning and laundry products. However, a given fragrance is typically
a mixture of several dozen to several hundred chemicals that may cause the allergic
contact dermatitis [5–9]. The last data indicated that more than 3000 chemical compounds
are used in fragrance mixtures but only 82 substances (54 single chemicals, 28 natural
extracts) were classified as contact allergens in humans by The Scientific Committee on
Consumer Safety [2]. A perfume usually contains 30 to 50 (sometimes even 200) ingredients
responsible for its unique fragrance, which come from nature or chemical synthesis. The
composition and scent of the essential oils depend not only on the plant’s origin, conditions
of collection, storage, transport and drying but also on the process of oil production. It
is particularly important to monitor contaminants that may be present in oils obtained
from plants as they may contain traces of toxic or carcinogenic pesticides [7,8]. Due to all
of the properties of essential oils, the high-quality natural oils are very expensive. Their
replacement by synthetic chemical compounds reduces the cost of perfume production,
https://www.mdpi.com/journal/molecules
Molecules 2021, 26, 3098 2 of 10

which mainly depends on the quality of oil used [6]. Owing to the high price of essential
oils, manufacturers are adulterating their products while maintaining high prices. Two
techniques of adulteration that are frequently used involve adding cheaper materials or
dilution with water or other solvents.
The most commonly counterfeited scents are branded perfumes and toilet waters, but
the imitations do not have the same quality or scent as the authentic ones [2,3]. Customers
at first glance are not able to recognize a counterfeit because its external features strongly
resemble those of the original even though it does not have the same molecular composition
or properties. However, in some situations consumers can avoid buying a counterfeit
simply by staying focused. Counterfeiters often use misspellings on the packaging to avoid
liability for trademark theft (e.g. Boos instead of Boss or J’ader instead of J’adore).
Considering that perfumes are complex mixtures of various compounds, the deter-
mination of their ingredients is a difficult task. Several methods have been suggested for
analysing fragrances such as chromatography [10–12], spectroscopy [13–16], mass spectrom-
etry [17,18], and electronic nose [19,20]. Due to the fact that most perfume components are
volatile or semi-volatile, gas chromatography coupled with mass spectrometry (GC–MS) is
the technique of choice for qualitative and quantitative analyses of perfume ingredients. In
recent years, GC–MS has been successfully employed in forensic reconstructions of crime
scenes, such as identifying traces of volatiles on the clothes of a sexual assault victim because
the fragrance may have been transferred between the victim and the attacker [21–23]. A
technique that plays an important role in the fragrance industry is the artificial electronic
olfactory system (EOS), especially concerning perfume counterfeiting, the quality control
of raw and finished products, and the quantification of perfume concentration in an un-
known sample. Spectroscopic techniques like Raman spectroscopy or NMR can be used for
perfume analysis, the former having been applied to determine fragrance composition [24].
Interestingly, up to now NMR spectroscopy has only been applied to characterize the
compositions of several perfumes and to separate the components of a given mixture based
on diffusion coefficients using diffusion ordered spectroscopy (DOSY) [14–16].
Herein, we present a fast, simple and relatively inexpensive method to detect counter-
feit perfumes. The goal of our study is to present the potential of NMR spectroscopy in the
direct compositional analysis original perfumes and their counterfeits. We envision that it
may be possible through the application of NMR spectroscopy with the aid of chemometric
analysis. Within our studies, the 1 H NMR spectra of original perfumes were compared with
those of fakes and inspired equivalents. To the best of our knowledge, the use of 1 H NMR
spectra has never been reported for this purpose before. Our results offer the perspective
for a fast, routine way to detect counterfeit perfumes by means of NMR spectroscopy.

2. Results and Discussion

Representative 1 H NMR spectra of the authentic, inspired and counterfeit perfumes
are shown in the Figure 1. The main compounds characterizing perfume samples with
their diagnostic 1 H signals, chemical shifts δH and multiplicities are reported in Table
S1 (see Supporting Information). Considering the usefulness of the applied approach to
a direct analysis of perfume composition, we compared our results with data obtained
by means of a GC–MS analysis for original Light Blue and its substitutes and counterfeit
version [25]. Most of the identified compounds were also found in samples analyzed by
NMR. Twenty-three molecules were identified on the basis of 1D and 2D NMR spectral
analysis, while 34 compounds were detected by the GC–MS approach.
 2021, 26, x FOR PEER REVIEW 3 of 11
es 2021, 26,Molecules 2021,
x FOR PEER 26, 3098
REVIEW 3 of 11 3 of 10

Figure 1. Representative 1H NMR spectra of authentic (A,A′), inspired (B,B′) and counterfeit (C,C′)
1 H NMR spectra 0 ), inspired (B,B0 ) and counterfeit (C,C0 ) samples in two spectral
Figure 1. Representative
Figure 1. Representative of authentic
1H NMR spectra (A,A
of authentic (A,A′), inspired (B,B′) and counterfeit (C,C′)
samples in two spectral region from −0.25 to 4.25 ppm and from 6 to 8.5 ppm.
samples in two spectral region from −0.25
region from −0.25 to 4.25 ppm and from 6 to 8.5 ppm. to 4.25 ppm and from 6 to 8.5 ppm.

The main constraint of our

The main study was
constraint of the
our limitedwas amount of authentic samples. So
The main constraint of our study was thestudy
limited amount the limited amount
of authentic of authentic
samples. So samples. So
far, two authentic far,samples
two of Light
authentic blue
samples and ofJ’adore
Light fragrances
blue and were
J’adore obtained
fragrances and
were tested.
obtained and tested.
far, two authentic samples of Light blue and J’adore fragrances were obtained and tested.
To evaluate chemical
To similarity,
evaluate theirsimilarity,
chemical spectra were theircalibrated
spectra with calibrated
were reference to withthereference
TMS to the TMS
To evaluate chemical similarity, their spectra were calibrated with reference to the TMS
signal, manuallysignal,
phased, and
manually the baseline
phased, was
and was corrected.
the baseline Then
wasThen by means
corrected. of AMIX
ThenofbyAMIX soft-
meanssoft- of AMIX software
signal, manually phased, and the baseline corrected. by means
ware spectra, they werethey
spectra, segmented into buckets
were segmented into and
bucketsnormalized to the total
and normalized sum ofsum
the
ware spectra, they were segmented into buckets and normalized to the to the
total total
sum of theof the integrals
integrals of all the buckets. After that, a lineararegression analysisanalysis
was conducted to com- to compare the
integrals of all of
theallbuckets.
the buckets.
After After
that, athat,
linear linear
regressionregression was conducted
analysis was conducted to com-
pare the bucketsbuckets
for both spectra.
for both For
spectra.each comparison,
For each the
comparison, coefficient
the of determination
coefficient of determination (R2 ) of
pare the buckets for both spectra. For each comparison, the coefficient of determination
(R2)2of the fit was calculated. The results for Light blue are
Light presented in Figure 2a, while
the fit
(R ) of the fit was was calculated.
calculated. The results The for
results
Lightfor blue are blue are presented
presented in Figure in2a,Figure
while2a, while those
those for J’adore for
are in Figure
J’adore are2b.
in The highest
Figure 2b. value
The of R2 was obtained 2 when two authentic
those for J’adore are in Figure 2b. The highest value of R was obtained when two authentic two authentic
highest 2 value of R was obtained when
samples of Lightsamples
blue were of compared
Light blue (R2 =2 compared
were 0.99, Figure (R2a)
2 = while, in the2a)case of J’adore
in thethecase of J’adore the
samples of Light blue were compared (R = 0.99, Figure 2a)0.99, Figure
while, in the casewhile,
of J’adore the
coefficient of determination
coefficient of (R 2)2 was 0.98. Given
determination (R that0.98.
2 ) was a goodGivencorrelation
that a was correlation
good obtained, was obtained,
coefficient of determination (R ) was 0.98. Given that a good correlation was obtained,
further PCA analyses
furtherwere PCAconducted
analyses on one
were authenticonsample
conducted for eachsample
one authentic of the fragrances
further PCA analyses were conducted on one authentic sample for each of thefor each of the fragrances
fragrances
tested. However, the
tested. slight differences
However, the observed
slight for
differences the two
observedJ’adore
for samples
the two suggest
J’adore that, suggest that,
samples
tested. However, the slight differences observed for the two J’adore samples suggest that,
for future applications
for future of applications
the developed of method,
the severalmethod,
developed batches several
of authentic
batches samples
of authentic samples
for future applications of the developed method, several batches of authentic samples
should be tested.should be tested.
should be tested.

