Comprehensive Liquid Chromatography and Other Liquid-Based

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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 Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04370 • Publication Date (Web): 09 Nov 2016 Downloaded from http://pubs.acs.org on November 10, 2016

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Analytical Chemistry

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Comprehensive Liquid Chromatography and other Liquid-based Comprehensive Techniques

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Coupled to Mass Spectrometry in Food Analysis

3 Francesco Cacciola1, Paola Donato1, Danilo Sciarrone2, Paola Dugo2,3,4, Luigi Mondello2,3,4*

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University of Messina, Via Consolare Valeria, 98125 Messina, Italy.

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Messina, Polo Annunziata, Viale Annunziata, 98168 Messina, Italy.

Dipartimento di Scienze Biomediche, Odontoiatriche e delle Immagini Morfologiche e Funzionali,

Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, University of

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Rome, Via Alvaro del Portillo 21, 00128 Rome, Italy

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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

Chromaleont s.r.l., c/o Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed

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*Corresponding author: L. Mondello, tel.+39-090-6766536; fax +39-090-358220.

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Email address: [email protected]

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Content:

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Introduction

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Impact of comprehensive two-dimensional liquid chromatography and mass spectrometry on

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food analysis

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Triacylglycerols in vegetable oils and marine organisms

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Phospholipids in milk and egg samples

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Carotenoids in vegetables and fruits

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Polyphenols in beverages and plant extracts

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Peptides in Saccharomyces cerevisiae and milk products

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Other comprehensive liquid-based chromatography methods

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Comprehensive two-dimensional supercritical fluid chromatography×liquid chromatography

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(SFC× LC) and liquid chromatography×supercritical fluid chromatography (LC×SFC)

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Comprehensive two-dimensional liquid chromatography×gas chromatography (LC×GC) and

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liquid chromatography coupled to comprehensive two-dimensional gas chromatography×gas

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chromatography (LC-GC×GC)

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Concluding remarks

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Introduction

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The analytical benefits of comprehensive two-dimensional chromatography methods (LC×LC) have

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been constantly exploited over the last twenty years. The power of LC×LC methods, along with

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recent advances in mass spectrometry (MS), enabled a much deeper insight into the true qualitative

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and quantitative composition of real-world food samples.

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LC×LC experiments are usually carried out on two analytical columns with complementary

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(orthogonal) selectivity. A transfer device (in most cases one or two-switching valves), positioned

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between the two dimensions, enables the isolation and re-injection of the chromatography eluate

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from the 1D to the 2D column, throughout the whole analysis. Separations in the 2D are usually

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carried out in a fast way, and ideally must end (not to incur in the so-called “wrap-around effects”)

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before the following re-injection step. The most striking advantage of LC×LC methods, over the

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one-dimensional (1D) counterparts, is the enhanced resolving power: in theory, the peak capacity

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(nc) is multiplicative of the nc values of both 1D and 2D. Such a value is practically never reached

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for a series of reasons e.g., lack of complete orthogonality, partial loss of 1D resolution, non-ideal

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chromatography conditions, etc. To this regards, some tricks have been exploited in the recent years

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to mitigate such an issue especially for reversed-phase×reversed-phase LC separations (RP-

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LC×RP-LC).

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Since its first application in 1978 over 70 original papers have been published for the analysis of

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real-world food samples and specifically 14 in the last two years. In most cases, the outstanding

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selectivity and sensitivity of LC×LC methodologies combined with MS detection made trace (ppb

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level), and ultra-trace (ppt level and lower) analysis feasible, thus reducing the need for tedious

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sample preparation processes. A critical descriptions of significant applications/evolutions are

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herein reported concerning the last two years on LC×LC and other liquid-based comprehensive

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two-dimensional chromatography techniques.

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Impact of comprehensive two-dimensional liquid chromatography and mass spectrometry on

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food analysis

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Unlike GC, LC is characterized by a much wider variety of different separation mechanisms,

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namely normal phase (NP), reversed phase (RP), size exclusion (SEC), ion exchange (IEX), affinity

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chromatography (AC) and hydrophilic interaction liquid chromatography (HILIC) which might be

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useful for tuning a higher number of potentially “orthogonal” combinations. However, the

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hyphenation of selected LC approaches may present some inconveniences, such as mobile phase

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immiscibility, that can lead to precipitation of buffers or salts. For such a reason, off-line techniques

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have been frequently exploited in the LC field, for the pre-treatment of complex samples1. Although

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it does show a plethora of advantages e.g. simplicity of operation, possibility of coupling different

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separation modes and no problems related with immiscible solvents, some pitfalls can be

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experienced in terms of time, sample contamination and software issues. Some of these negative

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aspects may be circumvented by using on-line LC×LC techniques. The latter are faster and more

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reproducible, but they need purpose-designed interfaces and they are more difficult to operate.

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Another requirement of an LC×LC separation is that any two components separated into different

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fractions in the 1D must remain separated in the 2D, and that elution profiles from both dimensions

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are preserved2-5. An LC×LC separation is considered “orthogonal” if the two separation

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mechanisms are independent of each other thus providing complementary selectivities. The sample

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components are spread out according to two different retention patterns, over a range as broad as

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possible in respect of retention factor variation6-9. Successful orthogonal separations can be

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achieved when suitable mobile and stationary phases are selected, taking into account the

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physicochemical properties of the food components including size and charge, hydrophobicity and

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polarity10-21.

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The interface techniques of LC×LC include different types of interface namely dual loop, stop-flow

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and vacuum evaporation. Due to its simple structure the dual loop interface is mostly used in

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LC×LC separations. Some remarkable implementations have been recently carried out in Peter ACS Paragon Plus Environment

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Schoenmakers and Dwight Stoll’s research groups. The former investigated an actively modulated

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LC×LC (LC/a×m/LC) aiming to overcome one of the limitations of contemporary LC×LC arising

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from the combination of diverse 1D and 2D column diameters: the capability of such an approach

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was evaluated for both SCX×RP and HILIC×RP-LC separations22-24. The latter developed a

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“selective” LC×LC system (sLC×LC) with the aim to break the long-standing link between the

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timescales of the

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technology25-27; a schematic of the instrument configuration for sLC×LC, which allows advantages

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similar to those derived from off-line LC×LC approach but without most of the major drawbacks of

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off-line work, is illustrated in Figure 1. Stop-flow mode is applied generally when the analysis

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speed of the 2D cannot keep up with the sampling frequency of the 1D. A longer 2D column with

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respect to the commonly employed ones, is usually employed in order to improve resolution as well

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as peak capacity28. Two main disadvantages of such an approach consists in a longer analysis time

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with respect to continuous LC×LC, and potential band broadening phenomena which may arise for

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both long parking periods and non-adequate peak focusing on the top of the 2D column1,29-30.

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Vacuum evaporation was utilized by Guan and co-workers31 for eliminating the incompatibility of

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mobile phases used in NP-LC×RP-LC separations: it allowed to condense the 1D eluents and the 2D

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solvent redissolved the residents at the inside wall of a loop for further separation in the 2D. The

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main pitfall is the potential sample loss risk for volatile components due to evaporation in the

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interface. More recently a newly developed vacuum evaporation assisted adsorption (VEAA)

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interface, allowing fast removal of NP-LC solvent in the vacuum condition and successfully solving

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the solvent incompatibility problem between NP-LC and RP-LC was constructed for preparative

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purposes32. A proof-of-principle experiment with a novel thermal modulation device with potential

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use in LC×LC systems has been recently reported by Verstraeten et al.33. Based on the thermal

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desorption concept used in comprehensive two-dimensional gas chromatography (GC×GC) systems

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pre-concentration of neutral analytes eluting from the 1D was performed in a capillary “trap”

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column packed with highly retentive porous graphitic carbon particles, placed in an aluminum low-

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D and

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D separations through novel implementation of existing valve

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thermal-mass LC heating sleeve. Remobilization of the trapped analytes was achieved by rapidly

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heating the trap column, by applying temperature ramps up to +1200 °C/min. Compared to the non-

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modulated signal, the presented thermal modulator yielded narrow peaks, and a concentration

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enhancement factor up to 18 was achieved. Even though such an approach was only tested in off-

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line mode it shows great promise for a further designing of on-line LC×LC separations based on

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valveless thermal modulation.

