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Comprehensive Liquid Chromatography and Other Liquid-Based Comprehensive Techniques Coupled to Mass Spectrometry in Food Analysis Francesco Cacciola,† Paola Donato,† Danilo Sciarrone,‡ Paola Dugo,‡,§,∥ and Luigi Mondello*,‡,§,∥

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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 § Unit of Food Science and Nutrition, Department of Medicine, University Campus Bio-Medico of Rome, Via Alvaro del Portillo 21, 00128 Rome, Italy ∥ Chromaleont s.r.l., c/o Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, University of Messina, Polo Annunziata, Viale Annunziata, 98168 Messina, Italy

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CONTENTS

Impact of Comprehensive Two-Dimensional Liquid Chromatography and Mass Spectrometry on Food Analysis Triacylglycerols in Vegetable Oils and Marine Organisms Phospholipids in Milk and Egg Samples Carotenoids in Vegetables and Fruits Polyphenols in Beverages and Plant Extracts Peptides in Saccharomyces cerevisiae and Milk Products Other Comprehensive Liquid-Based Chromatography Methods Comprehensive Two-Dimensional Supercritical Fluid Chromatography × Liquid Chromatography (SFC × LC) and Liquid Chromatography × Supercritical Fluid Chromatography (LC × SFC) Comprehensive Two-Dimensional Liquid Chromatography × Gas Chromatography (LC × GC) and Liquid Chromatography Coupled to Comprehensive Two-Dimensional Gas Chromatography × Gas Chromatography (LC-GC × GC) Concluding Remarks Author Information Corresponding Author Notes Biographies Acknowledgments References

ity. A transfer device (in most cases one or two-switching valves), positioned between the two dimensions, enables the isolation and reinjection of the chromatographic eluate from the first dimension (1D) to the second dimension (2D) column, throughout the whole analysis. Separations in the 2D are usually carried out in a fast way and ideally must end (not to incur in the so-called “wrap-around effects”) before the following reinjection step. The most striking advantage of LC × LC methods, over the one-dimensional (1D) counterparts, is the enhanced resolving power: in theory, the peak capacity (nc) is multiplicative of the nc values of both 1D and 2D. Such a value is practically never reached for a series of reasons, e.g., lack of complete orthogonality, partial loss of 1D resolution, nonideal chromatography conditions, etc. To this regard, some tricks have been exploited in the recent years to mitigate such an issue especially for reversed-phase × reversed-phase LC separations (RP-LC × RP-LC). Since its first application in 1978, over 70 original papers have been published for the analysis of real-world food samples and specifically 14 in the last two years. In most cases, the outstanding selectivity and sensitivity of LC × LC methodologies combined with MS detection made trace (ppb level) and ultratrace (ppt level and lower) analysis feasible, thus reducing the need for tedious sample preparation processes. Critical descriptions of significant applications/evolutions are herein reported concerning the last two years on LC × LC and other liquid-based comprehensive two-dimensional chromatography techniques.

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IMPACT OF COMPREHENSIVE TWO-DIMENSIONAL LIQUID CHROMATOGRAPHY AND MASS SPECTROMETRY ON FOOD ANALYSIS Unlike GC, LC is characterized by a much wider variety of different separation mechanisms, namely, normal phase (NP), reversed phase (RP), size exclusion (SEC), ion exchange (IEX), affinity chromatography (AC), and hydrophilic interaction

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he analytical benefits of comprehensive two-dimensional chromatography methods (LC × LC) have been constantly exploited over the last 20 years. The power of LC × LC methods, along with recent advances in mass spectrometry (MS), enabled a much deeper insight into the true qualitative and quantitative composition of real-world food samples. LC × LC experiments are usually carried out on two analytical columns with complementary (orthogonal) selectiv© 2016 American Chemical Society

Special Issue: Fundamental and Applied Reviews in Analytical Chemistry 2017 Published: November 9, 2016 414

DOI: 10.1021/acs.analchem.6b04370 Anal. Chem. 2017, 89, 414−429

Analytical Chemistry

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Figure 1. Schematic of instrument configuration for sLC × LC. Reprinted from J. Chromatogr. A, Vol. 1228, Groskreutz, S. R.; Swenson, M. M.; Secor, L. B.; Stoll D. R. Selective comprehensive multidimensional separation for resolution enhancement in high performance liquid chromatography. Part I: principles and instrumentation, pp. 31−40 (ref 25). Copyright 2012, with permission from Elsevier.

orthogonal separations can be achieved when suitable mobile and stationary phases are selected, taking into account the physicochemical properties of the food components including size and charge, hydrophobicity, and polarity.10−21 The interface techniques of LC × LC include different types, namely, dual loop, stop-flow, and vacuum evaporation. Due to its simple structure, the dual loop interface is mostly used in LC × LC separations. Some remarkable implementations have been recently carried out in Peter Schoenmakers’ and Dwight Stoll’s research groups. The former investigated an actively modulated LC × LC (LC/a × m/LC) aiming to overcome one of the limitations of contemporary LC × LC arising from the combination of diverse 1D and 2D column diameters: the capability of such an approach was evaluated for both SCX × RP and HILIC × RP-LC separations.22−24 The latter developed a “selective” LC × LC system (sLC × LC) with the aim to break the long-standing link between the time scales of the 1D and 2D separations through novel implementation of existing valve technology; 25−27 a schematic of the instrument configuration for sLC × LC, which allows advantages similar to those derived from off-line LC × LC approach but without most of the major drawbacks of off-line work, is illustrated in Figure 1. Stop-flow mode is applied generally when the analysis speed of the 2D cannot keep up with the sampling frequency of

liquid chromatography (HILIC) which might be useful for tuning a higher number of potentially “orthogonal” combinations. However, the hyphenation of selected LC approaches may present some inconveniences, such as mobile phase immiscibility, that can lead to precipitation of buffers or salts. For such a reason, off-line techniques have been frequently exploited in the LC field, for the pretreatment of complex samples.1 Although it does show a plethora of advantages, e.g., simplicity of operation, possibility of coupling different separation modes, and no problems related with immiscible solvents, some pitfalls can be experienced in terms of time, sample contamination, and software issues. Some of these negative aspects may be circumvented by using online LC × LC techniques. The latter are faster and more reproducible, but they need purpose-designed interfaces, and are more difficult to operate. Another requirement of an LC × LC separation is that any two components separated into different fractions in the 1D must remain separated in the 2D and that elution profiles from both dimensions are preserved.2−5 An LC × LC separation is considered “orthogonal” if the two separation mechanisms are independent of each other thus providing complementary selectivities. The sample components are spread out according to two different retention patterns, over a range as broad as possible with respect to retention factor variation.6−9 Successful 415



