J Am Oil Chem Soc (2012) 89:1537–1543
DOI 10.1007/s11746-012-2052-8
ORIGINAL PAPER
Quantitative Analysis of Lard in Cosmetic Lotion Formulation
Using FTIR Spectroscopy and Partial Least Square Calibration
Endang Lukitaningsih • Miftahus Sa’adah •
Purwanto • Abdul Rohman
Received: 13 November 2011 / Revised: 26 February 2012 / Accepted: 6 March 2012 / Published online: 18 March 2012
Ó AOCS 2012
Abstract Fourier transform infrared (FTIR) spectroscopy
in combination with chemometrics of partial least squares
(PLS) has been optimized for rapid determination of lard in
a binary mixture with palm oil in a cosmetic lotion for-
mulation. Lard, palm oil, and a binary mixture were
extracted from matrix samples using liquid–liquid extrac-
tion, evaporated with a vacuum rotary evaporator, and the
fat/oil yielded was further subjected to FTIR spectrometric
measurement using attenuated total reflectance (ATR) as a
sampling handling technique. The level of lard in the
mixture with palm oil in the lotion formulation was
quantified at frequency region of 1,200–1,000 cm-1. The
PLS calibration model reveals good correlation between
the actual value of lard (x-axis) and the FTIR predicted
value (y-axis) with a coefficient of determination (R2) of
[0.99. Furthermore, the classification between lotions with
and without lard in their formulation was performed using
principal component analysis using the same frequency
region used for quantification. The developed method was
subsequently used for analysis of cosmetic lotions com-
mercially available in the market. All samples analyzed did
not contain lard in their formulations.
Keywords Lard
Palm oil
Lotion
FTIR spectroscopy

