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Enantiomer identification in the flavour and fragrance fields by “interactive” combination of linear retention indices from enantioselective gas chromatography and mass spectrometry / Erica Liberto; Cecilia Cagliero; Barbara Sgorbini; Carlo Bicchi; Danilo Sciarrone; Barbara Zellner D’Acampora; Luigi Mondello; Patrizia Rubiolo. - In: JOURNAL OF
CHROMATOGRAPHY A. - ISSN 0021-9673. - 1195(2008), pp. 117-126.
Original Citation:
Enantiomer identification in the flavour and fragrance fields by “interactive” combination of linear retention indices from enantioselective gas chromatography and mass spectrometry
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Enantiomer identification in the flavour and fragrance fields by “interactive” combination of 1
linear retention indices from enantioselective GC and mass spectrometry. 2
3
Erica Liberto1, Cecilia Cagliero1, Barbara Sgorbini1, Carlo Bicchi1, Danilo Sciarrone2, 4
Barbara D’Acampora Zellner2, Luigi Mondello3, Patrizia Rubiolo1*, 5
6
1
1
Dipartimento di Scienza e Tecnologia del Farmaco, Facoltà di Farmacia, 7
Università degli Studi di Torino, Via Pietro Giuria 9, Turin 10125, Italy; 8
2
Dipartimento Farmaco-Chimico, Facoltà di Farmacia, 9
Università degli Studi di Messina, Viale Annunziata, Messina 98168, Italy 10
3
Campus-Biomedico, Via E. Longoni 47, Rome 00155, Italy 11
Address for correspondence: 12
Prof. Dr. Patrizia Rubiolo 13
Dipartimento di Scienza e Tecnologia del Farmaco, Facoltà di Farmacia, 14
Università degli Studi di Torino, Via Pietro Giuria 9, Turin 10125, Italy; 15
Fax: +39 011 6707687; e-mail: patrizia.rubiolo@unito.it 16
17 18
19
Abstract 20
This study describes the development of a gas chromatography-mass spectrometry (GC-MS) 21
library to identify optically-active compounds in the flavour and fragrance field using 22
enantioselective-GC with cyclodextrin (CD) derivatives as chiral selectors in combination with 23
mass spectrometry (ES-GC-MS). The library operates on the “interactive” combination of linear 24
retention indices (ITs ) in parallel to MS spectra, so as to identify enantiomers reliably and to 25
measure EE and/or ER unequivocally. Since mass spectrometry is not a selective probe to 26
discriminate optical isomers, mass spectra (or diagnostic ions in SIM mode) are used to locate the 27
enantiomer(s) in the chromatogram, and ITs to identify it(them) safely and reliably in particular in 28
complex mixtures. The library has been built up through the following steps 29
(a) selection of CD derivatives able to cover a wide range of racemate separations. Four 30
cyclodextrin derivatives were used: 2,6-di-O-methyl-3-O-pentyl-β-CD, 2,3-di-O-methyl-6-O-tert-31
butyldimethylsilyl-β-CD, 2,3-di-O-ethyl-6-O-tert-butyldimethylsilyl-β-CD, and 2,3-di-O-acetyl-6-32
O-tert-butyldimethylsilyl-β-CD. 33
(b) determination of the analytes ITs and evaluation of their stability and reliability at both intra- 34
and inter-laboratory level 35
(c) determination of the range within which the IT of an enantiomer has to fall to be correctly 36
identified, i.e. determination of a common retention index allowance (RIA) 37
(d) construction of the library, at the moment comprising the enantiomers of 134 racemates. A 38
record has been attributed to each enantiomer including ITs determined on the four CD coated 39
columns, mass spectrum, IUPAC chemical name, CAS number, molecular weight, and, when 40
separated, racemate enantiomer resolution on the CD investigated. 41
Some applications of the library are also reported. 42
43
Keywords: enantiomer identification, mass spectral library, enantioselective GC-MS, cyclodextrin, 44
linear retention indices, mass spectra, flavours, fragrances. 45
46 47
1. Introduction 48
The interaction of a compound with a biological system has long been shown to be stereoselective. 49
Enantiomer recognition and enantiomeric excess (EE) and/or ratio (ER) determination are a very 50
important task in flavour and fragrance fields, so as (i)to define the correlation between chemical 51
composition and organoleptic properties; (ii)to implement quality control and detect fraud or 52
adulteration of “natural” samples; (iii) to determine the biosynthetic pathway when the formation of 53
a compound is studied or to classify a sample; (iv) to determine the geographic origin of a “natural” 54
sample. 55
56
Cyclodextrin derivatives (CDs) have truly represented a milestone in enantioselective gas 57
chromatography (ES-GC); being first introduced by Sibilska and Koscielsky at the University of 58
Warsaw in 1983 for packed columns [1] and applied to capillary columns in the almost 59
contemporary works of Juvancz et al. [2] and Schurig et al. [3]. Moreover, Nowotny et al. first 60
proposed diluting CD derivatives in moderately polar polysiloxane (OV-1701) to provide them with 61
good chromatographic properties and a wider range of operative temperatures [4]. Since then, 62
several groups have investigated CD derivatives as chiral selectors forenantioselective-GC 63
applications, and several hundreds articles have been published dealing with the theory of chiral GC 64
recognition with CDs, synthesis of new CD derivatives, their enantioselectivity and applications, 65
many of them concerning the flavour and fragrance field [5 - 8]. 66
Chiral recognition of marker compounds in complex real-world samples, as those in the 67
flavour and fragrance field often are, generally requires a two-dimensional approach, 68
becauseenantioselective-GC may double the number of peaks of optically-active analytes. This 69
makes some parts of the total chromatograms even more complex and, as a consequence, increases 70
the probability of interferences with a correct EE and/or ER determination. Two complementary but 71
distinct approaches can therefore be adopted; the first and most popular one is to introduce a second 72
dimension in separation. Chiral recognition is here generally carried out by conventional heart-cut 73
GC-GC [9-12], where the first column, coated with a conventional phase, serves to locate the 74