Figure 2.Figure 2. The comparison

The comparison of the buckets
of the buckets from NMRfrom NMR for
spectra spectra for two authentic
two authentic samplessamples of Light
of Light blue blueJ’adore (b).
(a) and
Figure 2. The comparison of the buckets from NMR spectra for two authentic samples of Light blue
(a) and J’adore (b).
(a) and J’adore (b).
Due to the very large number of signals, the obtained NMR spectra were first analyzed
Due to the by a main
very large components analysisthe
number of signals, (PCA), which
obtained NMRexplains
spectrathewere
variance structure of a set of
first ana-
Due to the very large number of signals, the obtained NMR spectra were first ana-
lyzed by a mainvariables through
components linear(PCA),
analysis combinations of the principal
which explains components
the variance (PCs).
structure of aThe PCA scores
lyzed by a main components analysis (PCA), which explains the variance structure of a
discriminated the authentic perfume samples from the inspired
set of variables through linear combinations of the principal components (PCs). The PCA and counterfeit samples
set of variables through linear combinations of the principal 1components (PCs). The PCA
based on selected proton signals detected by H NMR. For
scores discriminated the authentic perfume samples from the inspired and counterfeit this purpose a full-spectrum
scores discriminated the authentic perfume samples 1from the inspired and counterfeit
samples based on PCA analysis
selected was conducted
proton as an input;
signals detected by H however, the only
NMR. For observedadifference
this purpose full- in chemical
samples based on selected proton signals detected by 1H NMR. For this purpose a full-
es 2021, 26, x FOR PEER REVIEW 4 of 11

Molecules 2021, 26, 3098 4 of 10

spectrum PCA analysis was conducted as an input; however, the only observed difference
in chemical composition referred to solvents. The outlying points belonged to ethyl alco-
hol, isopropyl composition
alcohol, diethylreferredphthalate and cedrol.
to solvents. The outlying(PCA points
analysis for fulltospectrum
belonged ethyl alcohol, isopropyl
bucket tables are shown in the Supplementary Information.) As a
alcohol, diethyl phthalate and cedrol. (PCA analysis for full spectrum consequence, the full
bucket tables are
spectrum was divided into three spectral ranges of chemical shifts.
shown in the Supplementary Information.) As a consequence, the full The first covered the spectrum was
range from 0.6divided
to 3.0 ppm,
into the
three second from
spectral 3.0 toof6.0
ranges ppm, and
chemical the last
shifts. The from 6.0 to 8.5the range from
first covered
ppm. The interpretation of thethe
0.6 to 3.0 ppm, results
secondfrom the3.0
from firsttointerval
6.0 ppm, didandnottheallow
last the
from original
6.0 to 8.5 ppm. The
samples to be distinguished
interpretation of the results from the first interval did not allow therange
from the inspired samples because all samples in this original samples to
of chemical shifts were very similar,
be distinguished from theso inspired
no statistically
samples significant
because all differences
samples inwere this ob-
range of chemical
served. shifts were very similar, so no statistically significant differences were observed.
Similar results Similar
were obtained for the
results were range from
obtained 3.0 range
for the to 6.0 from
ppm.3.0 Thetorespective
6.0 ppm. The clus-respective clus-
tering of PCA is shown
tering in Figure
of PCA 3A, and
is shown some outliers
in Figure 3A, and corresponding to the counterfeit
some outliers corresponding to the counterfeit
sample of Lightsample
blue andofthe inspired
Light samples
blue and the of Rush, J’adore,
inspired samples andofEuphoria were observed.
Rush, J’adore, and Euphoria were ob-
This model described
served. 78.7% of total
This model variation78.7%
described in accordance with PC1
of total variation = 65.92% and
in accordance withPC2PC1= = 65.92% and
12.78%. The loading plot of a principal component indicated buckets and therefore
PC2 = 12.78%. The loading plot of a principal component indicated buckets and therefore spec-
tral regions that contributed
spectral regions significantly to it. As
that contributed shown in Figure
significantly to it. As 3B,shown
signalsinthat were3B, signals that
Figure
responsible forwere
the similarity/dissimilarity between the observations
responsible for the similarity/dissimilarity betweencorresponded to wa- corresponded
the observations
ter (4.82 ppm),toisopropanol (4.07) isopropanol
water (4.82 ppm), and isopropyl myristate
(4.07) (4.97 ppm),
and isopropyl which
myristate (4.97were
ppm),ob-which were ob-
served in non-original
served inproducts.
non-original This was very characteristic
products. This was veryofcharacteristic
falsified andof lower-quality
falsified and lower-quality
products, because manufacturers
products, often use moreoften
because manufacturers wateruse or other solvents
more water or such
otheras isopropyl
solvents such as isopropyl
myristate
myristate to reduce to reduce
the cost of perfumethe cost of perfume
production. production.
Moreover, Moreover,that
we observed wealso
observed
ethyl that also ethyl
alcohol was
alcohol was responsible forresponsible
an outlyingfor an outlying
behaviour, whichbehaviour, whichthat
may indicate maypoorindicate that poor quality
quality
ethanol was usedethanol was used for
for production production
of falsified andofinspired
falsifiedfragrances.
and inspired As fragrances.
a consequence, As a consequence,
consumers buyconsumers
poor quality buygoods
poor atquality goods atprice.
an excessive an excessive price.plot
A PCA score A PCAbased score plot based on the
on the
proton spectra proton spectra is
of 26 samples ofshown
26 samples
in theisFigure
shown3.in the Figure 3.

Figure 3. PCAFigure
scores3.plot
PCA (A)scores plot
and the (A) and the corresponding
corresponding loading plot (B)loading plotfrom
generated the 1 H NMR
(B) generated from the 1H
spectra from the authentic
NMR
perfume (black spectra
color, from
different the authentic
shapes perfume
marked various (black
scents) andcolor, different
counterfeit (redshapes
color, marked
different various
shapes marked various
scents) and
scents) and inspired counterfeit
samples (red
(colors, color, different
different shapes various
shapes marked marked scents)
variousforscents) and inspired
the range sam-shifts from 3.0 to
of chemical
6.0 ppm. ples (colors, different shapes marked various scents) for the range of chemical shifts from 3.0
to 6.0 ppm.
As shown on the scores plot in Figure 3A, the authentic perfumes were not clearly
As shownseparated
on the scoresfromplot in other
each Figureor3A,fromthethe
authentic
inspired perfumes were notsamples.
and counterfeit clearly Therefore, a
separated fromPCAeachplot
other or from the inspired and counterfeit samples. Therefore,
obtained for the range of chemical shifts from 6.0 to 8.5 ppm a PCAproved to be the
plot obtained for the range of chemical shifts from 6.0 to 8.5 ppm proved to be
most important because it allowed not only branded samples to be distinguished the most from
important because it allowed
non-original notbut
ones, only branded
also enabled samples
originaltofragrance
be distinguished from to
compositions non-
be differentiated
original ones, but also enabled
among original
each other. fragrance
These results compositions
are shown into be differentiated
Figure 4. The totalamong
variation described
each other. These
by results are shown
the scores of PC1inversus
FigurePC24. Thewastotal variation
61.96%. Thisdescribed by the that
plot showed scores
the counterfeits
of PC1 versus PC2 was a61.96%.
differed lot from This
theplot showed
original that the
perfumes counterfeits
and differed
also occupied a lot from
a different region in the plot
the original perfumes
compared andto also occupied
the inspired a different
samples. This region
provedin thethe
that plot compared
adulterated to the had different
perfumes
chemical compositions in the range of chemical shifts that corresponded to unsaturated
compounds. It was particularly evident in the case of Light Blue, Si, Euphoria and Good
es 2021, 26, x FOR PEER REVIEW 5 of 11

