POTENSIAL USE OF FOURIER TRANSFORM INFRARED (FTIR)

SPECTROSCOPY FOR SIMULTANEOUS DETERMINATION

OF LARD AND VIRGIN COCONUT OIL IN CREAM
COSMETICS PREPARATIONS

By

Rohman, Aa,b,c Che Man, Y.Bb and Sismindaria,c

a
Integrated Research and Testing Laboratory, Gadjah Mada University, Yogyakarta,
Indonesia.

b
Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM, Serdang,
Selangor, Malaysia.

c
Department of Pharmaceutical Chemistry, Fac. of Pharmacy, Gadjah Mada University,
Yogyakarta, Indonesia.
ABSTRACT

Cosmetics preparations typically consist of mixture of several components of natural

origin or their derivatives. Their analysis is made rather difficult by their usually high

complexity. Fourier transform infrared (FTIR) spectroscopy combined with

chemometrics partial least square (PLS) was simultaneously used to analyze lard and

virgin coconut oil (VCO) in cream cosmetics formulations. The spectral bands

associated with lard, VCO, and their blends (ranging from 0 - 50 %) were recorded,

interpreted, identified, and quantified by relating them to those spectroscopically

representative of pure lard and pure VCO as references. For quantitative analysis of lard

and VCO, PLS regression was used to develop a calibration model at frequency regions

of 3020 – 3000 cm-1 and 1103 – 1092 cm-1 (for lard) and 924 – 930 cm-1 (VCO). The

equations obtained for the relationship between actual content and FTIR predicted

values of lard and VCO were y = 0.9996x + 0.0076 with coefficient of determination

(R2) of 0.999 and y = 0.998x + 0.047 with R2 of 0.998, respectively. The PLS

calibration model was cross-validated using laboratory made cosmetics samples. FTIR

can be used as a potential analytical technique to detect and to quantify the presence of

lard and VCO in cosmetics preparations with total analysis time at about 3 min.

Keywords: FTIR spectroscopy, Lard, Virgin coconut oil (VCO), Partial least square
(PLS), Cosmetics.
INTRODUCTION

The use of cosmetics has increased many folds nowadays, and their productions

have increased tremendously (Hans et al., 2008). Human exposure to cosmetic products

and their ingredients occurs primarily via the topical route like cream and lotion

formulations (Kroes et al., 2007). Several considerations are necessary regarding

suitability and safety of cosmetics because they are used nearly constantly and in direct

contact with the skin (Kajimura et al., 2008).

Lard obtained from the rendering of fatty porcine tissue is commonly used in

cosmetic products as viscosity-increasing agents in several cosmetic products. Lard is a

GRAS (generally recognized as safe) substance (Food and Drug Administration, 2006).

However, lard exposures in the skin were found to be associated with certain health

risks such as hypercholesterolemia and coronary health disease (Imaki et al., 1989).

Lard is also associated with the risks of breast, pancreas, and colon cancers (Reddy,

1992). Lard appears to act both at initiation and during promotion of carcinogenesis. A

survey conducted by Food and Agricultural Organization (FAO) showed that there is a

significant correlation between lard exposure and the incidents of cancer in different

organs such as breast, prostate, rectum, colon, and lungs (Rashood, et al., 1996).

Cosmetics products containing lard are prohibited to be used for the followers of some

religions.

Virgin coconut oil (VCO) is derived from fresh coconuts (rather than dried, as in

copra) and is extracted directly from coconut meat under mild temperature. A study

shows that extra virgin coconut oil is as effective and safe as mineral oil when used as a

moisturizer, with absence of adverse reactions (Agero and Verallo-Rowell, 2004).

 Over the past 20 years, Fourier transform infrared (FTIR) spectroscopy has

evolved to become an important tool for quantitative analysis, and its application to fats

and oils has been active area of research (Frederik et al., 2008). In the analytical field,

FTIR spectroscopy provides a highly effective choice. It is a fast and non-destructive

technique, sensitive, and free of chemical preparation (Blanco et al., 2007).

