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S0021-9673(13)00067-8
doi:10.1016/j.chroma.2013.01.012
CHROMA 353955
To appear in:
Journal of Chromatography A
Received date:
Revised date:
Accepted date:
24-7-2012
17-12-2012
4-1-2013
Please cite this article as: M.-J. Motilva, A. Serra, A. Maci`a, Analysis of
food polyphenols by ultra high-performance liquid chromatography coupled
to mass spectrometry: An overview, Journal of Chromatography A (2010),
doi:10.1016/j.chroma.2013.01.012
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Highlights
UHPLC opens new perspectives to determine phenolics in complex food samples
o Higher resolution, higher efficiency and faster separations are achieved in UHPLC
o UHPLC-MS allows developing rapid, sensitive and selective methods
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Maria-Jos Motilva*, Aida Serra, Alba Maci
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Agrria, Universitat de Lleida, Av/Alcalde Rovira Roure 191, 25198 Lleida, Spain
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Abstract
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Phenolic compounds, which are widely distributed in plant-derived foods, recently
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attracted much attention due to their health benefits, so their determination in food
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samples is a topic of increasing interest. In the last few years, the development of
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chromatographic columns packed with sub-2 m particles and the modern high resolution
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mass spectrometry (MS) have opened up new possibilities for improving the analytical
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methods for complex sample matrices, such as ingredients, foods and biological samples.
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In addition, they have emerged as an ideal tool for profiling complex samples due to its
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speed, efficiency, sensitivity and selectivity. The present review addresses the use of the
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compounds in food samples. Additionally, the different strategies to extract, quantify the
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phenolic compounds and to reduce the matrix effect (%ME) are also reviewed. Finally, a
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1. Introduction
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Over the last decade, polyphenols have had an increasing impact on answering key
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questions and understanding vital functions of biological systems. It is very well known that
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phenolic compounds have positive health effects because they are a source of natural
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antioxidants and have nutritional properties. Polyphenols are widely found in fruit,
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the natural ingredients used. Cocoa, apples, tea, berries, coffee, wine, jam, chocolate and
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onions are common sources of polyphenols in the human diet [1]. These samples are
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complex since they contain different classes of phenolic compounds, which differ in
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polarity and size, from simple phenolic compounds (phenolic acids) to oligomers
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samples are found at low concentration levels. The diverse chemical nature of phenols
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has hindered their analysis. Over the last decade, they have been an active search for
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new methods to determine the polyphenols in plant samples, foods, dietary supplements
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and, latterly HPLC tandem mass spectrometry (MS/MS) techniques have enabled
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The method of choice for qualitative and quantitative analysis of polyphenols is HPLC.
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Since its introduction in the 1970s, this technique has been used for all the phenolic
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groups and hundreds of applications in food analysis have been published [2-5]. In most of
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these studies, the stationary phase, solvent, and gradient have to be optimized previously
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polyphenols. The analyses of these complex samples by HPLC require high resolution and
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long analysis time, the later being an important limitation when a high number of samples
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have to be analyzed for research or quality control purposes. Speed is often an important
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factor in research to enable a higher number of samples to be analyzed. In the last few
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years, ultra-high performance LC (UHPLC) has opened up new possibilities for improving
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the analytical methods for complex sample matrices, such as ingredients, foods and
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biological samples. The development of ultra-high pressure pump systems and sub-2 m
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packing materials has allowed significant improvement in the separation, speed and
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efficiency of modern HPLC in the last decade. The innovative ultra-high pressure or
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UHPLC has made it possible to achieve five to ten-fold faster separations than with
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conventional LC systems, while maintaining or increasing resolution [6]. The narrow peaks
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produced by fast UHPLC require a small detection volume and fast acquisition rate to
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ensure the high efficiency of these peaks. While typical flow rates for the HPLC analyses
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of polyphenols lie in the 1.0 to 1.5 mL/min range and 150 mm and 4.6 mm as the length
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and inner diameter, respectively, the introduction of analytical columns with smaller inner
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diameters (1 and 2.1 mm) and shorter lengths (50 mm) minimizes the injection volume. By
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using these short columns containing stationary phases with smaller particle sizes lead to
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increase of the mobile phase linear velocity and this velocity is greatly expanded. Higher
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linear velocities can be used without sacrificing separation quality. Therefore, UHPLC has
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the advantages of improved resolution, higher peak efficiency, fast separations and
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The coupling of LC with MS is particularly suited to the analysis of food samples [2-5].
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better means of identifying and quantifying polyphenols in complex matrices, such as plant
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foods, food ingredients and formulated foods. This review focuses on the recently
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published literature for the analysis of phenolic compounds by the improved LC, UHPLC,
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samples, and also examine briefly their sample pre-treatment, and the strategies used for
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their quantification and to reduce the matrix effect (%ME). Finally, the future trends for
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the human diet. Their chemical structures are characterized by the presence of at least
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one aromatic ring with one or more hydroxyl functional groups attached. Phenolics range
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from simple small single aromatic-ring structures to the complex and weighty condensed
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proposed their own. For example, Crozier et al. [10] classified these compounds into
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food samples. Nonetheless, in this review, the phenolic classification is based on their
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skeleton structure, ranging from simple, small single aromatic-ring structures to the
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chalcones and lignans. Phenolic acids are further divided into benzoic acid derivatives,
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based on a C6-C1 skeleton (such as vanillic acid and gallic acid), cinnamic acid derivatives,
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which are based on a C6-C3 skeleton (such as caffeic acid and ferulic acid),
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hydroxyphenylacetic
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examples, vanillic acid is found in herbs, such as oregano and thyme, and olives; gallic
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acid in wine, tea, species (cloves) and chestnuts; caffeic acid in fruit (black chokeberry),
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herbs (thyme and oregano) and spices (ginger and thyme); ferulic acid in cereals,
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chocolate and herbs (thyme), and dihydrocaffeic acid and hydroxyphenylacetic acid in
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olives [11].
derivatives,
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Phenyl alcohols, hydroxytyrosol and tyrosol are present in olives and olive oil.
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Besides, these food samples contain other phenolic compounds, known as secoiridoids.
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These are exclusive to the Olea Europaea species, are not included in practically any
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classification, although the secoiridoid derivatives of oleuropein and ligstroside are the
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hydroxylation patterns, and are found in red wine. Chalcones contain a C6-C3-C6 basic
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structure lacking a heterocyclic C-ring and a group of phenolic compounds with an open-
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ring structure. As an example, high concentration levels of phloridzin are found in oregano.
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. The chalcones areespecially abundant in fruits (e.g. citrus fruit, apples, including cider),
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vegetables (e.g. tomatoes, shallots, bean sprouts, potatoes) and spices (e.g. licorice).
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Most of the chalcone contents in citrus fruits and various plants are mediated through the
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phenylpropanoid units linked by a hydrogen bridge. Flax and sesame seeds contain high
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levels of lignans. Lignans are also present in cereals and vegetable seed oils, and some
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composed of two aromatic rings linked by a three carbon bridge (C6 -C3 -C6 skeleton) and
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are the most abundant group of phenolic compounds. These are sub-classified into
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foodstuffs, e.g. the isoflavones daidzein and genistein in soy, or flavanones in citrus fruit,
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other flavonoids are extensively distributed in the diet, e.g. quercetin, the main flavonol in
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our diet, is present in many fruit and vegetables, such as onions and tea. Other vegetables
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that contain flavonols include broccoli and kale. Flavonols are the most extensive of the
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flavonoids, and the main dietary flavonols are kaempferol, quercetin, isorhamnetin and
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myricetin, and these are normally present as O-glycosides. Examples include kaempferol,
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which is found in tea, beans and spices (capers) and quercetin in vegetables (onions),
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chocolate, black elderberries, and also spices (capers and cloves). Most red and purple
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fruit, such as grapes, cherries and blueberries have important quantities of anthocyanins,
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which are responsible for the red, purple, and blue colour of many fruit, vegetables and
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peonidin, petunidin and malvidin. When these phenolic compounds are found as sugar
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Flavones are less common in fruit and vegetables. The most important rich dietary
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sources of flavones are parsley and celery [13]. Some cereals, such as millet and wheat,
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and herbs contain larges quantities of C-glycosides of flavones [9, 11]. Flavones, which
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include luteolin and apigenin, are also present in virgin olive oil, olives, herbs (sage, thyme
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and oregano), and vegetables (artichoke). Flavanones (eriodictyol and naringenin) and
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flavanonols (taxifolin) are also found in oregano and thyme. As it has been commented
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before, the isoflavones, such as genistein and daidzein, are found in soy and soy
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products.