inspired samples. This proved that the adulterated perfumes had different chemical com-
Molecules 2021, 26, positions
3098 in the range of chemical shifts that corresponded to unsaturated compounds. It 5 of 10
was particularly evident in the case of Light Blue, Si, Euphoria and Good Girl. Unlike coun-
terfeit perfumes, these inspired samples showed considerable similarity with what was
observed for authentic fragrances like Rush, Light Blue, J’adore, Euphoria and Si. The load-
Girl. Unlike counterfeit perfumes, these inspired samples showed considerable similarity
ing plot presented in Figure 4B indicates the signals that were associated to the similar-
with what was observed for authentic fragrances like Rush, Light Blue, J’adore, Euphoria and
ity/dissimilarity between the observations of this study. The farthest outlying points be-
Si. The loading plot presented in Figure 4B indicates the signals that were associated to
longed to diethyl phthalate (7.53 and 7.72 ppm), galaxolide (6.77 and 7.02 ppm), benzyl
the similarity/dissimilarity between the observations of this study. The farthest outlying
salicylate (6.88, 6.97, 7.27 and 7.87 ppm), 2-ethylhexyl trans 4-methoxycinnamate (over-
points belonged to diethyl phthalate (7.53 and 7.72 ppm), galaxolide3(6.77 and 7.02 ppm),
lapped with other signals 6.31 ppm and 7.63 ppm, characteristic coupling constant J = 16
benzyl salicylate (6.88, 6.97, 7.27 and 7.87 ppm), 2-ethylhexyl trans 4-methoxycinnamate
Hz) and α-hexylcinnamaldehyde (7.17, 7.37, 7.42, and 7.47 ppm). Hexyl cinnamal (α-hex-
(overlapped with other signals 6.31 ppm and 7.63 ppm, characteristic coupling constant
ylcinnamaldehyde) is a widely used fragrance chemical because its scent resembles jas-
3 J = 16 Hz) and α-hexylcinnamaldehyde (7.17, 7.37, 7.42, and 7.47 ppm). Hexyl cinnamal
mine. In the perfume and cosmetics industry, synthetic hexyl cinnamal is used; however,
(α-hexylcinnamaldehyde) is a widely used fragrance chemical because its scent resembles
it can be found naturally in chamomile oil. This ingredient may cause an allergic skin
jasmine. In the perfume and cosmetics industry, synthetic hexyl cinnamal is used; however,
reaction and it is labelled by The European Chemicals Agency as a skin sensitizer. In turn,
it can be found naturally in chamomile oil. This ingredient may cause an allergic skin
galaxolide is a synthetic
reaction andaroma
it isand known
labelled by as
Theone of the components
European in musk.asGalaxolide
Chemicals Agency a skin sensitizer. In turn,
is also used as galaxolide
a fixative that reduces the evaporation rate of the volatile components
is a synthetic aroma and known as one of the components in musk. of Galaxolide
perfumes. Benzyl salicylate
is also used as acts as a preservative
a fixative that reducesandthefragrance in one.
evaporation rateIt of
gives
the products
volatile components of
a sweet, floral aroma.
perfumes.Diethyl
Benzylphthalate similar
salicylate toaisopropyl
acts as myristate
preservative is used as
and fragrance in aone.
solvent
It gives products a
and fixative. 2-Ethylhexyl
sweet, floral aroma. Diethyl phthalate similar to isopropyl myristatean
trans-4-methoxycinnamate is a cinnamate ester used as is ul-
used as a solvent
traviolet light absorber to protect
and fixative. products.trans-4-methoxycinnamate is a cinnamate ester used as an
2-Ethylhexyl
ultraviolet light absorber to protect products.

Figure 4. PCA scores plot (A) and the corresponding loading plot (B) generated from the 1H NMR
Figure 4. PCA scores plot (A) and the corresponding loading plot (B) generated from the 1 H NMR spectra of the authentic
spectra of the authentic perfume (black color, different shapes marked various scents) and coun-
perfume (black color,
terfeit (reddifferent shapes shapes
color, different markedmarked
variousvarious
scents) scents)
and counterfeit (red samples
and inspired color, different
(colors,shapes marked various
different
scents) andshapes
inspired samples (colors, different shapes marked various scents) for the range
marked various scents) for the range of chemical shifts from 6.0 to 8.5 ppm. of chemical shifts from 6.0 to
8.5 ppm.
The significant differences in 1H NMR signals related to compounds between the au-
The significant differences 1 H NMR signals related to compounds between the
thentic, inspired and counterfeit samples wereindetermined using a one-way analysis of
authentic,
variance (ANOVA). It wasinspired and counterfeit
performed samplesfrom
on the integration wererectangular
determinedbuckets,
using aand
one-way analysis
of variance (ANOVA). It was performed on the integration from
the only significant differences observed between the authentic and non-original samples rectangular buckets,
and the only significant differences observed between the authentic
involved diethyl phthalate, galaxolide, benzyl salicylate, 2-ethylhexyl trans-4-methox- and non-original
samples involved diethyl phthalate, galaxolide, benzyl salicylate, 2-ethylhexyl trans-4-
ycinnamate and α-hexylcinnamaldehyde. The results for one selected fragrance were pre-
methoxycinnamate and α-hexylcinnamaldehyde. The results for one selected fragrance
sented in Figures 5 and 6 (for complete results see Supporting Information). The relative
were presented in Figures 5 and 6 (for complete results see Supporting Information). The
integration values from buckets showed significant differences in the means of the three
relative integration values from buckets showed significant differences in the means of
group of samples. In the case of the counterfeit sample, and to a lesser extent inspired 2, a
the three group of samples. In the case of the counterfeit sample, and to a lesser extent
slight differences among points was observed. It was probably caused by the more com-
inspired 2, a slight differences among points was observed. It was probably caused by
plex processing of spectra, which contained large signals from solvents that may have led
the more complex processing of spectra, which contained large signals from solvents that
may have led to signal distortion with low concentration in the aromatic region. As shown
in Figure 6, diethyl phthalate, galaxolide, benzyl salicylate (and also isopropyl myristate
in the case of Light blue) seemed to be the clearest indicators for discriminating authentic,
inspired and counterfeit samples. Additionally, in the case of galaxolide, 2-ethylhexyl
 characterized by a higher level of 2-ethylhexyl trans-4-methoxycinnamate in comparison
to the authentic and counterfeit samples. The counterfeit samples of J’adore, Euphoria, Rush
and Si were characterized by a higher level of benzyl salicylate. However, only in the case
of Rush fragrances was it able to differentiate not only authentic and inspired samples
from counterfeit but also authentic from inspired. The counterfeit and inspired samples
Molecules 2021, 26, 3098 6 of 10
of Euphoria showed higher integrations from diethyl phthalate, while in the case of Si,
Rush and J’adore its higher content was only observed in counterfeit samples. Moreover,
the counterfeit samples of Euphoria, J’adore, Rush, and Good girl demonstrated a higher
trans-4-methoxycinnamate
level of hexyl cinnamal. On the other hand, andaisopropyl myristate, the
high concentration differentiation
of galaxolide was between
ob- authentic
and inspired
served in counterfeit samples
samples of Goodwasgirl,
notEuphoria,
possible. and
The mean
J’adore,integration value forin2-ethylhexyl
while its content au- trans-4-
methoxycinnamate
thentic and inspired samples was at allowed the authentic
a comparable sample for
level (except to be discriminated
the from the counterfeit,
inspired 5 sample
while
of J’adore). These in the
results arecase of the inspired
presented samples, the
in the Supporting differences were not statistically significant.
Information.
On the basis of the α-hexylcinnamaldehyde, the differentiation between the authentic and
counterfeit, as well as between the counterfeit and inspired 2 and 3, proved possible.

es 2021, 26, x FOR PEER REVIEW 7 of 11

Figure 5. TheFigure 5. The of

comparison comparison
the meansof the meansvalues
integration of 1 H NMR
integration valuessignals
of 1H NMR signals for
for authentic, authentic,
inspired and counterfeit Light
Blue samplesinspired
obtainedand
by counterfeit
ANOVA. Note Lightthat
Bluefive
samples obtained
replications by ANOVA.
of each Note conducted
sample were that five replications
and the integration of five
of eachassample
spectra was taken were
an input conducted and the integration of five spectra was taken as an input for
for analysis.
analysis.

