Accepted Manuscript

Title: Analysis of food polyphenols by ultra high-performance

liquid chromatography coupled to mass spectrometry: An
overview
Authors: Maria-Jose Motilva, Aida Serra, Alba Maci`a
PII:
DOI:
Reference:

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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o Application of UHPLC-MSto determine phenolics in plant origin foods and feeds

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Analysis of food polyphenols by ultra high-performance liquid chromatography

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coupled to mass spectrometry: An overview

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Maria-Jos Motilva*, Aida Serra, Alba Maci

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Food Technology Department, XaRTA-TPV, Escola Tcnica Superior dEnginyeria

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Agrria, Universitat de Lleida, Av/Alcalde Rovira Roure 191, 25198 Lleida, Spain

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*Corresponding Author. Phone: +34 973 702817; Fax: + 34 973 702596;

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E-mail address: motilva@tecal.udl.es (Dr. Maria-Jos Motilva)

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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

33

chromatographic columns packed with sub-2 m particles and the modern high resolution

34

mass spectrometry (MS) have opened up new possibilities for improving the analytical

35

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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improved liquid chromatography (LC), ultra-high performance LC (UHPLC), coupled to MS

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or tandem MS (MS/MS) as the detector system for the determination of phenolic

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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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briefly outline future trends of UHPLC-MS methods is commented.

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Keywords: Food samples, MS, phenolic compounds, UHPLC

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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

54

antioxidants and have nutritional properties. Polyphenols are widely found in fruit,

55

vegetables, nuts, seeds, beverages, and also in manufactured foods as a component of

56

the natural ingredients used. Cocoa, apples, tea, berries, coffee, wine, jam, chocolate and

57

onions are common sources of polyphenols in the human diet [1]. These samples are

58

complex since they contain different classes of phenolic compounds, which differ in

59

polarity and size, from simple phenolic compounds (phenolic acids) to oligomers

60

(procyanidins and condensed tannins) and additionally, these compounds in these

61

samples are found at low concentration levels. The diverse chemical nature of phenols

62

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

64

and pharmaceutical preparations. The development of advanced separation techniques,

65

such as capillary electrophoresis (CE), high-performance liquid-chromatography (HPLC),

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and, latterly HPLC tandem mass spectrometry (MS/MS) techniques have enabled

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researchers to elucidate complex phenolic structures through the easy determination of

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accurate molecular weights and limited fragmentation patterns.

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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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to the chromatographic technique to determine different groups and subclass of

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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

77

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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reduced solvent consumption compared with conventional HPLC.

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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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The combination offers the possibility of taking advantage of chromatography as a

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separation technique and MS as an identification tool, since it provides a large amount of

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information about a complex mixture, enabling the screening, confirmation and

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quantification of hundred of components with one analysis [7].

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So, the coupling of

UHPLC with MS offers a potent alternative to conventional HPLC-MS, and provides a

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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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coupled to MS. Additionally, we focus on the determination of these compounds in food

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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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determining phenolic compounds by UHPLC-MS are briefly commented on.

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2. Phenolic compounds: their chemistry and occurrence in food

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Phenolic compounds, or also known as polyphenols, constitute an essential part of

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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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tannins [8, 9].

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There is no universal classification of polyphenols and several authors have

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proposed their own. For example, Crozier et al. [10] classified these compounds into

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flavonoids and non-flavonoids.

Figure 1 shows the most important families of dietary

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polyphenols, some characteristics examples, their molecular structure and occurrence in

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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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complex and weighty condensed tannins.

Non-flavonoid compounds include simple phenolic acids, phenyl alcohols, stilbenes,

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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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hydroxyphenylpropanoic acid derivatives, such as dihydrocaffeic acid. To give some

129

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

138

main phenolic compounds of virgin olive oil.

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Stilbenes, such as resveratrol, are composed of a C6-C2C6 skeleton with various

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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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formation of 4, 2, 4, 6-tetrahydroxychalcone (also known as naringenin chalcone) [12].

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Lignans, such as matairesinol and secoisolariciresinol, are formed of two

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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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vegetables and pulses contain trace level of these compounds.

