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Journal of Chromatography A
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a r t i c l e
i n f o
Article history:
Received 19 February 2008
Received in revised form 16 April 2008
Accepted 18 April 2008
Available online 24 April 2008
Keywords:
Enantiomer identication
Mass spectral library
Enantioselective GCMS
Cyclodextrin
Linear retention indices
Mass spectra
Flavours
Fragrances
a b s t r a c t
This study describes the development of a gas chromatographymass spectrometry (GCMS) library to
identify optically active compounds in the avour and fragrance eld using enantioselective GC with
cyclodextrin derivatives (CDs) as chiral selectors in combination with MS. The library operates on the
interactive combination of linear retention indices (IT values) in parallel to MS spectra, so as to identify enantiomers reliably and to measure EE and/or ER unequivocally. Since MS is not a selective probe
to discriminate optical isomers, mass spectra (or diagnostic ions in SIM mode) are used to locate the
enantiomer(s) in the chromatogram, and IT values to identify it(them) safely and reliably in particular in
complex mixtures. The library has been built up through the following steps:
(a) Selection of CD derivatives able to cover a wide range of racemate separations. Four
cyclodextrin derivatives were used: 2,6-di-O-methyl-3-O-pentyl--CD, 2,3-di-O-methyl-6-O-tertbutyldimethylsilyl--CD, 2,3-di-O-ethyl-6-O-tert-butyldimethylsilyl--CD, and 2,3-di-O-acetyl-6-Otert-butyldimethylsilyl--CD.
(b) Determination of the analytes IT values and evaluation of their stability and reliability at both intraand inter-laboratory level.
(c) Determination of the range within which the IT of an enantiomer has to fall to be correctly identied,
i.e. determination of a common retention index allowance (RIA).
(d) Construction of the library, at the moment comprising the enantiomers of 134 racemates. A record has
been attributed to each enantiomer including IT values determined on the four CD coated columns,
mass spectrum, IUPAC chemical name, CAS number, molecular weight, and, when separated, racemate
enantiomer resolution on the CD investigated.
Some applications of the library are also reported.
2008 Elsevier B.V. All rights reserved.
1. Introduction
The interaction of a compound with a biological system has
long been shown to be stereoselective. Enantiomer recognition and
enantiomeric excess (EE) and/or ratio (ER) determination are a very
important task in avour and fragrance elds, so as (i) to dene
the correlation between chemical composition and organoleptic
properties; (ii) to implement quality control and detect fraud or
adulteration of natural samples; (iii) to determine the biosynthetic pathway when the formation of a compound is studied or
118
100 ppm in hexane. The enantiomer stereochemistry was conrmed either through authentic samples or by combining literature
data and analysis of essential oils or fruit avour headspaces or
extracts.
3. Results and discussion
3.1. Basic approach
As already mentioned, the safest approach to identify unequivocally a given optical isomer by enantioselective GCMS, in
particular in complex mixtures, is to combine two identication
parameters (i.e. IT values and MS spectrum), one of them suitable to
distinguish between enantiomers (IT values). Unlike conventional
119
Fig. 1. Chiral test proles 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 butyrate, (7) menthol, (8) hydroxycitronellal, (9) -decalactone, (10) -dodecalactone; (a) (R) enantiomer, (b) (S) enantiomer, x and y: enantiomer conguration not
assigned.