Figure 6. Means Figure

comparison plot comparison
6. Means from ANOVA (Tukey
plot test, p < 0.05).
from ANOVA (Tukey test, p < 0.05).

The most common The methods

higher level of in
used 2-ethylhexyl
the perfume trans-4-methoxycinnamate
laboratory, such as GC orwas LC observed
chro- in the case
of authentic Rush, Euphoria and Si samples, while a lower content
matography, have the disadvantages of being time consuming, destructive, and not envi- was observed for in-
spired and counterfeit samples. Moreover, the inspired sample
ronmentally sound because of their use of solvents [26]. On the other hand, NMR spec- of Good girl fragrance was
characterized requires
troscopy is non-destructive, by a higher levelamount
a small of 2-ethylhexyl trans-4-methoxycinnamate
of solvent and is an excellent tool forin comparison to
the authentic
the precise structural and counterfeit
characterization samples.
of a pure compound,The counterfeit samples
but for mixtures, of J’adore, Euphoria, Rush
overlapping
to beSithe
signals appear and were characterized
main issue. This by a higher level
methodology notofonly
benzyl salicylate.
minimizes However,
analysis timeonly in the case
and cost but also provides adequate identification of fragrance components, such as non-
volatile ingredients with low thermostability, that cannot be identified by GC–MS. Our
results indicated that combination of 1H NMR spectroscopy with principal component
analysis offers the possibility for the simultaneous identification of counterfeit samples
Molecules 2021, 26, 3098 7 of 10

of Rush fragrances was it able to differentiate not only authentic and inspired samples
from counterfeit but also authentic from inspired. The counterfeit and inspired samples of
Euphoria showed higher integrations from diethyl phthalate, while in the case of Si, Rush
and J’adore its higher content was only observed in counterfeit samples. Moreover, the
counterfeit samples of Euphoria, J’adore, Rush, and Good girl demonstrated a higher level of
hexyl cinnamal. On the other hand, a high concentration of galaxolide was observed in
counterfeit samples of Good girl, Euphoria, and J’adore, while its content in authentic and
inspired samples was at a comparable level (except for the inspired 5 sample of J’adore).
These results are presented in the Supporting Information.
The most common methods used in the perfume laboratory, such as GC or LC chro-
matography, have the disadvantages of being time consuming, destructive, and not environ-
mentally sound because of their use of solvents [26]. On the other hand, NMR spectroscopy
is non-destructive, requires a small amount of solvent and is an excellent tool for the pre-
cise structural characterization of a pure compound, but for mixtures, overlapping signals
appear to be the main issue. This methodology not only minimizes analysis time and cost
but also provides adequate identification of fragrance components, such as non-volatile
ingredients with low thermostability, that cannot be identified by GC–MS. Our results
indicated that combination of 1 H NMR spectroscopy with principal component analysis
offers the possibility for the simultaneous identification of counterfeit samples without
the need for signal identification. Therefore, the attractiveness of our method is high and
might find further applications because the composition of the analysed mixture is not
required. However, the information on the composition of a given product is available
in the raw data and can be accessed if needed. Moreover, this methodology can be used
in laboratories equipped with spectrometers with proton frequencies between 400 and
700 MHz.