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On the other hand, flavonoids are characterized by a C15 phenylchromane core,

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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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flavonols, flavones, isoflavones, flavanones, anthocyanidins, flavanonols and flavanols.

Although the occurrence of some classes of flavonoids is restricted to a few

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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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cereals. The most common anthocyanidins are pelargonidin, cyanidin, delphinidin,

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peonidin, petunidin and malvidin. When these phenolic compounds are found as sugar

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conjugates, they are known as anthocyanins.

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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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Flavanols, the most structurally-complex sub-class of flavonoids, especially

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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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epicatechin) to complex structures including the oligomeric or procyanidins (such as

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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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(such as epigallocatechin). For example, catechins and epigallocatechin are present at

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high concentration levels in cocoa and tea.

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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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attached to a variety of polyol-, catechin- and triterpenoid cores (gallotannins, ellagitannins

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and complex tannins), or they are oligomeric and polymeric proanthocyanidins that can

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possess different inter-flavanyl coupling and substitution patterns (condensed tannins)

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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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pentagalloylglucose molecule, and complex tannins are defined as tannins in which a

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catechin unit is bound glycosidically to either a gallotannin or an ellagitannin unit [16].

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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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mesquite, cherry, oak and others [24].

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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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elucidation, and then quantification.

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3. Sample pre-treatment strategies: extraction techniques and preconcentration

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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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chromatographic analysis, has been limited to dilution probably to avoid time-consuming

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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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determination of flavanols in tea extracts by UHPLC-MS/MS without any sample

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preparation. The tea samples were only diluted 10-fold in water before analysis and they

were well dissolved. Guillarme et al. [25] evaluated the qualitative

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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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where polyphenols are dispersed as a microemulsion, a sample pre-treatment technique

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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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polyphenols by UHPLC-MS in plant tissues and foods samples. Liquid-liquid extraction

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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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3.1. Liquid-liquid extraction (LLE)

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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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acid [40-42], acetone/water/acetic [43, 44] or acetonitrile/water [45].

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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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important limitation of this sample pre-treatment technique.

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3.2. Solid-phase extraction (SPE)

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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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The stationary phase is available in a wide variety of chemistries, adsorbents, and

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sizes. For the

extraction and preconcentration of polyphenols, the reversed phase

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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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determination of phenolic compounds in food samples by UHPLC-MS. These cartridges

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with dual-phase materials could be a good strategy to food pre-treatment due to its

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improved selectivity and capacity.

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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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very interesting to extract and preconcentrate phenolic compounds in food samples

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because faster extractions are achieved and additionally the 96-well plate format allows

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increasing the sample throughput.

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3.3. Accelerated solvent extraction (ASE)

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The ASE technique has not been extensively applied to sample pre-treatment in the

281

chromatographic determination of food phenolic composition. The main application of ASE

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related to polyphenols is in the preparation of phenolic extracts from different plantsources

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to produce functional foods and nutraceuticals. ASE as the sample pre-treatment

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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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3.4. Other extraction techniques

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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

291

phenolic acids, flavones, flavonols in Burdock leaves, which is a popular vegetable in

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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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thermodynamic critical points to extract bioactive components from agricultural materials

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[55].

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Until now, these extraction techniques (MAE and SFE) combined with UHPLC-MS have

297

not been reported for the determination of phenolic compounds in food samples.

298

Nevertheless, new chromatographic equipments that combine the potential of supercritical

299

carbon dioxide (SFC) with UHPLC technology opens new perspectives for applying this

300

extraction technology to the analysis of phenols in food samples.

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4. UPLC and UHPLC

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Over recent decades, different advances in the development of silica-based particles

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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

306

separation, an advanced form of LC has been reported. This novel technique is known as

307

UHPLC, and it uses narrow-bore columns packed with very small particles, sub-2 m

308

(from 1.7 to 1.9 m), and mobile phase delivery systems operating at high back-pressures

309

(up to 1300 bar or 15000 psi). According to the van Deemter equation, as the particle size

310

decreases to less than 2.5 m, there is a significant gain in efficiency (in UHPLC the

311

particle size is 1.7 m) and it does not diminish at increased flow rates because the eddy

312

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

314

(particle size from 3 to 5 m) are improved resolution, higher peak efficiency, shorter

315

retention times, and reduced solvent consumption. As a consequence of the higher peak

316

efficiency, the peaks are narrower (sharper peaks), and the detection limits (LODs) are

317

lower. This fact means the sensitivity in UHPLC is higher than in comparison to the

318

conventional HPLC [58, 59].