Partial least squares
E. Lukitaningsih
M. Sa’adah
Purwanto
A. Rohman (&)
Faculty of Pharmacy, Gadjah Mada University,
Yogyakarta 55281, Indonesia
e-mail: abdulkimfar@gmail.com
E. Lukitaningsih
Purwanto
A. Rohman
Research Center of Halal Products, Gadjah Mada University,
Yogyakarta 55281, Indonesia
Introduction
In recent years, the use of personal care products in the
form of cream and lotion cosmetic products has increased
tremendously [1]. Human exposure to cosmetic formula-
tions and their ingredients occurs primarily via the topical
route such as cream and lotion [2]. As a consequence, some
considerations and care are necessary in relation to the
suitability, efficacy, effectiveness and safety of cosmetics
products, because cosmetics products are used nearly
constantly and come into direct contact with the human
skin [3].
In some cosmetics preparations, pork fat or lard (LD)
obtained from the rendering process of fatty porcine tissue
is commonly used as viscosity-increasing agents. Food and
drug administration (FDA) has compiled LD as one of the
generally recognized safe substances used for food and
cosmetics products [4]. However, cosmetics products
containing LD are prohibited to be used for the followers of
some religions, like Islam [5, 6]. Furthermore, among oil
ingredients used in lotions and other cosmetics products,
palm oil is the most commonly used. Therefore, in this
study, we used palm oil as the oil mixture in LD. For some
reasons especially the restrictions to exposure to LD, the
development of an analytical method offering fast and
reliable results is highly desirable. One of the developed
methods is Fourier transform infrared (FTIR) spectroscopy.
In the analytical field, FTIR spectroscopy provides a
highly effective choice. It is a fast and non-destructive
technique, sensitive, and does not need or is simple in
sample preparation and can be considered as a ‘‘green
analytical technique’’ because of the low use of chemical
reagents and solvents in its operation [7]. Due to its sim-
plicity in sample handling, an accessory of horizontal
attenuated total reflectance (HATR) is used for the analysis
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of fats and oils [8]. Previous research has shown the
potential of FTIR spectroscopy as a method for analysis of
LD, mainly in food systems such as in cakes and chocolate
products [9, 10] or LD in the mixture with other fats and
oils [11]. Guillen and Cabo [12] as well as Che Man et al.
[13] used FTIR spectroscopy to characterize lard and other
edible fats and oils. Almost all studies performed previ-
ously were based on food or food products.
In cosmetics, FTIR spectroscopy has been used for
determination of virgin coconut oil used as an emulsifying
system in cream formulation [14]. Recently, we also
explored the use of FTIR spectroscopy for simultaneous
analysis of lard with virgin coconut oil (VCO) [15]. Com-
pared with VCO, palm oil has the close similarity with LD as
described in score plot of principal component in PCA model
[13]. Besides, from research in the literature, there is no
report available related to the use of FTIR spectroscopy for
analysis of LD in a binary mixture with palm oil having a
similar profile to LD in lotion products. Therefore, the
objective of this research was to develop FTIR spectroscopy
combined with chemometrics of partial least squares (PLS)
and principal component analysis for quantification and
classification of LD in lotion formulations.
Materials and Methods
Lard Preparation
Lard (LD) was prepared according to Rohman and Che
Man [16]. LD was extracted by rendering the adipose tissue
of pig (Sus scrofa) obtained from various slaughter houses
around Jogjakarta, Indonesia. The rendering process was
done at 90–100 °C for 2 h in a conventional oven. The
melted fat was strained through triple-folded muslin cloth,
dried with anhydrous Na2SO4, and centrifuged at
3,000 rpm for 20 min. The fat layer was decanted, shaken
vigorously, and centrifuged again before being filtered
using Whatman filter paper. The filtered fats were stored in
tightly closed containers in the refrigerator until being used
for preparation of cream cosmetics. Palm oil and other
materials used were bought in Jogjakarta.
Preparation of Cosmetic Lotions
For each 50 g, the cosmetic lotions used consisted of
stearic acid (1.5 g); LD or palm oil as well as their blends
(5 g); cetyl alcohol (0.25 g); cera flava (0.25 g); liquid
paraffin (0.05 g) propyl paraben (0.1 g), butylated
hydroxytoluene (0.1 g); lanolin (0.1 g); dimetikon (0.1 g);
distilled water (39.4 g); Na2EDTA (0.5 g); Glycerin
(1.25 g); perfume (0.25 g) and triethanolamine (0.9).
Lotion formulation was prepared by weighing each
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J Am Oil Chem Soc (2012) 89:1537–1543
ingredient on an analytical balance. Cetyl alcohol, stearic
acid, propyl paraben, lanolin, dimetikon and oils used were
heated at about 70 °C (oil phase). Triethanolamine together
with water was heated at about 70 °C (water phase). The
oil phase was poured into the water phase and stirred with a
magnetic stirrer until it reached ambient temperature
(30 min). Perfume was added to the lotion obtained and
was further subjected to liquid–liquid extraction in order to
extract the fat/oil from the lotion formulation.
Calibration and Validation Samples
For calibration samples, nine laboratory prepared lotion
samples with certain LD concentrations were prepared. The
weight percentage ratios of lard and palm oil used were as
follows:
Formula Fat/oil F.1 F.2 F.3 F.4 F.5 F.6 F.7 F.8 F.9
Lard 0 0.6 1.2 1.8 2.4 3 3.6 4.4 5
Palm oil 5 4.4 3.8 3.2 2.6 2 1.4 0.6 0
F = formula
A series of nine lotion samples prepared independently in
our laboratory, which were different from calibration sam-
ples, were constructed as prediction or validation samples.
Lard, palm oil as well as their blends in lotion formulations
were extracted with chloroform, evaporated, and analyzed
using an FTIR spectrophotometer. The spectral regions
where the variations among them were observed were cho-
sen for developing PLS and PCA models.
Fat Extraction
To representative samples of lotion (10 g) were added 5 ml
of concentrated HCl and 20 ml of water and then shaken
vigorously. The filtrate was transferred to a separatory
funnel and extracted using 2 9 15 ml of chloroform. The
chloroform extracts were combined and drained into a
250-ml round-bottom flask and evaporated using a vacuum
rotary evaporator at 40 °C, until chloroform was com-
pletely removed. The lipid extracts obtained were further
determined using an FTIR spectrometer.
Instrumental Analysis
FTIR spectra were scanned using a FTIR spectrometer
ABB MB3000 FTIR spectrometer (Clairet Scientific,
Northampton, UK) equipped with a DTGS detector with a
resolution of 4 cm-1, number of scans 32 co-adding in the
400–4,000 cm-1 region. Spectra were acquired using
Horizon MB FTIR software version 3.0.13.1 (ABB, Can-
ada). The samples were placed in contact with a horizontal
J Am Oil Chem Soc (2012) 89:1537–1543
attenuated total reflectance (HATR) element (ZnSe crystal)
at a controlled ambient temperature (20 °C). All spectra