peak(s) of the optically-active racemate(s), and the second column, coated with a CD stationary 75
phase, separates its(their) enantiomers after on-line transfer through the heart-cut interface. When 76
the number of components to be investigated is very high, comprehensive GCxGC can also be used 77
but, in this case, column “geometry” must be inverted because of the high efficiency required by the 78
columns coated with CDs in order to give reliable separations [13, 14], the first column has to be 79
coated with the enantioselective CD stationary phase while the second one “distributes” the peaks 80
over the chromatographic plane [15, 16]. The second approach involves the use of a second 81
dimension in identification. In this case, the enantiomer is located and identified by MS detection 82
(or very rarely FT-IR). Single- or multiple-ion monitoring-MS (SIM-MS) carried out after a careful 83
choice of suitable diagnostic ions of the optically-active marker(s) of the sample under investigation 84
can be applied to “clean” the part of the chromatogram where the enantiomers elute, thus making 85
correct EE and/or ER determination possible. 86
In general, most GC-MS software takes insufficient account of the identification potential of 87
GC, because the identification power of mass spectrometry when used as detector for GC is 88
considered to be, and very often is, exhaustive. Retention indices (Is) are the most reliable and 89
effective tool for analyte identification by GC data. They were first introduced by Kovats for 90
isothermal analysis [17] and then by Van den Dool and Kratz for temperature programmed analysis 91
[18], the latter being better known as linear retention indices (ITs). Most GC-MS software packages 92
do not include ITs as identification criterion, and only some of them report ITs in the library as 93
“blind or inactive” data appearing in the legend of each proposed identification record, making 94
them only useful for further or additional confirmation. On the contrary, the “interactive” use of ITs 95
(i.e. their use as an active identification parameter) can be highly effective since it provides a 96
second “independent" tool to identify a compound, operating actively and simultaneously in parallel 97
to MS spectra. Moreover, ITs are based on a chemical property of an analyte of a completely 98
different nature compared to MS, which can orthogonally and synergically be combined with its 99
MS fragmentation pattern, i.e. its chromatographic interaction with a given chromatographic 100
separation system or better with a given stationary phase. 101
Mass spectrometry is well known to be unable to discriminate between optical isomers, not 102
being a selective chiral probe in this sense, and therefore giving indistinguishable spectra. As a 103
consequence it cannot be used alone to determine which enantiomer is present in a sample, or to 104
establish the predominant one or to measure its EE and/or ER. In enantioselective-GC-MS, a given 105
optical isomer can only unequivocally be identified through its IT obtained with a column coated 106
with a chiral selector suitable to separate it from its enantiomer. In the chiral recognition of 107
optically-active isomers in a complex mixture, the two identification parameters (i.e. ITs and MS 108
spectrum) must therefore be combined but, unlike conventional GC-MS analysis, mass spectra (or 109
diagnostic ion monitoring) are used to locate the two enantiomers in the chromatogram, and ITs for 110
their identification. 111
This study deals with the development of a MS library specific for the identification of 112
optically-active compounds in the flavour and fragrance field using “interactive” ITs in parallel to 113
MS spectra, so as to identify enantiomers reliably and, when necessary, to enable the measurement 114 of EE and/or ER unequivocally. 115 116 2. Experimental 117
2.1 Racemate standards and essential oils
118
Analyses of 134 racemate standards and pure enantiomers solubilised in cyclohexane at a 119
concentration of 100 ppm were carried out. The enantiomeric recognition of the marker compounds 120
characteristic of commercially available essential oils (e.o.) and extracts was also carried out, in 121
particular balm lemon, bergamot, boronia, cornmint, lavender, lemon, peppermint, and rosemary 122
e.o.s; apple flavour and apricot, peach and coconut headspace sampled by SPME were analysed. 123
2.2 Enantioselective-GC columns
124
The library has been built on the basis of ITs obtained on four columns (l: 25 m, i.d.: 0.25 mm, 125
df: 0.25 µm) coated with four cyclodextrin derivatives (CD) as chiral selectors diluted at 30% in PS-126 086: 127 2,6-di-O-methyl-3-O-pentyl-β-CD (2,6DM3PEN-β-CD) [19, 20] 128 2,3-di-O-methyl-6-O-tert-butyldimethylsilyl-β-CD (2,3DM6TBDMS-β-CD) [21] 129 2,3-di-O-ethyl-6-O-tert-butyldimethylsilyl-β-CD (2,3DE6TBDMS-β-CD) [22] 130 2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl-β-CD (2,3DA6TBDMS-β-CD) [21] 131 132
All columns were from MEGA (Legnano, Italy). Their performance were periodically tested 133
through the Grob test [23, 24] and a home made chiral test [25] consisting of limonene, 2-octanol, 134
camphor, isobornyl acetate, linalyl acetate, 2-methyl-(3Z)-hexenyl butyrate, menthol, 135
hydroxycitronellal, γ-decalactone and δ-decalactone racemates. 136
2.3. Enantioselective-GC-MS conditions
137
A Shimadzu QP2010 GC-MS system was used and results were elaborated with the Shimadzu 138
GCMS Solution 2.51 software (Shimadzu, Milan, Italy). 139
GC conditions: injection mode: split; split ratio: 1:20 for standard solutions, 1:50 for essential 140
oils; injection volume: 1µl. Temperatures: injection: 220°C, transfer line: 230°C; ion source: 200°C; 141
temperature programme: from 50°C to 220°C at 2°C/min (if not specified otherwise). Carrier gas: 142
He; flow rate: 1.0 ml/min. 143
2.4 Library setting-up
144
The library was created on the basis of the analysis of the 134 racemate standards and pure 145
enantiomers, on each column, recording their mass spectra, calculated linear retention indices (ITs ) 146
and enantiomer resolution (R). The IT determination was carried out by injecting an homologous 147
