Journal Pre-proof

Application of ATR-FTIR spectroscopy along with regression modelling for the

detection of adulteration of virgin coconut oil with paraffin oil

Amit, Rahul Jamwal, Shivani Kumari, Amit S. Dhaulaniya, Biji Balan, Dileep Kumar
Singh

PII: S0023-6438(19)31096-5
DOI: https://doi.org/10.1016/j.lwt.2019.108754
Reference: YFSTL 108754

To appear in: LWT - Food Science and Technology

Received Date: 6 June 2019

Revised Date: 17 October 2019
Accepted Date: 18 October 2019

Please cite this article as: Amit, , Jamwal, R., Kumari, S., Dhaulaniya, A.S., Balan, B., Singh, D.K.,
Application of ATR-FTIR spectroscopy along with regression modelling for the detection of adulteration
of virgin coconut oil with paraffin oil, LWT - Food Science and Technology (2019), doi: https://
doi.org/10.1016/j.lwt.2019.108754.

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 1 Title Page

2 Application of ATR-FTIR spectroscopy along with regression modelling for the

3 detection of adulteration of virgin coconut oil with paraffin oil

4 Running title - PO adulteration detection in VCO by ATR-FTIR

5 Amita#, Rahul Jamwala#, Shivani Kumaria, Amit S. Dhaulaniyaa, Biji Balana, Dileep Kumar

6 Singha*

7 *Corresponding author - Dileep Kumar Singh

8 Corresponding author Email - dileepksingh@gmail.com

9 Telephone Number - 011-27667191

10 Mobile Number - 09810258052

a
11 Department of Zoology, University of Delhi, New Delhi, Delhi-110007, India

#
12 Amit and Rahul Jamwal have equal contribution and both should be considered as First

13 Authors.

14

15

16

17

18

19

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20 Abstract

21 Fourier transform infrared (FTIR) spectroscopy equipped with Attenuated total reflection

22 (ATR) was employed along with chemometrics to develop the methodology for classification

23 and quantification of virgin coconut oil (VCO) from its adulterant paraffin oil (PO) in

24 proportions of 1 to 18% v/v PO. Out of a total of 128 samples, based on spectral data of 96

25 samples, Principal Component Analysis (PCA) was used on the selected informative region

26 (3000-2800 cm-1 and 1800-700 cm-1) and Linear Discriminant Analysis (LDA) was applied

27 on selected 13 wavenumbers obtained from the loading plot. LDA accurately classified 100%

28 of the initial groups as well as when cross-validated. For quantification, Principal Component

29 Regression (PCR) and Partial Least Square Regression (PLS-R) calibration models were

30 developed and compared for normal, first and second derivatives of the combined optimized

31 region and separate optimized regions 3000-2800 cm-1 and 1800-700 cm-1 to get a robust

32 calibration model. PLS-R model for 2nd derivative of optimized spectra (1800-700 cm-1)

33 showed best results for prediction with high precision and accuracy (RPD: 38.17, RE%:

34 0.05), the high R2 value of 0.999 and low root mean square error (RMSE) of 0.16% v/v. The

35 lowest limit of detection of PO in VCO was predicted as 1% v/v.

36 Keywords: FTIR Spectroscopy, adulteration, virgin coconut oil (VCO), paraffin oil (PO),

37 chemometrics

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44

2
45 Abbreviations

46 ATR: Attenuated Total Reflectance

47 FTIR: Fourier Transform Infrared

48 GC: Gas Chromatography

49 LDA: Linear Discriminant Analysis

50 PCA: Principal Component Analysis

51 PCR: Principal Component Regression

52 PLS-R: Partial Least Square Regression

53 PO: Paraffin Oil

54 R2: coefficient of determination

55 RPD: Residual Predictive Deviation

56 RE%: Relative Prediction Error

57 RMSEC: Root Mean Square Error of Calibration

58 RMSECV: Root Mean Square Error of Cross Validation

59 RMSEP: Root Mean Square Error of Prediction

60 SNV: Standard Normal Variate

61 VCO: Virgin Coconut Oil

62

63

64

65

66

67

68

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3
70 1. Introduction

71 In recent times, VCO has rapidly become one of the most worthwhile edible oil after olive oil

72 (Rohman, 2017). This is not only attributed to its better fragrance and flavour but also its

73 possible health advantages. VCO has a high proportion of medium-chain fatty acids like

74 capric, caprylic and caproic acids which are found to have antiviral and antimicrobial

75 attributes (Nasir, Abllah, Jalaludin, Shahdan, & Manan, 2018). VCO is the oil extracted from

76 mature and fresh kernel/meat parts of coconut fruit by natural or mechanical means, without

77 applying heat or chemical treatment (Agarwal, & Bosco, 2017). VCO is found rich in vitamin

78 E, pro-vitamin A and phenols, that are lost in RDB (refining, deodorizing and bleaching)

79 coconut oil because of UV radiation from sunlight at the time of copra drying process

80 (Dumancas et al., 2016). It was very late to recognize VCO as functional oil dissimilar to

81 RDB coconut oil and other edible oils which are pertinent for cooking motives in the

82 worldwide market particularly in South East Asia including the Philippine, Malaysia,

83 Thailand, and Indonesia (Ghani et al., 2018).

84 The adulteration of food matrices is the paramount concern for the buyers and food

85 processing corporations. From the judicial aspect, the quality standards were set by the

86 necessity of quality labels having the ingredients and chemical configuration of each food

87 item (Hong et al., 2017). The blending process involves the substitution of high-cost

88 ingredients with a cheaper one. VCO is routinely mixed with other edible or non-edible oils

89 of low remunerative cost. Even though the expansion of adulteration is on the lucrative

90 ground, the act can influence the quality of VCO (Marina, Che Man, Nazimah, & Amin,

91 2009; Xu et al., 2015).