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Table 1 shows the sample pre-treatment and chromatographic conditions for the

320

determination of phenolic compounds in food samples by using UHPLC-MS. Table 2

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shows the polyphenols that have been determined in different food samples by using the

322

advanced chromatographic technique UHPLC coupled to MS.

323

compared the use of HPLC with UHPLC coupled to MS, as the detector system, for

324

determining phenolic compounds in food samples, such as beverages [29], virgin olive oil

325

[26, 27], cocoa [43] or soy foods [33]. In these reports,the chromatographic separation in

326

UHPLC was between three and seven times faster than with conventional HPLC. As an

327

example, Ortega et al. [43] reduced the analysis time from 80 min in conventional HPLC to

328

12.5 min in UHPLC for the determination of flavanols in cocoa samples. Figure 2 shows

Different authors have

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329

the extracted ion chromatograms (EICs) of the studied flavanols. As it can be seen, the

330

efficiency of the peaks by UHPLC-MS/MS (Figure 2A) allowed the flavanols nonamers

331

(oligomers or procyanidins) to be identified while by HPLC-MS/MS (Figure 2B) allowed

332

until the flavanols hexamers. Additionally, the quantification limits (LOQs) in UHPLC (10-

333

90 g/L) were lower than in HPLC (20-200 g/L).

As mentioned above, the peak efficiency and then the chromatographic resolution

335

provided in UHPLC are higher than in conventional HPLC. As a consequence of these

336

facts, the coupling UHPLC technique by MS is less susceptible to matrix effects (ME). It

337

has been reported that an efficient UHPLC separation may contribute to a reduction in ion

338

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

340

consumes approximately 80% less organic solvent, and therefore it can also be

341

considered more cost-effective.

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5.Chromatographic conditions

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5.1. Analytical column

346

As it has previously been reported, the UHPLC system is based on the principal use

348

of the stationary phase, consisting of particles of less than 2 m, while HPLC columns are

349

typically filled with particles from 3 to 5 m. These UHPLC columns have an internal

350

diameter of 2.1 mm, a length of between 50 and 150 mm, and a particle size between 1.7

351

and 1.9 m, according to the commercial brand.

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For the analysis of phenolic compounds in food samples by this novel technique

353

coupled to MS, different analytical columns have been used, such as Ethylene Bridged

354

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]

357

and HT C18 [70] from Agilent; and Hypersil Gold [28, 48, 52] from ThermoFisher. All these

358

columns are based on reversed-phase mode, except for the BEH Amide, which uses

359

hydrophilic interaction chromatography (HILIC). For the analysis of phenolic compounds in

360

food samples, the most widely-used analytical column is the BEH C18. The particles in this

361

column are characterized by being hybrid with ethylene bridged and completely

362

endcapped.

12

Page 12 of 42

363

In the analytical columns based on the reversed-phase mode, the phenolic

364

compounds are eluted according to their polarity and molecule size. On the other hand,

365

when a hydrophilic interaction chromatography (HILIC) is used, the more polar compounds

366

are eluted after the non-polar compounds [46]. Guillarme et al. [25] compared four

367

different endcapped, deactivated analytical columns from two different providers for the

368

analysis of seven diastereoisomers favanols, and the obtained chromatograms are shown

369

in Figure 3. These columns were

370

Hypersil Gold C18 (conventional C18 material) (Figure 3B), Acquity BEH phenyl (a hybrid

371

BEH phenyl material) (Figure 3C), and Acquity BEH Shield C18 (a hybrid BEH C18 support

372

with a polar embedded group) (Figure 3D). The different separations provided confirmation

373

that endcapped, monomeric C18 columns are required for the separation of the studied

374

flavanols. In addition, these authors also demonstrated that the columns containing a polar

375

embedded group improved the selectivity of the studied diastereoisomers flavanols.

