Analytical

Methods
Accepted Manuscript

This is an Accepted Manuscript, which has been through the

Royal Society of Chemistry peer review process and has been
accepted for publication.

Accepted Manuscripts are published online shortly after

acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.

You can find more information about Accepted Manuscripts in the

Information for Authors.

Please note that technical editing may introduce minor changes

to the text and/or graphics, which may alter content. The journals
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.

www.rsc.org/methods
Page 1 of 29 Analytical Methods

Graphical Abstract
1
2
3
FFA Analysis
4
5
Methods
6

Analytical Methods Accepted Manuscript

7 Instrumental Classical Titration
8 Methods Methods
9
10
Electrochemical Spectroscopic
11 Colorimetric Methods Thermal Methods
12
Methods Methods
13
14 Copper soap
Voltammetric Methods Infrared Methods
15 Methods
16
17
Automated FI Electrical Conductivity Mid Infrared
18
19
Methods Methods Methods
20
21 Phenolphthalien based Direct Infrared
pH metric Methods
22 Methods Methods
23
24
Indirect Infrared
25
26
Methods
27
28 Near Infrared
29 Methods
30
31
32 Raman Methods
33
34
35
NMR Methods
36
37
38
39
40 Block diagram of the methods covered in the review for assessment of free
41
42
fatty acids in oils and fats.
 Analytical Methods Page 2 of 29

1
2
3
4 1 Analytical Approaches for free fatty acids assessment in oils and fats
5
6 2 S. A. Mahesar1, S. T. H. Sherazi1*, Abdul Rauf Khaskheli2, Aftab A. Kandhro3,
7
8
9 3 Sirajuddin1
10
1
4 National Centre of Excellence in Analytical Chemistry, University of Sindh,

Analytical Methods Accepted Manuscript

11
12
13 5 Jamshoro-76080, Pakistan
14
15 2
6 Department of Pharmacy, Shaheed Mohtarma Benazir Bhutto Medical University, Larkana-
16
17 7 77150, Pakistan
18
19 3
20
8 Dr. M. A. Kazi Institute of Chemistry, University of Sindh, Jamshoro-76080, Pakistan
21
22 9 Abstract
23
24
25 10 Basically, free fatty acids (FFAs) are produced by hydrolysis of oils and fats. Level of
26
27
28
11 FFA depends on time, temperature and moisture content because the oils and fats are
29
30 12 exposed to various environments such as storage, processing, heating or frying. As FFAs
31
32 13 are less stable as compared to neutral oil therefore they are more prone to oxidation and
33
34
35 14 produce rancidity. Thus, FFA is a key feature linked with the quality and commercial
36
37 15 value of oils and fats. American Oil Chemists' Society (AOCS), Association of Official
38
39 16 Analytical Chemists (AOAC) and European Commission Regulation (EEC) have
40
41
42 17 established almost identical standard methods for the assessment of FFA. These methods
43
44 18 are based on the titration where oils or fats are needed to be dissolved in hot neutralized
45
46
19 ethanol or ethanol/diethyl ether using phenolphthalein as an end point indicator.
47
48
49 20 Titrimetric procedures are however laborious and need large amount of chemicals and
50
51 21 solvents. Besides cost of chemicals, environmental issues further limit these procedures.
52
53
54
22 In addition, accurate detection of end point especially for highly colored crude oil by
55
56 23 colorimetric indicator is a difficult task. Despite all mentioned demerits, unfortunately
57
58
59
60 1
Page 3 of 29 Analytical Methods

1
2
3
24 titration method is still being used in most of the edible oil industries for the
4
5
6 25 determination of FFA. Due to lack of any comprehensive review on this very important
7
8 26 topic, we have made an attempt to present a review in order to discuss various available
9
10
27 methods with special emphasis on the instrumental methods due to their high sensitivity,

Analytical Methods Accepted Manuscript

11
12
13 28 accuracy and rapidity.
14
15 29 Keywords: Free fatty acids; vegetable oils; standard titration method; instrumental
16
17
18 30 methods
19
20 31 *Corresponding Author: S. T. H. Sherazi
21
22
23
24 32 Email: tufail.sarfaraz@yahoo.com Tel. No. +92-22-9213429 Fax +92-22-9213431
25
26
27 33
28
29
30
31 34
32
33
34 35
35
36
37
38 36
39
40
41 37
42
43
44
45 38
46
47
48
49
39
50
51
52 40
53
54
55
56 41
57
58
59
60 2
 Analytical Methods Page 4 of 29

1
2
3
42 1. Introduction
4
5
6 43 Vegetable oils or fats are extracted from the oilseeds in the crude form. Various refining
7
8 44 stages such as degumming, neutralization, dewaxing, bleaching and deodorization are
9
10
45 followed to convert extracted crude oils to edible oils. During processing, a number of

Analytical Methods Accepted Manuscript

11
12
13 46 parameters such as moisture, peroxide value, free fatty acids (FFAs), saponification value
14
15 47 (SV) and iodine value (IV) are monitored. Among these parameters FFA content is
16
17
18 48 considered as a very crucial factor and linked with the quality as well as economic value
19
20 49 of edible oils. 1 Generally FFAs are the hydrolysis product generating as a result of the oil
21
22 50 and fat oxidation during long time storage or processing at elevated temperature and
23
24
25 51 heating or frying. Initially, AOCS2, AOAC3 and European Commission Regulation
26
27 52 (EEC)4 have recommended standard methods for the determination of FFA. These
28
29
53 methods are almost similar and based on the titration of oil (3.5-56.4 g). Oils or fats are
30
31
32 54 dissolved in hot neutralized 95% ethanol (50-100 ml) or ethanol/diethyl ether (50-150
33
34 55 ml), against a strong base using phenolphthalein as an indicator. Although, these
35
36
37 56 titrimetric methods are very simple but extremely laborious and need large amount of
38
39 57 expensive chemicals associated with environmental issues. Furthermore, the analysis of
40
41 58 dark crude oils in the presence of colorimetric indicator is very problematic to find out
42
43
44 59 the accurate end point during titration.
45
46 60 To overcome such issues, a number of instrumental methods have been developed
47
48 61 as potential alternative to classical methods for more accurate and sensitive measurement
49
50
51 62 of FFA contents in the edible oils and fats. In general these methods can be categorized
52
53 63 into three major groups:
54
55
56
64 (a) Colorimetric methods
57
58
59
60 3
Page 5 of 29 Analytical Methods

1
2
3
65 (b) Spectroscopic methods
4
5
6 66 (c) Electrochemical methods
7
8 67 Generally, second and third group involves variety of sophisticated instruments with
9
10
68 diverse approach that compete with each other in terms of speed of analysis, amenability