120
Table 1
IT variation of three marker analytes as a consequence of different injection time intervals of the hydrocarbon standard mixture
Hydrocarbon Injection frequency: every week
2,6DM3PEN--CD
(S)-()-Limonene
(R)-(+)-Limonene
(1R, 2S, 5R)-()-Menthol
(1S, 2R, 5S)-(+)-Menthol
(R)--Decalactone
(S)--Decalactone
1061
1068
1350
1352
1610
1616
1061
1068
1350
1352
1610
1615
1060
1067
1348
1351
1608
1614
1060
1067
1348
1350
1608
1613
1059
1067
1348
1350
1607
1613
2
1
2
2
3
3
1061
1068
1350
1352
1610
1616
1061
1068
1350
1352
1610
1615
1060
1067
1348
1351
1608
1614
1
1
2
1
2
2
1061
1068
1350
1352
1610
1616
1061
1068
1350
1352
1610
1616
1061
1068
1350
1352
1610
1616
1061
1068
1349
1352
1610
1615
1061
1068
1349
1352
1610
1615
0
0
1
0
0
1
2, 3DM6TBDMS--CD
(S)-()-Limonene
(R)-(+)-Limonene
Menthol
(R)--Decalactone
(S)--Decalactone
1082
1097
1371
1635
1645
1081
1096
1370
1633
1643
1081
1096
1370
1633
1644
1081
1096
1370
1633
1644
1081
1096
1370
1633
1643
1
1
1
2
2
1082
1097
1371
1635
1645
1081
1096
1370
1633
1643
1081
1096
1370
1633
1644
1
1
1
2
2
1082
1097
1371
1635
1645
1082
1097
1371
1634
1645
1082
1097
1371
1635
1645
1082
1097
1371
1634
1645
1082
1097
1371
1635
1645
0
0
0
1
0
2,3DE6TBDMS--CD
(S)-()-Limonene
(R)-(+)-Limonene
(1R, 2S, 5R)-()-Menthol
(1S, 2R, 5S)-(+)-Menthol
(R)--Decalactone
(S)--Decalactone
1056
1072
1282
1285
1573
1588
1056
1072
1282
1285
1573
1588
1057
1073
1283
1285
1574
1588
1056
1072
1282
1285
1573
1588
1056
1072
1282
1284
1573
1587
1
1
0
1
1
1
1056
1072
1282
1285
1573
1588
1056
1072
1282
1285
1573
1588
1057
1073
1283
1285
1574
1588
1
1
0
0
1
0
1056
1072
1282
1285
1573
1588
1056
1073
1283
1285
1574
1588
1056
1073
1282
1285
1573
1588
1056
1073
1282
1285
1573
1588
1056
1072
1282
1284
1573
1588
0
1
1
1
1
0
2,3DA6TBDMS--CD
Limonene
(1R, 2S, 5R)-()-Menthol
(1S, 2R, 5S)-(+)-Menthol
(R)--Decalactone
(S)--Decalactone
1053
1383
1393
1809
1819
1053
1384
1394
1809
1819
1051
1382
1392
1807
1817
1051
1081
1091
1807
1816
1051
1081
1091
1806
1816
2
3
3
3
3
1053
1383
1393
1809
1819
1053
1384
1394
1809
1819
1051
1382
1392
1807
1817
2
2
2
2
2
1053
1383
1393
1809
1819
1053
1384
1394
1809
1819
1053
1384
1394
1810
1820
1053
1383
1393
1810
1819
1053
1083
1093
1810
1820
0
1
1
1
1
Section A and B: IsT calculated every consecutive days on hydrocarbons injected on the day A1/B1. Section C: IsT calculated every consecutive days on hydrocarbons injected
each day every ve analysis.
determination and evaluation of their reliability, and (c) denition of a correct procedure to select a suitable retention index
allowance (RIA) that included determination of the optimal injection amount and measurement of the average total analyte tailing
factor.
All results and considerations reported in the present article are
based on data resulting from enantioselective GCMS analyses of
134 racemates whose IT values were determined on four columns
coated with different CD chiral selectors (see below).
Table 2
Average IT s and IT s of chiral test components carried out over three weeks, on a period of 2 months randomly repeated for three couples of 2 consecutive days for each CD
column
2,6DM3PEN--CD
2,3DM6TBDMS--CD
2,3DE6TBDMS--CD
2,3DA6TBDMS--CD
Week 1 Week 2 Week 3 IT Week 1 Week 2 Week 3 IT Week 1 Week 2 Week 3 IT Week 1 Week 2 Week 3 IT
(S)-()-Limonene
(R)-(+)-Limonene
(S)-2-Octanol
(R)-2-Octanol
(S)-()-Camphor
(R)-(+)-Camphor
(X)-Isobornyl acetate
(Y)-Isobornyl acetate
(R)-()-Linalyl acetate
(S)-(+)-Linalyl acetate
(X)-2-Methyl-(3Z)-hexenyl butyrate
(Y)-2-Methyl-(3Z)-hexenyl butyrate
(1R, 2S, 5R)-()-Menthol
(1S, 2R, 5S)-(+)-Menthol
(X)-Hydroxycitronellal
(Y)-Hydroxycitronellal
(R)--Decalactone
(S)--Decalactone
(S)--Decalactone
(R)--Decalactone
1061
1068
1061
1068
1061
1067
0
1
1147
1146
1146
1
0
1184
1184
1184
1269
1273
1269
1273
1269
1273
0
0
1240
1240
1240
1243
1245
1350
1352
1243
1245
1349
1352
1243
1245
1349
1352
0
0
1
0
1438
1438
1438
1610
1616
1610
1616
1610
1616
0
0
1637
1636
1636
1082
1097
1128
1130
1193
1199
1257
1261
1254
1256
1246
1249
1081
1096
1128
1130
1193
1198
1256
1260
1254
1255
1245
1249
1082
1095
1128
1130
1193
1199
1256
1260
1254
1256
1245
1249
1
2
0
0
0
1
1
1
0
0
1
0
1371
1370
1371
1447
1450
1635
1646
1641
1646
1446
1449
1634
1645
1640
1645
1447
1450
1634
1645
1641
1645
1
1
1
1
1
1
Analyses were repeated three times each day for a total of 18 determinations for each column.