92 Palm oil, palm kernel oil and paraffin oil (liquid paraffin) are the major adulterants of VCO

93 remarkably in South East Asian countries (Sheeba, Rajesh, Vallabhan, Nampoori, &

94 Radhakrishnan, 2005; Raj, Swapna, & Sankararaman, 2018). Among all, the blending of PO

4
 95 is immensely perilous from a human health perspective since it can eventually cause shed

96 load fateful health issues like disorders of the intestine, mesenteric lymph glands and liver

97 (IARC, 2012) and even cancer (Libish et al., 2011). It has been reported that the blending of

98 VCO with up to 18-20 percent of the PO does not notably alter its flavour or color (Libish et

99 al., 2011).

100 There are very few reports about detection of PO adulteration in VCO oil, among which

101 development of fiber optic evanescent wave refractometer with limit of 2% PO adulteration

102 (Sheeba et al., 2005) and fiber optic sensing system with detection limit of 3% PO

103 adulteration in VCO (Libish et al., 2011) are prominent. There are no noticeable reports of

104 FTIR analysis of PO adulteration in VCO except one study which showed diminishing of

105 VCO spectra with the addition of PO but it lacked precision, as well as there, was no

106 quantification and detection of PO (Raj et al., 2018). FTIR is a highly reliable and sensitive

107 analytical technique for qualitative as well as quantitative analysis of fats and edible oil

108 adulteration (Antony, Sharma, Mehta, Ratnam, & Aparnathi, 2017). It is a swift, non-

109 calamitous technique with minimum sample preparation (Che Man et al., 2011; Arslan et al.,

110 2019; Andrade et al., 2019). So, this study attempts to first time explore the differentiation,

111 classification, and quantification of PO adulteration in VCO by utilizing ATR-FTIR spectra

112 combined with chemometrics.

113 Multivariate branch of chemometrics has been enormously utilized in the recent past for the

114 analysis of FTIR spectra (Varmuza & Filzmoser, 2016; Kar, Tudu, Jana, & Bandyopadhyay,

115 2019). In the present study, PCA has been applied for extracting principal components and

116 for the selection of most informative wavenumbers crucial for further analysis. LDA has been

117 employed for classifying and differentiating binary mixture of PO and VCO. For developing

118 a robust quantification methodology, PCR and PLS-R models were exploited and compared

119 for normal, first and second derivatives of the combined optimized spectral region (3000-

5
120 2800 cm-1 and 1800-700 cm-1) as well as for the two separate optimized informative spectral

121 regions 3000-2800 cm-1 and 1800-700 cm-1 respectively. These regions were optimized by

122 selecting specific spectral regions having the most important wavenumbers which are

123 assigned to the main absorbance bands explaining major characteristics of the oil under

124 investigation. Through this study, we have developed a robust methodology for the

125 quantitative analysis of VCO blending with different concentrations of PO for prediction of

126 the lowest detectable concentration.

127 2. Material and Methods:

128 2.1 Sample collection

129 A total of 4 brands of VCO were purchased from the local supermarket at Hudson lane, New

130 Delhi, India and PO (Sigma-Aldrich® for IR spectroscopy, Germany) was procured from an

131 authorized local vendor in New Delhi, India. To check the purity of these 4 VCO samples,

132 fatty acid composition was analyzed through Gas Chromatography (GC).

133 2.2 Fatty acid composition analysis by Gas Chromatography

134 The fatty acid composition of all 4 pure VCO brands was determined as fatty acid methyl

135 esters (FAME) using Gas Chromatography “Shimadzu GC-2010 (Shimadzu Corporation,

136 Tokyo, Japan)”, equipped with Flame ionization detector (FID). FAMEs were extracted

137 according to the method described by Masoodi, & Hakimi, 2017 with slight modifications.

138 Briefly, 45 mg of VCO oil from each brand was boiled with 1 ml of 0.5N methanolic sodium

139 hydroxide at 100 0C for 15 minutes in glass vials. The vials were cooled on ice before the

140 addition of 2 ml of boron trifluoride 14% methanol and the vials were again boiled for 15

141 minutes. The vials were cooled on ice, further 1 ml of isooctane and 2 ml of saturated

142 sodium chloride were added. The vials were shaken vigorously and left to stand for the layer

143 separation. The upper layer containing methyl esters were transferred to GC vials and 1µl

144 was injected into an RTX5 capillary column having an internal diameter of 0.25 mm, length

6
145 30 m and film thickness of 0.25µm (Restek Corporation, Bellefonte, PA, USA). The analysis

146 was performed with helium as carrier gas (total flow rate of 16.3 ml/min) with the column

147 oven temperature was programmed as follows: initial temperature 140 0C held for 5 min,

148 after which the temperature was increased to 280 0C at a rate of 4C/min. The temperature was

149 maintained at 280 0C for the subsequent 40 minutes. The detector and injector temperature

150 was maintained at 295 0C and 270 0C respectively. The individual fatty acids were

151 determined by comparing their retention times with standard FAME Mix (certified reference

152 material) (Sigma Chemicals, St. Louis, Missouri, USA).