Analytical Methods Accepted Manuscript

11
12
13 69 to automation and reduction of harmful solvents. Some instrumental methods provide a
14
15 70 significant gain in accuracy and sensitivity than classical titrimetric methods.
16
17
18 71 Several methods have been reported during the last sixty year. In 1950s,
19
20 72 spectrophotometric technique was well addressed and several workers published plenty
21
22 73 of articles on FFA using colorimetric methods. After that, the trend changed towards the
23
24
25 74 electrochemical techniques. In last two decades, attention was however focused on use of
26
27 75 infrared spectroscopic methods for the direct or indirect determination of FFA. These
28
29
76 methods were claimed to be environmental friendly as either chemicals are totally
30
31
32 77 avoided or require negligible amount of chemicals as well as samples. Fig. 1 shows the
33
34 78 block diagram of the methods developed for the determination of FFA during last few
35
36
37 79 decades. To the best of our knowledge there is no any comprehensive review available on
38
39 80 FFA analysis. Therefore, present review will explore many methods which were
40
41 81 developed in last few decades.
42
43
44 82
45
46 83
47
48 84
49
50
51 85
52
53 86
54
55
56
87
57
58
59
60 4
 Analytical Methods Page 6 of 29

1
2
3
88 2. Discussion
4
5
6 89 2.1. Colorimetric methods
7
8 90 Various calorimetric methods have been reported by several researchers.
9
10
91 2.1.1. Copper soap method

Analytical Methods Accepted Manuscript

11
12
13 92 In 1956, Ayers5 made his first effort to determine the FFA by calorimetric method.
14
15 93 According to his procedure, aqueous solution of copper or cobalt nitrate was used to
16
17
18 94 convert FFA into water-insoluble soaps. Colored soap precipitate was extracted with
19
20 95 chloroform and quantification was carried out by measuring absorbance at 675 and 527
21
22 96 nm for copper and cobalt, respectively. Although method was not proposed for FFA
23
24
25 97 analysis but it provided a base to develop colorimetric methods for FFA analysis in edible
26
27 98 oils. Later on Baker6 proposed an improved copper salt based method which was
28
29
99 designed particularly for FFA analysis in edible oils. The method involves the dissolution
30
31
32 100 of oil in benzene followed by mixing with aqueous cupric acetate solution. The mixture
33
34 101 was centrifuged and quantification of FFA was determined by measuring the absorption
35
36
37 102 of benzene layer at 640 nm. This method showed good correlation with titrimetric
38
39 103 method. The only limitation of this method was the dissolution of some copper salts of
40
41 104 saturated fatty acids in benzene. In view of the inconsistencies in working wavelength of
42
43
44 105 640 vs 675 nm 5,6, Bains et al.,7 examined the spectra and the effect on color of oil during
45
46 106 the measurement. He concluded that 670 nm was the optimum wavelength for copper
47
48 107 soaps in non-colored or colored oils. Twelve years later, Lowry and Tinsley8 reported a
49
50
51 108 substantial work to optimize the method in terms of sensitivity and reproducibility. The
52
53 109 conclusion of his work was the stabilization of the copper soap of saturated fatty acids in
54
55
56
110 benzene and improvement in the sensitivity by controlling the pH using pyridine. Due to
57
58
59
60 5
Page 7 of 29 Analytical Methods

1
2
3
111 the practical and health concerns of benzene, various authors used alternative to benzene
4
5
6 112 such as toluene 9, isooctane 10
and cyclohexane 11
. Gyrik et al., 12
proposed a new
7
8 113 analytical tool for the determination of total FFA content in thermally treated cooking oil.
9
10
114 The method was based on the colorimetric copper soap combined with the concept of

Analytical Methods Accepted Manuscript

11
12
13 115 optothermal window (OW) (with 632.8 nm HeNe laser used as a radiation source). The
14
15
16 116 results obtained by this method were compared to those acquired by conventional
17
18 117 spectroscopy; the correlation between the two methods was high for FFA concentrations
19
20 118 exceeding 2 mol/ml.
21
22
23 119
24
25 120 2.1.2. Automated FIA systems
26
27 121 The first automated system for FFA analysis in edible oils using copper soap method was
28
29
30 122 introduced in 1980s. 9 The automation was accomplished using a flow injection analysis
31
32 123 (FIA) system which was able to analyze up to 20 samples/h. In contrast to the previously
33
34
35
124 published methods, based on organic solvents, the quantification was done in the aqueous
36
37 125 phase. However, the system did not receive much attention, because of the complications
38
39 126 and stability problems. Later on an improved hard ware in FIA system using a single-
40
41
42 127 extraction step was reported.13 This new system offered enhanced stability and much
43
44 128 shorter analysis time (130 samples/h). The only shortcoming of the system was poor
45
46 129 sensitivity. Puchades et al.,14 improved the sensitivity of FIA systems through optimized
47
48
49 130 operating conditions, for example pH, flow rate, tubing length/diameter, and oil/solvent
50
51 131 proportion resulted in better analysis speed (75 samples/h) and lower limit of detection
52
53
132 (~0.01% FFA). Despite the mentioned improvements the method did not receive much
54
55
56
57
58
59
60 6
 Analytical Methods Page 8 of 29

1
2
3
133 attention due to the complications in the analysis and incompatible values as compared to
4
5
6 134 the official method.
7
8 135 A continuous liquid-liquid extraction system using a single-channel flow-reversal
9
10
136 injection mode was also proposed for the direct determination.15 The results provided by

Analytical Methods Accepted Manuscript

11
12
13 137 the proposed method were in accordance with those obtained by a manual procedure and
14
15 138 comparable to the standard acid-base titration method, but the sampling frequency was
16
17
18 139 lower (12 samples/h).
19
20 140 2.1.3. Phenolphthalein based method
21
22 141 Due to the shortcomings of the official method, Linares et al.,16 made an attempt to
23
24
25 142 automate the official method. He used the same reagents as the IUPAC official method,
26
27 143 whilst quantification was based on monitoring the decrease in the phenolphthalein (PHP)
28
29
144 intensity at 562 nm as a result of the reaction between KOH and FFA in the oil sample.
30
31
32 145 The analytical range of method was 0.15-0.81% FFA and sample throughput up to ~ 60
33
34 146 samples/h. Later on a modified method with similar analytical range was reported 17 using
35
36
37 147 3-7 ml of inexpensive 1-propanol as a diluent and carrier in comparison to the huge
38
39 148 amount of solvents in official method. The sample throughput was 30-100 samples/h.
40
41 149 A direct measurement of FFA in virgin olive oil without dilution was reported using
42
43
44 150 similar system.18 The system was equipped with an optical fiber beam connected to the
45
46 151 spectrophotometer set at 562 nm. The system had the same sensitivity as the previous
47
48 152 methods with a sample throughput of 12-60 samples/h. Afterward an indirect titrimetric
49
50
51 153 method was developed based on the complete extraction of acids from oil into reagent
52
53 154 (0.05 mol dm3 triethanolamine in the mixture of 50 % H2O + 50 % 2-PrOH) and indirect
54
55
56
155 titration of acids in BH+ form against aqueous alkali in the presence of a PHP indicator.19
57
58
59
60 7
Page 9 of 29 Analytical Methods

1
2
3
156 The benefits of proposed method included the lack of toxic solvents, analysis at room
4
5
6 157 temperature and no need for preliminary neutralization of acid admixtures.
7
8 158
9
10 159 Saad et al.,20 reported a single and two-line manifold FI methods using PHP and