1056
1072
1111
1112
1133
1141
1220
1222
1231
1237
1240
1244
1282
1285
1373
1374
1573
1588
1586
1588
1056
1073
1111
1113
1133
1141
1220
1222
1231
1237
1240
1244
1282
1285
1373
1374
1573
1588
1586
1588
1056
1071
1110
1112
1133
1141
1220
1223
1231
1237
1240
1244
1282
1285
1373
1374
1573
1588
1586
1588
0
1
1
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1052
1053
1053
1236
1237
1235
1241
1258
1242
1259
1242
1258
1
1
1321
1322
1321
1301
1303
1296
1298
1383
1393
1646
1651
1809
1819
1866
1875
1303
1304
1297
1299
1383
1394
1647
1652
1810
1820
1867
1876
1301
1303
1296
1298
1382
1392
1645
1651
1810
1820
1866
1875
2
1
1
1
1
2
2
1
1
1
1
1
121
Table 3
RIA window values expressed as average start-apex and apex-stop IT variation of the groups of racemate analysed with each CD column
Class
2,6DM3PEN--CD
2,6DM6TBDMS--CD
2,6DE6TBDMS--CD
2,6DA6TBDMS--CD
Start-apex
Apex-stop
Start-apex
Apex-stop
Start-apex
Apex-stop
Start-apex
Apex-stop
Esters
Alcohols
Ketones
Acids
Hydrocarbons
Aldehydes
Lactones
1.6
1.6
1.6
1.5
1.1
2.0
1.8
1.4
1.8
1.7
2.4
1.8
2.0
2.0
1.3
1.3
1.5
1.1
1.5
1.0
1.5
1.8
1.8
2.1
2.6
1.5
1.5
2.2
1.3
1.2
1.4
1.1
1.3
1.0
1.6
1.7
1.5
1.5
4.3
2.0
1.0
2.5
1.4
1.4
1.5
1.3
1.3
1.0
1.8
Mean
1.6
1.9
1.3
1.9
1.3
2.1
1.4
1.3
1.9
1.6
4.8
1.6
1.5
1.8
1.4
1.4
1.5
1.3
1.3
1.2
1.7
1.6
1.8
1.7
3.5
1.7
1.5
2.1
2.1
1.4
2.0
Table 4
Inter-laboratory average IT s and IT s of the chiral test components
2,6DM3PEN--CD
2, 3DM6TBDMS--CD
(S)-()-Limonene
(R)-(+)-Limonene
(S)-2-Octanol
(R)-2-Octanol
(S)-()-Camphor
(R)-(+)-Camphor
(X)-Isobornyl acetate
(Y)-Isobornyl acetate
(R)-()-Linalyl acetate
(S)-(+)-Linalyl acetate
(X)-2-Methyl-(3Z)-hexenyl butyrate
(Y)-2-Methyl-(3Z)-hexenyl butyrate
(1R, 2S, 5R)-()-Menthol
(1S, 2R, 5S)-(+)-Menthol
(X)-Hydroxycitronellal
(Y)-Hydroxycitronellal
(R)--Decalactone
(S)--Decalactone
(S)--Decalactone
(R)--Decalactone
2, 3DE6TBDMS--CD
Laboratory 1 Laboratory 2 I
Laboratory 1 Laboratory 2 I
1061
1068
1062
1069
1
1
1147
1147
1082
1096
1128
1130
1193
1199
1257
1260
1254
1256
1245
1249
1082
1097
1129
1131
1193
1199
1256
1261
1254
1256
1246
1249
0
1
1
1
0
0
1
1
0
0
1
0
1371
1371
1447
1450
1634
1645
1641
1645
1447
1450
1634
1645
1641
1645
0
0
0
0
0
0
1184
1185
1269
1273
1270
1274
1
1
1240
1241
1243
1245
1350
1352
1244
1245
1350
1352
1
0
0
0
1438
1438
1610
1616
1611
1616
1
0
1637
1637
2, 3DA6TBDMS--CD
Laboratory 1 Laboratory 2 I
1056
1072
1111
1112
1133
1141
1220
1222
1231
1237
1240
1244
1282
1285
1373
1374
1573
1588
1586
1588
1056
1072
1110
1112
1134
1142
1221
1224
1233
1239
1241
1245
1283
1285
1374
1375
1573
1587
1587
1589
0
0
1
0
1
1
1
2
2
2
1
1
1
0
1
1
0
1
1
1
1053
1237
1237
1242
1258