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the Acquity BEH C18 (a hybrid BEH C18) (Figure 3A),

In recent years, a comprehensive two-dimensional LC (2D-LC) has been exploited as

377

a powerful analytical technique for studying complex matrices, such as food samples. This

378

technique, which has a much greater resolving power compared to a single-dimensional

379

LC, is based on transferring all the fractions over the first dimension to the second

380

dimension. Typically, this system combines two analytical columns with different

381

separation mechanisms in order to achieve better separation, identification and

382

quantification. Good resolution of compounds prior to MS detection improves the reliability

383

of the MS results by decreasing the matrix suppression. In any LC x LC combination, the

384

speed of the second dimension determines the overall analytical time. This speed should

385

be as fast as possible, with good resolution. To achieve a fast speed, short columns

386

packed with small particles or monolithic columns are used. Thus, a combination of HPLC

387

with UHPLC offers a good configuration for a comprehensive 2D system and this

388

technique has also been used to analyze phenolic compounds by UHPLC-MS. Scoparo et

389

al. [62] and Kalili et al. [67, 68] used a comprehensive 2D-LC technique to determine

390

phenolic compounds in tea [62, 67], cocoa and apple [68] samples. The analytical columns

391

used, which combined HPLC x UHPLC, were size exclusion chromatography (SEC) and

392

reversed phase (BEH C18) to separate compounds according to their size and polarity,

393

respectively [62]; and HILIC and reversed phase (BEH C18) to separate the compounds

394

based on their hydrophobicity and polarity [67, 68]. On the other hand, Cavaliere et al. [42]

395

coupled two UHPLC columns (Zorbax Eclipse Plus C18) in series to obtain as high an

396

efficiency as possible to determine flavonols, flavanols, anthocyanidins and stilbenes in

397

grapes.

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Page 13 of 42

398

Nowadays, the requirements in LC are shifting more and more to faster separations.

399

The determination of food polyphenols and the effects of these compounds on human

400

health require sensitive and robust methods for the quantification of these compounds in

401

different food matrices. Rapid and high resolution analytical methods are also needed for

402

routine screening and quality assurance of polyphenol-rich foods.

In HPLC, in order to achieve high resolution it is necessary to increase the column

404

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

408

capability. In contrast, as it has previously been commented, in UHPLC a high resolution is

409

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

414

classes were identified in tomato fruit [39] and in red mustard greens [28] in an hour.

415

Figure 4 shows the UHPLC-DAD chromatogram (330 nm) for the determination of flavanol

416

glycosides and hydroxycinnamic acid derivatives in red mustard greens extract [28]. On

417

the other hand, few minutes such as 3 min were also reported for the determination of

418

minor amount of phenolic compounds in food samples by UHPLC-MS [27, 32, 44].

419

5.2. Mobile phase

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421

For the analysis of phenolic compounds by reversed-phase chromatography, acidified

422

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,

425

26, 30, 39, 43, 65], or trifluoroacetic acid (TFA) [44, 45]. Its concentration is kept as low as

426

possible in order to ensure satisfactory ionization, and it is between 0.05 and 0.2%, or in

427

some case 5 % [63]. The separation of

428

accomplished most effectively by gradient elution, by using methanol [25, 27, 31, 32, 34,

429

36, 54, 62, 64], acetonitrile [26, 28, 29, 33, 35, 37-41, 46-48, 51, 52, 59, 65-70] or

430

methanol/acetonitrile mix [42, 53] as an organic modifier in the phase B. Normally,

431

acetonitrile is preferred due to the lower pressure obtained than methanol.

phenolic compounds with similar polarity is

14

Page 14 of 42

432

On the other hand, the chromatographic HILIC mode

was used to analyze

phlorotannins, and the mobile phase was made up of ammonium acetate (phase A) and

434

acetonitrile (phase B). Phlorotannins were resolved with HILIC mode due to the high

435

polarity of these compounds, which eluted with little or no retention in the reverse-phase

436

due to the lack of interaction with the non-polar stationary phase [46]. In this study, a

437

gradient ranging from 5% to 35% of aqueous phase yielded a reasonable compromise

438

between run times, peak shape and sensitivity. In HILIC, water is introduced as the

439

stronger eluting solvent instead of the polar organic phase as in reversed mode.