3. Materials and Methods

3.1. Samples Preparation
In the present work, the eight authentic samples of perfumes (Dolce & Gabbana Light
Blue (n = 2), G. Armani Si, C. Herrera Good girl, Gucci Rush, Dior J’adore (n = 2), and C.
Klein Euphoria), six counterfeits and 14 inspired perfumes were analyzed (Table 1). The
Molecules 2021,
Molecules 26, x 2021,
2021, FOR
26,
Molecules
Molecules Molecules
PEER
Molecules
x 2021,
FOR
26, x PEER
26,
FOR counterfeit
2021,
REVIEW
2021, 26,
x FOR
PEER 26,
x PEER
REVIEWFOR
REVIEW and
x PEER
FOR
REVIEW inspired
PEER
REVIEWREVIEWsamples were purchased over the Internet. Inspired samples 8 of 11
8 of 11
8 of
were purchased from Yodeyma Paris, FM World, Refan and Eyfel. All samples for NMR
measurements were prepared in the same way. A volume of 50 µL of analysed perfumery
Molecules 2021,
Molecules 26, x 2021,
2021, FOR
26,
Molecules
Molecules Molecules
PEER
Molecules
x 2021,
FOR
26, 2021,
x PEER
26,
FOR 2021,
REVIEW26,
REVIEW
x PEER
FOR 26,
x PEER
FOR x PEER
FOR
REVIEWREVIEWPEER
REVIEWREVIEW 8 of 11
8 of 11
8 of
Molecules 2021,
Molecules 26, x 2021,
2021, FOR
26,
Molecules
Molecules PEER
Molecules
x 2021,
FOR
26, x PEER
26,
FOR product
REVIEW
Molecules
2021, 2021,
26,
x FOR
PEER 26,
x PEER
REVIEWFOR was
x PEER
FOR
REVIEW dissolved
REVIEWPEER
REVIEWREVIEW in 600 µL of deuterated chloroform (CDCl3 99.8% D) with 0.03% 8 of 11
8 of 11
8 of
Molecules 2021, 26, x FOR Molecules
PEER
Molecules 2021, Table
2021,
REVIEW26, x 26,
FOR1.
Table
x A list
1.
Table
FOR
PEER A of the
list
Table
1. A
PEER
REVIEW Table
of
1.theTable
considered
1. A
listAoflist
REVIEW the 1. A
list
considered list
perfumes
of theof the
perfumes
ofconsidered
the considered considered
with their
considered
with
perfumes perfumes
denotations.
perfumes
their
perfumes
withwith with with
their
denotations.
theirtheir their
denotations.
denotations. denotations.
denotations. 1 8 of 11
Molecules 2021, 26, x FOR PEER REVIEW
(v/v) tetramethylsilane (TMS) as an internal standard. Five repetitions of a H NMR
Molecules
Molecules 2021, 26, x PEER
experiment
2021, 26, x FOR FORwerePEER REVIEW for each NMR tube.
performed
REVIEW
Light Blue
Light Blue
Light Light Light
Blue BlueLight
J’adoreBlue BlueJ’adore
J’adore
J’adore Rush J’adore
J’adoreRushRushRush Euphoria
Rush Rush
Euphoria Good
Euphoria
Euphoria Euphoria
Euphoria Girl
Good Girl
Good Good Good
Girl Good
Si
Girl SiGirl
Girl Si Si
Table 1. A list of the
Molecules 2021, 26, Table
x FOR 1. A
Table list Table
1. Aof1.
Table Table
considered
the
list 1. A
1. A list
considered
Aoflist
the oflist
theofconsidered
perfumes the
perfumes
ofconsidered
the considered considered
withwith with perfumes
theirperfumes
theirwith
perfumes
perfumes with with
denotations.
denotations.
theirtheir theirtheir
denotations.
denotations. denotations.
denotations.
Table 1. PEER
A list
Table 1. AREVIEW
oflist
theofconsidered
Table
TabletheTable
Table1. A listperfumes
1. Aoflist
considered
1. A 1.
listAoflist
thethe with
perfumes
ofconsidered theirperfumes
ofconsidered
the considered
with
the considered denotations.
perfumes
their with with
theirtheir
denotations.
perfumes
perfumes withwith
theirtheir denotations.
denotations.
denotations.
denotations.
Authentic
Authentic Authentic
Authentic
Authentic Table
Authentic 1. A1.list
Table of Table
A list the considered
of the Table
1. ATable
considered
1. Aof
list perfumes
1.the
listA theofwith
oflist
perfumes
considered thewiththeir denotations.
considered
considered their
perfumes perfumes perfumes
denotations.
with with theirwith their their denotations.
denotations.
denotations.
Molecules 2021, 26, x FOR PEER REVIEW
Light Blue
LightLight Blue
Light
BlueLight Light
J’adore
Blue J’adoreBlueJ’adore
BlueJ’adore Rush
J’adore J’adore
RushRushRush Euphoria
Rush Rush
Euphoria EuphoriaEuphoria Good
Euphoria Euphoria
Good GirlGood Girl
Good GirlGoodGirl Good
Si Girl SiGirl Si Si
Light Blue
LightLight Blue
Light J’adore
Light
BlueBlue Light
Blue
J’adoreBlue
Table
J’adore
Light Blue Table
1. A
J’adore Rush
J’adore
1.
listA J’adore
ofRush
list
J’adore Rush theof the Euphoria
Rush
considered
considered
Rush
Rush Rush
Euphoria
perfumes perfumes
Euphoria
Euphoria with Good
Euphoria
with
their
EuphoriaGoodGood Good Girl
Euphoria
their Girl
denotations.
denotations.
GirlGood GirlGoodGirl Si
Good
Si Girl SiGirl Si Si
Inspired 1
Inspired 1 Light
Inspired
Inspired 1 1 Inspired
Inspired Blue1 1
LightLight Light
J’adore Blue
BlueBlueTableJ’adore Rush
J’adore J’adore Euphoria
RushRush Rush Euphoria Good Euphoria
Euphoria GoodGirl Good Good
Si
Girl Girl Girl Si
Authentic
Authentic Authentic
Authentic
Authentic
Authentic 1. A list of the considered perfumes with their denotations.
Authentic
AuthenticAuthentic
Authentic Authentic
Authentic Authentic Light Light
BlueBlue J’adore J’adore RushRush Euphoria Euphoria Good GoodGirl Girl
Inspired
AuthenticInspired 2 Inspired
2 Inspired
Inspired 2 Inspired2 2 2
Authentic
Authentic
Authentic Table 1. A list of the considered perfumes with their denotations.
Inspired 1 Inspired
Inspired 1 Inspired
Inspired Inspired 1 1 Light
1 Inspired
1 Inspired 1 Blue J’adore Rush Euphoria Good Girl
Inspired 1
Inspired 1 Inspired
Inspired
Inspired 1 3 3
1 Inspired 1
Authentic
Authentic 1
Inspired 3
Inspired 3 Inspired
Inspired
Inspired
Inspired 1 Inspired3 3
Inspired
Inspired 1 1 Light
1 Inspired 2 Blue J’adore Rush Euphoria Good Girl
Inspired 2 Inspired
Inspired 2 Inspired
Inspired Inspired
2 22 22
Authentic
2 Inspired
Inspired 2 Inspired
Inspired 2
Inspired Inspired
2 2
Inspired
Inspired 1
Inspired 1 3
Inspired
Inspired 4
Inspired 4 Inspired
4 Inspired
2 Inspired
Inspired 42 42 42
Inspired
Inspired
Authentic
Inspired 3 Inspired
Inspired 3 Inspired
Inspired
Inspired 3 Inspired
3 331 33 4
Inspired
Inspired
Inspired
3 Inspired
Inspired
5
Inspired
3 Inspired
Inspired
5 3 Inspired
Inspired
3 Inspired
Inspired
Inspired
Inspired 523 523
Inspired
Inspired
Inspired 3 Inspired5 5
Inspired
Inspired
3 Inspired
Inspired
Inspired 4 Inspired
Inspired 4 Inspired
Inspired 4 Inspired
4
Inspired 41 4 5
Inspired 4 Inspired
Inspired 4 Inspired
Inspired 4 4 423 43
Inspired
Inspired
Inspired
Counterfeit
Counterfeit
InspiredCounterfeit Counterfeit
Counterfeit
4 Counterfeit 4 Counterfeit
Inspired
Inspired 4 4
Inspired 5 Inspired
Inspired 5 Inspired
Inspired 5 5235 55
Inspired
Inspired
Inspired
5 Inspired
Inspired
Inspired 5 Inspired
Inspired 5 Inspired
Inspired 5 5
Inspired
Inspired 4 4
Inspired 5 Inspired Inspired
Inspired
5 3.2.
Inspired 53 5
Counterfeit
Counterfeit Counterfeit
Counterfeit
Counterfeit
Counterfeit
Inspired NMR
43.2. 3.2. Measurements
NMR NMRMeasurements
3.2. 3.2.
Measurements
3.2.
NMR NMR 3.2.
NMR NMR Measurements
Measurements
Measurements
Measurements
Counterfeit
Counterfeit Counterfeit
Counterfeit
Counterfeit
Counterfeit Inspired
Inspired 5 5 1 1
Counterfeit
Counterfeit Counterfeit
Counterfeit
Inspired 4 AllAllHH NMR
AllNMRAllspectra
1H NMR 1spectra
All
H NMR 1HAllwere
were
1
spectra
NMR H recorded
AllNMR
spectra 1 H
were NMR
recorded
spectra spectra
recorded
were using
spectra
wereusing
were
recorded ausing
Bruker
awere
recorded recorded
using Avance
a recorded
Bruker aAvance
Bruker
using using
Bruker IIusing
aAvance Plus
BrukerII Plus
aAvance
Bruker16.4
aAvance
II Bruker
16.4
Plus TPlus
spectrometer
IIAvance
16.4Avance
TIIspectrometer
T II
Plus
16.4 T II
Plus
spectrometer
16.4 Plus
16.4 16.4
T sp
T spectro
spectromet
Inspired (Bruker5 BioSpin, Rheinstetten, Germany) operating at the 1 H1 frequency of 700.08 1MHz
Counterfeit
Counterfeit (Bruker
(Bruker
3.2. NMR BioSpin,
BioSpin,(Bruker
Measurements
(Bruker
(Bruker
3.2. NMR
3.2. 3.2.
BioSpin,
Measurements
3.2.
NMR NMR (Bruker
Rheinstetten,
3.2.
NMR BioSpin,
Rheinstetten,
NMR
BioSpin, BioSpin,
Germany)
Measurements
Measurements
Rheinstetten,
Rheinstetten,
Measurements
Measurements Rheinstetten,
Rheinstetten,
Germany) operating
Germany) Germany) Germany)
operating Germany)
at theat
operating
operating H
operating
the operating
frequency
1
at the Hat the
1 at
frequency1theat
of the
1700.08
Hof
H frequency
H frequency H
700.08 frequency
MHz
frequency MHz
of 700.08
of 700.08 of MH7
Inspired The 53.2.
The
NMR
3.2. NMR
instrument Measurements
3.2.
instrument
The NMR
was
instrument
3.2.
Measurements
3.2. NMR
was
The
3.2.
NMR
equipped
The
equipped
NMR
Measurements
Measurements
instrument
was equippedwith
