Trends in Analytical Chemistry 52 (2013) 206–225

Contents lists available at ScienceDirect

Trends in Analytical Chemistry

journal homepage: www.elsevier.com/locate/trac

Review

Chiral separations in food analysis

Anna Rocco, Zeineb Aturki, Salvatore Fanali ⇑
Institute of Chemical Methodologies, Italian National Council of Research, Area della Ricerca di Roma I, Via Salaria km 29, 300 – 00015 Monterotondo (Rome), Italy

a r t i c l e i n f o a b s t r a c t

Keywords: Analysis and determination of enantiomers is attractive in food chemistry, as, for example, the presence
Adulteration of D-amino acids can indicate adulteration, as only the natural L-isomer should be present in fruit juices.
Amino acid We highlight the importance of analyzing enantiomers in food, describe the general principle of enantio-
Beverage analysis mer resolution and review the main applications in the past five years in food and beverage analysis. We
Chiral
consider high-performance separation methods (gas and liquid chromatography) and recently developed
Electromigration
Enantiomer
miniaturized tools (capillary electromigration and nano liquid chromatography).
Food analysis Ó 2013 Elsevier Ltd. All rights reserved.
High-performance liquid chromatography
Nano-liquid chromatography
Polyphenol

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
2. General principles of enantiomer separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
3. Selected applications dealing with chiral analysis in food chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
3.1. Enantiomer separation by gas chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
3.2. Chiral separations by high-performance liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
3.3. Chiral analysis by electromigration techniques and miniaturized liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
4. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

Abbreviations: ACN, Acetonitrile; ASE, Accelerated solvent extraction; AA, Amino acid; ADAM, 1-aminoadamantane; AQC, 6-aminoquinolyl-N-hydroxysuccinimidyl
carbamate; aFA, anteiso fatty acid; BGE, Background electrolyte; CCD, Contactless conductivity detection; CD, Cyclodextrin; CDampy, 6-deoxy-6-[N-(2-methylamino)pyr-
idyl)]-b-cyclodextrin; CDen, 6-deoxy-6-[1-(2-amino)ethylamino]-b-cyclodextrin; CDD, Circular dichroism detector; CEC, Capillary electrochromatography; CE, Capillary
electrophoresis; CLC, Capillary liquid chromatography; CLE, Chiral ligand exchange; CS, Chiral selector; CSP, Chiral stationary phase; CMT, Counter-migration technique; DAD,
Diode-array detector; DS, Dietary supplement; EIE, Extracted ion electropherogram; EKC, Electrokinetic chromatography; ECD, Electron-capture detector; ECNI, Electron-
capture negative ion; EI, Electron ionization; FAME, Fatty acid methyl ester; FID, Flame-ionization detector; FLEC, (+)-1-(9-fluorenyl)ethyl chloroformate; FMOC, 9-
fluorenylmethyl-chloroformate; FITC, Fluorescein isothiocyanate; NBD-F, 4-fluoro-7-nitro-2,1,3-benzoxadiazole; GC, Gas chromatography; GCB, Graphitized carbon black; b-
TBDM, heptakis(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-b-cyclodextrin; HT, Hesperetin; HPLC, High-performance liquid chromatography; HS-b-CD, Highly-sulfated-b-
cyclodextrin; HP-b-CD, Hydroxypropyl-b-CD; IT, Ion trap; LIF, Laser-induced fluorescence; LE, Ligand exchange; LLE, Liquid-liquid extraction; LOD, Limit of detention; LOQ,
Limit of quantification; MZ, meso-zeaxanthin; MEKC, Micellar electrokinetic chromatography; mCD, Modified cyclodextrin; CD3NH2, 3-monodeoxy-3-monoamino-b-CD;
MRM, Multiple-reaction monitoring; nano-LC, Nano-liquid chromatography; NAC, N-acetyl-cysteine; OPA, o-phthalaldehyde; Orn, Ornithine; PFT, Partial filling technique;
QuEChERS, Quick, easy, cheap, effective, rugged and safe; RI, Refractive index; SIM, Selected ion monitoring; SPE, Solid-phase extraction; SPME, Solid-phase microextraction;
SBSE, Stir-bar sorptive extraction; SFC, Supercritical fluid chromatography; TAS, Total analysis system; TBDMS, t-butyldimethylsilyl; TCA, Trichloroacetic acid; TQ, Triple
quadrupole.
⇑ Corresponding author. Tel.: +39 06 90 67 22 56; Fax: +39 06 90 67 22 69.
E-mail address: salvatore.fanali@cnr.it (S. Fanali).