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catechins, are present in cocoa derivatives (e.g. dark chocolate and milk chocolate [11]
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and other food sources, such as red fruit, including red grapes and, indirectly, in red wine
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[14]. Tea also represents a rich source of flavanols, also providing a small quantity of
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quercetin. Flavanols range from the simple monomers (catechin and its isomer
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dimers, trimers, etc) and polymeric proanthocyanidins, which are also known as
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condensed tannins. Additionally, the simple monomers, catechin and epicatechin can be
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hydroxylated to form the gallocatechins and can also undergo esterification with gallic acid
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Finally, tannins can also be classified into condensed tannins, complex tannins and
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hydrolysable tannins, which include gallotannins and ellagitannins. Tannins are defined as
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either galloyl-esters and their derivatives, in which galloyl moieties or their derivatives are
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and complex tannins), or they are oligomeric and polymeric proanthocyanidins that can
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[15]. In more detail, condensed tannins are polymeric flavonoids consisting of flavanol
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(catechin) units; gallotannins are hydrolysable tannins with a polyol core (referring to a
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compound with multiple hydroxyl groups) substituted by 10-12 gallic acid residues;
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ellagitannins are also hydrolyzable tannins derived from pentagalloylglucose but, unlike
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gallotannins, they contain additional C-C bonds between adjacent galloyl moieties in the
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Although some tannins are extremely astringent [17], they are abundant in many edible
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plants species, their presence being notable in fruit, leaves and bark [16, 18]. Fruit, such
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as pomegranates, persimmons and berries (e.g. cranberries) [19], strawberries and red
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raspberries [20]; herbs and spices, such as cumin, thyme, vanilla, hops (used to make
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beer bitter) and cinnamon [21, 22], and other products such as nuts [23], legumes and
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chocolate [18], contain tannins. Additionally, smoked foods (smoked fish and meat) may
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have tannins on their surface due to their presence in the woods used in smoking e.g.
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Given the complexity of phenolic structures and the plant tissues and foods that
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contain them, their analysis requires a series of steps: extraction, isolation, structural
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The aim of this section is to present a brief summary of the sample pre-treatment
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techniques used for the determination of phenolic compounds in food samples by UHPLC-
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MS. However, in some studies the technique of pre-treatment of the sample, prior to
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sample preparation. This practice was only applicable to aqueous liquid foods, in which
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the polyphenols
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preparation. The tea samples were only diluted 10-fold in water before analysis and they
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reported the contamination of the ESI source and eventual clogging of the heated capillary
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was avoided . However, in vegetable oils, such as virgin olive oil [26, 27], or sauces [28],
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previous its chromatographic analysis is necessary and it is more complex than a simple
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sample dilution .
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Table 1 shows the reported sample pre-treatment methodologies for the determination of
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(LLE) and solid-phase extraction (SPE) are the sample pre-treatment techniques most
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frequently used.
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The extraction of polyphenols by off-line LLE has been performed using different
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solvents, such as water [29-31], hot water [25, 32], methanol [33-39], methanol/formic
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During the LLE process, polyphenols can be degraded by enzyme action when the
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collected plant material is fresh. It is thus advisable to use dry, lyophilized, or frozen
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samples that are generally ground into a powder prior to LLE. This extraction technique,
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which is tedious, uses a large volume of solvents and a long time to extract the target
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compounds. Often, this time required for LLE is enormous higher compared to the UHPLC
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separations. Normally, the use of LLE requires a one or two hours to extract the
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compounds and the UHPLC separations are achieved in few minutes. This fact is the most
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With SPE, many of the problems associated with LLE extraction can be prevented.
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SPE is more efficient than LLE, yields quantitative extractions that are easy to perform, is
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rapid, and can be automated. Moreover, solvent use and lab time are reduced.
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category materials, silica gels modified with hydrophobic alkyl- or aryl-bonded silica
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(mainly based on 18) [46, 47] and polymeric sorbents [28, 48] are the most used . For the
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analysis of oil samples (feed oils), diol cartridges have also been reported [28]. On the
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other hand, mixed-mode cartridges, which provide dual modes of retention, both reversed-
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phases and ion-exchange retention modes, nowadays have not been reported for the
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with dual-phase materials could be a good strategy to food pre-treatment due to its
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The most important limitation of SPE extraction is the need to evaporate or remove
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some of the elution solvent, usually by nitrogen stream, to preconcentrate the analytes.
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Recently, the use of the micro-elution SPE (SPE)plate instead of cartridges as the device
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format allows a small sample volume to be loaded onto the plate, eluting the retained
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analytes in a small volume and avoiding the evaporation step to preconcentrate the
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analytes, prior to chromatographic analysis [49, 50]. Therefore, this technique allows a
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rapid isolation of the target analytes, since the extraction time is considerably reduced.
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However the SPE has not been applied so far to extract phenolic compounds in food
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samples by UHPLC-MS. The main applications of SPE are reported for the analysis of
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biological samples, where only a few amount of sample (for example urine, plasma or
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tissues) is necessary to load. Therefore, the use of these microplates (SPE) could be
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because faster extractions are achieved and additionally the 96-well plate format allows
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The ASE technique has not been extensively applied to sample pre-treatment in the
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combined with UHPLC-MS has been reported for the determination of anthocyanidins in
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red cabbage [51] and phenolic acids in rosemary [52], using ethanol/water as the
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extraction solution.
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The microwave assisted extraction MAE is a simple and rapid technique that can be
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completed in a few minutes. MAE technique has been used for sample pre-treatment of
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China and Japan [53], and isoflavones in Traditional Chinese Medicine (TCM) [54].
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Supercritical fluid extraction (SFE) utilizes the solvent properties of fluids near their
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[55].
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Until now, these extraction techniques (MAE and SFE) combined with UHPLC-MS have
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not been reported for the determination of phenolic compounds in food samples.
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carbon dioxide (SFC) with UHPLC technology opens new perspectives for applying this
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have been made with the aim of improving the separation efficiency of the HPLC analysis.
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As a result of these improvements in the packing material used for the chromatographic
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separation, an advanced form of LC has been reported. This novel technique is known as
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UHPLC, and it uses narrow-bore columns packed with very small particles, sub-2 m
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(from 1.7 to 1.9 m), and mobile phase delivery systems operating at high back-pressures
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(up to 1300 bar or 15000 psi). According to the van Deemter equation, as the particle size
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decreases to less than 2.5 m, there is a significant gain in efficiency (in UHPLC the
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particle size is 1.7 m) and it does not diminish at increased flow rates because the eddy
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diffusion and mass transfer resistance in the mobile phase are reduced [56, 57].