1244
1260
2
2
1321
1322
1302
1303
1296
1298
1383
1393
1646
1652
1810
1820
1866
1876
1302
1304
1297
1298
1385
1394
1648
1654
1813
1823
1869
1878
0
1
1
0
2
1
2
2
3
3
3
2
122
Table 5
List of compounds included in the library
Hydrocarbons
-Phellandrene
-Pinene
-Citronellene
-Phellandrene
-Pinene
Camphene
Caryophyllene
Limonene
Sabinene
Heterocyles
Ambroxide
Menthofuran
Rose oxide
Esters
-Terpinyl acetate
Bornyl acetate
Bornyl isovalerate
Butyl butyrolactate
cis-2-Methyl-3-hexenylbutyrate
cis-Carvyl acetate
Dihydrocarvyl acetate
Dimethyl methylsuccinate
Ethyl 2-methylbutyrate
Ethyl 2-phenylbutyrate
Ethyl 3-hydroxybutyrate
Ethyl 3-hydroxyhexanoate
Ethyl 3-methyl-3-phenylglicidate
Isobornyl acetate
Isobornyl isobutyrate
Lavandulyl acetate
Linalyl acetate
Linalyl cinnamate
Linalyl propionate
Menthyl acetate
Methyl 3-hydroxyhexanoate
Methyl dihydrofarnesoate
Neomenthyl acetate
Nopyl acetate
Propylene glycolbutyrate
Styrallyl acetate
Lactones
Aerangis lactone
3-Methyl--decalactone
-Decalactone
-Dodecalactone
-Heptalactone
-Hexalactone
-Nonalactone
-Octalactone
-Undecalactone
-Decalactone
-Dodecalactone
-Decalactone
-Dodecalactone
-Heptalactone
-Hexalactone
-Nonalactone
-Octalactone
-Pentadecalactone
-Pentalactone
-Tetradecalactone
-Undecalactone
Massoia decalactone
Massoia dodecalactone
Whyskey lactone
Ketones
1,8-Epoxy p-menthan-3-one
3,6-Dimethylocta 2-en-6-one
3-Methylcyclohexanone
3-Oxocineole
-Damascone
-Ionone
-Irone
123
Fig. 2. Extract ion proles (77, 99, 114 m/z) of (): Cocus nucifera L. fruit head space sampled by SPME, and (- - -) -octalactone standard solution analysed on a 2,3DA6TBDMS-CD column.
Grob test to evaluate their performance. IT s of the chiral test components were then determined by injecting both the hydrocarbon
standard mixture and three times the chiral test. Table 4 reports IT
values of the chiral test components and IT values between the
two laboratories. These results show that the IT reproducibility is
very high and that the system proposed can be used at an interlaboratory level provided that rigorously standardised conditions
are applied.
3.2.5. Creation of the library
The library was then created by attributing a record to
each enantiomer. Each record includes retention indices on four
columns, mass spectrum, IUPAC chemical name, CAS number,
molecular weight, and, when separated, racemate enantiomer resolution on the chiral selector investigated and, in the included data
base, relative retention and tailing factor together with the original
source of the each enantiomer (see below). Table 5 reports the list
of the compounds at present included in the library.
