In-Depth Assessment of Analytical Methods for Olive Oil Purity

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An In-Depth Assessment of Analytical Methods for Olive Oil Purity, Safety and Quality Characterization Noelia Tena, Selina C Wang, Ramon Aparicio-Ruiz, Diego Luis Garcia-Gonzalez, and Ramon Aparicio J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 20 Apr 2015 Downloaded from http://pubs.acs.org on April 26, 2015

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Journal of Agricultural and Food Chemistry

An In-Depth Assessment of Analytical Methods for Olive Oil Purity, Safety and Quality Characterization

Tena Noelia1, Wang Selina C2, Aparicio-Ruiz Ramón1, García-González Diego L.1, Aparicio Ramón1.

1

Instituto de la Grasa (CSIC), University Pablo de Olavide - Building 46, Ctra. de Utrera, km. 1 E– 41013, Sevilla, Spain.

2

Olive Center, University of California - Davis, Davis, California 95616, United States.

*Corresponding author: Tel: +34 954611550

Fax: +34 954616790

E-mail: [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT

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This paper evaluates the performance of the current analytical methods (standard and

3

widely used otherwise) that are used in olive oil for determining fatty acids, triacylglycerols,

4

mono and di acylglycerols, waxes, sterols, alkyl esters, erytrodiol and uvaol, tocopherols,

5

pigments, volatiles and phenols. Other indexes that are commonly used, such as free acidity

6

and peroxide value, are also discussed in relation to their actual utility assessing quality and

7

safety and their possible alternatives. The methods have been grouped based on their

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applications: (i) purity and authenticity; (ii) sensory quality control; and (iii) unifying methods

9

for different applications. The speed of the analysis, advantages and disadvantages, and

10

multiple quality parameters are assessed. Sample pre-treatment, physicochemical and data

11

analysis, and evaluation of the results have been taken into consideration. Solutions based on

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new chromatographic methods or spectroscopic analysis and their analytical characteristics

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are also presented.

14 15

Keywords: Olive oil, quality, authentication, analytical methods, trade standards

16 17 18 19 20 21 22 23 24 25

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Introduction

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Virgin olive oil, the most valuable olive oil category, is highly appreciated around the

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world for its healthy properties, principally due to the presence of monounsaturated fatty acid

29

(oleic acid 18:1 ω-9) and antioxidant compounds (phenols, tocopherols, and chlorophyll

30

pigments). Its high price is also explained by its genuine organoleptic properties, which make

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it an excellent ingredient in many recipes. Olive oils, including those that are lower grade than

32

virgin olive oil are generally more expensive than the most commonly consumed vegetable

33

oils, thus the difference in price makes it a very attractive product for fraudsters. The possible

34

adulterations can be classified into two types; (i) mixture of different categories of olive oil

35

and (ii) mixture with other vegetable oils. In order to detect these adulterations and to

36

guarantee the quality and safety of olive oil, this product has been at the forefront of the

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implementation of multiple standard methods. Thus, olive oil has become one of the most

38

strictly regulated food products.

39

The components of olive oil are numerous and they are usually clustered into major

40

and minor compounds. Due to the high number of chemical compounds, which results in a

41

highly complex matrix, the number of analytical methods used to characterize them is also

42

large. Table 1 shows the chemical composition of three virgin olive oils - from three cultivars

43

and producer countries – characterized by only a few chemical compounds of those ones

44

commonly used in traceability

45

Currently the regulatory bodies and associations for olive oil present an extensive

46

collection of analytical methods to characterise olive oils and olive-pomace oils and to avoid

47

possible frauds. These organizations are, among others, International Olive Council (IOC),

48

European Commission (EC), Codex Alimentarius Commission, (CODEX STAN), German

49

Society for Fat Science (DGF), Association of Official Analytical Chemistry (AOAC),

50

American Oils Chemists' Society (AOCS), International Union of Pure and Applied

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51

Chemistry (IUPAC), Federation of Oil Seeds and Fats Association (FOSFA). Olive oil is

52

subjected to worldwide trade, and international regulation is provided by EC1, IOC2 and the

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CODEX STAN.3 EC regulations are enforced in EU countries, while the member countries of

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IOC and Codex standards sign voluntary agreements that include the limits established for

55

each quality and purity criteria including precision values. The limits adopted for each

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analytical parameter can sometimes vary from the EU regulations to IOC trade standards,

57

consequently harmonization is always in progress to minimize hurdles to international trade.4

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The IOC trade standards are updated once a year, while EU often needs more time to publish

59

regulations. For this reason IOC is usually more proactive in including methods and also it

60

can standardize and validate methods to determine parameters that have no associated

61

detection limits (e.g., phenols). Thus, IOC has approved, or has under revision, 18 standard

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methods to characterise olive oil and pomace olive oil,2 16 of them being based on liquid or

63

gas chromatographic techniques. These methods are, however, time consuming, they need a

64

considerable amount of solvents, and an expert analyst is often required for their

65

implementation. Although the standard methods are developed for specific purposes,

66

advances in technology and knowledge have led to the approval of new methods that

67

sometimes overlap existing ones. This is the case for the determination of waxes content by

68

capillary gas chromatography,5 which overlapped with the method approved in 2010 to

69

determine the content of waxes, fatty acid methyl esters and fatty acid ethyl esters by capillary

70

gas chromatography.6 This wide range of possible standard methods is sometimes perceived

71

as a disadvantage by newcomers to the olive oil sector instead of an advantage when selecting

72

a method for their own needs. As there are several standard methods to solve the same

73

problem (i.e., to detect the same adulterant or to identify and quantify the same chemical

74

compounds), the analysts often have difficulties establishing which standard method is the

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most suitable for their purposes. The extensive collection of standard methods is partially due

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to the fact that the regulatory bodies add the new methods to the list of the standard methods

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keeping the previous ones. This additive—instead of substitutive—policy seems to imply that

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not all the standard methods satisfy completely the requested expectations. Consequently, a

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considerable investment in optimizing current methods and developing new ones is important

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in order to keep pace with changing technologies and sophisticated adulterations. These new

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methods should be easy to implement, robust, have an adequate precision, be amenable to

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being validated, and be capable of detecting any fraud and malpractices in olive oil

83

production.

84

In addition to the difficulties of analysts in choosing the most adequate standard

85

method, they may have problems in applying the methods due to lack of experience. For

86

example, they may have problems in the identification of the chemical compounds, due to

87

possible shifts in the retention time, small changes in column composition, temperature

88

program or low resolution of peaks. Thus, in the particular case of analysts that work with

89

olive oil for the first time, they often need to discuss their results with other analysts, expert in

90

the field, to identify any problem and to confirm that they are working properly.

91

Not only is the expertise of the analyst a critical issue. There are also other factors

92

such as the quality of equipment, reagents, environment (e.g., temperature, humidity, air

93

pollution), among others that can make the results difficult to reproduce. Validated analytical

94

methods are thus essential for the quality performance of analytical laboratories. Today,

95

analysts must be aware of the important role of quality assurance of their laboratories. The

96

results of the laboratories are directly related to the use of validated and accredited methods.

97

Regarding laboratory accreditation, the complexity of the standard methods is again a

98

problem. A better justification for the existing methods through a detailed description of their

99

application and the avoidance of any overlap between them is desirable to facilitate the

100

accreditation of laboratories.

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Understanding all the drawbacks associated with the considerable complexity of

102

standard methods, the olive oil sector is demanding the development of a rapid and universal

103

method that can be used for all the determinations required to confirm olive oil authenticity

104

and/or quality. Despite the difficulties implementing the current standard methods, certain

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improvements have being achieved. For example, the compilation of information about the

106

validation of the standard methods7 has allowed a better monitoring of the reproducibility and

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robustness of the methods. Additionally, the optimization of some standard methods has been

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carried out by improving columns, using different solvents, changing chromatographic

109

conditions and reducing the pretreatment of samples.

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The improvement of the standard methods requires time, and the alternative to the

111

current situation could be the design of a global procedure that allowed the detection of

112

different adulterations. Thus the strategy of some current research studies is to combine

113

standard methods and emerging technologies—including fingerprinting approaches—into a

114

single comprehensive analytical strategy. The analytical techniques that have been suggested

115

to achieve this aim are spectroscopic techniques—near-infrared (NIR) spectroscopy,8 Fourier

116

transform mid-infrared spectroscopy (FTIR),9 FT-Raman spectroscopy,10 fluorescence

117

spectroscopy11

118

techniques—isotope ratio mass spectrometry12 —and “omic” techniques.14 In these areas, the

119

fraudsters do not have much information on how to avoid being detected in, but analysts may

120

have problems in interpreting the information with plausible chemical explanations. These

121

techniques can be helpful if they are combined with multivariate statistical techniques. Even

122

then, the conclusions obtained from the analysis ideally should be supported by chemical or

123

biochemical explanations. These new techniques, which provide a wide range of information

124

about different chemical compounds in only one analysis, need to be validated with blind

125

samples prior to being proposed as standards.

nuclear

magnetic

resonance

(NMR)

spectroscopy13—spectrometric

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In this paper the standard methods have been analysed from a point of view of the

127

analysts and the potential problems that they may have when applying them. The practical

128

aspects and the possible alternatives (non standard methods) for each determination have been

129

discussed as well. A critical review about the chemical compounds analysed by these methods

130

and the concentration range of these compounds in olive oil is presented, together with the

131

utility of these determinations in the characterization and authentication of olive oil.

132 133

Methods for the control of olive oil purity and authenticity

134

The chemical compounds used for determining olive oil authenticity are numerous and

135

in order to explain their analytical methods, the compounds have been clustered into the sets

136

of major and minor compounds. The group of major compounds is primarily made up of

137

triacylglycerols (TAGs) or glycerol esters of fatty acids (FAs); fatty acids account for 94-95%

138

of the total weight of TAGs. Table 2 shows some examples of application of these major and

139

minor compounds in detecting adulterations.

140

The broad and heterogeneous set of minor compounds includes several series of

141

chemical compounds with a lipid structure (e.g., waxes), compounds that are not related to

142

lipids from a chemical-structure viewpoint (e.g., pigments, volatiles), and compounds that

143

have the common characteristic of being obtained from unsaponifiable matter. The

144

compounds that are present in unsaponifiable matter, which rarely represent more than 2% by

145

weight of total olive oil, are regarded as the olive oil fingerprint. Thus, this fingerprint is very

146

useful in authentication and geographical identification of olive oil, among other applications.