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As well as LC×LC techniques, also mass spectrometry play a fundamental role in the field of food

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analysis16,18. Food products in fact are very complex mixtures containing many nutrients of organic

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(lipids, carbohydrates, proteins, vitamins) and inorganic (water, minerals, oxygen) nature but also

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xenobiotic substances that can come from technological processes, agrochemical treatments or

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packaging materials e.g. residues of pesticides, drugs, toxins, mutagenic compounds, migrants from

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packaging, metals and inorganic compounds of toxicological concern. To this regard, the great

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technological advances made in the MS field, over the last decade, apparently diminished the need

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for a high-resolution chromatography step. Such a statement is not completely true since the

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LC×LC-MS hyphenation, in its various combinations, generates valuable and extremely powerful

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analytical tools capable of providing a profound view on the overall composition of food products.

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Also it may be a valuable tool for the assessment of food quality and authenticity, the control of

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technological processes, the determination of nutritional value and the detection of molecules with a

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possible beneficial effect on human health.

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The main LC×LC-MS applications to food bioactive molecules can considered essentially as

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“untargeted” ones and have been applied to triacylglycerols (TAGs)34-46, phospholipids (PLs)47-51,

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carotenoids52-58, polyphenols59-104, and peptides105-108. As far as ionization modes are concerned the

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majority of such applications utilized electrospray ionization (ESI) and atmospheric pressure

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chemical ionization (APCI); the former for phospholipids, polyphenols, and peptides, the latter for

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TAGs and carotenoids. It is especially towards analyzers that much advances in MS instrumentation

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over the last ten years has been carried out, at some point over-shadowing those achieved the ones ACS Paragon Plus Environment

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in LC×LC. In fact quadrupoles (single stage, q-MS34-37,45-47,52-54,57,58,79-81 or triple stages, QqQ-

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MS83,104), ion trap (IT)-MS67,90,95-97,101,102 and time-of-flight (ToF)-MS48,63-65,82 analyzers and hybrid

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MS e.g. Q-(or QqQ)-IT41-44, Q-ToF39,41,49-51,85,86,88,90-93,98-100,107 or IT-ToF55,56,66,72,74,105,106,108 have

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been widely employed allowing to attain a very significant gain in sensitivity and speed for food

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applications. We will not go into details for the instrumental developments and/or operation of all

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analyzers, inasmuch the main objective of this review is to highlight the potential benefits arising

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from the use of LC×LC-MS for handling specific case-studies.

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Considering the hyphenation of MS to LC×LC separations, some significant aspects should be

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considered. For example optimal ESI performance is attained at flow rates of 0.2-0.4 mL/min,

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which is entirely compatible with the recent trend of 2D UHPLC (sub-3 or sub-2 µm d.p.), thus

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requiring the reduction in column diameter from conventional 4.6 to 2.1 mm I.D.65,70,77-

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79,85,86,88,89,93,97,99,100,103,104

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mostly used in 4.6 mm I.D. formats; when coupled to the higher flow rates used for fast 2D analysis

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they implied flow splitting prior to MS detection34-37,52-54,59,62,66,70,80,103. Superficially porous phases,

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due to their relatively high permeability and optimal thermal conductivity properties, were viable to

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be used in both 4.6 and 2.1 mm formats, although clearly the latter was preferred from the

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perspective of hyphenation to MS40,45,46,55-58,66,67,71-73,75,76,78,81,83,87,89,95-98,101,102,106. Another critical

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aspect to be considered when dealing with fast 2D UHPLC analyses is the sufficiently high

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acquisition speed. Peak widths in the order of a few seconds are often encountered in the reported

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works requiring fast acquisition rates, in the order of 5-10 Hz and higher, which are mandatory to

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obtain the requisite ~ 15 data points across each peak. To this regard, the latest generation of ToF-

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based analyzers largely dominating the field, provided the appropriate scanning speeds but also

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effective structural elucidations through tandem MS data (Q-ToF and IT-ToF accounts for over

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40% of the overall food applications).

. Monolithic columns, especially employed in former applications, were


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Triacylglycerols in vegetable oils and marine organisms

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TAGs are the major components of naturally occurring fats and oils from animal and vegetable

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sources whose chemical properties they affect to a large extent. As TAGs represent primary

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constituents of the human diet, their disproportion may lead to several human pathologies such as

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coronary heart diseases, dyslipidaemia, obesity, etc.109. Furthermore, deficiency of long-chain

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polyunsaturated fatty acids (PUFAs), necessary for the biosynthesis of cellular membranes,

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substantially impairs vital cell membrane functions110. They consist of three FA moieties esterified

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to a glycerol backbone resulting thus characterized by a large number of individual species. TAGs-

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specific features are represented by total carbon number (CN), structure of FAs (presence of

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unsaturations, and length), position of attack to the glycerol skeleton (sn). TAGs from vegetable oils

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generally present saturated FAs in the primary positions (sn-1 and sn-3) and unsaturated fatty acids

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in sn-2 position with the exclusion of carbon chains longer than 18 carbon units. In TAGs from fish

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oil, the nature of the fatty acid esterified in sn-2 position strictly depends upon the fish species (i.e.,

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salmon oil contains a greater percentage of PUFA at sn-2 position, whereas menhaden oil shows a

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random distribution of them)111,112.

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Due to the great complexity of the sample, the separation power of LC×LC has often been exploited

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in such applications, with non-aqueous reversed-phase liquid chromatography (NARP-LC) and

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silver ion LC (Ag-LC) being common choices34-42,45,46. In the former approach, TAGs are separated

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on the basis of increasing partition number (PN), PN=CN-2DB, where CN is the total carbon

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number of the three FAs, and DB is the number of double bonds. The numbers and the positions of

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the double bonds, along with chain length, affect retention. Under NARP-LC conditions, TAGs

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with the same PN are difficult to separate, thus representing critical pairs. In Ag-LC applications,

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the elution order relates to increasing DB numbers and to the position or configuration of the double

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bonds within each FA. Under Ag-LC operational conditions, the resolution of TAGs with the same

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DB number is critical. In terms of detection, besides evaporative light scattering (ELS), APCI- and

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ESI-MS systems have often been employed. When using ESI-MS, TAGs normally require the ACS Paragon Plus Environment

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addition of an electrolyte, such as ammonium formate or ammonium acetate, to produce an adduct

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ion ([M+NH4]+). With regards to APCI-MS, the technique is the most popular because it produces

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intense diacylglycerol-like fragment ions [DAG]+, due to the loss of an FA. However, due to the

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complexity of many TAG mixtures, the power of the LC separation process is fundamental for

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reliable peak assignment, whatever detector type is used. In all applications, Ag-LC was employed

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as the 1D separation mode, and NARP-LC as the 2D. The 1D column consisted of a micro-bore

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column (1 mm ID)34-37,40-42,45, or a narrow-bore column (2.1 mm ID)

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column39,43,44,46.

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The potential of off-line LC×LC coupling of NARP and silver-ion chromatography was

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successfully demonstrated in some recent works39,45,46. In the former one, Holčapek et al. exploited

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the orthogonality of both separation modes for complex TAG mixtures containing FAs with

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different acyl chain lengths, different number, positions and geometry of DBs and different

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regioisomeric positions of FAs on the glycerol skeleton39. The Ag-LC mode enabled at least the

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partial resolution of regioisomeric TAG mixtures including cis-/trans-regioisomers, as illustrated on

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two examples of randomization mixtures. In the other two works coming from Mondello’s research

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group, off-line LC×LC was successfully applied to marine organisms45,46. In recent years, there has

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been an increasing interest about the composition of dietary supplements containing fish oil, such as

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mackerel, tuna, salmon, and menhaden oils. These oils usually contain high concentrations of long

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chain (C18, C20, and C22) monoenoic and polyenoic fatty acids (MUFA and PUFA), specifically

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of the omega-3 biosynthetic family. The presence of high levels of the long-chained omega-3 FAs,

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eicosapentaenoic (EPA or Ep), and docosahexaenoic (DHA or Dh), has been reported as one of the

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major benefits of consuming fish with the diet108,109. Aiming to unravel such a complexity for the

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first time off-line results were compared to stop-flow ones, in terms of peak capacity and analysis

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time45. Figure 2 reports the off-line Ag+-LC×NARP-LC-APCI-q-MS contour plot of the TAG

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fraction of the menhaden oil sample. From the comparison of on- and off-line modes, the latter

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procedure outperforms the former because of the higher peak capacity values, viz. 2160, allowing to ACS Paragon Plus Environment

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or conventional I.D.

