Forensic Science International 280 (2017) 130138

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Forensic Science International

journal homepage: www.elsevier.com/locate/forsciint

Differentiation of red lipsticks using the attenuated total reection

technique supported by two chemometric methods
Marta Gadysz, Magorzata Krl* , Pawe Koscielniak
Laboratory for Forensic Chemistry, Department of Analytical Chemistry, Faculty of Chemistry, Jagiellonian University in Krakw, Gronostajowa 2, 30-387
Krakw, Poland

A R T I C L E I N F O A B S T R A C T

Article history:
Received 19 June 2017 The main challenge for the identication and differentiation of lipsticks for forensic purposes is the fact
Received in revised form 2 September 2017 that they have a similar chemical composition in particular, samples of the same hue. The objective of
Accepted 25 September 2017 the presented research was to solve this problem using a nondestructive analytical technique
Available online 6 October 2017 attenuated total reection spectroscopy (ATR). 38 red lipsticks produced by 20 different manufacturers
were examined in optimized experimental conditions. To facilitate discrimination and provide more
Keywords: extensive analyses of the obtained data, two chemometric techniques: principal component analysis and
Red lipsticks cluster analysis were used. Ultimately, nine groups of investigated lipstick samples with comparable
Differentiation
chemical compositions were differentiated. Moreover, lipstick smears on six different interfering
Attenuated total reection
surfaces were analyzed and spectral subtraction was performed in order to identify individual samples.
Principal component analysis
Cluster analysis The established approach succeeded in identifying the index number and the manufacturer of the
samples by using an in-lab built ATR spectra library. The developed method demonstrates a great
potential for the differentiation and identication of red lipsticks with a very similar hue. It also seems to
have good prospects for future application in forensic science investigations.
2017 Elsevier B.V. All rights reserved.

1. Introduction occurring in smaller quantities are antioxidant materials and

Lipstick residues and smears may be signicant forensic
evidence in investigations, in particular in cases of rape, sexual
assault, murder and burglary. They may be found left on a variety of
surfaces for instance, tissue paper, cigarette butts, glasses,
drinking cups, clothing etc., and constitute indirect evidence
linking a suspect with the victim or crime scene [14]. Neverthe-
less, identication of lipstick samples and differentiation between
them can be a major challenge, because the majority of lipsticks
have a similar composition. They are composed of three main
ingredients: oils (typically 4070%) a mixture of castor,
vegetable, mineral or lanolin oil; waxes (approximately 15%)
especially beeswax or carnauba; and colouring agents (roughly 8%)
organic dyes such as erythrosine, amaranth, rhodamine,
tartrazine, derivatives of uorescein, and inorganic pigments,
particularly titanium dioxide and iron oxides. Other constituents
* Corresponding author at: Laboratory for Forensic Chemistry, Department of
Analytical Chemistry, Faculty of Chemistry, Jagiellonian University in Krakw,
Gronostajowa 2, 30-387 Krakw, Poland.
E-mail address: krolm@chemia.uj.edu.pl (M. Krl).
perfumes [3,4].
There have been many publications regarding lipstick exami-
nation, reporting results from simple optical [5] to modern
analytical methods (i.e. CE [6], HPLC [7,8], UHPLCMS [9],
GCMS [10], ICP-OES [11], ICPMS [12], AAS [11,13], LIBS [14,15],
XRF [16]). However, most of them have focused on (aspects of) the
negative impact of the lipstick components on humans and the
environment. Among other things, the content of azo [6], xanthene
[7,17] or other organic colorants [8,9], as well as hazardous
nitrosamines [10] and heavy metals (e.g. Pb) [1116,1820] in
lipsticks have been studied. In the case of forensic lipstick
examination, the most common technique is nondestructive
Raman spectroscopy [2,3,21,22]. It should be pointed out here,
however, that in every mentioned article, Raman spectroscopy has
been accompanied by problems associated with uorescence. An
in-situ method of surface enhanced resonance Raman scattering
(SERRS) detection of the colorants in lipstick smears on both glass
and cotton surfaces was reported [23], but a surfactant was
required to obtain SERRS spectra of the dyes and pigments in such
waxy samples. Moreover, the technique of neutron activation
analysis (NAA) a nondestructive and highly sensitive but also
very time-consuming and expensive method was employed to
study the presence of trace elements in samples of lipsticks [24].