147 148

Olive oil major compounds

149

Fatty acid methyl esters and trans fatty acids

150

Fatty acids (FAs) are the main components of olive oil. They are not present, however,

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151

as free fatty acids; when they are, they occur only in small amounts. Fatty acids are usually

152

forming part of glycerides (mono-, di- and tri-acylglycerols) and phospholipids through ester

153

bonds. FAs present in olive oil are linear chain of 16–24 carbon atoms. FAs, which were

154

some of the very early compounds to be analysed by gas chromatography (GC), were of

155

maximum importance in the detection of adulterants until the end of the 60s. At that time seed

156

oils with a modified fatty acid composition similar to olive oil appeared. The analytical

157

performances, however, were improved and made the method suitable to determine trans-

158

isomers of fatty acids because trans isomers of fatty acids (TFAs) appears as a result of

159

certain manipulations, for instance isomerization reactions that occur during the heating

160

processes of olive oil.15 The interest in determining the trans isomers of fatty acids is

161

explained by the fact that their determination allows the detection of refining oils in virgin

162

olive oil. Furthermore, GC analysis of fatty acids is also carried out as a tool for the

163

calculation of the theoretical composition of TAGs with the final objective of determining the

164

value of the theoretical ECN42 (Equivalent Carbon Number),16 and for the detection of the

165

presence of hazelnut oil by means of the Global Method.17

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The first step of FA analysis is derivatization by forming methyl esters (FAMEs),

167

since they are more volatile and nonpolar than free fatty acids, and this makes them easier to

168

elute through chromatographic columns. Among the methods for preparing FAMEs18 the

169

trans-esterification with methanolic solution of potassium hydroxide at room temperature19,20

170

is the most commonly used. However, another method is suggested for method for VOOs

171

with acidity higher than 3.3% and crude olive pomace, which is based on the esterification

172

and trans-esterification with sodium methylate in methanol in acid conditions at high

173

temperature.18 Perhaps the first method can be also suggested for the determination of trans-

174

fatty acid and the determination of ECN42 if samples are always firstly purified through a

175

silica gel solid-phase extraction cartridge, and the sample spiked with hexane—olive oil:

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hexane (1:4); e.g. 0.15 g of olive oil in 0.6 mL of hexane—is pulled down with hexane/diethyl

177

ether (87:13) as in other standards.18

178

GC analysis can be undertaken with chromatographic columns with diverse

179

characteristics. For instance, phases with the highest polarity allow excellent separations of

180

polyunsaturated FAs. Phases with lower polarity are used for the separation of saturated and

181

monoenoic compounds with the same chain length although it may produce peaks that

182

interfere in the determination of trans isomers of linolenic acid. An HP-88 capillary column –

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coated with 88% cyanopropyl aryl siloxane (100 m × 0.2 mm i.d. × 0.2 µm film thickness) –

184

or a Varian Chrompack CP-Sil 88 column – coated with cyanopropyl polysiloxane (100 m ×

185

0.25 mm i.d. × 0.2 µm film thickness) – can provide an accurate quantification of TFA

186

isomers previously derivatized as methylesters. These columns are too long for FAs and can

187

be substituted by others of 50 m × 0.20-0,32 mm i.d. 0.1× 0.2 µm film thickness with a cross-

188

linked stationary phase of cyanopropylsiloxane allowing also the trans-fatty acids

189

determination (Table 3A). However, columns shorter than 50 m allow determining FAs but

190

with loss of trans-fatty acids.

191

The stationary phase polymerizes in aged columns, and the variation of retention times

192

for the esters of FAs is observed. Better peak resolution is obtained if the carrier gas is

193

hydrogen instead of helium. Furthermore, no silver ion pre-fractionation of TFA (by TLC,

194

SPE, or HPLC) is required prior to the GC analysis.21

195

The use of WCOT columns of fused silica introduced remarkable improvements in

196

separations with higher resolution, higher precision in analysis both in quality and quantity,

197

higher sensitivity, a shorter analysis time, reduction in the preparation of analysis procedures,

198

etc. Most phases are now chemically bonded to tube walls, which decrease loss in the column

199

and, in turn, increase duration and temperature resistance and improve the resolution.

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200

The determination of trans fatty acids can present a problem of overestimation. Small

201

quantities of trans oleic acid could be formed when the sample is introduced into the gas

202

chromatographic injector. This problem is more pronounced when automatic samplers with

203

packing materials are used. To minimize this problem it is necessary to replace the injector

204

insert by a clean insert, deactivate the insert, use lower injector temperature from 250 ºC to

205

30-50 ºC or remove the packing materials if the injection is manual.

206

Alternative methods based on spectroscopic techniques have been developed to

207

analyze FAs and trans-fatty acids although they cannot determine individual FAs and TFAs

208

as GC method does. Thus, 1H-NMR methodology shows a good performance for FAs

209

quantification despite the fact that results for individual saturated compounds often deviate

210

from GC results because of the sensitivity of 1H-NMR (400 MHz) integration; total amounts

211

of saturates are, however, accurately determined.22 Quantification of the sum of saturated fatty

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acids (SFAs), the monounsaturated oleic acid (MUFA), and the polyunsaturated linoleic and

213

linolenic acids (PUFAs) can be obtained by means of mathematical equations using

214

appropriate signal intensities as variables.22 Additionally linolenic acid can be quantified from

215

the signal of the methyl protons of the linolenyl chain at δ 0.96.

216

TFAs can also be determined by IR spectroscopy, which is a rapid tool for detecting

217

isomers.23 The intensities of Raman spectroscopy bands near 1656 cm-1 and 1670 cm-1 have

218

also been related to the content of cis and trans isomers.24

219 220

Triacyglycerols

221

Triacylglycerols (TAG) consist of a glycerol moiety with each hydroxyl group

222

esterified to a fatty acid. Each individual TAG species has three characteristics: (i) the total

223

carbon number (CN), which is the sum of the alkyl chain lengths of each one of the 3 FAs; (ii)

224

the degree of unsaturation in each FA; and (iii) the position and configuration of the double

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bonds in each FA.25 Twenty TAGs have been identified and independently quantified in olive

226

oil, but only five are present in significant proportions.18

227

One of the main aims of TAG analysis is identifying TAGs that are biosynthesized and

228

those that are synthesized by means of FFAs esterification with glycerol. The analytical

229

approach is based on the use of pancreatic lipase, an enzyme that has the ability to hydrolyze

230

the 1- and 3-position of natural TAGs, thereby producing 2-monoacylglycerols. The initial

231

determination of palmitic and stearic acids at the 2-position has been currently substituted by

232

determination of the content of 2-glyceryl monopalmitate.26,27

233

Thus, the possible combinations of FA molecules and their positions on the glycerol

234

backbone of TAGs makes the analysis of the TAG composition a very challenging task either

235

with GLC (gas-liquid chromatography) or RP-HPLC (reversed phase liquid chromatography).

236

Table 3A summarizes the RP-HPLC conditions (column dimension, stationary and mobile

237

phases, kind of detector, and flow-rate) and GLC conditions (columns, detectors and

238

chromatographic conditions) used for the determination of olive oil TAG profiles.

239

TAGs separation is based on the number of carbon atoms and their unsaturation when

240

RP-HPLC is used because of the good resolution of RP-18 column. In the case of the

241

presence of oxidized compounds, which interfere in the trilinolein (LLL) determination,

242

samples should be purified by means of silica gel. Thus, in the case of olive-pomace oil, the

243

European Commission recommends to use the SEP PAK silica cartridge (Waters).28 The IOC

244

recommends making the purification by SPE according to IUPAC 2.507.29

245

This procedure, however, implies checking FA compositions in the oil after its

246

purification in a long and tedious process during which mistakes can occur. This test is

247

recommended by the European Commission in the case of the olive pomace oil and the IOC

248

recommends this test in the determination of the 2-glyceryl monopalmitate.26

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249

The purified oil is resolved in acetone, but sometimes the impurities of acetone can

250

produce disturbances in the baseline of the chromatogram (region 12-15 min); if this happens

251

a good solution is using another source of acetone or a mixture of propionitrile/acetone (25/75

252

v/v).

253

The RP-18 column resolution depends on the particle diameter. Experience shows that

254

columns with 3 µm have no loss of resolution while saving time and solvents. Another

255

empirical aspect concerns samples of raw olive-pomace oils, which should be resolved in

256

acetone and then filtered, with 0.2 µm pore size, to remove precipitates that would shorten the

257

column life.

258

Regarding to the mobile phase, critical peaks are effectively separated with

259

acetone/acetonitrile in isocratic elution, although with this phase, saturated long-chain TAGs

260

are not resolved enough. As a consequence, some researchers30,31 carried out experiments on

261

isocratic elution with propionitrile as previously investigated.32 Although propionitrile (HPLC

262

grade) is expensive and highly toxic, it reduces dramatically the baseline drift when compared

263

with the methods based on an acetone/acetonitrile mixture. The result is a better separation of

264

TAGs clustered as ECN42, and it is used for the detection of the admixture of hazelnut oil to

265

olive oil.33

266

The refractive index (RI) is the most appropriate detector, despite its drawbacks of low

267

sensitivity and of different response towards saturated and highly unsaturated TAGs and the

268

numerous external variables that affect baseline stability.34 Concerning other detectors, UV

269

detector, which has not been very widely used because it presents problems with

270

isomerization and conjugation of double bonds, has good sensitivity and allows the use of

271

elution gradients that improve the resolution.

272

The alternative of using GLC (Table 3A) is scarcely applied today despite the fact that

273

it also offers attractive possibilities as an efficient separation method, good quantitative

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recovery and reproducibility, an adequate time for analysis and the availability of a flame

275

ionization detector (FID), a simple but universal linear response detector. But the GLC

276

technique is not free of problems such as, for example, the injection system and the column

277

deterioration. Selectivity in GLC depends on the length and chemical nature of the column

278

stationary phase. For example, columns with phenyl-methyl-silicone phase are capable of

279

reaching temperatures of about 360-370 ºC for a long time, and of separating TAGs by carbon

280

atom number; unsaturated positional isomers cannot be separated by this phase unless sample

281

derivatization followed by a reduction had previously been done.18

282

Another alternative is the analysis by HPLC-MS because MS can identify partially

283

resolved HPLC peaks35 so giving much more information on the position of the three fatty

284

acid molecules at TAGs. Although few ionization techniques can be coupled to HPLC, the

285

identification of positional isomers36 can be carried out by APCI (atmospheric pressure

286

chemical ionization) coupled to HPLC while the identification of individual acyls can be done

287

without electrospray ionization (ESI) without reference materials. A rapid and simple sample

288

preparation – no analyte purification, chemical modification or derivatization is required37 –

289

has increased the interest in applying MALDI-TOF-MS (matrix-assisted laser desorption

290

ionization time-of-flight mass spectrometry); LDI-TOF-MS being proposed for detecting the

291

presence of sunflower oils in olive oil.38

292 293

Olive oil minor compounds

294

As already mentioned, the set of minor components, which are used for the

295

determination of purity and authenticity of olive oil designations, consists of compounds that

296

derive from TAGs and other liposoluble compounds, and the compounds that derive from the

297

unsaponifiable matter.

298

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299

Diacylglycerols and Monoacylglycerols

300

Diacylglycerols (DAG), which are present in a range of 1% to 3% in olive oils, are

301

found as 1,2- and 1,3-isomers; 1,2-isomers being attributed to the incomplete biosynthesis of

302

TAGs (Kennedy pathway) whereas 1,3-isomers are attributed to enzymatic or chemical

303

hydrolysis of TAGs.