153 2.3 Adulteration experiment and sample preparation

154 For adulteration experiment, three brands of VCO were blended with different concentration

155 of PO, forming the ‘calibration set’, for classification studies by PCA and LDA as well as to

156 build calibration models. To evaluate the prediction capability of the calibration models, the

157 leftover one VCO brand was blended with the PO to prepare the ‘external set’. Libish et al.

158 (2017) have found that the blending of VCO with up to 18-20 percent of the PO does not

159 notably alter its flavour or color. So keeping this fact in mind, the upper limit of PO

160 adulterants has been taken up to 18% v/v. As depicted in Table 1, a total of 7 admixtures

161 (VCO/PO) were prepared as 1%, 3% and then in arithmetic progression with an increment of

162 3%, up to 18% v/v, generating a total of 128 samples. Out of the total, the first 3 brands of

163 VCO were used to prepare 96 samples (12 pure VCO samples and 84 VCO/PO blend

164 samples) referred to as “calibration set”. And the remaining forth VCO brand was blended

165 with PO in the same proportions to make an “external set” of 32 samples (4 pure VCO

166 samples and 28 VCO/PO blend samples) for regression model prediction. All samples were

167 vortexed to ensure complete homogenization and stored at -200C until analysis.

168 2.4 FTIR spectra acquisition

7
169 “A Thermo-Scientific®–Nicolet iS50 FTIR spectrophotometer (Waltham, Massachusetts,

170 USA)” equipped with a diamond crystal cell Attenuated total reflection (ATR) accessory and

171 a deuterated triglycerine sulphate (DTGS) detector was used to gather FTIR spectra in the

172 mid-infrared region (4000 to 400 cm−1). All the spectra were recorded at a resolution of 4

173 cm−1 with 32 scans and the data spacing of 0.482 cm−1. Approximately 2 µl of the sample

174 was loaded with the aid of a Pasteur pipette on the beam splitter i.e. KBr (Potassium

175 bromide), developing a thin film. The obtained spectra were deducted from the background

176 spectrum and a fresh reference spectrum was recorded after every three scans. All the

177 recorded spectra were analyzed with “OMNIC 9.2.41 software (Thermo scientific)”

178 (https://www.thermofisher.com/order/catalog/product/833-036200).

179 2.5 Spectral Data Pre-treatment

180 Before multivariate data analysis, to eliminate the baseline drift and variability associated

181 with the intensity, “Unscrambler X 10.5.1” (https://www.camo.com/unscrambler/) has been

182 used for smoothening of spectra by applying Savitzky-Golay method having a second stage

183 polynomial and 7 points window size (Savitzky & Golay, 1964). Smoothening will be high if

184 the frame size is large and polynomial order is low. Furthermore, few pre-treatments that

185 include mean centring coupled with standard normal variate (SNV) was also applied to the

186 data (Zhang, Saha, & Vishwanathan, 2012; Wójcicki, Khmelinskii, Sikorski, & Sikorska,

187 2015; Jovic, Smolić, Primožič, & Hrenar, 2016).

188 Because there are differences in the composition and viscosity of pure VCO and PO

189 adulterated oil samples (Zhang, Li, Jia, Zhang, & Zhang, 2015; Nasir et al., 2018), it may

190 produce baseline offset and slope in spectra that may affect the development of robust

191 calibration model. The most frequent method to correct the baseline offset and slope is the

192 application of different derivatives of the spectra.

8
193 The first derivative is the measurement of the slope of the spectral curve at each point which

194 is not affected by baseline offsets in the spectrum. Thus, the first derivative is a very effective

195 way to remove baseline offsets. The second derivative measures the change in the slope of

196 the curve which ignores the offset and remains unaffected by linear slant that may be present

197 in the spectra. Hence it is also a very efficient method for removing both the baseline offset

198 and slope from spectral data (Chen, Zhang, Ma, Tuchiya, & Miao, 2015). The first and

199 second derivatives were obtained using the Savitzky-Golay method with a segment of 7 point

200 window and a second-order polynomial. These treatments were carried out using the

201 “Unscrambler X version 10.5.1 software”.

202 2.6 Statistical analysis using Chemometrics

203 For paraffin oil adulteration, calibration set of samples having seven different concentrations

204 of adulterant (1 to 18% v/v PO), were used for PCA to investigate contrast among samples

205 using “Unscrambler X 10.5.1”. PCA alters numerous likely concurrent variables into a few

206 uncoordinated factors that are termed as principal components (PCs) and therefore decreases

207 the size of the dataset (Vasconcelos, Coelho, Barros, & de Almeida, 2015). PCA allowed the

208 recognition of the most informative variables (wavenumbers) corresponding with the VCO

209 and its different adulterants with PO. The rejection of futile variables is necessary to get

210 sounder and less complicated results.

