Recents analytical techniques advances in the carotenoids and their derivatives determination in various matrixes.

Recent Analytical Techniques Advances in the Carotenoids and Their

Derivatives Determination in Various Matrixes

Daniele Giuffrida,

*

Paola Donato,

Paola Dugo,

and Luigi Mondello

†_{Dipartimento di Scienze Biomediche, Odontoiatriche e delle Immagini Morfologiche e Funzionali, University of Messina, Via}

Consolare Valeria, 98125 Messina, Italy

§_{Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, University of Messina, Polo Annunziata-Viale}

Annunziata, 98168 Messina, Italy

¶_{Chromaleont s.r.l., c/o Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, Polo Annunziata, University of}

Messina, Viale Annunziata, 98168 Messina, Italy

‡_{Department of Medicine, University Campus Bio-Medico of Rome, Via Álvaro del Portillo 21, 00128 Rome, Italy}

ABSTRACT: In the present perspective, different approaches to the carotenoids analysis will be discussed providing a brief

overview of the most advanced both monodimensional and bidimensional liquid chromatographic methodologies applied to the carotenoids analysis, followed by a discussion on the recents advanced supercritical fluid chromatography × liquid chromatography bidimensional approach with photodiode-array and mass spectrometry detection. Moreover a discussion on the online supercriticalfluid extraction-supercritical fluid chromatography with tandem mass spectrometry detection applied to the determination of carotenoids and apocarotenoids will also be provided.

KEYWORDS: carotenoids analysis, mono- and multidimensional chromatography, LC-PDA-MS, supercriticalfluid extraction-supercritical fluid chromatography-MS

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Carotenoids are widely distributed natural pigments produced mainly by plants and microorganisms commonly found in many foods and food products. Historically they have been used as food colorants, but recently their importance has grown due to the beneficial health properties that have been ascribed to them.1 Chemically they belong to the tetraterpene family and their structure is usually based on a hydrocarbon C40 skeleton

(carotenes) having a long unsaturated system which acts as the chromophore; some carotenoids derivatives, like the apocar-otenoids and norcarapocar-otenoids or longer carapocar-otenoids with 45 or 50 carbons, are not tetraterpenes. Quite often also oxygen atoms are present in their structure commonly as hydroxyl, epoxy, or keto groups giving rise to various xanthophylls structures (Figure 1), although other oxygen containing functions might sometime also be present. Moreover, when the hydroxyl function is present, it is often esterified with fatty acids; in fact, the esterification provides greater stability to the molecule. The long π conjugated system present in the carotenoid chemical structure is very sensitive to light, heat, and oxygen and carotenoids isomers and degradative products may easily be produced; therefore, great care should be taken in the carotenoids analysis to avoid analytical errors.

Carotenoids oxidative and enzymatic cleavage products called apocarotenoids are also widely distributed in plants where they act as bioactive molecules2 (Figure 2); apocar-otenoids are generated by cleavage of a fragment from one side from the usual C40 carotenoid structure. Recently their

occurrence in food has gained interest due to the health related properties that have been attributed to them.3,4

Carotenoids analyses have usually been performed after a saponification step which removed chlorophylls and undesir-able lipids and provided an easier chromatographic compounds separation, but recently the trend is toward the study of the native carotenoids composition which was lost if the saponification step was carried out in the matrix before the

Received: January 17, 2018 Revised: March 1, 2018 Accepted: March 13, 2018 Published: March 13, 2018

Figure 1. Chemical structures of four common carotenoids. Hydrocarbon carotenoids, lycopene and β-carotene; oxygenated carotenoids, zeaxanthin and violaxanthin.

pubs.acs.org/JAFC

chromatographic analysis, thus possibly leading to analytical errors.5

Guides to carotenoid analyses in foods are available in the literature.5−11 Open column chromatography (OCC), thin layer chromatography (TLC), and high-performance thin-layer chromatography (HPTLC) are still used in separations of carotenoid extracts, using acetone as the most traditional solvent for carotenoid extraction followed by partition solvents. High-performance liquid chromatography (HPLC) can nowa-days be considered the most commonly used methodology for carotenoid separations;12,13in particular, the C30columns have