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the 1D. A longer 2D column with respect to the commonly employed ones, is usually employed in order to improve resolution as well as peak capacity.28 Two main disadvantages of such an approach includes a longer analysis time with respect to continuous LC × LC and potential band broadening phenomena, which may arise for both long parking periods and nonadequate peak focusing on the top of the 2D column.1,29,30 Vacuum evaporation was utilized by Guan and co-workers31 for eliminating the incompatibility of mobile phases used in NP-LC × RP-LC separations: it allowed the 1D eluents to condense and the 2D solvent to redissolve the residents at the inside wall of the loop for further separation in the 2D. The main pitfall is the potential sample loss risk for volatile components due to evaporation in the interface. More recently, a newly developed vacuum evaporation assisted adsorption (VEAA) interface, allowing fast removal of NP-LC solvent in the vacuum condition and successfully solving the solvent incompatibility problem between NP-LC and RP-LC, was constructed for preparative purposes.32 A proof-of-principle experiment with a novel thermal modulation device with potential use in LC × LC systems has been recently reported by Verstraeten et al.33 On the basis of the thermal desorption concept used in comprehensive two-dimensional gas chromatography (GC × GC) systems, preconcentration of neutral analytes eluting from the 1D was performed in a capillary “trap” column packed with highly retentive porous graphitic carbon particles, placed in an aluminum low-thermal-mass LC heating sleeve. Remobilization of the trapped analytes was achieved by rapidly heating the trap column, by applying temperature ramps up to +1200 °C/min. Compared to the nonmodulated signal, the presented thermal modulator yielded narrower peaks, and a concentration enhancement factor up to 18 was achieved. Even though such an approach was only tested in off-line mode, it shows great promise for further design of online LC × LC separations based on valveless thermal modulation. In addition to LC × LC techniques, mass spectrometry also plays a fundamental role in the field of food analysis.16,18 Food products in fact are very complex mixtures containing many nutrients of organic (lipids, carbohydrates, proteins, vitamins) and inorganic (water, minerals, oxygen) nature but also xenobiotic substances that can come from technological processes, agrochemical treatments, or packaging materials, e.g., residues of pesticides, drugs, toxins, mutagenic compounds, migrants from packaging, metals, and inorganic compounds of toxicological concern. To this regard, the great technological advances made in the MS field, over the past decade, apparently diminished the need for a high-resolution chromatography step. Such a statement is not completely true since the LC × LC-MS hyphenation, in its various combinations, generates valuable and extremely powerful analytical tools capable of providing a profound view on the overall composition of food products. Also, it may be a valuable tool for the assessment of food quality and authenticity, the control of technological processes, the determination of nutritional value, and the detection of molecules with a possible beneficial effect on human health. The main LC × LC-MS applications to food bioactive molecules can be considered essentially as “untargeted” ones and have been applied to triacylglycerols (TAGs),34−46 phospholipids (PLs),47−51 carotenoids,52−58 polyphenols,59−104 104 and peptides.105−108 As far as ionization modes are concerned, the majority of such applications utilized electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), the former for phospholipids, polyphenols,

and peptides and the latter for TAGs and carotenoids. Many advances in MS instrumentation over the last ten years have especially been carried out toward analyzers, at some point overshadowing those achieved in LC × LC. In fact, quadrupoles (single stage, q-MS,34−37,45−47,52−54,57,58,79−81 or triple stages, QqQ-MS83,104), ion trap (IT)-MS,67,74,90,95−97,101,102 and timeof-flight (ToF)-MS48,63−65,82 analyzers and hybrid MS, e.g., Q(or QqQ)-IT,41−44 Q-ToF,39,41,49−51,85,86,88,90−93,98−100,105,107 or IT-ToF,55,56,66,72,106,108 have been widely employed to allow attainment of a very significant gain in sensitivity and speed for food applications. We will not go into details of the instrumental developments and/or operation of all analyzers, inasmuch the main objective of this Review is to highlight the potential benefits arising from the use of LC × LC-MS for handling specific case studies. Considering the hyphenation of MS to LC × LC separations, some significant aspects should be considered. For example, optimal ESI performance is attained at flow rates of 0.2−0.4 mL/min, which is entirely compatible with the recent trend of 2 D ultra high pressure liquid chromatography (UHPLC) (sub-3 or sub-2 μm d.p.), thus requiring the reduction in column diameter from conventional 4.6 to 2.1 mm I.D.65,70,77−79,85,86,88,89,93,97,99,100,103,104 Monolithic columns, especially employed in the former applications, were mostly used in 4.6 mm I.D. formats; when coupled to the higher flow rates used for fast 2D analysis, they implied flow splitting prior to MS detection.34−37,52−54,59,62,66,70,80,103 Superficially porous phases, due to their relatively high permeability and optimal thermal conductivity properties, were viable to be used in both 4.6 and 2.1 mm formats, although clearly the latter was preferred from the perspective of hyphenation to MS.40,45,46,55−58,66,67,71−73,75,76,78,81,83,87,89,95−98,101,102,106 Another critical aspect to be considered when dealing with fast 2 D UHPLC analyses is the sufficiently high acquisition speed. Peak widths in the order of a few seconds are often encountered in the reported works requiring fast acquisition rates, in the order of 5−10 Hz and higher, which are mandatory to obtain the required ∼15 data points across each peak. In this regard, the latest generation of ToF-based analyzers, largely dominating the field, provided the appropriate scanning speeds but also effective structural elucidations through tandem MS data (Q-ToF and IT-ToF accounts for over 40% of the overall food applications). Triacylglycerols in Vegetable Oils and Marine Organisms. TAGs are the major components of naturally occurring fats and oils from animal and vegetable sources whose chemical properties they affect to a large extent. As TAGs represent primary constituents of the human diet, their disproportion may lead to several human pathologies such as coronary heart disease, dyslipidaemia, obesity, etc.109 Furthermore, deficiency of long-chain polyunsaturated fatty acids (PUFAs), necessary for the biosynthesis of cellular membranes, substantially impairs vital cell membrane functions.110 They consist of three FA moieties esterified to a glycerol backbone and thus characterized by a large number of individual species. TAGs-specific features are represented by total carbon number (CN), structure of FAs (presence of unsaturations and length), and position of attack to the glycerol skeleton (sn). TAGs from vegetable oils generally present saturated FAs in the primary positions (sn-1 and sn-3) and unsaturated fatty acids in the sn-2 position with the exclusion of carbon chains longer than 18 carbon units. In TAGs from fish oil, the nature of the fatty acid esterified in the sn-2 position strictly depends upon the fish 416



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Figure 2. Contour plot of the off-line Ag+-LC × RP-LC-APCI-MS plot of the TAG fraction in a menhaden oil sample.

The potential of off-line LC × LC coupling of NARP and silver-ion chromatography was successfully demonstrated in some recent works.39,45,46 In the former one, Holčapek et al. exploited the orthogonality of both separation modes for complex TAG mixtures containing FAs with different acyl chain lengths, different numbers, positions, and geometry of DBs, and different regioisomeric positions of FAs on the glycerol skeleton.39 The Ag+-LC mode enabled at least the partial resolution of regioisomeric TAG mixtures including cis-/transregioisomers, as illustrated on two examples of randomization mixtures. In the other two works coming from Mondello’s research group, off-line LC × LC was successfully applied to marine organisms.45,46 In recent years, there has been an increasing interest about the composition of dietary supplements containing fish oil, such as mackerel, tuna, salmon, and menhaden oils. These oils usually contain high concentrations of long-chain (C18, C20, and C22) monoenoic and polyenoic fatty acids (MUFAs and PUFAs), specifically of the omega-3 biosynthetic family. The presence of high levels of the longchained omega-3 FAs, eicosapentaenoic (EPA or Ep) and docosahexaenoic (DHA or Dh), has been reported as one of the major benefits of consuming fish with the diet.108,109 Aiming to unravel such a complexity for the first time, off-line results were compared to stop-flow ones, in terms of peak capacity and analysis time.45 Figure 2 reports the off-line Ag+LC × NARP-LC-APCI-q-MS contour plot of the TAG fraction of the menhaden oil sample. From the comparison of on- and off-line modes, the latter procedure outperforms the former because of the higher peak capacity values, viz. 2160, allowing one to identify a number of triacylglycerols as high as 253 in menhaden oil. On the other hand, the major bottleneck of the off-line approach is the longer analysis time, mainly attributable to the collection and reinjection of the fractions to be transferred from the 1D to the 2D. A very interesting application