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The major advantages of UHPLC (1.7 m particle size) over conventional HPLC
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(particle size from 3 to 5 m) are improved resolution, higher peak efficiency, shorter
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retention times, and reduced solvent consumption. As a consequence of the higher peak
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efficiency, the peaks are narrower (sharper peaks), and the detection limits (LODs) are
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lower. This fact means the sensitivity in UHPLC is higher than in comparison to the
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Table 1 shows the sample pre-treatment and chromatographic conditions for the
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shows the polyphenols that have been determined in different food samples by using the
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compared the use of HPLC with UHPLC coupled to MS, as the detector system, for
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determining phenolic compounds in food samples, such as beverages [29], virgin olive oil
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[26, 27], cocoa [43] or soy foods [33]. In these reports,the chromatographic separation in
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UHPLC was between three and seven times faster than with conventional HPLC. As an
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example, Ortega et al. [43] reduced the analysis time from 80 min in conventional HPLC to
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12.5 min in UHPLC for the determination of flavanols in cocoa samples. Figure 2 shows
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the extracted ion chromatograms (EICs) of the studied flavanols. As it can be seen, the
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efficiency of the peaks by UHPLC-MS/MS (Figure 2A) allowed the flavanols nonamers
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until the flavanols hexamers. Additionally, the quantification limits (LOQs) in UHPLC (10-
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facts, the coupling UHPLC technique by MS is less susceptible to matrix effects (ME). It
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has been reported that an efficient UHPLC separation may contribute to a reduction in ion
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suppression when this is only produced by the coelution of two different compounds [60,
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61]. Additionally, another advantage of UHPLC over conventional HPLC is that UHPLC
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consumes approximately 80% less organic solvent, and therefore it can also be
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5.Chromatographic conditions
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5.1. Analytical column
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As it has previously been reported, the UHPLC system is based on the principal use
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of the stationary phase, consisting of particles of less than 2 m, while HPLC columns are
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typically filled with particles from 3 to 5 m. These UHPLC columns have an internal
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diameter of 2.1 mm, a length of between 50 and 150 mm, and a particle size between 1.7
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For the analysis of phenolic compounds in food samples by this novel technique
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coupled to MS, different analytical columns have been used, such as Ethylene Bridged
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Hybrid (BEH) C18 [26, 27, 30-35, 40, 41, 44, 45, 47, 53, 62, 63], and (BEH) C8 [29, 64],
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High Strength Silica (HSS) T3 [36, 37, 43, 65], BEH Shield C18 [25, 38, 66], and BEH
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Amide [46] from Waters; Zorbax SB C18 [51, 54, 67-69], Zorbax Eclipse Plus C18 [39, 42]
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and HT C18 [70] from Agilent; and Hypersil Gold [28, 48, 52] from ThermoFisher. All these
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columns are based on reversed-phase mode, except for the BEH Amide, which uses
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food samples, the most widely-used analytical column is the BEH C18. The particles in this
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column are characterized by being hybrid with ethylene bridged and completely
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endcapped.
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compounds are eluted according to their polarity and molecule size. On the other hand,
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when a hydrophilic interaction chromatography (HILIC) is used, the more polar compounds
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are eluted after the non-polar compounds [46]. Guillarme et al. [25] compared four
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different endcapped, deactivated analytical columns from two different providers for the
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analysis of seven diastereoisomers favanols, and the obtained chromatograms are shown
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Hypersil Gold C18 (conventional C18 material) (Figure 3B), Acquity BEH phenyl (a hybrid
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BEH phenyl material) (Figure 3C), and Acquity BEH Shield C18 (a hybrid BEH C18 support
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with a polar embedded group) (Figure 3D). The different separations provided confirmation
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that endcapped, monomeric C18 columns are required for the separation of the studied
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flavanols. In addition, these authors also demonstrated that the columns containing a polar
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a powerful analytical technique for studying complex matrices, such as food samples. This
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LC, is based on transferring all the fractions over the first dimension to the second
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dimension. Typically, this system combines two analytical columns with different
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speed of the second dimension determines the overall analytical time. This speed should
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be as fast as possible, with good resolution. To achieve a fast speed, short columns
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packed with small particles or monolithic columns are used. Thus, a combination of HPLC
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with UHPLC offers a good configuration for a comprehensive 2D system and this
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technique has also been used to analyze phenolic compounds by UHPLC-MS. Scoparo et
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al. [62] and Kalili et al. [67, 68] used a comprehensive 2D-LC technique to determine
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phenolic compounds in tea [62, 67], cocoa and apple [68] samples. The analytical columns
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used, which combined HPLC x UHPLC, were size exclusion chromatography (SEC) and
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reversed phase (BEH C18) to separate compounds according to their size and polarity,
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respectively [62]; and HILIC and reversed phase (BEH C18) to separate the compounds
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based on their hydrophobicity and polarity [67, 68]. On the other hand, Cavaliere et al. [42]
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coupled two UHPLC columns (Zorbax Eclipse Plus C18) in series to obtain as high an
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grapes.
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Nowadays, the requirements in LC are shifting more and more to faster separations.
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The determination of food polyphenols and the effects of these compounds on human
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health require sensitive and robust methods for the quantification of these compounds in
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different food matrices. Rapid and high resolution analytical methods are also needed for
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length, but as a consequence the retention time and the band broadening are also
405
increased. A strategy to reduce the analysis time could be the use of other materials such
406
as monolithic columns, which operate at higher flow rates with lower backpressures than
407
conventional columns, but unfortunately, these columns did not increase the separation
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achieved by reducing the size of the stationary phase particles without increasing the
410
retention time or band broadening. Therefore, the use of UHPLC is opening up new
411
possibilities for improving the analytical methods for the determination of phenolic
412
compounds in complex food samples that usually require high resolution and long analysis
413
time. As examples, more than a hundred phenolic compounds with various structural
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classes were identified in tomato fruit [39] and in red mustard greens [28] in an hour.
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Figure 4 shows the UHPLC-DAD chromatogram (330 nm) for the determination of flavanol
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glycosides and hydroxycinnamic acid derivatives in red mustard greens extract [28]. On
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the other hand, few minutes such as 3 min were also reported for the determination of
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minor amount of phenolic compounds in food samples by UHPLC-MS [27, 32, 44].
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water and organic modifier solvent are used as phases A and B, respectively. The pH of
423
the phase A of the mobile phase is normally kept below three by the addition of a small
424
amount of formic acid [25-29, 31-38, 40-42, 46-48, 51-54, 62-64, 66-70], acetic acid [25,
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26, 30, 39, 43, 65], or trifluoroacetic acid (TFA) [44, 45]. Its concentration is kept as low as
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possible in order to ensure satisfactory ionization, and it is between 0.05 and 0.2%, or in
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accomplished most effectively by gradient elution, by using methanol [25, 27, 31, 32, 34,
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36, 54, 62, 64], acetonitrile [26, 28, 29, 33, 35, 37-41, 46-48, 51, 52, 59, 65-70] or
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phlorotannins, and the mobile phase was made up of ammonium acetate (phase A) and
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acetonitrile (phase B). Phlorotannins were resolved with HILIC mode due to the high
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polarity of these compounds, which eluted with little or no retention in the reverse-phase
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due to the lack of interaction with the non-polar stationary phase [46]. In this study, a
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between run times, peak shape and sensitivity. In HILIC, water is introduced as the
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stronger eluting solvent instead of the polar organic phase as in reversed mode.
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With the improved LC, UHPLC, the flow-rate used is lower than in conventional HPLC, and
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it is about 0.4 ml/min due to the low inner diameter (ID) and length of the column. As a
442
consequence of the use of the lower inner diameter (ID), the sample volume injection is
443
444
conventional HPLC.
445
an
us
cr
ip
t
433
446
UHPLC-MS combines the separation of LC with the selectivity and sensitivity of the
448
449
compounds in complex matrixes, such as food samples. In the last few years, different
450
food samples have been analyzed to determine their phenolic compounds by using the
451
improved UHPLC coupled to MS [ 25-48, 51-54, 62-70]. The food samples analyzed were
452
soy foods [33], cocoa [43, 44, 68], olive oil [26, 27], beverages [25, 29, 32, 34, 40, 41, 52,
453
62, 63, 66, 67, 70], vegetables [36, 38, 39, 46, 48, 51, 53, 64, 65], fruit [29, 42, 48, 68],
454
milk-based food products [41], spices [28, 45, 52], and herbs used in traditional Chinese
455
Ac
ce
pt
ed
447
456
The ionization technique used in these studies was electrospray (ESI), being an
457
excellent tool for identifying phenolic compounds. Collision-induced dissociation (CID) has
458
proven to be the most important tool for elucidating flavonoid structures. The CID of
459
deprotonated flavonoids has provided key information about the basic flavonoid skeletons.
460
For example, for the analysis of flavonoid glycosides, this ionization technique gives
461
information on the glycoside molecular masses due to their prominent [M-H]- ions and
462
463
Another example for the analysis of anthocyanidins is the MS spectra that reflect the
464
cleavage of the glycosidic bonds directly linked to the flavylium ring, thus giving also the
465
corresponding aglycone.