become the most prevalent selection.14−16 The serial connection of more then one column has been proposed as an alternative to one single column liquid chromatography (LC).17,18Sometime, monodimensional chromatography is not sufficient for an optimal carotenoids separations in tricky samples and a proposed alternative was also the use of multidimensional separation mechanisms.19−21Although lately, supercritical fluids have been used for both the carotenoids separations (supercritical fluid chromatography, SFC) and the carotenoid extraction (supercriticalfluid extraction, SFE),22−24 only very recently the direct online extraction and determi-nation of carotenoids, by a supercritical fluid extraction-supercritical fluid chromatography-mass spectrometry (SFE-SFC-MS) methodology was reported,25and a supercriticalfluid chromatography-triple quadrupole/mass spectrometry ap-proach for the apocarotenoids determination was also recently reported.26In the present perspective, the different approaches to the carotenoids analysis described above will be discussed providing a brief overview of the most advanced both monodimensional and bidimensional liquid chromatographic methodologies applied to the carotenoids analysis, followed by a discussion on the recents advanced supercritical fluid chromatography × liquid chromatography bidimensional approach with photodiode-array (PDA) and MS detection, and then a discussion on online supercritical fluid extraction-supercritical fluid chromatography with tandem mass spec-trometry (MS/MS) detection applied to the determination of carotenoids and apocarotenoids.

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MONODIMENSIONAL CHROMATOGRAPHY

High-performance liquid chromatography (HPLC) can nowa-days be considered the most commonly used methodology for carotenoid separations and identification with photodiode-array

(PDA) and mass spectrometry detection (MS),12−15 with column dimensions usually of 250 mm × 4.6 mm i.d. and particle sizes of 5 or 3μm.

Many types of stationary phase have been used, including normal phase (NP) and reversed phase (RP) materials. Normal-phase HPLC of xanthophylls is commonly performed using a silica or silica-based nitrile-bonded column and the mobile phase usually consists of an apolar hydrocarbon solvent to which a more polar solvent is added as modifier. Reversed-phase separation on C18column has also been broadly used for

carotenoids because of the hydrophobic interactions taking place and for the solvent and polarity range suitability with the carotenoids.

Carotenoids chromatography on reversed-phase C18columns

is frequently performed using acetonitrile and methanol with the addition of a stronger less polar solvent as modifier, such as methyl-tert-butyl ether (MTBE). Better performances in carotenoids separations on C18 column using ultra

high-performance liquid chromatography (UHPLC) have recently been reported.9,10,13,17 This technology make use of narrow-bore columns (2.1 mm i.d.) packed with very small particles (sub-2-μm stationary phase thickness) and mobile phase delivery systems operating at high pressure; in fact in UHPLC systems the back-pressure can reach up to 103.5 MPa, much higher than the back-pressure usually obtained in conventional HPLC systems, which is around 35−40 MPa. UHPLC features over conventional HPLC are quicker run times, higher sensitivity, and lower mobile phase waste. However, reversed-phase C₃₀ columns are nowadays the preferred alternative for carotenoids analysis. The higher hydrophobicity of the C₃₀ stationary phase compared with the C18 one has provided an improved resolution for

carotenoids. Triacontyl-bonded (C30) stationary phases has,

for example, successfully been used in the separation of a standard mixture of epoxycarotenoids isomers, employing a gradient elution of methanol, methyl-tert-butyl ether (MTBE), and water.16 Serial connection of more than one column has been suggested in the separation of carotenoids in saponified red orange essential oil.17The advantages of coupling two C30

columns to increase the peak capacity was shown; in fact, a peak capacity of 79 was reached with two C₃₀coupled columns, in comparison to 61 obtained using a single column. This novel overtures was also employed in the characterization of the carotenoids in orange juice.18 The use of this methodology afforded the identification of 44 different carotenoids. Among them, several violaxanthin diesters have been directly identified in orange juice for thefirst time.

As far as the general carotenoids detection is concerned in the carotenoids analyses in HPLC and UHPLC, the UV−vis instruments have been the most common detectors, having the carotenoids very characteristic UV−vis spectra, considering the position of the absorption maxima (λ max) the shape (spectral fine structure % III/II), and eventually the presence of a cis band in the spectrum which enables the differentiation among trans (E) and cis carotenoids isomers (Z) (spectral fine structure % AB/AII). In the carotenoids analysis, it should be taken into consideration that two pigments with different structures but identical chromophores will have the same UV− vis spectra and therefore it sometime occurs that, for example, two carotenoids show the same UV−vis spectra but have different molecular ion (m/z) values or the opposite might also occur; therefore, a great help in the carotenoids identifications has been the online use of both detection systems (PDA and

Figure 2. Different positions of eccentric zeaxanthin oxidative cleavages sites leading to different apozeaxanthinals. 1, apo-14′-zeaxanthinal; 2, apo-12′-zeaxanthinal; 3, apo-10′-zeaxanthinal; and 4, apo-8′-zeaxanthinal.