440

With the improved LC, UHPLC, the flow-rate used is lower than in conventional HPLC, and

441

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

also reduced. The sample volume injection is around 3 l, in comparison to 20 l in the

444

conventional HPLC.

445

6. Applications of UHPLC-MS for the analysis of food samples

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433

446

UHPLC-MS combines the separation of LC with the selectivity and sensitivity of the

448

MS detector to permit the identification of individual compounds from a mixture of

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

medicine (TCM) [30, 31, 35, 37, 54, 69].

Ac
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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

fragmentation products of the aglycone arising from Retro-Diels-Alder reactions [71-73].

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

analyzed anthocyanins in the negative mode, and their MS spectra proved to be a

481

valuable tool differentiating anthocyanins from non-anthocyanin polyphenols, such as

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

spectra of non-anthocyanin polyphenols. Figures 5A and 5B show the MS spectrum of an

485

anthocyanidin (delphinidin-glucoside) and a flavanol (quercetin-glucoside), respectively in

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

VIS detector, especially when an anthocyanin is co-eluting with another compound, or

490

when the anthocyanin is present at low concentration levels.

Ac
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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

reported as a well-established and powerful platform for both high-resolution metabolite

510

profiling and rapid fingerprinting of crude plant extracts.

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501

In the literature, the combinations reported for the determination of phenolic

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.

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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

These authors used a triple quadrupole to determine isoflavones in soy food.

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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

As an example, Figure 6 shows the extracted ion chromatograms (EICs) of different

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

During Q-TOF analysis, a solution of the reference compound, leucine-enkephalin, was

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

solution [60, 67, 68].

ed

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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

damping gas and fragmentation.

Ac
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556

564

Arapitsas et al. [51] used an UHPLC-ESI-Q-linear ion trap to determine anthocyanins

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

sensitivity, mass accuracy and rapid full scanning mode.

In order to achieve optimum ionization efficiency, the different parameters of the

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

particularly their aglycones, needed higher collision energies to obtain diagnostically

581

relevant product ions.

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575

582
6.1.

Quantification and matrix effect of phenolic compounds by UHPLC-MS in food

an

583

samples

584
585

In the different analytical methods reported in the literature for the analysis of phenolic

587

compounds by UHPLC-MS, some of them are only based on identifying without

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

solutions when these

592

tentatively identified by comparing the information obtained with available bibliographic

593

data.

pt

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586

Ac
ce

were commercially available. Otherwise, phenolic peaks were

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

compounds. Another strategy proposed by various authors to obtain phenolic standards is

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

virgin olive oil in order to obtain the standards.

19

Page 19 of 42

The ME is considered to be either an unexpected suppression or enhancement of the

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

determination of phenolic compounds in food samples by UHPLC-MS. In this strategy, the

612

signal of the analyte in a standard solution was compared to that of a post-extraction

613

spiked sample at the same concentration (matrix-matched standard).

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

alterations are reduced. Therefore, MS detection proceeded by an efficient UHPLC

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

reduction in ion suppression [60, 61].

an

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614

As well as UHPLC as the strategy to obtain more efficient chromatographic

623

separations, on-line orthogonal two-dimensional LC (2D-LC) has also been reported to

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

achieved, and that the ionization alterations are decreased.

Ac
ce

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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

components for the determination of phenolic compounds in food samples. By using an

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

(LODs) increased [26, 65].

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

literature, both an isotopically-labelled standard [41, 64] and a deuterium-labelled standard

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

were prepared by adding different amounts of standard phenolic compounds to food

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

normalized peak area (measured area x dilution factor).

an

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646

In food analyses, the determination of isobaric compounds in natural products is often

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

analysis of these compounds, it is very important to obtain a good resolution in order to

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

achieved is better because these compounds have different accurate mass.

Ac
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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

phenolic compounds in food samples by UHPLC-MS, the sensitivity was 3 orders of

672

magnitude higher than those reported using HPLC-UV detection [25, 26, 32, 33, 42, 65].