15
Page 15 of 42
466
For the analysis of flavanols in cocoa samples, singly charged molecular ions, [M-H]-,
467
are the most abundant ions for monomers until tetramers at m/z 289.1, 577.3, 865.5 and
468
115.7, respectively. For the oligomers pentamers and hexamers, the doubly charged ions
469
at m/z 720.4 and 864.5 are the predominant ions. In addition, doubly charged ions are the
470
most abundant ions for heptamers through nonamers as well as for dodecamers, while
471
triply charged species are the most intense ions for decamers and undecamers [43, 44,
472
68].
Normally, ESI ionization in the negative mode is the most widely used for analyzing
474
phenolic compounds since the pseusomolecular ion obtained is more sensitive than in the
475
positive mode [25-29, 31, 32, 34-36, 38, 40, 43, 46, 52, 53, 62, 65, 66, 68]. However, in
476
some cases, positive mode is also used [34, 45]. On the other hand, the anthocyanins [41,
477
51] and isoflavonoids [33, 64] are normally analyzed in the positive mode. The isoflavones,
478
which are methoxylated compounds, do not ionize at all due to the absence of a free
479
hydroxyl group in the negative ion mode. There is an exception. Recently, Sun et al. [48]
480
481
482
flavonol glycosides. These authors reported that the doubled ions of [M-2H]- and [M-
483
2H+H2O]- are unique to anthocyanins, while a single molecular ion [M-H]- dominates the
484
485
486
positive mode, and Figure 5C and 5D in negative mode. As it can be seen in these figures,
487
the negative ionization can be a useful strategy to differentiate these two phenolic
488
compounds. This strategy is more reliable than the traditional methodology that uses UV-
489
490
Ac
ce
pt
ed
an
us
cr
ip
t
473
491
Once the ions have been produced, they are separated according to their masses. To
492
this end, different analyzers have been reported for the analysis of phenolic compounds,
493
and the MS [39, 40, 46, 47, 54, 63] and tandem MS (MS/MS) [25-36, 38, 41-45, 51-53, 62,
494
64-70] modes have been used. The mode MS only uses a single analyzer, and time-of-
495
flight (TOF) [39, 40, 47, 54, 63] and orbitrap [46] have been used. Recently, Steevensz et
496
al. [46] used the analyzer orbitrap to determine phorotannins in brown algae. This analyzer
497
is very sensitive and it is able to reach up to 100,000 resolution. Unfortunately, there are
498
still few applications of this analyzer to analyze phenolic compounds in the food field.
499
When a single analyzer is used, TOF is the most commonly reported. TOF techniques can
500
record an accurate full- scan spectrum throughout the acquisition range and this technique
16
Page 16 of 42
is an excellent tool for the unequivocal target and non-target identification and confirmation
502
in food analysis. The availability of full-scan mass spectra throughout each chromatogram
503
and accurate-mass measurements has provided qualitative information that can be used
504
to determine unknown phenolic compounds found in food samples. This analyzer has
505
been employed to analyze tea [40], tomato extracts [39], herbs from TCM [54] and wine
506
[63]. Recently, Funari et al. [62] applied the UHPLC-TOF metabolite profiling method to
507
rapidly determine the chemical composition of the phenolic compounds of different crude
508
plant extracts. Therefore, UHPLC in combination with TOF, as the high resolution MS, is
509
510
cr
ip
t
501
512
compounds by UHPLC are based on triple quadrupole (QqQ) [25-28, 31-34, 36, 38, 41-43,
513
47, 62-66, 70], as the same two analyzers, and Q-TOF [30, 35, 37, 44, 45, 53, 67, 68], Q-
514
linear ion trap [36, 51] and linear trap-Orbitrap [48] as the hybrid systems.
an
us
511
Among tandem MS techniques, the most widely employed are triple quadrupole
516
(QqQ) instruments. Triple quadruple uses two scanning quadrupole modules, Q1 and Q3,
517
units on either side of a quadrupole used as a collision cell, Q2. This system can be run in
518
one of four modes for a variety of experiments, such as the daughter scan mode, parent
519
scan mode, neutral loss scan mode, and the most sensitive selective reaction monitoring
520
(SRM). The last one is used for quantification purposes and the others are used to
521
fragment the molecule and thus identify an analyte. Churchwell et al. [33] were the first to
522
use UHPLC coupled to MS for the analysis of phenolic compounds in food samples.
523
Ac
ce
pt
ed
515
524
From then, triple quadrupole has also been used for the determination of phenolic
525
acids [26-28, 34, 36, 52, 62, 66, 70], flavanols [32, 42, 43, 62, 70], flavonols [25, 34, 36-38,
526
41, 42, 62, 65, 70], flavones [27, 31, 34, 42, 64-66], anthocyanins [41, 42, 48], flavanone
527
[64], stilbenes [42], isoflavones [33, 64, 65], lignands [27] in different food matrices, such
528
as soy foods [33], fruit [29, 42, 48], olive oil [26, 27], wine [29, 70], tea [26, 29, 32, 62, 66],
529
vegetables [36, 38, 56, 64, 65], milk-products [41], cocoa [43] and infusions [31, 34, 52].
530
531
phenolic compounds, vanillic acid (Figure 6A), p-coumaric acid (Figure 6B), ferulic acid
532
(Figure 6C) and hydroxytyrosol (Figure 6D) for the analysis of different virgin olive oils. As
533
it can be seen in this figure, these phenolic compounds were determined in 3 min [27].
534
On the other hand, the hybrid instruments combine the advantages of both different
535
mass analyzers, offering great potential for the screening, the selectivity, the confirmation,
17
Page 17 of 42
536
and above all, the structural elucidation of unknown compounds in complex matrices, such
537
as food samples, due to the accurate mass of a characteristic fragment ion in studied
538
compounds.
In Q-TOF systems, the quadrupole (first analyzer) selects ions that are held in an ion
540
accumulator until they are released in a burst into the TOF flight tube (second analyzer).
541
The TOF analyzer can analyze much larger masses, and the accumulator allows
542
accumulation of a specific ion to increase sensitivity or allow much more precise mass
543
measurements for accurate mass determination. Cooper et al. [44] were the first to use
544
UHPLC coupled to the hybrid Q-TOF system to determine flavanols in cocoa samples.
545
Since then, seven more methodologies have been reported for analyzing different food
546
matrices, such as tea [67], species [45], cocoa [68 ], apples [68], vegetables [53] and
547
herbs from TCM [30, 35, 37]. The phenolic compounds to be analyzed were phenolic acids
548
[35, 45, 53, 67], flavanols [30, 53, 67, 68], flavonols [37, 45, 67, 68], flavones [35, 53, 57].
549
550
injected into the lock-spray probe automatically at a flow rate of around 10 l/min. This
551
standard compound was ionized into an [M-H]+ ion weighing 556.2771 Da in the positive
552
mode or an [M-H]- of 554.2615 in the negative mode, and this was used as the lock mass
553
for real-time recalibration of the mass axis and to ensure accurate mass measurement [30,
554
35, 40]. Other calibration solutions reported were 0.1% phosphoric acid [44] and NaF
555
ed
an
us
cr
ip
t
539
One of the newest combinations is the quadrupole linear ion trap, which combines a
557
quadrupole module with a linear ion trap [38, 39, 51]. The ion trap analyzers are more
558
sensitive than quadrupole ones. The linear IT combines the separating capability of the
559
quadrupole analyzer with the tandem capability of an ion trap. Trapping electrode tings are
560
added to each end of the quadrupole rods to create the linear trap. The analyzer can be
561
run in a normal scanning quadrupole mode for separation and detection of mass ions, or
562
the end electrodes can be turned on to retain a specific ion in the trap for collision with the
563
Ac
ce
pt
556
564
565
in red cabbage. By using this analyzer, the specific determination of the fragmentation
566
patterns of these phenolic compounds was obtained. Over the last two years,
567
anthocyanins and flavonols have been determined in tomato extracts [39] by using this
568
sensitive hybrid system. Recently, the hybrid system linear trap and orbitrap has been
569
used for the analysis of fruit [48] and vegetables [28, 48] for the analysis of anthocyanins
18
Page 18 of 42
570
[28, 48] and flavanols [28]. As mentioned above, this analyzer has a high potential due to
571
its sensitivity.