FTIR spectroscopy with attenuated total reflectance (ATR) has been used to

analyze fats and oils. Generally, an ATR accessory is used with the FTIR spectrometer

for oil and fat study, due to its ability to handle liquid samples easily (Yang et al.,

2005). Previous research has shown the potential of FTIR spectroscopy as a method for

analysis of lard (Syahariza et al., 2005; Che Man et al., 2005). Guillen and Cabo (1997)

also used FTIR spectroscopy to characterize lard and other edible oils. Che Man and

Mirghani (2001) have developed a FTIR spectroscopic method for detecting lard in

mixtures of other animal fats. FTIR combined with principal component analysis has

been used for analysis of adulteration of VCO with palm kernel olein (Manaf et al.,

2007).

Almost all studies performed previously were based on food or food products

and no existing scientific records or data which are available to analyze lard and VCO

simultaneously in cosmetics preparations. Therefore, in this study, we attempt to

develop FTIR to be used as a rapid analytical technique for quantitative analysis of lard

and VCO in cosmetics preparations.

MATERIALS AND METHODS

VCO, stearic acid, lanolin, cetyl alcohol, and triethanolamin were obtained from

local market in Jogjakarta. Lard was prepared according to Che Man et al (2005).

1. Preparation of cream cosmetics formulation

The cosmetic mixture studied consisted of stearic acid (3.0 % w/w); lard or VCO

or their blends (50.0 w/w); lanolin (4.0 % w/w); distilled water (39.5 % w/w); cetyl

alcohol (1.0 % w/w); and triethanolamine (2.5 % w/w).

Cream formulation was prepared by weighing each ingredient on an analytical

balance. Triethanolamine together with water was heated at about 700C (water

phase). Cetyl alcohol, stearic acid, lanolin, lard/VICO/or their blends were heated at

about 70°C (oil phase). Oil phase was poured into water phase and stirred with

magnetic stirrer until reached ambient temperature (30 min).

2. Calibration standard and validation sets

To make a calibration curve, lard and virgin coconut oil were blended as follows: 0

% lard-50 % VCO; 10 % lard-40 % VCO; 20 % lard-30 % VCO; 30 % lard-20 %

VCO; 40 % lard-10 % VCO; and 50 % lard-0 % VCO, in chloroform.

An amount of laboratory prepared cosmetic samples containing all components in

cosmetics mixture studied with certain lard concentrations were also prepared for

validation sets.

3. Fat extraction

Representative samples of cream formulation (25 g) were added with 2 ml of

concentrated HCl and 18 ml of water and shaked vigorously. The filtrate was

transferred to a separatory funnel and extracted using 2 x 15 ml of chloroform. The

chloroform extracts were combined and drained into a 250 ml round-bottom flask
 and concentrated with a rotary evaporator at 40oC. The concentrated lipid extract

was quantitatively transferred to a vial and made up to a final volume of 25.0 ml

with chloroform. All chemicals and solvents were of analytical grade.

4. Instrumental Analysis

FTIR spectra of all samples were acquired on the software Nicolet Omnic (version

7.0, Thermo Nicolet). A Thermo Nicolet 6700 instrument (Thermo Nicolet Corp.,

Madison, WI), equipped with a multibounce plate ZnSe attenuated total reflectance

(ATR) crystal, detector of deuterated triglycine sulfate (DTGS), and a germanium

on potassium bromide substrate beamsplitter, was used to measure the spectra of oil

samples. Drops of oil samples were placed on a multibounce plate ZnSe ATR

crystal at controlled ambient temperature. Measurements were taken at 64 scans and

at a resolution of 4 cm-1 with strong apodization. After each scan, the plate was

carefully cleaned by wiping with acetone and hexane and dried with soft tissue

before filling in with the next sample. To avoid any spectral variation from day to

day to a minimum, an air background spectrum was collected before each sample

spectrum. The instruments were maintained at constant humidity to minimize water

vapor interference. All spectra were recorded from 4,000 to 650 cm-1 and done

triplicate.

5. Statistical Analysis

Chemometric analysis including partial least square was carried out using the

program TQ Analyst version 6.0 (Thermo Nicolet, Madison, WI) software package.