instrument
Measurements
Measurements
witha
was 5 a
with mm
was
5 mm broadband
equippedequipped
a were broadband
5recorded
mm with
broadband BBI
with
a 5 BBIa
mmprobe.
5 mm
probe.
BBI
1 H1 NMR
broadbandbroadband
probe. H NMR H spectra
BBI BBI
spectra
probe. were
probe.1were
H 1H NM
NMR sp
TNMR spectra T were
1
Counterfeit All 1The
3.2. NMR H
All The
H 1instrument
1instrument
Measurements
NMRAllNMR
3.2. 3.2.
NMR
All 3.2.
1NMR
spectra All was
NMR
1H
spectra
H All
were
Measurements
NMR was
equipped
1H
NMR
were equipped
Measurements
Measurements
NMR
recorded
spectra
recorded with
spectra
using with
a awere
using a a5 recorded
5recorded
mm
Bruker mm
broadband
Bruker broadband
Avance
using
aAvanceusing
II BBI
Plus
aAvance BBI
probe.
aAvance
Bruker
II Bruker
16.4
Plus probe.
Avance
1
16.4 HPlus
TNMR
Avance
1 H
spectrometer
II NMR
Plus spectra
II
spectrometer Plus
16.4 spectr
T we
16.4 sp
All 1acquired
H NMR H NMR
1spectra All 1spectra
were
All
H 1spectra
NMR H were
recorded
NMRspectrawere
recorded
using
spectra
were awere using
Bruker
recorded using
aAvance
recorded Bruker
using BrukerII
using
adataPlusadata
Bruker IIAvance
16.4
Bruker PlusIIspectrometer
Tover 16.4
Avance II 16.4
T
Plus II Plus
16.4spectro
spectromet 16.4
Tove sp
acquired
acquiredAll
with1H
All
a
withNMR
acquired All
H 1H
NMR
calibrated
acquired
a
with spectra
NMR
acquired
calibrated
with
a spectra
90°
with
a were
spectra
with
pulse
a
90° recorded
were afor
calibrated
calibrated
calibrated pulse
90° were
recorded
calibrated
32
90°for
pulse scansusing
recorded
90°
32
pulse
for ascans
using
90° Bruker
using
pulse
collecting
pulse
scansfor
32 32forascans
Bruker
32
collecting foraAvance
64 Bruker
32
scansK 64Avance
scans
collecting
collecting K 64II Plus
Avance
points
collecting
K II
64 16.4
Plus
collecting II
64
points
dataK data KTover
Plus
16.4
points 64
aspectrometer
16.4
KTover
spectral
data
points a Tover
points spectro
spectromet
data
spectral
a pointsaMH
spectr sp
Counterfeit (Bruker
All 1BioSpin,
(Bruker H BioSpin,
(Bruker
(Bruker
NMR (Bruker
BioSpin,
3.2.
spectra
All 1(Bruker
Rheinstetten,
NMR
All
H 1BioSpin,
Rheinstetten,
3.2. NMR
BioSpin,
NMR H 1BioSpin,
Germany)
Measurements
All
were NMR H NMR
recorded
spectra spectra Rheinstetten,
Rheinstetten,
Germany)
Measurements
Rheinstetten,
Rheinstetten, spectra
wereusing operating
Germany)
were awere
recorded Germany)
operating
Germany)
Bruker
recorded Germany)
at
recorded
using the
Avanceat
operating
operating using H
operating
1
the at
using
aoperating
1BrukerII
a operating
frequency
1 H
theat
Plusa
Bruker 1
1H Avance Hat
frequency
the
Bruker
16.4 the
H at
of
frequency
1
T
Avance
1the
700.08
Hof
frequency
Avance
spectrometer
II Plus II H
frequency
1
700.08
of
Plus
16.4 frequency
MHz
II of
700.08
PlusMHz
16.4
TMHz of
700.08
16.4
T
spectro 7
sp
(Bruker
width(Bruker
of
width BioSpin,
BioSpin,
(Bruker
(Bruker
14,097
of
widthwidth Hz
14,097
of Rheinstetten,
(Bruker
BioSpin,
(zg,
width
ofHz
14,097 (Bruker
BioSpin,
width BioSpin,
Rheinstetten,
Bruker,
of
(zg,
14,097
Hz of
14,097
Bruker,
Hz
(zg, Germany)
BioSpin,Rheinstetten,
Germany).
Hz
(zg,
Bruker, Germany)
Rheinstetten,
Rheinstetten,
14,097 Hz
(zg,
Germany).
Bruker, operating
Rheinstetten,
Germany)
(zg, The
Bruker,
Germany).Germany)
Bruker, Germany)
operating
repetition
The
Germany).Germany). at operating
operating
repetition
The the
Germany)
Germany).
The at
time
repetition Hat
the
The offrequency
time
repetitionoperating
the
Theat
8.27
repetition
of
time atof
frequency
1the
H
s,
8.27
time H of
1theat
frequency
repetition
including
time
s,of
8.27
1700.08
the
Hof
frequency
time
of
including
8.27
s,
1H
700.08
a of
s, MHz
frequency
offrequency
relax-
8.27
including aof
700.08
8.27
s, of
700.08
s,
includ
relax-
including a MH7a
inc
rela
The instrument
The
(Bruker instrument
The The was
The The
equipped
instrument
instrument
BioSpin, instrument
instrument
was equipped
was
(Bruker
Rheinstetten, was with
was
equipped
BioSpin, a
with
equipped
Germany) was
5 mm
equipped
a
withequipped
5 broadband
mm
with
a
Rheinstetten, 5 with with
broadband
mma 5 mma 5 BBI
broadband a
mm 5 mm
probe.
BBI
broadband broadbandbroadband
1
probe.
BBI H NMR
BBI 1
probe. HBBINMR
probe.1H BBI
spectra
probe.
1H
NMR probe.
spectra
NMR1were
H NMR
spectra1 H
were NM
spectr sp
we
(Bruker BioSpin, Rheinstetten, 5Toperating Germany) Germany)
aat the H
operating operating
afrequency at theat
Hof the
700.08
HNMR H
frequency frequency
MHz of 7
1 1 1
(Bruker 3.2. NMR
BioSpin, Measurements
All Rheinstetten,
H Germany) operating at1aH the 1frequency of 700.08
6a NMR 5Tspectra were recorded using Bruker Avance II Plus 16.4
1
The instrument
The instrument
The was
The All
equipped
The 1 H
instrument
was
instrument equippedNMR
1
instrument
was with
wasspectra
a7s,
with
equippedwas mm
equippedwere1mm
with recorded
broadband
a7 equipped with with
broadband
a7of5T 5T using
BBI
1mm probe.
a7 relaxation
5longest
1mm
BBI Bruker
broadband NMR
broadband
probe. 1Avance
HBBINMR spectra
BBI 1II
probe. Plus
probe.
spectra 1were
H 16.4
1H
NMRwereTNM sp
sp
ation delay
ation
acquired
The ofinstrument
delay
ation
with 6a calibrated
ation s,
of
delay was
ation ation was
6a calibrated
s,
delay
of was
6a of
s,
acquired delay
calculated
delay equipped
of
6acalculated
was
90° s, was of
as
s, 6afor
was
calculated
with
pulse as 1with
calculatedwas
of as
calibrated
32 the
calculated
scans
a7 as
of 5T mm
calculated
longest
the
90° as1mm
broadband
longest
theof
pulse
1collecting asthebroadband
7 relaxation
of
longest
for
64 Tthe
32
K
BBI
ofrelaxation
the
time
longest
scans
data
BBI
probe.
longest
to
time
relaxation probe.
to
time
collecting
points over
H
relaxation
ensure
relaxation ensure
time
Kto 64
H NMR
complete
time
Kto
ensure spectra
time
to
complete
ensure spectr
to
ensur
1comple
wee
com
The acquired
instrument with
acquired
acquired
The acquired
waswith
The with
The
equipped
instrument
(Bruker with
instrument
instrument was 90° calibrated
calibrated
calibrated with
waspulse
90°a
equipped
BioSpin, was
5 90°for
pulse
mm
equipped 90°
32
pulse
for
equipped pulse
scans
32
broadband
with
Rheinstetten, for
with
a 5 32for
scans
with
mma 5 32
collecting
scans
BBI
Germany) a
mm 5scans
broadband mm 64
collecting
collecting
probe. collecting
K
broadband 1data
64 K
broadband
H
operating 64
NMR
BBI dataK
BBI 64
points
probe.
atdata
points
BBI
spectra1a
1probe.
the data
over
Hpoints
probe.
H NMR data
1spectral
overpoints
a1were
Hspectral
over
a
NMR
frequency points
H ove
NM
spectra
spectr sp
sp
acquired
acquired
magnetizationwith a
with
acquired
acquired
magnetization (Bruker
3.2.
calibrated
acquired
a
with
recovery. NMR
acquired
All
calibrated
with
a
magnetization
recovery.
1BioSpin,
An aMeasurements
90°
with
H NMRpulse
with
a
90°
calibrated
calibrated
magnetization Rheinstetten,
exponential
An a
90°for
calibrated
spectra
pulse
recovery.
exponential 32
calibrated
90°for
pulse
recovery. scans
were90°
32
pulse
line
An for An Germany)
collecting
90°
pulse
recorded
scans
for
32
broadening pulse
32for
scans 32
collecting
scans
exponential
exponential
line broadening 64
for
using
of operating
scans
line K
32 a
64data
scans
collecting
collecting line
0.05 64
Hz data
Kat
points
collecting
Bruker
K 64 the
collectingover
Avance64
points
data
broadening
broadening
of 0.05 was
Hz K data H
K
points
applied
wasof a
64frequency
over
0.05 spectral
data
II K
Plus
points
of
applied a data
over
0.05
to
Hz points
16.4
spectral
theover
Hz
was
toa of
points
Tove
was
the a
spectr
app 7
sp
sp
width ofwidth
width
acquired ofmagnetization
magnetization
14,097
with Hz
14,097
width
aof (zg,
width
ofHz
14,097
calibrated
recovery.
width
Bruker,
of
(zg,
14,097
Hz
acquired 90°
recovery.
of
14,097 14,097
Bruker,
Hz
(zg, Germany).
Hz
(zg,
Bruker,
with
pulse HzAnGermany).
AnGermany).
(zg,
Bruker,
afor
exponential
exponential
(zg,
calibrated The
32Bruker,
scans Bruker,
The
Germany).
90°
line
repetition
Germany). line
broadening
Germany).
repetition
The
pulse
collecting The broadening
time
repetition
for The of
Ktime
64repetition
32 The
8.27
repetition
of
time
scans
data
of
ofrepetition
s,0.05
8.27
time
of 0.05
Hz
including
time
s,of
8.27
collecting
points
Hz
s,was
time
of
including
over 8.27 was
s,applied
of
a8.27
Krelax-
aincluding
64 data
spectral s,applied
a8.27 as,to
1includ
relax-
1including inc
rela
points at
Molecules 2021, 26, 3098 8 of 10