The library with interactive IT values described here is dedicated
to GC/MS systems adopted here, and operates together with the
related data collecting software (see Section 2.3) [27]. This software
is provided with an automated retention index calculation option.
IT calculation rst requires the creation of the reference index table
obtained by injecting an homologous series of standards (in this
case the C9 C25 n-alkane series). IT values of enantiomers or of
real-world sample components are then determined relatively to
the reference index table. Moreover, up to ve distinct retention
index values (i.e. determined on ve stationary phases) for each
compound whose mass spectrum is appended to the mass spectral
library can be introduced in each record, and a column selection tool
is available in order to display in the similarity search result window only the IT values determined on the investigated stationary
phase.
Fig. 3. Enantioselective GCMS prole of Lavandula angustifolia P. Mill. essential oil analysed on a 2,6DM3PEN--CD column.
124
Fig. 4. Enantioselective GCMS prole of linalool in Lavandula angustifolia essential oil with a 2,6DM3PEN--CD column. Library search with inactive (1) and active (2)
RIA.
125
unequivocal identication. If RIA is not operative, IT values are displayed but they do not actively operate for identication: the
enantiomers are therefore located by MS but not distinguished.
These considerations are clearly illustrated by the enantioselective
GCMS analysis of linalool in lavender e.o with a 2,6DM3PEN--CD
column (Figs. 3 and 4) where R enantiomer was found to elute as
rst and to be the most abundant (ER >95%) [29].
3.3.2. Unresolved racemates
Fig. 5 shows the reported results after library search when
linalyl acetate is analysed with the identical column used above
for the same e.o. IT values and RIA are active but specic enantiomers are not identied because they are not separated. However,
library search displays the resolution obtained with the four chiral columns enabling us to nd the one separating them. Fig. 6
Fig. 7. Enantioselective GCMS prole of -pinene in Lavandula angustifolia essential oil analysed on a 2,3DE6TBDMS--CD column. Library search with narrow (1) and wide
(2) RIA.
126
reports the enantioselective GC prole of the same e.o. analysed on a 2,3DE6TBDMS--CD where linalyl acetate is base-line
separated, R-isomer eluted as rst and was prevailing (ER > 99%)
[29].
3.3.3. Poorly separated enantiomers
The choice of a correct RIA is fundamental for those racemates
whose resolution on a given column is below 1.5, i.e. when they
partially overlap. In this case, too wide a RIA may include both
enantiomers making their identication problematic, even if their
IT values are different; on the other hand, if RIA is too narrow,
the risk that IT falls outside the window is concrete, in particular
if the chromatographic system is not perfectly stable. The rst
possibility is clearly illustrated in Fig. 7 for -pinene enantiomer
identication in the same lavender essential oil when it is analysed
with a 2,3DE6TBDMS--CD column and a RIA between 3 and +3
is applied. On the other hand Fig. 7, shows that the general RIA
adopted for this library, from 1 to + 2, provides a correct enantiomer identication showing that the R isomer elutes as rst with
an ER above 75%.
4. Conclusions
The results reported here demonstrated the reliability of the
adopted approach for an unequivocal enantiomer identication by
enantioselective GCMS in the avour and fragrance eld and the
fundamental importance of combining IT values and mass spectra
actively for the correct identication of an enantiomer. IT values
have also been demonstrated to be highly stable, thus affording the
adoption of a RIA value common to all class of racemates investigated between +1 and 2 IT units, provided that the reference
hydrocarbon standard mixture is injected every ve analyses when
the library is created. The library has also been shown to operate
effectively in the analysis of real-world samples and at present it
includes the enantiomers of 134 racemates analysed on columns
coated with four different CD derivatives. The number of records is
being constantly increased although its implementation takes time
because of the difculty to nd new racemates and the related pure
enantiomers.
Acknowledgements
This research was carried out within the project entitled:
Sviluppo di metodologie innovative per lanalisi di prodotti
agroalimentari (FIRB Cod.: RBIP06SXMR 002) of the Ministero
dellIstruzione, dellUniversita` e della Ricerca (MIUR) (Italy).
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