https://doi.org/10.1016/j.forsciint.2017.09.019
0379-0738/ 2017 Elsevier B.V. All rights reserved.
 M. Gadysz et al. / Forensic Science International 280 (2017) 130138
Another technique described in the literature used to differentiate
lipstick is TLC [1,4,25]. However, although the information
obtained by the TLC analytical methods was usually sufcient
for lipstick differentiation from the forensic point of view, these
methods have the important drawback of destructive sample
preparation.
One of the techniques, which to the best of our knowledge
has not yet been applied to lipstick examination is attenuated total
reection spectroscopy (ATR). Two signicant advantages of ATR,
namely that the sample is not destroyed and no sample
preparation is required, have led to this technique being widely
employed in forensic science investigations [2629]. Consequent-
ly, the aim of the presented research was to develop the rst ATR-
based procedure enabling differentiation of red lipsticks. Addi-
tionally, chemometric methods such as principal component
analysis (PCA) and cluster analysis (CA) were applied in order to
facilitate discrimination and provide more extensive analyses of
the obtained data. Lastly, the in-lab built ATR spectra library,
enabling identication of all investigated samples, was tested by
analyzing traces of random lipsticks on six different, potentially
interfering surfaces.
2. Experimental
The solutions (isopropanol and methanol) used throughout the
experiments were supplied by Sigma-Aldrich (Germany). 38 red
lipsticks (for 5 randomly selected 5 items of the same index
number were bought) of a very similar hue representing
20 different manufacturers were either purchased in local shops
or donated by Inglot Sp. z o.o. (cosmetics company). Detailed
information about all of them is presented in Table 1.
The experiments were carried out using a Thermo Nicolet
iS50 FTIR (Thermo Fisher Scientic Co, Waltham, MA, USA) with a
Smart Orbit micro-ATR accessory. All spectra were collected from
650 to 4000 cm
1 by putting small amounts of a sample
(approximately 10 mg) on the ZnSe crystal and carefully pressing
it with an ATR pressure tower. After each measurement, the surface
of the crystal was cleaned with 50% v/v isopropanol and then with
methanol in order to avoid contamination. A new background was
collected prior to the analysis of each new sample.
Processing of the obtained spectra was carried out using
Thermo Electrons OMNIC 9 software. All statistical analyses were
performed using Statistica 12.5 PL software (StatSoft, Tulsa, OK,
USA) and OriginPro 2017 software (OriginLab Corporation, North-
ampton, MA, USA).
3. Applied chemometric/computational methods
3.1. Principal component analysis
PCA is a well-known multivariate statistical method enabling
reduction of the dimensionality of the original dataset by creating a
linear combination of variables called principal components (PCs).
However, the determination of the optimal number of components
that should be chosen is a crucial step. Inappropriate estimation of
PCs could lead to loss of information. The Scree test, Kaisers
stopping rule and the percentage of cumulative variance are the
most frequently used strategies [30], and these were applied in this
study. Furthermore, PCs are a very useful tool for demonstrating
the relationships between analyzed samples.
3.2. Cluster analysis
In CA, samples are assembled in high dimensional space. At
the beginning, each sample constitutes its own separate cluster,
and then two objects, which are the closest to each other, are
131
combined. This procedure is accomplished repeatedly until all
samples are arranged into one cluster. It is worth pointing out
that several methods for measuring similarity and combining
clusters are available. Their choice depends on the investigated
samples and this should be evaluated experimentally [31]. In this
study, Euclidean distances were calculated and Wards method
was employed. Furthermore, in the literature, over 20 different
rules for stopping an obtained dendrogram are known. In this
article, Mojenas stopping rule was considered to be the most
efcient.
According to Mojenas stopping rule, one should select the
number of groups where the following inequality (Eq. (1)) is
fullled
di1 > d ksd 1
where d0,dn, dn
1 are distances corresponding to n, n
1, . . .
1 clusters. The terms d, sd, and k are the mean, the standard
deviation of the d values, and a constant, respectively [32]. As
stated by Milligan and Cooper [33], the value of k should be 1.25.
3.3. Correlation method
Correlation coefcients (CC) were computed as Pearson
correlation coefcients by means of Thermo Scientic OMNIC
software. It seems important to note that the used algorithm
additionally included elimination of the effects of baseline
variation.
4. Results and discussion
Firstly, the parameters of the experiments were optimized. It is
known that increasing the number of scans reduces the signal-to-
noise level of data, and enables one to distinguish small peaks from
noise, and that lower resolution can have an effect on the
differentiation of peaks. Therefore, 16, 20 and 60 scans, and
resolutions of 4 and 2 cm
1 were tested. Despite the fact that only
slight differences were observed between registered spectra,
20 scans per second with 4 cm
1 spectral resolution for both
background and samples were considered to be optimal.
The homogeneity (intra-variability) of lipsticks was investigat-
ed by analyzing spectra obtained from three different parts of
several randomly selected lipstick samples (L3, L10, L14, L21, L31,
L32, L34 and L36) during one day. As demonstrated in exemplary
Fig. 1a presenting the results obtained for three lipsticks, no visual
differences were found on the spectra. Consequently, it was
considered that the samples were homogeneous and their
composition was stable during one day.
Additionally, during three days the inter-variability of lipstick
was examined using 5 of the previously selected lipsticks. The
similarity of the spectra was calculated as correlation coefcient
(L10: 99.90  0.04; L31: 99.91  0.04; L32: 99.94  0.01; L34:
99.83  0.06; L36: 99.92  006). The presented results provided
the basis for claiming that the analyzed samples of the lipstick
exhibited almost unchanged chemical composition also within a
few days. Moreover, both above-mentioned experiments proved
the high intra- and inter-repeatability of the proposed ATR-FTIR
method.
Finally, the variation of chemical composition of lipstick
samples within the same series was also investigated during
one day by analyzing ve items (ae) of ve samples (L10, L31, L32,
L34 and L36) from the same manufacturer with the same index
number. As it can be seen in exemplary Fig. 1b the obtained
spectra for samples L36 ae almost completely overlapped.
Therefore, it was ascertained that the chemical composition of
analyzed lipsticks that were both produced by the same
132 M. Gadysz et al. / Forensic Science International 280 (2017) 130138