211 In terms of qualitative investigation, PCs imparting to the variation in the dataset were

212 exposed to discriminant analysis using “IBM SPSS Statistics 20” (https://www.ibm.com/in-

213 en/products/spss-statistics) to determine the possibility of a sample of a formerly determined

214 cluster. LDA is a statistical approach applied to get a linear amalgamation of forms with the

215 ability to distinguish observation classes. The consequential pattern can be applied as a linear

216 classifier (Vasconcelos et al., 2015).

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217 Furthermore, a calibration set of data was used to develop the calibration models using

218 “Unscrambler X 10.5.1”. An external set was used to predict the lowest limit of detection

219 (LOD) of PO in VCO. PCR and PLS-R are widely known statistical techniques frequently

220 applied as quantitative prediction methods based on spectral data (Lim, Abdul Mutalib,

221 Khaza’ai, & Chang, 2018). These models are specially devised for the cases having more

222 probably correlated predicting variables than the number of samples. These cases are very

223 often in the statistical analysis of spectroscopic data of different food matrices. Both models

224 are exploited and compared for three different optimized informative spectral regions which

225 include one combined optimized region (3000-2800 cm-1 and 1800-700 cm-1) and two

226 separate optimized regions 3000-2800 cm-1 and 1800-700 cm-1 respectively. The optimization

227 process was performed by selecting specific spectral regions having the most important

228 wavenumbers which are assigned to the main absorbance bands explaining major

229 characteristics of the oil under investigation. Through the optimization process, specific

230 spectral regions were examined for their ability to offer a high correlation between actual and

231 predicted values using PCR and PLS-R regression models (Rohman, Setyaningrum, &

232 Riyanto, 2014; Pereira, Leite, Andrade, Bell, & Anjos, 2019). But some regions of the

233 spectrum having low signal to noise ratio have to be neglected as these regions have very

234 little or no information (Vasconcelos et al., 2015).

235 The performance of the models was explained and evaluated by using the R2, RMSE

236 (signifies the standard deviation of the residuals) and the relative prediction errors (RE %) of

237 both calibration and prediction (external) data sets. The model with a higher R2 value (closer

238 to 1) and lower RMSE and RE% values is relatively more significant. Both RMSE and RE%

239 are indicators that depict how well the model will predict. Furthermore, accuracy and the

240 precision of the predictive model was assessed by measuring the total standard error of

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241 prediction (SEP), bias (BIAS) and the residual predictive deviation (RPD) of the prediction

242 (external) dataset (Andrade, Vicentini, Bell, & Anjos, 2019).

243 3. Results and Discussion

244 3.1 FTIR Spectra Analysis and oil composition

245 FTIR spectra of VCO in the ‘informative’ region displays numerous well resolved

246 characteristic peaks (bond vibrations) at specific wavenumber (cm-1) that can be assigned to

247 functional groups of its fatty acids as shown in Table 2 (Che Man et al., 2011). As illustrated

248 by the FTIR spectra of VCO and different adulterants of PO (Fig. 1), 3000-2800 cm-1 and

249 1800-700 cm-1 are the two major ‘informative’ spectral regions. Predominantly, 2954 cm-1,

250 2924 cm-1 and 2852 cm-1 from first informative region and 1743 cm-1, 1465 cm-1, 1417 cm-1,

251 1377 cm-1, 1228 cm-1, 1155 cm-1, 1111 cm-1, 962 cm-1, 872 cm-1 and 721 cm-1 from second

252 informative region manifest coherent differences in spectra. These differences are attributed

253 to the different component compositions of two oils. VCO is largely composed of

254 triacylglycerols (Rohman et al., 2010) while the PO is entirely composed of n-alkanes (C16-

255 C20) (Zhang et al., 2015). From GC analysis, fatty acid compositions of all 4 VCO brands

256 were found to be similar (Table 3) (Rohman and Che Man, 2009; Dorni, Sharma, Saikia, &

257 Longvah, 2018). It was found that all 4 VCO brands were pure and there was no prior

258 adulteration with either PO or any other vegetable oil. In paraffin oil (n-alkane) spectra, two

259 strong intensity bands at around 3000-2850 cm-1 due to –C–H stretching and at 1470-1450

260 cm-1 due to –C–H bending are present while one medium intensity band at around 1390-1370

261 cm-1 owing to–C–H(–CH3) bending and one weak band at around 721 cm-1 which

262 corresponds to the out-of-plane vibration of cis –HC=CH- group from unsaturated fatty acyl

263 chains and the overlapping CH2 rocking vibrations are also observed as depicted in Table 2

264 (Naranjo & Baliga, 2012; Danezis, Tsagkaris, Camin, Brusic, & Georgiou, 2016; Leite,

265 Pereira, Andrade et al., 2019).

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266 3.2 Principal Component Analysis

267 Every spectrum was selected for two major ‘informative’ spectral regions from 3000 to 2800

268 cm-1 and from 1800 to 700 cm-1 as mentioned earlier. The rest of the regions which are left

269 out are found to have a little signal to noise ratio which was susceptible to instigate

270 misclassification. PCA was then applied to the combined informative spectral region (3000-