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identify a number of triacylglycerols as high as 253 in menhaden oil. On the other hand, the major

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bottleneck of the off-line approach is the longer analysis time, mainly attributable to the collection

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and reinjection of the fractions to be transferred from the 1D to the 2D. A very interesting

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application was tuned by Wei et al. who reported a couple of novel mixed-mode single

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chromatographic column for determination of TAGs in edible oils43,44. Such columns, namely

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phenyl-hexyl and octyl-sulfonic combine the features of traditional C18/silver-ion and C8/silver-ion

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columns, providing hydrophobic interactions with TAGs under acetonitrile conditions and can offer

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π-π interactions with TAGs under methanol conditions. Compared to conventional off-line LC×LC

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employing two different chromatographic columns (C18 and silver-ion column) and using elution

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solvents comprised of two phases (usually reversed-phase/normal-phase) for TAG separation, such

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a method, involving a single column, can be achieved by simply altering the mobile phase between

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acetonitrile and methanol, exhibiting a much higher selectivity for the separation and quantification

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of TAGs with enhanced efficiency and speed. Such a technique has a great potential as a routine

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analytical method for analysis of edible oil quality and authenticity control.

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Phospholipids in milk and egg samples

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Phospholipids (PLs) are an important class of health-promoting bio-molecules playing an important

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functional, structural and metabolic role in the human body. The two main classes of PLs are

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glycerophospholipids, which consist of a glycerol backbone esterified with two fatty acids (FAs) at

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sn-1 and sn-2 positions, while the sn-3 position is occupied by a phosphate group attached to a polar

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head of various structure, and sphingolipids, comprised of a sphingosine backbone, consisting of 18

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carbon atoms, attached to the phosphate group. Since PLs are ubiquitous components in food it is

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highly recommendable to increase dietary intake of specific PLs for the prevention of diseases: a

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systematic study of the PL structure of foods may help in understanding the role of PLs in nutrition

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and health studies.


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From an analytical point of view, due to their polarity of PLs, NP-LC methods have been widely

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employed

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phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylcholine (PC), sphingomyelin

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(SM), lysophosphatidylcholine (Lyso-PC). Each PL class is composed of a mixture containing

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many molecular species, characterized by different FAs; for such structural features, RP-LC

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techniques have been exploited for the separation of PLs, on the basis of FA chain length and

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degree of unsaturation. Based on the difficulty of employment of a single technique for elucidation

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of different PL classes and molecular species within a specific PL class in 2013 Dugo et al.

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demonstrating the analytical advantages on the coupling of orthogonal separation mechanisms in PL

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analysis47. In particular a silica hydrophilic interaction LC (HILIC) 1D column, and a 2D C18 were

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used for the analysis of a Folch-extracted cow milk sample using ESI-MS for structural elucidation.

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The main aim of the study was the enhanced resolving power but also the possibility to

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advantageously use such a system for inexpensive detectors such as q-MS or ELS without the need

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for tandem MS detection (each 2D peak corresponded to a single PL species, eluted according to

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increasing PN values). A further improvement of the stop-flow methods was reported the same year

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by Wang et al. who employed an intermediate column to trap the components eluting from the

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HILIC column; these components were then eluted from the trap column using a make-up flow48. A

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very interesting application of HILIC×RP-LC to food analysis was reported in 2015 by Sun et

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al.49,50: a through characterization of the PI, PE and PC classes with the localization of double bond

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positions along the fatty acyl chains of these PL molecular species were achieved through the

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combination of the HILIC×RP-LC set-up to in-line ozonolysis-MS analysis. The TIC

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chromatogram after 2D-LC/MS analysis of the PE class in egg yolk and the O3-MS spectrum of

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PE(18:0_22:6) in the egg yolk sample are illustrated in Figure 3a and 3b. Such a work is an

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extension of a previous one carried out by the same authors who had only focused on PC molecular

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species51. The ozonolysis device is composed of a gas-permeable, liquid-impermeable Teflon tube

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passing through a glass chamber filled with ozone gas, which is then placed in-line between the

with

retention

related

to

the

polar

head,

i.e.,


phosphatidylinositol

(PI),

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LC×LC and the MS detector51. The eluting PL molecules in the LC mobile phase passed through

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the device where they rapidly reacted with the ozone that penetrated through the tubing wall. This

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comprehensive method was successfully applied to an egg yolk PL extract, which revealed the

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detailed structures of the PL molecules. The additional level of structural detail for phospholipid

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analyses that can be generated by this approach will be complementary to other experimental

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methods used in lipidomics.

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Carotenoids in vegetables and fruits

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Carotenoids are the most common pigments in nature and are usually characterized by a C40

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tetraterpenoid structure, with a symmetrical skeleton. Due to the presence or absence of oxygen in

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the structure, carotenoids are usually divided into two groups namely hydrocarbon carotenoids

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(carotenes) and the oxygenated counterparts (xanthophylls): the latter usually occur in a free form,

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or in a more stable, fatty-acid esterified form. They do show an extreme instability, which leads to

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several molecular modifications: as a result a high number of possible structures can arise, making

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the NP-LC×RP-LC mode an intriguing choice for such a separation challenge. The first example of

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LC×LC carotenoid analysis was reported by Dugo et al., who elucidated the free carotenoid

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composition of orange essential oil and juice52. The authors employed a silica column, operated

303

under NP conditions, in the 1D; a monolithic RP column (C18) was used in the 2D, with both PDA

304

and MS detection. Under NP-LC conditions, free carotenoids are separated into groups of different

305

polarity, from the non-polar carotenes up to the highly-polar polyols. In the RP-LC mode,

306

carotenoids elute according to their increasing hydrophobicity, and decreasing polarity. The

307

complementary information gathered from PDA and MS detection were of the utmost importance,

308

given the limited availability of commercial reference materials, and the fact that many carotenoids

309

present very similar UV/vis or MS spectra, which hampers reliable peak identification. Additional

310

information can also be attained by considering specific peak positions in the 2D plots, for

311

carotenoids belonging to the same class. Other studies have dealt with the analysis of the native ACS Paragon Plus Environment

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312

carotenoid composition of Citrus and Capsicum samples53-56. In all these cases, the saponification

313

step was avoided, thus preventing artefact formation. Instead of a silica column, a micro

314

cyanopropyl (under NP-LC conditions) was employed in the 1D, allowing separation in groups of

315

different polarity, from hydrocarbons to free xanthophylls. In the 2D, the elution order was largely

316

dependent on the FA chain length so, specifically, retention increased with chain length. The use of

317

APCI-MS, in the positive mode, was very helpful in the identification process because protonated

318

ions were generated, giving in turn typical molecular losses and complementary ions. For example,

319

a carotenoid diester generates an [M+H]+ ion, which undergoes the loss of one or two FAs, as well

320

as water molecules, enabling the identification of the FAs bound to the carotenoid structure.

321

In 2012 Cacciola et al.56 presented a comparison of a conventional NP-LC×RP-LC and a NP-

322

LC×UHP-RP-LC set-ups for elucidation of the carotenoid pattern in a Capsicum annuum extract. In

323

the latter case, two columns of the same stationary phase (C18) were serially coupled with different

324

gradient and modulation times (1.50 and 1.00 min). Despite the doubling of the stationary phase

325

length, with respect to the “conventional” NP-LC×RP-LC set-up, the NP-LC×RP-UHPLC method

326

with a 1.50 min modulation time (and gradient), greatly suffered the reduced number of fractions

327

transferred from the 1D. On the other hand, among the two NP-LC×RP-UHPLC set-ups tested, the

328

one at 1.00 min modulation time yielded the best results in terms of performance due to increased

329

1

330

Cacciola et al. for analysis of the analysis of the carotenoid content in Pouteria sapote (red

331

mamej)57 and various overripe fruits58. A typical 2D plot of a saponified carotenoid extract of red

332

mamey sample obtained by NP-LC×UHP-RP-LC (wavelength 450 nm) is shown in Figure 4. In

333

total, 23 compounds belonging to 17 different carotenoid chemical classes were positively separated

334

and identified57. Additionally, a new carotenoid named as Iso-3-deoxycapsanthin in which the

335

hydroxyl group is placed on the C2 carbon atom and not on the C4 carbon atom of the β-ring, was

336

formulated in consideration of both the PDA, MS and location on the NP-LC×UHP-RP-LC

337

retention plane. In the other work, the obtained results on seelcted overripe fruits, namely hybrid

D sampling. A similar NP-LC×RP-UHPLC set-up was later employed in two works carried out by


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338

persimmon-apple, banana pulp, banana peel and nectarine showed that no post-climacteric

339

carotenoid losses occurred with respect to normal ripeness stage highlithing how such matrices still

340

could represent an important source of bioactives for uses either in animal feed production, or to the

341

recovery of purified molecules for nutraceutical purposes58.