304

The official method39 allows the determination of DAGs and TAGs together. TAGs

305

are clustered on the basis of their carbon atom number while in the case of DAGs the peaks

306

are resolved according to their carbon atom number and their structure, the 1,2 isomers having

307

lower retention time than 1,3 isomers. The method does not require particular experience of

308

the analyst. Thus, 1 mL of the internal standard (dinonadecanoine 0.1% w/v in methyl-

309

tertbutyl-ether) is added to a sample of 100 mg. Later, a dried aliquot of 20-30 µL is silylated

310

with 200 µL of the silylation reagent, consisting of a 9:3:1 (V/V/V) mixture of anhidre

311

pyridine/hexamethyl disilazane/trimethylchlorosilane. After a complete silylation, 2 mL of n-

312

heptane is added and 0.5-1.0 µL is injected into a GC instrument equipped with a capillary

313

column of 3-7 m length × 0.25–0.32 mm i.d. (coated with SE52 or SE54 stationary phase).

314

Experience has taught that it is advisable to purify the sample by column chromatography or

315

SPE prior to adding the internal standard that could also be 1,3-dipalmitoyl rac-glycerol. The

316

silylation process can be critical. The solution of silylation reagent should be freshly made. It

317

is not easy to detect the final of the reaction; it would take about 20 minutes. If a slight

318

opalescence is observed after the silylation, it does not mean any anomaly. However the

319

formation of a white floc or the appearance of a pink color is indicative of the presence of

320

moisture. A rapid warming applied to the bottom of the conical flask to the GC detector can

321

eliminate the moisture. If after that the floc is not eliminated, the silylation reagent is

322

deteriorated and the test must be repeated.

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The method for the determination of the percentage of 2-glyceryl monopalmitate

324

(Table 3A) has some drawbacks such as the fact that it is a lengthy and tedious method (e.g.,

325

when sample acidity is > 3%, it has to be previously neutralized). The neutralization of the oil

326

is necessary because the activity of the pancreatic lipase depends on the pH, which should be

327

adjusted to 8.3. Furthermore, the separation between phases after lipase pancreatic digestion

328

is not automatic; it should be taken into account that lipase pancreatic is not stable and may

329

lose activity easily. The thick interface between phases (diethyl ether and aqueous phase),

330

after the centrifugation step, makes it difficult to collect an aliquot of the organic phase for

331

preparing the silanized derivatives. Moreover, on-column injection shows well-known

332

problems such as a large broad solvent front, low repeatability and tailing peaks.

333 334

Waxes

335

The waxes, which usually referred to wax esters because they are fatty acids esterified

336

to long-chain alcohols,20 contain even numbers of carbon atoms from C36 to C46 in olive oil.

337

Since waxes are in the epidermal cells of olives40, their concentration characterizes olive oil

338

pomace categories.

339

The official method for determining waxes5 uses lauryl arachidate in hexane as

340

internal standard and on-column GC injection. The standard method also suggests using

341

palmityl palmitate or myristyl stearate as internal standards. Aragón et al. 201141 propose to

342

use these internal standards in heptane to avoid the impurities that can be present in some

343

commercial hexane and to increase the time of its evaporation. Concerning sample

344

purification, SPE cartridges have replaced silica gel columns because the former requires

345

smaller amounts of sample that reduces the elution solvent volume. SPE cartridges, however,

346

need of much more attention from the analyst. Based on this change, new methods have been

347

proposed as alternatives to the standard method.42 They are more rapid with lower

348

consumption of organic solvents, and no difference concerning the recovery factors. 15



349

However, there is not much information about the precision and limits of quantification yet.

350

With respect to the chromatographic column, the standard method suggests using semipolar

351

columns with phenyl-methyl-silicone because this allows for a high resolution while resistant

352

at high temperatures. Columns, however, should be conditioned, prior to being used, with a

353

gradual heat until a temperature of 350 ºC is reached. It is important to note that in the last

354

standard method43 the sum of the contents of waxes is computed from C42 to C46, excluding

355

C40, unlike previous norms.5,6

356 357

Unsaponifiable Fraction

358

As mentioned earlier, the unsaponifiable matter fails to react with sodium hydroxide

359

and potassium hydroxide to produce soaps but remain soluble in classic solvents (e.g.,

360

hexane, petroleum ether, diethyl ether) after saponification. Thus, any method for determining

361

unsaponifiable matter involves olive oil saponification and unsaponifiable matter separation

362

by means of extraction with an appropriate solvent, such as diethyl or petroleum ether.

363

Although the determination of unsaponifiable matter seems simple, this type of analysis still

364

presents problems because of the lack of accuracy and precision in the results. Some

365

difficulties are the impossibility of extracting all of the unsaponifiable matter and the

366

formation of emulsions due to a too vigorous shaking. These emulsions can be destroyed

367

adding small quantities of ethanol. Other problems are soap hydrolysis, loss of unsaponifiable

368

matter during solvent drying, evaporation and incomplete saponification.

369

Although diethyl ether is the preferred solvent, it has a number of limitations. For

370

example, if soaps pass into the solvent together with the unsaponifiable matter, it is wise to

371

separate the soaps by washing the ether extract with an aqueous solution of sodium

372

hydroxide, which can provoke soap hydrolysis and liberate acids. After solvent evaporation at

373

a low temperature and reduced pressure, it is necessary to dry the unsaponifiable residue in

16


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374

order to ensure the elimination of any traces of solvent and water. A drying process should be

375

carried out to avoid possible volatile compounds to be present in the unsaponifiable fraction.

376

Water elimination is usually performed by treating the unsaponifiable extract with water-free

377

sodium sulfate followed by filtration. Finally, any remaining moisture traces are eliminated by

378

vacuum drying with acetone or benzene. Sometimes incomplete saponification may occur44,

379

in which case, error can be introduced due to the saponifiable portion of TAGs soluble in

380

ether and hexane. When incomplete saponification is thought to have happened the

381

unsaponifiable residue containing the nonsaponified segment has to be saponified and

382

extracted again following the same procedure.

383

Fractionation of the unsaponifiable components into several groups of constituents

384

(e.g., hydrocarbons, tocopherols, and sterols) can be carried out by several procedures, most

385

of them being chromatographic such as column chromatography and thin-layer

386

chromatography (TLC) with two different supports, alumina and silica gel (Table 3B). The

387

latter, which has traditionally been the chosen method for the complete fractionation of the

388

unsaponifiable matter, can be applied to unsaponifiables extracted from petroleum ether and

389

containing FFAs. In this case, firstly the silica gel must be treated with diluted potassium

390

hydroxide to retain the FFAs and avoid interference with the other fractions. In the separation

391

by thin-layer chromatography, the temperature is an important factor to have a good

392

separation; the optimum temperature is 20-25 ºC.

393 394

Currently HPLC has been described to carry out the fractionation of the unsaponifiable components, as described in the section focused on sterols.

395 396

Hydrocarbons

397

The hydrocarbons, which are present in olive oils, include from high amounts of

398

squalene (C30H50), an unsaturated linear triterpenic polymer of isoprene, to small quantities of

17



399

saturated, linear (from C-15 to C-35), branching and terpenic compounds. Steroidal

400

hydrocarbons (sterenes or steradienes) with two double bonds in the ring system are present in

401

refined olive oils (ROOs), while small quantities of polycyclic aromatic hydrocarbons (PAHs)

402

have been quantified in olive-pomace oils.

403

The need of detecting the presence of refined edible oils in virgin olive oils (VOOs)

404

resulted in a standard,45 Lanzón (1990)46 being the pioneer in suggesting stigmasta-3,5-diene

405

(derived from β-sitosterol) as a marker for detecting admixtures of VOO with refined

406

vegetable oils. The method is based on the isolation of unsaponifiable matter, separation of

407

steroidal hydrocarbon fraction with a 15 g silica gel chromatography column, and analysis by

408

on-column GC with a column of 25 m coated with 5% phenyl-methyl silicone phase, as firstly

409

proposed by Lanzón (1990).46 The separation by silica gel chromatographic can result in

410

chromatograms with interfering peaks because the silica has low acidity. If this occurs, the

411

silica gel should be treated by heating for a minimum of four hours at 550 ºC or extra pure

412

silica gel 60 (Merck, ref. 7754) could be used.

413

Technological advancements have allowed for the preparation of fraudulent mixtures

414

of desterolised vegetable oils in refined olive oil. In 2001 the IOC published the method to

415

determine sterenes (campestadienes and stigmastadienes)47 to detect desterolised seed oils in

416

refined oils. This determination is based on the isolation of unsaponifiable matter, separation

417

of sterene fraction with silica gel chromatographic column impregnated with silver nitrate,

418

and analysis by capillary GC. The critical part of this determination is the optimization of the

419

volumes used to extract each fraction (three different fractions are extracted from the silica

420

gel column). The chromatograms obtained from each fraction can be used for identification if

421

the volumes have been optimized correctly. Another problem with this determination is that in

422

some cases 2,4 and 3,5 diene isomers are separated into two different peaks, which entails the

423

computation of the sum of area to know the total concentration. In these cases the column

18


Page 18 of 62

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424

should be changed with another that is less polar or has bigger internal diameter. This method

425

can be applied to the quantification of stigmastadiene only if the concentration is higher than

426

4 mg/kg. If the concentration of stigmastadiene is within 0.01 and 4 mg/kg, the IOC method45

427

should be used. The presence of PAHs in olive-pomace oils—arising from pyrolysis

428

processes, environmental and natural sources—compelled IOC48 to set up an analytical

429

method with suitable sensitive, selective and accurate analytical parameters for routine

430

analysis to determine the PAHs concentration, expressed as benzo(a)pyrene on wet weight. It

431

consists of an extraction step (e.g., saponification with alcoholic KOH and liquid–liquid

432

partitioning) followed by one or more purification procedures (column chromatography, TLC,

433

SPE) to end with the analytical determination by GC coupled to flame ionization detection

434

(FID) or mass-spectrometry (MS), or by HPLC with spectrofluorometric detection. The last

435

has proven to successfully separating PAHs from the other hydrocarbons (PAHs co-elute with

436

other hydrocarbons in silica TLC) with a good quantitative response.49,50

437 438

Sterols

439

The major chemical series of the olive oil unsaponifiable matter is that of sterols,

440

which are usually grouped into 4-demethylsterols or phytosterols, 4,4-dimethylsterols or

441

triterpenic alcohols, and 4-monomethylsterols or methylsterols; all of them with analogous

442

chemical structure.

443

The fact that the concentrations of some sterols are characteristic of the genuineness of

444

vegetable oils made them of interest for purity and authenticity control olive oil designations.

445

Thus, for example, brassica seed oils have high content of brassicasterol while olive oils are

446

characterized by high concentration of ∆5-avenasterol. These differences induced IOC to set

447

up a method that allowed for their quantification. Results are expressed as percent of the total

448

area of sterols, although the illegal process of removing sterols without forming fatty acid

449

trans-isomers51 suggests giving the information in absolute concentration. In addition to 19



450

qualitative differences in the sterol profiles of vegetable oils, some researchers52,53 have

451

pointed out that the pedoclimate (soil, climate, etc.) of the orchards and the cultivars affect the

452

olive oil sterolic composition up to the point that some authentic olive oils can be qualified as

453

non-genuine. The current proposal of decision trees2,54 has been approved to be applied in

454

those anomalous oils to overcome the number of wrong classifications by means of the

455

implementation of equations that combine values of several chemical parameters.