0165-9936/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.trac.2013.05.022
 A. Rocco et al. / Trends in Analytical Chemistry 52 (2013) 206–225
1. Introduction
Food analysis is a very important branch of analytical chemis-
try, able to provide information about chemical composition, pro-
cessing, quality control (QC) and contamination of foodstuffs,
ensuring compliance with food and trade laws. To assure product
authenticity and safety is one of the main demands in the food
field, and furthermore aims to satisfy increased consumer concern
about what is in their food.
As is well known, many components of natural products can be
chiral, having one or more chiral center and allowing the existence
at least of one pair of enantiomers.
Enantiomers are compounds possessing identical physical
chemical properties with a molecular asymmetry: the two com-
pounds are not superimposable mirror images. Two enantiomers
rotate polarized light in opposite directions so they are classified
as dextro (+) or levo (). In the case of amino acids (AAs), they
are reported as L- or D-stereoisomers. Also, modern nomenclature
of enantiomers makes use of R and S symbols considering the sub-
stituent groups around the asymmetric carbon. Obviously, when
enantiomers are consumed, they may interact with the chiral envi-
ronment present in the human body (e.g., enzymes, proteins, and
receptors) to produce different effects.
In food components, diverse activity has been shown for enan-
tiomers. For example, L-AAs, compared with D-enantiomers, can
give a different taste to foodstuffs, and carvone or linalol enantio-
mers exhibit different aromas.
Food originating from natural products is often characterized by
a definite enantiomeric composition, biological processes being
mostly stereospecific. This concept is the basis of the pursuit and
the development of several enantioseparative methods applied to
food analysis.
Chiral analysis in the food field includes:
(1) determination of aroma components and development of
flavors closely approximating natural flavors;
(2) identification of markers to assess authenticity and adultera-
tion of foods and beverages;
(3) evaluation of processing and storage time effects;
(4) age dating (i.e. by means of D-AAs);
(5) investigation of health-promoting compounds;
(6) control and monitoring of fermentation processes, and
microbiological activity in general;
(7) analysis of chiral metabolites; and,
(8) biotransformation of persistent pollutants (e.g., pesticides)
[1–3].
Consequently, knowledge of the enantiomeric composition of a
certain foodstuff is of paramount importance in determining both
quality and genuineness. Modern and ever more sophisticated ana-
lytical methods are needed for appropriate chiral analysis in food.
Analytical methods need to be optimized to achieve fast separation
at high separation efficiency and enantioresolution, and enhanced
sensitivity, and, last, but not least, to give the possibility of cor-
rectly identifying analyzed compounds [e.g., using mass spectro-
metric (MS), polarimetric and/or circular dichroism detectors
(CDDs)]. In addition to the reported features, it is necessary to con-
sider the need for useful chiral stationary phases (CSPs) or chiral
selectors (CSs) operating with different resolution mechanisms.
Chiral compounds have been separated utilizing analytical
techniques that have also been used for preparative purposes
[e.g., gas chromatography (GC), supercritical fluid chromatography
(SFC) and high-performance liquid chromatography (HPLC)].
Recently, miniaturized techniques, such as capillary electrophore-
207
sis (CE) and capillary LC (CLC) or nano-LC, were also applied for
enantiomeric analysis in food chemistry [4].
At present, the greatest number of published methods for chiral
analysis is LC. To explain these data, it is necessary to consider the
following aspects:
(1) the instrumentation is possessed by a large number of
laboratories;
(2) many data are available in literature;
(3) dedicated research work for developing modern instrumen-
tation and new stationary phases is available; and,
(4) there is great interest from companies involved in the
market.
Although LC has been widely accepted in recent decades, CE has
been demonstrated to be a powerful tool in chiral analysis, offering
high separation efficiency and high resolution utilizing cheaper ap-
proaches. Usually, only a few mg of the CS is added to the back-
ground electrolyte (BGE) (e.g., cyclodextrins or their derivatives,
antibiotics, metal complexes with chiral AAs or peptides). Alterna-
tive to CE, capillary electrochromatography (CEC), a hybrid tech-
nique, can offer advantages derived from the high electric field of
CE and those of LC (high stereoselectivity due to the use of station-
ary phases).
In this review, we provide a short discussion about the
principles of chiral separation mechanisms and resolution. We
present the main determinations of chiral compounds achieved
in food chemistry with modern separation techniques, including
GC, HPLC, CE, CEC and nano-LC.
2. General principles of enantiomer separation
Enantiomer separation can be achieved using a chiral com-
pound possessing the capacity to interact with the two isomers
selectively. Such interaction can produce stable compounds (for-
mation of diastereoisomers by means of a chiral derivatization
agent) or labile diastereomeric complexes (formed with the use
of a CS and characterized by less strong bonds involved). In both
cases, the physico-chemical properties of the two diastereomeric
forms differ, enabling the separation of diastereoisomers or enan-
tiomers, respectively, in the two approaches. In the first case, the
chiral derivatization agent acts before the separation through a real
chemical reaction and the resolution method is named indirect,
while, in the second method, called direct resolution, the CS is
present in the environment of the separation system.
Fig. 1. Three points interaction model. CS is the chiral selector; b, c, d and a0 , b0 , c0 , d0
are the interaction sites of the CS and the two enantiomers, respectively.
208 A. Rocco et al. / Trends in Analytical Chemistry 52 (2013) 206–225
From a mechanistic point of view, the separation of the two
enantiomers is generated when, between the two compounds
and a selected CS, a three-point interaction takes place. As a sum-
mary and to give an idea about the enantioselective mechanism,
Fig. 1 shows the interactions between the two enantiomers (#1
and #2) and the CS. As can be observed, both enantiomers interact
with the CS but, due to the spatial configuration of the substitu-
ents, they have a different complex stability (#1 has three points
of proper interaction, while #2 only two). In a chromatographic
process, the enantiomers therefore reach the detector selectively
retarded, where the enantiomer forming more stable diastereo-
meric complex exhibits a longer retention time (#1 > #2). In a
modern interpretation of interaction between enantiomers and
the CS, a repulsive effect is also considered responsible for the dis-
crimination process.
In the direct approach, the CS can be employed in different ways
[e.g., bonded to the stationary phase or to the capillary wall (CEC,
open-tubular CEC) or as mobile-phase additive (HPLC, nano-LC,
CE)]. In LC, most of the CSs used are bonded or adsorbed on station-
ary phases. Among them, a large number of applications have been
carried out utilizing cyclodextrins (CDs) or, e.g., their derivatives,
proteins, polysaccharides, macrocyclic antibiotics, chiral synthetic
polymers, or chiral ion exchangers [5].
Even if a large number of CSPs is available, it is practically
impossible to find a universal stationary phase, considering the dif-
ferent enantioresolution mechanisms involved. Recently, research
was oriented towards development of modified cellulose or amy-
lose CSPs, capable of solving a large number of enantiomers. Most
of these CSPs can be used with various mobile phases, e.g. normal
phase, organic polar mode, or reversed phase, and are usually per-
formed with porous particles. A further improvement has con-
cerned the use of core-shell particles, which offer reduced
analysis time and increased separation efficiency [6].
In selecting the most appropriate separation technique to be
used for chiral separations, first the characteristics of analytes to
be separated must be considered (e.g., their volatility and solubil-
ity). For instance, if compounds possess adequate volatility, the
chiral separation can be performed by GC. Alternatively, the
compounds can be derivatized in order to increase their volatility.
In GC, excellent results have been obtained using CSPs based on
modified CDs coated or bonded on the capillary wall [7]. When
the enantiomers are thermolabile and non-volatile, LC and/or
electro-driven techniques can be advantageously taken into
account.
Since this review is focused on chiral separations in food analy-
sis, a detailed description of commercialized stationary phases will
not be given.
3. Selected applications dealing with chiral analysis in food
chemistry
In this section, we present and discuss the main applications of
chiral separations in food chemistry, reported in literature in
2008–13, taking into account the separation techniques employed.
In particular, we consider studies concerning the determination
of chiral constituents or contaminants in food matrices. However,
we should mention that such molecules are also studied for their
metabolic pathway and pharmacological properties, so including
other matrices, such as biological fluids, which are reviewed only
marginally. Furthermore, we do not consider analyses confirming
the presence of contaminants in water and soil, as they practically
contaminate all the food web.
Data containing additional information (e.g., experimental
conditions, stationary and/or mobile phases used, and detectors)
are available in Tables 1–3.
3.1. Enantiomer separation by gas chromatography
Gas chromatography (GC) plays a very important role in food
analysis for not only identifying volatile compounds (e.g., aroma
components) but also determining food constituents [e.g., fatty
acids (FAs)] or contaminants (e.g., pesticides) [2]. Concerning chiral
separations, GC is applied to the determination of the enantiomeric
composition of drugs, metabolites, pesticides, flavors and fra-
grances [7]. Today, in food analysis, GC chiral separations represent
a new way to assess quality, genuineness, nutritional properties,
and age dating of food and drinks, to find alternative sources of
flavoring, and to study the toxicity and the bioaccumulation of
contaminants in foodstuffs. Some examples are given in order to
better explain the potential and the way to perform GC in these
fields.
Brückner’s group [8] made an attempt to correlate quantities of
D-AAs, in particular D-proline, with the storage time of bottled
wines analyzing white, red, ice and sparkling wines. AAs, extracted
by means of a Pasteur pipettes equipped with plugs of glass wool
and filled with strong acid cation resin, were converted into vola-
tile N(O)-pentafluoropropionyl AA 2-propyl esters and analyzed
with a Chirasil-L-Val fused-silica capillary column (N-propionyl-L-
valine tert butyl amide polysiloxane) coupled to a mass spectrom-
eter (MS). A different approach was instead used to determine the
presence of D-AAs and D-pipecolic acid in kidney and adzuki beans
[9]. The authors preferred the indirect method to perform the
chiral separation, transforming the analytes in their N-ethoxycar-
bonyl/(S)-1-phenylethylamide diastereomeric derivatives using
both non-polar DB-5 and intermediately-polar DB-17 columns.
The use of conventional achiral stationary phases was preferred
due to their good thermal stability and thus long-term durability.
Another class of chiral compounds studied for ascertaining bio-
logical processes or proposed as markers for authenticity controls
are anteiso and 2- and 3-hydroxy FAs. Anteiso FAs (aFA) are
long-chain carboxylic acids with a methyl branch on the (n2)-
carbon. Even if present in small traces, the 12-methyltetradecanoic
acid (a15:0) and the 14-methylhexadecanoic acid (a17:0) are the
most common aFAs occurring in food. Their nutritional importance
was recently reconsidered, since it was reported that they suppress
the proliferation of human cancer cells. Because bioactivity can be
related to the spatial configuration of a chiral molecule, it was fun-
damental also to assess the configuration of aFA. Vetter et al. [10]
examined the enantiomeric distribution of both a15:0 and a17:0 in
the neutral and polar lipids of aquatic food samples and cheese
(gilthead sea-bream fillet, brown trout fillet, see bass fillet, seal
oil, cow mozzarella and camembert cheese). aFAs were extracted
employing accelerated solvent extraction (ASE) followed by lipid
fractionation by solid-phase extraction (SPE) and transformation
into the corresponding FA methyl esters (FAMEs). Finally, the
enrichment of aFAMEs was carried out by RP-HPLC. Enantiosepara-
tions were performed studying the effect of different amounts of
the selected CSP (b-TBDM) dissolved in different achiral polysilox-
anes. Best results were achieved employing a capillary column
comprising 66% b-TBDM in OV-1701 (14% cyanopropylphenyl,
86% dimethyl polysiloxane). The GC system was coupled with elec-
tron-ionization MS (EI-MS). The prevalence of the (S)-enantiomers
of a15:0 and a17:0 was demonstrated in both neutral and polar li-
pid fractions, while the amounts of (R)-enantiomers, detectable in
all samples, were higher in the polar lipids than in the neutral lip-
ids. This phenomenon was correlated to the formation of (R)-aFA in
the polar lipids by means of microorganisms.
The same group paid attention on the chiral analysis of 2- and
3-hydroxy FAs (OH-FAs), which are present at trace levels in foods
[11]. While 2-OH-FAs are present in mammalian tissues, yeast,
bacteria, and plants, 3-OH-FAs are mainly exclusive to the lipids
of microorganisms. In biological samples, the enantiopure form is
Table 1
Chiral separations by GC
Analytes
c- and d-lactones, a-ionone, linalool,
nerolidol, ethyl 2-methylbutyrate, 2-
methylbutyric acid, 2-methylbutanol
Monoterpenes (133 compounds which
comprise mono- and sesquiterpenes,
their oxygenated derivatives; chiral:
a-pinene, sabinene, camphene, b-
pinene, limonene, b-phellandrene,
neomenthol)
Linalool, wine lactone, b-citronellol, cis-
rose oxide
Simeconazole
Ethyl 2-methylbutanoate, linalool and 4-
hydroxy-2,5-dimethyl-3(2H)-furanone
(i.e. furaneol), methyl jasmonate
Cypermethrin and cis-bifenthrin
Chiral: -thujene, camphene, -pinene, -
pinene, sabinene, -phellandrene, -
phellandrene, limonene, citronellal,
linalool, terpinen-4-ol and aterpineol
Matrix
Peach, coconut, apricot, raspberry,
strawberry, melon
Needles and berries of the Juniperus
communis and J. oxycedrus
Sour citrus fruits (Yuzu, Sudachi and
Kabosu)
Cucumber, tomato, apple, pear, wheat,
rice
Strawberry
Tea
Mandarin essential oils
Chiral selector Experimental Mode/ Sample preparation Ref.
conditions Detection
Columns coated with 6I–VII-O-TBDMS-2I– He carrier gas T MS LODs: rapid TAS by on-line combining [16]
VII
-3I–VII-O-acetyl-b-CD (AcAc-CD) and 6I– program 50–220°C 1–3 ppb headspace-SPME with enantioselective
VII
-O-TBDMS-2I–VII-ethyl-3I–VII-O-methyl- GC–MS
b-CD (EtMe-CD), both diluted at 30% in
PS086. For each chiral selector three
different column dimensions used:
25 m  0.25 mm I.D.  0.25 lm film
thickness, for AcAc-CD and
25 m  0.25 mm I.D., 0.15 lm film
thickness, for EtMe-CD, and two 0.10 mm
I.D.  0.10 lm film thickness narrow bore
columns 11.7 m for AcAc-CD, and 11.3 m
for EtMe-CD, and 5 m long, respectively.
Cyclodex-B capillary column He gas carrier T MS Hydrodistillation and headspace-SPME [17]
30 m  0.256 mm I.D., 0.25 lm film program 40–180°C (three beds 50/30 lm divinylbenzene-
A. Rocco et al. / Trends in Analytical Chemistry 52 (2013) 206–225
thickness carboxenpolydimethylsiloxane)
GC  GC: BC-WAX column He carrier gas FID Peel: extraction with the solvent mixture [18]
30 m  0.25 mm I.D., 0.25 lm film Different T program (pentane–diethyl ether, 2:1 by volume).
thickness  b-DEX 225 column Volatile components isolated by solvent
30 m  0.25 mm I.D., film thickness of assisted flavor evaporation technique.
0.25 lm Juice: squeezed, saturated with brine to
inhibit enzymatic reactions and to give
the salting-out effect, extracted with the
solvent mixture. Volatile components
prepared under the same conditions used
for peel extracts.
BGB-172 column 30 m  0.25 mm He carrier gas T EI-MS Preparative HPLC for single enantiomer. [14]
I.D.  0.25 lm film thickness (20% tert- program 150–240°C LODs: 0.4– Sample smashed, homogenized with
butyldimethylsilyl-b-CD in 15% diphenyl- 0.9 lg/kg acetonitrile; addition of anhydrous
and 85% dimethyl-polysiloxane) magnesium sulfate (MgSO4) and sodium
chloride (NaCl); vortexed; centrifuged;
SPE.
through oven transfer adsorption– RPLC: linear gradient FID LODs: Steam distillation-solvent extraction of [19]
desorption (TOTAD) interface for on-line methanol/water, 0.02– volatile compounds of methyl jasmonate
coupling RPLC–GC (RPLC–TOTAD–GC) from 25:75, v/v up to 0.07 mg/L treated samples.
RPLC: 150 mm  4.6 mm I.D., 10 um-C6 100% methanol. GC:
column; GC: fused-silica column coated He carrier gas T
with permethylated -CD (Chirasil–Dex) program 40–180°C
25 m  0.25 mm I.D. a 0.25 lm film
thickness
BGB-172 chiral column 30 m  0.25 mm He carrier gas T ECD Ground tea powder soaked with hot [13]
I.D.  0.25 lm film thickness (20% tert- program 160–230°C water; ultrasonic extraction with
butyldimethylsilyl-b-CD in 15% diphenyl- acetone; partition with hexane; upper
and 85% dimethylpolysiloxane, v/v) solution collected for evaporation;
petroleum ether used to reconstitute the
residues. Clean-up with sandwich glass
cartridge.
For chiral analysis: Megadex DETTBS- H2 carrier gas T isotope Dilution in hexane [20]
(diethyl-tert-butyl-silyl -CD) program 50–200°C ratio MS FID
25 m  0.25 mm I.D., 0.25 lm film (for chiral)
thickness
209
(continued on next page)
 210
Table 1 (continued)

Analytes Matrix Chiral selector Experimental Mode/ Sample preparation Ref.

conditions Detection
Sotolon Dry white wine Fused silica column coated with a 20% b- He gas carrier T MS LOD: LLE with CH2Cl2 and concentrated with [21]
CD solution in 35% program 50–210°C 1 mg/L nitrogen
phenylmethylpolysiloxane
30 m  0.25 mm I.D., 0.25 lm film
thickness
Medium-to-high volatility racemates in Different essential oils (bergamot, lemon, 6I–VII-O-TBDMS-3I–VIIO-ethyl-2I–VII-O- He carrier gas T MS Hydrodistillation and dilution [22]
the flavor and fragrance field orange, bitter orange, lavender, methyl–CD (MeEt-CD, 4) and 6I–VII-O- program 50–220°C
(Hydrocarbons, Heterocycles, esters, peppermint, rosemary and sage) TBDMS-2I–VIIO-ethyl-3I–VII-O-methyl–CD
lactones, alcohols, ketones, aldehydes, (EtMe-CD, 3)
acids) Compared with: 6I–VII-O-TBDMS-2I–VII, 3I–
VII
-O-methyl–CD (MeMe-CD) and 6I–VII-O-
TBDMS-2I–VII, 3I–VIIO-ethyl–CD (EtEt-CD)
(25 m  0.25 mm I.D.) columns coated
with 0.15 um film of CD diluted at 30% in