342 343

Polyphenols in beverages and plant extracts

344

Polyphenols are widely distributed in nature and have drawn considerable attention in the last

345

decades due to the maintenance of optimal human health and the reduction of chronic diseases113-115.

346

Due to their enormous structural variety i.e. phenolic acids, flavan-3-ols, flavanones, flavones,

347

flavonoids, lignans, among others116 they occur in nature in very complex mixtures thus requiring

348

more powerful separation systems for their analysis. RP-LC×RP-LC and HILIC×RP-LC, have been

349

employed for addressing several types of real-world food samples namely beer and wines28,59-62,66-

350

68,70,71,73,75,83,87,100

351

82,85,88,90-93,95-102

352

So far RP-LC×RP-LC techniques were mostly employed for their separations since fully

353

compatible solvents are employed and an equally generic and steep mobile phase range in each

354

repeated 2D run named ”full gradient” has been widely adopted. It offers a high bandwidht effect

355

and very narrow peak widht can be achieved with remarkable 2D nC. Since the gradient is kept as

356

the same the probability of “wrap-around” phenomena may arise for some strongly retained

357

compounds; futhermore the analytes coming from the 1D column have also weak retention on RP-

358

column wheras the the analytes eluted after instead do show stronger retention on the 2D column.

359

As a result the compounds do not fill the available LC×LC plane and tend to cluster more or less

360

along the diagonal line.

361

To improve the orthogonality and maximize the utilization of the RP-LC×RP-LC space other three

362

gradient approaches have been investigated, for handling real-world food samples20:

, tea and tea-like beverages72,86,87,95,104, vegetable and fruit extracts63,64,74,76,77,79-

.


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363

a) “segmented gradient”75,78,81 although less steep that the “full gradient”, it provides significant

364

bandwidth suppression effects. The probability of “wrap-around” effects is also reduced because the

365

concentration of the organic solvent can be adjusted to suit the sample retention, thus resulting in

366

increased fraction coverage.

367

b) “parallel gradient”62,73,: it shows a quasi-isocratic elution with larger bandwidths compared to a

368

repetitive gradient run. The advantages of such an approach are the longer 2D elution time as post-

369

gradient equilibration is not necessary within the individual fraction cycles and the possibility to be

370

employed in highly correlated RP-LC×RP-LC systems. The gradient needs to be programmed

371

according to the retention characteristics of the 1D elution pattern.

372

c) “shift gradient”83: the use of a 2D gradient alleviates bandwidth suppression effects and the

373

continuous change of the gradient reduces the likelihood of “wrap-around” phenomena; as for the

374

“segmented gradient” the concentration of the organic solvent can be adjusted to suit the sample

375

retention, resulting in remarkable 2D nC.

376

Another recent promising column combination in LC×LC employs the use of HILIC and RP

377

conditions in the 1D and 2D, respectively. The combination of HILIC and RP-LC presents higher

378

orthogonality with respect to RP-LC×RP-LC, although the hyphenation of these two sepration

379

modes is more complicated due to the rela,tive elution strengths of the mobile phase employed and

380

the need to down-schedule the flow-rates in the 1D is highly beneficial for allowing proper “peak

381

focusing” on the top of the 2D column85,86,88,89,91-98,100-103.

382

In most of these applications, hyphenation of the LC×LC set-up as front-end separation to MS

383

proved to be clearly beneficial allowing to reduce co-elutions and minimizing matrix effects63-

384

67,72,74,7983,85,86,88,91-93,95-102

385

In the span of the last two years 11 papers have been published in the field of LC×LC applied to

386

polyphenol analysis (4 for RP-LC×RP-LC and 7 for HILIC×RP-LC. A novel LC×LC-PDA-QqQ-

387

MS set-up was very recently reported by Donato et al. for the analysis of wine components83.

388

Correlation between the two chromatographic separation modes was decreased by designing a 60-s

.


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Page 16 of 52

389

shift gradient program in the 2D, and the increase in orthogonality was evaluated quantitatively

390

utilizing a number of metrics. The system was employed for the analysis of a red wine sample,

391

without preliminary clean-up procedures. Figure 5A shows the LC×LC analysis of the wine sample

392

obtained with a cyanopropyl column in 1D and a C18 column in the 2D and with the optimized “full

393

gradient” elution program. The number of separated and positively identified polyphenols greatly

394

increased compared to the 1D-LC in a number up to 35. Despite the gain in separation power, some

395

important coelutions still occurred e.g. procyanidin B1 and procyanidin B2 (peaks 5 and 6 with the

396

same UV and MS spectra and the same fragment ions), laricitrin-glucoside and syringetin-glucoside

397

(peaks 38 and 39, with the same UV spectra but different MS spectra), Due to the clear lack of

398

orthogonality with peaks mainly concentrated around the diagonal line of the LC×LC plot a “shift

399

gradient” was further investigated and the resulting LC×LC plot is shown in Figure 5B. A visual

400

inspection of the LC×LC plot already shows a better peak spreading, with no diagonal-line

401

distribution with respect to the plot in Figure 5A, and also reduced background noise as a

402

consequence of the reduced pressure turbulence with an increased number of separated and

403

identified compounds increased up to 43 (> 23% with respect to the “full gradient” approach).

404

Accurate quantitation of trace level compounds was possible, by using multiple reaction monitoring

405

(MRM) targeted analysis. Sensitivity of the method developed for the analysis of a red wine sample

406

was well-suited for the determination of selected antioxidants e.g. trans-resveratrol and regulated

407

contaminants e.g. monuron. The estimated limits of detection and of quantification were 0.3 µg L−1

408

and 1 µg L−1, respectively, well below the minimum detection limit (10 µg L−1) set by current

409

regulation. On the other hand, a thoroughly profiling of the main metabolites from several licorice

410

(Glycyrrhiza glabra) samples collected at different locations achieved by HILIC×RP-LC-IT-MS

411

was recently developed by

412

separation capabilities allowing to separate as much as 89 different metabolites in a single sample

413

grouped according to their chemical classes. Figure 6 shows the HILIC×RP-LC-PDA licorice

414

metabolites profiles obtained for five licorice samples collected from China (A), Iran (B), Crotone

Montero et al102. Such a set-up was shown to possess powerful


16

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415

(Italy, C), Azerbaijan (D) and Villapiana (Italy, E). Triterpene saponins were the most abundant

416

metabolites followed by glycosylated flavanones and chalcones, whereas glycyrrhizic acid, as

417

expected, was confirmed as the main component in all the studied samples. The usefulness of this

418

method is to generate patterns that could be potentially employed to confirm the authenticity and

419

geographical origin of unknown or suspected licorice samples.

420 421

Peptides in Saccharomyces cerevisiae and milk products

422

Shotgun proteomics is one of the most common strategies for the analyses of complex protein

423

mixtures and combines proteolytic digestion of biological samples with analysis through LC/MS to

424

overcome many problems related to direct protein level identification. Generally hydrolized

425

proteins are very complex mixtures and conventional lD-LC analyses are not capable to separate all

426

the analytes occurring in the mixture. The first LC×LC-Q-ToF-MS analysis was exploited by

427

Cortes and co-workers105 who tuned a system composed of a cation-exchange column in the 1D and

428

two parallel C18 columns in the 2D for the analysis of α-casein and dephosphorylated α-casein.