MS) coupled to the chromatography system. Mass detectors giving information about structural features enormously contribute in the carotenoids characterization providing information on their molecular mass and their fragmentation pattern. The use of atmospheric pressure chemical ionization (APCI) for the carotenoids analysis has rapidly grown; in fact, it efficiently ionizes not only xanthophylls and carotenes but also carotenoids esters. The possibility of a rapid switchover between positive and negative ionization modes in the APCI probe during the same analytical run allows the collection of a greater number of qualitative information about a sample in a single run, and this is of great help especially for the carotenoids esters identification; in fact, in the negative mode the quasi-molecular ion species is dominating the MS spectrum, whereas fragment ions are mainly occurring in the APCI positive mode due to in source fragmentation. Thus, positive and negative APCI ionization modes are providing complementary informa-tion that can greatly help, for example, in the identification of carotenoid esters regioisomers.9,10,18,27The high selectivity and sensitivity provided by tandem mass spectrometry (MS/MS) brings advantages in the carotenoids analyses; the use of specific multiple reaction monitoring (MRM) experiments in which specific transition are monitored offers not only qualitative information but also allows for the individual quantifications of carotenoids in very low concentration, compared to the spectrophotometric methods commonly used for the carotenoids quantifications, normally carried out by external calibration with the respective standard.9,10,28

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CHROMATOGRAPHY

Carotenoids Separation by Comprehensive Liquid Chromatography (LC × LC). Multidimensional liquid chromatography (MD-LC) can be considered as a possible alternative for a superior compounds separation in those cases where monodimensional systems show limitation.21 Compre-hensive 2-D chromatography systems are characterized by the fact that the entire sample to be analyzed is subjected to two online diverse chromatographic separation steps, thus increas-ing very much the overall separation power and peak capacity. All compounds eluting from the first dimension (1D) separation are sequentially transferred into the second dimension (2D) for a further separation. The columns of the first and second dimension analyses are connected via an automated switching multiport valve system that is able to transfer subsequently small aliquots eluting from the first column into the second column and of which technical aspects are beyond the scope of this perspective. The second dimension analysis should be completed before the successive transfer from thefirst column occurs. The best performances of comprehensive system take place when the two separation mechanisms operating in the two different dimensions have complementary selectivity, so-called“orthogonal” systems. The most orthogonal setup could be considered the normal phase (NP)× reversed phase (RP) one. The final visualization of the comprehensive analysis is a 2-D contour plot in which the separated compounds are scattered over the plane and each one is represented by an ellipse-shaped peak, defined by double-axis retention time coordinates; moreover, the software normally allows also for a 3D visualization. The first development of a comprehensive liquid chromatography (LC × LC) method-ology for the study of the native carotenoid composition in a very complex matrix was applied to a sample of red orange

essential oils.19 Free carotenoids and carotenoid esters were characterized. In this study a comprehensive NP-LC× RP-LC-PDA/APCI-MS methodology was set up using a cyano microbore column (250 mm × 1.0 mm i.d., 5 μm particle size) in thefirst dimension (NP) and a monolithic C18 column (4.6 mm i.d.) in the second dimension (RP) that were coupled by a two position 10-port switching valve. Compounds were separated in the first dimension (1D) according to their polarity, from hydrocarbons to free xanthophylls; the analytes were separated in the second dimension (2D) according to their hydrophobicity, the elution order being largely dependent on the fatty acid chain esterified to the xanthophyll so, specifically, retention increased with chain length. In total, 40 different carotenoids were characterized and among them, 16 carotenoid monoesters and 21 carotenoid diesters were identified in the native carotenoid composition of the red orange essential oil. A further step in the application of liquid comprehensive chromatography to the native carotenoids analysis was achieved by combining normal phase separation in thefirst dimension and reversed phase ultra high-performance liquid chromatog-raphy (UHPLC) in the second dimension for the study of the native carotenoids composition in an other very complex matrix like a red chilli peppers carotenoid extract.20 In this study, a novel NP-LC× RP-LC application has been worked out, using a microbore (1.0 mm i.d.) cyano column for thefirst dimension separation, interfaced by two six-port, two position switching valves to two serially coupled C18 column packed with fused-core particles (30 mm× 4.6 mm i.d., 2.7 μm particle size) in the second dimension. The fused-core technology provides a packing material with particles having an overall size of 2.7μm, consisting of a silica nucleus encircled by a thin (0.5 μm) porous shell of stationary phase. This was thefirst work that reported the use of UHPLC conditions in the second dimension performed on octadecylsilica columns packed with 2.7 μm particles. Thirty-three components belonging to ten different chemical classes were identified by this methodology (Figure 3A). The application of the UHPLC technology in this