series of n-alkanes containing 17 n-hydrocarbons (C9-C25) purchased from Supelco (Bellefonte, 148
PA), each at 100 ppm in hexane. The enantiomer stereochemistry was confirmed either through 149
authentic samples or by combining literature data and analysis of essential oils or fruit flavour 150
headspaces or extracts. 151
152
3. Results and discussion 153
3.1 Basic approach 154
As already mentioned, the safest approach to identify unequivocally a given optical isomer by 155
enantioselective-GC-MS, in particular in complex mixtures, is to combine two identification 156
parameters (i.e. ITs and MS spectrum), one of them suitable to distinguish between enantiomers 157
(ITs). Unlike conventional GC-MS analysis, mass spectra (or monitoring by diagnostic ions (SIM)) 158
are used to locate the two enantiomers in the chromatogram, and ITs for their identification. 159
The present study was divided into two main steps: the first one concerns the building up of the 160
library using the approach described above and the evaluation of its reliability, and the second one 161
involves its application to its everyday use in a routine laboratory. 162
163
3.2 Development of the library
164
The library has been created through the following steps (a) choice of a set of chiral selectors 165
(CD derivatives) able to cover a wide range of racemate separations, (b) analyte IT determination 166
and evaluation of their reliability, and (c) definition of a correct procedure to select a suitable 167
retention index allowance (RIA) that included determination of the optimal injection amount and
168
measurement of the average total analyte tailing factor. 169
All results and considerations reported in the present article are based on data resulting from 170
enantioselective-GC-MS analyses of 134 racemates whose ITs were determined on four columns 171
coated with different CD chiral selectors (see below): 172
173
3.2.1. Selection of the chiral selector set
174
Four cyclodextrin derivatives were chosen as chiral selectors to build up this library (see 175
experimental § 2.2). The choice of a relatively large number of chiral selectors (columns) was 176
mainly due to the fact that a derivatised CD with a universal enantioselectivity has not yet been 177
found, therefore a number of derivatives suitable to cover most of the usual separation in the flavour 178
and fragrance field had to be used. This lack is due to the intrinsic mechanism of chiral recognition 179
with CD derivatives in gas chromatography that is based on a host-guest interaction of each 180
enantiomer of a racemate with the CD selector, depending the separation on the rather low 181
difference in the energy of interaction of each enantiomer with the CD chiral selector [13, 14]. On 182
the basis of the authors’ experience, laboratories involved with enantioselective GC in the flavour 183
and fragrance field should be provided with at least two columns coated with different CD 184
derivatives, which enables to separate at least 80% of the most common racemates in this field. 185
The selected columns were periodically tested through the Grob test [23, 24] to evaluate their 186
chromatographic performance over time and a home made chiral test [25] containing racemates 187
with different volatility, structure and polarity to control their enantioselectivity. Its composition is 188
reported in paragraph 2.2. Figure 1 reports the chiral test profiles carried out on the four columns 189
investigated. 190
3.2.2. Analyte IT determination and reliability 191
As mentioned above, ITs are the identifying parameter and can actively and successfully be 192
used only if they are highly stable over time; ITs repeatability over time is fundamental for a reliable 193
identification. The development of this library required some fundamental investigations, not 194
necessary in routine work, to evaluate the reliability of the system. The frequency of injection of the 195
hydrocarbon reference mixture to obtain the highest IT repeatability was first studied. The intra-196
laboratory IT stability versus the frequency of injection of the hydrocarbons was determined by a 197
series of periodical experiments carried out on some of the components of the chiral test on all 198
columns investigated. Table 1 reports the IT variation of the marker analytes by injecting 199
hydrocarbon standard mixture at different intervals. These results clearly show that the highest IT 200
stability is obtained when the hydrocarbons are injected every five analyte injections. This 201
frequency was then adopted for the creation of the library . Less frequent hydrocarbon injections are 202
required for routine analysis. 203
Control analyses were carried out over a period of two months, they were randomly repeated for 204
three couples of two consecutive days for each CD column. Analyses were repeated three times 205
each day for a total of 18 determinations for each column. Table 2 reports the average ITs and ∆ 206
ITof each enantiomer calculated over the whole period investigated. These results show that ITs are 207
highly repeatable and their ∆ IT
s never exceed two units. 208
3.2.3. Determination of the retention index allowance, RIA
209
The RIA window determination, i.e. the range within which the IT of an analyte has to fall to be 210
correctly identified, is a key point for an univocal identification. A “friendly” operating library 211
should be based on a unique RIA window to be automatically applied to all enantiomers when 212
analysed on all columns investigated. The ideal RIA should be “narrow” enough to include only one 213
of the two enantiomers; RIA’s choice is therefore strongly conditioned by the enantiomer resolution 214
obtained with a given chiral selector and it is critical, in particular, for those enantiomers whose 215
resolution on a given column is below 1.5, i.e. where partial peak overlapping occurs. On the other 216
hand, RIA cannot be too “narrow” to avoid that the IT of a given analyte falls outside the range 217
because of retention variation. A reliable RIA therefore requires not only highly stable ITs , as 218