429

Such a set-up was later implemented by Donato et al. who used of RP-LC conditions in both

430

dimensions (both consisting of partially porous particles), thus avoiding the use of salt

431

concentrations in the 1D, and a single column switching valve as an interface in place of a trap and a

432

secondary column106. In 2014 Zhao et al. investigated an LC×LC-Q-ToF-MS system for the

433

analysis of Saccharomyces cerevisiae tryptic digests evaluating two different set-up. Both set-up do

434

present a Porous Graphitic Carbon (PGC) stationary phase in the 1D and a C18 stationary phase in

435

the 2D107. PGC is a two-dimensional form of graphite which has sufficient stability throughout the

436

entire pH range and it is compatible with a large array of solvent systems. Differences of the two

437

set-up lie in the pH values: in the first set-up 1D analyses were carried out with a mobile phase at

438

pH=10, in the second set-up 1D analyses were carried out with a mobile phase at pH=2. With the 1D

439

mobile phase at pH=10 a total of 9700 distinct peptides from the 2152 non-redundant proteins were

440

positively identified, whereas with the 1D mobile phase at pH=2, 7277 peptides and 1895 proteins ACS Paragon Plus Environment

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Page 18 of 52

441

were determined: 1552 of these proteins common to both sets. These results showed how

442

hydrophobic peptide coverage of the PGCpH10-RP system would be superior to that found using the

443

PGCpH2-RP system (Figure 7).

444

Very recently Sommella et al. (2015) developed a LC×UHPLC system in two different set-up for

445

the characteriztion of milk peptide fractions, generated by enzymatic hydrolysis in the products

446

during fermentation108. Identification of peptides was carried out by means of IT-ToF-MS equipped

447

with ESI interface in positive ionisation mode. 1D stationary phase for both set-up was a C18

448

microbore column whereas two types of C18 stationary phases for 2D separations were employed,

449

namely 1.7 µm (100 Å) for the first set-up and 1.9 µm (80 Å) for the second set-up. The two set-up

450

used the same chromatographic conditions: 1D mobile phase had a pH~9, on the other hand 2D

451

mobile phase had a pH~2. The UHPLC 2D gradient for both the set-up was in continuous shifting

452

mode. The differences of the two approaches are mainly appreciable for the polar peptides that are

453

more retained on the 2D C18 column with 1.7 µm (100 Å) particle size, with respect to the 2D C18

454

column with 1.9 µm (80 Å) particle size. The choice of a different pH in the LC×UHPLC set-up as

455

well as a continuous shifting gradient in 2D ensured a good employment of the separation space,

456

and a satisfactory selectivity. The combination of the two C18 columns allowed to obtain high peak

457

capacity values, in particular column with 1.9 µm (80 Å) particle size possessing excellent kinetic

458

and thermodynamic properties, while column with 1.7 µm (100 Å) particle size provided the

459

highest value of peak capacity.

460 461

Other comprehensive liquid-based chromatography methods

462

Comprehensive two-dimensional supercritical fluid chromatography×liquid chromatography

463

(SFC× LC) and liquid chromatography×supercritical fluid chromatography (LC×SFC)

464

NP or RP separation modes commonly employed in the 1D or 2D of LC×LC can be replaced by

465

supercritical fluid chromatography (SFC)117-122. Exposed to atmospheric pressure, the expansion of

466

carbon dioxide (CO2) produces 1D fractions in solvents compatible with the 2D mobile phases. A ACS Paragon Plus Environment

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467

very interesting property of supercritical fluids is the low viscosity which brings shorter separation

468

kinetics (retention times and re-equilibration times), and limited pressure drop in the system that

469

make possible the use of serially-coupled columns. The most common SFC×RP-LC interface

470

designed for food analysis is based on the “solvent displacement method”. After the 1D analysis the

471

effluent is depressurized through a back-pressure restrictor, mixed with water in a T-junction, and

472

transferred to the interface. The addition of water is advantageous for two reasons: avoid

473

interferences from residual CO2 gas after the transfer from the 1D, and achieve effective focusing of

474

the analytes in the trap. The characteristics of the packing materials, used as the loop stationary

475

phase, obviously affect the trapping of the analytes, while the flow rate of the make-up water has

476

influence on the performance of the back-pressure restrictor. In the earlier work, an SFC×RP-LC

477

system comprised of a cyanopropyl column in the 1D, separations and a 2D C18 in the 2D was

478

investigated for the analysis of a lemon oil sample117. As an interface, a two-position ten-port

479

switching valve equipped with two C18 packed loops was used. A make-up flow of water was

480

added to the SFC effluent, prior to fraction collection in the packed loops, to obtain good “peak

481

focusing” of the analytes. The subsequent two SFC×RP-LC food applications came from the same

482

research group, and were directed to the analysis of fatty acids118 and TAGs119 in fish oil. In the

483

former one118, SFC×RP-LC and NP-LC×2RP-LC systems were investigated and compared. In the

484

SFC×RP-LC system, two strongly-acidic cation-exchange columns, individually loaded with silver

485

ions, were serially-coupled in the 1D, whereas an SB (stable bond) C18 column was employed in

486

the 2D. For the NP-LC×2RP-LC set-up an SB cyanopropyl column, and two SB C18 ones were

487

employed for the 1D and 2D, respectively. Overall, the SFC×RP-LC approach provided significantly

488

higher peak capacity, mainly to the high degree of orthogonality, based on the extent of

489

unsaturation and hydrophobicity. A similar set-up, differing for the employment of two serially-

490

coupled Ag columns in the 1D, and a longer monolithic C18 column (10 cm) in 2D, was later

491

investigated by the same research group for the SFC×RP-LC separation of fish oil TAGs, both in


19


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Page 20 of 52

492

the off- and on-line mode119; the best results were achieved with the off-line approach because of

493

the higher nC achieved in the 2D separation, even at the expense of longer analysis time.

494

The feasibility of using the opposite combination viz. RP-LC or NP-LC in the 1D and SFC in the

495

2

496

bodies of Ganoderma lucidum120,121. In the first work a C18 column, eluted with an ACN gradient,

497

was used in the 1D, while an amino column was employed in the SFC dimension, eluted with ACN

498

as modifier119. The use of ACN reduced the level of baseline noise compared to the use of neat CO2.

499

The resultant contour plot of the RP-LC×SFC separation for blackberry-sage oil sample is reported

500

in Figure 8.The off-line separation was completed in 280 min yielding a practical nC of 2400

501

(roughly 57% of the theoretical one). In the second work an NP-LC×SFC set-up with a cyanopropyl

502

column as the 1D, and a monolithic C18 column as the 2D, connected by a two-position ten-port

503

switching valve121. Such a platform allowed within 2 h analysis to obtain a nC value of about 350.

504

The most recent work concerning the hyphenation of SFC with RP-LC was carried out in

505

Mondello’s research group for the carotenoid and chlorophyll characterization in different sweet

506

bell peppers (Capsicum annuum L.)122. The 1D consisted of a sub-2 µm SB C18 column operated

507

with an SFC mobile phase in an ultra-performance convergence chromatography system, whereas

508

the 2D was performed in RP-LC mode with a C30 column combined with PDA and MS detection.

509

This approach allowed the determination of 115 different compounds belonging to chlorophylls,

510

free xanthophylls, free carotenes, xanthophyll monoesters, and xanthophyll diesters, and proved to

511

be a significant improvement in the pigments determination compared to the conventional 1D-LC

512

approach so far applied to the carotenoid analysis in the studied species. Moreover, the present

513

study also aimed to investigate and to compare the carotenoid stability and composition in overripe

514

yellow and red bell peppers collected directly from the plant, thus also evaluating whether

515

biochemical changes are linked to carotenoid degradation in the non-climacteric investigated fruits.