acquired with a calibrated 90◦ pulse for 32 scans collecting 64 K data points over a spectral
width of 14,097 Hz (zg, Bruker, Germany). The repetition time of 8.27 s, including a
relaxation delay of 6 s, was calculated as 7 T1 of the longest relaxation time to ensure
complete magnetization recovery. An exponential line broadening of 0.05 Hz was applied
to the raw data prior to Fourier transformation. All samples were run at 300 K. The spectra
were calibrated at 0 ppm from the TMS peak, which was used as a chemical shift standard.
All spectral regions were individually corrected using a fifth-order baseline function.

3.3. Statistical Analysis

Statistical analysis was performed using AMIX 3.9.14 (Bruker, Rheinstetten, Germany)
and OriginPro 2020 9.7.0.188 (OriginLab Northampton, MA, USA). The variation of the data
was explored by principal component analysis (PCA), which was used for unsupervised
pattern recognition, allowing the observation of trends and similarities between samples.
The spectral region from 0 to 8.5 ppm of 1 H NMR spectra was chosen as the input data for
PCA analysis. Prior to chemometric analysis the NMR spectra were manually phased, the
baseline was corrected and each spectrum calibrated to the TMS signal at 0 ppm. AMIX
software was used to segment the NMR spectra into buckets. The width of the buckets was
user-defined and equal to 0.05 ppm for 1 H NMR data. The spectra were normalized to
the total sum of the integrals of all the buckets. A one-way ANOVA was performed using
the OriginPro 2020 to determine significant differences in compound levels. For an easier
chemometric comparison, rectangular bucket integrals were used as input for one-way
ANOVA analysis. The Tukey test was performed to reveal pair-wise differences between
means (p < 0.05).

4. Conclusions
The first approach to a rapid and reliable discrimination of perfumes based on proton
nuclear magnetic resonance spectroscopy was demonstrated. This method is characterized
by a very fast and simple sample preparation that allows the discrimination of authentic,
counterfeit and inspired perfumes on the basis of the synergic combination of 1 H NMR
spectroscopy and chemometric techniques. Our research showed that 1 H NMR fingerprints
may serve as an alternative method to identify counterfeit products. However, the number
of samples was a limitation of the study, but in our opinion it indicated an overall trend
in the discrimination of perfume. Furthermore, the small differences observed for two
authentic J’adore samples suggest that the comparison of authentic samples from different
batches is necessary. We believe that the developed method possesses great potential, and
further studies on the application of NMR spectroscopy in such studies are ongoing in
our laboratory.

Supplementary Materials: The following are available online. Figure S1: The comparison of the
means integration values of 1 H NMR signals for authentic, inspired and counterfeited Light Blue
samples obtained by ANOVA. (Tukey test, p < 0.05). Figure S2: The comparison of the means
integration values of 1 H NMR signals for authentic, inspired and counterfeited Good girl samples
obtained by ANOVA. (Tukey test, p < 0.05). Figure S3: The comparison of the means integration
values of 1 H NMR signals for authentic, inspired and counterfeited Good girl samples obtained by
ANOVA. (Tukey test, p < 0.05). Figure S4: The comparison of the means integration values of 1 H
NMR signals for authentic, inspired and counterfeited Si samples obtained by ANOVA. (Tukey
test, p < 0.05). Figure S5: The comparison of the means integration values of 1 H NMR signals for
authentic, inspired and counterfeited Si samples obtained by ANOVA. (Tukey test, p < 0.05). Figure S6:
The comparison of the means integration values of 1 H NMR signals for authentic, inspired and
counterfeited Rush samples obtained by ANOVA. (Tukey test, p < 0.05). Figure S7: The comparison
of the means integration values of 1 H NMR signals for authentic, inspired and counterfeited Rush
samples obtained by ANOVA. (Tukey test, p < 0.05). Figure S8: The comparison of the means
integration values of 1 H NMR signals for authentic, inspired and counterfeited J’adore samples
obtained by ANOVA. (Tukey test, p < 0.05). Figure S9: The comparison of the means integration
values of 1 H NMR signals for authentic, inspired and counterfeited J’adore samples obtained by
Molecules 2021, 26, 3098 9 of 10

ANOVA. (Tukey test, p < 0.05). Figure S10: The comparison of the means integration values of 1 H
NMR signals for authentic, inspired and counterfeited Euphoria samples obtained by ANOVA. (Tukey
test, p < 0.05). Figure S11: The comparison of the means integration values of 1 H NMR signals for
authentic, inspired and counterfeited Euphoria samples obtained by ANOVA. (Tukey test, p < 0.05).
Figure S12: 1 H NMR spectra of authentic, inspired and counterfeited samples of Light blue in spectral
region from 0 to 11 ppm. Figure S13: Expanded region of 1 H NMR spectrum of inspired Light blue
sample in the region of chemical shift from 3.85 ppm and to 5.15 ppm. Figure S14: 1 H NMR spectra
of authentic, inspired and counterfeited samples of Good girl in spectral region from 0 to 11 ppm.
Figure S15: 1 H NMR spectra of authentic, inspired and counterfeited samples of Si in spectral region
from 0 to 11 ppm. Figure S16: 1 H NMR spectra of authentic, inspired and counterfeited samples of
Rush in spectral region from 0 to 11 ppm. Figure S17: 1 H NMR spectra of authentic, inspired and
counterfeited samples of J’adore in spectral region from 0 to 11 ppm. Figure S18: 1 H NMR spectra
of authentic, inspired and counterfeited samples of Euphoria in spectral region from 0 to 11 ppm.
Figure S19: PCA loading plot generated from the 1 H NMR spectra from the authentic perfume and
counterfeited and inspired samples for the range of chemical shifts from 0 to 10 ppm. Figure S20:
The comparison of the buckets from NMR spectra for two authentic samples of Light blue. Figure S21:
The comparison of the buckets from NMR spectra for two authentic samples of J’adore. Table S1:
The main compounds assigned in 1 H NMR spectra of perfume samples, their diagnostic 1 H signals,
chemical shifts δH and multiplicities.
Author Contributions: The study was conceived by B.P.-S., G.C. and Ł.A., B.P.-S. and G.C. designed
and performed the experiments. B.P.-S. and G.C. analyzed the data, B.P.-S. wrote the paper, B.P.-
S., G.C. and Ł.A. read and revised manuscript critically. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in the article and in the
Supplementary Material.
Acknowledgments: This work was supported by funds from Lodz University of Technology. Au-
thors express their gratitude to Schimar Ltd. Łódź for providing authentic samples of perfumes.
Conflicts of Interest: All authors disclose no potential competing interest.
Sample Availability: Not applicable.