572
Modern high resolution MS (HR-MS), such as TOF, Q-TOF and Orbitrap, has
573
emerged as an ideal tool for profiling complex samples, such as food samples, due to its
574
576
MS/MS, such as ion mode, capillary voltage, cone voltage and collision energy were
577
optimized. Nitrogen was used as the desolvation gas [70], and both nitrogen and argon
578
were employed as the collision gas [27, 43, 65]. The fragmentation of phenolic acids
579
yielded product ions more efficiently at lower collision energies, while flavonoids, and
580
581
us
cr
ip
t
575
582
6.1.
an
583
samples
584
585
In the different analytical methods reported in the literature for the analysis of phenolic
587
588
quantifying these compounds in food samples (see Table 1). Generally, the phenolic
589
compounds were identified in these samples by comparing their retention time and DAD,
590
MS and tandem MS fragmentation spectra with those obtained from pure standard
591
592
593
data.
pt
ed
586
Ac
ce
594
On the other hand, apart from identifying phenolic compounds, some authors also
595
have quantified them and different strategies have been reported to its quantification and
596
to reduce the matrix effect (ME). In MS it is very important to quantify each analyte with
597
the respective calibration curve because the ionization can vary depending on the
598
molecule structure. But when commercial phenolic standards were not available, the
599
phenolic compounds were tentatively quantified with respect to other similar phenolic
600
601
the use of the semipreparative LC [31, 35, 37, 54, 64] applied to the isolation of individual
602
phenols from phenolic extracts. Suarez et al. [ 26] isolated the secoiridoid derivates (the
603
most abundant phenolic compounds in olive oil), and the lignan acetoxypinoresinol from
604
Page 19 of 42
606
analytes response induced by the co-eluting matrix and it can heavily affect the
607
reproducibility, linearity, and accuracy. In order to minimize inaccuracies and errors in the
608
quantification of phenols, the matrix effect (ME) has been tested and evaluated in
609
numerous studies [26, 27, 31, 34, 38, 41-43, 63, 65] during the validation of the analytical
610
methods. To evaluate the ME, the post-extraction addition was the main strategy for the
611
612
613
ip
t
605
On the other hand, several operational strategies have been suggested to minimize or
615
reduce the interferences of co-eluting matrix compounds. Due to the more efficient
616
chromatographic separations (high resolution) and high peak efficiency (narrow peaks)
617
obtained in UHPLC, the analyte co-elution is nearly restricted and thus the ionization
618
619
separation was reported to be less susceptible to matrix effects, when this was only
620
produced for the co-elution of two different compounds, and might contribute to a
621
an
us
cr
614
623
624
compensate for signal suppression and to improve qualitative and quantitative data for the
625
analysis of phenolic compounds in food matrices due to its increased resolution [74-76].
626
This fact is due to that sufficient chromatographic resolution is attained, minimum co-
627
elution of compounds with close m/z ratios and similar fragmentation pathways are
628
Ac
ce
pt
ed
622
629
The use of an extensive clean-up procedure prior to UHPLC-MS analysis, such as off-
630
line LLE and off-line SPE is other reported strategy to reduce the presence of interfering
631
632
extensive sample pre-treatment in combination with UHPLC, the %ME was reported to be
633
low (lower than 20%) for the determination of phenolic compounds in food samples. Some
634
authors diluted the sample 1:2 after the sample pre-treatment to decrease even more
635
these results, but this dilution produced a decrease of sensitivity and the detection limits
636
637
The use of an internal standard (IS) has also been employed to compensate the
638
signal alteration and it was the most widely calibration approach used. In the literature,
639
different reports have used an IS to reduce inaccuracies in the quantification [38, 39, 63,
20
Page 20 of 42
640
64]. On the other hand, the use of stable isotope-labeled IS has also been a suggested
641
practice to compensate the ME in food analysis, and these IS have been reported to be
642
the best choice by the fact of having similar ionization properties of the analyte. In the
643
644
[29, 69] have been used to reduce signal variability and improve precision. However, these
645
compounds are not always available and most of them are very expensive.
Another calibration approach to reduce the ME reported for the determination of
647
phenolic compounds by UHPLC-MS in food samples was external calibration [26, 27, 31,
648
32, 34-36, 42-44, 52, 54, 65, 66]. When this calibration was used, the calibration curves
649
650
samples (matrix-matched standard) [26, 27, 35, 36, 44], or also by adding different
651
amounts of standard phenolic compounds in organic solvent [10, 31, 32, 34, 42, 43, 52,
652
54, 65, 66]. Cavaliere et al. [42] quantified phenolic compounds in grape berries by
653
external calibration and the concentrations were corrected for the estimated ME. They
654
evaluated the ME as the plot between the dilution factors of the sample respect to the
655
an
us
cr
ip
t
646
657
a difficult task. The isobaric compounds have the same nominal mass but with different
658
molecular formula. When a low resolution MS, such as quadrupole, is used for the
659
660
select correctly the precursor ion. If these compounds are not well resolved in the MS1, a
661
mixed product ion spectrum will be obtained and this will lead to wrong conclusions or
662
wrong quantifications. The use of UHPLC could help to resolve isobaric compounds due to
663
its high resolution in comparison to the traditional HPLC. On the other hand, the use of
664
modern high resolution MS techniques (HR-MS), such as TOF, Q-TOF, ion trap or
665
orbitrap, where the exact mass instead of nominal mass is obtained, the selectivity
666
Ac
ce
pt
ed
656
667
As it has been previously commented, the UHPLC systems provide higher sensitivity
668
than HPLC systems since sharper peaks (higher efficiency) are obtained. Apart of this
669
higher sensitivity, when the MS as the detector system is coupled to this chromatographic
670
technique, the sensitivity is increased even more. It was reported that the determination of
671
672
magnitude higher than those reported using HPLC-UV detection [25, 26, 32, 33, 42, 65].
673
674
compounds and other compounds from food matrix) and sensitivity to determine different
21
Page 21 of 42
675
phenolic compounds in different food samples, such as virgin olive oil [26], carob flour [65],
676
tea extracts [25, 34] and cocoa samples, at low g/l concentration levels.
677
678
679
It is well known that phenolic compounds are an important food source of natural
681
antioxidants, and different positive health effects have been reported for them. However,
682
today they are not detailed intake values for all polyphenols, because there are no
683
684
685
food composition databases as the main requirement for establishing daily intake of
686
polyphenols.
ip
t
680
us
cr
Based on these requirements, the UHPLC-MS offers a very suitable and good
688
689
690
formulated foods. This fact is due to the improvements made in the preparation of sub-2
691
692
in less time. Faster separations are achieved while the resolution is maintained or
693
increased. The use of these columns packed with sub-2 m particles required the use of
694
UHPLC systems because generates pressures that exceed the limits of standard HPLC
695
instruments. Nevertheless, in the last few years, the use of the core-shell columns (totally
696
porous) allows achieving high efficiency separations in a reduced analysis time by using
697
the conventional HPLC system. Therefore, this technology seems to be very promising
698
Ac
ce
pt
ed
an
687
699
Additionally, the use of UHPLC columns, allows achieving higher efficiency of the
700
peaks. This fact is less susceptible to matrix effects when this is only produced for the
701
702
703
conditions directly.
704
705
706
certainly for the rapid determination of analytes at low concentration levels in complex food
707
matrices. This coupling (UHPLC- MS) is suitable for identifying phenolic compounds,
708
when standard compounds are available, but it has limited utility for identifying novel
709
compounds, since structural elucidations require MS/MS analysis for identification and
22
Page 22 of 42
710
confirmation. For this purpose, high mass-accuracy TOF and high sensitivity QqQ together
711
with the improved LC, are useful tools for determining phenolic compounds in food
712
samples.
The continuous improvements in the UHPLC system open new possibilities in the
713
analysis
of
food
polyphenols.