D, was demonstrated in two recent works for the separation of blackberry-sage oil and fruiting


20

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516

Comprehensive two-dimensional liquid chromatography×gas chromatography (LC×GC) and

517

liquid chromatography coupled to comprehensive two-dimensional gas chromatography×gas

518

chromatography (LC-GC×GC)

519

Among the chromatographic combinations, the hyphenation of liquid chromatography to gas

520

chromatography in a comprehensive way (LC×GC), or, more recently, to comprehensive two-

521

dimensional gas chromatography (LC-GC×GC), has proven to be worthy of attention123-125. The

522

reason for this attractiveness, in the first case, is related to the fact that a complete separation of

523

complex mixtures using 1D-GC is often difficult to be achieved. Being real-world samples usually

524

characterized by an heterogeneous nature, which comprising a variety of chemical families and

525

constituents, present in a wide range of concentrations, the LC pre-separation allows to isolate more

526

homogenous groups of compounds prior to GC analysis in order to avoid to exceed the capability of

527

a monodimensional GC system. A detailed map of the entire sample could be then obtained using

528

LC×GC: the high degree of orthogonality resulting from the complementary nature of the two

529

dimensions afford very high resolving power. As all the comprehensive techniques, ordered

530

structures are detailed in LC×GC chromatograms allowing group-wise integration or, if necessary,

531

target compound analysis. As an alternative, the LC-GC×GC mode, which has recently experienced

532

a wide diffusion in different analytical fields, allows a deeper investigation within the single

533

chemical families thanks to the possibility, once separated in LC, to optimize the GC×GC

534

parameters for each chemical class, separately. The further nowadays “natural” hyphenation of the

535

last GC system to a mass spectrometer generates a very powerful three (LC×GC-MS) or four-

536

dimensional (LC-GC×GC-MS) analytical method, enabling an improved identification capability

537

thanks to the generation of highly pure spectra compared to those generated from GC–MS analysis.

538

Furthermore, in many situations the LC pre-separation can be exploited to perform a purification

539

step, avoiding the introduction of non-volatile components in the GC system. As a consequence, the

540

goal achievable by coupling LC to GC consists in the exploitation of the high selectivity of LC

541

stationary phases with the high separation power of GC. Besides the positive features, several points ACS Paragon Plus Environment

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Page 22 of 52

542

have to be taken into account as drawbacks. From an instrumental point of view, the introduction of

543

a large volume of liquid effluent into a GC injector represents one of the most important issues.

544

Nowadays the advent of commercially-available fully automated on-line LC-GC interfaces has

545

greatly reduced issues deriving from a high degree of manual operations, as loss of sample during

546

the transfer and evaporative steps, as well as contamination during the transfer of the LC fraction to

547

the GC injector123,124. When LC and GC are coupled the resulting analytical method is greatly

548

influenced by the differences in analysis times of the two dimensions: the inability to perform the

549

GC run in a simultaneous manner with LC requires the latter to be operated in the stop-flow mode.

550

As a consequence, one of the main limitations in a comprehensive LC×GC approach is the GC total

551

run time (analysis + cooling) which generates very long analysis times (typically >3hours)125 in

552

proportion with the number of LC fractions transferred. Moreover, another concern related to the

553

stop-flow mode is that a possible band-broadening effect could be enhanced by the frequent

554

stopping/start of the column flow.

555

Since the first LC×GC set-up, dealing with the analysis of volatile organic compounds (VOCs) in

556

water126, only few papers have been further published, all related with food analysis. LC×GC

557

methods for the investigation of edible oils and fats have been described by de Koning et al.127,128

558

and Janssen et al.129,130. All these applications featured deep studies on intact TAGs and FAMEs

559

derived thereof, in butter, olive oil and other edible oils. An automated LC×GC instrument,

560

combined to a ToF-MS and a FID, was used comparing two types of interface, namely a six-port

561

switching valve and a dual side-port 100 µL syringe, reporting similar results for both the

562

interfaces127. An AgLC column was employed for TAG separation according to the number of

563

double bonds (0 DB to ≥ 3 DB), while a GC separation based on carbon number was afforded in

564

the 2D. As an example Figure 9 shows an LC×GC group-type separation (fingerprinting) of an olive

565

oil sample.The highly informative and ordered 2D plot together with the ToF-MS data allowed an

566

easy identification: the presence of un-even TAGs denoting the addition of animal fat was reported,

567

as for trans fatty acids between TAGs with 0 and 1 DB. In a further application for FAME analysis, ACS Paragon Plus Environment

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568

TAGs eluted from an AgLC column were transferred to an autosampler vial for a TAG-to-FAME

569

conversion: once completed, the samples were injected into the GC system. The Ag-LC×GC

570

experiment for the FAMEs analysis was performed on a polar column providing information on the

571

FA chain lengths as well as on the number and location of the double bonds. Fast GC and a reduced

572

number of LC fractions were applied in order to reduce the total LC×GC analysis time from 10 to

573

about 2.5 hours127 . More recently, the use of LC as a pre-separation step before a GC×GC analysis

574

has been reported in different food-related fields as for the analysis of edible oils131 , mineral oil

575

(MO) contamination investigations132-134, and terpene analysis135. The wax fraction of different

576

edible oils was investigated by means of LC-GC×GC and compared to a LC-GC approach131. In

577

particular the phytol esters, geranylgeraniol esters and the straight-chain esters of palmitic acid, and

578

the unsaturated C(18) acids were studied. A 600 µL wax ester fraction was isolated on a 250 × 2

579

mm i.d. 5 µm particle size LC silica column and transferred into the GC×GC injector through a

580

press-fit connector via a 40 cm x 0.53 mm i.d. × 0.03 µm f.t. laboratory-made pre-column (OV-

581

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

582

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

583

olive oil and the geranylgeraniol esters 22:0 and 24:0 in a variety of oils were described. The

584

authors reported a weakness of GC×GC consisting in a serious degradation of the diterpene esters

585

due to the increased elution temperatures related with the higher resistance of the system associated

586

with the presence of the narrow-bore second dimension column compared to monodimensional GC.

587

Despite the GC×GC separation power, LC-GC was finally considered as the most suitable approach

588

for quantitative routine analysis of marker wax esters. Later on, a 3D prototype AgLC×GC×GC was

589

described for the analysis of FAMEs, derived from TAGs separated in AgLC128, providing some

590

stereospecific information. The authors concluded that in the case of highly complex fractions

591

containing TAGs with three and more double bonds (which cannot be separated by AgLC), even 3D

592

comprehensive chromatography does not provide sufficient selectivity. Information for routine

593

analysis for food labelling purposes can be obtained with GCFAME×GCFAME in about 2 hours even if ACS Paragon Plus Environment

23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 52

594

for deeper information AgLCTAG×GCFAME×GCFAME should be considered. In the last case, it must

595

be considered that the total time required would reach 72 hours since 36 AgLC fractions should be

596

subjected to GCFAME×GCFAME. A LC-GC×GC method was developed by Biederman and co-

597

workers for characterizing mineral oil aromatic hydrocarbons (MOAH) in contaminated sunflower

598

oil in terms of aromatics ring number and degree of alkylation132. The possible sources of food

599

contamination (i.e. lubricating oil, extender oil from handle, tar from wood furnace, and distillate

600

aromatic extract oil) were investigated based on the MOAH profile, thanks to the characteristic

601

different number of rings and rate of alkylation. Mineral oil saturated hydrocarbons (MOSH) and

602

MOAH were pre-fractionated directly from the LC outlet on a 25 cm × 2 mm i.d. 5 µm particle size

603

LC column packed with Lichrospher Si 60, and reconcentrated to 20-100 µL. Ten microliters of

604

each fraction were transferred to a programmed temperature vaporizing injector (PTV) connected to

605

a 1 m × 0.53 mm i.d. deactivated pre-column plus a 20 m × 0.25 mm i.d. × 0.12 µm f.t. of PS-255

606

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

607

extracting characteristic ions, together with the addition of standards and MS spectra, the aromatics

608

of a given ring type and differing in alkylation were localized in the 2D plot (Figure 10). The

609

improved separation achieved after the LC pre-separation step was highlighted, as in the case of

610

steranes and hopanes. In fact, these 4- and 5-ring saturated hydrocarbons were coeluted with the

611

highly alkylated two- and three-ring aromatics in direct GC×GC applications. A problem was

612

reported dealing with an increased retention in 2D for n-alkanes, benzenes and 2-ring components,

613

producing a partial overlap within different classes when high concentrated samples are analyzed.