study has shown great potential in resolution and rapidity for the second dimension chromatographic step. Future improve-ments in comprehensive 2-D liquid chromatography will probably come from the development of new stationary phases, new automated systems with reduced dead volumes, higher pressure check valves, and compatibility with hyphenation of different detectors.

Carotenoids Separation by Comprehensive Super-critical Fluid Chromatography × Liquid Chromatogra-phy (SFC× LC). As it has been previously described, NP x RP set up in comprehensive liquid chromatography is considered

Figure 3. Summary representation indicating the improvements in terms of compounds identification, solvents, and time saving in going from a comprehensive NP-LC× RP-LC setup (A) to a SFC × RP-LC approach (B) in the separation of carotenoids in chilli peppers.*Data from reference20.**Authors unpublished work.

to be one of the most powerful combination because it greatly enhances the orthogonality of the system. However, this combination is not free from some drawbacks like the (i) immiscibility of the mobile phases between the two dimensions that has been partially overcome by the use of a microbore column and very slowflow rate in the order of 10 μL/min in thefirst dimension, (ii) the peak focusing at the head of the secondary column, that has also been in part circumvented by employing a lowflow rate in the first dimension (iii) the long analytical time and (iv) the relatively high solvent consumption. A feasible option is to replace thefirst (1D) NP-LC dimension by supercriticalfluid chromatograpgy (SFC); this combination lessens the solvent immiscibility problems and brings many advantages that are characterizing the use of supercriticalfluid carbon dioxide, like a fast rate of separation and high resolution together with a high orthogonality toward RP-LC. Supercritical CO2 is considered particularly suitable for carotenoids

separation because of its low polarity; in SFC quite often a proportion of an organic solvent is added to the mobile phase as modifier, in order to widen the affinity of the mobile phase for the different compounds and also little variation in the density of thefluid are achieved by small changes in its pressure or temperature which can further ameliorate the separation. Moreover, additional benefits in SFC compared to LC, are the use of a much more ecological mobile phase with the reduction of organic solvent utilization and costs. An online SFC× RP-LC comprehensive separation system was developed for the characterization of native carotenoids in a red chilli pepper extract, with photodiode array and quadrupole time-of-flight (Q TOF) mass spectrometry (MS) detection, for thefirst time, by using a cyano microbore column (250 mm× 1.0 mm i.d., 5.0 μm particle size) in the first dimension SFC separation (1_D),

and a C18column (50 mm× 2.1 mm i.d., 1.7 μm particle size)

for the second dimension (2_{D) UHPLC separation (author}

unpublished work). In this work two fully automated two-position six-port switching valves equipped with two packed octadecyl silica cartridges for effective trapping and focusing of the analytes after elution from1D were used with the addition of a water makeupflow to the SFC effluent prior to entering the loops that permitted the efficient focus the solutes on the sorbent material and to reduce interferences of expanded CO2

gas on the second dimension separation. Compared to the previously described NP× RP-LC approach,20the SFC× RP-LC platform afforded an higher identification power; in fact up to 50 components belonging to 15 different chemical classes were successfully identified in the sample tested. Moreover, the SFC × RP-LC system greatly reduced the organic solvent consumption both in the first and second dimensions by, respectively, half and about an 11th and the analysis time also by half (Figure 3B). It is predictable that the use of supercritical fluids in comprehensive approachs will be further exploited by the academic community.