271 2800 cm-1 and 1800 to 700 cm-1) to determine the resemblance and disparity between VCO

272 and its PO adulterant in various ratios. Generally, the first few PCs describe the majority of

273 the observed variance. Results of PCA showed distinct segregation and clustering between

274 pure VCO and different PO adulterants. First, two PCs with an explained variance of 94%

275 and 4% covered the total variance of 98% with the clear splitting of data and no overlapping

276 of samples as shown in the score plot (Fig. 2). From the plot, it is evident that samples having

277 the same percentages of adulteration were grouped in the same cluster. As the percentage

278 adulteration of PO increases, samples move away from VCO. The samples with a higher

279 concentration of VCO are situated on the positive side of the PC1 (VCO pure and PO 1 to

280 6%), while those higher in PO are located on the negative side (PO 9 to 18%). These results

281 indicate that the negative variation of PC1 is related to the increase in the concentration of PO

282 present in the blend. This clearly shows that PCA grants qualitative discrimination between

283 the different concentrations of PO adulterants and pure VCO under analysis.

284 The loading plot (Fig. 3) depicted the specific wavenumbers assigned to different functional

285 groups responsible for the separation of different concentrations of PO adulterants with VCO.

286 The absolute value of specific wavenumber loading demonstrates the usefulness of the

287 contribution of each wavenumber. Further apart a wavenumber is from the origin of variable

288 point, greater the contribution of that wavenumber to the PCA model (Marina, Che Man, &

289 Amin, 2010). The loading plot is used to identify the variables that have the largest effect on

290 a particular component (Che Man, Rohman, & Mansor, 2011). In our study (Fig. 3), PC1

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291 (first component) is showing positive loading for peaks including 721 cm-1 assigned for C-H

292 in cis C=C bonds, 872 cm-1 for =CH2 bending, 962 cm-1 for trans –HC=CH– out of plane

293 bending, 1111 cm-1 for –C–O stretching, 1155 cm-1 for strong –C–O stretching and –CH2–

294 bending, 1228 cm-1 for –C–O stretching, 1417 cm-1 for =C–H (cis-) bending (rocking) and

295 1743 cm-1 for very strong –C=O stretching vibration (Table 2). These loading values are

296 more relevant for the discrimination of VCO from its PO adulterants as the above mentioned

297 functional groups with their peculiar bending and stretching vibrations assigned to these

298 wavenumbers are characteristic of the triacylglycerol structure of the VCO. These peaks are

299 not prominent in case of PO. So, the most important frequencies contributing to the

300 separation of VCO from its PO adulterants based on PC1 are 721 cm-1, 872 cm-1, 962 cm-1,

301 1111 cm-1, 1155 cm-1, 1228 cm-1, 1417 cm-1 and 1743 cm-1. While, with respect to PC2

302 (second component), wavenumbers 1465 cm-1, 2954 cm-1, 1417 cm-1, 2924 cm-1 and 1377cm-
1
303 have negative loadings (Fig. 3) and these wavenumbers are assigned to similar functional

304 groups (–C–H (–CH2,CH3) with strong intensities as the change in dipole moment with

305 respect to distance for the C-H stretching is greater than that for others. Whereas 1228 cm-1,

306 1155 cm-1 and 1111 cm-1 have small positive loading due to the presence of –C-O stretching

307 vibrations. Hence, PC1 accounts for the most variation in FTIR spectra absorbances while

308 PC2 accounts for the next largest variation. These prominent wavenumbers can be exploited

309 for further classification and differentiation of VCO from its PO adulterants.

310 3.3 Linear Discriminant Analysis

311 For further classification and discriminative analysis of PO adulterants, LDA was applied on

312 a subset of samples (VCO 100%, PO 1%, and PO 3%) using absorbances at the 13

313 wavenumbers mentioned in the loading plot section. This subset of the calibration set (VCO

314 100%, PO 1%, and PO 3%) was created by 12 replications of every class. For determining if

315 the samples are competent for further analysis, different parameters like Eigenvalue and

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316 Wilks’ Lambda are calculated. Both the discriminant functions with Eigenvalue >1 and p

317 values <0.001 are significant and demonstrate 96.4% and 6.6% variance of the adulterant oil

318 respectively. All groups scatter plot, as deduced by LDA using discriminant function 1 and 2

319 for spectral data of VCO 100%, PO 1%, and PO 3%, also explains the total variance (Fig. 4).

320 The group centroid represented in the plot depicts that function 1 and function 2 are

321 differentiating between VCO and PO adulterants. The confusion matrix resulting from the

322 LDA classified 100% of the initial groups as well as classified correctly when cross-validated

323 as shown in Table 4. In cross-validation, each case is classified by the functions derived from

324 all cases except that case. From the outcome, it has been observed that the aliquots of PO 1%

325 class belong to the lowest concentration of the PO used. Thus, 1% v/v was the lowest

326 detected PO concentration in VCO. This detected concentration is not the constraint of the

327 experimental procedure pursued instead it is because of this lowest concentration of PO

328 adulteration used in our work.