Analytical Methods Accepted Manuscript

11
12 160 bromothymol blue (BTB) as indicators. The method was based on the monitoring of the
13
14
15
161 changes of absorbance of the indicators at 562 nm for PHP and 627 for BTB. Various FI
16
17 162 parameters were optimized such as carrier and reagent concentration, length of reaction
18
19 163 coil, flow-rate, injected volume and size of mixing chamber. The single-line manifold
20
21
22 164 with PHP as indicator was recommended for the determination of samples with acidity
23
24 165 higher than 0.4, while for lower acidities (< 0.4) a two-line manifold with BTB was
25
26 166 recommended. The method was linear over the range 0.4-10.0 FFA for single-line
27
28
29 167 manifold and 0.11-0.50 FFA for the two-line manifold. Sample throughputs were 35-74
30
31 168 and 21-46 samples/h for single-line and two-line manifolds, respectively.
32
33 169
34
35 170 A non-aqueous single-line manifold system was built by modification of an HPLC for
36
37
38 171 FIA.21 Oil samples were injected without pre-treatment into a n-propanol solution
39
40 172 containing KOH and PHP. The linear concentration range was calculated as 0.091.50
41
42
43
173 and 0.071.40 FFA% for corn and sunflower oils, respectively. The results were
44
45 174 comparable with those obtained by the AOCS method. The benefits of proposed method
46
47 175 were its simplicity and high sample throughput.
48
49
50 176
51
52 177 Makahleh and Saad22 reported a single line FIA method attached with capacitively
53
54 178 coupled contactless conductivity detector (C4D). Methanol mixed with 1.5 mM sodium
55
56
57 179 acetate (pH 8) 80:20 (v/v) was used as a carrier stream at a flow rate of 1.0 mL min1.
58
59
60 8
 Analytical Methods Page 10 of 29

1
2
3
180 Good agreement was found between the standard non-aqueous titrimetry method and the
4
5
6 181 proposed method. The proposed method offers high sampling rate of 4060 sample/h
7
8 182 and considerably low cost automated system that needs minimum human intervention
9
10
183 over long periods of time. Recently a visual and green titrimetric method was proposed

Analytical Methods Accepted Manuscript

11
12
13 184 using PHP as an indicator.23 The sample was dissolved in a waterethanol mixture (1:1
14
15 185 v/v) and titrated with a 0.02 mol L-1 aqueous NaOH solution. The results obtained by
16
17
18 186 proposed approach were comparable with AOCS titration method.
19
20 187
21
22 188 2.1.4. Miscellaneous spectrophotometric methods
23
24
25 189 A new approach for the FFA determination in non aqueous medium without titration was
26
27 190 reported in the literature.24,25 The method utilized phenol red as fatty acid indicator,
28
29
191 which was solubilized in reverse micelles formed by AOT [sodiumbis(2-ethylhexyl)
30
31
32 192 sulfosuccinate] in isooctane. Quantification was achieved by monitoring the changes in
33
34 193 absorbance at 560 nm (disappearance of the red color). Although the method was
35
36
37 194 economical (small amounts of samples and solvents) but found to be lacking in accuracy.
38
39 195 Therefore, no any additional efforts were made for its improvement. Rejeb and Gargouri
40
41 26
196 reported a new method for the determination of olive oil acidity. The method was
42
43
44 197 based on the measurement of polyunsaturated free fatty acids reacting with lipoxygenase.
45
46 198 Hydroperoxy-fatty acids enzymatically formed were easily determined by UV
47
48 199 spectrophotometry at 234nm. The proposed method was successfully applied for the
49
50
51 200 determination of acidity in olive oil samples and good agreement was obtained between
52
53 201 the proposed enzymatic method and classical titration method. This method was accurate,
54
55
56
202 simple, sensitive, inexpensive, and reliable for acidity determination of olive oil with a
57
58
59
60 9
Page 11 of 29 Analytical Methods

1
2
3
203 high sample throughput. Recently, Fedosov et al.,27 developed a novel microtitration
4
5
6 204 method for determining the FFA in small samples of oil (5150 mg). The method was
7
8 205 based on the optical signal of pyranine (aqueous pK 7.3), which changed its absorbance
9
10
206 and fluorescence. All reactants were dissolved in a medium with universal solubility,

Analytical Methods Accepted Manuscript

11
12
13 207 which allowed accurate optical measurements on conventional equipment. Titration
14
15 208 curves for FFA standards stipulated the selection of the neutralization point. Comparison
16
17
18 209 of fluorescence and absorbance of pyranine pointed to somewhat better signal to noise
19
20 210 properties of the fluorescent signal when working with heavily pigmented oil samples.
21
22 211 Blind examination of different experimental mixtures (FFA = 0.1540 %) revealed a
23
24
25 212 good correspondence between the pyranine method and official method. Furthermore, it
26
27 213 was reported that the small size of optical cells could allow further minimization of the
28
29
214 pyranine assay if adapting it to an automated procedure.
30
31
32 215
33
34 216 2.2. Electrochemical methods
35
36 217 2.2.1. Voltammetric technique
37
38
39
218 Voltammetry is an important class of electrochemical methods widely used in analytical
40
41 219 chemistry and various industrial processes. In voltammetry, information about an analyte
42
43 220 is obtained by measuring the current as the potential is varied.28 It is commonly used for
44
45
46 221 compounds that are readily oxidized/reduced to provide highly selective and sensitive
47
48 222 quantitative methods. Electrochemically FFA are weakly active and can be reduced or
49
50 223 oxidized. The voltammetric behavior of quinone in the presence of acids in buffered
51
52
53 224 protic solvents was first reported by Takamura and Hayakawa.29 It was reported that
54
55 225 reduced form of quinone (dissolved in ethanol) gave a very strong voltammetric signal in
56
57
226 presence of acids. For FFA analysis in edible oils the behavior of quinone has been
58
59
60 10
 Analytical Methods Page 12 of 29

1
2
3
227 utilized by various authors30-34 to develop HPLC and FIA methods. The FIA system
4
5
6 228 coupled with voltammetric detection had high sensitivity, better reproducibility, low
7
8 229 solvent consumption and excellent throughput of 60 samples/h. Unfortunately this
9
10
230 technique did not receive much attention. Li et al.,35 proposed a new method using