mentioned in paragraphs 3.2.2. and 3.2.4, but also highly inert columns to avoid peak tailing that 219
can affect enantiomer ITs and/or resolution, in particular with polar analytes. RIA is therefore 220
directly related to the tailing factor of a peak and is determined by measuring the ITs at the start, 221
apex and stop points of the peak; the window is defined through the following expressions: 222
ITx - (ITap - ITstart) < ITx < ITx + (ITstop - ITap) 223
where ITx is the IT of the analyte considered, ITap is the IT at the apex of the peak, ITstart is the IT at the 224
peak starting point and ITstop is the IT at the peak final point. This approach gives two sub-window 225
values (the first one before the peak apex, the second one after the peak apex) limiting identification 226
mistakes with asymmetrical peaks. A correct RIA is also conditioned by the injected analyte 227
concentration that should be such to avoid column overloading in particular with CD stationary 228
phases because of the peak defocusing phenomenon [26]. 229
The unique RIA for the present library was obtained from the average RIA of each class of 230
compounds, in its turn determined from the individual RIA of each of the enantiomers of 134 231
racemates with different structures, polarities and volatilities analysed three times on the four 232
enantioselective columns adopted. Table 3 reports the average RIA window values of the different 233
groups of compounds analysed with each column. The good RIA homogeneity between the 234
different classes of compounds allowed us to adopt a unique average RIA value of –1 and +2 for all 235
analytes analysed on the investigated columns. 236
3.2.4 Interlaboratory ITs reliability
237
The ITreliability was also tested by an inter-laboratory round robin test carried out in both the 238
authors’ laboratories. The chiral test was simultaneously analysed with two sets of four CD columns 239
under rigorously controlled conditions (see experimental). Each CD column was first submitted to a 240
reconditioning cycle (2 hours) and to a Grob test to evaluate their performance. ITs of the chiral test 241
components were then determined by injecting both the hydrocarbon standard mixture and three 242
times the chiral test. Table 4 reports ITs of the chiral test components and ΔITs between the two 243
laboratories. These results show that the IT reproducibility is very high and that the system proposed 244
can be used at an inter-laboratory level provided that rigorously standardised conditions are applied. 245
3.2.5 Creation of the library
246
The library was then created by attributing a record to each enantiomer. Each record includes 247
retention indices on four columns, mass spectrum, IUPAC chemical name, CAS number, molecular 248
weight, and, when separated, racemate enantiomer resolution on the chiral selector investigated and, 249
in the included data base, relative retention and tailing factor together with the original source of the 250
each enantiomer (see below). Table 5 reports the list of the compounds at present included in the 251
library. 252
The library with interactive ITs described here is dedicated to GC/MS systems adopted here, 253
and operates together with the related data collecting software (see experimental § 2.3) [27]. This 254
software is provided with an automated retention index calculation option. IT calculation first 255
requires the creation of the reference index table obtained by injecting an homologous series of 256
standards (in this case the C9-C25 n-alkane series). ITs of enantiomers or of real-world sample 257
components are then determined relatively to the reference index table. Moreover, up to five 258
distinct retention index values (i.e. determined on five stationary phases) for each compound 259
whose mass spectrum is appended to the mass spectral library can be introduced in each record, 260
and a column selection tool is available in order to display in the similarity search result window 261
only the ITs determined on the investigated stationary phase. 262
The correct elution order of the enantiomers of a racemate was determined by analysing pure 263
standards either commercially available or supplied by other laboratories, and natural sources 264
containing identified enantiomer confirming the results with literature data. The determination of 265
the correct elution order of δ-octalactone enantiomers on the four columns is here reported as an 266
example of the latter approach. Coconut fresh fruits were analysed by HS-SPME-ES-GC-qMS on 267
the four CD columns and δ-octalactone located in the four chromatograms through its MS spectrum. 268
(Figure 2). The absolute R configuration was attributed to the identified peak on the basis of 269
literature data reporting the elution order on the same stationary phases [28]. 270
3.3 Application of the library to real world samples
272
The reliability of the library was then evaluated through the chiral recognition of some 273
components of Lavandula angustifolia P. Mill (lavender) e.o. These examples are also reported to 274
show how the long and careful work required for the library development resulted in a very easy 275
and quick operation in the everyday work. Since the MS spectra of optical isomers are not 276
distinguishable, their EE and/or ER can directly be determined from the TIC integration, provided 277
that: (a) when complex matrices are analysed, their peaks are perfectly separated and/or diagnostic 278
ions suitable to discriminate them in extract ion mode from other co-eluting peaks are present in 279
their mass spectra or that a suitable spectral deconvolution programme is available, and (b) a 280
dramatically high difference of enantiomer abundances do not influence the mass spectrum relative 281
ion abundances. Moreover, the fact that each record also includes the enantiomer resolution of a 282
racemate on the four columns enables the operator to find the column(s) (within the four 283
investigated) able to separate from an unresolved racemate, provided that it has been identified 284
through its mass spectrum. 285