Despite
the
important
advances
in
fast
liquid
715
chromatography, food matrices are very complex, and although multi-residue methods
716
with minimal sample manipulation are demanded, sample extraction and clean-up
717
treatments must be carefully developed to reduce total analysis time. The use of the SPE
718
as the sample pre-treatment technique by using plates as the device format instead of
719
cartridges is a fast technique that allows a lower sample volume load. This speed to
720
extract the target phenolic compounds and clean-up the sample matrix is achieved
721
us
cr
ip
t
714
722
724
(UPC2), which uses columns packed with sub-2 m particle and CO2 as the mobile phase,
725
726
samples.This new technique, so far, not been applied for the determination of polyphenols
727
729
Acknowledgement
730
pt
728
ed
an
723
This work was supported by the Spanish Ministry of Education and Science financing
731
the
733
734
A. Serra grant.
735
736
737
project
AGL2009-13517-C13-02
and
also
by
the
Catalan
Government
Ac
ce
732
8. References
738
[1] J.A.M. Kyle, G.G. Duthie, in: O. Andersen, K. Markham (Eds.), Flavonoids in Foods in
739
740
2006, p. 219.
741
742
[3] J. Valls, S. Milln, M.P. Mart, E. Borrs, Ll. Arola, J Chromatogr. A 1216 (2009) 7143.
743
744
Page 23 of 42
745
746
747
748
[7] D. Guillarme, J. Schappler, S. Rudaz, J.L. Veuthey, Trends Anal. Chem. 29 (2010)
749
750
751
15.
[8] J.B. Harbone, in: P.M. Dey, J.B. Harborne (Eds.), Methods in Plant Biochemistry (Vol.
I, Plant Phenolics), Academic Press, 1989.
[9] M. Gonzlez-Castejn, A. Rodrguez-Casado. Pharmacol. Res. 64 (2011) 438.
753
[10] A. Crozier, I.B. Jaganath, M.N. Clifford, Nat. Prod. Rep. 26 (2009) 1001.
754
755
758
759
760
cr
[13] USDA
Database
for
the
flavonoid
us
757
125.
content
of
selected
foods.
http://www.nal.usda.gov/fnic/foodcomp/Data/Flav/flav.html, 2012.
an
756
ip
t
752
762
763
764
ed
761
766
767
metabolites: Occurrence, structure and role in the human diet. Blackwell Publishing
768
Ac
ce
pt
765
769
770
771
772
773
[22] K.V. Peter. Handbook of Herbs and Spices Vol. 1. CRC Press. Boca Raton, 2001.
774
775
776
777
778
6882.
[26] M. Surez, A. Maci, M.P. Romero, M.J. Motilva, J. Chromatogr. A 1214 (2008) 90.
24
Page 24 of 42
779
780
[27] M.I. Alarcn Flores, R. Romero-Gonzlez, A. Garrido Frenich, J.L. Martnez Vidal,
Food Chem. 134 (2012) 2465.
[28] L. Lin, J. Sun, P. Chen, J. Harnly, J. Agric. Food Chem. 59 (2011) 12059.
782
783
784
[31] X. Deng, G. Gao, S. Zheng, F. Li, J. Pharm. Biomed. Anal. 48 (2008) 562.
785
786
[33] M. Churchwell, C.N. Twaddle, L.R. Meeker, D.R. Doerge, J. Chromatogr. B, 825
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
cr
us
792
an
791
[35] Y. Zhou, F.F.K. Choi, Z.Z. He, J.Z. Song, C.F. Qiao, X. Liu, L.S. Ding, S.L. Gesang,
[37] H. Zhang, H. Yang, M. Zhang, Y. Wang, J. Wang, L. Yau, Z. Jiang, P. Hu, J. Food
Comp. Anal. 25 (2012) 142.
790
1271.
ed
789
pt
788
(2005) 134.
Ac
ce
787
ip
t
781
810
[45] N. Kumar, P. Bhandari, B. Singh, S.S. Bari, Food Chem. Toxicol. 47 (2009) 361.
811
[46] A.J. Steevensz, S.L. Mackinnon, R. Hankinson, C. Craft, S. Connan, D.B. Stengel,
812
25
Page 25 of 42
813
814
[47] C.S. Funari, P.J. Eugster, S. Martel, P.A. Carrupt, J.L. Wolfender, D.H.S. Silva, J.
Chromatogr. A, 1259 (2012) 167-178.
815
[48] J. Sun, L. Lin, P. Chen, Rapid Commun. Mass Spectrom. 26 (2012) 1123.
816
[49] C.R. Mallet, Z. Lu, R. Fisk, J.R. Mazzeo, U.D. Neue, Rapid Commun. Mass
817
818
819
821
822
[53] Z. Lou, H. Wang, S. Zhu, M. Zhang, Y. Gao, C. Ma, Z. Wang, J. Chromatogr. A 1217
825
826
cr
[54] G. Du, H.Y. Zhao, Q.W. Zhang, G.H. Li, F.Q. Yang, Y. Wang, Y.C. Li, Y.T. Wang, J.
us
824
(2010) 2441.
Chromatogr. A 1217 (2010) 705.
[55] J. Shi, L.S. Kassama, Y. Kakuda in: J Shi (Ed.), Supercritical Fluid Technology for
827
Extraction
828
830
Bioactive
Components
in
Functional
Food
Ingredients
and
[56] E. Barcelo-Barrachina, E. Moyano, M.T. Galcern, J.L. Lliberia, B. Bag, M.A. Cortes,
J. Chromatogr. A 1125 (2006) 195.
829
of
an
823
ip
t
820
832
[58] C.C, Leandro, P. Hancock, R.J. Fussell, B.J. Keely, J. Chromatogr. A 1103 (2006) 94.
833
[59] X. Li, Z. Xiong, X. Ying, L. Cui, W. Zhu, F. Li, Anal. Chim. Acta 580 (2006) 170.
834
835
837
838
839
840
841
842
843
844
845
846
pt
Ac
ce
836
ed
831
26
Page 26 of 42
847
A.J. Steevensz, S.L. Mackinnon, R. Hankinson, C. Craft, S. Connan, D.B. Stengel, J.E.
848
849
850
851
852
[69] G.D. Zheng, K. Li, Y.S. Li, E.H. Liu, J. Sep. Sci. 35 (2012) 174.
854
855
ip
t
853
857
858
859
862
863
us
an
861
(2010) 1067.
[76] H. Trufelli, P. Palma, G. Famiglini, A. Cappiello, Mass Spectrom. Rev. 30 (2011) 491.
860
cr
856
Ac
ce
pt
ed
864
27
Page 27 of 42
Figure Captions
864
865
866
867
868
Figure 2. Extracted ion chromatograms of flavanols for the analysis of cocoa sample by
870
ip
t
869
871
Figure 3. UHPLC chromatograms obtained for the analysis of eight flavanols with various
873
RP columns packed with sub-2 m particles. (A) Acquity BEH C18 (50 mm x 2.1 mm ID,
874
1.7 m), (B) Hypersil GOLD (50 mm x 2.1 mm ID, 1.9 m), (C) Acquity BEH phenyl (50
875
mm x 2.1 mm ID, 1.7 m), and (D) Acquity BEH Shield RP18 (50 mm x 2.1 mm ID, 1.7
876
m). Peak assignation: (1) catechin, (2) epicatechin, (3) gallic acid, (4) catechin gallate, (5)
877
epicatechin gallate, (6) epigallocatechin, (7) gallocatechin gallate, and (8) epigallocatechin
878
gallate [25].
an
us
cr
872
879
881
glycosides and hydroxycinnamic acid derivates of red mustard greens extract [28].
880
ed
882
884
(A, C) and the flavanol quercetin-3-O-glucoside (B, D) in positive and in negative mode
885
respectively [48].
Ac
ce
886
pt
883
887
Figure 6. Extracted ion chromatograms (EICs) of the phenolic compounds (A) vanillic
888
acid, (B) p-coumaric, (C) ferulic acid and (D) hydroxytyrosol for the analysis of different
889
890
28
Page 28 of 42
890
Table 2. Food samples and phenolic compounds which have been analyzed by UHPLC-
891
MS.