Table 1
List of lipsticks examined during the present study.

Code of lipstick Manufacture Index numbera Seriesb Comment

L1 INGRID 289 Wonder shine
L2 INGRID 288 Wonder shine
L3 MISSLYN M20.94 Rouge a levres High passion
L4 MISSLYN M20.87 Rouge a levres Cayun pepper
L5 GOSH 005 Velvet touch lipstick Matt, classic red
L6 p2 030 Full color lipstick Challenge authority
L7 BourjoisParis 13 Rouge Edition Rouge jet set
L8 e 02 Longlasting lipstick All you need is red
L9 REVLON 720 Super lustrous lipstick Fire & Ice
L10a LOREAL Paris 330 Color riche Matte, cocorico
L10b LOREAL Paris 330 Color riche Matte, cocorico
L10c LOREAL Paris 330 Color riche Matte, cocorico
L10d LOREAL Paris 330 Color riche Matte, cocorico
L10e LOREAL Paris 330 Color riche Matte, cocorico
L11 MAYBELLINE New York 955 Color sensational Craving coral, matte
L12 SEPHORA B06 Rouge Baume SPF20 Soft red
L13 SEPHORA R05 Rouge Cream Hot Tango
L14 SEPHORA N 62 Rounge Brilliance Stay Together
L15 SEPHORA N 19 Color lip last Pure red-matt
L16 CATRICE 430 Ultimate color Hotn Spice
L17 CATRICE 440 Ultimate color Hungs-and Hibis-Kisses
L18 CATRICE 140 Ultimate stay Behind the red curtain
L19 LOVELY 8 Creamy color
L20 WIBO 1 Matte Intense
L21 MISS SPORTY 301 My BFF My cheeky cherry
L22 EVELINE COSMETICS 486 Aqua Platinum
L23 EVELINE COSMETICS 710 Color edition red in love
L24 RIMMEL 510 Moisture renew Mayfair red lady
L25 ASTOR 203 Perfect stay fabulous
L26 MAX FACTOR 715 Color elixir Ruby tuesday
L27 MANHATTAN 550 Soft rouge lipstick Watermelon
L28 INGLOT 176 Lipstick
L29 INGLOT 180 Lipstick
L30 INGLOT 429 Lipstick matte
L31a INGLOT 44 Slim gel lipstick
L31b INGLOT 44 Slim gel lipstick
L31c INGLOT 44 Slim gel lipstick
L31d INGLOT 44 Slim gel lipstick
L31e INGLOT 44 Slim gel lipstick
L32a INGLOT 21 AMC lip pencil matte
L32b INGLOT 21 AMC lip pencil matte
L32c INGLOT 21 AMC lip pencil matte
L32d INGLOT 21 AMC lip pencil matte
L32e INGLOT 21 AMC lip pencil matte
L33 INGLOT 64 AMC lip paint
L34a INGLOT 12 HD lip tint matte
L34b INGLOT 12 HD lip tint matte
L34c INGLOT 12 HD lip tint matte
L34d INGLOT 12 HD lip tint matte
L34e INGLOT 12 HD lip tint matte
L35 MANHATTAN 45 M Xtreme last & shine
L36a MANHATTAN 45 N Perfect creamy & care
L36b MANHATTAN 45 N Perfect creamy & care
L36c MANHATTAN 45 N Perfect creamy & care
L36d MANHATTAN 45 N Perfect creamy & care
L36e MANHATTAN 45 N Perfect creamy & care
L37 MANHATTAN 45 F Perfect creamy & care
L38 MANHATTAN 90 A Perfect creamy & care
a
The index number is a number assigned to each lipstick in a given series.
b
Series name refers to cosmetics collection. The same series name indicates the same collection of lipsticks.