456

The chromatographic methods are mostly devoted to determining the composition of

457

4-demethylsterols or phytosterols. Generally speaking, the determination of sterols comprises

458

the sum of both possible forms (free and esterified) as their sterol acetates55 as shown in

459

Figure 1. Nevertheless, there is a method for determining the individual concentration of free

460

and esterified sterols independently with the particular objective of detecting the presence of

461

hazelnut oil.56 For the determination of both forms together, the unsaponifiable material is

462

usually purified by TLC on silica gel with hexane/diethyl ether (65:35 v/v). The experience

463

has demonstrated that two developments must carry out in order to obtain a satisfactory

464

separation. The plate can be impregnated with alcoholic KOH for the purpose of retaining the

465

FFAs in the unsaponifiable fraction. Three large bands would appear on the TLC plate. The

466

first one contains 4,4-dimethylsterols or triterpenic alcohols, some phytols, and aliphatic

467

alcohols. The second is composed of the remaining phytols and methyl sterols, and the third

468

band contains sterols, erythrodiol, and uvaol. However, the individual separation of sterols is

469

not exempt from difficulties is the amount of the unsaponifiable fraction in diethyl ether to be

470

deposited on TLC excesses 200 µL. An inadequate development of TLC and defective

471

scraping on TLC of the sterol band are other habitual problems the analysts deal with.

472

4,4-Dimethylsterols or triterpenic alcohols, the first fraction, are analyzed by

473

saponifying olive oil with KOH after the addition of an internal standard like C-20 or C-21

474

and by extracting the unsaponifiable matter with diethyl ether and later purifying it on a plate.

20


Page 20 of 62

Page 21 of 62


475

No analytical problems have been observed for their GC determination in a nonpolar column

476

(100 dimethylpolysiloxane or a 5% phenyl-95% dimethylpolysiloxane).57

477

The methyl sterols, the second fraction, are separated on TLC with a mobile phase of

478

cyclohexane/ethylic acetate (85:15, v/v). GC analysis of methyl sterols can be carried out

479

under the same conditions as for 4-demethylsterols or phytosterols (EC 19911 and

480

henceforth). These compounds are quantified together with triterpenic alcohols in the same

481

chromatogram.57

482

Erythrodiol and uvaol (triterpene dialcohols) are determined by scraping their TLC

483

band with the band of 4-demethylsterols or phytosterols,58 the third fraction, and subsequent

484

GC analysis. The use of capillary columns yields a significant chromatographic resolution of

485

those diols. Results are still given in percentages while absolute values (concentrations) are

486

preferred as they would diminish substantially the current high number of false positive

487

(genuine VOO classified as adulterated).

488

Analysts, however, have simplified the analysis because the problems of using TLC.

489

Thus, the most reliable and widely used technique is the off-line HPLC-GC coupling that

490

comprises separation of the fractions of sterols or other unsaponifiable components (e.g.,

491

branching and terpenic hydrocarbons) by HPLC with a silica gel column, collection of the

492

fraction, elimination of the solvent, further derivation, and injection onto GC. Figure 2 shows

493

a typical HPLC chromatogram in which the peaks assigned to the different sterol fractions are

494

shown.59 Figures 3 y 4 display the correct separation and peak resolution of the compounds

495

named 4,4-dimethylsterols and 4-monomethylsterols after applying the described HPLC-GC

496

based method.

497 498

Aliphatic alcohols

499

Free and esterified aliphatic alcohols are a linear chain made of even and odd number

500

of 20-32 carbon atoms. It is important to note that phytol (present in VOOs at low 21



501

concentrations) is an artifact generated from chlorophyll decomposition by alkaline treatment

502

during saponification. The standard method60 begins by adding the internal standard (1-

503

eicosanol) to 5 g of the sample prior to the saponification with ethanolic KOH and then

504

extraction with ethyl ether. The alcoholic fraction is separated from the unsaponifiable matter

505

by depositing it on a silica gel plate, or in a column of the same material, and eluting with

506

hexane:diethyl ether (60:40 or 70:30, v/v). The silylized derivatives can be analyzed with SE-

507

30 or SE-54 or SE-52 capillary nonpolar columns. Because EU regulations61 (and followings)

508

proposed the determination of wax content as an alternative to the quantification of aliphatic

509

alcohols, the analytical method has not been modified since it became a standard method.

510 511

Tocopherols

512

Tocopherols (heteroaromatic acid compounds with high molecular weight) are lipid-

513

soluble compounds easily oxidized in the presence of light, oxygen, alkaline pH, or traces of

514

transition metal ions. The detection method62 employs the direct analysis using normal-phase

515

HPLC and fluorescence or UV detection. Tocopherols are directly analyzed through olive oil

516

injection, dissolved in a hexane:isopropanol (99.8:0.2, v/v) mobile phase, in a Si-60 5 µm

517

LiChrosorb column with a length of 250 mm and an internal diameter of 4 mm. The

518

fluorescence detector characteristics are λext = 290 nm and λem = 330 nm.63 SPE has also been

519

reported as an adequate procedure for sample preparation prior to HPLC analysis of

520

tocopherols in VOO.64 Luminescent methods have also been used for the determination of

521

total tocopherols in VOO without prior separation.65 IOC2 recommends using the ISO 9936

522

method66 to determine α-tocopherol. A portion of oil is dissolved in n-heptane and the

523

individual tocopherols are separated by HPLC-fluorescence or HPLC-UV. The content of

524

each tocopherol is calculated by using calibration curves.

525 526 22


Page 22 of 62

Page 23 of 62

527


Methods for olive oil sensory quality control

528

Virgin olive oil (VOO) stands out as a gourmet product because of its rich flavor, and

529

its classification into quality categories basically relies on the sensory assessment, together

530

with some chemical indices that do not provide information about aroma or taste. The sensory

531

assessment by panel test is not, obviously, a chemical method, but some analytical techniques

532

attempt to analyze the chemical compounds actually responsible for flavor and give objective

533

chemical information that can explain the sensory perception.

534

Numerous problems make the sensory interpretation of instrumental analysis difficult

535

to accomplish. This sensory interpretation can be even harder if those analyses do not provide

536

information on the compounds that cause a particular attribute. Thus, instrumental approaches

537

can be grouped as those that have a casual relationship with sensory quality (henceforth,

538

apparently causal relationship), and those that have a causal relationship. The former methods

539

do not analyze flavor compounds that are responsible for attributes, but compounds whose

540

concentrations are, or seem to be, mathematically correlated with some sensory parameters.

541

Mathematical correlation, however, can fail under certain circumstances, and in those cases,

542

the relationship between sensory and chemical information would be dissociated.

543 544

Apparently causal methods for sensory quality control

545

Although methods are validated prior to being proposed as standards not always the

546

validation step is carried out under the strictest conditions which can result in overstated and

547

over-optimistic analytical parameters (e.g., reproducibility, robustness) that usually present

548

umpteen exceptions when the method is widely applied by analysts. The problem is not only

549

circumscribed to methods based on multivariate information but also to methods based on

550

parameters with a weak or casual relationship with sensory quality. The fact is that a

551

successful control of olive oil adulteration by means of statistical procedures encouraged

23



552

some analysts to think that a strong mathematical relationship can prevail over the lack of any

553

scientific evidence explaining the relationship between sensory assessment and chemical

554

compounds (e.g., pyropheophytins, ethyl esters).

555 556

Pigments

557

Although pigments are related to VOO color,67 the determination of some of their

558

compounds has been proposed as a method for determining VOO quality and adulteration68, 69

559

although its fundamentals in this application is not quite clear.

560

In analytical terms, the method can be focused on determining a particular compound

561

(pyropheophytin a) in a rapid way69,70 or the maximum number of pigments71. In the first

562

case, the analyst can use two methods69,70, although one is more focused on the determination

563

of pheophytins70 than pyropheophytins,69 because both methods use a reverse-phase solid-

564

phase extraction (RP-SPE). The first, however, elutes with petroleum ether (65-95 ºC) and the

565

second with petroleum ether (40-60 ºC): ethyl ether (9:1) for removing lipids. Acetone is used

566

in both methods to collect the pigments. The critical point in both methods is the collection of

567

the analytes in 0.2-0.3 mL of acetone and later the injection in the HPLC instrument because

568

the high volatility of acetone suggests making this step as rapid as possible.

569

When the objective is to have information of a complete profile of pigments, SPE

570

packs can be responsible for the degradation of some pigments – mostly carotenoids – despite

571

their well-known ability in cleanness and free acidity removal. In that case, it is suggested to

572

employ a method that is lengthy and tedious but excellent and well-tested: liquid-liquid

573

extraction, with hexane and dimethylformamide (DMF).71

574 575 576

24


Page 24 of 62

Page 25 of 62

577


Diacylglycerols

578

The difference in the formation of DAG isomers has not only been proposed for olive

579

oil authenticity but also for distinguishing VOOs from extra-VOOs (EVOOs),72 despite the

580

fact that the relationship of DAGs with sensory quality is merely a result of casual with the

581

apparent causal relation in the case of VOO defects resulting from hydrolytic processes; for

582

example, before processing of olives (e.g., fusty), or when vertical containers are used for

583

decanting, among others.

584

The method proposed by Gertz and Fiebig (2006)72 uses a silica gel column

585

chromatography to which 100 mg of the olive oil spiked with 1 mL of toluene is transferred

586

and then the column is washed with isooctane:diisopropylether (0.85:0.15) in a first step. In a

587

second step the DAGs are eluted with diethyl ether and the solvent removed with a rotary

588

evaporator prior to be silylated at room temperature. Then 1 mL of acetone is added to the

589

solution and 1-2 µL are injected into a gas chromatograph. Pérez-Camino et al. (2001),73

590

however, suggested using SPE to which an aliquot of 500 µL of the dehydrated olive oil

591

solution in hexane (0.2 mg/mL) and 200 µL of the internal standard are transferred. The

592

method provided by IOC39 allows separating the 1,2 isomer from the 1,3 isomer of

593

diacylglycerols although saturated and unsaturated DAGs are eluted together. This method

594

suggests analyzing the olive oil sample by GC once it is purified. The suggested

595

chromatographic conditions consist in a direct on-column injection with a capillary column

596

fused silica 3-7 m coated with SE 52 or SE 54 liquid phase (5% diphenyl dimethyl

597

polysiloxane) (Table 3A).

598 599

Fatty Acid Alkyl Esters

600

Alkyl esters are made up of methyl and ethyl esters of fatty acids previously present in

601

their free form. They are a consequence of inappropriate practices in the harvesting and

602

storing of the olives prior to olive oil extraction because of the rupture of the olive drupe and 25



603

its contact with microorganisms (yeasts) and lipolytic and pectolytic enzymes. Consequently,

604

it seems that presence of the ethyl esters of fatty acids (FAEEs), which are present in the

605

waxy fraction of olive oils,75 indicates that olive oil could have been obtained from unhealthy

606

olives when harvested or because of inadequate processing of the olives. However, the

607

relationship of fatty acid alkyl esters with soft-deodorized oil76 is casual as well as its

608

relationship with sensory quality.