329 3.4 Prediction of PO adulteration by regression models based on FTIR spectra

330 For quantification, selected informative spectral regions from the FTIR spectra were used to

331 build PCR and PLS-R models. In most of the linear regression and prediction issues, the

332 independent variables may be highly collinear known as multicollinearity. PCR solves the co-

333 linearity issue with few numbers of factors. Though, PLS-R may even solve the co-linearity

334 with fewer factors than PCR. Simulations have shown that PLS-R gives its minimal root

335 mean square error (RMSE) with a smaller number of factors than PCR (Yeniay & Goktas,

336 2002). These regression methods depend on two steps which are known as calibration and

337 prediction. For the calibration step, a statistical model was constructed to set up a relation

338 between the FTIR spectral observations (predictor variables) and the different concentrations

339 of VCO and PO adulterants (response variable), using the calibration set of samples. And in

340 the prediction step, the constructed model was utilized to measure the concentration of an

14
341 external set of samples that were not used in model development (Wu, Li, & Tu, 2015). The

342 optimal number of factors (latent variables for PLS-R and PCR, respectively) was decided by

343 employing the Leave-One-Out method for cross-validation. It is calculated from the plot

344 between the number of factors and the RMSECV which gives an optimal number of factors

345 for both the models (Rohman, Che Man, Ismail, & Hashim, 2017). The optimum number of

346 factors is one of the main aspects which reduce the RMSECV value. The competence of the

347 developed models for prediction of the percentage PO blending with VCO for an external set

348 of samples was examined by the RMSEP value. The relationship between the number of

349 factors and the RMSEC value is inversely proportional to each other. A model constructed

350 with a more number of factors would result in overfitting, leading to a very low RMSEC but

351 very high RMSEP values.

352 The prediction capability of the model is measured from the R2 and the RMSEP (root mean

353 square error of prediction). The lesser the RMSEP value, the higher the extent of prediction

354 accuracy given by the model and vice-versa for the R2 value of prediction (Rohman et al.,

355 2017). For every model, R2, RMSE, and RE% were measured for both calibration and

356 prediction datasets, whereas BIAS, SEP and RPD (must be above 6.5) were measured for

357 prediction dataset. Table 5 represents the number of factors consequent to the least RMSEC

358 and RMSECV values of the constructed models for the combined optimized spectral region

359 as well as for the separate optimized regions. To predict the lowest limit of detection,

360 regression models (PCR & PLS-R) were developed and compared for normal, first derivative

361 and second derivative (Fig. 5) of the combined optimized spectral region and both the

362 separate optimized regions. Models were selected based on the lowest RMSE, RE%, and

363 highest R2. Fig. 6 and Fig. 7 depict the graphs of the measured concentration of PO blending

364 versus the predicted concentration from the FTIR spectra, which manifests the accuracy and

365 significance of the developed models. Table 5 also demonstrates the different quality aspects

15
366 (accuracy and precision) of the two calibration models in forms of R2, RE%, RPD and RMSE

367 values.

368 The relationship between measured and predicted values of PO based on FTIR spectra shows

369 R2 values ranging from 0.970 to 0.999 for calibration and from 0.972 to 0.999 when the

370 external set was used for prediction. And RMSEC value ranged from 0.142 to 1.065% v/v for

371 calibration and from 0.162 to 1.031% v/v for prediction (Table 5). The second derivative

372 PLS-R models for the optimized region (1800-700 cm-1) gave the best results based on the R2

373 value of 0.999, the RMSEC value of 0.142% v/v and RE% of 0.047 for the calibration

374 dataset. Concerning the precision of the selected model, it resulted in low positive BIAS

375 (0.009), indicating a low contribution to the SEP value (0.165). These results along with

376 obtained RMSEP value (0.162%), prediction RE% (0.05) and RPD (38.17) showed excellent

377 accuracy of the selected model (Williams, 2001; Pereira et al., 2019). This can also be

378 explained from the findings of the loading plot as most of the informative wavenumbers are

379 obtained from the same optimized region (1800-700 cm-1) which is giving the best results for

380 the regression model developed. Therefore, it can be concluded that the PLS-R model for the

381 optimized informative spectral region (1800-700 cm-1) can be implemented for prediction of

382 trace amount of PO (1% v/v) adulteration in VCO.

383 4. Conclusion

384 Through this present study, for the first time, it has been established that ATR-FTIR

385 spectroscopy combined with multivariate chemometric tools can be used for the detection of

386 VCO with PO adulteration. This whole technique is swift, needs no redundant sample

387 preparation and does not use perilous chemicals. PCA and LDA were able to differentiate and

388 classify PO adulterants up to 1% v/v. Further, calibration models (PLS-R and PCR) were

389 developed on the combined optimized informative spectral region as well as on the separate

390 optimized regions of the calibration set of data for the quantification of the PO in VCO. The

16
391 best calibration model was derived from 2nd derivative spectra of the optimized region (1800-

392 700 cm-1) which gave the highest R2 value of 0.999 and the lowest RMSEP value of 0.162%

393 v/v. The performance of the calibration models was analyzed by using an external set of data

394 which gave low relative error and high (above 6.5) residual predictive deviation, resulting in

395 high accuracy and precision. Therefore, the lowest limit of detection of PO in VCO was

396 predicted as 1% v/v. In conclusion, the mid-infrared FTIR spectroscopy combined with

397 regression modelling can be used as an excellent tool for the detection of PO adulteration in

398 VCO.

399 Acknowledgment: We are extremely grateful to International Atomic Energy Agency,

400 Vienna, Austria, for its compassionate assistance for funding through research project named

401 “field-deployable analytical methods to access the authenticity, safety, and quality of food in

402 India” (IAEA Project contract number - 22125). We are thankful to the University of Delhi,

403 Delhi, for assisting with the infrastructure needed for the work. Lastly, we would like to

404 thank the Council of Scientific and Industrial Research (CSIR) Government of India, for

405 providing fellowship to the first author.