Analytical Methods Accepted Manuscript

11
12
13 231 polypyrole-modified electrode in linear potential sweep voltammetric mode. In this
14
15 232 method, the behavior of naphtoquinone in the presence of FFA was investigated in
16
17
18 233 ethanol/1,2-dichloroethane (3:1) solution containing 0.1 M LiClO4. A linear calibration
19
20 234 graph was obtained in the range of 5.01066103 M for FFA (R = 0.993), with
21
22 235 sensitivity of 2.41102 A L/mol and a limit of detection 1.2106 M. The method was
23
24
25 236 accurate, superior in sensitivity and capable to analyze 45 samples/h.
26
27 237
28
29 238 2.2.2. Electrical Conductivity method
30
31 239
32
33 240 Recently a new approach for the determination of FFA in edible oils based on the
34
35
241 electrical conductivity (EC) value change of a 0.04 M potassium hydroxide (KOH)
36
37
38 242 solution during KOHFFA mixed reaction was reported.36 The EC value changes of the
39
40 243 KOH solution layers were determined, and the FFA was determined from calibration
41
42
43 244 plot. Various parameters were optimized like the concentration of KOH solution, holding
44
45 245 time, the types of edible oils, setting temperature, and the ratio of oil to KOH solution
46
47 246 (m/v). The method was validated using standard addition and the AOCS method. The
48
49
50 247 results and analytical performance of the EC procedure was better than that of the AOCS
51
52 248 method.
53
54 249
55
56
57 250
58
59
60 11
Page 13 of 29 Analytical Methods

1
2
3
251 2.2.3. pH-metric methods
4
5
6 252 In general the low polarity and high viscosity of oils have made it impractical to use pH
7
8 253 metric methods for the determination of FFA. Lapshina et al.,37 carried out pH metric
9
10
254 measurements by extracting FFA from oil into a polar solvent. In order to facilitate

Analytical Methods Accepted Manuscript

11
12
13 255 extraction, the author suggested the use of a weak base triethanolamine (TEA) in an
14
15 256 appropriate solvent mixture (diethyl ether 80%, chloroform 19% and water 1%) to
16
17
18 257 convert carboxylic acids into their salts. Although the method was workable but showed
19
20 258 poor reproducibility because of instability of the electrode and the basic reagent in the
21
22 259 solvent mixture. Later on the method was modified by replacing the triethanolamine with
23
24
25 260 a 1:1 (v:v) mixture of iso-propanol and water containing 0.2 M TEA and 0.02 M
26
27 261 KNO3.38-41 Rather than extracting FFA, this solvent mixture effectively makes an
28
29
262 emulsion in the range of pH measurement. Soon after, Velasco-Arjona and Luque de
30
31 42
32 263 Castro proposed a fully automated method using robotic station for the determination
33
34 264 of AV in olive oil without titration based on pH measurements of emulsion. The excellent
35
36
37 265 correlation with potentiometric titration demonstrated the convenience of the automated
38
39 266 method. This method was able to analyze 15 samples/h. Various publications on this
40
41 267 approach were reported for acidity measurements in oilseeds.43-45 A new concept of flow
42
43
44 268 titration was proposed using sequential injection analysis (SIA) with a diode array
45
46 269 spectrophotometric detector linked with a multivariate curve resolution-alternating least
47
48 270 squares (MCR-ALS) in the presence of alizarine indicator.46 Total waste generated in this
49
50
51 271 method was 3.33 ml (0.41 ml of sample, 0.25 ml of indicator and 3 ml of carrier). The
52
53 272 results of the proposed method compared well with tritrimetric method and the frequency
54
55
56
273 of the analysis was 12 samples/h.
57
58
59
60 12
 Analytical Methods Page 14 of 29

1
2
3
274 2.3. Spectroscopic methods
4
5
6 275 2.3.1. Infrared methods
7
8 276 2.3.1.1. Direct Infrared methods
9
10
277 The possibility of utilizing FTIR spectroscopy for FFA analysis has been examined by

Analytical Methods Accepted Manuscript

11
12
13 278 number of scientists over the last two decades. Lanser et al.,47 first time reported a semi
14
15 279 quantitative method for the analysis of FFA in soybean oils by computer-assisted FTIR
16
17
18 280 using deconvolution technique. He collected the spectra of oils using transmission cell,
19
20 281 and observed the spectral changes in the carbonyl region in the range 2000-1600 cm1, and
21
22 282 correlated to the FFA content obtained by the standard AOCS method. The method was
23
24
25 283 based on developing a calibration plot using oleic acid as standard into FFA-free soybean
26
27 284 oil. A major restriction to make the method more quantitative was the spectral variation
28
29
285 of ester band among various oils, because the carboxylic acid C=O absorption appears on
30
31
32 286 the shoulder of triglyceride ester linkage absorption.
33
34 287 Later on an accurate method was developed based on the measurement of the carboxylic
35
36
37 288 acid C=O band at 1711 cm-1 using transmission approach, covering an analytical range of
38
39 289 0.28% FFA.48 To avoid the matrix effects noted earlier47, the ratioed out calibration and
40
41 290 sample spectra against the spectrum of a FFA-free oil of the same type of oil were
42
43
44 291 analyzed rather than a background spectrum of air. This approach gave results
45
46 292 comparable in precision and accuracy to that of the AOCS reference titration method.
47
48 293 Through macroprogramming the method was completely automated, this approach takes
49
50
51 294 an analysis time less than 2 min.
52
53 295
54
55
56
57
58
59
60 13
Page 15 of 29 Analytical Methods

1
2
3
296 After the above preliminary studies, a new procedure was reported for FFA in olive oil
4
5
6 297 based on attenuated total reflectance (ATR) FTIR spectroscopic measurements using
7
8 298 partial least-squares regression (PLSR).49 The developed method was applicable to
9
10
299 samples of different categories of olive oil with analysis time of 5 min/sample. Che Man

Analytical Methods Accepted Manuscript

11
12
13 300 et al.,50-51 described transmission FTIR spectroscopic methods using 200 m NaCl
14
15 301 windows for low level of FFA contents in palm olein (PO) and 100 m BaF2 windows for
16
17
18 302 higher FFA in crude palm oil (CPO). The PLS calibration models were developed to
19
20 303 correlate FTIR and chemical method. The developed method drastically reduced the
21
22 304 analysis time to less than 2 min/sample. Verleyen et al.,52 followed up more rigorously
23
24
25 305 research on the work previously reported47 and developed effective calibrations for
26
27 306 different oils (corn, soybean, sunflower, palm, palm kernel, and coconut oils). It was
28
29
307 concluded the calibration models developed for the six oils differed significantly and
30
31
32 308 indicated the need to develop a calibration for each specific oil or fat.
33
34 309 Inon et al.,53 developed the chemometric based method using PLS multivariate calibration
35
36
37 310 and net analyte signal (NAS) preprocessing for the determination of FFA in commercial
38
39 311 olive oil samples of different types and origins. The limit of detection (LOD), sensitivity
40
41 312 and selectivity of the methodology developed were evaluated in terms of the net analyte
42
43
44 313 signal and found LOD value of 0.072%, with sensitivity of 0.077 in terms of per unit
45
46 314 concentration.
47
48
49 315 Due to the practical limitations of transmission spectroscopy for the quantification of
50
51 316 higher FFA, a simple and direct methodology was developed for the determination of
52
53
317 higher FFA contents (>30) by single bounce (SB) ATR FTIR spectroscopy in poultry
54
55
56 318 feed lipids54, deodorizer distillates and crude oils.55 The accuracies of the methods were
57
58
59
60 14
 Analytical Methods Page 16 of 29