609

The method COI/T.20/DOC. No 31,43 which allows the determination of waxes as

610

well (Table 3A), is based on solid–liquid chromatography (LC) by traditional glass column

611

for isolating the fraction containing alkyl esters and waxes. The method, although very

612

simple—it only requires adding a suitable amount of internal standard to 500 mg of the

613

sample prior to transfer into LC—may pose problems in its implementation because of the

614

difficulty in having similar packing of the silica gel columns in repeatability studies. The

615

difficulty becomes greater with the change of LC packed with 15 g of silica gel6 to 3 g of

616

silica gel43, which reduces the sample pre-concentration. Furthermore, the flow rate of

617

hexane:diethyl ether (99:1) of about 15 drops every 10 seconds is not an easy matter for

618

inexperienced analysts. Experience also suggests that 15-20 mL of hexane should be added

619

instead of 10 mL immediately after the sample is transferred into the column, which helps to

620

achieve a better chromatogram resolution. Furthermore, in samples with low concentration of

621

alkyl esters, they can be masked by the peak tail of the solvent (n-heptane or isooctane),

622

which suggests using n-hexane, which is much more volatile.

623

An interesting alternative is the use of a method based on GC-(EI)MS77 where there is

624

no sample preparation and FAAEs are directly thermo-desorbed and cryo-focused in the

625

cooled injector of a GC–MS (EI) instrument. The detection limit of the method competes with

626

the standard, but the new one is faster, simpler, requires a lower amount of organic solvents

26


Page 26 of 62

Page 27 of 62


627

and significantly enhances method repeatability. However, it is a new method that requires

628

further studies to fully understand its performance parameters and potential problems.

629 630

Free fatty acids

631

Free fatty acids (FFAs) arise from of the separation of fatty acids from TAGs in the

632

olive because of the action of enzymes, further stimulated by light, water and heat. Thus,

633

FFAs content is a marker of TAGs hydrolysis, and hence it indicates how fresh and how well

634

handled the olives were before being milled. The content of FFAs or free acidity is usually

635

determined by titration following well-established standards78,79 although the amount of

636

sample1 seems to be high (i.e., 20 g when acidity is < 1.0%) and could be reduced to half.

637

There is also the alternative of substituting the solvents for others that are much more

638

amicable, like ethanol:water (1:1).80

639

Determination can be carried out by GC as FAMEs as well. FFA methyl esters are

640

prepared by derivatization with trimethylsilyldiazomethane because methylation enhances the

641

volatility and reduces activity of FFAs, although methylation can be omitted if a highly polar

642

capillary column (e.g., coated with 88% cyanopropyl-aryl-siloxane or 100% cyanopropyl

643

polysiloxane) is used. The procedure also requires the addition of an internal standard (e.g.,

644

tridecanoic or nonadecanoic FAs) and a purification step by SPE filled with an amino

645

stationary phase. Medium-polarity thermostable columns (e.g., coated 65% phenyl-35%

646

dimethyl-polysiloxane) have also been used with success.

647

Although FFA determinations can be easily and rapidly achieved by Fourier transform

648

infrared spectroscopy (FTIR), this approach is not an official method yet. Lanser et al.

649

(1991)81 used peaks near 1745 and 1711 cm−1 to construct a model allowing the determination

650

of the free fatty acid content in crude oils. The C=O carbonyl groups of esters absorb at ~

651

1746 cm−1, while the carboxylic acid group of free fatty acids has its characteristic band at

27



652

1711 cm−1. Later, an FTIR instrument and ATR accessory were successfully used to

653

determine FFA content in oils and fats.82 However, it is FTIR with transmission cells that has

654

been much more successful.83 In this procedure the free fatty acids that are present in the

655

sample are derivatized to the corresponding free fatty acid salt after reacting with sodium

656

carbodiimide (a weak base) in methanol. The resulting salt presents a measurable

657

spectroscopic band in a region without interference and consequently this band is very easy to

658

measure and calibrate.84,85

659 660

Oxidation products

661

The peroxide value (PV) is an indicator of the primary oxidation status of olive oil,

662

mainly because of the presence of hydroperoxides86. Despite the importance of PV as a

663

quality index, the standard method (ISO 2009)78 is laborious, time consuming, requires the

664

use of organic solvents and, even more importantly, its accuracy depends strongly on the

665

experience of the analyst.88 Its relevance justifies the need for reliable methods that use

666

amicable solvents (i.e., isooctane) that do not perturb the interpretation of the results, and can

667

also be fast and non-destructive, with high degree of automation to provide near-real-time

668

measurements for assessing olive oil quality. In this context, vibrational spectroscopy offers

669

alternative analytical tools87; for example, disposable IR Cards have successfully been applied

670

to determine PV of edible oils.89,90

671

A shift in double-bond configuration occurs during the formation of peroxy radicals

672

and hydroperoxides because the normal methylene-interrupted configuration is transformed

673

into a conjugated form. The absorbance in ultraviolet light of secondary oxidative compounds

674

(conjugated dienes and trienes) is determined according to standard methods,91 which can be

675

easily implemented by analysts. New instrumentation, however, has better characterized the

28


Page 28 of 62

Page 29 of 62


676

exact wavelengths at which the conjugated dienes and trienes absorb (232 nm and 268 nm)

677

when using isooctane and 232 nm and 270 when cyclohexane is the solvent.

678 679

Causal methods for sensory quality control

680

The sensations perceived when a VOO is consumed are evaluated by sensory

681

assessment implemented in the so-called panel test.92 The official method, however, is

682

questioned by numerous VOO sectors, because the difference between virgin and extra-virgin

683

olive oils depends on the presence of defects, whatever their level of perception. It is there

684

where the method fails: when the panel test analyzes oils that could not have any defect for

685

some panel tests while others have been able to detect defects at very low intensity of the

686

sensory perception, which is enough to qualify olive oils as virgin instead of extra-virgin.

687

Reasons can be found in many aspects -limit of detection, subjectivity, inadequate training,

688

too high sensitivity of some assessors for some odors - but none of them is convincing by

689

itself to explain the errors in the analysis. Therefore, an objective measurement of VOO

690

sensory quality should follow another strategy based on Analytical Chemistry.

691

From a chemical perspective, the flavor of VOO is explained by the occurrence of a

692

series of minor chemical compounds that have a significant sensory impact. Thus, “I smell,

693

therefore there are volatiles” is the sentence, which emulating Descartes’ phrase, summarizes

694

the scientific support explaining that there is a causal relationship between volatile

695

compounds and aroma, and, for a similar reason, between phenols and VOO taste. The

696

methods for quantifying those chemical compounds have as their main objective to contribute

697

to the explanation of what the assessors perceive when they smell and taste VOOs.

698 699

Phenols

700

The prevalent classes of phenols found in VOO are phenolic alcohols, phenolic acids,

701

flavonoids, lignans, and secoiridoids, some of them having antioxidant properties.93 However, 29



702

and despite, the interest of these compounds from sensory and health viewpoints, no

703

harmonized and widely accepted analytical method exists today because their results are not

704

comparable.94 There are several approaches, one of them being supported by IOC,95 while

705

another96 is still under evaluation. The first one is based on the individual separation of

706

phenols by HPLC while the second one provides the total content of o-diphenols by a more

707

rapid and cost effective colorimetric method. Their results, however, do not seem to be

708

comparable. The colorimetric method has a low specificity, as the color reaction can occur

709

with any oxidizable phenolic hydroxyl group.97 However, it could be a valid method to

710

determine the concentration of total phenols as required in the recent health claim on

711

polyphenols98 once the method is improved and corrected.

712

Regardless the method applied, the first step is the extraction of the phenol fraction

713

from the oil matrix removing the interfering components (e.g., lipids, pigments). The

714

extraction of the phenol fraction can be carried out by SPE (e.g., C18 or diol) or liquid-liquid

715

extraction (LLE). If SPE is used, the addition of hexane or other organic solvents to the oil

716

before extraction99 makes the sample flow through the SPE cartridge easier with no effect on

717

the recovery of phenols. However, the IOC method95 uses a LLE with methanol:water (80:20,

718

v/v). Some authors100,101 have pointed out that the recovery of “bitter” phenolic compounds

719

increases as follows: SPE C18 (with methanol) < SPE diol (with methanol) < LLE (60%

720

aqueous methanol). LLE also seems to be more appropriate for the extraction of oxidized

721

phenolic compounds.102

722

In the separation step, reversed-phase HPLC has been the most successful approach

723

despite the fact that the elution time is generally long, more than 40 min. Separation is carried

724

out using a column RP C18 (250 × 4 or 4.6 mm i.d.) with water–methanol or water–

725

acetonitrile mixtures enriched with acetic, phosphoric or sulfuric acid (up to 3%, pH < 2) to

726

suppress phenol dissociation and improve peak asymmetry. Analysts, however, should pay

30


Page 30 of 62

Page 31 of 62


727

attention to the various pKa values of individual phenols. Furthermore, degassing should be

728

constant to avoid bubbles that disrupt the long runs. The starting composition of eluent should

729

use small amount of organic phase (approx. 5%). By the end of the run, this amount is usually

730

rather high, though it should never reach 100%. The water quality used for the analysis is

731

essential for avoiding higher backpressures expected at certain ratios. The conditioning times

732

from run to run that are needed to ensure reproducible retention data are long.

733

Ultra high performance liquid-chromatography (UHPLC), with columns packed with

734

1.7 µm particles, coupled to MS/MS (LLE-UPLC-MS/MS) has been applied with success up

735

to the point that its analytical parameters, as LOD and LOQ, are better than HPLC-DAD and

736

HPLC-FLD with the exception of vanillic acid and pinoresinol.103 Although it is much more

737

rapid (18 min) than the IOC standard method and methodologies under evaluation by IOC,

738

the instrumentation is not affordable by most of laboratories.

739

The identification and quantification techniques depend on the aim of the study and

740

available facilities. In the last decade LC-MS techniques have found extended application,

741

though routine analysis is still less demanding regarding instrumentation. Thus, a good

742

detection system can be an in-tandem diode array detector (DAD) with fluorescence detector

743

(FLD). It is well known that UV detection at 280 nm is the standard wavelength, though

744

others (225, 240, 254 nm) are also useful for constituents of olive oil polar fractions, while

745

flavonoids are detected at higher wavelengths (340 nm).

746

UV spectrophotometry (spectrum region between 200-290 nm) is extremely helpful in

747

the identification of phenols and monitoring the purity of HPLC peaks, although

748

hydroxycinnamic acids have a characteristic maximum at 310–332 nm. Differences in

749

maximum wavelength and molar absorption values among the various phenols affect

750

quantification.93 Spectrofluorimetric, in-tandem or not with DAD, is a valuable tool as it is

751

more specific and sensitive than spectrophotometric detection.104,105 However, there is a

31



752

certain interest in modifying the IOC standard by adding a step of hydrolysis prior to the

753

quantification with HPLC-DAD and suggesting the colorimetric method under revision96 as a

754

routine method for determining the phenolic content for verifying health claims.98 This, once

755

again, increases confusion among analysts since it does not produce comparable results and it

756

is contrary to the current demand of analysts for a reduced number of standard methods.