Three examples involving the identification and ER determination of well resolved racemates, 286
unresolved racemates, and poorly resolved compounds are here discussed. 287
3.3.1 Well resolved racemates
288
Two different strategies for library searches can be applied depending if RIA option is operated 289
or not. Enantiomers can be distinguished through “interactive” ITs only when the retention index 290
allowance (RIA) is operative. This parameter indicates the IT window where to run the search; only 291
the spectra of analytes with ITs falling in the selected window are considered, thus making possible 292
their unequivocal identification. If RIA is not operative, ITs are displayed but they do not “actively” 293
operate for identification: the enantiomers are therefore located by MS but not distinguished. These 294
considerations are clearly illustrated by the enantioselective-GC-MS analysis of linalool in lavender 295
e.o with a 2,6DM3PEN-β-CD column (Figures 3 and 4) where R enantiomer was found to elute as 296
first and to be the most abundant (ER >95%) [29]. 297
3.3.2. Unresolved racemates
298
Figure 5 shows the reported results after library search when linalyl acetate is analysed with the 299
identical column used above for the same e.o. ITs and RIA are active but specific enantiomers are 300
not identified because they are not separated. However, library search displays the resolution 301
obtained with the four chiral columns enabling us to find the one separating them. Figure 6 reports 302
the enantioselective-GC profile of the same e.o. analysed on a 2,3DE6TBDMS-β-CD where linalyl 303
acetate is base-line separated, R-isomer eluted as first and was prevailing (ER > 99%)[29]. 304
3.3.3. Poorly separated enantiomers
305
The choice of a correct RIA is fundamental for those racemates whose resolution on a given 306
column is below 1.5, i.e. when they partially overlap. In this case, too wide a RIA may include both 307
enantiomers making their identification problematic, even if their ITs are different; on the other 308
hand, if RIA is too “narrow”, the risk that IT falls outside the window is concrete, in particular if the 309
chromatographic system is not perfectly “stable”. The first possibility is clearly illustrated in Figure 310
7 for α-pinene enantiomer identification in the same lavender essential oil when it is analysed with a 311
2,3DE6TBDMS-β-CD column and a RIA between -3 and +3 is applied. On the other hand Figure 7, 312
shows that the general RIA adopted for this library, from -1 to + 2, provides a correct enantiomer 313
identification showing that the R isomer elutes as first with an ER above 75%. 314
315
4. Conclusions 316
The results here reported demonstrated the reliability of the adopted approach for an 317
unequivocal enantiomer identification by enantioselective-GC-MS in the flavour and fragrance field 318
and the fundamental importance of combining ITs and mass spectra actively for a correct 319
identification of an enantiomer. ITs have also been demonstrated to be highly stable, thus affording 320
the adoption of a RIA value common to all class of racemates investigated between +1 and – 2 IT 321
units, provided that the reference hydrocarbon standard mixture is injected every five analysis when 322
the library is created. The library has also been shown to operate effectively in the analysis of real 323
world samples and at present it includes the enantiomers of 134 racemates analysed on columns 324
coated with four different CD derivatives. The number of records is being constantly increased 325
although its implementation takes time because of the difficulty to find new racemates and the 326
related pure enantiomers. 327
328
Acknowledgements 329
This research was carried out within the project entitled: ”Sviluppo di metodologie 330
innovative per l'analisi di prodotti agroalimentari” (FIRB Cod.: RBIP06SXMR_002) of the 331
Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) (Italy).
332 333 334
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Chromatogr. 15 (1992) 367. 361
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Chromatogr. 16 (1991) 209. 367
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Chromatogr. 14 (1991) 317. 369
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(2007) 413. 371
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Captions to figures 375
Figure 1. Chiral test profiles carried out on the four columns investigated. 1: limonene, 2: 2-octanol, 376
3: camphor, 4: isobornyl acetate, 5: linalyl acetate, 6: 2-methyl-(3Z)-hexenyl butyrate, 7: menthol, 377
8: hydroxycitronellal, 9: γ-decalactone, 10: δ-dodecalactone; a: (R) enantiomer, b: (S) enantiomer, x 378
and y: enantiomer configuration not assigned. 379
380
Figure 2. Extract ion profiles (77, 99, 114 m/z) of ( ___): Cocus nucifera L. fruit head space sampled 381
by SPME, and (---) δ-octalactone standard solution analysed on a 2,3DA6TBDMS-β-CD column. 382
383
Figure 3. Enantioselective-GC-MS profile of Lavandula angustifolia P. Mill. essential oil analysed 384
on a 2,6DM3PEN-β-CD column. 385
386
Figure 4. Enantioselective-GC-MS profile of linalool in Lavandula angustifolia essential oil with a 387
2,6DM3PEN-β-CD column. Library search with “inactive” (1) and “active” (2) RIA. 388
389
Figure 5. Identification of linalyl acetate in Lavandula angustifolia essential oil. 390
391
Figure 6. Enantioselective-GC-MS profiles of linalyl acetate in Lavandula angustifolia essential oil 392
(___ ) and of its racemate standard ( ---) analysed on a 2,3DE6TBDMS-β-CD column. 393
394
Figure 7. Enantioselective-GC-MS profile of α-pinene in Lavandula angustifolia essential oil 395
analysed on a 2,3DE6TBDMS-β-CD column. Library search with narrow (1) and wide (2) RIA 396
Table 1: ITLRI variation of three marker analytes as a consequence of different injection time intervals of the hydrocarbon standard mixture. Section A and B: IT LRIs calculated every consecutive days on hydrocarbons injected on the day A1/B1. Section C: IT LRIs calculated every consecutive days on hydrocarbons injected each day every five analysis.