Pomace oil
Sunflower oil
Refined oil
Non-Alcoholic
beverages
Fruit juices
Tea
Ac
ce
Chamomile
Red cabbage
Vegetables
Carob flour
Tomato
Cauliflower
Lettuce
[27]
[27]
[27]
[27]
Phenolic acids
Flavanols
Flavonols
Phenolic acids
Flavanols
Phenolic acids
Flavonols
Flavones
Phenolic acids
Flavones
Flavonols
[ 29]
[ 25, 32, 40, 62, 67]
[ 29, 62, 66, 67 ]
[ 62, 67]
[ 66]
Anthocyanidins
Phenolic acids
Flavones
Flavonols
Isoflavones
Phenolic acids
Phenolic alcohols
Flavanone
Flavonol
Flavone
Flavonols
Phenolic acids
Flavonols
[ 46]
ed
Wine
pt
Alcoholic
beverages
Soybean oil
ip
t
Reference
[ 33]
[ 43, 44, 68]
[ 68]
[68]
[ 26, 27]
[26, 27]
[26]
[26, 27]
[26]
cr
Oils
Phenolic compounds
Isoflavones
Flavanols
Phenolic acids
Flavonols
Phenolic acids
Flavones
Lignans
Alcohol acids
Secoiridoid derivates
Phenolic acids
Phenyl alcohols
Flavones
Phenolic acids
Phenyl alcohols
Flavones
Phenolic acids
Phenyl alcohols
Flavones
Phenolic acids
Phenyl alcohols
Flavones
us
Specific food
Soy supplement
Cocoa derivates and
chocolate
an
Food group
Soy
Cocoa
[ 34]
[55, 65]
[ 39]
[ 38]
[ 36]
29
Page 29 of 42
Algae
Burdock leaves
Hongcaitai or Zicaitai
Red radish
Grape berries
Fruits
Apple
Yogurt
an
Ice-cream
Mustard
Species and
herbs
892
893
pt
Traditional Chinese
Medicine (TCM)
Ac
ce
Herbs
ed
Rose
Rosemary
[46]
[53]
[48]
[48]
us
Blueberry
Milk-based food
products
[64]
[42]
[29]
cr
Grape
Isoflavones
Flavanones
Flavones
Phlorotannins
Phenolic acids
Flavones
Flavonols
Anthocyanins
Anthocyanins
Flavonols
Flavanols
Anthocyanidins
Stilbenes
Phenolic acids
Phenolic acids
Flavonols
Anthocyanins
Anthocyanins
Flavonols
Anthocyanins
Flavonols
Anthocyanidins
Flavonols
Hydroxycinnamic acid
derivates
Flavonols
Phenolic acids
Phenolic acids
Isoflavones
Flavanols
Phenolic acids
Flavanone
Flavones
Flavonol
ip
t
Mung bean
[68]
[48]
[41]
[41]
[28]
[45]
[52]
[59]
[30, 44]
[35, 69]
[69]
[31, 35, 69]
[37]
30
Page 30 of 42
ip
t
cr
us
Table 1. Sample pre-treatment and chromatographic conditions for the determination of phenolic compounds in food
samples by using UHPLC-MS methodologies.
Sample pretreatment stratregy
LLE (Methanol)
Cocoa
Chocolate
Flavanols
LLE
(acetone/water/acetic
acid, 70/28/2, v/v/v)
Phenolic acids
Flavones
Lignans
Secoiridoide
derivates
LLE
(Methanol/water,
80/20, v/v)
Japanese
green tea
Flavanols
Red cabbage
Anthocyanins
MS method
(analyzer)
QqQ
Identification/
quantification
Identification
[ 33]
A) (water/THF/TFA,
98/2/0.1, v/v/v)
B) Acetonitrile + 0.1%
formic acid
Q-TOF
Quantification
[ 44]
BEH C18
(100 x 2.1 mm, 1.7
m)
QqQ
Quantification
[ 26]
LLE
(methanol / water /
CHCl3, 2.5/1/1, v/v/v)
+
Methanol / 0.1%
formic acid (4/1, v/v)
BEH C18
(150 x 2.1 mm, 1.7
m)
TOF
Identification
[ 40]
ASE
(water / ethanol /
formic acid, 84/5/1,
v/v/v)
Zorbax SB C18
(100 x 2.1 mm, 1.8
m)
A) 5 % formic acid
B) Acetonitrile
Q-Ion trap
Identification
[ 51]
Mobile phase
BEH C18
(50 x 1 mm, 1.7 m)
Isocratic:
Acetonitrile / 0.1%
formic acid (40/60, v/v)
BEH C18
(50 x 2.1 mm, 1.7 m)
ep
te
Soy foods
Stationary phase
an
Phenolic
compounds
Isoflavones
Ac
c
Food sample
Ref.
Page 31 of 42
ip
t
LLE
(methanol / water /
formic acid, 70/30/1,
v/v/v)
White wine
Grapefruit juice
Green tea
infusion
Phenolic acids
LLE (Water)
Traditional
Chinese
Medicine
Flavone
Traditional
Chinese
Medicine
Flavanols
LLE
(water)
Carob flour
Flavone
Flavonol
Isoflavone
Rose species
Flavonol
Phenolic acids
Milk-based
food products
Flavonols
Anthocyanins
cr
Flavonols
Flavanols
Anthocyanins
Stilbenes
QqQ
Identification
Quantification
[ 42]
QqQ
Quantification
[ 29]
BEH C18
(100 x 2.1 mm, 1.7
m)
QqQ
Quantification
[ 31]
BEH C18
(50 x 2.1 mm, 1.7 m)
Q-TOF
Identification
[ 30]
LLE
(acetone/water,
80/20, v/v)
HSS T3
(100 x 2.1 mm, 1.8
m)
QqQ
Quantification
[ 65]
LLE
(acetonitrile/water,
80/20, v/v)
BEH C18
(100 x 2.1 mm, 1.7
m)
A) 0.05 % TFA
B) Acetonitrile
Q-TOF
Identification
[45]
LLE
(Methanol/formic acid
(9/1, v/v)
BEH C18
(100 x 2.1 mm, 1.7
m)
A) water/formic acid
(9/1, v/v)
B) acetonitrile
QqQ
Quantification
[ 41]
an
us
BEH C8
(150 x 2.1 mm, 1.7
m)
ep
te
Ac
c
Grape berries
Page 32 of 42
ip
t
LLE
(acetone/water,
70/30, v/v)
Off-line
Comprehensive 2D
cr
Flavanols
Phenolic acids
Flavonols
Q-TOF
Identification
[ 68]
QqQ
Quantification
[ 43]
QqQ
Quantification
[ 32]
A) 0.05-0.01 % acetic
acid
B) Methanol
QqQ
Identification
[ 25]
LLE
(Methanol)
BEH C18
(100 x 2.1 mm, 1.7
m)
QqQ
Quantification
[ 34]
LLE
(Methanol)
TOF
Ion Trap
Quantification
[ 39]
HT C18
(50 x 2.1 mm, 1.8 m)
A) Water / acetonitrile /
formic acid
(99/1/0.1, v/v/v)
B) Water / acetonitrile /
formic acid
(1/99/0.1, v/v/v)
QqQ
Quantification
[ 70]
us
Cocoa
Apple
Flavanols
LLE
(acetone/water/acetic
acid, 70/29.5/0.5,
v/v/v)
Tea
Flavanols
LLE
(Hot water)
Tea
Flavanols
LLE
(Boiling water)
Chamomile
extract
Flavone
Phenolic acids
Flavonols
Tomato
extracts
Phenolic acids
Flavanone
Flavonol
Flavone
Red wine
Phenolic acids