614

The need not to overload the second dimension column owing to the limited sample capacity

615

worsened the detection limit for the less abundant classes of MOAH, particularly the 5-ring

616

components. In order to overcome this problem and achieve lower detection limits, the removal of

617

the benzenes and the 2-rings MOAH was proposed exploiting the LC step. Seven sunflower oils

618

were investigated: the most contaminated sample presented 500 mg/kg for the 1- and 2-ring

619

aromatics and 186 and 22 mg/kg for the 4- and 5-ring components, respectively suggesting a ACS Paragon Plus Environment

24

Page 25 of 52

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


620

contamination with MO less refined or even crude. The concentration of benzenes, 2-ring

621

components (naphthalenes and benzothiophenes) and heavier aromatics was roughly one third of

622

the MOAH while the 4-ring components accounted for about 5%. As a consequence of such results,

623

the authors reported health risk concerns about the possible concentration of 5-ring components,

624

including the largely alkylated benzopyrenes. The occurrence of MO migration from recycled paper

625

and board used in food packaging was explored coupling an LC silica column separation to a

626

GC×GC system with MS and FID for identification and quantification purposes, respectively133.

627

LC-GC transfer occurred by the retention gap technique and partially concurrent eluent evaporation

628

through the Y-interface123,124: exploiting the LC step MOSH and MOAH fractions were analyzed

629

separately in GC×GC. The instrument configuration and GC conditions were the same reported in

630

reference 132. The authors concluded that further investigations were required in order to measure

631

the proportion of MO possibly released from recycled fibers respect to the same contamination

632

resulting from cardboard boxes and bags used for packing foods printed with inks based on MO.

633

Mondello and co-workers compared the results of two different laboratories on sixteen commercial

634

baby food samples using both LC-GC and LC-GC×GC134. A silica LC column was used to isolate

635

the MOSH fraction in both the LC-GC and LC-GC×GC methods while two LC-GC interfaces were

636

used, namely a retention gap technique using a Y-interface and a dual side-port 100 µL

637

syringe123,124. Various degrees of MOSH contamination (from 0.3 mg/kg to circa 14 mg/kg) were

638

found, not only in the meat and fish products, but also in the fruit ones. The same type of

639

contamination was also detected in a lab-made fruit-based baby food, and thus, the single

640

ingredients were analyzed: corn starch and sugar were identified as sources of contamination. The

641

results were confirmed, exploiting an off-line LC-GC×GC-quadrupole MS system based on specific

642

locations of the analytes in the 2D plot together with their highly pure MS spectra. Zoccali and co-

643

workers investigated the sesquiterpene hydrocarbon fraction of different Citrus essential oils with

644

different LC and GC combinations135. A highly detailed qualitative and quantitative report was


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 52

645

attained for different samples, in which several constituents were reported for the first time thanks

646

to the enhanced sensitivity afforded by the cryogenically-modulated GC×GC.

647 648

Concluding remarks

649

Comprehensive two-dimensional liquid and liquid-based chromatography methods have been

650

constantly investigated in the last two decades. The power of such innovative methods greatly

651

benefited from the increasing use of mass spectrometry thus enabling a much deeper insight into the

652

true determination of real-world food samples. From a chromatographic stand-point, notably

653

implementations have been lately directed towards the implementation of UHPLC methods,

654

allowing very rapid analysis in the second dimension, without sacrificing separation efficiency.

655

The extent to which mass spectrometry (especially Q-ToF- and IT-ToF-MS platforms) was a

656

powerful aid in unravelling eluting components has been witnessed by several implemented food

657

applications as reported in this review. It is reasonable to believe that the development of novel

658

stationary phases, e.g. capillary columns at nano-flow rate gradients, and commercial instruments

659

with reduced dead volumes, and high pressure valves, will undoubtedly enhance the performance of

660

LC×LC methodologies. Further, as witnessed by the RP-LC×RP-LC applications for polyphenol

661

analysis, the development of more sophisticated and user-friendly software, allowing reliable and

662

quick integration of 2D peaks, will ultimately be a valid tool for a widespread routine use of mass

663

spectrometry data for quantitative analysis.

664 665 666 667 668 669 670 ACS Paragon Plus Environment

26

Page 27 of 52

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671

Biographies for Reviews

672

Francesco Cacciola is Assistant Professor of Food Chemistry at the University Messina, Italy. He

673

graduated in Pharmacy from the same University in 2004, and after graduation, from February 2005

674

to August 2006, he was visiting scientist at the University of Pardubice (Czech Republic) under the

675

supervision of Prof. Ing. Pavel Jandera. He received his Ph.D. in “Food and Safety Chemistry” at

676

the University of Messina in 2009, defending a thesis entitled “Employment of High Resolution

677

HPLC Techniques for the Analysis of Complex Matrices”. In 2009 he was awarded a scholarship to

678

work for one year as post-doctoral fellow at the “Center for Food Satefy and Applied Nutrition”,

679

Food and Drug Administration in College Park, Maryland, USA, under the supervision of Dr.

680

Jeanne Rader. His research interests include the characterization of food bioactive molecules by

681

liquid chromatography, “comprehensive” multidimensional liquid chromatography and hyphenation

682

to mass spectrometry.

683 684

Paola Donato is Associate Professor of Analytical Chemistry at the University of Messina, Italy.

685

She received a Degree in Pharmaceutical Chemistry and Technology from the same University in

686

2000 followed by a Doctoral Degree in “Pharmaceutical Sciences” in 2004 discussing a thesis

687

entitled “Effects of Complexation with α- and β-Cyclodextrins on the Chemical-Physical Properties

688

and Antioxidant Activity of 3-hydroxyflavones”. From 2010 to 2014 she was Assistant Professor of

689

Analytical Chemistry at the University “Campus Bio-Medico” in Rome. She has been presenting

690

author and invited lecturer in several scientific national and international conferences, schools, and

691

seminars. Her research is mainly focused on the development of prototype instrumentation and

692

advanced liquid chromatographic and mass spectrometric techniques (hyphenated and

693

multidimensional “comprehensive”) and their application to the study of natural complex matrices

694

(biological samples and foodstuffs).


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 52

697

Danilo Sciarrone is Associate Professor of Analytical Chemistry at the University of Messina, Italy.

698

He received a Degree in Pharmaceutical Chemistry and Technology from the same University in

699

2004. In 2008 he received his Ph.D. in "Food Chemistry and Safety", collaborating actively in the

700

implementation of projects carried out by the research group in the field analytical and food

701

chemistry. In 2012 he received the "Leslie Ettre Award" for the most original and interesting

702

research by capillary gas chromatography with an emphasis on the environment and on food

703

security. From December 2010 to July 2015 he was Assistant Professor of Analytical Chemistry at

704

the University of Messina. His interests include the application of chromatographic techniques such

705

as LC, LC×LC, GC, GC×GC, LC×GC, GC-MS, MD-GC, SPME-GC, GC chiral, and the

706

development of innovative techniques and fast chromatography.

707 708

Paola Dugo is Full Professor of Food Chemistry at the University of Messina, Italy. She received a

709

Degree in Chemistry in 1991 and a Ph.D. in Pharmacognosy in 1996 both from the same

710

University. In 1993 she carried out research at the University of Leeds (United Kingdom), with

711

Prof. K.D. Bartle. From 1995 to 2000 she was Assistant Professor of Food Chemistry and then

712

Associate Professor of Food Chemistry until 2011 at the University of Messina. She is Editor of

713

“Journal of Chromatography A”, Elsevier, and member of the Editorial board of the “Flavour and

714

Fragrance Journal”, Wiley. In 2015 she became part of the "Power list" published by the

715

International Scientific Journal “The Analytical Scientist”. In 2016 she received the “HTC-14

716

award” for the most innovative contribution in the field of hyphenated techniques in

717

chromatography and separation technology. Her research focuses on innovative chromatographic

718

techniques and multidimensional techniques (“heart-cutting” and “comprehensive”) in combination

719

with mass spectrometry for the study of complex natural matrices and particularly lipids in food and

720

biological samples.