1
2
3
319 checked by comparison to a conventional AOCS titrimetric procedure. Yu et al.,56-57
4
5
6 320 reported a new approach for the automated determination of FFA in edible oils using
7
8 321 spectral reconstitution (SR) technique. The data obtained by this technique was accurate
9
10
322 and reproducible. The developed automated method was capable of analysing ~90

Analytical Methods Accepted Manuscript

11
12
13 323 samples/h; a rate appropriate with the throughput required by commercial contract or
14
15 324 high-volume process control laboratories.
16
17
18 325
19
20 326 2.3.1.2. Indirect infrared methods
21
22 327 For fats and oils that may have undergone significant thermal stress or extensive
23
24
25 328 oxidation, an indirect FTIR spectroscopic method was developed for FFA based on ATR
26
27 329 approach in order to remove the matrix effect.48 The 1% KOH/methanol was used to
28
29
330 extract the FFA and convert them to their potassium salts. The carboxylate anion absorbs
30
31
32 331 at 1570 cm1, well away from interfering absorptions of carbonyl commonly present in
33
34 332 oxidized oils. To overcome the matrix effects the indirect method was more sensitive and
35
36
37 333 accurate than the direct determination of FFA, although the method complicated the
38
39 334 analysis. An automated flow injection system was developed for the rapid determination
40
41 335 of the FFA content in olive, sunflower, and corn oils.58 The method was based on the
42
43
44 336 indirect approach48 to avoid the matrix effects linked with direct measurements of
45
46 337 carboxylic acid band at 1711 cm-1. This automated method was able to analyze ~40
47
48 338 samples/h.
49
50
51 339
52
53 340 A transmission spectroscopic method using potassium phthalimide (K-phthal) as a weak
54
55
56
341 base to convert the FFA present in oils to their carboxylate salts without causing oil
57
58
59
60 15
Page 17 of 29 Analytical Methods

1
2
3
342 saponification was reported.59 The method was automatable, and capable of analyzing
4
5
6 343 about 60 samples/h. Later on a sensitive and rapid methodology for the quantification of
7
8 344 a low levels (<0.005%) of FFA in edible oils was described using sodium hydrogen
9
10
345 cyanamide (NaHNCN) to convert FFA to their carboxylate salts.60-61 The feasibility of

Analytical Methods Accepted Manuscript

11
12
13 346 employing a portable variable filter array (VFA) IR spectrometer equipped with a
14
15 347 transmission flow cell for FFA analysis was reported by Li et al.62 The indirect approach
16
17 60-61
18 348 was employed to convert the FFA to their salts as described earlier. Based on this
19
20 349 measurement, a VFA-IR spectrometer provided an economical instrumental means for at-
21
22 350 line monitoring of FFA levels in crude and refined edible oils capable of analyzing ~20
23
24
25 351 30 samples/h. Aryee et al.,63 determined FFA content in fish oils employing same
26
27 352 approach. It was reported that FTIR method is a flexible and viable instrumental
28
29
353 alternative to the standard titrimetric method.
30
31
32 354
33
34 355 2.3.2. Near infrared methods
35
36
37 356 The near infrared (NIR) methods for the analysis of FFA in oils and fats were reported
38
39 357 before the mid infrared (MIR) methods. Frankel et al.,64 used NIR spectroscopy for the
40
41 358 first time to determine the quality of the soybeans and its oil stored at different moisture
42
43
44 359 levels. He observed that NIR analysis at 2260 nm showed a good correlation coefficient
45
46 360 of 0.864 with titratable FFA. Furthermore, it was reported that NIR analysis is most
47
48 361 suitable and rapid to evaluate hydrolytic deterioration in stored soybeans. This
49
50
51 362 methodology was used to evaluate the factors affecting the food quality of soybeans for
52
53 363 domestic and foreign markets.
54
55 364
56
57
58
59
60 16
 Analytical Methods Page 18 of 29

1
2
3
365 After the initial work of Frankel et al.,64 there was no any further work carried out on NIR
4
5
6 366 for ten years. Majority of the methods were developed on mid IR range. In 1997, a direct
7
8 367 method for the determination of FFA in fish oil was reported.65 PLS and multiple linear
9
10
368 regression (MLR) was used for calibration and described that first derivative

Analytical Methods Accepted Manuscript

11
12
13 369 mathematical treatment provided better results as compared to the second derivative and
14
15 370 N-point smoothing for NIR spectra. The calibration equation for FFA obtained from PLS
16
17
18 371 was found better as compared to the MLR.
19
20 372
21
22
23 373 CheMan et al.,66 developed a calibration for the determination of FFA in crude palm oil
24
25 374 and its fractions based on the NIR reflectance approach. For the preparation of FFA
26
27
28 375 calibration, oil was hydrolyzed with 0.15% (w/w) lipase in an incubator at 60C (200
29
30 376 rpm). The optimized calibration models were constructed with MLR analysis based on
31
32 377 C=O overtone regions from 18502050 nm. Calibrations were validated with an
33
34
35 378 independent set of 810 samples. The developed method was rapid, and its accuracy was
36
37 379 generally good for raw-material quality control. The method was capable to analyze 12
38
39
380 samples/h. The hydrolytic degradation of lipids in fish oil was monitored in
40
41
42 381 transflectance mode by an NIR monochromator instrument in the range of 1100
43
44 382 2500 nm.67 For FFA analysis PLS regression calibration model was performed. The
45
46
47
383 accuracy of the developed model was tested using a validation set. It was reported that
48
49 384 NIR spectroscopy with PLS regression could be successfully used to monitor hydrolytic
50
51 385 degradation of lipids in the fish oil stored under industrial conditions.
52
53
54 386
55
56
57
58
59
60 17
Page 19 of 29 Analytical Methods

1
2
3
387 A novel method was developed for the estimation of FFA content directly in high-oleic
4
5
6 388 sunflower seeds.68 For the calibration a sample set of different varieties from the harvest
7
8 389 of 2004 and 2005 was used. The developed NIR spectroscopic calibration was calculated
9
10
390 with a modified PLS algorithm, standard normal variate (SNV), detrend scatter correction

Analytical Methods Accepted Manuscript

11
12
13 391 and the 2nd derivative of the spectra of ground sunflower seeds. The results obtained by
14
15 392 this method demonstrated the efficiency and cost effectiveness of the NIR method for the
16
17
18 393 evaluation of FFA content in sunflower seeds.
19
20 394
21
22 395 Ng et al.,69 described a transmission based method using PLS and forward stepwise
23
24 396 multiple linear regression (FSMLR) techniques for FFA in frying oils. PLS model
25
26
27 397 provided improved results compared to FSMLR model when derivative as spectral
28
29 398 treatment used in the region of 700-1100 nm. The best correlations between the NIR and
30
31
399 wet chemical method for FFA was 0.943. Where as in the longer wavelength region
32
33
34 400 (1100-2500 nm) FSMLR model was better than PLS model. The developed method was
35
36 401 capable to analyze 20 samples/h. Many authors used NIR calibration for FFA
37
38 70 71
39
402 quantification in the oxidation and interesterification of oil to replace the traditional
40
41 403 methods. Furthermore it was reported that NIR spectroscopy might be adapted for real-
42
43 404 time quality control purposes. Recently, Yu et al.,72 reported an automatic determination
44
45
46 405 of acid value in edible oils by NIR spectrometer coupled with continuous samples
47
48 406 injection cell using edible oils as raw materials. The R2 and the RMSEP of the calibration
49
50 407 for acid value were 0.9873 and 0.114 mg/g in the range of 5500-4600 cm-1 with baseline
51
52
53 408 correction point at 6524 cm-1 and 4823 cm-1 and SNV. By this method analysis rate could
54
55 409 be achieved as 90 samples/h.
56
57
410
58
59
60 18
 Analytical Methods Page 20 of 29