Hydrocarbon Injection frequency: every week
Hydrocarbon Injection frequency: every three days
Hydrocarbon Injection frequency: every five analyses Anal. A1 Anal. A2 Anal. A3 Anal. A4 Anal. A5 ∆ ITLR I Anal. B1 Anal. B2 Anal. B3 ∆ ITLR I Anal. C1 Anal. C2 Anal. C3 Anal. C4 Anal. C5 ∆ ITLR I 2,6DM3PEN-β-CD (S)-(-)-Limonene 1061 1061 1060 1060 1059 2 1061 1061 1060 1 1061 1061 1061 1061 1061 0 (R)-(+)-Limonene 1068 1068 1067 1067 1067 1 1068 1068 1067 1 1068 1068 1068 1068 1068 0 (1R, 2S, 5R)-(-)-Menthol 1350 1350 1348 1348 1348 2 1350 1350 1348 2 1350 1350 1350 1349 1349 1 (1S, 2R, 5S)-(+)-Menthol 1352 1352 1351 1350 1350 2 1352 1352 1351 1 1352 1352 1352 1352 1352 0 (R)-γ-decalactoneDecalactone 1610 1610 1608 1608 1607 3 1610 1610 1608 2 1610 1610 1610 1610 1610 0 (S)-γ-decalactoneDecalactone 1616 1615 1614 1613 1613 3 1616 1615 1614 2 1616 1616 1616 1615 1615 1 2, 36DM6TBDMS-β-CD (S)-(-)-Limonene 1082 1081 1081 1081 1081 1 1082 1081 1081 1 1082 1082 1082 1082 1082 0 (R)-(+)-Limonene 1097 1096 1096 1096 1096 1 1097 1096 1096 1 1097 1097 1097 1097 1097 0 Menthol 1371 1370 1370 1370 1370 1 1371 1370 1370 1 1371 1371 1371 1371 1371 0 (R)-γ-Decalactonedecalactone 1635 1633 1633 1633 1633 2 1635 1633 1633 2 1635 1634 1635 1634 1635 1 (S)-γ-Decalactonedecalactone 1645 1643 1644 1644 1643 2 1645 1643 1644 2 1645 1645 1645 1645 1645 0 2,63DE6TBDMS-β-CD (S)-(-)-Limonene 1056 1056 1057 1056 1056 1 1056 1056 1057 1 1056 1056 1056 1056 1056 0 (R)-(+)-Limonene 1072 1072 1073 1072 1072 1 1072 1072 1073 1 1072 1073 1073 1073 1072 1 (1R, 2S, 5R)-(-)-Menthol 1282 1282 1283 1282 1282 0 1282 1282 1283 0 1282 1283 1282 1282 1282 1 (1S, 2R, 5S)-(+)-Menthol 1285 1285 1285 1285 1284 1 1285 1285 1285 0 1285 1285 1285 1285 1284 1 (R)-γ-Decalactonedecalactone 1573 1573 1574 1573 1573 1 1573 1573 1574 1 1573 1574 1573 1573 1573 1 (S)-γ-Decalactonedecalactone 1588 1588 1588 1588 1587 1 1588 1588 1588 0 1588 1588 1588 1588 1588 0 2,63DA6TBDMS-β-CD Limonene 1053 1053 1051 1051 1051 2 1053 1053 1051 2 1053 1053 1053 1053 1053 0 Formattato Formattato
(1R, 2S, 5R)-(-)-Menthol 1383 1384 1382 1081 1081 3 1383 1384 1382 2 1383 1384 1384 1383 1083 1 (1S, 2R, 5S)-(+)-Menthol 1393 1394 1392 1091 1091 3 1393 1394 1392 2 1393 1394 1394 1393 1093 1 (R)-γ-Decalactonedecalactone 1809 1809 1807 1807 1806 3 1809 1809 1807 2 1809 1809 1810 1810 1810 1 (S)-γ-Decalactonedecalactone 1819 1819 1817 1816 1816 3 1819 1819 1817 2 1819 1819 1820 1819 1820 1
Table 2: Average IT LRIs and Δ IT LRIs of chiral test components carried out overover three weeks of chiral test components,.Control analyses were carried out overon a period of two months, they were randomly repeated for three couples of two consecutive days for each CD column. Analyses were repeated three times each day for a total of 18 determinations for each column.
2,6DM3PEN-β-CD 2,3DM6TBDMS-β-CD 2,3DE6TBDMS-β-CD 2,3DA6TBDMS-β-CD
Week 1 Week 2 Week 3 ∆ ITLRI Week 1 Week 2 Week 3 ∆ ITLRI Week 1 Week 2 Week 3 ∆ ITLRI Week 1 Week 2 Week 3 ∆ ITLRI (S)-(-)-Limonene 1061 1061 1061 0 1082 1081 1082 1 1056 1056 1056 0 1052 1053 1053 1 (R)-(+)-Limonene 1068 1068 1067 1 1097 1096 1095 2 1072 1073 1071 1 (S)-2-Octanol 1147 1146 1146 1 1128 1128 1128 0 1111 1111 1110 1 1236 1237 1235 2 (R)-2-Octanol 1130 1130 1130 0 1112 1113 1112 1 (S)-(-)-Camphor 1184 1184 1184 0 1193 1193 1193 0 1133 1133 1133 0 1241 1242 1242 1 (R)-(+)-Camphor 1199 1198 1199 1 1141 1141 1141 0 1258 1259 1258 1 (X)-Isobornyl acetate 1269 1269 1269 0 1257 1256 1256 1 1220 1220 1220 0 1321 1322 1321 1 (Y)-Isobornyl acetate 1273 1273 1273 0 1261 1260 1260 1 1222 1222 1223 1 (R)-(-)- Linalyl acetate 1240 1240 1240 0 1254 1254 1254 0 1231 1231 1231 0 1301 1303 1301 2 (S)-(+)-Linalyl acetate 1256 1255 1256 0 1237 1237 1237 0 1303 1304 1303 1 (X)-2-Methyl-(3Z)-hexenyl butyrate 1243 1243 1243 0 1246 1245 1245 1 1240 1240 1240 0 1296 1297 1296 1 (Y)-2-Methyl-(3Z)-hexenyl butyrate 1245 1245 1245 0 1249 1249 1249 0 1244 1244 1244 0 1298 1299 1298 1 (1R, 