species (i.e., salmon oil contains a greater percentage of PUFA at the sn-2 position, whereas menhaden oil shows a random distribution of them).111,112 Due to the great complexity of the sample, the separation power of LC × LC has often been exploited in such applications, with nonaqueous reversed-phase liquid chromatography (NARP-LC) and silver ion LC (Ag+-LC) being common choices.34−42,45,46 In the former approach, TAGs are separated on the basis of increasing partition number (PN), PN = CN-2DB, where CN is the total carbon number of the three FAs and DB is the number of double bonds. The numbers and the positions of the double bonds, along with chain length, affect retention. Under NARP-LC conditions, TAGs with the same PN are difficult to separate, thus representing critical pairs. In Ag+-LC applications, the elution order relates to increasing DB numbers and to the position or configuration of the double bonds within each FA. Under Ag+-LC operational conditions, the resolution of TAGs with the same DB number is critical. In terms of detection, besides evaporative light scattering (ELS), APCI- and ESI-MS systems have often been employed. When using ESI-MS, TAGs normally require the addition of an electrolyte, such as ammonium formate or ammonium acetate, to produce an adduct ion ([M + NH4]+). With regard to APCI-MS, the technique is the most popular because it produces intense diacylglycerol-like fragment ions [DAG]+, due to the loss of an FA. However, due to the complexity of many TAG mixtures, the power of the LC separation process is fundamental for reliable peak assignment, whatever detector type is used. In all applications, Ag+-LC was employed as the 1D separation mode and NARP-LC as the 2D. The 1 D consisted of a microbore column (1 mm I.D.),34−37,40−42,45 a narrow-bore column (2.1 mm I.D.),38 or a conventional I.D. column.39,43,44,46 417



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of these PL molecular species was achieved through the combination of the HILIC × RP-LC setup to in-line ozonolysis-MS analysis. The TIC chromatogram after 2DLC/MS analysis of the PE class in egg yolk and the O3-MS spectrum of PE(18:0_22:6) in the egg yolk sample are illustrated in Figure 3a,b. Such a work is an extension of a

was tuned by Wei et al., who reported a couple of novel mixedmode single chromatographic columns for determination of TAGs in edible oils.43,44 Such columns, namely, phenyl-hexyl and octyl-sulfonic, combine the features of traditional C18/ silver-ion and C8/silver-ion columns, providing hydrophobic interactions with TAGs under acetonitrile conditions and can offer π−π interactions with TAGs under methanol conditions. Compared to conventional off-line LC × LC employing two different chromatographic columns (C18 and silver-ion column) and using elution solvents comprised of two phases (usually reversed-phase/normal-phase) for TAG separation, such a method, involving a single column, can be achieved by simply altering the mobile phase between acetonitrile and methanol, exhibiting a much higher selectivity for the separation and quantification of TAGs with enhanced efficiency and speed. Such a technique has a great potential as a routine analytical method for analysis of edible oil quality and authenticity control. Phospholipids in Milk and Egg Samples. Phospholipids (PLs) are an important class of health-promoting biomolecules playing an important functional, structural, and metabolic role in the human body. The two main classes of PLs are glycerophospholipids, which consist of a glycerol backbone esterified with two fatty acids (FAs) at sn-1 and sn-2 positions, while the sn-3 position is occupied by a phosphate group attached to a polar head of various structure, and sphingolipids, comprised of a sphingosine backbone, consisting of 18 carbon atoms, attached to the phosphate group. Since PLs are ubiquitous in food, it is highly recommended to increase dietary intake of specific PLs for the prevention of diseases: a systematic study of the PL structure of foods may help in understanding the role of PLs in nutrition and health studies. From an analytical point of view, due to the polarity of PLs, NP-LC methods have been widely employed with retention related to the polar head, i.e., phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylcholine (PC), sphingomyelin (SM), and lysophosphatidylcholine (Lyso-PC). Each PL class is composed of a mixture containing many molecular species, characterized by different FAs; for such structural features, RP-LC techniques have been exploited for the separation of PLs, on the basis of FA chain length and degree of unsaturation. On the basis of the difficulty of employment of a single technique for elucidation of different PL classes and molecular species within a specific PL class, in 2013, Dugo et al. demonstrated the analytical advantages of the coupling of orthogonal separation mechanisms in PL analysis.47 In particular, a silica hydrophilic interaction LC (HILIC) 1D column and a 2D C18 were used for the analysis of a Folch-extracted cow milk sample using ESIMS for structural elucidation. The main aim of the study was the enhanced resolving power but also the possibility to advantageously use such a system for inexpensive detectors such as q-MS or ELS without the need for tandem MS detection (each 2D peak corresponded to a single PL species, eluted according to increasing PN values). A further improvement of the stop-flow methods was reported the same year by Wang et al., who employed an intermediate column to trap the components eluting from the HILIC column; these components were then eluted from the trap column using a makeup flow.48 A very interesting application of HILIC × RP-LC to food analysis was reported in 2015 by Sun et al.:49,50 a thorough characterization of the PI, PE, and PC classes with the localization of double bond positions along the fatty acyl chains

Figure 3. (a) TIC of 2D-LC/MS analysis of the PE class in egg yolk PL extract in positive ion mode; (b) O3 -MS spectrum of PE(18:0_22:6) in an egg yolk sample. Reproduced from Sun, C.; Zhao, Y. Y.; Curtis, J. M. J. Agric. Food Chem. 2015, 63, 1442−1451 (ref 49). Copyright 2015 American Chemical Society.

previous one carried out by the same authors who had only focused on PC molecular species.51 The ozonolysis device is composed of a gas-permeable, liquid-impermeable Teflon tube passing through a glass chamber filled with ozone gas, which is then placed in-line between the LC × LC and the MS detector.51 The eluting PL molecules in the LC mobile phase passed through the device where they rapidly reacted with the ozone that penetrated through the tubing wall. This comprehensive method was successfully applied to an egg yolk PL extract, which revealed the detailed structures of the PL molecules. The additional level of structural detail for PL analysis that can be generated by this approach will be complementary to other experimental methods used in lipidomics. Carotenoids in Vegetables and Fruits. Carotenoids are the most common pigments in nature and are usually characterized by a C40 tetraterpenoid structure, with a symmetrical skeleton. According to the presence or absence of oxygen in the structure, carotenoids are usually divided into two groups, namely, hydrocarbon carotenoids (carotenes) and the oxygenated counterparts (xanthophylls): the latter usually occur in a free form or in a more stable, fatty-acid esterified 418



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Figure 4. Contour plot (λ = 450 nm) of the NP-LC × UHP-RP-LC-PDA analysis of carotenoids in a saponified red mamey. Reproduced from Cacciola, F.; Giuffrida, D.; Utczas, M.; Mangraviti, D.; Dugo, P.; Menchaca, D.; Murillo, E.; Mondello, L. Food Anal. Meth. 2016, 9, 2335−2341 (ref 58). Copyright 2016 Springer.