757

Other alternatives to quantify the phenolic fractions have been studied. Mass

758

spectrometry (MS), for its part, offers a high volume of data on fragments derived from

759

phenolic compounds. Thus, mass spectrometry can be also used to perform quantitative

760

analysis of phenol composition on the basis of fragment abundance. Finally, Nuclear

761

Magnetic Resonance (NMR) and NMR-MS are powerful tools for the structural elucidation of

762

isolated phenolic compounds that are not detectable by other means,106 though NMR cannot

763

yet be used in routine analysis.

764 765

Volatile compounds

766

Any proposal to replace sensory assessment with an analytical procedure presently has

767

two challenges. The first is the determination of the chemical compounds responsible for

768

VOO aroma from the large set of currently identified compounds107,108. The second challenge

769

is to explain those important VOO sensory attributes that are described with considerable

770

vagueness such as fruitiness and green. Therefore, the establishment of the relationship

771

between VOO chemical compounds and sensory attributes is the most complex aspect of the

772

global study of olive oil flavor. However, the relationship between volatile compounds and

773

sensory defects is already well-established.109 Once the markers for a sensory defect are

774

identified, a series of tasks should be implemented: (i) calculate the thresholds of the markers,

775

(ii) select the better analytical evaluation (e.g., Trap, SPME), (iii) produce reference materials

776

to facilitate the implementation of the method for analytical evaluation. All these tasks

32


Page 32 of 62

Page 33 of 62


777

(Figure 5) should be carried out in order to develop an analytical method for aroma

778

authentication.

779

The separation and quantification of volatiles is usually carried out by GC, although

780

aroma sensors,110 which do not require a sample preparation step, are gaining in popularity.

781

The sensors are applicable within a screening context, since aroma sensors do not supply

782

individual information of volatiles but instead produce a response curve resulting from all the

783

volatiles present in a static headspace.111 Table 4 describes the analytical techniques, chemical

784

phases and sensors that are commonly used in the analysis of volatiles. In addition to the

785

described methods, in-tandem GC-olfactometry (GC-O) is a valuable tool for the selection of

786

aroma-active components. In GC-O human subjects (assessors) assign the sensory properties

787

of the different peaks/zones of the chromatogram when sniffing GC effluents. Many key

788

aroma compounds occur at very low concentrations but still have sensory relevance due to

789

low odor thresholds108 and they are perceived by panelists and consumers.112

790 791

Unifying methods

792

Researchers have always been looking for unifying methods that allow reduction of the

793

tremendous laboratory workload, given that the current methods offer partial solution for

794

determining the presence of adulterants in olive oil. A difference between theoretical and

795

empirical TAGs is an indication of a mixture of different oils and hence of the presence of an

796

extraneous edible oil in olive oil. The practical implementation of this theory is the so-called

797

Global Method, in which an analytical step for determining FAMEs and TAGs is followed by

798

a mathematical procedure that makes this standard17 different from the other previously

799

approved methods to date.

800

Another proposal of a unifying method was provided by Mariani and co-workers in

801

1991,114 in which sterols, triterpenic alcohols, squalene and tocopherols are quantified

33



802

simultaneously. This method is based on the concurrent determination of these compounds

803

avoiding the use the TLC for separating the unsaponifiable fraction, which is tedious. The

804

procedure is something similar to that already described for the simultaneous determinations

805

of waxes, FA methyl esters and FA ethyl esters.6

806

Regarding the unifying method for the sensory quality assessment there is a certain

807

interest for a mimetic application of a method for quantifying volatiles of wine to olive oil,

808

when it is obvious than the first has an aqueous matrix and the second is a lipid. The recent

809

method based on the quantification of ethyl esters for assessing quality has raised the interest

810

on quantifying ethanol, which is involved in the formation of ethyl esters. However the

811

relationship between ethyl esters and VOO quality is casual and there is not a kinetic equation

812

that explains the formation of ethyl esters in VOO. Furthermore, the odor threshold of ethanol

813

is so high in a lipidic matrix that it does not contribute to VOO aroma.

814

The aforementioned unifying methods satisfy one of the requirements of analysts

815

working on olive oil control, that is to obtain complete chemical information from an oil

816

without use of extensive resources. Therefore such methods will presumably be welcomed by

817

control labs. However, these methods still need to be fully validated and experience is

818

currently insufficient to propose amendments or detect potential problems. It is also unclear

819

whether these methods should be proposed as alternatives to the current standard methods or

820

as additional options for more experienced analysts.

821 822

Further research in perfecting methods

823

The described methods have solved many problems and have made olive oil one of the

824

most protected foodstuffs though they still need to be improved with further research. The

825

required improvements need to take into account current problems, which are somewhat

826

different from those encountered in the past, and to be stricter in repeatability and

34


Page 34 of 62

Page 35 of 62


827

reproducibility considering the recent technical advances. The greater ability of current

828

techniques for identifying compounds in olive oil also sets the stage for the evaluation of new

829

purity and quality parameters in addition to validating the current ones. Searching new

830

parameters is of particular interest in quality assessment where the development of alternative

831

methods based on volatile compounds for the assessment of sensory characteristics is

832

required. New research based on chemical and sensory approaches would make the current

833

sensory assessment dispensable to some extent, alleviating the workload associated with panel

834

tests. Researchers also strive to develop methods for assessing health benefits of virgin olive

835

oil (e.g., analyzing phenols) to respond the current demand of industry and consumers.

836

Another challenge is to develop rapid approaches that may serve as alternatives to lengthy

837

chromatographic methods. Thus, spectroscopic techniques may provide effective solutions in

838

cases of complex analytical tasks such as identification of geographical origins. The

839

establishment of open databank and data-sharing platforms fed with spectroscopic and

840

chromatographic data is also being pursued to facilitate the implementation of practical and

841

standardized applications. This task is arduous, requires time and needs to keep pace with the

842

development of new accurate methods and the rise of new challenges.

843

35



844

References

845 846

(1) EC. Commission Regulation (EEC) 2568/91 on the characteristics of olive oil and olive-

847

residue oil and on the relevant methods of analysis. Off. J. Eur. Communities 1991,

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(2) International Olive Council (IOC) T.15/Doc. No 3/Rev. 7. Trade standard applying to olive oils and olive-pomace oils. Madrid, Spain, 2013. (3) Codex Alimentarius Commission (CODEX STAN) 33-1981. Codex standard for olive oils and olive pomace oils, 1981. (4) Aparicio, R.; Conte, L. S.; Fiebig, H. J. Olive oil authentication. In: Handbook of Olive Oil Aparicio, R.; Harwood, J. Ed.; Springer Science, 2013 pp: 589-653. (5) International Olive Council (IOC) T.20/doc. No. 18/Rev.2. Determination of wax content by capillary column gas chromatography. Madrid, Spain, 2003.

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(6) International Olive Council (IOC) COI/T.20/DOC. 28 Determination of the content of

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waxes, fatty acid methyl esters and fatty acid ethyl esters by capillary gas

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chromatography. Madrid, Spain, 2010.

860 861

(7) International Olive Council (IOC) T.20/DOC. NO 42-2/Rev.1. Precision values of the methods of analysis adopted by the international olive council. Madrid, Spain, 2011.

862

(8) Laroussi-Mezghani, S.; Vanloot, P.; Molinet, J.; Dupuy, N.; Hammamib, M.; Grati-

863

Kamoun, N.; Artaud, J. Authentication of Tunisian virgin olive oils by chemometric

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865

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(9) Rohman, A.; Man, Y.B.C.;Yusof, F.M. The Use of FTIR Spectroscopy and chemometrics

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(11) Guzmán, E.; Baeten, V.; Fernández Pierna, J.; A. García-Mesa, J.A. Evaluation of the

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(77) Boggia, R.; Borgogni, C.; Hysenaj, V.; Leardi, R.; Zunin, P. Direct GC-(EI) MS determination of fatty acid alkyl esters in olive oil. Talanta, 2014, 119, 60-67. (78) International Organization of Standardization (ISO) 660, Animal and vegetable fats and oils – Determination of acid value and acidity. Geneva, 2009. (79) American Oil Chemists Society (AOCS), official method Cd 3d–63. Acid value. AOCS Press, Champaign. 1999.

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(80) Aricetti, J.A.; Tubino, M. A. Visual titration method for the determination of the acid

1055

number of oils and fats: a green alternative. J. Am. Oil Chem. Soc. 2012, 89, 113–

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(81) Lanser, AC.; List, GR.; Holloway, RK.; Mounts, TL. FTIR estimation of free fatty acid

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content in crude oils extracted from damaged soybeans. J. Am. Oil Chem. Soc. 1991,

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68, 448–449.

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(82) Ismail, AA.; van de Voort, FR.; Emo, G.; Sedman, J. Rapid quantitative determination of

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free fatty acids in fats and oils by Fourier transform infrared spectroscopy. J. Am. Oil

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Chem. Soc. 1993, 70, 335–341.

1063

(83) Li, D.; Sedman, J.; García-González, DL.; van De Voort, FR. Automated acid content

1064

determination in lubricants by FTIR spectroscopy as an alternative to acid number

1065

determination. J. ASTM Int. 2009, 6, 1–12.

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acids in edible oils with the use of a variable filter array IR spectrometer. J. Am. Oil

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Chem. Soc. 2008, 85, 599-604.

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(85) Yu , X.; Du, S.; van de Voort, FR.; Yue, T.; Li, Z. Automated and simultaneous

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determination of free fatty acids and peroxide values in edible oils by FTIR

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spectroscopy using spectral reconstitution. Anal. Sci. 2009, 25, 627–632.

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(86) Choe, E.; Min, DB.; Mechanisms and factors for edible oil oxidation. Comprehensive Rev Food Sci. Food Safety, 2006, 5,169-186.

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infrared spectra of edible oils under oxidative conditions. J. Agric. Food Chem. 2000,

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80, 2028-2036.

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(88) Van de Voort, FR.; Ismail, AA.; Sedman, J.; Dubois, J.; Nicodemo, T. The determination

1078

of peroxide value by Fourier transform infrared spectroscopy. J. Am Oil Chem. Soc.

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1994, 71, 921-926.

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(89) Ma, K.; van de Voort, FR.; Ismail, AA.; Sedman, J. Quantitative determination of

1081

hydroperoxides by FTIR spectroscopy using a disposable IR card. J. Am. Oil Chem.

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Soc. 1998, 75, 1095–1101.

1083

(90) Pizarro, C.; Esteban-Díez, I.; Rodríguez-Tecedor, S.; González-Sáiz, JM. Determination

1084

of the peroxide value in extra virgin olive oils through the application of the stepwise

1085

orthogonalisation of predictors to mid-infrared spectra. Food Control, 2013, 34,158-

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167.

1087 1088

(91) International Olive Council (IOC) T20/Doc. No 19/Rev. 3. Method of analysis spectrophotometric investigation in the ultraviolet. Madrid, Spain 2010.

1089

(92) International Olive Council (IOC) T.20/Doc. No 15/Rev. 6. Sensory analysis of olive oil:

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method for the organoleptic assessment of virgin olive oil, Madrid, Spain 2013.