28

Page 29 of 52

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723

Luigi Mondello is Full Professor of Analytical Chemistry at the University of Messina, Italy. In

724

1991 he received a Degree in Chemistry from the same University. In 1993 he carried out research

725

at the University of Leeds (United Kingdom), with Prof. K.D. Bartle. Since 1996 to 2000 he was

726

Assistant Professor of Food Chemistry and then until 2005 Associate Professor of Food Chemistry

727

at the University of Messina. He is Editor in Chief of “Journal of Essential Oil Research”, Taylor &

728

Francis, Associate Editor of “Journal of Separation Science”, John Wiley-VCH, and Associate

729

Editor of Food Analytical Methods, Springer. He is present in the Analytical Scientist’s “Power

730

List”, and has been awarded several prizes e.g. HTC Award, COLACRO Medal, Silver Jubilee

731

Medal, Liberti Medal, TASIAs, IFEAT Medal. His research is focused on the development of

732

multidimensional chromatographic instrumentation and software (GC×GC, LC×LC, LC-GC×GC,

733

LC-GC-GC-GC prep., SFC×UPLC), coupled to state-of-the-art MS detection, for the study of

734

complex matrices constituents and contaminants.

735 736

Acknowledgements

737

The authors wish to thank the “University of Messina” for support through the “Research and

738

Mobility” Project.

739

The table of content (TOC) graphic was designed by using the following articles: Top: Reprinted

740

from J. Chromatogr. A, Vol. 1458, Donato, P.; Rigano, F.; Cacciola, F.; Schure, M.; Farnetti, S.;

741

Russo, M.; Dugo, P.; Mondello L., Comprehensive two-dimensional liquid chromatography-tandem

742

mass spectrometry for the simultaneous determination of wine polyphenols and target contaminants,

743

pp. 54-62 (ref. 83). Copyright 2016, with permission from Elsevier; Bottom left: Reproduced from

744

Mazzi Leme, G.; Cacciola, F.; Donato, P.; Cavalheiro, A. J.; Dugo, P.; Mondello L. Anal. Bioanal.

745

Chem. 2014, 406, 4315-4324. (ref. 81) Copyright 2014 Springer; Bottom right: Reprinted from J.

746

Chromatogr. A, Vol. 1428, Montero, L.; Sánchez-Camargo, A.P.; García-Canas, V.; Tanniou, A.;

747

Stiger-Pouvreau, V.; Russo, M.; Rastrelli, L.; Cifuentes, A.; Herrero, M.; Ibánez, E., Anti-

748

proliferative activity and chemical characterization bycomprehensive two-dimensional liquid ACS Paragon Plus Environment

29


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Page 30 of 52

749

chromatography coupled tomass spectrometry of phlorotannins from the brown macroalga

750

Sargassum muticum collected on North-Atlantic coasts, pp.115-125 (ref. 101), Copyright 2016,

751

with permission from Elsevier.

752 753

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87. ACS Paragon Plus Environment

33


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977

Figure legends

978

Figure 1. Schematic of instrument configuration for sLC×LC. Reprinted from J. Chromatogr. A,

979

Vol. 1228, Groskreutz, S. R.; Swenson, M. M.; Secor, L. B.; Stoll D. R. Selective comprehensive

980

multi-dimensional

981

chromatography. Part I: principles and instrumentation, pp. 31-40 (ref. 25). Copyright 2012, with

982

permission from Elsevier.

983

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

984

menhaden oil sample.

985

Figure 3. (a) TIC of 2D-LC/MS analysis of the PE class in egg yolk PL extract in positive ion

986

mode; (b) O3-MS spectrum of PE(18:0_22:6) in an egg yolk sample. Reproduced from Sun, C.;

987

Zhao, Y. Y.; Curtis, J. M. J. Agric. Food Chem. 2015, 63, 1442-1451 (ref. 49). Copyright 2015

988

American Chemical Society.

989

Figure 4. Contour plot (λ=450 nm) of the NP-LC×UHP-RP-LC-PDA analysis of carotenoids in a

990

saponified red mamey. Reproduced from Cacciola, F.; Giuffrida, D.; Utczas, M.; Mangraviti, D.;

991

Dugo, P.; Menchaca, D.; Murillo, E.; Mondello, L. Food Anal. Meth. 2016, 9, 2335-2341 (ref. 58).

992

Copyright 2016 Springer.

993

Figure 5. Contour plot (λ=280 nm) of the RP-LC×RP-LC-PDA analysis of a red wine sample

994

obtained with a cyanopropyl column in 1D and a C18 column in 2D under optimized “full gradient”

995

(A) and SG “shift gradient” program conditions (B). Reprinted from J. Chromatogr. A, Vol. 1458,

996

Donato, P.; Rigano, F.; Cacciola, F.; Schure, M.; Farnetti, S.; Russo, M.; Dugo, P.; Mondello L.

997

Comprehensive two-dimensional liquid chromatography-tandem mass spectrometry for the

998

simultaneous determination of wine polyphenols and target contaminants, pp. 54-62 (ref. 83).

999

Copyright 2016, with permission from Elsevier.

separation

for

resolution

enhancement

in

high

performance

liquid

1000

Figure 6. Contour plot (λ=280 nm) of the HILIC×RP-LC-PDA licorice metabolites profiles

1001

obtained for licorice samples collected from China (A), Iran (B), Crotone (Italy, C), Azerbaijan (D)

1002

and Villapiana (Italy, E) Reprinted from Anal. Chim. Acta, Vol. 913, Montero, L.; Ibaňez, E.; ACS Paragon Plus Environment

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1003

Russo, M.; di Sanzo, R.; Rastrelli, L.; Piccinelli, A.L.; Celano, R.; Cifuentes, A.; Herrero, M.

1004

Metabolite profiling of licorice (Glycyrrhiza glabra) from different locations using comprehensive

1005

two-dimensional liquid chromatography coupled to diode array and tandem mass spectrometry

1006

detection, pp.145-159 (ref. 102). Copyright 2016, with permission from Elsevier.

1007

Figure 7. (A) Venn diagrams of protein and peptide identifications and distributions of sequence-

1008

unique peptides identified from tryptic digests of yeast protein lysates according to (B) peptide HI,

1009

(C) peptide molecular weight, and (D) peptide length (number of amino acid residues) between the

1010

2D PGCpH2-RP and 2D PGCpH10-RP systems. The percentage values above the orange columns in

1011

B−D represent the percentage increases in the number of peptides identified by the 2D PGCpH10-RP

1012

platform over the 2D PGCpH2-RP platform for the corresponding parameter. Reproduced from

1013

Zhao, Y.; Szeto, S.S.W.; Kong, R.P.W.; Hin Law, C.; Li, G.; Quan, Q.; Zhang, Z.; Wang, Y.; Chu,

1014

I.K. Anal. Chem. 2014, 86, 12172-12179 (ref. 106). Copyright 2014 American Chemical Society.

1015

Figure 8. Contour plot of the SFC×NP-LC-UV separation of a blackberry-sage oil sample.

1016

Reprinted from J. Chromatogr. A, Vol. 1220, Stevenson, P.G.; Tarafder, A.; Guiochon, G. pp. 175-

1017

178 (ref. 120). Copyright 2012, with permission from Elsevier.

1018

Figure 9. Comprehensive normal-phase LC×GC-FID separation of an olive oil. Reprinted from J.

1019

Chromatogr. A, Vol. 1000, Janssen, H.-G.; Boers, W.; Steenbergen, H.; Horsten, R.; Floter, E.

1020

Comprehensive two-dimensional liquid chromatography×gas chromatography: evaluation of the

1021

applicability for the analysis of edible oils and fats, pp. 385-400 (ref. 129), Copyright 2003, with

1022

permission from Elsevier.

1023

Figure 10. GC×GC-MS plots of extracted ions representing selected alkylated species of the most

1024

important aromatic compounds. Reproduced from Comprehensive two-dimensional GC after HPLC

1025

preseparation for the characterization of aromatic hydrocarbons of mineral oil origin in

1026

contaminated sunflower oil, Biedermann, M.; Grob, H. J. Sep. Sci., Vol. 32, (ref 132). Copyright

1027

2009 Wiley.

1028 ACS Paragon Plus Environment

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For Table of Contents Only

1030

1031


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Figure 1 254x190mm (300 x 300 DPI)


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Figure 2 208x152mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3 176x234mm (300 x 300 DPI)


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Figure 4 254x190mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5A 201x156mm (300 x 300 DPI)


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Figure 5B 201x157mm (300 x 300 DPI)



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Figure 6 219x188mm (300 x 300 DPI)


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Figure 7 244x170mm (300 x 300 DPI)



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Figure 8 187x185mm (300 x 300 DPI)


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Figure 9 244x140mm (300 x 300 DPI)



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Figure 10 216x186mm (300 x 300 DPI)


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