Flavanols
Flavonols
HSS T3
(100 x 2.1 mm, 1.8
m)
Cocoa
an
Zorbax SB C18
(50 x 4.6 mm, 1.8 m)
Ac
c
ep
te
BEH C18
(100 x 2.1 mm, 1.7
m)
Page 33 of 42
ip
t
Off-line
Comprehensive 2D
Traditional
Chinese
Medicine
Isoflavone
Microware-assisted
extraction (MAE)
(65% Ethanol)
Green and
white
cauliflower
Flavonol
LLE
(Methanol)
Rosemary
Phenolic acids
ASE
(ethanol/water)
Burdock leaves
Phenolic acids
Flavonol
Flavone
Ultrasonic and
microware assisted
technologies (MAE)
Red wine
Flavanols
Phenolic acids
LLE
(dichlorometane)
Traditional
Chinese
Medicine
Phenolic acids
Flavone
LLE
(Methanol)
Sage tea
Hydrolysable
tannins
cr
LLE
(acetone/water,
70/30, v/v)
Q-TOF
Identification
[ 67]
Zorbax SB C18
(50 x 4.6 mm, 1.8 m)
TOF
Quantification
[ 54]
QqQ
Quantification
[ 38]
Hypersil Gold
(50 x 2.1 mm, 1.9 m)
A) % formic acid
B) Acetonitrile + 0.1%
formic acid
QqQ
Quantification
[ 52]
BEH C18
(150 x 2.1 mm, 1.7
m)
Q-TOF
Identification
[ 53]
BEH C18
(100 x 2.1 mm, 1.7
m)
BEH C18
(100 x 2.1 mm, 1.7
m)
A) 5 % formic acid
B) Acetonitrile
TOF
Quantification
[ 63]
Q-TOF
Quantification
[ 35]
QqQ
Quantification
[ 66]
us
Flavanols
Phenolic acids
Flavonols
Flavones
ep
te
an
Zorbax SB C18
(50 x 4.6 mm, 1.8 m)
Ac
c
Green Tea
Page 34 of 42
ip
t
Anthocyanins
Flavonol
LLE (Methanol/water,
(60/40, v/v)
+
Off-line SPE (HLB)
Lettuce
Phenolic acids
LLE
(Methanol)
HSS T3
(100 x 2.1 mm, 1.8
m)
Mung bean
sprouts
Flavanones
Flavones
Green tea
Black tea
Phenolic acids
Flavanol
Flavone
Flavonol
Linear Orbitrap
Identification
[ 28]
A) Water / methanol /
formic acid
(94.9/5/0.1, v/v/v)
B) Water / methanol /
formic acid
(39.9/60/0.1, v/v/v)
QqQ
Quantification
[ 36]
BEH C8
(150 x 2.1 mm, 1.7
m)
A) 10 mM formic acid
B) Methanol
QqQ
Quantification
[ 64]
Off-line
Comprehensive 2D
QqQ
Identification
[ 62]
BEH Amide
(100 x 2.1 mm, 1.7
m)
A) 10 mM ammonium
acetate pH 9
B) Acetonitrile
Orbitrap
Identification
[ 46]
an
us
LLE
(Ethanol/water,
50/50, v/v)
ep
te
LLE
(Ethanol/water,
70/30, v/v)
cr
Red Mustard
greens
BEH C18
(50 x 2.1 mm, 1.7 m)
Brown
macroalgae
Phlorotannins
Traditional
Chinese
Medicine
Phenolic acids
Flavanone
Flavone
LLE
(methanol)
Zorbax SB C18
(50 x 4.6 mm, 1.8 m)
QqQ
Identification
[ 69]
Traditional
Chinese
Medicine
Flavonol
LLE
(methanol)
HSS T3
(100 x 2.1 mm, 1.8
m)
Q-TOF
Identification
[ 37]
Ac
c
LLE
(dichloromethane)
+
Off-line SPE (C18)
Page 35 of 42
ip
t
LLE
(methanol/water/formic
acid, 60/40/1, v/v/v)
+
Off-line SPE (HLB)
Lippia Species
Flavanone
LLE
(ethanol)
+
Off-line SPE (C18)
BEH C18
(150 x 2.1 mm, 1.7
m)
Olive oil
Pomace oil
Sunflower oil
Refined oil
Soybean oil
Phenolic acids
Phenyl alcohols
Flavones
cr
Anthocyanins
Linear Ion
Trap-Orbitrap
Identification
[ 48]
TOF
Identification
[47]
QqQ
Quantification
[27]
an
us
Blueberry
Hongcaitai
Red radish
Ac
c
ep
te
BEH C18
(100 x 2.1 mm, 1.7
m)
Page 36 of 42
Figure 1
Classification
Examples
Molecular structure
O
HO
Caffeic acid
OH
HO
Simple phenolic
acids
O
OH
Ferulic acid
HO
HO
Gallic acid
OH
HO
Phenyl alcohols
Hydroxytyrosol
Stilbenes
Resveratrol
ip
t
HO
OH
HOO
HO
OH
Grapes, wine
OH
HO
HO
Chalcones
Phloridzin
OH
OH
Fruits, herbs
cr
NON-FLAVONOIDS
H3C-O
HO
OH
HO
us
HO
Matairesinol
CH3
OH
Lignans
H3C
Flaxseed
OH
OH
Secoisolariciresinol
H3C-O
HO
HO
OH
Flaxseed
an
H3C-O
OH
HO
Kaempferol
Spices
OH
OH
OH
Flavonols
HO
Quercetin
HO
OH
HO
OH
Cyanidin
Anthocyanidins
Blackberries, strawberry
OH
+
O
HO
O-CH3
OH
OH
Malvidin
+
O
HO
HO
O-CH3
OH
ed
HO
Luteolin
Flavones
OH
O-CH3
pt
Flavanone
OH
OH
HO
Apigenin
Naringenin
ce
Ac
O-CH3
OH
HO
OH
HO
OH
O
HO
HO
OH
HO
Herbs (thyme)
OH
O
HO
Taxifolin
OH
Herbs (oregano)
OH
HO
HO
Genistein
Isoflavones
OH
HO
Daidzein
OH
OH
Catechin
OH
O
HO
OH
Flavanols
OH
OH
Epigallocatechin
HO
HO
OH
Tea
OH
HO
OH
Condensed tannins
HO
HO
Proanthocyanidins
OH
OH
HO
HO
O
OH
HO
OH
OH
HO
OH
OH
HO
OH
HO
Complex tannins
Acutissimin A
OH
Red wine
O
O
HO
O
HO
O
O
OH
OH
O
O
OH
OH
HO
HO
OH
OH
OH
HO
OH
O
Hydrolisable
tannins
Gallotannins
OH
HO
HO
O
O
HO
HO
HO
O
O
HO
O
O
OO
OH
OH
OH
O
HO
Ellagitannins
OH
HO
HO
O
HO
O
OH
HO
O
O
O
HO
OH
OH
O
O
HO
HO
HO
HOOH
OH
OH
HO
OH
HO
O
HO
OH
HO
HO
HO
OH
O
HO
OH
OH
OH
OH
HO
OH
OH
OH
HO
HO
O
O
OO
HO OH
HO
OH
Figure 1
FLAVONOIDS
+
O
HO
Eriodictyol
Flavanonols
OH
+
O
HO
Page 37 of 42
cr
Figure 2
A) UHPLC-MS/MS
20
10
20
10
20
20
ce
pt
10
10
20
100
10
Ac
Abundance (%)
Tetramer
ed
10
10
20
20
30
40
30
40
30
40
30
40
30
40
Tetramer
1
10
10
20
100
Pentamer
4
10
20
100
Hexamer
5
10
20
100
Heptamer
Heptamer
40
100
Hexamer
30
Pentamer
100
20
Trimer
100
40
100
Trimer
100
30
100
20
Dimer
Dimer
10
100
100
0
us
10
M
an
100
Abundance (%)
100
B) HPLC-MS/MS
20
41
10
20
100
Octamer
10
20
Figure 2
100
Nonamer
0
10
20
Page 38 of 42
Ac
ce
pt
ed
M
an
us
cr
Figure 3
Figure 3
Page 39 of 42
Ac
ce
pt
ed
M
an
us
cr
Figure 4
Figure 4.
Page 40 of 42
Ac
ce
pt
ed
an
us
cr
ip
t
Figure 5
Figure 5
Page 41 of 42
Figure 6
cr
ip
t
an
us
ce
pt
ed
Ac
Figure 6
Page 42 of 42