2S, 5R)-(-)-Menthol 1350 1349 1349 1 1371 1370 1371 1 1282 1282 1282 0 1383 1383 1382 1 (1S, 2R, 5S)-(+)-Menthol 1352 1352 1352 0 1285 1285 1285 0 1393 1394 1392 2 (X)-Hydroxycitronellal 1438 1438 1438 0 1447 1446 1447 1 1373 1373 1373 0 1646 1647 1645 2 (Y)-Hydroxycitronellal 1450 1449 1450 1 1374 1374 1374 0 1651 1652 1651 1 (R)-γ-Decalactone 1610 1610 1610 0 1635 1634 1634 1 1573 1573 1573 0 1809 1810 1810 1 (S)-γ-Decalactone 1616 1616 1616 0 1646 1645 1645 1 1588 1588 1588 0 1819 1820 1820 1 (S)-δ-Decalactone 1637 1636 1636 1 1641 1640 1641 1 1586 1586 1586 0 1866 1867 1866 1 (R)-δ-Decalactone 1646 1645 1645 1 1588 1588 1588 0 1875 1876 1875 1 Formattato Formattato Formattato Formattato Formattato Formattato Formattato Formattato Formattato
Table 3 - RIA window values expressed as average start-apex and apex-stop ITLRI variation of the groups of racemate analysed with each CD column
2,,6DM3PEN- β-CD 2,, 6DM6TBDMS-β-CD 2,, 6DE6TBDMS-β-CD 2,, 6DA6TBDMS-β-CD Average Start-apex RIA Average Apex-stop RIA Class Start-apex Apex-stop Start-apex Apex-stop Start-apex Apex-stop Start-apex Apex-stop Esters 1,.6 1,.4 1,.3 1,.8 1,.3 1,.7 1,.4 1,.3 1,.4 1,.6 Alcohols 1,.6 1,.8 1,.3 1,.8 1,.2 1,.5 1,.4 1,.9 1,.4 1,.8 Ketones 1,.6 1,.7 1,.5 2,.1 1,.4 1,.5 1,.5 1,.6 1,.5 1,.7 Acids 1,.5 2,.4 1,.1 2,.6 1,.1 4,.3 1,.3 4,.8 1,.3 3,.5 Hydrocarbons 1,.1 1,.8 1,.5 1,.5 1,.3 2,.0 1,.3 1,.6 1,.3 1,.7 Aldehydes 2,.0 2,.0 1,.0 1,.5 1,.0 1,.0 1,.0 1,.5 1,.2 1,.5 Lactones 1,.8 2,.0 1,.5 2,.2 1,.6 2,.5 1,.8 1,.8 1,.7 2,.1 Mean 1,.6 1,.9 1,.3 1,.9 1,.3 2,.1 1,.4 2,.1 1,.4 2,.0
Table 4: Interlaboratory average ITLRIs and ΔITLRIs of the chiral test components. 2,6DM3PEN- β-CD 2, 36DM6TBDMS- β-CD 2, 36DE6TBDMS- β-CD 2, 36DA6TBDMS- β-CD Lab 1 Lab 2 ∆ ITLR I Lab 1 Lab 2 ∆I T
LRI Lab 1 Lab 2
∆
ITLRILab 1 Lab 2
∆ ITLRI (S)-(-)-Limonene 1061 1062 1 1082 1082 0 1056 1056 0 1053 1053 0 (R)-(+)-Limonene 1068 1069 1 1096 1097 1 1072 1072 0 (S)-2-Octanol 1147 1147 0 1128 1129 1 1111 1110 1 1237 1237 0 (R)-2-Octanol 1130 1131 1 1112 1112 0 (S)-(-)-Camphor 1184 1185 1 1193 1193 0 1133 1134 1 1242 1244 2 (R)-(+)-Camphor 1199 1199 0 1141 1142 1 1258 1260 2 (X)-Isobornyl acetate 1269 1270 1 1257 1256 1 1220 1221 1 1321 1322 1 (Y)-Isobornyl acetate 1273 1274 1 1260 1261 1 1222 1224 2 (R)-(-)- Linalyl acetate 1240 1241 1 1254 1254 0 1231 1233 2 1302 1302 0 (S)-(+)-Linalyl acetate 1256 1256 0 1237 1239 2 1303 1304 1 (X)-2-Methyl-(3Z)-hexenyl butyrate 1243 1244 1 1245 1246 1 1240 1241 1 1296 1297 1 (Y)-2-Methyl-(3Z)-hexenyl butyrate 1245 1245 0 1249 1249 0 1244 1245 1 1298 1298 0 (1R, 2S, 5R)-(-)-Menthol 1350 1350 0 1371 1371 0 1282 1283 1 1383 1385 2 (1S, 2R, 5S)-(+)-Menthol 1352 1352 0 1285 1285 0 1393 1394 1 (X)-Hydroxycitronellal 1438 1438 0 1447 1447 0 1373 1374 1 1646 1648 2 (Y)-Hydroxycitronellal 1450 1450 0 1374 1375 1 1652 1654 2 (R)-γ-decalactoneDecalactone 1610 1611 1 1634 1634 0 1573 1573 0 1810 1813 3 (S)-γ-decalactoneDecalactone 1616 1616 0 1645 1645 0 1588 1587 1 1820 1823 3 (S)-δ-decalactoneDecalactone 1637 1637 0 1641 1641 0 1586 1587 1 1866 1869 3 (R)-δ-decalactoneDecalactone 1645 1645 0 1588 1589 1 1876 1878 2 Formattato Formattato Formattato Formattato
Table 5 List of compounds included in the library
Hydrocarbons Menthyl acetate 3-Methylcyclohexanone 3-Hexanol
α-−Phellandrene Methyl 3-hydroxyhexanoate 3-Oxocineole 3-Octanol
α-−Pinene Methyl dihydrofarnesoate α-Damascone 4-Methyl-1-phenylpentanol β-Citronellene Neomenthyl acetate α-Ionone 6-Methyl-5-hepten-2-ol
β-Citronellene Nopyl acetate β-Irone α-Terpineol
β-−Phellandrene Propylene glycolbutyrate Camphor Borneol
β-−Pinene Styrallyl acetate Camphorquinone Ciscis- Myrtanol
Camphene Lactones Carvone Citronellol
Caryophyllene Aerangis lactone Fenchone Fenchyl alcohol
Limonene 3-Methyl-γ-decalactone Isomenthone Geosmin
Sabinene δ-Decalactone Menthone Isoborneol
Heterocyles δ-Dodecalactone Methyl cyclopentenolone Isomenthol
Ambroxide δ-Heptalactone Nootkatone Isopinocampheol