406 Conflict of interest

407 The authors have no conflict of interest.

408

409

410

411

412

413

17
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23
 Table 1. Adulteration experiment and Sample preparation

Calibration Set
VCOa branded oil samples POb adulterant
Brand 1 (4 Samples) Sample of VCO adulterated with PO 1-18% c (7×4) =28

Brand 2 (4 Samples) Sample of VCO adulterated with PO 1-18% (7×4) =28

Brand 3 (4 Samples) Sample of VCO adulterated with PO 1-18% (7×4) =28

Total 12 pure VCO Total 84 VCO/PO blends

External Set
Brand 4 (4 Samples) Sample of VCO adulterated with PO 1-18% (7×4) =28

Total 4 pure VCO Total 28 VCO/PO blends

Grand Total 96 Calibration set + 32 External set = 128 samples

a
VCO: Virgin Coconut Oil
b
PO: Paraffin Oil

c
1%, 3% and then in arithmetic progression with an increment of 3%, up to 18% v/v
Table 2. Wavenumbers of bands on FTIR spectra in mid-infrared region, along with the assigned
functional groups, vibrational modes and their intensities for (a) VCO (Guillen & Cabo, 1997;
Che Man et al., 2011) and (b) Paraffin oil (alkanes) (Naranjo & Baliga, 2012).

Wavenumbers (cm-1) Functional Assignment Intensities

groups
(a)
2954 –C–H (–CH3) Stretching(asymmetrical) Medium
2924 –C–H (–CH2) Stretching(asymmetrical) Very strong
2852 –C–H (–CH2) Stretching(symmetrical) Very strong
1743 –C=O (ester Stretching Very strong
group)
1465 –C–H (–CH2, Bending (scissor) Medium
CH3)
1417 =C–H (cis-) Bending (rocking) Weak
1377 –C–H (–CH3) Bending(symmetrical) Medium
1228 –C–O Stretching Medium
1155 –C–O Stretching Strong
–CH2– Bending
1111 –C–O Stretching Medium
962 –HC=CH– (trans-) Out of plane bending Weak
872 =CH2 Bending (wagging) Weak
721 –(CH2)n– Bending (rocking) Medium

(b)
3000-2850 –C–H Stretching Strong
1470-1450 –C–H Bending(scissor) Strong
1390-1370 –C–H(–CH3) Bending Medium
721 –(CH2)n– Bending (rocking) and Weak
–HC=CH- out-of-plane vibration
 Table 3. Major Fatty acid composition of 4 VCO Brands determined by Gas Chromatography
Analysis

(%)

Major Fatty VCOa RSDb VCOa RSDb VCOa RSDb VCOa RSDb
% % % %
acids (%) Brand 1 Brand 2 Brand 3 Brand 4

Caprylic 5.07±0.04 0.82 4.83±0.02 0.43 5.72±0.02 0.35 5.04±0.01 0.34

acid (C8:0)

Capric acid 5.34±0.04 0.74 4.42±0.01 0.22 5.1±0.01 0.19 4.82±0.01 0.31
(C10:0)

Lauric acid 50.44±0.03 0.06 45.64±0.04 0.08 48.12±0.02 0.05 47.65±0.02 0.04
(C12:0)

Myristic 20.95±0.01 0.07 23.13±0.02 0.08 21.6±0.01 0.07 22.87±0.01 0.06

acid (C14:0)

Palmitic 8.03±0.02 0.24 10.9±0.02 0.18 9.15±0.01 0.16 8.87±0.02 0.23

acid (C16:0)

Stearic acid 4.88±0.02 0.42 5.93±0.02 0.42 5.63±0.02 0.37 5.57±0.01 0.27
(C18:0)

Oleic acid 3.03±0.02 0.87 3.2±0.01 0.47 2.96±0.02 0.67 3.04±0.02 0.65
(C18:1)

a
VCO = Virgin Coconut Oil
b
RSD = Relative Standard Deviation (%)
Data represents means ± SD (standard deviation) (n=3)
Table 4. Confusion matrix for the classification of pure VCO, PO and different VCO adulterants
with PO