A. Rocco et al. / Trends in Analytical Chemistry 52 (2013) 206–225

PS086 (polymethylphenylpolysiloxane,
15% phenyl)
Ala, Val, Thr, Glya, Ile, Pro, Leu, Ser, GABAa, white, red, Ice and sparkling wines Chirasil-L-Val fused silica capillary He carrier gas T MS LOD: Sample adjusted to pH 2.3 with 0.1 M HCl, [8]
Asx, Met, Phe, Glx, Tyr, Orn, Lys ano column (N-propionyl-L-valine tert butyl program 70–190°C 0.05–0.1% ion exchanger SPE, conversion into N(O)-
chiral AA amide polysiloxane), 25 m  0.25 mm D- pentafluoropropionyl 2-propyl esters
I.D., 0.12 lm film thickness enantiomer
(mg/L)
Ala, Val, Leu, ILe, Pro, a-aminobutyric Kidney and adzuki bean Indirect method He gas carrier T FID MS Extraction with acidified (pH 6 2) water, [9]
acid, b-aminoisobutyric acid, pipecolic Achiral columns: DB-5 (SE-54 bonded) program 80–290°C T LODs: 2.9– diethyl ether and ethyl acetate in
acid, norleucine, norvaline, 2,3- and DB-17 (OV-17 bonded) fused-silica ramp 150–300°C 79.4 ng/mL sequence. Dilution with distilled water
diaminopropionic acid, N- capillary columns, 30 m  0.25 mm I.D., and subjected to the N-
methylaspartic acid 0.25 lm film thickness Ultra-2 (5% ethoxycarbonylation with subsequent
phenyl–95% methylpolysiloxane bonded diastereomeric amidation.
phase cross-linked capillary column,
25 m  0.20 mm I.D., 0.11 lm film
thickness
Twenty-nine volatile compounds (chiral: ‘Chilliwack’, ‘Tulameen’, ‘Willamette’, Cyclosil B column 30 m  0.25 mm I.D., He carrier gas T MS LOD: SBSE [23]
ketone, a-ionone, a-pinene, linalool, ‘Yellow Meeker’, and ‘Meeker’raspberries 0.25 lm film thickness. program 40–220°C 1 lg/kg.
terpinen-4-ol, d-octalactone, d-
decalactone, and 6-methyl-5-hepten-
2-ol)
2-hydroxydecanoic acid (2-OH-10:0), Bovine, human, mare and goat milk, goat Indirect method He carrier gas T ECNI-MS Extracted saponifiable lipids converted [11]
2-hydroxytetradecanoic acid (2-OH- cheese, goat cream cheese, Emmental, Factor Four VF-5 ms column 30 m, program 40–300°C LODs: 0.02– into methyl esters, and the resulting
14:0), c-decalactone, c-dodecalactone, hand and feta cheese, cow feta cheese, 0.25 mm I.D., 0.25 lm film thickness 0.42 mg/ FAMEs separated into OH-FAMEs (minor
d-decalactone, d-dodecalactone, and d- cow mozzarella, buffalo mozzarella, St. 100 g fraction) and derivatized with (R)-()-R-
tetradecalacton; 2-hydroxydodecanoic John’s Wort oil, cedarnut oil, walnut oil, methoxy-R-trifluoromethylphenylacetyl
acid (2-OH-12:0), 2- avocado oil, brain of pork, bovine, goat, chloride (Mosher’s reagent)
hydroxyhexadecanoic acid (2-OH- and sheep.
16:0), 2-hydroxyoctadecanoic acid
(2-OH-18:0), 2-hydroxyeicosanoic
acid (2-OH-20:0), 3-hydroxydecanoic
acid (3-OH-10:0), 3-
hydroxydodecanoic acid (3-OH-12:0),
3-hydroxytetradecanoic acid (3-OH-
14:0), 3-hydroxyhexadecanoic acid
(3-OH-16:0), 3-hydroxyoctadecanoic
acid (3-OH-18:0)
Chiral: a-ionone, a-pinene, linalool, RBDV-resistant transgenic and wild type Cyclosil B column 30 m  0.25 mm I.D., He carrier gas T MS SBSE [24]
terpinen-4-ol, d-octalactone, and d- ‘Meeker’ raspberries 0.25 lm film thickness program 40–220°C
 decalactone
Sotolon
Optically active compounds in the flavor
and fragrance
Linalool, linalyl propionate, c-
lactones(C6, C7, C8, C11, C12, C14,
C15), d-octalactone, a-
hexachlorohexane, trichlorfon, trans-
chlordane, heptachlor, limonene, 2-
octanol, camphor, isobornyl acetate,
linalyl acetate, 2-methyl-(3Z)-hexenyl
butyrate, menthol, hydroxycitronellal,
c-decalactone and d-decalactone
In bold type are evidenced those experimental parameters which gave the best results.
White wines
Balm lemon, bergamot, boronia,
cornmint, lavender, lemon, peppermint,
rosemary essential oils; apple flavor and
apricot, peach and coconut extracts
Lavender and bergamot essential oils
Precolumn: fused silica column coated He carrier gas T MS Samples with addition of anhydrous [25]
with polar phase BP20; 2 m  0.25 mm program 50–210°C sodium sulfate extracted three times with
I.D., 0.25 lm film thickness; fused silica CH2Cl2; dried over anhydrous sodium
column coated with a solution of 20% b- sulfate and concentrated under a nitrogen
CD in (35% phenyl)-methylpolysiloxane, stream. Preparative-HPLC for sotolon
30 m  0.25 mm I.D., 0.25 lm film enantiomers with Chiralpak AS-H model
thickness 250  20 mm, 5 lm.
Four columns coated with four CD He carrier gas T MS Headspace-SPME [15]
derivatives as chiral selectors diluted at program 50–220°C
30% in PS-086:
I) 2,6-di-O-methyl-3-O-pentyl-b-CD
(2,6DM3PEN-b-CD) II) 2,3-di-O-methyl-
6-O-tert-butyldimethylsilyl-b-CD
(2,3DM6TBDMS-b-CD) III) 2,3-di-O-ethyl-
6-O-tert-butyldimethylsilyl-b-CD
(2,3DE6TBDMS-b-CD) IV) 2,3-di-O-acetyl-
6-O-tert-butyldimethylsilyl-b-CD
(2,3DA6TBDMS-b-CD), 25 m  0.25 mm
I.D., 0.25 lm film thickness
A. Rocco et al. / Trends in Analytical Chemistry 52 (2013) 206–225
Columns coated with 30% of 2,3-di-O- He carrier gas MS Hydrodistillation and dilution [26]
ethyl-6-O-tert-butyldimethylsilyl-b-CD in Different T program
PS-086 25 m  0.25 mm I.D., 0.15 lm and
0.25 lm film thickness; 5 m  0.25 mm
I.D., 0.15 lm film thickness; 1, 2, 5 and
10 m  0.10 mm I.D., 0.10 lm film
thickness
211
 212
Table 2
Chiral separations by HPLC

Analytes Matrix
Cyflumetofen Cucumber, tomato, apple
Lignans Sesame seeds (whole and hulled),
milled whole camelina seeds
(Camelina sativa), pumpkin seeds
(without shells), and hemp seeds
(whole and hulled), rye bran, wheat
bran, oat bran, raspberries,
strawberries, lingonberries, sea
buckthorn, cranberries, linseeds
(Linum flavum), bilberries,
cloudberries, blackberries, red
Chiral selector
Chiralpak AD-H column
250 mm  4.6 mm I.D., 5 lm particle
size
Chiralcel OD-R 250 mm  4.6 mm
I.D., 10 lm particle size, equipped
with a OD-RH guard column
(4.0  10 mm);
Chirobiotic V 250 mm  2.1 mm I.D.,
5 lm particle size, equipped with a
Symmetry C18 guard column
(2.1  10 mm, 3.5 lm)
Experimental conditions
T 25°C n-hexane and 2-propanol
(95:5, v/v)
T 30°C different mixture of MeOH/
0.1% AcOH (v/v) T 20°C
Mode/Detection
DAD
k = 234 nm LODs: 0.33–
0.5 mg/kg for each enantiomer
Triple-quadrupole MS
Sample preparation Ref.
Sample extraction with ACN, NaCl [38]
and anhydrous MgSO4, purified with
ENVI-Carb cartridge (apple) or
Pesticarb/NH2 cartridge (cucumber,
tomato), and filtered
After milling, samples extraction by [30]
ASE; enzymatic hydrolysis
performed adding 480 IU of b-
glucuronidase/sulfatase; extraction
with EtOAc; evaporated to dryness;
residue dissolved in MeOH/0.1%
acetic acid (AcOH) 20/80 (v/v), and
then filtered.