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(93) Tsimidou, MZ. Analytical Methodologies: Phenolic compounds related to olive 0il taste

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issues. In: Handbook of olive oil: Analysis and Properties, 2nd edition. Aparicio, R.;

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Harwood JL. Eds.; Springer, New York, 2013 pp 311-334.

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(94) Romero, C.; Brenes, M. Comment on addressing analytical requirements to support

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health claims on “Olive oil polyphenols” (EC Regulation 432/212). J. Agric. Food

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1097 1098 1099 1100

(95) International Olive Council (IOC) T.20/Doc No 29. Determination of biophenols in olive oils by HPLC. Madrid, Spain, 2009. (96) International Olive Council (IOC) Decision No DEC-17/97-V/2009. Identification and quantification of the phenolics in olive oil. Madrid, Spain, 2009.

1101

(97) Carrasco-Pancorbo, A.; Cerretani, L.; Bendini, A.; Segura-Carretero, A.; Gallina-Toschi,

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T.; Fernandez-Gutierrez, A.. Analytical determination of polyphenols in olive oils. J.

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Sep. Sci. 2005, 28, 837–858.

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(98) European Comunities (EC) No 432/2012 L 136/1-40. Establishing a list of permitted

1105

health claims made on foods, other than those referring to the reduction of disease risk

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and to children’s development and health. Off. J. Eur. Commun. 2012.

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(99) Papoti, VT.; Tsimidou, MZ. Looking through the qualities of a fluorimetric assay for the

1108

total phenol content estimation in virgin olive oil, olive fruit or leaf polar extract. Food

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Chem. 2009 112, 246–252.

1110

(100) Pirisi, FM.; Cabras, P.; Falqui, CC.; Migliorini, M.; Muggelli, M. Phenolic compounds

1111

in virgin olive oil. 2. Reappraisal of the extraction, HPLC separation, and

1112

quantification procedures. J. Agric. Food Chem. 2000, 48, 1191–1196.

1113

(101) Hrncirik, K.; Fritsche, S. Comparability and reliability of different techniques for the

1114

determination of phenolic compounds in virgin olive oil. Eur. J. Lipid Sci. Technol.

1115

2004, 106, 540–549.

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1116

(102) Armaforte, E.; Mancebo-Campos, V.; Bendini, A.; Salvador, MD.; Fregapane, G. et al.

1117

Retention effects of oxidized polyphenols during analytical extraction of phenolic

1118

compounds of virgin olive oil. J. Sep. Sci. 2007, 30, 2401–2406.

1119

(103) Suárez, M.; Macià, A.; Romero, MP.; Motilva, MJ. Improved liquid chromatography

1120

tandem mass spectrometry method for the determination of phenolic compounds in

1121

virgin olive oil. J. Chromatogr. A 2008, 1214, 90–99.

1122 1123

(104) Brenes, M.; García, A.; Ríos, JJ.; García, P.; Garrido, A. Use of 1-acetoxypinoresinol to authenticate Picual olive oils. Int. J. Food Sci Technol. 2002, 37, 615–625.

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(105) Selvaggini, R.; Servili, M.; Urbani, S.; Esposto, S.; Taticchi, A. et al. Evaluation of

1125

phenolic compounds in virgin olive oil by direct injection in high-performance liquid

1126

chromatography with fluorometric detection. J. Agric. Food Chem. 2006, 54, 2832–

1127

2838.

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(106) Pérez-Trujillo, M.; Gómez-Caravaca, AM.; Segura-Carretero, A.; Fernández-Gutiérrez,

1129

A.; Parella, AT. Separation and identification of phenolic compounds of extra virgin

1130

olive oil from Olea europaea L by HPLC-DAD-SPE-NMR/MS. Identification of a

1131

new diastereoisomer of the adehydic form of oleuropein aglycone. J. Agric. Food

1132

Chem. 2010, 58, 9129–9136.

1133

(107) Aparicio, R.; Morales, MT.; García-González, DL. Towards new analyses of aroma

1134

and volatiles to understand sensory perception of olive oil. Eur. J. Lipid Sci. Technol.

1135

2012, 114, 1114–1125.

1136

(108) Morales, MT.; Aparicio-Ruiz, R.; Aparicio, R. Chromatographic methodologies:

1137

compounds for olive oil odor issues. In: Handbook of olive oil: Analysis and

1138

Properties 2nd edition Aparicio, R.; Harwood, JL. Eds.; Springer, New York, 2013 pp

1139

261-310.

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1140 1141 1142 1143

(109) Morales, M.T.; Luna, G.; Aparicio, R. Comparative study of virgin olive oil sensory defects. Food Chem. 2005, 91, 293–301. (110) García-González DL, Aparicio R Sensors: from biosensors to the electronic nose. Grasas y Aceites, 2002, 53, 96–114.

1144

(111) García-González, DL.; Aparicio, R. Coupling MOS sensors and gas chromatography to

1145

interpret the sensor responses to complex food aroma: Application to virgin olive oil.

1146

Food Chem. 2010, 120, 572–579.

1147 1148

(112) García-González, DL.; Vivancos, J.; Aparicio, R. Mapping brain activity induced by olfaction of virgin olive oil aroma. J. Agric. Food Chem. 2011, 59, 10200–10210.

1149

(113) International Olive Council (IOC) T.20/Doc. No 17/Rev. 1. Determination of trans

1150

unsaturated fatty acids by capillary column gas chromatography. Madrid, Spain, 2001.

1151

(114) Mariani, C.; Fedeli, E.; Grob, K. Valutazione dei componenti minori liberi ed

1152 1153 1154

esterificati nelle sostanze grasse. Riv. Ital. Sostanze Gras. 1991, 68, 233-242. (115) Aparicio, R. Final report project G6RD-CT2000-00440 (MEDEO). Commission of the European Communities (Growth Program), Brussels, Belgium, 2004.

1155 1156 1157 1158

48


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Page 49 of 62

1159


FIGURE CAPTIONS

1160 1161

Figure 1. Chromatogram of 4-demethylsterols of a sample of virgin olive oil. Note: 1,

1162

Cholesterol; 2, internal standard; 3, Brassicasterol; 4, 24-Methylenecholesterol; 5, Campesterol; 6,

1163

Campestanol; 7, Stigmasterol; 8, ∆7-Campesterol; 9, ∆5,23-Stigmastadienol; 10, Chlerosterol; 11, β-

1164

Sitosterol; 12, Sitostanol; 13, ∆5-Avenasterol; 14, ∆5-24-Stigmastadienol; 15, ∆7-Stigmastenol; 16, ∆7-

1165

Avenasterol.

1166 1167

Figure 2. Sterols obtained in fractionation of the unsaponifiable matter by HPLC.59

1168 1169

Figure 3. Chromatogram of 4,4-dimethylsterols of a sample of virgin olive oil. Note: 1, internal

1170

standard (5-α-cholestanol); 2, Taraxerol; 3, Dammaradienol; 4, β-amyrin; 5, Butyrospermol; 6,

1171

Cycloartenol; 7, 24-Methylene-cycloartanol.

1172 1173

Figure 4. Chromatogram of 4-monomethylsterols of a sample of virgin olive oil. Note: 1,

1174

internal standard (5-α-cholestanol); 2, Obtusifoliol; 3, Gramisterol, 4, Cycloeucalenol; 5,

1175

Citrostadienol.

1176 1177

Figure 5. Different approaches to explain the sensory assessment from chemical compounds.

1178

49



Table 1. Three Monovarietal Olive Oils from Cultivars Arbequina (Spain), Coratina (Italy) and Koroneiki (Greece) Characterized by Fatty Acids (%), 4-Demethylsterols (mg/kg), Diols (mg/kg), and Aliphatic Alcohols (mg/kg). Note: C16:1 is the Sum of C16:1n-9 and C16:1n-7; b, C18:1 is the sum of C18:1n-9 and C18:1n-7.

Compounds C16:0 C16:1a C17:0 C17:1n-8 C18:0 C18:1b C18:2n-6 C18:3n-3 C20:0 C20:1n-9 C22:0 Campesterol Stigmasterol β-Sitosterol Δ5-Avenasterol Cholesterol 24-Methylen cholesterol Campestanol ∆7-Campesterol Chlerosterol Sitostanol ∆5,24-Stigmastadienol ∆7-Stigmastenol ∆7-Avenasterol Erythrodiol+Uvaol Docosanol Tetracosanol Hexacosanol Octacosanol

Arbequina

Coratina

Koroneiki

12.54±3.76 1.36±0.30 0.12±0.01 0.20±0.04 1.40±0.36 72.12±3.44 11.38±0.17 0.69±0.11 0.40±0.07 0.35±0.05 0.12±0.00 61.01±8.89 14.85±6.94 1269.10±195.73 289.02±60.25 2.17±0.54 6.84±0.73 7.24±0.28 1.99±0.35 1.99±0.19 18.29±0.83 12.13±0.19 2.72±0.23 6.88±0.47 17.92±4.78 11.36±2.23 17.79±1.85 40.60±4.19 30.67±1.98

9.13±0.28 0.44±0.06 0.06±0.01 0.11±0.02 2.36±0.14 80.8±1.00 6.07±0.66 0.68±0.02 0.37±0.01 0.42±0.02 0.12±0.00 46.76±0.68 10.99±0.96 1157.60±5.86 98.20±5.27 3.60±0.80 4.05±1.20 5.89±0.42 4.73±0.57 14.06±1.04 18.87±5.25 6.79±1.26 3.18±0.32 4.26±0.55 26.80±5.40 23.52±3.05 33.17±4.70 52.52±9.54 26.77±4.55

11.21±0.57 0.95±0.07 0.25±0.1 0.15±0.05 2.59±0.24 77.75±1.76 5.63±1.10 0.55±0.13 0.5±0.09 0.55±0.07 0.27±0.10 43.25±0.68 6.95±0.79 831.1±6.11 187.1±8.74 3.59±0.16 4.71±0.22 5.49±1.23 1.10±0.05 12.77±0.94 8.63±1.89 6.39±0.48 2.91±0.29 4.37±0.31 27.67±3.29 13.16±3.98 35.69±6.23 73.48±9.96 26.71±5.28

50


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Table 2. Utility of Some Chemical Parameters in Olive Oil Authenticity and Approximate Percentage of Detection of Adulterant Oils. Note: GM, Global Method 17; FAMES, Fatty Acid Methyl Esters.