Abbreviations

GC–MS gas chromatography coupled with mass spectrometry

EOS electronic olfactory system
DOSY diffusion ordered spectroscopy
PCA principal component analysis
TMS tetramethylsilane

References
1. Dronova, O.; Smagorinskiy, B.P.; Yastrebov, V. Counteraction to e-commerce crimes committed with the use of online stores. In
Big Data-Driven World: Legislation Issues and Control. Technologies; Kravets, A.G., Ed.; Springer: Cham, Switzerland, 2019; Volume
181, pp. 121–131. [CrossRef]
2. Cano, M.; Borrego, V.; Roales, J.; Idigoras, J.; Lopes-Costa, T.; Mendoza, P.; Pedrosa, J.M. Rapid discrimination and counterfeit
detection of perfumes by an electronic olfactory system. Sens. Actuators B Chem. 2011, 156, 319–324. [CrossRef]
3. Abedi, G.; Talebpour, Z.; Jamechenarboo, F. The survey of analytical methods for sample preparation and analysis of fragnances
in cosmetics and personal care products. Trends Anal. Chem. 2018, 102, 41–59. [CrossRef]
4. Heydorn, S.; Menné, T.; Johansen, J.D. Fragrance allergy and hand eczema—A review. Contact Derm. 2003, 48, 310–316. [CrossRef]
[PubMed]
5. Steinemann, A. Fragranced consumer products: Exposures and effects from emissions. Air Qual. Atmos. Health 2016, 9, 861–866.
[CrossRef] [PubMed]
Molecules 2021, 26, 3098 10 of 10

6. Basketter, D.A.; Lemoine, S.; McFadden, J.P. Skin sensitisation to fragrance ingredients: Is there a role for household clean-
ing/maintenance products? Eur. J. Dermatol. 2015, 25, 7–13. [CrossRef]
7. Fortineau, A.D. Chemistry Perfumes Your Daily Life. J. Chem. Educ. 2004, 81, 45–50. [CrossRef]
8. Deska, M.; Girek, T.; Herman, B. Środki konserwujace ˛ w preparatach i kosmetycznych i bezpieczeństwo ich stosowania. Prace
Nauk. Akad. Im. Jana Długosza W Cz˛estochowie Tech. Inform. Inżynieria Bezpieczeństwa 2016, 4, 87–108. [CrossRef]
9. Rudzki, E.; Parapura, K.; Czubalska, M. Alergia na perfumy. Alerg. Astma Immunol. 2002, 7, 165–169.
10. van Asten, A. The importance of GC and GC–MS in perfume analysis. Trends Analyt. Chem. 2002, 21, 698–708. [CrossRef]
11. Debonneville, C.; Chaintreau, A. Quantitation of suspected allergens in fragrances. Part II: Evaluation of comprehensive gas
chromatography conventional mass spectrometry. J. Chromatogr. A 2004, 1027, 109–115. [CrossRef]
12. Casabianca, H.; Graff, J.B.; Jame, P.; Perrucchietti, C.; Chastrette, M. Application of hyphenated techniques to the Cromatographic
authentication of flavors in food-products and perfumes. J. High. Resolut. Chromatogr. 1995, 18, 279–292. [CrossRef]
13. Cabaleiro, N.; de la Calle, I.; Bendicho, C.; Lavilla, I. Fast screening of terpenes in fragrance-free cosmetics by fluorescence
quenching on a fluoresceine bovine serum albumin probe confined in a drop. Anal. Chim. Acta 2012, 719, 61–67. [CrossRef]
[PubMed]
14. Hoffman, R.E.; Arzuan, H.; Pemberton, C.; Aserin, A.; Garti, N. High-resolution NMR “chromatography” using a liquids
spectrometer. J. Magn. Reson. 2008, 194, 295–299. [CrossRef]
15. Pemberton, C.; Hoffman, R.E.; Aserin, A.; Garti, N. NMR chromatography usingmicroemulsion systems. Langmuir 2011, 27,
4497–4504. [CrossRef] [PubMed]
16. Talzi, V.P. A 13 C and 1 H NMR analysis of perfumes. Russ. J. Appl. Chem. 2006, 79, 107–116. [CrossRef]
17. Haddad, R.; Catharino, R.R.; Marques, L.A.; Eberlin, M.N. Perfume fingerprinting by easy ambient sonic-spray ionization mass
spectrometry: Nearly instantaneous typification and counterfeit detection. Rapid Commun. Mass Spectrom. 2008, 22, 3662–3666.
[CrossRef]
18. Chingin, K.; Gamez, G.; Chen, H.; Zhu, L.; Zenobi, R. Rapid classification of perfumes by extractive electrospray ionization mass
spectrometry (EESI-MS). Rapid Commun. Mass Spectrom. 2008, 22, 2009–2014. [CrossRef]
19. Wilson, A.D.; Baietto, M. Applications and advances in electronic-nose technologies. Sensors 2009, 9, 5099–5148. [CrossRef]
20. Poprawski, J.; Boilot, P.; Tetelin, F. Counterfeiting and quantification using an electronic nose in the perfumed cleaner industry.
Sens. Actuators B Chem. 2006, 116, 156–160. [CrossRef]
21. Gherghel, S.; Morgan, R.M.; Blackman, C.S.; Karu, K.; Parkin, I.P. Analysis of transferred fragrance and its forensic implications.
Sci. Justice 2016, 56, 413–420. [CrossRef]
22. Gherghel, S.; Morgan, R.M.; Arrebola-Liébanas, J.; Romero-González, R.; Blackman, C.S.; Garrido-Frenich, A.; Parkin, I.P. Devel-
opment of a HS-SPME/GC-MS method for the analysis of volatile organic compounds from fabrics for forensic reconstruction
applications. Forensic Sci. Int. 2018, 290, 207–218. [CrossRef] [PubMed]
23. Gherghel, S.; Morgan, R.M.; Arrebola-Liébanas, J.F.; Blackman, C.S.; Parkin, I.P. Fragrance transfer between fabrics for forensic
reconstruction applications. Sci. Justice 2019, 59, 256–267. [CrossRef]
24. Godinho, R.B.; Santos, M.C.; Poppi, R.J. Determination of fragrance content in perfume by Raman spectroscopy and multivariate
calibration. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2016, 157, 158–163. [CrossRef] [PubMed]
25. Borowiecka, J.; Wesołowski, W. Analiza składników wyrobów perfumeryjnych z rodziny owocowej przeznaczonych dla kobiet
technika˛ GC/MS. Bromat. Chem. Toksykol. 2010, 4, 572–580.
26. Bombarda, I.; Dupuy, N.; Le Van Da, J.P.; Gaydou, E.M. Comparative chemometric analyses of geographic origins and composi-
tions of lavandin var. Grosso essential oils by mid infrared spectroscopy and gas chromatography. Anal. Chim. Acta 2008, 613,
31–39. [CrossRef] [PubMed]