manufacturer and had the same index number were characterized
by very good repeatability.
4.1. Visual inspection of spectra
As seen in Fig. 2, a typical spectrum of a lipstick sample can be
divided into three main ranges. Generally, the rst range (between
3740 and 3100 cm
1) includes one broad band, which is attributed
to water and hydroxyl groups. The second range (from 3050 to
2775 cm
1) takes account of symmetric (2850 cm
1) and
asymmetric (2916 cm
1) C

H stretching vibrations and asym-
metric CH3 stretching vibrations (2954 cm
1). The last range
(below 1800 cm
1) contains many peaks. CO stretching vibra-
tions (1730 cm
1), CH2 scissor deformation (1465 cm
1), CO
stretching and C

H bending vibrations (1172 cm
1), and CH2
rocking mode (712 cm
1) are just some of them. All of the
mentioned peaks could be attributed to various oils and waxes,
especially castor oil and beeswax two common ingredients in
lipsticks [3]. Therefore, it can be supposed that the most
informative and distinguishing range is below 1800 cm
1.
 M. Gadysz et al. / Forensic Science International 280 (2017) 130138 133

Fig. 2. Typical lipstick spectrum, represented by L13.

are distinctive and consequently they can be effortlessly distin-

Fig. 1. Investigation of a) homogeneity of lipsticks L21, L14 and L3; b) variability of
lipsticks from the same manufacturer and with the same index number: L36 ae.
guished.
Concisely, according to PCA analysis, red lipsticks can be divided
into the following six groups: G1 {L34}, G2 {L32}, G3 {L31, L33}, G4
{L10}, G5 {L15, L25} and G6, which includes all the remaining
samples.
In an attempt to nd the most efcient grouping of lipsticks,
cluster analysis was performed in order to classify lipstick samples
in the G6, based on similarities of their ATR spectra in the range
from 1800 to 650 cm
1 (the ngerprint region). Accordingly, the
dendrogram presented in Fig. 4 was created, whereas the results
obtained from Mojenas stopping rule are shown in Table I in the
ESM. They indicated that the value of 1.19 should be used as the
stopping distance.
As a consequence, the dendrogram was divided into the
following four groups of lipstick samples: G6a {L1, L8, L20, L27,
L28, L29, L30, L35, L36a, L37, L38,}, G6b {L4, L5, L13, L17, L21, L22,

4.2. Differentiation of red lipsticks

4.2.1. Chemometric methods

A primary discrimination of lipstick samples was performed
using the PCA technique, taking into account all 38 of the collected
ATR spectra registered in the range from 4000 to 650 cm
1. Fig. I in
the Electronic Supplementary Material (ESM) shows a plot for
deciding on the number of variables a scree test. Interpretation
of this plot indicates that only the rst three components should be
retained in the present study (the line drops precipitously),
because sufcient useful chemical information is provided by
these three components. They also complied with the Kaiser
criterion, which only recommends components with eigenvalues
greater than unity. Furthermore, approximately 90% of the total
variance in the spectra was explained by them: 70.17%, 10.38%,
8.14% for the rst, second, and third PC, respectively. Thus, it is
reasonable to base inferences solely on these PCs.
The PCA score plot of the rst three PCs, demonstrating the
separation of samples, is presented in Fig. 3. Additionally, in Fig. II
in the ESM, two-dimensional plots are presented to make the
visual analysis easier. As seen, most of the lipsticks, except L10, L15,
L25, L31, L32, L33 and L34, were classied into one multi-element
class. Additionally, it was noted that lipstick L15 is very similar to
L25 and lipstick L31 is comparable to L33. Moreover, lipsticks L10,
L32 and L34 are each completely separated from all other lipsticks. Fig. 3. The PCA score plot of the rst three PCs for all investigated lipsticks, taking
This nding suggests that some of the analyzed lipstick samples into account ATR spectra registered in the range from 4000 cm
1 to 650 cm
1.
134 M. Gadysz et al. / Forensic Science International 280 (2017) 130138
Fig. 4. Cluster analysis of lipsticks from G6, taking into account ATR spectra registered in the range from 1800 cm
1 to 650 cm
1.
L26}, G6c {L2, L3, L6, L11, L14, L16, L18, L23, L24} and G6d {L7, L9,
L12, L19}. Ultimately, as a result of chemometric analysis, nine
groups of red lipstick samples with comparable chemical
compositions were created: G1G5 and G6 ad.
It is worth noting that according to chemometric classication,
all the investigated lipstick samples manufactured by Manhattan
were classied into one group (G6 a). On the other hand, the
lipstick samples from Sephora were categorized into various
groups (G6 b, c, G5), while some of lipstick samples manufactured
by Inglot were placed in group G6 a and some in G1G3.
4.2.2. Visual classication
A visual comparison of the analyzed spectra was also
performed. In the rst step of differentiation between lipstick
samples, one should look at both the relative intensities and the
degree of resolution of bands from 3050 to 2775 cm
1. As can be
seen in Fig. 5, this region enables differentiation between all
groups, except between G2 and G6, and between G4 and G5.
Further distinction between groups was based on the bands
occurring below 1800 cm
1. As is evident in Fig. 6a, the most
characteristic feature for G5 is a lack of band at 1745 cm
1
corresponding to the carbonyl group. Moreover, in G4 one can see
a weak doublet at 1375 and 1363 cm
1, whereas in G5 there is
only a band at 1363 cm
1. Both of these bands are due to CH3
deformation [34].
The differentiation of G2 from G6 is presented in Fig. 6b. On the
face of it, spectra obtained for these groups are completely unlike
each other in the range from 1800 to 650 cm
1. The most
noteworthy difference is strong absorption near 1000 and
1100 cm
1 in G2. It is highly possible that these peaks correspond
to phosphates and ethers or alcoholethers, respectively, which
are found in many plasticizers [34].
Moreover, the differences between G6 ad are selected in Fig. 6c
only representative spectra of lipstick samples from the groups
have been presented. As far as one can see, the region where
differences are most clearly visible is from 1400 to 800 cm
1. In this
region, a little broad band (especially in G6 ac) at 1150 cm1
characteristic for C