PLS was employed for developing a calibration model. The spectral regions where

the variations were observed were chosen for developing the PLS model. A
 Microsoft Excel 2003 spreadsheet was used to correlate the FTIR-predicted and

chemical data.

6. Validation

The “leave-one-out” cross-validation procedure was used to verify the calibration

model. The standard error of prediction (SEP) and R2 were used as the validity

criteria for the calibration. The validation step was further investigated using the

mean difference (MD) and standard deviation of difference (SDD) for repeatability

and accuracy.

RESULTS AND DISCUSSION

Figure 1 shows FTIR spectra of lard and virgin coconut oil (VCO) at the

frequency 4000 – 650 cm-1. Both of spectra look very similarly and showed a typical

characteristic of absorption bands for common oils (Safar et al., 1994). Peak (a) in the

region 3007 cm-1 is due to C-H stretching vinylic, and this peak only appeared in the

lard. The stretching vibrations of methylene (-CH2-) and methyl (-CH3) groups are

observable in frequencies 2922 (b) and 2852 cm-1 (c), respectively. Methylene and

methyl groups also observable in the regions 1465 cm-1 (e) and 1375 cm-1 (f) due to

their bending vibrations (Pavia et al., 2001).

The carbonyl (C=O) absorption of the triacylglycerol ester linkage is observed

at frequency 1740 cm-1 (d) with strong intensity due to the great difference of

electronegativity of carbon and hydrogen atoms. The bands at 1235 (g), 1160 (h), 1117

(i), 1098 (j) and at 721 cm-1 (l) are results from the overlapping of the methylene

rocking vibrations and the out of plane bending vibration of cis-disubstituted olefins. A
peak at 962 cm-1 (k) has been associated with bending vibration of cis disubstituted

olefinic groups. This peak is only observed in VCO (Guillen and Cabo, 1997).

The big differences of the two spectra are assigned with arrows for lard (a and j)

and circle (k) for VCO as shown in Figure 1. A peak at 3006 cm-1 (a) is present in lard,

but is absent in the spectrum of VCO. It is due to cis C=CH stretching (Che Man and

Setyowaty, 1999). This is not surprising because lard has a higher iodine value than that

of VCO (Table 1). The high iodine value denotes higher content of unsaturated fatty

acids, which in turn contributes to a high absorbance value in the region of cis C=CH

stretching. Compared with VCO, lard has more unsaturated fatty acid contents, oleic

and linoleic acids (Table 1), which support the presence of peak at 3006 cm-1. This

feature differentiates lard from VCO. Because of its higher value of iodine value than

VCO, lard exhibits a clear peak at frequency 3006 cm-1, while VCO has no clear band

in this region (Manaf et al., 2007).

Another peak used to distinguish lard and VCO is the region at frequency 1117

– 1097 cm-1, in which lard showed overlap of two peaks having maximum absorption at

frequency 1117 cm-1 (i) and 1098 cm-1 (j), while VCO shows only one absorption at

1117 cm-1 and at region 927 cm-1 for VCO at which lard was absent. The spectrum of

lard was in agreement with that reported by Syahariza et al (2005) and Jaswir et al

(2003), meanwhile the spectrum of VCO was consistent with that reported by Manaf et

al (2007). The overlay of combined spectra of lard, VCO and their blends ranging from

0 % to 50 % lard/VCO is illustrated in Figure 2.

Figure 1. FTIR spectra of pure lard and pure VCO. The assigned peak with arrow are absorption bands which are significant in
differentiation of lard and with circle in differentiation of VCO.
Table 1. Comparison of fatty acid composition (%) of lard and VCO

Fatty acid Lard* VCO**

Capric (C10:0) 0.1 3.4 – 15.0

Lauric (C12:0) 0.1 41.0 – 46.0
Myristic (C14:0) 1.5 13.0 – 23.0
Palmitic (C16:0) 26.0 4.2 – 12.0
Stearic (C18:0) 5.0 – 14.0 1 – 4.7
Oleic (C18:1) 41.0 – 51.0 3.4 – 12.0
Linoleic(C18:2) 2.0 – 15.0 0.9 – 3.7
Iodine value 46.0 – 70.0 4.9

* Source: O’Brien (1998)

**Source: Che Man et al (1997

Furthermore, frequency ranges make a difference between lard and VCO i.e.