A. Rocco et al. / Trends in Analytical Chemistry 52 (2013) 206–225

gooseberries, blackcurrant
Lutein (L), Raw and cooked fish, seafood, cooked Chiralpak AD column (amylose Linear gradient: hexane (solvent A) DAD Lyophilization, homogenization, [35]
zeaxanthin (Z), egg yolk derivative coated on silica-gel) and hexane:isopropanol (1:1, solvent LOD: 0.2 pmol centrifugation, extraction, dryness,
Meso- B), from 90% A and 10% B for 55 min, repeated extraction.
zeaxanthin followed by a 1 min 100% A, held at Preparative HPLC for L and Z peaks
100% A for 10 min, a 4 min gradient
to 90% A and 10% B, held for 15 min.
Malathion Chinese cabbage, sugar beet and two Cellulose tris(3,5-dimethyl- T 10°C n-hexane/2-propanol (99/1, v/ UV k = 230 nm Triturated sample extraction; filtered [39]
crops (paddy rice/Oryza sativa, phenylcarbamate) (CDMPC) coated v) LOD: 0.05 lg/g for each through anhydrous sodium sulfate;
wheat/Triticum aestivum Linn) onto aminopropylated spherical gel enantiomer concentrated to near dryness;
column 250 mm  4.6 mm I.D. additional cleanup step through a
column of anhydrous Na2SO4/
alumina neutral + activated carbon/
anhydrous Na2SO4
Difenoconazole Cucumber, tomato and soil Chiralcel OJ-H (cellulose tris-(4- T 20°C n-hexane/ethanol 90/10 (v/v) DAD Samples smashed, homogenized, [36]
stereoisomers methylbenzoate)), Chiralcel OD-H k = 220 nm, optical rotation extracted with ACN, MgSO4 and NaCl,
and their (cellulose tris-(3,5-dimethylphenyl - dispersion (ORD) centrifuged, applied to SPE and after
hydroxylated carbamate)), Chiralpak AS-H LODs (DAD): 0.01–0.03 lg g1 filtered
metabolite (Amylose tris-[(S)–a-
difenoconazole methylbenzylcarbamate]) and
alcohol (CGA- Chiralpak AD-H (Amylose tris (3,5-
205375) dimethylphenylcarbamate)) columns
250 mm  4.6 mm I.D., 5 lm particle
size
Benalaxyl, Cucumber, tomato, grape, apple cellulose tris (3,5- T 25°C ACN/0.1% formic acid (45/55, TQ-MS/MS, UV (k = 220 nm), Extraction with ACN containing 1% of [40]
furalaxyl, and dimethylphenylcarbamate) (Lux v/v) CDD LOD (MS): 1 lg/kg for acetic acid, vortexed, addition of
metalaxyl Cellulose-1), cellulose tris (4-chloro- each enantiomer MgSO4 and CH3COONa, centrifuged.
(fungicides) 3-methylphenylcarbamate) (Lux Extract subjected to SPE for cleanup
Cellulose-4), cellulose tris (4- and filtered
methylbenzoate) (Lux Cellulose-3),
and amylose tris (2-chloro-5-
methylphenylcarbamate) (Lux
Amylose-2), columns
150 mm  2.0 mm id, 3 lm particles
size; Amylose tris (3,5-dimethyl
phenylcarbamate) (Chiralpak AD-RH)
column 150 mm  2.1 mm I.D., 3 lm
particles size
Epoxiconazole Grape, soil Lux Cellulose-1, cellulose tris-(3,5-
dimethylphenylcarbamate) (CDMPC)
150 mm  2.0 mm I.D., 3 lm particles
size
Myclobutanil Cucumber, soil cellulose-based Chiralcel OD-RH
column 150 mm  4.6 mm I.D., 5 lm
particle size Chiralpak AD-RH and AS-
RH, 150 mm  4.6 mm I.D., 5 lm
particle size
Tetraconazole Cucumber, musk melon, soils Chiralcel1OJ-H (cellulose tris-(4-
methylbenzoate)) column
250  4.6 mm I.D., 5 lm particle size
Chiralcel OD-RH (cellulose tris-(3,5-
dimethylphenylcarbamate))
column150  4.6 mm I.D.
Dinotefuran Rice, tomato, apple ChromegaChiral CCA column
250 mm  4.6 mm I.D., 5 lm particle
size
Tebuconazole Cabbage, cucumber, and soils HPLC: Lux amylose-2 column
250  4.6 mm I.D., 5 lm particle size
LC: amylose-2 column 150  2.0 mm
I.D, 3 lm
Ala, Arg, Asn, Asp, Japanese black vinegar, fermented Indirect method
Cys, Gln, Glu, milk, yogurt ODS column Ascentis Express C18
His, Ile, Leu, Lys, 100 mm  2.1 mm I.D., 2.7 lm
Met, Phe, Pro, particle size
Ser, Thr, Tyr, Trp,
Val, Glya
a
no chiral
Myclobutanil, Strawberry Lux Cellulose-1 [cellulose tris (3,5-
fenbuconazole dimethylphenylcarbamate)]
150 mm  2.0 mm I.D., 3 lm particle
size
Proteic AAs Sake Indirect method:
Develosil ODS-UG-5 column
250 mm  6 mm I.D.
Shim-pac amino-Na column 6.0
id  100 mm I.D., with Shim-pack
ISC-30 4.0 id  50 mm as guard
column
Famoxadone Spinach Cellulose-1 (tris(3,5-
dimethylphenylcarbamate) of
cellulose), Cellulose-2 (tris(3-chloro-
4-methylphenylcarbamate) of
cellulose) and Amylose-2 (tris(5-
ACN/water (90/10, v/v)
T 40°C ACN/water (70/30, v/v)
T 20°C n-hexane and ethanol (90/10,
v/v)
ACN and water (60/40, v/v)
T 30°C n-hexane/ethanol/methanol
(85/5/10, v/v/v)
T 30°C ACN-water (40/60, v/v)
T 25°C ACN-water (90/10, v/v)
T 40°C stepwise gradient 5 mM
heptafluorobutyric acid/deionized
water (A) and Methanol (B), from 20%
to 80% B
T 25°C ACN/0.1% formic acid solution
(60:40, v/v)
T 40°C linear gradient: from 100%
50 mM sodium acetate buffer (A), to
67% methanol (B) or isocratic
93% 20 mM sodium citrate buffer (pH
3.2)/7% ethanol (A), 600 mM sodium
citrate containing 200 mM borate
(pH 10.0) (B), and 200 mM sodium
hydrate (C).
85% methanol and 15% (v/v) 5 mmol/
L ammonium acetate solution added
to 0.1% formic acid
TQ-MS/MS LODs:
0.005 mg kg1 (grape) and
0.025 mg kg1 (soil)
UPLC/TQ-MS/MS
LODs: 0.61 lg/kg for the two
enantiomers
DAD
LODs: 0.06–0.12 lg/g
depending on matrices optical
rotation dispersion detector
DAD k = 270 nm
LODs: 0.15 mg/kg (rice and
tomato), 0.05 mg/kg (apple)
DAD
LOD: 0.01 lg/g
TQ-MS/MS LODs: 0.001–
0.003 lg/g,
UPLC/CDD, UV k = 235 nm
LODs (CDD): 11–64 pmol/
injection
LODs (UV): 4.9–23 pmol/
injection
MS/MS
CDD
LOD (MS): 2 lg/kg for each
enantiomer
Fluorescence detector
excitation k = 340 emission
k = 450 nm
LODs: 0.006–20 lM for OPA-
NAC pre-column
derivatization; 0.01–20 lM for
the FLEC/ADAM pre-column
derivatization, 1.5 lM (dl-Cys)
for the OPA-NAC post-column
derivatization
MS/MS
LOD: 0.3 lg/kg for each
enantiomer
Extraction with ethyl acetate, [41]
evaporated to near dryness and
completed with a nitrogen stream.
Redissolved in acetone/petroleum
ether before SPE (Florisil).
Extraction with ACN, MgSO4 and [42]
NaCl, clean-up SPE (GCB),
concentrated to almost dryness,
redissolved in ACN and filtered
Extraction with ACN, anhydrous [43]
MgSO4 and NaCl, SPE.
Removal of the solvent in vacuo,
sample dissolved in hexane and
filtered
Extraction with ACN, NaCl and [44]
anhydrous MgSO4; SPE with
ENVICarb Cartridge. Eluate collected
A. Rocco et al. / Trends in Analytical Chemistry 52 (2013) 206–225
and concentrated dryness,
reconstituted in ethanol and filtered
Extraction with aqueous NaCl and [45]
ethyl acetate; cleaned up by SPE on
NH2 cartridge. Evaporated to dryness
with nitrogen gas, and reconstituted
with mobile phase
Removal of high molecular weight [37]
proteins with a 10 kDa cut-off
membrane centrifugal filter device or
with aqueous sulfosalicylic acid
solution. SPE (Bond Elute SCX). The
eluent evaporated under reduced
pressure and redissolved in
deionized water. Derivatization with
NBD-F
Extraction with ACN containing [46]
acetic acid, MgSO4 and sodium
acetate; dispersive SPE with PSA, C18,
and anhydrous MgSO4 for cleanup.
Dilution with water and filtered.
Alternatively AOAC Official Method
2007.01
Pre-column derivatization with o- [47]
phthalaldehyde (OPA) and N-acetyl-
cysteine (NAC); asparagine, histidine,
lysine, and proline in sake measured
with pre-column derivatization with
(+)-1-(9-fluorenyl)ethyl
chloroformate (FLEC) and 1-
aminoadamantane (ADAM)
Cysteine measured as dl-cysteine by
the post-column derivatization with
OPA and NAC;
Extraction with ACN, NaCl and [48]
anhydrous MgSO4; dispersive SPE.
Dilution with water.
(continued on next page)
213
 214
Table 2 (continued)

Analytes Matrix Chiral selector Experimental conditions Mode/Detection Sample preparation Ref.
chloro-2-methylphenylcarbamate) of
amylase) columns 150 mm  2.0 mm
I.D., 3 lm particle size
Homoeriodictyol, Lemon, grapefruit, and tomato, rat Chiralcel OJ-RH column ACN/ water/ phosphoric acid (22/78/ UV Enzymatic hydrolysis by means [34]
isosakuranetin, serum and urine 150 mm  4.6 mm I.D., 5 lm particle 0.1, v/v/v) crude preparation of Helix pomatia
and taxifolin size protected by a Chiralcel OJ-RH glucuronidase type H-2 in sodium
guard column (0.4 cm  1 cm, 5 lm acetate buffer (pH 4.8), ascorbic acid
particle size) and water. Samples centrifuged, the
supernatant collected and dried.
Reconstituted with mobile phase,
centrifuged, and the supernatant
used for HPLC
Tigloylshikonin ‘‘Shikon color,’’ root extract from Chiralcel-OJ-H column T 40°C. DAD Extraction with ethanol, following [49]
Lithospermum erythrorhizon Siebold 250 mm  4.6 mm n-hexane/2-propanol/acetic acid (95/ k = 515 nm evaporation or commercial root
and Zuccarini 5/0.3, v/v/v) extract diluted, filtered (0.45 lm)
and purified with a RP-HPLC. Purified

A. Rocco et al. / Trends in Analytical Chemistry 52 (2013) 206–225

tigloylshikonin dissolved in MeOH,
subjected to saponification, and to
RPLC/ESI-MS analysis to check
saponification
Proteic AA Soy, misos and fish sauce Sumichiral OA-2500 5 mM citric acid in methanol Fluorescence detector Samples deproteinized with [50]
250 mm  4.6 mm I.D. excitation k = 470 sulfosalycilic acid, defatted with
emission k = 530 nm diethyl ether. Clean up with C18 SPE,
followed by SCX SPE. Sample
dissolved in boric acid and reacted
with NBD-F
Tryptophan, Cooked ham (low fat content), LCxLC: C18-Atlantis column, T 20°C C18: ammonium acetate UV Milled meat mixture prepared as a [28]
tyrosine, minced meat (beef meat), dry cured 150  4.60 mm I.D., 5 lm particle buffer (20 mM, pH 6)/methanol (94/ k = 260 nm steak of 1 cm thickness; vacuum-
phenylalanine, Iberian ham, smoked salmon, and soft size, with a Supercosil-C18 Pelliguard 6, v/v) Methanol/water (90/10, v/v) LODs: 0.16–3 mg/L packaged in laminated film bags of
m-tyrosine, o- cheese pre-column 20  4.4 mm; teicoplanin low gas permeability; transported in
tyrosine, and 5- Chirobiotic-T column, 150  21 mm, insulated polystyrene boxes and
OH-tryptophan 5 lm irradiated under an electron-beam
radiation source. Free AAs extraction
with ascorbic acid solution and
filtered
Naringin, Pummelo Chiralpak IB (cellulose tris-3,5- n-hexane/ethanol doped with 0.5% UV Albedo homogenized in methanol/ [51]
neohesperidin dimethylphenylcarbamate) column, TFA (60/40 v/v) k = 292 nm water and Celite; filtered; addition of
250 mm  4.6 mm I.D., 5 lm particle CDD 96% ethanol; dried and then filtered
size
Bisacetylenic Carrots Daicel Chiralpak AD-H column Linear gradient: hexane (A) and UV Minced carrots sequentially [31]
oxylipins 250  4.6 mm I.D., 5 lm particle size isopropanol (B) from 5% up to 25% B k = 210 nm extracted with n-pentane, and ethyl
acetate. Filtration, separation of n-
pentane extractables (A), the ethyl
acetate extractables, and the non-
soluble residue. Fraction A subjected
to further fractionation onto column
filled with silica gel.
Taxifolin Tu fu ling (Rhizoma smilacis glabrae) Chiralcel OJ-RH column T 25°C ACN/water/ phosphoric acid DAD Tu fu ling: extraction with ethanol, [33]
and apple (Malus  domestica) 150  4.6 mm I.D., 5 lm particle size, (15/85/0.5, v/v/v) k = 288 nm adjusted to pH 2.0 with formic acid.
protected by a Chiralcel OJ-RH guard LOQ: 0.5 lg/mL (LOD not Extracts defatted with petroleum
column (0.4  1 cm, 5 lm particle reported) ether, dried, reconstituted in mobile
size) MS phase.
Apple: sample extracted with
methanol, using a Thomas tissue
grinder.
Hydrolysis of glycosides to aglycones
 by b-glucuronidase from Helix
pomatia Type HP-2. Samples
reconstituted in mobile phase,
centrifuged and injected
Catechin and Buckwheat groats Chiralpak 1A column T 40°C hexane/ethanol (8/2 or 5/5, v/ UV Phenolic compounds extracted with [52]
epicatechin v) containing 0.5% TFA k = 280 nm methanol from the powdered groats
under reflux, subjected to Sephadex
LH-20 chromatography. The fraction
of buckwheat groats phenolic
compounds dried and then dissolved
in methanol.
Salsolinol Dried banana chip and grape seed Cyclobond I 2000 (b-CD-bonded) T 4°C. IT-MS/MS Samples extraction with ice-cold [53]
column 250  2.1 mm I.D., 5 lm 150 mM ammonium acetate buffer LOD: 15 ng/mL for each 0.1M HCl solution. Ice-cold ethanol
particle size, connected to a C18 (pH 5.5) enantiomer added to precipitate proteins;
guard column (20  2.1 mm, 5 lm) filtration
Finopril Chinese cabbage (Brassica pekinensis) (R,R) Whelk-O 1 column n-hexane/isopropanol 95/5(v/v) UV Homogenized samples extracted [54]
250 mm  4.6 mm I.D. k = 225 nm with ACN; addition of NaCl and
CDD liquid phase layer was separation;
k = 234 nm addition of anhydrous sodium sulfate
LOQs (UV): 0.005 (S-) and and evaporation to dryness; dilution