Oil Samples Predicted Group Membership Total

a b
VCO 100% PO 1% PO 3%
VCO 100% 12 0 0 12
Count PO 1% 0 12 0 12
PO 3% 0 0 12 12
Original
VCO 100% 100.0 0 0 100.0
% PO 1% 0 100.0 0 100.0
PO 3% 0 0 100.0 100.0
VCO 100% 12 0 0 12
Count PO 1% 0 12 0 12
PO 3% 0 0 12 12
Cross-validated
VCO 100% 100.0 0 0 100.0
% PO 1% 0 100.0 0 100.0
PO 3% 0 0 100.0 100.0

a
VCO: Virgin Coconut Oil
b
PO: Paraffin Oil
 Table 5. PCR and PLS-R models for (a) Combined informative region (3000-2800 cm-1 and 1800-700 cm-1) (b) 3000-2800 cm-1 (c) 1800-700 cm-1

a
R2 RMSE g
Model b c d
RE % h
RE% i
Spectra Factor Calibration Validation Prediction RMSEC RMSECV RMSEP RPD BIAS SEP
(Cal) (Pred)

(a) Combined informative spectral region (3000- 2800 cm-1 and 1800-700 cm-1)
e
PCR Normal 5 0.971 0.968 0.973 1.045 1.117 1.007 0.127 6.128 0.125 -0.0147 1.023
st
1 derivative 4 0.983 0.982 0.982 0.797 0.832 0.810 0.111 7.645 0.1125 -0.040 0.822
2nd derivative 4 0.979 0.978 0.978 0.892 0.926 0.914 0.118 6.767 0.1195 -0.043 0.927
f
PLS-R Normal 5 0.998 0.998 0.998 0.214 0.237 0.227 0.059 27.395 0.057 0.028 0.229
1st derivative 4 0.990 0.989 0.990 0.607 0.646 0.620 0.097 10.018 0.0984 -0.036 0.629
2nd derivative 5 0.999 0.998 0.998 0.172 0.205 0.218 0.051 28.475 0.058 0.011 0.221

(b) 3000- 2800 cm-1

PCR Normal 3 0.976 0.975 0.980 0.944 0.987 0.864 0.121 6.062 0.116 0.011 0.878
st
1 derivative 4 0.984 0.983 0.984 0.773 0.812 0.775 0.1099 7.958 0.110 -0.014 0.788
2nd derivative 4 0.988 0.987 0.988 0.665 0.702 0.666 0.1019 9.415 0.102 -0.02 0.677
PLS-R Normal 3 0.979 0.977 0.983 0.890 0.931 0.804 0.1179 7.58 0.112 0.017 0.817
1st derivative 4 0.985 0.984 0.985 0.739 0.778 0.738 0.1074 9.975 0.1073 -0.014 0.750
2nd derivative 5 0.998 0.997 0.998 0.250 0.297 0.268 0.0625 23.106 0.0647 0.005 0.273

(c) 1800-700 cm-1

PCR Normal 5 0.970 0.966 0.972 1.065 1.140 1.031 0.128 5.997 0.126 -0.019 1.048
st
1 derivative 5 0.987 0.986 0.982 0.702 0.734 0.815 0.1047 7.629 0.112 -0.068 0.826
2nd derivative 6 0.998 0.998 0.998 0.204 0.214 0.225 0.056 27.61 0.059 0.008 0.225
PLS-R Normal 5 0.998 0.998 0.998 0.224 0.243 0.230 0.059 27.008 0.059 0.004 0.223
1st derivative 4 0.994 0.993 0.993 0.448 0.495 0.514 0.083 12.164 0.089 -0.049 0.519
2nd derivative 5 0.999 0.999 0.999 0.142 0.155 0.162 0.047 38.174 0.050 0.009 0.165

a 2
R : Coefficient of determination; bRMSEC: Root mean square error of calibration: cRMSECV; Root mean square error of cross validation; dRMSEP; Root mean square error of
prediction; ePCR: Principal component regression; fPLS-R: Partial least square regression; gRE%: Relative Prediction Error; hRPD: Residual Predictive Deviation; iSEP: Standard Error
of Prediction
Figure Captions

Figure 1. FTIR spectra of pure virgin coconut oil and different percentage of paraffin oil

depicting major peaks with assigned wavenumbers (cm-1).

Figure 2. Score plot of VCO and VCO/PO blends from PCA analysis of FTIR spectra for the

combined fingerprint region.

Figure 3. The loading plot for first and second principal components utilizing peak absorbance

values at selected frequencies.

Figure 4. All groups scatter plot as deduced by discriminant analysis using discriminant function

1 and 2 for FTIR spectral data of pure VCO, PO 1% and PO 3%.

Figure 5. Overlay FTIR spectra of VCO and its various PO blends in the region of 4000-400

cm-1 for (a) normal, (b) first derivative (c) second derivative respectively.

Figure 6. Partial least squares regression (PLS-R) calibration model of 2nd derivative of

optimized regions (1800 cm-1 to 700 cm-1) for relationship between actual (Reference Y) versus

FTIR predicted adulteration level of Paraffin oil in Virgin coconut oil (Predicted Y).

Figure 7. Partial least squares regression (PLS-R) prediction for 2nd derivative of optimized

regions (1800 cm-1 to 700 cm-1).

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Highlights

• ATR-FTIR spectroscopy along with chemometrics was applied for the detection of PO

adulteration in VCO.

• PCA and LDA successfully differentiated and classified PO adulterants from VCO.

• Outstanding R2 (0.999), RMSEC (0.142) and RMSEP (0.162) values obtained for PLS-R.

• The lowest limit of detection of PO in VCO was predicted as 1% v/v.

• This whole technique is swift and needs no redundant sample preparation.