O stretch in nonaromatic ethers is observable.
Moreover, the existence of a siloxane group is most likely due to a
band occurring at 1108 cm
1, which is most visible in spectra from
G6 b. Besides, strong absorption in the range from 1100 to
1000 cm
1 is characteristic for G6 a, b and, as was mentioned
before, these bands are connected with phosphates and ether/
alcoholethers [34]. It is noteworthy that there are also three
unique bands for G6 d at approximately 1387, 1376 and
1363 cm
1.
Summarizing, visual comparison of the obtained spectra led to
conrmation of the classication ascertained according to the
chemometric results.
4.2.3. Correlation method
In order to make the analysis more objective as well as to verify
the existence of an unequivocal dependence between the
composition of the lipstick samples and the manufacturer (or
series), the correlation method was applied.
At rst, the variability of the same lipstick sample, s, was
ascertained as the mean CC (correlation coefcient) value
calculated for ve items (a-e) of the ve samples (L10, L31, L32,
L34 and L36) of lipsticks of the same index number, the same series
and the same manufacturer. The mentioned samples were
representatives of different PCA groups so they characterized by
different chemical composition. It is worth pointing out that the
CCs value were evaluated by analyzing samples on three successive
days and they ranged between 0.04% and 0.33%. On this basis the
following assumptions related to ATR spectra of each examined
pair of lipstick samples have been made:
 if CC  100% 3s, it is highly possible that the spectra
correspond to the same lipsticks,
 if CC < 100% 3s, the spectra are considered as coming from
different lipsticks.
In short, the similarity limit in the case of the present study is a
value of 99.01%.
Comparing the spectra of lipsticks produced by different
manufacturers, it was ascertained that they are usually characterized
by CCs considerably less than 99.00% (see Table II in ESM), e.g., for L15
(Sephora) and L3 (Misslyn), the CC = 55.81% or for L6 (p2) and L20
(Wibo), the CC = 92.58%. There were a few cases where the
compositions of two lipsticks originating from different
 M. Gadysz et al. / Forensic Science International 280 (2017) 130138
Fig. 5. Differentiation between groups based on the range from 3050 cm
1 to 2775 cm
1.
manufacturers were very similar, e.g., for L3 (Misslyn) and L22
(Eveline Cosmetics) CC = 99.45%, for L29 (Inglot) and L4 (Misslyn)
CC = 99.55%.
If the investigated samples were from the same manufacturer
but from different series, most of them could also be easily
distinguished. It can be seen in Table 2 that CCs between spectra of
lipstick samples produced by Inglot, Manhattan and Sephora were
not higher than 98.50%. Moreover, it is evident that lipsticks
manufactured by Inglot are characterized by the most variable
composition (CC from 96% to 24%).
The obtained results conrmed the theoretical assumption that
lipstick samples from the same manufacturer and from the same
series are more similar to each other than lipstick samples from
different series (see CC for L28 and L29 from Inglot, or L36 and
L37 from Manhattan). On the other hand, some exceptions can be
observed: sample L35 from the Xtreme last & shine series is
undistinguished (CC between 99.11% and 99.49%) from the samples
from the Perfect creamy & care series. Moreover, the data suggest
that a comparison of samples with a similar index number (from
the same manufacturer and from the same series) can cause
problems. In these cases, the CCs between samples were calculated
to be greater than 99.01%, and in consequence, they were not
differentiable with the use of the presented method. The
discrimination of such lipsticks samples can be problematic and
135
they may differ to a very limited extent, e.g. only slightly in the
concentration of colour additives. However, further data are
needed in order to conrm this assumption. It has not escaped our
notice that there were also lipsticks produced by the same
manufacturer in the same series with an index number differing
only by one, e.g., L1 Ingrid no. 289 and L2 Ingrid no. 288, which
revealed, without any doubt, two different spectra (CC = 98.52%).
It seems important to reiterate that samples from the same
manufacturer, the same series and having the same index number
(for instance L36 ae), as was mentioned before, were considered
to be indistinguishable. The CC between spectra was much higher
than 99.01%.
4.3. Analysis of lipsticks traces on various surfaces preliminary
examination
Due to the fact that lipstick samples can be found left on various
surfaces, it is justied to investigate the possibility of identifying
lipstick traces in the form of such smears.
Firstly, one of the samples (L28) was left as a lipstick mark on
various surfaces in order to assess which surfaces caused
interfering bands. The surfaces studied included: envelope, paper
(80 g/m2), tissue (100% cellulose), cigarette butts, and a transparent
plastic cup, as well as the white collar of a shirt. As can be seen in
136 M. Gadysz et al. / Forensic Science International 280 (2017) 130138