673

The coupling of UHPLC with MS provides sufficient selectivity (between phenolic

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

7. Conclusions and future perspectives

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

validated and standardised methods

684

determination of polyphenol content is important for the development of comprehensive

685

food composition databases as the main requirement for establishing daily intake of

686

polyphenols.

ip
t

680

us

cr

for their determination in food samples. The

Based on these requirements, the UHPLC-MS offers a very suitable and good

688

alternative to conventional HPLC-DAD or HPLC-MS, and provides a better means of

689

identifying and quantifying polyphenols in complex matrices, such as food ingredients or

690

formulated foods. This fact is due to the improvements made in the preparation of sub-2

691

m packing materials of the analytical columns, which provide high-efficiency separations

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

and could be a good competitor for UHPLC.

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

coelution of two different compounds, and may contribute to a reduction in ion

702

suppression. It is important to remark, the ability to transfer existing HPLC to UHPLC

703

conditions directly.

704

The combination of UHPLC with an MS detector appears to be a suitable approach

705

that fulfils key requirements in terms of sensitivity, selectivity, and peak-assignment

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

through avoiding the post-extraction solvent evaporation and reconstitution steps.

us

cr

ip
t

714

722

The recent developed technology ultra-performance convergence chromatography

724

(UPC2), which uses columns packed with sub-2 m particle and CO2 as the mobile phase,

725

could be a complementary strategy to determine phenolic compounds in food

726

samples.This new technique, so far, not been applied for the determination of polyphenols

727

in foods, but it opens new perspectives for future applications.

729

Acknowledgement

730

pt

728

ed

an

723

This work was supported by the Spanish Ministry of Education and Science financing

731
the

733

(Interdepartmental Commission for Research and Technological Innovation) through the

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

Flavonoids. Chemistry, Biochemistry and Applications, CRC Press, Boca Ratn,

740

2006, p. 219.

741

[2] M. Caveri, F. Bianchi, C. Corradini, J Chromatogr. A 970 (2002) 3.

742

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743

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744

L. Drahos, K. Vkey, J Chromatogr. A 1259 (2012) 74.

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746

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758
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Nutraceuticals. Processing Technologies. Taylor & Francis, 2007, pp. 3.

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Bioactive

Components

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Ingredients

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838
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842
843
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845
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848

Melanson, Phytochem. Anal. (2012) in press

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P. Arapitsas, P.J.R. Sjberg, C. Turner, Food Chem. 109 (2008) 219.

850

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852
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854

[70] L. Jaitz, K. Siegl, R. Eder, G. Rak, L. Abranko, G. Koellensperger, S. Hann, Food

855

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ip
t

853

[71] E. Hvattum, D. Ekeberg, J. Mass Spectrom. 38 (2003) 43.

857

[72] U. Justesen, J. Mass Spectrom. 38 (2001) 169.

858

[73] M. Stobiecki, Phytochem. 54 (2000) 237.

859

[74] A. Cappiello, G. Famiglini, P. Palma, H. Trufelli, J. Liq. Chromatogr. Rel. Technol. 33

862
863

us

an

861

(2010) 1067.

[75] F. Gosetti, E. Mazzucco, D. Zampieri, M.C. Gennaro, J Chromatogr. A 1217 (2012)

3929.

[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

Figure 1. Classification of dietary polyphenols, some examples, their molecular structure

867

and its occurrence in foods.

868
Figure 2. Extracted ion chromatograms of flavanols for the analysis of cocoa sample by

870

(A) UHPLC-MS/MS and (B) HPLC-MS/MS [43].

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

Figure 4. UHPLC-DAD chromatogram at 330 nm for the determination of flavonol

881

glycosides and hydroxycinnamic acid derivates of red mustard greens extract [28].

880

ed

882

Figure 5. MS spectrum in full-scan mode of the anthocyanidin delphinidin-3-O-glucoside

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

virgin olive oils [27].