because protonated ions were generated, giving in turn typical molecular losses and complementary ions. For example, a carotenoid diester generates an [M + H]+ ion, which undergoes the loss of one or two FAs, as well as water molecules, enabling the identification of the FAs bound to the carotenoid structure. In 2012, Cacciola et al.56 presented a comparison of conventional NP-LC × RP-LC and NP-LC × UHP-RP-LC setups for elucidation of the carotenoid pattern in a Capsicum annuum extract. In the latter case, two columns of the same stationary phase (C18) were serially coupled with different gradient and modulation times (1.50 and 1.00 min). Despite the doubling of the stationary phase length, with respect to the “conventional” NP-LC × RP-LC setup, the NP-LC × RPUHPLC method with a 1.50 min modulation time (and gradient) greatly suffered from reduced number of fractions transferred from the 1D. On the other hand, among the two NP-LC × RP-UHPLC setups tested, the one at 1.00 min modulation time yielded the best results in terms of performance due to increased 1D sampling. A similar NP-LC × RP-UHPLC setup was later employed in two works carried out by Cacciola et al. for analysis of the carotenoid content in Pouteria sapote (red mamey)57 and various overripe fruits.58 A typical 2D plot of a saponified carotenoid extract of red mamey sample obtained by NP-LC × UHP-RP-LC (wavelength 450 nm) is shown in Figure 4. In total, 23 compounds belonging to 17 different carotenoid chemical classes were positively separated and identified.57 Additionally, a new carotenoid named as Iso-3-deoxycapsanthin, in which the hydroxyl group is placed on the C2 carbon atom and not on the C4 carbon atom of the β-ring, was formulated in consideration of both the PDA and MS and location on the NP-LC × UHP-RP-LC

form. They do show an extreme instability, which leads to several molecular modifications: as a result, a high number of possible structures can arise, making the NP-LC × RP-LC mode an intriguing choice for such a separation challenge. The first example of LC × LC carotenoid analysis was reported by Dugo et al., who elucidated the free carotenoid composition of orange essential oil and juice.52 The authors employed a silica column, operated under NP conditions, in the 1D; a monolithic RP column (C18) was used in the 2D, with both PDA and MS detection. Under NP-LC conditions, free carotenoids are separated into groups of different polarity, from the nonpolar carotenes up to the highly polar polyols. In the RP-LC mode, carotenoids elute according to their increasing hydrophobicity and decreasing polarity. The complementary information gathered from PDA and MS detection were of the utmost importance, given the limited availability of commercial reference materials and the fact that many carotenoids present very similar UV/vis or MS spectra, which hamper reliable peak identification. Additional information can also be attained by considering specific peak positions in the 2D plots, for carotenoids belonging to the same class. Other studies have dealt with the analysis of the native carotenoid composition of Citrus and Capsicum samples.53−56 In all these cases, the saponification step was avoided, thus preventing artifact formation. Instead of a silica column, a micro cyanopropyl (under NP-LC conditions) was employed in the 1D, allowing separation in groups of different polarity, from hydrocarbons to free xanthophylls. In the 2D, the elution order was largely dependent on the FA chain length so, specifically, retention increased with chain length. The use of APCI-MS, in the positive mode, was very helpful in the identification process 419



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Figure 5. Contour plot (λ = 280 nm) of the RP-LC × RP-LC-PDA analysis of a red wine sample obtained with a cyanopropyl column in 1D and a C18 column in 2D under optimized “full gradient” (A) and SG “shift gradient” program conditions (B). Reprinted from J. Chromatogr. A, Vol. 1458, Donato, P.; Rigano, F.; Cacciola, F.; Schure, M.; Farnetti, S.; Russo, M.; Dugo, P.; Mondello, L. Comprehensive two-dimensional liquid chromatography-tandem mass spectrometry for the simultaneous determination of wine polyphenols and target contaminants, pp. 54−62 (ref 83). Copyright 2016, with permission from Elsevier.

Polyphenols in Beverages and Plant Extracts. Polyphenols are widely distributed in nature and have drawn considerable attention in the last decades due to the maintenance of optimal human health and the reduction of chronic diseases.113−115 Due to their enormous structural variety, i.e., phenolic acids, flavan-3-ols, flavanones, flavones, flavonoids, lignans, among others116 they occur in nature in very complex mixtures thus requiring more powerful separation systems for their analysis. RP-LC × RP-LC and HILIC × RP-

retention plane. In the other work, the obtained results on selected overripe fruits, namely, hybrid persimmon-apple, banana pulp, banana peel, and nectarine, showed that no postclimacteric carotenoid losses occurred with respect to normal ripeness stage highlighting how such matrices still could represent an important source of bioactives for uses either in animal feed production or for the recovery of purified molecules for nutraceutical purposes.58 420



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Figure 6. Contour plot (λ = 280 nm) of the HILIC × RP-LC-PDA licorice metabolites profiles obtained for licorice samples collected from China (A), Iran (B), Crotone (Italy, C), Azerbaijan (D), and Villapiana (Italy, E). Reprinted from Anal. Chim. Acta, Vol. 913, Montero, L.; Ibaňez, E.; Russo, M.; di Sanzo, R.; Rastrelli, L.; Piccinelli, A. L.; Celano, R.; Cifuentes, A.; Herrero, M. Metabolite profiling of licorice (Glycyrrhiza glabra) from different locations using comprehensive two-dimensional liquid chromatography coupled to diode array and tandem mass spectrometry detection, pp.145−159 (ref 102). Copyright 2016, with permission from Elsevier.

LC × LC plane and tend to cluster more or less along the diagonal line. To improve the orthogonality and maximize the utilization of the RP-LC × RP-LC space, three other gradient approaches have been investigated, for handling real-world food samples:20 (a) Although less steep than the “full gradient”, the “segmented gradient”75,78,81 provides significant bandwidth suppression effects. The probability of “wrap-around” effects is also reduced because the concentration of the organic solvent can be adjusted to suit the sample retention, thus resulting in increased fraction coverage. (b) The “parallel gradient”62,73 shows a quasi-isocratic elution with larger bandwidths compared to a repetitive gradient run. The advantages of such an approach are the longer 2D elution time as postgradient equilibration is not necessary within the individual fraction cycles and the possibility to be employed in highly correlated RP-LC × RPLC systems. The gradient needs to be programmed according

LC, have been employed for addressing several types of realworld food samples namely beer and wines,28,59−62,66−68,70,71,73,75,83,87,100 tea and tea-like beverages,72,86,87,95,104 vegetable and fruit extracts.63,64,74,76,77,79−82,85,88,90−93,95−102 So far RP-LC × RP-LC techniques were mostly employed for their separations since fully compatible solvents are employed and an equally generic and steep mobile phase range in each repeated 2D run named ”full gradient” has been widely adopted. It offers a high bandwidht effect and very narrow peak widht can be achieved with remarkable 2D nc. Since the gradient is kept as the same the probability of “wraparound” phenomena may arise for some strongly retained compounds; futhermore the analytes coming from the 1D column have also weak retention on RP-column wheras the analytes eluted after instead do show stronger retention on the 2 D column. As a result, the compounds do not fill the available 421



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Figure 7. (A) Venn diagrams of protein and peptide identifications and distributions of sequence-unique peptides identified from tryptic digests of yeast protein lysates according to (B) peptide HI, (C) peptide molecular weight, and (D) peptide length (number of amino acid residues) between the 2D PGCpH2-RP and 2D PGCpH10-RP systems. The percentage values above the orange columns in (B−D) represent the percentage increases in the number of peptides identified by the 2D PGCpH10-RP platform over the 2D PGCpH2-RP platform for the corresponding parameter. Reproduced from Zhao, Y.; Szeto, S. S. W.; Kong, R. P. W.; Hin Law, C.; Li, G.; Quan, Q.; Zhang, Z.; Wang, Y.; Chu, I. K. Anal. Chem. 2014, 86, 12172−12179 (ref 107). Copyright 2014 American Chemical Society.