Table 2
Spectra correlation coefcients between samples from the same manufacturer: a)
Inglot; b) Manhattan; c) Sephora. Lipsticks samples from the same series are bolded.

a)
L28 100.0
L29 99.57 100.0
L30 96.44 96.70 100.0
L31 91.67 91.50 87.83 100.0
L32 90.24 90.90 95.44 83.29 100.0
L33 79.95 79.48 74.51 88.50 65.70 100.0
L34 27.11 25.90 24.67 40.22 17.65 53.76 100.0
L28 L29 L30 L31 L32 L33 L34

b)
L27 100.0
L35 98.46 100.0
L36a 97.79 99.11 100.0
L37 98.24 99.49 99.65 100.0
L38 97.93 99.26 99.83 99.78 100.0
L27 L35 L36a L37 L38

c)
L12 100.0
L13 94.87 100.0
L14 95.95 96.69 100.0
L15 62.50 57.46 55.17 100.0
L12 L13 L14 L15

In striving to nd the most efcient approach to identication of

lipstick marks, two spectral subtractions were performed: (1) in
the whole range of registered spectra, (2) in the ngerprint region.
The effectiveness of the proposed approach to the identication of
lipsticks marks was veried by analyzing six different traces of red
lipstick (L7, L12, L18, L23, L28 and L35) smudges on all the above-
mentioned surfaces.
Overall, the procedure was carried out in a few steps by:
measuring the surface spectrum and the spectrum of the lipstick
on the same surface, then subtracting one from the other in the
whole range of the rejected spectrum and in the ngerprint region
(from 1800 to 650 cm
1), and subsequently identifying the
spectrum by using an in-lab built ATR spectra library. The obtained
results indicate that the second approach solely enables one to
connect lipstick marks with the manufacturer and index number
(see Table 3). It should be highlighted that the described
subtraction approach did not yield pure spectra of lipstick samples
additional or negative bands appeared in some cases.
Nevertheless, a comparison of the spectral subtraction result with
the spectra collected in the in-lab built library enabled in every
case without exception the successful identication of lipstick
samples.

5. Conclusions

ATR spectroscopy was successfully used for the differentiation

Fig. 6. Differentiation of lipsticks according to ATR spectra registered in the range
from 1800 cm
1 to 650 cm
1: a) G4 represented by L25, G5 represented by L10; b)
G2 represented by L32, G6 represented by L12; c) G6a represented by L35, G6b
represented by L26, G6c represented by L3, and G6d represented by L19.
Fig. III in the ESM, all of the analyzed surfaces, except the
transparent plastic cup, provided additional bands to the lipstick
spectra.
In order to isolate lipstick spectra from surface spectra, Thermo
Electrons OMNIC software was used. This is one of the most
difcult issues in the identication of samples in forensic science.
of 38 red lipsticks of a very similar hue. Although no unambiguous
trends could be identied within manufacturers or series and the
differentiation of lipsticks is a complicated issue, combining the
two chemometric methods (PCA and CA) enabled the distinguish-
ing of nine groups from investigated lipsticks of similar ATR
spectra. Similarities cannot be assumed, in advance, on the basis
of manufacturer, series or index number compatibility. However,
visual analysis revealed bands that were characteristic for
appropriate groups and conrmed the classication obtained
by chemometric techniques. Furthermore, the approach sup-
ported by the correlation coefcient enabled deeper differentia-
tion of all studied lipstick spectra. And, importantly, the
differentiation could be achieved non-destructively in a matter
of minutes.
 M. Gadysz et al. / Forensic Science International 280 (2017) 130138 137

Table 3
A comparison of the two approaches for the identication of lipsticks in the form of lipstick marks smudged on interfering surfaces. Correct identication of lipsticks is
indicated by +.