A. Rocco et al. / Trends in Analytical Chemistry 52 (2013) 206–225

0.006 (R-) mg/kg with petroleum ether/ethyl acetate;
cleanup through SPE (anhydrous
sodium sulfate, neutral aluminum
oxide + activated carbon and then
anhydrous sodium sulfate). Eluate
evaporated and dissolved in
isopropanol.
Arabinose, ribose, Honey, natural sweeteners, Oporto Chiralpak AD-H (Daicel) column T 25°C Chiral: hexane–ethanol–TFA DAD/RI Samples solved in the mobile phase [29]
mannose, wine 250 mm  4.6 mm I.D. ((7:3):0.1, v/v); LODs (RI): 0.7–5.1 ppm mixture using vortex and
fucose, xylose, carbohydrate analysis column isocratic elution mobile phase water– ultrasounds and then filtered
lyxose, glucose 300 mm  3.9 mm I.D, 10 lm particle acetonitrile (1:9, v/v)
and fructose (a- size
and b forms of
each D- and L-
enantiomer)
Benalaxyl Tomato, tobacco, sugar beet, Cellulose tris-(3,5-dimethylphenyl- T 20°C n-hexane and 2-propanol (90/ DAD Triturated sample extracted with [55]
capsicum, and the soil carbamate) (CDMPC) column 10 for the soil and 98/2, v/v, for plant k = 210 nm ethyl acetate, anhydrous sodium
250 mm  4.6 mm I.D samples) LOD: 0.05 lg/g for each sulfate, and sodium chloride. The
enantiomer mixture filtered through fiberglass.
Rinsed with ethyl acetate then
evaporated. Dissolved with
petroleum ether and then passed
through SPE (anhydrous Na2SO4/
silica gel activated carbon/anhydrous
Na2SO4). The extracted solution
collected and dried.
Hesperidin, Fruit juice rat Hesperetin: Chiralpak AD-RH column T 25°C ACN/water/ phosphoric acid DAD or MS (phosphoric acid Glycoside epimers: dilution of fruit [32]
naringin and 150 mm  4.6 mm I.D., 5 lm particle (42/58/0.01, v/v/v) replaced with formic acid) juice with water, sodium acetate
eriocitrin and size ACN/water/phosphoric acid (30/70/ k(hes) = 298 nm buffer (pH 4.8), ascorbic acid and
aglycones Naringenin: Chiralcel OD-RH column 0.04, v/v/v) k(nar) = 292 nm crude preparation of H. pomatia type
hesperetin, 150 mm  4.6 mm I.D., 5 lm ACN/ water/ phosphoric acid (20/80/ k(hes) = 288 nm HP-2 followed by incubation.
naringenin and Eriodictyol: Chiralpak OJ-RH column 0.04, v/v/v), LOD: 0.5 lg/mL Addition of ice-cold ACN to
eriodictyol 150 mm  4.6 mm I.D., 5 lm precipitate proteins. The supernatant
collected and evaporated to dryness.
The residue reconstituted with
mobile phase, vortexed and
centrifuged.

(continued on next page)

215
216 A. Rocco et al. / Trends in Analytical Chemistry 52 (2013) 206–225

predominant, so that the evaluation of enantiomeric composition

[27]
Ref.

could be considered a valuable tool for authenticity control. In or-

der to improve the selectivity and the sensitivity of the developed
Extrusion was carried out on ground

method, the extracted saponifiable lipids were converted into

thermally hydrolyzed. After cooling,

diastereomers production with OPA

and 1-thio-b-D-glucose tetraacetate FAMEs, which were separated into OH-FAMEs (minor fraction)
hydrochloric acid and proteins
material, samples dissolved in

and derivatized enantioselectively with (R)-(a)-R-methoxy-R-trif-

Sample preparation

pH adjusted to pH = 7, then

luoromethylphenylacetyl chloride [(R)-()-MTPA-Cl, Mosher’s

reagent]. Consequently, the resulting diastereomeric compounds
were separated employing an achiral column and detected with
high-sensitivity electron-capture negative-ion MS (GC/ECNI-MS)
in selected-ion monitoring (SIM) mode, achieving limits of detec-
tion (LODs) in the 0.02–0.42 mg/100 g range. The method was
applied to the determination of 2- and 3-OH-FAs in several food
matrices, such as bovine, human and goat milk, several kinds of
cheese, cow and buffalo mozzarella, and vegetal oils. The potential
for using enantiomeric composition as a marker for authenticity
control was suggested for distinguishing buffalo mozzarella from
Mode/Detection
Fluorescence detector

cow mozzarella or feta cheese made from bovine milk instead of

sheep milk.
Several applications of chiral GC relate to the study of toxicity,
the processes of absorption, distribution, and degradation of chiral
agrochemicals (e.g., pesticides, fungicides, and hormones) in
organisms, vegetables and environment, which are often enantio-
selective [12].
Kuang et al. [13] evaluated the species-specific risk of exposure
(9.5 mM, pH 7.05)/methanol (60/40,

to two pyrethroids, namely cypermethrin and cis-bifenthrin,

Linear gradient: phosphate buffer
Experimental conditions

widely used in China for tea plantations. Both insecticides are sold
as racemic mixtures, even if each enantiomer shows different
insecticidal activity. Chiral analyses employed a chiral capillary
v/v) (A) and ACN (B)

column containing 20% tert-butyldimethylsilyl-b-CD (BGB-172)

coupled with an electron-capture detector (ECD). Analyzing
contaminated tea samples, it found that the patterns of
enantiomeric fractions of cypermethrin residues varied depending
on the type of tea, and the possible preferential degradation was
investigated.
The same chiral column was used by Li et al. [14] for the enan-
250 mm  4.6 mm I.D., 5 lm particle

tioselective determination of triazole fungicide simeconazole in

several vegetables and other foodstuffs (i.e. cucumber, tomato, ap-
ple, pear, wheat and rice). Simeconazole was extracted employing
Kromasil octyl (C-8) column
Chiral selector

a clean-up/enrichment procedure based on the modification of

QuEChERS method (quick, easy, cheap, effective, rugged and safe).
A homogenizer was used to improve the extraction efficiency com-
Indirect method

bined with a double-layered cartridge column [graphitized carbon

black (GCB) and silica modified with amino-groups] to remove pig-
In bold type are those experimental parameters that gave the best results.

ments and FAs from extracts. Ion-trap MS (GC–ITMS) with EI was

used for qualitative and quantitative determination of the simeco-
size

nazole enantiomers, using the acquisition method of multiple-

reaction monitoring (MRM). LOD values for both enantiomers were
estimated to be 0.4–0.9 lg/kg, depending on the food matrix.
As mentioned above, the peculiar role of GC for food analysis re-
lates to the determination of aroma and flavor components and
chemistry, mainly applied to establishing the authenticity of fla-
Matrix

vored foods and beverages, recognizing markers of essential oils,

and finding alternative sources of flavoring constituents.
Liberto et al. [15] developed a GC-mass spectral library com-
Full fat soya

bined ‘‘interactively’’ with linear retention indices for enantio-

meric identification of compounds present in flavors and
fragrances. The library was prepared by analyzing 134 racemate
standards and collecting the data obtained employing four differ-
ent CD-based chiral columns, able to cover a wide range of enan-
tioseparations, which were orthogonally integrated with linear
Table 2 (continued)

Analytes

retention indices. The library was applied to the chiral analysis

Proteic AAs

of some components of Lavandula angustifolia P. Mill (lavender)