to the retention characteristics of the 1D elution pattern. (c) In the “shift gradient”,83 the use of a 2D gradient alleviates bandwidth suppression effects, and the continuous change of the gradient reduces the likelihood of “wrap-around” phenomena; as for the “segmented gradient”, the concentration of the organic solvent can be adjusted to suit the sample retention, resulting in remarkable 2D nc. Another recent promising column combination in LC × LC employs the use of HILIC and RP conditions in the 1D and 2D, respectively. The combination of HILIC and RP-LC presents higher orthogonality with respect to RP-LC × RP-LC, although the hyphenation of these two separation modes is more complicated due to the relative elution strengths of the mobile phase employed, and the need to down-schedule the flowrates in the 1D is highly beneficial for allowing proper “peak focusing” on the top of the 2D column.85,86,88,89,91−98,100−103 In most of these applications, hyphenation of the LC × LC setup as front-end separation to MS proved to be clearly beneficial allowing one to reduce coelutions and minimize matrix effects.63−67,72,74,79,83,85,86,88,91−93,95−102 In the span of the last two years, 11 papers have been published in the field of LC × LC applied to polyphenol analysis (4 for RP-LC × RP-LC and 7 for HILIC × RP-LC). A novel LC × LC-PDA-QqQ-MS setup was very recently reported by Donato et al. for the analysis of wine components.83 Correlation between the two chromatographic separation modes was decreased by designing a 60 s shift gradient program in the 2D, and the increase in orthogonality was evaluated quantitatively utilizing a number of metrics. The system was employed for the analysis of a red wine sample, without preliminary cleanup procedures. Figure 5A shows the LC × LC analysis of the wine sample obtained with a cyanopropyl column in 1D and a C18 column in the 2D and with the optimized “full gradient” elution program. The number of separated and positively identified polyphenols

greatly increased compared to the 1D-LC in a number up to 35. Despite the gain in separation power, some important coelutions still occurred, e.g., procyanidin B1 and procyanidin B2 (peaks 5 and 6 with the same UV and MS spectra and the same fragment ions), laricitrin-glucoside, and syringetin-glucoside (peaks 38 and 39, with the same UV spectra but different MS spectra). Due to the clear lack of orthogonality with peaks mainly concentrated around the diagonal line of the LC × LC plot, a “shift gradient” was further investigated and the resulting LC × LC plot is shown in Figure 5B. A visual inspection of the LC × LC plot already shows a better peak spreading, with no diagonal-line distribution with respect to the plot in Figure 5A, and also reduced background noise as a consequence of the reduced pressure turbulence with an increased number of separated and identified compounds increased up to 43 (>23% with respect to the “full gradient” approach). Accurate quantitation of trace level compounds was possible, by using multiple reaction monitoring (MRM) targeted analysis. Sensitivity of the method developed for the analysis of a red wine sample was well-suited for the determination of selected antioxidants, e.g., trans-resveratrol and regulated contaminants, e.g., Monuron. The estimated limits of detection and of quantification were 0.3 and 1 μg L−1, respectively, well below the minimum detection limit (10 μg L−1) set by current regulation. On the other hand, a thorough profiling of the main metabolites from several licorice (Glycyrrhiza glabra) samples collected at different locations achieved by HILIC × RP-LC-ITMS was recently developed by Montero et al.102 Such a setup was shown to possess powerful separation capabilities allowing one to separate as much as 89 different metabolites in a single sample grouped according to their chemical classes. Figure 6 shows the HILIC × RP-LC-PDA licorice metabolites profiles obtained for five licorice samples collected from China (A), Iran (B), Crotone (Italy, C), Azerbaijan (D), and Villapiana (Italy, E). Triterpene saponins were the most abundant 422



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high peak capacity values, in particular a column with 1.9 μm (80 Å) particle size possessing excellent kinetic and thermodynamic properties, while a column with 1.7 μm (100 Å) particle size provided the highest value of peak capacity.

metabolites followed by glycosylated flavanones and chalcones, whereas glycyrrhizic acid, as expected, was confirmed as the main component in all the studied samples. The usefulness of this method is to generate patterns that could be potentially employed to confirm the authenticity and geographical origin of unknown or suspected licorice samples. Peptides in Saccharomyces cerevisiae and Milk Products. Shotgun proteomics is one of the most common strategies for the analyses of complex protein mixtures and combines proteolytic digestion of biological samples with analysis through LC/MS to overcome many problems related to direct protein level identification. Generally hydrolyzed proteins are very complex mixtures and conventional 1D-LC analyses are not capable of separating all the analytes occurring in the mixture. The first LC × LC-Q-ToF-MS analysis was exploited by Cortes and co-workers105 who tuned a system composed of a cation-exchange column in the 1D and two parallel C18 columns in the 2D for the analysis of α-casein and dephosphorylated α-casein. Such a setup was later implemented by Donato et al., who used RP-LC conditions in both dimensions (both consisting of partially porous particles), thus avoiding the use of salt concentrations in the 1D, and a single column switching valve as an interface in place of a trap and a secondary column.106 In 2014, Zhao et al. investigated an LC × LC-Q-ToF-MS system for the analysis of Saccharomyces cerevisiae tryptic digests evaluating two different setups. Both setups present a porous graphitic carbon (PGC) stationary phase in the 1D and a C18 stationary phase in the 2D.107 PGC is a two-dimensional form of graphite which has sufficient stability throughout the entire pH range, and it is compatible with a large array of solvent systems. Differences of the two setups lie in the pH values: in the first setup, 1D analyses were carried out with a mobile phase at pH = 10; in the second setup, 1D analyses were carried out with a mobile phase at pH = 2. With the 1D mobile phase at pH = 10, a total of 9700 distinct peptides from the 2152 nonredundant proteins were positively identified, whereas with the 1D mobile phase at pH = 2, 7277 peptides and 1895 proteins were determined: 1552 of these proteins common to both sets. These results showed how hydrophobic peptide coverage of the PGCpH10-RP system would be superior to that found using the PGCpH2-RP system (Figure 7). Very recently, Sommella et al. (2015) developed a LC × UHPLC system in two different setups for the characteriztion of milk peptide fractions, generated by enzymatic hydrolysis in the products during fermentation.108 Identification of peptides was carried out by means of IT-ToF-MS equipped with an ESI interface in positive ionization mode. 1D stationary phase for both setups was a C18 microbore column whereas two types of C18 stationary phases for 2D separations were employed, namely, 1.7 μm (100 Å) for the first setup and 1.9 μm (80 Å) for the second setup. The two setups used the same chromatographic conditions: 1D mobile phase had a pH ∼ 9; however, 2D mobile phase had a pH ∼ 2. The UHPLC 2D gradient for both of the setups was in continuous shifting mode. The differences of the two approaches are mainly appreciable for the polar peptides that are less retained on the 2D C18 column with 1.7 μm (100 Å) particle size, with respect to the 2 D C18 column with 1.9 μm (80 Å) particle size. The choice of a different pH in the LC × UHPLC setup as well as a continuous shifting gradient in 2D ensured a good employment of the separation space and a satisfactory selectivity. The combination of the two C18 columns allowed one to obtain