1
2
3
411 2.3.3. Raman spectrometry
4
5
6 412 Fourier transform (FT) Raman spectrometry in combination with PLS regression
7
8 413 was used for direct reagent-free determination of FFA content in olive oils and olives.
9
10
414 Oils were directly investigated in a simple flow cell, while olives were measured in a

Analytical Methods Accepted Manuscript

11
12
13 415 dedicated sample cup, which was rotated eccentrically to the horizontal laser beam during
14
15 416 spectrum acquisition. External and internal (leave-one-out) validation were used to assess
16
17
18 417 the predictive ability of the PLS calibration models for FFA content in oil and olives in
19
20 418 the range 0.206.14 and 0.153.79%, respectively.73 The results obtained by the proposed
21
22 419 procedures could be used for screening of good quality olives before processing, as well
23
24
25 420 as, for the on-line control of the production of oil.
26
74
27 421 El-Abassy reported a visible Raman spectroscopic method for determining the
28
29
422 FFA in extra virgin olive oil using PLS. For the calibration, oleic acid was used to
30
31
32 423 increase the FFA content in extra virgin olive oil up to 0.80%. The calibration curve of
33
34 424 actual FFA% obtained by titration versus predicted values based on the Raman spectral
35
36
37 425 regions 1600-945 cm-1 provided a robust and precise model with high accuracy for the
38
39 426 prediction of the FFA%. Although the methods were simple and accurate, but high cost
40
41 427 of the instrument restricted the use of this technique in the routine analysis.
42
43
44
428
45
46 429 2.3.4. Nuclear magnetic resonance spectroscopy
47
48 430 Nuclear magnetic resonance (NMR) spectroscopy is increasingly applied to the analysis
49
50 431 of food and food components. In the field of edible oils and fats, NMR provides useful
51
52
53 432 information on the lipid class composition (the relative amount of triacylglycerols,
54
55 433 diacylglycerols, FFA, sterols, phospholipids, and other minor components). The
56
57
434 determination of FFA by NMR was first studied on the lipids extracted from albacore
58
59
60 19
Page 21 of 29 Analytical Methods

1
2
3
435 tuna (Thunnus alalunga) using high-resolution l3C NMR spectroscopy.75 FFA carbonyl
4
5
6 436 resonances were detected at the lower field of the 13C NMR carbonyl region and used for
7
8 437 its quantitative determination. NMR data agreed with those obtained using UV
9
10
438 spectrophotometry, showed that NMR is a suitable method to follow lipolytic alterations.

Analytical Methods Accepted Manuscript

11
12
13 439 It was mentioned the method could be used to monitor the changes in fish lipids and oils
14
15 440 during processing or storage. Sun and Moreira76 studied the correlation between NMR
16
17
18 441 proton relaxation times and the increase in FFA in soybean oil degradation. Soybean oil
19
20 442 was degraded by heating only and by frying corn tortilla dough for 10 min/h up to 50 h.
21
22 443 Both AOCS and NMR methods were used to determine the extent of oil degradation.
23
24
25 444 Results showed that FFA increased as heating time increased. The longitudinal relaxation
26
27 445 time (T1) and the transverse relaxation time (T2) decreased as oil degradation increased.
28
29
446 There was a linear relationship between NMR and AOCS measurements. The number of
30
31
32 447 methods reported on 13C NMR77,78, 31P NMR79, and 1H NMR for FFA in vegetable oils.78,
33
34 80
448 Although the developed methods were easy, rapid and simple and could be used as a
35
36
37 449 promising tool for online quality control, but did not received much attention from the
38
39 450 industry due to the high price of the NMR equipment and technical skills.
40
41 451
42
43 452 2.4. Thermogravimetric methods
44
45
46 453 Thermometric methods are also titrimetric methods that use the change of temperature
47
48 454 resulting from a chemical reaction to determine the end point of the titration. The
49
50 455 principle is the same as that for potentiometric auto-titration; the only difference is the
51
52
53 456 type of sensor (detector) usually thermistors are used. Vaughan and Swithenbank81
54
55 457 reported first considerable advances in determining the end point of the acid/base reaction
56
57
458 in non-aqueous medium. A novel titrimetric procedure has been reported for the
58
59
60 20
 Analytical Methods Page 22 of 29

1
2
3
459 determination of FFA in edible oils using an automated thermometric titration with KOH
4
5
6 460 in isopropanol as titrant.82 The end point was indicated by the strongly exothermic base-
7
8 461 catalyzed reaction between acetone and chloroform. The procedure was accurate,
9
10
462 sensitive, easy to use, fast, and highly reproducible but did not get much attention.

Analytical Methods Accepted Manuscript

11
12
13 463
14
15 464 3. Conclusion
16
17
18 465 The above described instrumental techniques proposed considerable merits over classical
19
20 466 titration methods in terms of speed, automation and reduction of costly as well as toxic
21
22 467 chemicals. Calorimetric methods have poor sensitivity and need frequent calibration.
23
24
25 468 Electrochemical methods require titrant standardization while accuracy and
26
27 469 reproducibility depends on the conditions of the electrodes. Optimization of several
28
29
470 parameters is also very essential. The discussion reflects that none of the methods
30
31
32 471 described provides specificity to FFA measurement as all are based on measuring total
33
34 472 acidity rather than FFA. Among the spectroscopic methods, FTIR methods have proved
35
36
37 473 to be more beneficial in terms of simplicity, economy, environment friendly nature and
38
39 474 speed of analysis, especially when using direct approach for the determination of FFA in
40
41 475 vegetable oils. However, selection of the method for FFA determination depends on the
42
43
44 476 availability of equipment, skilled worker, purpose of study and number of samples.
45
46 477
47
48 478
49
50
51 479
52
53 480
54
55
56
481
57
58
59
60 21
Page 23 of 29 Analytical Methods

1
2
3
482 Conflict of Interest
4
5
6 483 The authors declare that there are no conflicts of interest.
7
8 484
9
10
485 Acknowledgement

Analytical Methods Accepted Manuscript

11
12
13 486 The authors would like to thank the National Centre of Excellence in Analytical
14
15 487 Chemistry, University of Sindh, Jamshoro, Pakistan for the financial support.
16
17
18 488
19
20
21 489
22
23 490
24
25
26 491
27
28
29 492
30
31
32 493
33
34 494
35
36
37 495
38
39
40 496
41
42
43 497
44
45 498
46
47
48 499
49
50
51 500
52
53
54 501
55
56
502
57
58
59
60 22
 Analytical Methods Page 24 of 29