Lipstick Code The range of spectral subtraction

4000650 cm
1 1800650 cm
1

Cigarette butts Envelope Paper Tissue White collar of shirt Cigarette butts Envelope Paper Tissue White collar of shirt
L7




+ + + + +
L12




+ + + + +
L18




+ + + + +
L23




+ + + + +
L28




+ + + + +
L35




+ + + + +

Table 4
Discriminating power of PCA, CA and CC analysis.
Method Discriminating power (DP)
PCA 0.29
CA 0.51
Correlation method 0.93
To sum up, on the basis of the performed PCA, CA and CC
differentiations, the discriminating power (DP) of the ATR analysis
of lipstick samples has been calculated according to the following
equation: DP = 1
2 M/(N(N
1)) (where M is number of matching
pairs; N is total number of analysed lipsticks). The obtained DP
values were listed in Table 4.
These results conrmed the above-mentioned conclusion that
PCA and CA enables some preliminary classication whereas the
correlation coefcient is the best choice for the lipsticks
discrimination.
Ultimately, by using the developed method and in-lab built ATR
spectra library, it was possible to detect and connect some lipsticks
left as lipstick marks with their manufacturer and index number,
even on interfering surfaces. This nding conrms that the ATR
technique is a useful tool in lipstick differentiation and identica-
tion. All things considered, it seems reasonable to assume that the
proposed method shows promise for future application in forensic
science investigations as a good alternative to routinely used
methods.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at https://doi.org/10.1016/j.forsciint.2017.
09.019.
References
[1] S. Singh, V. Saran, M. Mishra, A.K. Gupta, Forensic examination of lipstick by
the various physio-chemical and instrumental method, Int. J. Soc. Relev.
Concern 3 (2015) 17.
[2] M. Lpez-Lpez, N. zbek, C. Garca-Ruiz, Confocal Raman spectroscopy to
trace lipstick with their smudges on different surfaces, Talanta 123 (2014)
135139.
[3] F. Salahioglu, M.J. Went, Differentiation of lipsticks by Raman Spectroscopy,
Forensic Sci. Int. 223 (2012) 148152.
[4] B. Joshi, K. Verma, J. Singh, A comparison of red pigments in different lipsticks
using thin layer chromatography (TLC), J. Anal. Bioanal. Tech. 4 (2013) 14.
[5] Y. Ehara, Y. Marumo, Identication of lipstick smears by uorescence
observation and purge-and-trap gas chromatography, Forensic Sci. Int. 96
(1998) 110.
[6] D. Li, Z. Wang, L. Wang, X. Xu, H. Zhang, Ultrasonic extraction coupled with
capillary electrophoresis for the determination of azo dyes in lipsticks using
ionic liquid as dynamic coating and background electrolyte, Chin. J. Chem. 29
(2011) 147152.
[7] S. Scalia, S. Simeoni, Assay of xanthene dyes in lipsticks by inverse supercritical
uid extraction and HPLC, Chromatographia 53 (2001) 490494.
[8] S.C. Rastogi, V.J. Barwick, S.V. Carter, Identication of organic colourants in
cosmetics by HPLC-diode array detection, Chromatographia 45 (1997) 215
228.
[9] Y. Xian, Y. Wu, X. Guo, Y. Lu, H. Luo, D. Luo, Y. Chen, Simultaneous
determination of 11 restricted dyes in cosmetics by ultra-high-performance
liquid chromatography/tandem mass spectrometry, Anal. Methods 5 (2013)
19651974.
[10] Q. Ma, H.-W. Xi, C. Wang, H. Bai, G.-C. Xi, N. Su, L.-Y. Xu, J.-B. Wang,
Determination of ten volatile nitrosamines in cosmetics by gas chromatogra-
phy tandem mass spectrometry, Chinese J. Anal. Chem. 39 (2011)
12011207.
[11] . Ferreira Batista, A. dos Santos Augusto, E.R. Pereira-Filho, Chemometric
evaluation of Cd, Co Cr, Cu, Ni (inductively coupled plasma optical emission
spectrometry) and Pb (graphite furnace atomic absorption spectrometry)
concentrations in lipstick samples intended to be used by adults and children,
Talanta 150 (2016) 206212.
[12] N.M. Hepp, Determination of total lead in 400 lipsticks on the U.S. market