essential oil, to demonstrate its effectiveness for, e.g., QC
purposes. Fig. 2 shows the enantioselective GC-MS profile of Lav-
andula angustifolia P. Mill. essential oil. The same group, recently
 A. Rocco et al. / Trends in Analytical Chemistry 52 (2013) 206–225
proposed a statistical multivariate method to detect the authen-
ticity of fruit-flavored foods and beverages based on a rapid total
analysis system (TAS), which combined on-line headspace solid-
phase microextraction (SPME) with enantioselective GC-MS
[16]. They achieved LOD values lower than both the range of con-
centrations and the odor thresholds of the two markers taken as
representative, c-decalactone and d-decalactone, between 1 ppb
and 3 ppb. Since they used different CD-based chiral columns to
determine the characteristic chiral compounds responsible for
flavor in different fruits, they called their approach ‘‘one chiral
selector for one problem’’, where the problem refers to the deter-
mination of authenticity of fruit-flavored foods and beverages.
Table 1 shows chiral separations obtained with GC in the past five
years and related to food analysis.
3.2. Chiral separations by high-performance liquid chromatography
LC, particularly HPLC, is the widely employed separative tech-
nique for both preparative and analytical purposes, due to the
several advantages it offers, such as high selectivity, separation
efficiency, large variety of stationary phases and columns commer-
cially available, and appropriateness for non-volatile, polar, non-
polar, and thermally labile compounds. Furthermore, HPLC can
be used as a multidimensional technique, so increasing its selectiv-
ity and allowing on-line clean-up of samples. Finally, coupling with
a highly sensitive detector, such as MS, makes HPLC one of the
most powerful analytical tools for solving complex analytical tasks.
This justifies the widespread use of this technique in food analysis,
where, in chiral analysis, it is still more advantageous. Preparative
HPLC obtains the single enantiomer of interest, which is not always
commercially available.
Recently, the expansion of the food industry means that serious
attention must be paid to the potential risk of significant change in
nutritive value food components due to processing treatments.
Csapó et al. [27] evaluated the effect of extrusion processes,
operating at different temperatures and screw speed, on loss and
racemization of AAs in dry full-fat soy. On increasing the extrusion
temperature, they observed an increase of the extent of racemisa-
tion and enhancement of processes that altered the structure of the
AAs. Comparing the effect of extrusion on soya with respect to corn
(evaluated in a previous study), the process resulted in higher
amounts of D-AAs relative to dry matter for soya than in the case
of corn. This outcome was attributed to the higher protein content
of full-fat soya. Loss of L-AAs due to phenomena other than racemi-
sation (e.g., Maillard reaction) was also greater for soya, lysine
being the essential AA mainly lost.
Instead, Guillén-Casla et al. [28] proposed a method based on
bidimensional LC for the determination of the possible occurrence
of D-AAs in electron-beam irradiated foodstuffs. To be precise, they
selected three AAs found to be sensitive to radiation: tryptophan,
tyrosine, and phenylalanine and their decomposition products. Ex-
tracted AAs were first separated in an achiral column (C18), while
their chiral resolution was achieved with a teicoplanin-based col-
umn. This combination allowed on-line clean-up, and prevented
degradation of proteins, racemisation of L-enantiomers, and degra-
dation of tryptophan. LODs of 0.16–3 mg/L were obtained for each
enantiomer. Decomposition products were detected, e.g., m- and o-
tyrosine and 5-OH-tryptophan in soft cheese, while D-AAs were not
detected in irradiated food samples. Finally, it was concluded that
losses in the AA content were due to decomposition rather than
racemisation processes and they were comparable to those ob-
tained in a cooking treatment.
Concerning the use of HPLC to assess the authenticity of food
and its nutritional characteristics, Lopes and Gaspar [29] developed
a method for the simultaneous separation of anomers and
enantiomers of some underivatized monosaccharides. For the
217
separation of carbohydrates (including isomers and epimers), a
carbohydrate-analysis column was used. Subsequently, all the
stereoisomers of each monosaccharide were separated employing
a Chiralpak AD-H column and refractive index (RI) for detection,
achieving LOD values of 0.7–5.1 ppm. The method was applied to
the analysis of honey, natural sweeteners and Oporto wine. It
was possible to assign unequivocally the absolute stereochemistry
and prevalent anomeric forms of monosaccharides contained in
the matrices, defining their characteristic profiles.
Quite often, even if the amount of health-promoting com-
pounds in food is known, little importance has given to the enan-
tiomeric composition of such compounds when they are chiral.
Instead, this aspect is fundamental, especially considering the met-
abolic pathway in the human body. Only recently, for example,
lignans, compounds commonly occurring in plants, with demon-
strated antioxidant, antiviral, antidiabetic activity, were exten-
sively determined in oilseed species, bran extracts of cereal, and
seed and/or whole berry extracts, also considering their enantio-
meric composition [30]. For enantiomeric composition, two differ-
ent chiral columns (Chiralcel OD-R and Chirobiotic V) were used,
depending on the type of lignans, both coupled to a triple-quadru-
pole (TQ) MS. In most samples, secoisolariciresinol, pinoresinol,
medioresinol, and syringaresinol were present as mixtures of two
enantiomers.
Another class of phytochemicals with biological activity not yet
exhaustively elucidated are bisacetylenic oxylipins, of which car-
rots are a source. An extract made from Daucus carota L. was
screened for bisacetylenic oxylipins and their structures were
determined by means of LC-MS and 1D/2D NMR spectroscopy
[31]. Furthermore, to determine the stereochemistry and the abso-
lute configuration of the naturally-occurring isomers of falcarindi-
ol, the synthesis of all its possible stereoisomers was carried out.
Chiral HPLC analysis was performed employing a column packed
with Daicel Chiralpak AD-H material in normal-phase conditions
and the unequivocal assignment of the (Z)-(3R,8S)-configuration
for falcarindiol was assessed. In addition, the study allowed the
identification of nine additional bisacetylenes.
Vega-Villa et al. carried out a series of works on the enantio-
meric distribution of several flavonoids in food and in rats after
administration for pharmacokinetic studies [32–34]. In the most
recent, the stereospecific disposition of homoeriodictyol, isosaku-
ranetin, and taxifolin in lemon, grapefruit, and tomato, respec-
tively, was carried out with a previously validated method,
employing a Chiralcel OJ-RH column equipped with a Chiralcel
OJ-RH guard column.
The selective uptake of meso-zeaxanthin (MZ), a xanthophyll, in
the macula of the retina of the human eye is considered important
in protecting against the development of age-related macular
degeneration. Together with lutein and zeaxanthin, MZ operates
as filter against light damage. However, unlike lutein and zeaxan-
thin, which are supplied with food, MZ is practically absent in our
diet, but it is formed from lutein. Rasmussen et al. quantified the
amount of lutein, zeaxanthin and MZ in commonly consumed fish
and seafood and in egg yolk from USA and Mexico, where MZ is
added as colorant [35]. The samples were analyzed first into a re-
versed-phase HPLC system for carotenoid analysis. In such a sys-
tem, zeaxanthin and MZ eluted in one peak. For their separation,
the lutein and zeaxanthin peaks were collected from the re-
versed-phase HPLC system and injected into a Chiralpak AD col-
umn, which separated all-trans zeaxanthin, MZ and lutein. While
the three carotenoids were not detected in fish and seafood, in eggs
from Mexico, MZ contributed 28% of the total xanthophylls with
the total xanthophyll level being approximately twice that mea-
sured in egg yolk from the US. It was therefore concluded that
modification of poultry diet is a strategy to increase intakes of xan-
thophylls in the human diet.
Table 3

218
Chiral separations obtained with CE, CEC and nano-LC

Analytes Matrix Chiral selector Experimental conditions Mode/detection Sample preparation Ref.
Lipoic acid Dietary supplements TM-b-CD 100 mM phosphate buffer (pH CZE-DAD Extraction with 70% EtOH [60]
(tablets or capsules) 7.0) + 8 mM TM-b-CD k = 200 nm (sulfonated
capillary)
23 different AAs Citrulline in food Sepapak 2 24 cm  100 lm 0.5 M ammonium formate pH 2.5/ CEC-DAD Samples were diluted in water, [74]
supplements I.D., 5 lm particles size H2O/ACN (1/19/80, v/v/v) Nano-LC-DAD filtrated and subjected to
k = 210, 260 nm derivatization with FMOC
LODs for citrulline
7.5  107 M
DL- isocitric acid, citric acid (isomer of Fruit juice samples D-quinic acid Ni(II) 20 mM acetic acid, 20 mM NiSO4, CLE-CE-UV Dilution (deionized water), [61]
isocitric acid) (orange, pineapple, 80 mM D-quinic acid (pH 5) k = 200 nm (sulfonated centrifugation and filtration
grape, apple) containing 30% ACN capillary)
Lactic acid, a-hydroxy-butyric acid, 2- Milk and yogurt Vancomycin 10 mM Tris, 4.4 mM maleic acid, CE-CCD LLE (ACN), centrifugation and [62]
hydroxycaproic acid, 2-hydroxyoctanoic 0.03 mM CTAB pH 7.35 with 5 mM LODs for L- and D-lactic 2.8, evaporation
acid, 2-hydroxydecanoic acid, aspartic vancomycin 2.4 lmol/L
acid,
Carnitine Dietary food Succ-c-CD 0.5 M ammonium formate buffer CE-MS For drinks, homogenization and [64]

A. Rocco et al. / Trends in Analytical Chemistry 52 (2013) 206–225

supplements (drinks, (pH 2.5) with 0.2% (m/v) succ-c-CD LODs for Carn enantiomers dilution with water.
biscuits, capsules, 10 ng/mL For biscuits, tablets and capsules, the
tablets) samples were grinded, homogenized
and extracted with water.
Hesperetin Human urine phenyl-carbamate-2-propyl- 1% (v/v) TEAA pH 4.5 in MeOH/ Nano-LC-UV LLE extraction with ethyl acetate, [76]
b-CD CSP capillary column water 70/30 (v/v). Flow rate 400 nL/ k = 205 nm
24 cm  100 lm I.D. min LOD: 0.1 lg/mL
Carnitine Infant formulas Succ-c-CD 0.5 M ammonium formate buffer Chiral CE-MS with PFT. Dilution of milk powder samples in [63]
(pH 2.5) with 10 mM (m/v) succ-c- LOD for D-Carn16 ng/mL water and subjected to ultrafiltration
CD, PFT 600 s  50 mbar which corresponds about
100 ng/g of D-Carn in real
samples
Arg, Lys, Orn Dietary supplements, HS-b-CD 50 mM phosphate buffer (pH 2.0) CZE-DAD Dilution of DE in water. Filtration of [66]
wines Ac-c-CD containing 5% m/v HS-b-CD and 2% k = 260 nm wine samples.
m/v Ac-c-CD. In-capillary derivatization method
In-capillary derivatization with AQC
procedure: injection 50 mbar for 5 s
of analyte, 2 s of ACN and 3 s of AQC
Orn Food supplements c-CD 100 mM borate buffer (pH 10.0), EKC-DAD Dilution in deionized water, dilution in [67]
1 mM c-CD. k = 240 nm 100 mM borate at pH 10.0 and pre-
LOD: 1.6  107 M capillary derivatization with FITC
Orn Beers c-CD 50 mM ammonium carbonate (pH CZE-ESI-MS (IT) Samples degassed, their pH adjusted to [68]
10.0), 0.5 mM c-CD. LOD: 2.5  109 M 10.0, filtrated and pre-capillary
Sheath liquid: 50/50 v/v derivatization with FITC.
isopropanol/25 mM ammonium
carbonate. Flow rate 3.3 lL/min
10 different D- and L-AAs (D/L Glu, D/L Asp, D/L Transgenic and non- Modified CD 50 mM ammonium hydrogen CZE-ESI-MS (TOF) Soybean: Extraction with TCA, protein [70]
Ala, D/L Asn, D/L Arg) transgenic soybean CDen carbonate pH 9.0, 0.5 mM CD3NH2 precipitation (sodium deoxycholate)
CDampy Sheath liquid: and derivatization (FITC)
CD3NH2 50/50 v/v water/2-propanol. Flow Vinegar: derivatization (FITC)
rate 0.24 mL/h.
Catechin, gallocatechin, methylxanthines Green tea HP-b-CD 25 mM borate-phosphate buffer CD-MEKC Infusion of tea leaves with 60 mL water [73]
(pH 2.5), 90 mM SDS, 25 mM HP-b- k = 200 nm for 5 min at 85°C. Infusion was
CD. filtrated.
Orn, Arg, Lys, Asn, Gln, Gly Fermented foods HS-b-CD 50 mM phosphate buffer (pH 2.0) EKC-DAD Beer samples were degassed, Wine and [65]
(rose wine, beer, Ac-c-CD containing 5% m/v HS-b-CD and 2% k = 260 nm vinegar did not require any
vinegar) m/v Ac-c-CD. LOD 9.0  106 M pretreatment. Derivatization with AQC
10 different D- and L-AAs (D/L Glu, D/L Asp, D/L Transgenic and non- b-CD 100 mm sodium tetraborate (pH MEKC-LIF Extraction with TCA, protein [69]
Ala, D/L Asn, D/L Arg transgenic yeasts 10.0), 30 mM SDS, 20 mM b-CD LODs 1.0–5.3 nM precipitation (sodium deoxycholate)
 A. Rocco et al. / Trends in Analytical Chemistry 52 (2013) 206–225 219

As well as GC, several HPLC works assisted with risk assessment

[77]

[71]

[75]
for chiral agrochemicals, where the method most employed for
their extraction included the use of QuEChERS. Li et al. [36] com-
Infusion in hot water and dilution with

bined the small-scale extraction of QuEChERS with a conventional

with 5 M NaOH, adjusting the pH at

Centrifugation, supernatant treated
Centrifugation and filtration of the

acetonitrile extraction to enhance removing food colorings, such as

chlorophyll, carotene, and water soluble materials, for the determi-
and derivatization with (FITC)

9.0: Derivatization with FITC

nation of difenoconazole stereoisomers and their hydroxylated
metabolite, difenoconazole alcohol, in some vegetables and soil.
Among four different polysaccharide-type chiral stationary phases
examined, Chiralcel OJ (cellulose tris(4-methylbenzoate))
water (1:2, v/v)

exhibited higher resolving ability than Chiralcel OD (cellulose

tris(3,5-dimethylphenylcarbamate), Chiralpak AD (Amylose tris
samples.