1
2
3
4 503 References
5
6 504 1. R. D. OBrien, Fats and Oils: Formulating and Processing for Applications,
7
8
9 505 Technomic Publishing Company, Lancaster, PA, 1998.
10
506 2. Official Methods and Recommended Practice of the American Oil Chemists

Analytical Methods Accepted Manuscript

11
12
13 507 Society, 4th edn. Champaign, IL, 1989.
14
15
16 508 3. Official methods of analysis of the Association of Official Analytical Chemists,
17
18 509 15th edn. USA, 1990.
19
20 510 4. Determination of the free fatty acids in European Commission Regulation (EEC)
21
22
23 511 No. 2568/91, Off. J. Eur. Comm. No L 248 (5.9.91) p. 6.
24
25 512 5. C. W. Ayers, Anal. Chim. Acta, 1956, 15, 7783.
26
27
28
513 6. D. A. Baker, J. Am. Oil Chem. Soc., 1964, 41, 2122.
29
30 514 7. G. S. Bains, S.V. Rao and D.S. Bhatina, J. Am. Oil Chem. Soc., 1964, 41, 831
31
32 515 832.
33
34
35 516 8. R. R. Lowry and I. J. Tinsley, J. Am. Oil Chem. Soc., 1976, 53, 470472.
36
37 517 9. L. Ekstrom, J. Am. Oil Chem. Soc., 1981, 58, 935938.
38
39 518 10. D. Y. Kwon and J. S. Rhee, J. Am. Oil Chem. Soc., 1986, 63, 8992.
40
41
42 519 11. M. Brenardez, L. Pastoriza, G. Sampedro, J. J. Herrera and M. L. Cabo, J. Agric.
43
44 520 Food Chem., 2005, 53, 19031906.
45
46 521 12. M. Gyorik, Z. Ajtony, O. Doka, A. Alebic-Juretic, D. Bicanic and A. Koudijs,
47
48
49 522 Instrum. Sci. Technol., 2006, 34, 119128.
50
51 523 13. J. S. Canham and G. E. Pacey, J. Am. Oil Chem. Soc., 1987, 64, 10041007.
52
53
54
524 14. R. Puchades, A. Suescun and A. Maquieira, J. Sci. Food Agric., 66, 1994, 473
55
56 525 478.
57
58
59
60 23
Page 25 of 29 Analytical Methods

1
2
3
526 15. Z. Zhi, A. Rios and M. Valcarcel, Anal. Chim. Acta, 1996, 318, 187194.
4
5
6 527 16. P. Linares, M. D. Luque de castro and M. Valcarcel, Anal. Chim. Acta, 1989,
7
8 528 225, 431436.
9
10
529 17. P. G. Nouros, A. G. Constantinos and M. G. Polissiou, Anal. Chim. Acta, 1997,

Analytical Methods Accepted Manuscript

11
12
13 530 351, 291297.
14
15 531 18. E. Mariotti and M. Mascini, Food Chem., 2001, 73, 235238.
16
17
18 532 19. E. Kardash and Y. I. Turyan, Croat. Chem. Acta, 2005, 78, 99103.
19
20 533 20. B. Saad, C. W. Ling, M. S. Jab, B. P. Lim, A. S. Mohamad Ali, W. T. Wai and M.
21
22 534 I. Saleh, Food Chem., 2007, 102, 14071414.
23
24
25 535 21. H. F. Ayyildiz, H. Kara and S. T. H. Sherazi, Lipids, 2011, 46, 11811190.
26
27 536 22. A. Makahleh and B. Saad, Anal. Chim. Acta, 2011, 694, 9094.
28
29
537 23. J. A. Aricetti and M. Tubino, J. Am. Oil Chem. Soc., 2012, 89, 21132115.
30
31
32 538 24. P. A. Walde, J. Am. Oil Chem. Soc., 1990, 67, 110115.
33
34 539 25. P. A. Walde and C. Nastruzzi, Food Chem., 1991, 39, 249256.
35
36
37 540 26. I. B. Rejeb and M. Gargouri, Anal. Lett., 2011, 44, 14541462.
38
39 541 27. S. N. Fedosov, J. Brask and X. Xu, J. Am. Oil Chem. Soc., 2012, 89, 21552163.
40
41 542 28. C. G. Zoski, Handbook of Electrochemistry. Elsevier Science. 2007.
42
43
44 543 29. K. Takamura and Y. Hayakawa, Microchem. J., 1972, 17, 546555.
45
46 544 30. F. Kusu and K. Takamura, JAOAC Int., 1994, 77, 16861689.
47
48 545 31. T. Fuse, F. Kusu and K. Takamura, Bunseki Kagaku, 1995, 44, 2933.
49
50
51 546 32. T. Fuse, F. Kusu and K. Takamura, J. Pharm. Biomed. Anal., 1997, 15, 1515
52
53 547 1519.
54
55
56
548 33. T. Fuse, F. Kusu and K. Takamura, J. Chromatogr. A, 1997, 764, 177182.
57
58
59
60 24
 Analytical Methods Page 26 of 29

1
2
3
549 34. A. Kotani, F. Kusu and K. Takamura, Anal. Chim. Acta, 2002, 465, 199206.
4
5
6
7
550 35. S. G. Li, H. Zhang and W. T. Xue, Eur. J. Lipid Sci. Technol., 2007, 109, 1088
8
9 551 1094.
10

Analytical Methods Accepted Manuscript

11
12 552 36. X. Yu, C. Yang and S. Du, J. Gao, Food Anal. Meth., 2012, 5, 14531458.
13
14 553 37. T. M. Lapshina, Y. I. Turyan and S. I. Danilchuk, J. Anal. Chem. USSR, 1991,
15
16
17 554 46, 1150840.
18
19 555 38. Y. I. Turyan, O. Y. Berzin, I. Kuselman and A. Shenhar, J. Am. Oil Chem. Soc.,
20
21
22
556 1996, 73, 295301.
23
24 557 39. O. Y. Berezin, Y. I. Turyan, I. Kuselman and A. Shenhar, J. Am. Oil Chem. Soc.,
25
26 558 1996, 73, 17071711.
27
28
29 559 40. O. Y. Berezin, Y. I. Turyan, L. Kogan, I. Kuselman and A. Shenhar, J. Am. Oil
30
31 560 Chem. Soc., 1997, 74, 13391341.
32
33 561 41. I. Kuselman, Y. I. Turyan, O. Y. Berezin, L. Kogan, and A. Shenhar, J. AOAC
34
35
36 562 Int., 1998, 81, 873879.
37
38 563 42. A. Velasco-Arjona and M. D. Luque de Castro, J. Am. Oil Chem. Soc., 1998, 75,
39
40 564 18491853.
41
42
43 565 43. I. Kuselman, Y. I. Turyan, I. Burenko and B. Anisimov, Talanta, 1999, 49, 629
44
45 566 637.
46
47
48
567 44. Y. I. Turyan, E. Kardash and I. Garibyan, J. Am. Oil Chem. Soc., 2008, 85, 91
49
50 568 92.
51
52 569 45. E. O. Gerasimenko and Y. I. Turyan, Food Chem., 2012, 132, 15621565.
53
54
55 570 46. V. del Ro, M. S. Larrechi and M. P. Callao, Talanta, 2010, 81, 15721577.
56
57
58
59
60 25
Page 27 of 29 Analytical Methods

1
2
3
571 47. A. C. Lanser, G. R, List, R. K. Holloway and T. L. Mounts, J. Am. Oil Chem.
4
5
6 572 Soc., 1991, 68, 448449.
7
8 573 48. A. A. Ismail, F. R. van de Voort and J. Sedman, J. Am. Oil Chem. Soc., 1993, 70,
9
10
574 335341.