using a validated microwave-assisted digestion, inductively coupled plasma-
mass spectrometric method, J. Cosmet. Sci. 63 (2012) 159176.
[13] S. Gunduz, S. Akman, Investigation of lead contents in lipsticks by solid
sampling high resolution continuum source electrothermal atomic absorption
spectrometry, Regul. Toxicol. Pharm. 65 (2013) 3437.
[14] M.A. Gondal, Z.S. Seddigi, M.M. Nasr, B. Gondal, Spectroscopic detection of
health hazardous contaminants in lipstick using Laser Induced Breakdown
Spectroscopy, J. Hazard. Mater. 175 (2010) 726732.
[15] A. dos Santos Augusto, E. Ferreira Batista, E.R. Pereira-Filho, Direct chemical
inspection of eye shadow and lipstick solid samples using laser-induced
breakdown spectroscopy (LIBS) and chemometrics: proposition of classica-
tion models, Anal. Methods 8 (2016) 58515860.
[16] E. Da Silva, A. Matthews David, A. Pejovi c-Mili
c, The quantication of total lead
in lipstick specimens by total reection X-ray uorescence spectrometry, X-
Ray Spectrom. 44 (2015) 451457.
[17] Ch.Ch. Wang, A.N. Masi, L. Fernandez, On-line micellar-enhanced spectro-
uorimetric determination of rhodamine dye in cosmetics, Talanta 75 (2008)
135140.
[18] I. Al-Saleh, S. Al-Enazi, Trace metals in lipsticks, Toxicol. Environ. Chem. 93
(2011) 11491165.
[19] I. Al-Saleh, S. Al-Enazi, N. Shinwari, Assessment of lead in cosmetic products,
Regul. Toxicol. Pharm. 54 (2009) 105113.
[20] P. Piccinini, M. Piecha, S.F. Torrent, European survey on the content of lead in lip
products, J. Pharm. Biomed. Anal. 76 (2013) 225233.
[21] P. Gardner, M.F. Bertino, R. Weimer, E. Hazelrigg, Analysis of lipsticks using
Raman spectroscopy, Forensic Sci. Int. 232 (2013) 6772.
[22] F. Salahioglu, M.J. Went, S.J. Gibson, Application of Raman spectroscopy for the
diff ; ;erentiation of lipstick traces, Anal. Methods 5 (2013) 53925401.
[23] C. Rodger, V. Rutherford, D. Broughton, P.C. White, W.E. Smith, The in-situ
analysis of lipsticks by surface enhanced resonance Raman scattering, Analyst
123 (1998) 18231826.
[24] G. Misra, V.K. Mittal, Neutron activation analysis of lipsticks using g-ray
spectrometry, J. Appl. Spectrosc. 71 (2004) 270274.
[25] A.F.L. bin Abdullah, Y. Marimuthu, Ch. Kah Haw, N.F. binti Mohamad Said, N.Z.
M. Muslim, N.F.N. Hassan, M.H. Yaacob, Y. Chong Hooi, Forensic discrimination
of lipsticks by thin layer chromatography and gas chromatographymass
spectrometry, Malays. J. Forensic Sci. 2 (2011) 2228.
[26] J. Manheim, K.C. Doty, G. McLaughlin, I.K. Lednev, Forensic hair differentiation
using attenuated total reection fourier transform infrared (ATR FT-IR)
spectroscopy, Appl. Spectrosc. 70 (2016) 11091117.
[27] M.C.A. Marcelo, K.C. Mariotti, M.F. Ferrao, R.S. Ortiz, Proling cocaine by ATR-
FTIR, Forensic Sci. Int. 246 (2015) 6571.
[28] M. Szafarska, M. Wo zniakiewicz, M. Pilch, J. Zieba-Palus, P. Koscielniak,
Computer analysis of ATR-FTIR spectra of paint samples for forensic purpose, J.
Mol. Struct. 924926 (924) (2009) 504.
[29] C.M. Orphanou, The detection and discrimination of human body uids using
ATR FT-IR spectroscopy, Forensic Sci. Int. 252 (2015) e10e16.
138 M. Gadysz et al. / Forensic Science International 280 (2017) 130138

[30] B.R. Kowalski (Ed.), Chemometrics: Mathematics and Statistics in Chemistry,
Reidel, Dordrecht, 1984.
[31] D.L. Massart, L. Kaufman, The Interpretation of Analytical Chemical Data by the
Use of Cluster Analysis, John Wiley & Sons, New York, 1983.
[32] R. Mojena, Hierarchical grouping methods and stopping rules: an evaluation,
Comput. J. 20 (1977) 359363.
[33] G.W. Milligan, M.C. Cooper, An examination of procedures for determining the
number of clusters in a data set, Psychometrika 50 (1985) 159179.
[34] G. Socrates, Infrared and Raman characteristic group frequencies, Tables and
Charts, John Wiley and Sons, Ltd., Chichester, 2001.