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OTHER COMPREHENSIVE LIQUID-BASED CHROMATOGRAPHY METHODS Comprehensive Two-Dimensional Supercritical Fluid Chromatography × Liquid Chromatography (SFC × LC) and Liquid Chromatography × Supercritical Fluid Chromatography (LC × SFC). NP or RP separation modes commonly employed in the 1D or 2D of LC × LC can be replaced by supercritical fluid chromatography (SFC).117−122 Exposed to atmospheric pressure, the expansion of carbon dioxide (CO2) produces 1D fractions in solvents compatible with the 2D mobile phases. A very interesting property of supercritical fluids is the low viscosity which brings shorter separation kinetics (retention times and re-equilibration times) and limited pressure drop in the system that makes possible the use of serially coupled columns. The most common SFC × RPLC interface designed for food analysis is based on the “solvent displacement method”. After the 1D analysis, the effluent is depressurized through a back-pressure restrictor, mixed with water in a T-junction, and transferred to the interface. The addition of water is advantageous for two reasons: avoiding interferences from residual CO2 gas after the transfer from the 1 D and achieving effective focusing of the analytes in the trap. The characteristics of the packing materials, used as the loop stationary phase, obviously affect the trapping of the analytes, while the flow rate of the makeup water has influence on the performance of the back-pressure restrictor. In the earlier work, an SFC × RP-LC system comprised of a cyanopropyl column in the 1D, separations, and a 2D C18 in the 2D was investigated for the analysis of a lemon oil sample.117 As an interface, a twoposition ten-port switching valve equipped with two C18 packed loops was used. A makeup flow of water was added to the SFC effluent, prior to fraction collection in the packed loops, to obtain good “peak focusing” of the analytes. The subsequent two SFC × RP-LC food applications came from the same research group and were directed to the analysis of fatty acids118 and TAGs119 in fish oil. In the former one,118 SFC × RP-LC and NP-LC × 2RP-LC systems were investigated and compared. In the SFC × RP-LC system, two strongly acidic cation-exchange columns, individually loaded with silver ions, were serially coupled in the 1D, whereas an SB (stable bond) C18 column was employed in the 2D. For the NP-LC × 2RPLC setup, an SB cyanopropyl column and two SB C18 ones were employed for the 1D and 2D, respectively. Overall, the SFC × RP-LC approach provided significantly higher peak capacity, mainly to the high degree of orthogonality, based on the extent of unsaturation and hydrophobicity. A similar setup, differing for the employment of two serially coupled Ag columns in the 1D and a longer monolithic C18 column (10 cm) in 2D, was later investigated by the same research group for the SFC × RP-LC separation of fish oil TAGs, both in the offand online mode;119 the best results were achieved with the offline approach because of the higher nc achieved in the 2D separation, even at the expense of longer analysis time. The feasibility of using the opposite combination, viz., RPLC or NP-LC in the 1D and SFC in the 2D, was demonstrated in two recent works for the separation of blackberry-sage oil and fruiting bodies of Ganoderma lucidum.120,121 In the first 423



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Comprehensive Two-Dimensional Liquid Chromatography × Gas Chromatography (LC × GC) and Liquid Chromatography Coupled to Comprehensive TwoDimensional Gas Chromatography × Gas Chromatography (LC-GC × GC). Among the chromatographic combinations, the hyphenation of liquid chromatography to gas chromatography in a comprehensive way (LC × GC) or, more recently, to comprehensive two-dimensional gas chromatography (LC-GC × GC) has proven to be worthy of attention.123−125 The reason for this attractiveness, in the first case, is related to the fact that a complete separation of complex mixtures using 1D-GC is often difficult to achieve. With realworld samples usually being characterized by a heterogeneous nature, which comprises a variety of chemical families and constituents, and present in a wide range of concentrations, the LC preseparation allows one to isolate more homogeneous groups of compounds prior to GC analysis in order to avoid exceeding the capability of a monodimensional GC system. A detailed map of the entire sample could then be obtained using LC × GC: the high degree of orthogonality resulting from the complementary nature of the two dimensions affords very high resolving power. As with all the comprehensive techniques, ordered structures are detailed in LC × GC chromatograms allowing group-wise integration or, if necessary, target compound analysis. As an alternative, the LC-GC × GC mode, which has recently experienced a wide diffusion in different analytical fields, allows a deeper investigation within the single chemical families thanks to the possibility, once separated in LC, of optimizing the GC × GC parameters for each chemical class, separately. The further nowadays “natural” hyphenation of the last GC system to a mass spectrometer generates a very powerful three (LC × GC/MS) or fourdimensional (LC-GC × GC/MS) analytical method, enabling an improved identification capability thanks to the generation of highly pure spectra compared to those generated from GC/ MS analysis. Furthermore, in many situations, the LC preseparation can be exploited to perform a purification step, avoiding the introduction of nonvolatile components in the GC system. As a consequence, the goal achievable by coupling LC to GC consists of the exploitation of the high selectivity of LC stationary phases with the high separation power of GC. Besides the positive features, several points have to be taken into account as drawbacks. From an instrumental point of view, the introduction of a large volume of liquid effluent into a GC injector represents one of the most important issues. Nowadays, the advent of commercially available fully automated online LC-GC interfaces has greatly reduced issues deriving from a high degree of manual operations, as loss of sample during the transfer and evaporative steps, as well as contamination during the transfer of the LC fraction to the GC injector.123,124 When LC and GC are coupled, the resulting analytical method is greatly influenced by the differences in analysis times of the two dimensions: the inability to perform the GC run in a simultaneous manner with LC requires the latter to be operated in the stop-flow mode. As a consequence, one of the main limitations in a comprehensive LC × GC approach is the GC total run time (analysis + cooling) which generates very long analysis times (typically >3 h)125 in proportion with the number of LC fractions transferred. Moreover, another concern related to the stop-flow mode is that a possible band-broadening effect could be enhanced by the frequent stopping/start of the column flow.

work, a C18 column, eluted with an ACN gradient, was used in the 1D, while an amino column was employed in the SFC dimension, eluted with ACN as modifier.120 The use of ACN reduced the level of baseline noise compared to the use of neat CO2. The resultant contour plot of the RP-LC × SFC separation for the blackberry-sage oil sample is reported in Figure 8.The

Figure 8. Contour plot of the SFC × NP-LC-UV separation of a blackberry-sage oil sample. Reprinted from J. Chromatogr. A, Vol. 1220, Stevenson, P. G.; Tarafder, A.; Guiochon, G. pp. 175−178 (ref 120). Copyright 2012, with permission from Elsevier.

off-line separation was completed in 280 min yielding a practical nc of 2400 (roughly 57% of the theoretical one). In the second work, there was an NP-LC × SFC setup with a cyanopropyl column as the 1D and a monolithic C18 column as the 2D, connected by a two-position ten-port switching valve.121 Such a platform allowed, within a 2 h analysis, one to obtain a nc value of about 350. The most recent work concerning the hyphenation of SFC with RP-LC was carried out in Mondello’s research group for the carotenoid and chlorophyll characterization in different sweet bell peppers (Capsicum annuum L.).122 The 1D consisted of a sub-2 μm SB C18 column operated with an SFC mobile phase in an ultraperformance convergence chromatography system, whereas the 2D was performed in RP-LC mode with a C30 column combined with PDA and MS detection. This approach allowed the determination of 115 different compounds belonging to chlorophylls, free xanthophylls, free carotenes, xanthophyll monoesters, and xanthophyll diesters and proved to be a significant improvement in the pigments determination compared to the conventional 1D-LC approach so far applied to the carotenoid analysis in the studied species. Moreover, the present study also aimed to investigate and to compare the carotenoid stability and composition in overripe yellow and red bell peppers collected directly from the plant, thus also evaluating whether biochemical changes are linked to carotenoid degradation in the nonclimacteric investigated fruits. 424



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Figure 9. Comprehensive normal-phase LC × GC-FID separation of an olive oil. Reprinted from J. Chromatogr. A, Vol. 1000, Janssen, H.-G.; Boers, W.; Steenbergen, H.; Horsten, R.; Flöter, E. Comprehensive two-dimensional liquid chromatography × gas chromatography: evaluation of the applicability for the analysis of edible oils and fats, pp. 385−400 (ref 129), Copyright 2003, with permission from Elsevier.