Analytical Methods Accepted Manuscript

11
12
13 575 49. E. Bertran, M. Blanco, J. Coello, H. Iturriaga, S. Maspoch and I. Montoliu, J.
14
15 576 Am. Oil Chem. Soc., 1999, 76, 611616.
16
17
18 577 50. Y. B. Che Man and G. Setiowaty, Food Chem., 1999, 66, 109114.
19
20 578 51. Y. B. Che Man, M. H. Moh and F. R. van de Voort, J. Am. Oil Chem. Soc., 1999,
21
22 579 76, 485490.
23
24
25 580 52. T. Verleyen, R. Verhe, A. Cano, A. Huyghebaert and W. De Greyt, J. Am. Oil
26
27 581 Chem. Soc., 2001, 78, 981984.
28
29
582 53. F. A. In, J. M. Garrigues, S. Garrigues, A. Molina-Daz and M. Dela Guardia,
30
31
32 583 Anal. Chim. Acta, 2003, 489, 5975.
33
34 584 54. S. T. H. Sherazi, S. A. Mahesar, M. I. Bhanger, F. R. Van De Voort and J.
35
36
37 585 Sedman, J. Agric. Food Chem., 2007, 55, 49284932.
38
39 586 55. S. Naz, S. T. H. Sherazi, F. N. Talpur, S. A. Mahesar and H. Kara, JAOAC Int.,
40
41 587 2012, 95, 1570573.
42
43
44 588 56. X. Yu, S. Du, F. R. van de Voort, T. Yue and Z. Li, Anal. Sci., 2009, 25, 627
45
46 589 632.
47
48 590 57. X. Yu, F. R. van de Voort, J. Sedman and J. Gao, Anal. Bioanal. Chem., 2011,
49
50
51 591 401, 315324.
52
53 592 58. M. J. A. Caada. A. R. Medina and B. Ledle, Appl. Spectrosc., 2001, 55, 356
54
55
56
593 360.
57
58
59
60 26
 Analytical Methods Page 28 of 29

1
2
3
594 59. A. Al-Alawi, F. R. van de Voort and J. Sedman, J. Am. Oil Chem. Soc., 2004, 81,
4
5
6 595 441446.
7
8 596 60. A. Al-Alawi, F. R. van de Voort and J. Sedman, Spectrosc. Lett., 2005, 38, 389
9
10
597 403.

Analytical Methods Accepted Manuscript

11
12
13 598 61. A. Al-Alawi, F. R. van de Voort, J. Sedman and A. Ghetler, JALA, 2006, 11, 23
14
15 599 29.
16
17
18 600 62. Y. Li, D. L. Garcia-Gonzalez, X. Yu and F. R. van de Voort, J. Am. Oil Chem.
19
20 601 Soc., 2008, 85, 599604.
21
22 602 63. A. N. A. Aryee, F. R. van de Voort and B. K. Simpson, Process Biochem., 2009,
23
24
25 603 44, 401405.
26
27 604 64. E. N. Frankel, A. M. Nash and J. M. Snyder, J. Am. Oil Chem. Soc., 1987, 64,
28
29
605 987992.
30
31
32 606 65. Z. Hui-Zhen and L. Tung-Ching, J. Agric. Food Chem., 1997, 45, 35153521.
33
34
35 607 66. Y. B. Che Man and M. H. Moh, J. Am. Oil Chem. Soc., 1998, 75, 557562.
36
37 608 67. D. Cozzolino, I. Murray, A. Chree and J. R. Scaife, Food Sci. Technol., 2005, 38,
38
39
609 821828.
40
41
42 610 68. C. R. Moschner and B. Biskupek-Korell, Eur. J. Lipid Sci. Technol., 2006, 108,
43
44 611 606613.
45
46
47
612 69. C. L. Ng, R. L. Wehling and S. L. Cuppett, J. Agric. Food Chem., 2007, 55, 593
48
49 613 597.
50
51 614 70. J. A. Gerde, C. L. Hardy, C. R. Hurburgh Jr and P. J. White, J. Am. Oil Chem.
52
53
54 615 Soc., 2007, 84, 519522.
55
56 616 71. L. P. Houmoller, D. Kristensen and H. Rosager, Talanta, 2007, 71, 868873.
57
58
59
60 27
Page 29 of 29 Analytical Methods

1
2
3
617 72. X. Yu, J. Zhang, Q. Li, C. Xu and J. Gao, Trans. Chinese Soc. Agric. Machin.,
4
5
6 618 2012, 43, 150154.
7
8 619 73. B. Muik, B. Lendl, A. Molina-Da z and M. J. Ayora-Caad, Anal. Chim. Acta,
9
10
620 2003, 487, 211220.

Analytical Methods Accepted Manuscript

11
12
13 621 74. R. M. El-Abassy, P. Donfack and A. Materny, J. Am. Oil Chem. Soc., 2009, 86,
14
15 622 507511.
16
17
18 623 75. R. Sacchi, I. Medina, S. P. Aubourg, I. Giudicianni, L. Paolillo and F. Addeo, J.
19
20 624 Agric. Food Chem., 1993, 41, 12471253.
21
22 625 76. X. Sun and R. G. Moreira, J. Food Process Preserv., 1996, 20, 157167.
23
24
25 626 77. S. Ng, J. Am. Oil Chem. Soc., 2000, 77, 749755.
26
27 627 78. R. Kumar, V. Bansal, A. K. Tiwari, M. Sharma, S. K. Puri, M. B. Patel and A. S.
28
29
628 Sarpal, J. Am. Oil Chem. Soc., 2011, 88, 16751685.
30
31
32 629 79. F. M. Dayrit, O. E. M. Buenafe, E. T. Chainani and I. M. S. De Vera, J. Agric.
33
34 630 Food Chem., 2008, 56, 57655769.
35
36
37 631 80. J. K. Satyarthi, D. Srinivas and P. Ratnasamy, Energ. Fuel, 2009, 23, 22732277.
38
39 632 81. G. A. Vaughan and J. J. Swithenbank, Analyst, 1965, 90, 594599.
40
41 633 82. K. S. Thomas, J. Am. Oil Chem. Soc., 2003, 80, 2124.
42
43
44 634
45
46 635
47
48
49 636
50
51
52
53
54
55
56
57
58
59
60 28