(3,5-dimethylphenylcarbamate) and Chiralpak AS-H (Amylose

tris-[(S)–a-methylbenzylcarbamate]), working in normal-phase
conditions. The combination of anion-exchange SPE with GCB/Pes-
LODs catechin and epicatechin

ti-Carb multi-layers, resulted in clean-up that improved removal of

Nano-LC-UV-MS k = 205 nm

the visible pigments in cucumber sample and gave higher extrac-

0.028 and 0.011 lg/mL,

tion efficiency of studied compounds in all the different sample

(z-cell) nano-ESI-Q-IT

matrices. LOD values for both enantiomers were estimated at

LODs 2–8 ng/mL

0.01–0.03 lg/g.
respectively.

Recent developments in LC answered the need for high separa-

k = 214 nm
k = 240 nm

CLE-CE-UV
EKC-DAD

tion efficiency and rapid analysis. As an alternative to HPLC, ultra-

high-pressure LC (UPLC), was proposed for working at higher
pressure conditions to carry out fast analysis with columns packed
with sub-2-lm porous particles or sub-3-lm core-shell particles.
4/11/95 v/v/v 500 mM ammonium

Eto et al. [37] performed the analysis of D-AAs and L-AAs using
ammonium acetate, 3 mM ZnSO4
50 mM borate buffer (pH 8.35)

UPLC coupled with a circular dichroism detector (CDD) in less than

formate, pH 3.5/water/MeOH

6 min. 4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was se-

100 mM boric acid, 5 mM

and 6 mM L-Arg (pH 8.0)

lected as pre-column derivatization reagent in order to enhance

containing 3 mM b-CD

the sensitivity and the selectivity of UV and CDD detection. Fig. 3

shows the chromatograms of the AA enantiomers (after derivatiza-
tion) obtained by UPLC.
LODs of NBD-derivatized AAs with CDD were 11–64 pmol/injec-
tion, while, with UV detection, they were relatively lower – 4.9–
23 pmol/injection. The use of a CDD did not require chromato-
graphic separation of enantiomers, and AAs were separated on
ODS column Ascentis Express C18. Their enantiomeric ratio (%D)
vancomycin-modified silica-
Complexation with Zn (II) L-

25 cm  75 lm I.D., 5 lm

was calculated by the g-factor value (calculated from UV and

CDD peak heights). The validated method was applied to deter-
mine D-AAs and L-AAs in fermented foods, namely Japanese black
diol CSP, column

vinegar, fermented milk, and yogurt.

particles size

Table 2 summarizes significant works in the period 2008–13.

arginine
b-CD

3.3. Chiral analysis by electromigration techniques and miniaturized

liquid chromatography
Green and black teas,

CE offers great versatility in chiral discrimination, providing

Orange juice
Rice vinegar

unique separation efficiency and high resolution. Moreover, this

technique shows other features, including high speed of analysis
coffee

and minimal sample and solvent consumption. The very small

amount of CS used during the analysis makes this technique more
cost effective than HPLC, where chiral chromatographic columns
are too expensive. Despite these advantages, an important aspect
Arg, Pro, Ala, Leu, Ser, Phe, Asn, Glu, Asp

to consider is the limited concentration sensitivity due to the very

Catechin, epicatechin, ascorbic acid

small sample volume injected and the short path length of the
detector (usually UV).
Several strategies, including the use of sample-pre-concentra-
tion procedures, such as off-line and on-line techniques, and the
employment of alternative detection systems, have been assumed
for sensitivity enhancement. Chiral analysis by CE coupled with
17 dansyl-AAs

fluorescence, conductivity and MS detectors have been performed.

Often, a pre-concentration technique is combined with a highly
sensitive detector to improve the method sensitivity [56].
Among fluorescence detectors, LIF systems have mostly been
employed in the chiral analysis of AAs after derivatization with a
220 A. Rocco et al. / Trends in Analytical Chemistry 52 (2013) 206–225
Fig. 2. Enantioselective GC-MS profile of Lavandula angustifolia P. Mill. essential oil analyzed on a 2,6-di-O-methyl-3-O-pentyl-b-CD (2,6DM3PEN-b-CD)-coated column.
(From [15] with permission).
fluorescent reagent (e.g., FITC) allowing very low LODs in complex
matrices.
The coupling of CE with MS detectors provides another impor-
tant advantage – unambiguous identification and confirmation of
components in complex mixtures. A significant problem to take
into account with this coupled technique is the introduction of a
non-volatile CS used in the BGE (e.g., CDs and derivatives of CDs)
that causes contamination of the ion source, resulting in a consid-
erable loss of sensitivity and high background noise. Two different
strategies can be employed to overcome this limitation – the
partial filling technique (PFT) and the counter-migration technique
(CMT). In the PFT, the capillary is partially filled with the CS, avoid-
ing the detection window, whereas, in CMT, the capillary is com-
pletely filled with charged CS that migrates, in the opposite
direction to the detection system. This approach is also useful for
the analysis of neutral compounds that are transported by the
charged CS acting as ‘‘carrier’’. Another approach is the use of CSPs
(CEC mode), where the CS is fixed to the support, so avoiding con-
tamination of the ion source [57]. So far, no CEC-MS method has
been developed for the analysis of chiral components in food
products.
Different CE modes, including CD-modified capillary zone elec-
trophoresis (CD-CZE), CD electrokinetic chromatography (CD-EKC),
CD-modified micellar electrokinetic chromatography (CD-MEKC),
ligand exchange CE (LE-CE) and more recently capillary electro-
chromatography (CEC), have been employed to develop chiral
methods for the discrimination of components and beverages.
Among the wide variety of CSs, neutral, derivatized and synthe-
sized CDs have mainly been used in chiral food analysis [3,58,59].
Concerning food analysis by electromigration techniques, we
selected the papers described in this review on the basis of the dif-
ferent CE strategies and or detection systems employed. We dis-
cussed the enantiomeric analysis of different classes of chiral
compounds in different food matrices, highlighting how adultera-
tion, fermentation, processing, or storage processes can be identi-
fied by the enantiomeric ratio of the analyzed food components.
Organic acids have a great involvement in the analysis of foods
and beverages because they can influence the flavor or the taste of
food products. Furthermore, they, generally, naturally occur as
single enantiomers, but they are often detected in food products
as racemates that indicate their use as additives. The enantiomeric
analysis of these compounds is then important for establishing
authenticity and QC. The enantioseparation of lipoic acid by CZE
in dietary supplements was performed using a sulfonated capillary
and trimethyl-b-CD. Lipoic acid, an antioxidant, naturally occurs as
the (R)-enantiomer, while the synthetic acid is racemic. A rapid
CZE method with simple sample pretreatment assessed the con-
tent of the two enantiomers in nine dietary supplements, and
found the racemic mixture of lipoic acid in most of the commercial
samples. (R) Lipoic acid was detected only in three supplements, as
labeled [60].
A chiral LE-CE method was developed to evaluate the ratio of
citric acid to D-isocitric acid in order to detect fruit-juice adultera-
tion. The D- and L- isocitric acid enantiomers were separated using
D-quinic acid as CS and Ni(II) as central ion. The amounts of DL-iso-
citric and citric acid were estimated in different fruit juices obtain-
ing ratios for orange juices in accord with the Code of Practice [61].
Five a-hydroxy acids, including lactic, a-hydroxy-butyric, 2-
hydroxycaproic, 2-hydroxyoctanoic and 2-hydroxydecanoic acids,
were chirally separated by CE with contactless conductivity detec-
tion (CCD). Vancomycin (5 mM) was employed as CS, filling the
whole capillary. The use of this detector avoided applying the
PFT procedure, necessary for UV detection. The method developed
allowed enantioseparation of all the a-hydroxy acids with Rs val-
ues ranging of 2.6–3.8. The determination of lactic-acid enantio-
mers in milk and yogurt samples, with LLE sample pre-treatment
before CE analysis, was achieved LODs of 2.8 lmol/L and
2.4 lmol/L for L-lactic and D-lactic acids, respectively. In fresh milk
samples, neither of the two enantiomers was detected, while, in
yogurt samples, L-lactic acid was present in different amounts. In
one yogurt sample analyzed 10 days after opening, D-lactate was
found to be present, indicating microbial contamination [62].
Another interesting class of compounds, very important be-
cause involved in human health, are AAs. As reported by Herrero
et al. [58], quality, authenticity and genuineness of foods and bev-
erages can be assessed using the enantiomeric ratio of chiral AAs. A
chiral CE-MS2 method was developed for the determination of
DL-carnitine in infant formulas. Carnitine, a non-protein AA, is an
essential nutrient for infants because they are unable to synthesize
it. For this reason, this compound has to be supplemented in
baby-food products. However, the use of racemic carnitine has to
be avoided because, while L-carnitine is fundamental in the
metabolism of long-chain FAs, D-carnitine has a toxic influence
on biochemical processes. Carnitine was derivatized with
9-fluorenylmethyl-chloroformate (FMOC) reagent and separated
into its enantiomers using succinyl-c-CD (Succy-c-CD) as CS. The
 A. Rocco et al. / Trends in Analytical Chemistry 52 (2013) 206–225
Fig. 3. CDD chromatograms of NBD-D-amino acids (A) and NBD-L-amino acids (C). UV chromatogram of NBD-D-amino acids (B) and NBD-L-amino acids (D). Peak identification is as follows: (1) NBD-Asn, (2) NBD-OH, (3) NBD-Gln,
(4) NBD-Ser, (5) NBD-Asp, (6) NBD-Gly, (7) NBD-His, (8) NBD-Glu, (9) NBD-Thr, (10) NBD-F, (11) NBD-Arg, (12) NBD-Ala, (13) NBD-Pro, (14) NBD-Met, (15) NBD-Lys, (16) NBD-Val, (17) NBD-Trp, (18) NBD-Cys, (19) NBD-Phe, (20)
NBD-Ile, (21) NBD-Leu, (22) NBD-Tyr. (From [37] with permission).