Menthofuran δ-Hexalactone Piperitone Isopulegol
Rose oxide δ-Nonalactone Pulegone Lavandulol
Esters δ-Octalactone Verbenone Linalool
α-Terpinyl acetate δ-Undecalactone Aldehydes Linalool oxide
Bornyl acetate ε-Decalactone Citronellal Menthol
Bornyl isovalerate ε-Dodecalactone Cyclamen aldehyde Neoisomenthol Butyl butyrolactate γ-Decalactone Hydroxycitronellal Neomenthol
Ciscis- 2-Methyl-3-hexenylbutyrate
γ-Dodecalactone Myrtenal Nerolidol
cis -Carvyl acetate γ-Heptalactone Perillyl aldehyde Octan-1,3-diol Dihydrocarvyl acetate γ-Hexalactone Alcohol Patchouli alcohol Dimethyl methylsuccinate γ-Nonalactone α- Bisabolol Perillyl alcohol Ethyl 2-methylbutyrate γ-Octalactone 1-Octen-3-ol Terpinen-4-ol Ethyl 2-phenylbutyrate γ-Pentadecalactone 1-Phenyl ethanol Tetrahydrolinalool Ethyl 3-hydroxybutyrate γ-Pentalactone 1-Phenyl-1-propanol Transtrans- Myrtanol Ethyl 3-hydroxyhexanoate γ-Tetradecalactone 1-Phenyl-2-pentanol Viridiflorol
Ethyl 3-methyl-3-phenylglicidate
γ-Undecalactone 2-Butanol
Acids
Isobornyl acetate Massoia decalactone 2-Heptanol Citronellic acid Isobornyl isobutyrate Massoia dodecalactone 2-Hexanol 2-Methylbutyric acid Lavandulyl acetate Whyskey lactone 2-Methylbutanol 2-Phenyl propionic acid
Linalyl acetate Ketones 2-Octanol Chrysanthemic acid
Linalyl cinnamate 1,8-Epoxy p-menthan-3-one 2-Pentanol Linalyl propionate 3,6-dimethylocta Dimethylocta
2-en-6-one
Figure 1. Chiral test profiles carried out on the four columns investigated. 1: limonene, 2: 2-octanol, 3: camphor, 4: isobornyl acetate, 5: linalyl acetate, 6: 2-methyl-(3Z)-hexenyl
15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 0.25 0.50 0.75 1.00 1.25 1.50 Intensity (x1 000 000) TIC min 2,6DM3PEN-β-CD 1b 1a 2 3 5 6y 6x 4x 4y 7a 7b 8 9a 9b 10 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 0.25 0.50 0.75 1.00 1.25 1.50 min 2,3DM6TBDMS-β-CD TIC 1b 1a 2b 2a 3b 3a 6x 6y 5a 4x, 5b 4y 7 8x 8y 9a 10b 9b, 10a 0.25 0.50 0.75 1.00 1.25 1.50 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 TIC min 2,3DE6TBDMS-β-CD 1b 1a 2b 2a 3b 3a 4x 4y 5b 5a 6x 6y 7a 7b 8x 8y 9a 9b,10a, 10b min 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 0.25 0.50 0.75 1.00 1.25 TIC 2,3DA6TBDMS-β-CD 1 2 3b 3a 6x 6y 5a 5b 4 7b 7a 8a 8b 10b 10a 9a 9b
46.25 46.50 46.75 47.00 47.25 47.50 47.75 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 Intensity (x10 000) Scan:MIC (99,77,114)
Figure 2. Extract ion profiles (77, 99, 114 m/z) of ( ): Cocus nucifera L. fruit head space sampled by SPME, and ( ) d octalactone standard solution analysed on a 2,3DA6TBDMS-b-CD column.
(R )- (- )-L in a lo o l L in a ly l a ce ta te 10.0 15.0 20.0 25.0 30.0 35.0 40.0 1.0 2.0 3.0 4.0 Intesity (x1 000 000) TIC min (S) -( -) -L in a lo o l (1R )-(+ )-a P in en e (1S) -( -) -a P in en e
Figure 3. Enantioselective GC-MS profile of Lavandula angustifolia P. Mill. essential oil analysed on a 2,6DM3PEN-b-CD column.
Figure 4. Enantioselective GC-MS profile of linalool in Lavandula angustifolia essential oil with a 2,6DM3PEN-β-CD column.
A. Well resolved racemates
I T= 1 212 I T = 122 2 R = 5.3 26.50 27.00 27.50 28.00 1.0 2.0 3.0 4.0 Intensity (x1 000 000) TIC min 1. Search with “inactive” RIA
98% 1212 (R)-(-)-LINALOOL 98% 1222 (S)-(+)-LINALOOL
2. Search with “active” RIA
B. Unresolved racemates
Figure 5. Identification of linalyl acetate in Lavandula angustifolia essential oil.
98% 1240 (R)-(-)-LINALYL ACETATE 98% 1240 (S)-(+)-LINALYL ACETATE
95% 1231 (R)-(-)-LINALYL ACETATE 27.00 27.50 28.00 0.5 1.0 1.5 2.0 2.5 3.0 Intensity (x1 000 000) TIC min I T =1231
Figure 6. Enantioselective GC-MS profiles of linalyl acetate in Lavandula angustifolia essential oil ( ) and of its racemate standard ( ) analysed on a 2,3DE6TBDMS-b-CD column.
97% (1R)-(+)-α PINENE 97% (1S)-(-)-α PINENE 1. Narrow RIA 97% (1R)-(+)-α PINENE 97% (1S)-(-)-α PINENE 2. Wide RIA
C. Poorly resolved racemates
10.5 10.6 10.7 10.8 10.9 11.0 0.25 0.50 0.75 1.00 1.25 1.50 1.75 Intensity (x100 000) TIC I T= 921 I T= 924 R = 1.2 min