3020 – 3000 cm-1 and 1103 – 1092 cm-1 (for lard) and 924 – 930 cm-1 are used in

making calibration and validation models for quantitative analysis purpose.

1 

2  Figure 2. FTIR spectra lard, VCO, and teir blends with concentration range from 0 - 50 % .
11 

 
PLS calibration an cross-validation

The power of PLS is based on its ability to use spectral information from

broad spectral frequencies and to correlate spectral changes in the concentration of a

component of interest while simultaneously accounting for other spectral

contributions that may perturb the spectrum. A PLS calibration model was developed

based on the calibration standard that included the different weighted amounts of lard

blended with VCO. The relationship between actual lard and VCO concentration

data against the PLS of FTIR predicted data is shown in Figure 3A and 3B. A good

linear regression of the actual values against FTIR predicted values of lard and VCO

is obtained, y = 0.999x + 0.007; with the coefficient of determination (R2) = 0.999

and standard error of prediction (SEP) = 0.364 (lard) and y = 0.998x + 0.047 with R2

of 0.998, and SEP = 0.827 respectively.

For the validation procedure, 10 laboratory made samples of cream cosmetics

preparation containing lard and VCO in range of 0.0 - 50.0 % was used to minimize

the prediction error and to provide an estimate of the overall accuracy of predictions.

Using the relationship between actual lard/VCO content (%) in samples and FTIR

predicted values of lard/VCO content (%), a linier regression was obtained with

equation y = 0.797x + 1.224 and R2 = 0.991 and SEP = 1.21 (lard); and y = 0.853x –

0.056 with R2 = 0.986 and SEP = 1.84 (VCO) as shown in Figure 4.

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 A 

B 

Figure 3. A plot of actual concentrations versus FTIR predicted concentration lard

using PLS at frequency regions 3020 – 3000 cm-1 and 1103 – 1092 cm-1
(A); and at frequency 924 – 930 cm-1 for VCO (B).

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 A
FTIR predicted value of lard (%) 

Actual value of Lard  (%)

B
FTIR predicted value of VCO

Actual value of VCO (% w/w)

Figure 4. A plot of actual concentrations versus FTIR predicted concentration lard,

for validation, using PLS at frequency regions 3020 – 3000 cm-1 and 1103
– 1092 cm-1 (A); and at frequency 924 – 930 cm-1 for VCO (B).

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 The statistical results calculated from the cross validation as mean difference

(MD) and standard deviation of difference (SDD) for repeatability and accuracy for

the determination of lard content are shown in Table 2.

Table 2 Repeatability and accuracy of FTIR predicted values of lard and VCO
contents obtained by cross validation*

Lard VCO
Statistics Actual PLS Actual PLS
data FTIR data FTIR
MDa 2.325 2.130
SDDa 1.399 1.634
MDr - 2.338 - 2.074
SDDr - 1.267 - 1.603

*MD, mean difference; SDDr, standard deviation of difference; a, accuracy; r,

repeatability.

CONCLUSIONS

It can be concluded that FTIR spectroscopy combined with ATR and chemometrics

partial least square (PLS) can be used to analyze the lard and VCO contents

simultaneously in cream cosmetic formulations. The results can be extended to

various types of topical cosmetic preparations using oils as a base in their

formulations. The tedious time and chemical consuming reagents and solvents can be

avoided and it is environmentally friendly. The developed method is rapid. Once the

database is established and the model developed, each individual analysis can be

achieved in about 3 minutes.

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ACKNOWLEDGMENT

The first author acknowledge to Directorate for higher education, National Education

Ministry, Republic of Indonesia for financial sponsorship to pursue Ph.D program in

Halal Product Research Institute, Universiti Putra Malaysia (UPM), Malaysia.

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