221
222 A. Rocco et al. / Trends in Analytical Chemistry 52 (2013) 206–225
Fig. 4. CE-MS/MS extracted ion electropherogram (EIE) for a biscuit and the corresponding MS/MS spectra for the peaks of L- and D-Carn (carnitine). (From [64] with
permission).
chiral separation was performed using 0.5 mM formate buffer at
pH 2.5 with 10 mM of Succ-c-CD applying a PFT. CE-MS2 experi-
ments, using an ion-trap (IT) analyzer confirmed the identity of
DL-carnitine, achieving a 100-fold sensitivity enhancement with
respect to UV detection and obtaining a LOD of 100 ng/g for
D-carnitine. The determination of L-carnitine and its enantiomeric
impurity D-carnitine was assessed in 14 infant-supplement
products obtaining successful results [63]. The same method was
applied to the determination of DL-carnitine in dietary food
supplements, including drinks, biscuits and tablets. Fig. 4 shows
the extracted ion electropherogram of the carnitine enantiomers
in a biscuit-sample extract. The LOD was about 10 ng/mL, allowing
the determination of the enantiomeric impurity (D-carnitine) up to
0.025% in food products [64].
Ornithine (Orn) is another non-protein AA found in different
foodstuffs. A EKC-DAD method was developed for the fast enantio-
meric separation of Orn in a mixture of different AAs, with a
previous derivatization step with 6-aminoquinolyl-N-hydroxy-
succinimidyl carbamate (AQC). A phosphate buffer 50 mM at pH
2.0 containing a dual CD system, composed of 5% (m/v) HS-b-CD
and 2% (m/v) acetylated-c-CD, allowed the complete enantiosepa-
ration of Orn and 18 protein AAs. The method was applied to the
analysis of DL-Orn in several fermented beverages, such as beer,
rosé wine and vinegar. The non-protein AA was determined in beer
and wine but not in vinegar [65]. In order to accelerate the deriv-
atization procedure, Martínez-Girón et al., in a following study,
performed an in-capillary derivatization procedure with AQC,
where plugs of ACN, the derivatization reagent (10 mM) and
sample dissolved in borate (1:1) were injected in tandem. The
method succeeded in the determination of Orn, Arg and Lys in
several dietary supplements and wine samples [66].
Fast derivatization of Orn with FITC using an ultrasound probe
was proposed, reducing the derivatization time from 16 h to
10 min. Orn enantiomers were completely separated within
9 min in basic conditions (100 mM borate buffer pH 10) using
1 mM of neutral c-CD. LOD values of 1.6  107 M were obtained
for D-Orn and L-Orn. The chiral method developed was applied to
the analysis of Orn in 10 food supplements. The L-Orn content
found in the analyzed samples was in accord with the labeled con-
tent. D-Orn was not detected, demonstrating the safety and the
good quality of the food products that were prepared using only
the L-enantiomer [67].
A recent chiral CE-ESI-ITMS method was optimized for the
enantiomeric determination of Orn, previously derivatized with
FITC, in beers. The chiral separation was achieved using a BGE com-
posed of 50 mM ammonium carbonate pH 10 containing 0.5 mM
c-CD. Due to the very low concentration of the CD, the capillary
was completely filled, introducing the CS into the MS without
any significant decrease in sensitivity. The method provided LOD
values of 2.5  109 M for both Orn enantiomers, obtaining a sen-
sitivity enhancement of two orders of magnitude with respect to
the previous CE-UV method. The analysis of 16 beer samples sub-
mitted to different fermentation processes was performed, obtain-
ing for D-Orn values of 1.5–10% [68].
The chiral analysis of several AAs was also performed to distin-
guish conventional and transgenic yeasts. A MEKC-LIF method,
 A. Rocco et al. / Trends in Analytical Chemistry 52 (2013) 206–225
previously developed to identify and to quantify D-AAs and L-AAs in
conventional and transgenic maize, was used. The AAs were deriv-
atized with FITC and resolved into their enantiomers in a BGE com-
posed of 100 mM sodium tetraborate, 30 mM SDS and 20 mM b-CD
at pH 10.0. The D-Arg and L-Arg, Asn, Ala, Glu and Asp were sepa-
rated in less than 30 min with efficiencies up to 800 000 plates/m
and high sensitivity (LODs as low as 40 nM). From this study, it
was confirmed that the genetic modification made autolysis of
yeasts faster, with different amounts of L-AAs and D-AAs being ob-
served [69].
In a method developed to distinguish wild from transgenic
soybeans, five different chiral AAs, including Ala, Asn, Glu, Asp
and Arg, were separated by CE-LIF and CE-TOF-MS using three
modified CDs (mCDs), namely 6-deoxy-6-[1-(2-amino)ethyl-
amino]-b-CD (CDen), 6-deoxy-6-[N-(2-methylamino)pyridine)]-b-
CD (CDampy) and 3-monodeoxy-3-monoamino-b-CD (CD3NH2).
The resolving power of the mCDs was compared with b-CDs and
c-CDs. The optimal separation conditions were obtained employ-
ing 0.5 mM CD3NH2. LOD values achieved with chiral CE-MS were
in the nM range, and comparable to those obtained by CE-LIF.
CE-TOF-MS was applied to determination of D-AAs and L-AAs in
samples of wild and transgenic soybeans, resulting in different
profiles. Vinegar samples were also analyzed, with only L-AAs
detected [70].
The enantioseparation of 17 dansyl-AAs was obtained with a
chiral LE-CE method using Zn(II) L-arginine complex as the CS.
The method developed was applied to the analysis of some AA
enantiomers in rice-vinegar samples. Since the contents of AAs de-
pend on the time and the activity of fermentation, they could be
used as markers for the QC of rice vinegar [71].
Another important work concerning the analysis of vinegars,
that should be mentioned, even if it was published more than
the five years covered in this review, was performed by Carlavilla
et al. [72]. A chiral MEKC-LIF method for the determination of
AAs was developed, allowing the profiles of L-AAs and D-AAs in
balsamic, sherry, white wine and cider vinegars. In particular,
D-AAs, present in fermentation processes, were detected and
quantified, giving information to characterize the different
vinegars [72].
Flavonoid compounds, naturally occurring in vegetables, fruits,
and spices, are widely present in food and beverages. Their health-
related properties, including prevention of cancer and cardiovascu-
lar diseases, and reduction of cholesterol and hypertension, are
well known.
In the latest studies, particular attention was paid to the class of
catechins present in chocolate, tea and food plants. Several works
based on CD-MEKC were evaluated to separate catechin and epi-
catechin enantiomers and to assess the epimerization of these
compounds subjected to thermal manufacturing processes. A
recent paper by Gotti et al. described the characterization and
the differentiation of green-tea samples based on the thermal epi-
merization of catechins. A chiral CD-MEKC method using hydroxy-
propyl-b-CD (HP-b-CD) as CS was developed for the separation of
catechin and gallocatechin and applied to the analysis of green-
tea samples. A BGE containing 25 mM borate-phosphate buffer
(pH 2.5) with 90 mM SDS and 25 mM HP-b-CD was employed for
the enantioresolution of catechin and gallocatechin and simulta-
neous separation of methylxanthynes. Studies of thermal epimer-
ization of ()-epicatechin and epigallocatechin to ()-catechin
and ()-gallocatechin were performed. Since ()-gallocatechin is
not a naturally-occurring enantiomer but a compound originating
from the thermal process, it was identified for the first time as a
marker of tea samples subjected to thermal treatment. The method
was applied to the analysis of more than 20 tea samples of
different origin that had undergone to different manufacturing
processes. The chiral method combined with chemometric studies
223
discriminated between the different tea varieties on the basis of
native and non-native catechins [73].
In addition to CE, recently, new miniaturized techniques
including CEC and nano-LC were applied to food analysis. The pa-
pers published most recently showed CEC and nano-LC to be
attractive methods for chiral separations. As CE techniques, they
offer advantages, such as low consumption of solvent and sample,
high resolving power, high separation efficiency and good sensi-
tivity, especially when coupled with MS. In the literature, some
papers reported the use of CEC in the chiral analysis of
compounds involved in food, such as a-hydroxyacids and herbi-
cides, but only one concerning separation of AAs was applied to
food matrices.
The enantiomeric separation of FMOC-AAs by nano-LC and CEC
was performed using a cellulose tris(3-chloro-4-methylphenylcar-
bamate) as CSP. The AAs were derivatized by FMOC and separated
first with nano-LC by optimizing the mobile-phase composition.
CEC experiments were also carried out, using the same column
employed in nano-LC and a mobile phase composed by 0.5 M
ammonium formate, pH 2.5/H2O/ACN (1/19/80, v/v/v), allowing
the chiral discrimination of 20 FMOC AAs out of 23 tested. With
the CEC method, better separation efficiency and resolution were
achieved. Finally, the content and the enantiomeric purity of the
non-protein AA citrulline in food supplements were determined.
LODs of 7.5  107 M were obtained for D-citrulline and L-citrul-
line, allowing the detection of D-citrulline at values up to about
0.15% [74].
In order to assess the possible adulteration of orange juices, a
chiral nano-LC-MS method was developed for the analysis of D-
AAs and L-AAs [75]. The compounds were derivatized with FITC
and enantioseparated using a column packed with a vancomycin-
based stationary phase. The analytes were focused on a C18
cartridge before the chiral separation. LODs as low as 8 ng/mL were
obtained. The optimized nano-LC-MS method was applied to the
determination of the AAs in fresh and commercial orange juices
and only the L-enantiomers were found in both cases.
As evidence of the importance of determining food compo-
nents in biological fluids, a study used nano-LC to determine hes-
peretin (HT) enantiomers in human urine after ingestion of juice
of blood oranges. Taking into account the chiral nature of HT, the
study of the stereospecific kinetics of in-vitro and in-vivo metab-
olism could be a useful tool for further understanding stereose-
lective biotransformations in the human body. The chiral
separation of HT was achieved using a column packed with phe-
nyl-carbamate-propyl-b-CD stationary phase, employing a mo-
bile phase composed by a mixture of triethylammonium acetate
buffer (1%, v/v, pH 4.5) and water/methanol (30:70, v/v). The
method developed was successfully applied to the determination
of HT enantiomers in urine samples, subjected to LLE, obtained
from a male volunteer, after the ingestion of 1 L of a commercial
blood-orange juice [76].
Table 3 shows recent chiral separations in food analysis
obtained with electrodriven techniques and nano-LC.
4. Concluding remarks
This review presented the state of the art of enantiomer separa-
tion and determination in foodstuffs. The data discussed were from
publications appearing in 2008–13 and considering the separation
techniques used most. Among them, GC, HPLC, electromigration
techniques (mainly CZE and CEC) and nano-LC were considered.
Concerning the resolution modes, the direct resolution method
was used most because it gave better results. The main problems
related to the chiral separations were discussed, together with
the importance of analyzing a certain type of compound. Consider-
224 A. Rocco et al. / Trends in Analytical Chemistry 52 (2013) 206–225
ing the data discussed, as expected, a large number of works was
carried out with conventional techniques (e.g., GC and HPLC), but
miniaturized techniques, especially CE, all demonstrated their
capability of chiral separations for practical application to food
analysis. These techniques make use of limited volumes of mobile
phases and small amounts of expensive CSs, so they possess great
potential for further applications because they are cheap and eco-
friendly. Despite these features, further work is needed (i.e. to
study new stationary phases and to improve sensitivity). Recently,
in HPLC and nano-LC, core-shell particles achieved separations
with high efficiency in a short time due to the homogeneity and
the thinness of the material. The use of such particles can offer
obvious advantages for miniaturized techniques, so it is a challeng-
ing issue for further studies.
Acknowledgements
This review was partially supported by the research Grant
awarded by Ministry of University and Scientific Research, Italy
(PRIN Project 20098y822F_002 ‘‘Miniaturized analytical tech-
niques coupled with electrospray mass spectrometers: develop-
ment and applications to the analysis of compounds of
nutraceutical interest’’).
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