were ratioed against a background of an air spectrum. After
every scan, a new reference air background spectrum was
taken. These spectra were recorded as absorbance values at
each data point in triplicate.
Chemometrics
PLS and PCA were accomplished using Horizon MB FTIR
software version 3.0.13.1 (ABB, Canada). The leave-one-
out cross-validation procedure was used to verify the cal-
ibration model. The values of the root mean square error of
calibration (RMSEC) and coefficient of determination (R2)
were used as the validity criteria for the calibration model.
The predictive ability of PLS calibration model was further
used to calculate the validation or prediction samples.
Results and Discussion
Spectra Analysis
Figure 1 shows FTIR spectra of LD and palm oil in the
mid-infrared (MIR) region (4,000–650 cm-1). The selec-
tion of a palm oil as oil mixture in this study (lotion for-
mulation) was based on its popularity as one of new
Fig. 1 FTIR spectra of lard and
palm oil extracted from lotion
formulation. The assigned peak
with the circle constitutes
absorption bands which are
significant in differentiation
1539
components used in cosmetic lotions, especially in Indo-
nesia. Both spectra look very similar and typical charac-
teristic of absorption bands appear for common edible fats
and oils. Each peak in the FTIR spectra corresponds to a
functional group responsible for IR absorption [17]. The
peak assignment together with functional groups respon-
sible for peak absorption in Fig. 1 is shown in Table 1.
Both spectra appear very similar, however, they
revealed slight differences in terms of band intensities and
the exact frequencies at which the maximum absorbance
were generated for each fat and oil, due to the different
nature and composition of the evaluated fats and oils [12],
especially at wavenumber regions of 3,006 (a), 1,117
(k) and 1,098 cm-1 (l). The frequency region of
3,006 cm-1 was attributed to cis-olefinic C–H, while fre-
quencies of 1,117 and 1,098 cm-1 come from the stretch-
ing vibrations of the ester linkage in triacylglycerols.
Determination of the fatty acid composition of the studied
samples (palm oil and lard) revealed that both oils have
quite a high amount of oleic acid [13]. However, compared
to palm oil, lard contained twice as many linolenic acyl
groups as palm oil. This was reflected in the lard spectrum,
in which a sharper band was observed at frequency
3,006 cm-1 compared to the palm oil spectrum. Fats with a
high proportion of linolenic and linoleic acyl groups show
a higher frequency for this band compared to a high pro-
portion of oleic acyl groups [19].
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Table 1 Functional groups responsible for peak absorption in FTIR spectra of lard and palm oil [18]
Assignment Frequency (cm-1)
(a) 3,005
(b) 2,954
(c) and (d) 2,924 and 2,852
(e) and (f) 1,743
(g) 1,465
(h) 1,417
(i) 1,377
(j) 1,228
(k) and (l) 1,117 and 1,098
(m) 962
(n) 721
Lard and palm oil spectra show overlapping of two
peaks having maximum absorption at frequency regions of
1,117 cm-1 (k) and 1,098 cm-1 (l). These peak heights
were shown to be inversely related with the proportion of
saturated acyl groups and oleic acyl groups, respectively
[19]. Lard had an approximately equal proportion of sat-
urated acyl groups and oleic acyl group which was
reflected in the lard spectra, in which peaks of 1,117
(k) and 1,100 cm-1 (l) appeared, as having the same
height. Palm oil in contrast, having a higher concentration
of saturated acyl groups than oleic acyl group, demon-
strated unequal heights of peaks at 1,117 (k) and
1,100 cm-1 (l). In addition, lard and palm oil were also can
be differentiated at frequency of 3,006 cm-1 (a). In this
region, lard had a higher absorbance than palm oil. These
wavenumbers in which the peak intensities (absorbances)
of lard and palm oil were slightly different were further
selected to be optimized for analysis of lard in lotion
formulations.
Quantification of Lard in Cosmetic Lotions
Quantification of LD in a mixture with palm oil as the oil
base in cosmetic lotions was performed using multivariate
calibration of PLS at the combined frequency region of
1,200–1,000 cm-1. This region was chosen due to its
ability to offer the higher value of R2 and the lower value
of RMSEC, compared with other frequency regions
(namely 3,100–3,000 cm-1 and the combined frequency
regions of 3,100–3,000 cm-1 and 1,200–1,000 cm-1). The
main advantage of PLS is due to its ability to develop the
correlation between FTIR spectra and an analyte of inter-
est, even when no differences are visually observable in
FTIR spectral data [8]. The PLS calibration model was
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Vibrations of functional group
C–H vinylic (stretching)
Methyl (CH3–) group (stretching)
Asymmetric and symmetric methylene (–CH2–) group (stretching)
Carbonyl (C=O) of the triacylglycerol ester linkage with strong
intensity due to the great difference in electro-negativity between
carbon and hydrogen atoms (stretching)
Methylene (–CH2–) group (bending)
Methylene (–CH2–) group (bending)
Methyl (–CH3) group (bending)
C–O (stretching) from ester linkage
C–O (stretching) from ester linkage
Trans-olefins
Overlapping of the methylene (–CH2) rocking vibration and out of
plane vibration of cis-disubstituted olefins
developed based on the calibration standard that included
the different weighted amounts of LD blended with palm
oil in lotion formulation. FTIR spectra with different
concentration of LD and palm oil used for making cali-
bration model were shown in Fig. 2. Using three PCs, the
relationship between actual value (x-axis) against FTIR
predicted value (y-axis) of LD in the mixture with palm oil
in lotion formulation is shown in Fig. 3 with R2 of 0.99059.
The PLS calibration model was further subjected to
cross validation using the ‘‘leave-one-out’’ technique. The
root mean square error of cross validation (RMSECV)
obtained is relatively low (1.72% v/v). For the validation/
prediction procedure, another samples prepared in the
laboratory were used to minimize the prediction error and
to provide an estimate of the overall accuracy of predic-
tions. The value of root mean square error of prediction
obtained was 1.43%. Verification of spectral regions used
for carrying out the PLS calibration was done by calcu-
lating the predicted residual error sum of squares (PRESS)
value for different principal components (PCs) or factors.
PRESS was calculated from prediction errors in calibration
standard samples by cross validation and subsequently
plotted with the number of factors exploited in calibration
model. The optimal number of factors is three corre-
sponding to the point at which PRESS reaches a minimum
or begins to level off [20]. These results indicated that
FTIR spectroscopy combined with PLS is a reliable tech-
nique for detection and quantification of LD with detection
limit as little as 1% v/v.
Classification of Cosmetic Lotions Containing Lard
Classification of cream cosmetics with and without LD in
their formulation was performed using principal component
J Am Oil Chem Soc (2012) 89:1537–1543 1541