LC silica column and transferred into the GC × GC injector through a press-fit connector via a 40 cm × 0.53 mm I.D., 0.03 μm f.t. laboratory-made precolumn (OV-1701-OH). The column set consisted of a PS-255 20 m × 0.25 mm I.D., 0.12 μm f.t. used as 1D and a SOP-50 1.5 m × 0.15 mm I.D., 0.075 μm f.t. as 2D column. The various classes of wax esters in olive oil and the geranylgeraniol esters 22:0 and 24:0 in a variety of oils were described. The authors reported a weakness of GC × GC consisting in a serious degradation of the diterpene esters due to the increased elution temperatures related to the higher resistance of the system associated with the presence of the narrow-bore second dimension column compared to monodimensional GC. Despite the GC × GC separation power, LCGC was finally considered as the most suitable approach for quantitative routine analysis of marker wax esters. Later on, a 3D prototype Ag+-LC × GC × GC was described for the analysis of FAMEs, derived from TAGs separated in Ag+-LC,128 providing some stereospecific information. The authors concluded that, in the case of highly complex fractions containing TAGs with three and more double bonds (which cannot be separated by Ag+-LC), even 3D comprehensive chromatography does not provide sufficient selectivity. Information for routine analysis for food labeling purposes can be obtained with GCFAME × GCFAME in about 2 h; however, for more detailed information, Ag+-LCTAG × GCFAME × GCFAME should be considered. In the latter case, it must be considered that the total time required would reach 72 h since 36 Ag+-LC fractions should be subjected to GCFAME × GCFAME. A LC-GC × GC method was developed by Biederman and Grob for characterizing mineral oil aromatic hydrocarbons (MOAH) in contaminated sunflower oil in terms of aromatics ring number and degree of alkylation.132 The possible sources of food contamination (i.e., lubricating oil, extender oil from handle, tar from wood furnace, and distillate aromatic extract oil) were investigated on the basis of the MOAH profile, thanks to the characteristic different numbers of rings and rate of alkylation. Mineral oil saturated hydrocarbons (MOSH) and MOAH were prefractionated directly from the LC outlet on a 25 cm × 2 mm I.D., 5 μm particle size LC column packed with

Since the first LC × GC setup, dealing with the analysis of volatile organic compounds (VOCs) in water,126 only a few papers have been further published, all related with food analysis. LC × GC methods for the investigation of edible oils and fats have been described by de Koning et al.127,128 and Janssen et al.129,130 All these applications featured deep studies on intact TAGs and FAMEs derived thereof, in butter, olive oil, and other edible oils. An automated LC × GC instrument, combined to a ToF-MS and a FID, was used to compare two types of interfaces, namely, a six-port switching valve and a dual side-port 100 μL syringe, reporting similar results for both of the interfaces.127 An Ag+-LC column was employed for TAG separation according to the number of double bonds (0 DB to ≥3 DB), while a GC separation based on carbon number was afforded in the 2D. As an example, Figure 9 shows an LC × GC group-type separation (fingerprinting) of an olive oil sample. The highly informative and ordered 2D plot together with the ToF-MS data allowed an easy identification: the presence of uneven TAGs denoting the addition of animal fat was reported, as for trans fatty acids between TAGs with 0 and 1 DB. In a further application for FAME analysis, TAGs eluted from an Ag+-LC column were transferred to an autosampler vial for a TAG-to-FAME conversion: once completed, the samples were injected into the GC system. The Ag+-LC × GC experiment for the FAMEs analysis was performed on a polar column providing information on the FA chain lengths as well as on the number and location of the double bonds. Fast GC and a reduced number of LC fractions were applied in order to reduce the total LC × GC analysis time from 10 to about 2.5 h.127 More recently, the use of LC as a preseparation step before a GC × GC analysis has been reported in different foodrelated fields as for the analysis of edible oils,131 mineral oil (MO) contamination investigations,132−134 and terpene analysis.135 The wax fraction of different edible oils was investigated by means of LC-GC × GC and compared to a LC-GC approach.131 In particular, the phytol esters, geranylgeraniol esters, and the straight-chain esters of palmitic acid and the unsaturated C(18) acids were studied. A 600 μL wax ester fraction was isolated on a 250 × 2 mm I.D., 5 μm particle size 425



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Figure 10. GC × GC/MS plots of extracted ions representing selected alkylated species of the most important aromatic compounds. Reproduced from Comprehensive two-dimensional GC after HPLC preseparation for the characterization of aromatic hydrocarbons of mineral oil origin in contaminated sunflower oil, Biedermann, M.; Grob, H. J. Sep. Sci., Vol. 32, (ref 132). Copyright 2009 Wiley.

Lichrospher Si 60 and reconcentrated to 20−100 μL. Ten microliters of each fraction were transferred to a programmed temperature vaporizing injector (PTV) connected to a 1 m × 0.53 mm I.D. deactivated precolumn plus a 20 m × 0.25 mm I.D., 0.12 μm f.t. of PS-255 followed by a 1.5 m × 0.15 mm I.D., 0.075 μm f.t. of SOP-50. Exploiting a series of 2D plots extracting characteristic ions, together with the addition of standards and MS spectra, the aromatics of a given ring type and differing in alkylation were localized in the 2D plot (Figure 10). The improved separation achieved after the LC preseparation step was highlighted, as in the case of steranes and hopanes. In fact, these 4- and 5-ring saturated hydrocarbons were coeluted with the highly alkylated two- and threering aromatics in direct GC × GC applications. A problem was reported dealing with an increased retention in 2D for nalkanes, benzenes, and 2-ring components, producing a partial overlap within different classes when highly concentrated samples are analyzed. The need not to overload the second dimension column owing to the limited sample capacity worsened the detection limit for the less abundant classes of MOAH, particularly the 5-ring components. In order to overcome this problem and achieve lower detection limits, the removal of the benzenes and the 2-rings MOAH was proposed, exploiting the LC step. Seven sunflower oils were investigated: the most contaminated sample presented 500 mg/ kg for the 1- and 2-ring aromatics and 186 and 22 mg/kg for the 4- and 5-ring components, respectively, suggesting a contamination with MO less refined or even crude. The concentration of benzenes, 2-ring components (naphthalenes and benzothiophenes), and heavier aromatics was roughly onethird of the MOAH while the 4-ring components accounted for about 5%. As a consequence of such results, the authors reported health risk concerns about the possible concentration of 5-ring components, including the largely alkylated

benzopyrenes. The occurrence of MO migration from recycled paper and board used in food packaging was explored by coupling an LC silica column separation to a GC × GC system with MS and FID for identification and quantification purposes, respectively.133 LC-GC transfer occurred by the retention gap technique and partially concurrent eluent evaporation through the Y-interface:123,124 exploiting the LC step, MOSH and MOAH fractions were analyzed separately in GC × GC. The instrument configuration and GC conditions were the same as reported in ref 132. The authors concluded that further investigations were required in order to measure the proportion of MO possibly released from recycled fibers with respect to the same contamination resulting from cardboard boxes and bags used for packing foods printed with inks based on MO. Mondello and co-workers compared the results of two different laboratories on 16 commercial baby food samples using both LC-GC and LC-GC × GC.134 A silica LC column was used to isolate the MOSH fraction in both the LC-GC and LC-GC × GC methods while two LC-GC interfaces were used, namely, a retention gap technique using a Y-interface and a dual side-port 100 μL syringe.123,124 Various degrees of MOSH contamination (from 0.3 to about 14 mg/kg) were found, not only in the meat and fish products but also in the fruit ones. The same type of contamination was also detected in a lab-made fruit-based baby food, and thus, the single ingredients were analyzed: corn starch and sugar were identified as sources of contamination. The results were confirmed, exploiting an off-line LC-GC × GCquadrupole MS system based on specific locations of the analytes in the 2D plot together with their highly pure MS spectra. Zoccali and co-workers investigated the sesquiterpene hydrocarbon fraction of different Citrus essential oils with different LC and GC combinations.135 A highly detailed qualitative and quantitative report was attained for different samples, in which several constituents were reported for the 426