Chemical parameters

Sterols

Sterols + ∆ECN42

Related regulations

58

COI/T.20/Doc. No 30

COI/T.20/Doc. No 3058 16 COI/T.20/Doc. No 20

19

FAMES + Sterols

FAMES + TAGs

COI/T.20/Doc. No 24 COI/T.20/Doc. No 3058 17

COI/T.20/Doc. No 25

Informing variables

Adulterant oil

Approx. % detection

Brassicasterol; Apparent β-sitosterol

Rapeseed oil

1.0 -1.5%

Apparent β-sitosterol; Campesterol

Mustard seed oil

1.0 – 1.5%

Apparent β-sitosterol; ∆ECN42; Campesterol; Stigmasterol

Corn oil

1.0 - 1.5%

Apparent β-sitosterol; ∆ECN42

Safflower oil Sesame oil Soybean oil

1.0 – 4.0%

Cotton oil

3.5 - 5.0%

Sunflower oil

0.5 – 2.5%

Behenic acid; Stigmasterol

Peanut oil

3.0 - 5.0 %

Miristic acid; Stigmasterol

Palm oil Palm kernel oil

3.5 -10.0%

Campesterol; Stigmasterol; ∆ECN42 Apparent β-sitosterol; ∆ECN42; 7 ∆ -Stigmastenol

Results from applying GM

Hazelnut oil

51


115

< 8%


Page 52 of 62

Table 3A. The Most Common Methods for Quantifying Fatty Acids and Derived Acyl Lipids. Compounds

Fatty acids trans fatty acids

Technique

GCa-FIDb

c

HPLC -RI

d

Triacyglycerols

Triacylglycerols Diacylglycerols

2-glyceryl monopalmitate (%)

Sample pre-treatment

Reference

Column: Capillary 50-100 m×0.25-0.32 mm×0.1-0.3 µm Stationary phase: Cross-linked cyanopropylsilicone COI/T20/Doc. No 17 Carrier gas: Hydrogen or helium Rev 1 Temperature program: 165 ºC (15 min) to 200ºC at 5ºC/min Injection mode: Split

Purification with silica gel extraction cartridge. Cold methylation with KOH.

0.12 g oil in 0.5 mL hexane is charged into column (SPE-cartridge: 1 g of Si) and solution pulled through COI/T20/ Doc.No20 then, eluted with 10 mL hexane-diethylether (87:13v/v). Rev 3 The purified oil is dissolved in acetone at 5%.

Oven temperature: 25 ºC Mobile phase: acetone/acetonitrile (1:1 v/v) or propionitrile (flow-rate 0.6 to 1.0 mL/min) Column: RP-18 (25cm x 4 mm i.d.) (5 μm) with 22 to 23% carbon in form of octadecylsilane Detector: RI

HPLC-RI

Oven temperature: 20 ºC e 29 Purification by SPE (IUPAC method 2507). The purified COI/T20/Doc. No25 Mobile phase: propionitrile (flow-rate 0.6 mL/min) oil is dissolved in acetone at (5%). Rev 1 Column: RP-18 (25cm x 4.5 mm i.d.) (4 μm) Detector: RI

GC-FID

Capillary column: 3-7 m×0.25-0.32 mm×0.10-0.15 µm. C58 and C60 loss by thermal degradation can be avoided by short column 5 m should be enough Requires silylation. Gives information clustered into Phase: SE52, SE54 (5% diphenyl dimethyl polyxilosane) COI/T.20/Doc No 32 their carbon numbers. Carrier gas: Hydrogen or helium Temperature program: 80 ºC -1min- to 220 ºC at 20 ºC/min to 340 -10 min- at 5 ºC/min. Injection mode: On-column

GC-FID

GLC or SPE separation after hydrolytic reaction with pancreatic lipase. Silylation should be applied.

Waxes

Method details

GC-FID

Capillary column: 12 m×0.32 mm×0.10-0.30 µm Phase: Methylpolisiloxane or 5% phenylmethylpolysiloxane. Carrier gas: Hydrogen or helium COI/T.20/Doc No 23 Temperature program: (e.g. 60 ºC -1min- to 180 ºC at 15 ºC/min to 340 ºC -13min- at 5 ºC/min Injection mode: On-column Capillary column: 8-15 m×0.25-0.32 mm×0.1 -0.3 µm COI/T.20/Doc No 18 Phase: 5% Phenylmethylpolysiloxane, liquid phase SE52, Rev 2 SE54.

Fractionation by LC on hydrated silica gel column (15 g of silica). 52


Page 53 of 62


Carrier gas: Hydrogen or helium Temperature program: e.g. 80 ºC -1min- to 240 ºC at 20 ºC/min to 325 ºC -6 min- at 5 ºC/min to 340 ºC -10min- at 20 ºC/min. Injection mode: On-column Note: a, Gas Chromatography; b, Flame Ionization Detector; c, High Performance Liquid Chromatography; d, Refractive Index; e, Solid Phase Extraction.

53



Page 54 of 62

Table 3B. The Most Common Methods for Determining Minor Compounds. Chemical series

Technique

GCa-FIDb

Stigmastadienes

Aliphatic sterenes

hydrocarbons

and

GC-FID

Aliphatic alcohols

Method details

Capillary column: 25-30 m×0.25-0.32 mm×0.15-0.30 µm. Phase: 5% Methylpolysiloxane COI/T.20/Doc No 11 Carrier gas: Hydrogen with quality N-50 or helium Rev 2 Operation conditions: Temperature gradient (235 ºC -6 min- to 285 ºC at 2 ºC/min) Injection mode: Split (ratio 1:15) or on column

Fractionation of unsaponifiable-matter on LC Si-column.

Column: Capillary (25-30 m×0.25-0.32 mm×0.15-0.30 µm) Phase: 5% Phenylmethylpolysiloxane (see section 2.2.3.1) Fractionation of unsaponifiable-matter on LC COI/T.20/Doc No 16 Carrier gas: Hydrogen or helium Rev 1 Si-column impregnated with silver nitrate. Temperature program: 235 ºC -6 min- to 285 ºC at 2 ºC/min. Injection mode: Split (ratio 1:15) or on column

d

Capillary column: 20-30 m×0.25-0.32 mm×0.15-0.30 µm) Phase: 5% Diphenyl- 95% dimethylpolysiloxane (SE-52 or COI/T.20/Doc No 30 SE-54) Rev 1 Carrier gas: Hydrogen or helium Operation conditions: Isothermal 260 ± 5 ºC Injection mode: Split (ratio from 1:50 to 1:100)

GC-FID

Fractionation of unsaponifiable-matter on TLC or HPLC. Silylation should be applied.

GC-FID

Capillary column: 20-30 m×0.25-0.32 mm×0.10-0.30 µm. Phase: SE-52, SE-54 Fractionation of unsaponifiable-matter on TLC COI/T.20/Doc No 26 Carrier gas: Hydrogen or helium or HPLC. Temperature program: 180 ºC -8 min- to 260 ºC -15 minSilylation should be applied. at 5 ºC/min. Injection mode: Split (ratio from 1:50 to 1:100)

c

Sterols and triterpene dialcohols (erythrodiol+uvaol)

Reference

Sample pre-treatment

54


Page 55 of 62


Oven temperature: 20 ºC Mobile phase: n-Hexane/isopropanol (99:1 v/v) (flow-rate 1.0 mL/min) Detector: Fluorescence λex: 295 nm; λem: 330 nm Detector: Ultraviolet λex: 292 nm (not recommended)

e

Tocopherols

Waxes, Fatty Acid Methyl esters, Fatty Acid Ethyl Esters

Free Fatty alcohols, Free Tocopherols Free Sterols, Free Triterpenic Alcohols, Methyl Sterols, Sterols, Triterpenic Esters

HPLC -F (Si column)

0.10 g oil in 10 mL n-heptane.

ISO 9936

Fractionation by LC on hydrated silica gel 6 column 15 g of silica . GC-FID Fractionation by LC on hydrated silica gel column 3 g of silica43.

GC-FID

Silylation reaction. Fractionation by LC on silica gel column.

Capillary column: 8-12 m×0.25-0.32 mm×0.1 -0.3 µm Phase: Liquid phase SE52, SE54 COI/T.20/Doc No 28 Carrier gas: Hydrogen or helium Rev 1 Temperature program: e.g. 80 ºC -1min- to 140 ºC at 20ºC/min to 335 ºC -20min- at 5 ºC/min. COI/T.20/Doc No 31 Injection mode: On-column

Mariani et al 1991

56

116

Capillary column: 15 m×0.32 mm×0.1 µm Phase: Methylsilicone at 5% diphenyl Carrier gas: Hydrogen Temperature program: e.g. 80 ºC -1min- to 180 ºC at 20 ºC/min to 330 ºC at 6.5 ºC/min Injection mode: On-column

Note: a, Gas Chromatography; b, Flame Ionization Detector; c, See for the methodology of free and esterified sterols; d, Thin Layer Chromatography; e, High Performance Liquid Chromatography. ;

55



Page 56 of 62

Table 4. Advantages and Disadvantages of Most Common Analytical Techniques, Chemical Phases and Sensors Used to Study the Sensory Attribute of VOO. Technique

Description

Advantages

Disadvantages

Dynamic headspace- Gas Chromatography with Tenax

An inert gas (e.g., N2) sweeps headspace of sample, which is stirred or bubbled. Volatiles are trapped in Tenax. Trap is thermally desorbed in GC. Heating the trap to a high temperature while passing a current of inert gas through it.

High adsorption capacity. Useful for almost all kind of volatiles. Good recovery factors. Good repeatability. No artifacts.

Less sensitive to some acids. Temperature and flow rate must be controlled. Possible impurities of the adsorbent. An analysis per sample.

Static headspace- Gas Chromatography with SPME

Rapid, cheap, easy to use. All the steps in a single process when using automatic injector. Various kinds of fibers. Good repeatability.

A SPME fiber is exposed to sample vapor phase. Volatiles adsorbed on fiber are desorbed in GC injection port.

Metal Oxide Semiconductor (MOS) n-type (oxidizing compounds) p-type (reducing compounds)

They are a ceramic former heated by wire and coated with a metal oxide semiconducting film.

Conducting Polymer (CPSs) Polypyrroles Polyanilines

They are based on a measurable change in electrical conductivity when are exposed to volatile compounds

Acoustic Sensors Surface acoustic wave (SAW) Bulk acoustic wave (BAW)

They are based on the propagation of acoustic waves produced by piezoelectric materials in a multilayer structure.

Robust. Quite good sensitivity. Simple signal processing. Low cost. Can be doping with metals to improve sensitivity and selectivity and diminish humidity and temperature dependence. Easy to manufacture. Low power consumption. Robust. Can work at room temperature. Can be doped producing sensors to specific series of volatiles or to a particular application. High sensitivity. Short response time. Low power consumption. Small size. Robustness.

56


Differences in quantification of low molecular weight molecules. Less number of volatiles at low concentrations. Some of the disadvantages of the static headspace. Competitive effect between volatiles in SPME absorption Sensitivity affected by humidity. Temporary blinding effect. Sensor drifts. Poor specificity and selectivity with compounds with high molecular weight. Nonlinear response to some chemical compounds. Non-reproducible. Too long response time (20–40 s). Drift with over time or changes in temperature. Extremely sensitive to moisture. Temperature and humidity dependence. Poor reproducibility in the deposition of the coating material. A certain level of noise because of the oscillator high frequency.

Page 57 of 62


FIGURE 1

57



FIGURE 2

58


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FIGURE 3

59



FIGURE 4

60


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Page 61 of 62


FIGURE 5.

Reference Materials Trap-GC Thresholds of markers

Analytical evaluation

Other instrumentation

Causal explanation Sensory assessment

Pyropheophytins

Casual explanation

Freshness

Analytical evaluation

Ethyl esters

Alcoholic & Butyric Fermentation

61



TOC graphic

62


Page 62 of 62

In-Depth Assessment of Analytical Methods for Olive Oil Purity

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