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FLUID EXTRACTION-SUPERCRITICAL FLUID CHROMATOGRAPHY-MASS SPECTROMETRY (SFE-SFC-MS)

Although lately, supercriticalfluids have been used for both the carotenoids separations (SFC) and the carotenoid extraction (SFE),22−24only very recently the direct online extraction and determination of carotenoids by a supercriticalfluid extraction-supercritical fluid chromatography-mass spectrometry

(SFE-SFC-MS) methodology was reported.25 Supercritical carbon dioxide (CO2) offer peculiar features like low viscosity, high

density, and a high diffusion coefficient that makes it suitable for both the supercriticalfluid extraction and chromatography. The recently developed supercritical fluid extraction-chroma-tography-mass spectrometry methodology,25 allowed for the determination of targeted native carotenoids in red habanero pepper. In total, 21 analytes were extracted and identified by the developed methodology in less than 17 min, including free carotenoids, carotenoids monoesters, and carotenoids diesters, in a very fast“green” and efficient way. The online SFE-SFC conditions were optimized using CO2and MeOH and the SFC

separation were performed on a novel fused-core Ascentis Express C30 column, (150 mm × 4.6 mm i.d. and 2.7 μm particles) having a sub-2-μm stationary phase, in an approach that could be considered as a ultrahigh-performance super-criticalfluid chromatography (UHPSFC) methodology.

In Figure 4 is reported a schematic representation of this novel SFE-SFC-MS system, which operates in three different

modes A, B, and C. (A) Static extraction mode: during this mode the totalflow is splitted between the analytical column and the extraction vessel. (B) Dynamic extraction mode: during this step another valve diverts the totalflow into the extraction vessel (in the opposite direction compared to the static extraction) in order to transfer the extracted analytes into the analytical column. (C) Analysis mode: during this step the total flow is entirely directed into the analytical column. The reported methodology was extremely innovational confronted to the traditional solid−liquid extraction and conventional LC, which required much longer analytical time and solvent waste; moreover, being completed automated, drastically reduces the possible operator errors to occur and the possible analytes losses. Also very recently, the same system was used for the SFC-APCI (±)/QqQ/MS investigation on the apocarotenoids presence in red habanero chilli peppers,26 which had been previously determined in some food and biological matrixes by liquid chromatography.29,30The different apocarotenoids were detected by selective ion monitoring (SIM) of their radical anions generated in the negative ionization mode and, for the free apocarotenoids, also by comparison with the different generated standards mixtures. The transitions used in the MS/ MS experiments were selected on the basis of the product ion scan (PIS) experiments carried out on the various available standards using various collision energies both in positive and negative modes, before the multiple reaction monitoring

Figure 4. SFE-SFC-MS system: (A) static extraction mode, (B) dynamic extraction mode, and (C) analysis mode. Reprinted with permission from ref25. Copyright 2017 Wiley.

(MRM) experiments were made, in order to further confirm the reported compounds identifications. In this study, 25 different apocarotenoids were identified, 14 were free apocarotenoids and 11 were apocarotenoids fatty acids esters. The methodology allowed for all the separations to occur in less than 5 min. The detected Apo-10′-, Apo-14′-, and Apo-15-capsorubinals and different Apo-8′-capsorubinal and Apo-10′-zeaxanthinals fatty acid esters had not been previously identified in any Capsicum species and, to the best of the authors knowledge, in any food matrix. The reported highly sensitive hyphenated system could be regarded as a convenient tool for a rapid apocarotenoids detection and could be applied to the study on the occurrence of these important metabolites in different food, food products, and biological fluids.

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Corresponding Author

*Phone: +39-090-3503996. E-mail:dgiuffrida@unime.it.

ORCID

Daniele Giuffrida:0000-0002-0636-4345

Notes

The authors declare no competingfinancial interest.

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OCC, open column chromatography; TLC, thin layer chromatography; HPTLC, high-performance thin-layer chro-matography; HPLC, high-performance liquid chrochro-matography; LC, liquid chromatography; SFC, supercriticalfluid chromatog-raphy; SFE, supercriticalfluid extraction; MS, mass spectrom-etry; PDA, photo-diode-array; LC× LC, comprehensive liquid chromatography; UHPLC, ultrahigh-performance liquid chro-matography; Q TOF, quadrupole time of flight; MS/MS, tandem mass spectrometry; QqQ/MS, triple quadrupole mass spectrometry; NP, normal phase; RP, reversed phase

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