Fig. 2 The FTIR spectra of lard extracted from prepared lotion formulations with different concentrations used for preparing PLS calibration
model

Fig. 3 The scatter plot for

relationship between actual
value versus FTIR predicted
value of lard extracted from
prepared lotion formulations (%
v/v) using PLS calibration at
frequency regions of
1200–1000 cm-1

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Fig. 4 The PCA score plot for

classification of lotion products
containing lard or palm oil in
their formulations

Fig. 5 An example of FTIR

spectra of lotion formulations
commercially available in
supermarkets and pharmacy

analysis (PCA). Principal component analysis (PCA) is one reduced dimensions defined by the principal components
of the unsupervised pattern recognition techniques used in (PC). This technique is useful when there is a correlation
multivariate analysis. PCA projects the original data in present among data [21]. In this study, PCA was

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accomplished using FTIR spectra absorbances of lotions
containing lard and palm oil at the frequency region of
1,200–1,000 cm-1.
Figure 4 demonstrates the score plot of PCA of lard and
palm lotions representing the projection of samples defined
by the first component (PC 1), the second component (PC
2) and the third component. Using this projection both
lotions are well separated meaning that PCA can accom-
plish the classification between lotions containing lard and
palm oil. Furthermore, unknown lotion sample suspected of
containing one of both lard and palm oil can be assessed.
The decision was made to whether the unknown samples
fall close to lard or palm oil lotions. The closer the dis-
tance, the higher the possibilities of unknown samples
falling into one of the two groups.
The developed method was further used for the analysis
of lotion preparations commercially available in some
supermarkets and pharmacies in Yogyakarta. The FTIR
spectra of selected lotion samples are shown in Fig. 5. All
samples evaluated do not contain lard in their formulation.
From this conclusion, it can be seen that at frequency
region of 1,200–1,000 cm-1 there are no two peaks having
similar peak heights which are characteristics of a lard
spectrum. However, if the commercial samples were
spiked with lard, the corresponding peaks appear.
Conclusions
It can be concluded that FTIR spectroscopy using HATR
and chemometrics of partial least squares can be used to
analyze the presence of LD in cosmetic lotion formula-
tions. The results can be extended to various types of
topical cosmetic preparations using oils as a base in their
formulations. The tedious time and the uses of excess
reagents and solvents can be avoided.
Acknowledgments This research was fully supported by a project
grant from Gadjah Mada University through Fundamental Research
with the contract number: 085/Dir.Keu/KN/DIPA-UGM/2011, awar-
ded to Dr. Abdul Rohman.
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