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first time thanks to the enhanced sensitivity afforded by the cryogenically modulated GC × GC.

spectrometric techniques (hyphenated and multidimensional “comprehensive”) and their application to the study of natural complex matrices (biological samples and foodstuffs).

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CONCLUDING REMARKS Comprehensive two-dimensional liquid and liquid-based chromatography methods have been constantly investigated in the last two decades. The power of such innovative methods greatly benefited from the increasing use of mass spectrometry, thus enabling a much deeper insight into the true determination of real-world food samples. From a chromatographic standpoint, lately, notable implementations have been directed toward the implementation of UHPLC methods, allowing very rapid analysis in the second dimension, without sacrificing separation efficiency. The extent to which mass spectrometry (especially Q-ToF- and IT-ToF-MS platforms) was a powerful aid in unravelling eluting components has been verified by several implemented food applications as reported in this Review. It is reasonable to believe that the development of novel stationary phases, e.g., capillary columns at nanoflow rate gradients, and commercial instruments with reduced dead volumes and high pressure valves will undoubtedly enhance the performance of LC × LC methodologies. Further, as supported by the RP-LC × RP-LC applications for polyphenol analysis, the development of more sophisticated and user-friendly software, allowing reliable and quick integration of 2D peaks, will ultimately be a valid tool for a widespread routine use of mass spectrometry data for quantitative analysis.

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Danilo Sciarrone is Associate Professor of Analytical Chemistry at the University of Messina, Italy. He received a Degree in Pharmaceutical Chemistry and Technology from the same University in 2004. In 2008, he received his Ph.D. in “Food Chemistry and Safety”, collaborating actively in the implementation of projects carried out by the research group in the field analytical and food chemistry. In 2012, he received the “Leslie Ettre Award” for the most original and interesting research by capillary gas chromatography with an emphasis on the environment and on food security. From December 2010 to July 2015, he was Assistant Professor of Analytical Chemistry at the University of Messina. His interests include the application of chromatographic techniques such as LC, LC × LC, GC, GC × GC, LC × GC, GC/MS, MD-GC, SPME-GC, GC chiral, and the development of innovative techniques and fast chromatography. Paola Dugo is Full Professor of Food Chemistry at the University of Messina, Italy. She received a Degree in Chemistry in 1991 and a Ph.D. in Pharmacognosy in 1996 both from the same University. In 1993, she carried out research at the University of Leeds (United Kingdom), with Prof. K.D. Bartle. From 1995 to 2000, she was Assistant Professor of Food Chemistry and then Associate Professor of Food Chemistry until 2011 at the University of Messina. She is Editor of “Journal of Chromatography A”, Elsevier, and member of the Editorial board of the “Flavour and Fragrance Journal”, Wiley. In 2015, she became part of the “Power list” published by the International Scientific Journal “The Analytical Scientist”. In 2016, she received the “HTC-14 award” for the most innovative contribution in the field of hyphenated techniques in chromatography and separation technology. Her research focuses on innovative chromatographic techniques and multidimensional techniques (“heart-cutting” and “comprehensive”) in combination with mass spectrometry for the study of complex natural matrices and particularly lipids in food and biological samples.

AUTHOR INFORMATION

Corresponding Author

*Tel.: +39-090-6766536. Fax: +39-090-358220. E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies

Luigi Mondello is Full Professor of Analytical Chemistry at the University of Messina, Italy. In 1991, he received a Degree in Chemistry from the same University. In 1993, he carried out research at the University of Leeds (United Kingdom), with Prof. K.D. Bartle. From 1996 to 2000, he was Assistant Professor of Food Chemistry and then until 2005 Associate Professor of Food Chemistry at the University of Messina. He is Editor in Chief of “Journal of Essential Oil Research”, Taylor & Francis, Associate Editor of “Journal of Separation Science”, John Wiley-VCH, and Associate Editor of “Food Analytical Methods”, Springer. He is presently on the Analytical Scientist’s “Power List” and has been awarded several prizes, e.g., HTC Award, COLACRO Medal, Silver Jubilee Medal, Liberti Medal, TASIAs, and IFEAT Medal. His research is focused on the development of multidimensional chromatographic instrumentation and software (GC × GC, LC × LC, LC-GC × GC, LC-GC-GC-GC prep., SFC × UPLC), coupled to state-of-the-art MS detection, for the study of complex matrices constituents and contaminants.

Francesco Cacciola is Assistant Professor of Food Chemistry at the University Messina, Italy. He graduated in Pharmacy from the same University in 2004, and after graduation, from February 2005 to August 2006, he was visiting scientist at the University of Pardubice (Czech Republic) under the supervision of Prof. Ing. Pavel Jandera. He received his Ph.D. in “Food and Safety Chemistry” at the University of Messina in 2009, defending a thesis entitled “Employment of High Resolution HPLC Techniques for the Analysis of Complex Matrices”. In 2009, he was awarded a scholarship to work for one year as postdoctoral fellow at the “Center for Food Satefy and Applied Nutrition”, Food and Drug Administration in College Park, Maryland, USA, under the supervision of Dr. Jeanne Rader. His research interests include the characterization of food bioactive molecules by liquid chromatography, “comprehensive” multidimensional liquid chromatography, and hyphenation to mass spectrometry. Paola Donato is Associate Professor of Analytical Chemistry at the University of Messina, Italy. She received a Degree in Pharmaceutical Chemistry and Technology from the same University in 2000 followed by a Doctoral Degree in “Pharmaceutical Sciences” in 2004 discussing a thesis entitled “Effects of Complexation with α- and β-Cyclodextrins on the Chemical−Physical Properties and Antioxidant Activity of 3Hydroxyflavones”. From 2010 to 2014, she was Assistant Professor of Analytical Chemistry at the University “Campus Bio-Medico” in Rome. She has been presenting author and invited lecturer in several scientific national and international conferences, schools, and seminars. Her research is mainly focused on the development of prototype instrumentation and advanced liquid chromatographic and mass

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ACKNOWLEDGMENTS The authors wish to thank the “University of Messina” for support through the “Research and Mobility” Project. REFERENCES

(1) Guiochon, G.; Marchetti, N.; Mriziq, K.; Shalliker, R. A. J. Chromatogr. A 2008, 1189, 109−168. (2) Davis, J. M.; Giddings, J. C. Anal. Chem. 1983, 55, 418−424. (3) Giddings, J. C. Anal. Chem. 1984, 56, 1258A−1270A. (4) Giddings, J. C. J. Chromatogr. A 1995, 703, 3−15.

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