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

[ 29, 63, 70]

[ 28, 63]
[ 63, 70]

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

Virgin olive oil

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)

Virgin olive oil

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)

A) 0.2 % acetic acid

B) acetonitrile

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)

A) 0.1 % formic acid

B) acetonitrile + 0.1%
formic acid

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

LLE (Water and

methanol/water)

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]

A) 7.5 mM formic acid

B) Acetonitrile

QqQ

Quantification

[ 29]

BEH C18
(100 x 2.1 mm, 1.7
m)

A) 0.1 % formic acid

B) Methanol

QqQ

Quantification

[ 31]

BEH C18
(50 x 2.1 mm, 1.7 m)

A) 0.2 % acetic acid

B) Acetonitrile

Q-TOF

Identification

[ 30]

LLE
(acetone/water,
80/20, v/v)

HSS T3
(100 x 2.1 mm, 1.8
m)

A) 0.05 % acetic acid

B) Acetonitrile

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]

A) 0.1% formic acid

B) Acetonitrile /
methanol (50/5, v/v)
+ 0.1% formic acid

an

us

Zorbax Eclipse Plus

C18
(100 x 2.1 mm, 1.8
m)
(x2)

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

A) 0.1% formic acid

B) Acetonitrile

Q-TOF

Identification

[ 68]

A) 0.2 % acetic acid

B) Acetonitrile

QqQ

Quantification

[ 43]

A) 0.1 % formic acid

B) Methanol

QqQ

Quantification

[ 32]

BEH Shield C18

(50 x 2.1 mm x 1.7
m)

A) 0.05-0.01 % acetic
acid
B) Methanol

QqQ

Identification

[ 25]

LLE
(Methanol)

BEH C18
(100 x 2.1 mm, 1.7
m)

A) 0.1 % formic acid

B) Methanol

QqQ

Quantification

[ 34]

LLE
(Methanol)

Zorbax Eclipse Plus

C18
(150 x 4.6 mm, 1.8
m)

A) 0.5 % acetic acid

B) Acetonitrile

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)

Alcohol and malolactic

fermentation

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

Dilution with boil water

cr

LLE
(acetone/water,
70/30, v/v)

A) 0.1 % formic acid

B) acetonitrile

Q-TOF

Identification

[ 67]

Zorbax SB C18
(50 x 4.6 mm, 1.8 m)

A) 0.1 % formic acid

B) methanol

TOF

Quantification

[ 54]

BEH Shield C18

(150 x 1.0 mm, 1.7
m)

A) 0.1 % formic acid /

acetonitrile (95/5,
v/v)
B) 0.1 % formic acid /
acetonitrile (40/60,
v/v)

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)

A) 0.1 % formic acid

B) Acetonitrile /
methanol (20/80,
v/v)

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]

A) 0.1 % formic acid

B) Acetonitrile

Q-TOF

Quantification

[ 35]

BEH Shield C18

(150 x 2.1 mm, 1.7
m)

A) 0.1 % formic acid

B) Acetonitrile with
0.1% formic acid

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)

Hypersil Gold AQ C18

(200 x 2.1 mm, 1.9
m)

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

A) 0.1 % formic acid

B) Methanol

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

A) 0.1 % formic acid

B) Acetonitrile + 0.1%
formic acid

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)

A) 0.1 % formic acid

B) Acetonitrile

QqQ

Identification

[ 69]

Traditional
Chinese
Medicine

Flavonol

LLE
(methanol)

HSS T3
(100 x 2.1 mm, 1.8
m)

A) 0.1 % formic acid

B) Acetonitrile

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)

Hypersil Gold C18

(200 x 2.1 mm, 1.9
m)

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

Off-line SPE (Diol)

cr

Anthocyanins

A) 0.1 % formic acid

B) Acetonitrile + 0.1%
formic acid

Linear Ion
Trap-Orbitrap

Identification

[ 48]

A) 0.1 % formic acid

B) Acetonitrile + 0.1%
formic acid

TOF

Identification

[47]

A) 0.01% formic acid

B) Methanol

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

Occurrence in foods [11]

Molecular structure
O
HO

Caffeic acid

Fruit, thyme, spices

OH
HO

Simple phenolic
acids

O
OH

Ferulic acid

HO

Spices (cloves), chesnut,

vegetables (chicory)

HO

Gallic acid

OH
HO

Phenyl alcohols

Hydroxytyrosol

Stilbenes

Resveratrol

ip
t

HO

OH

HOO

Olive, olive oil

HO

OH

Grapes, wine

OH
HO

HO

Chalcones

Phloridzin

OH

OH

Fruits, herbs

cr

NON-FLAVONOIDS

Cereals, cocoa, herbs

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

Cocoa, fruits, vegetables (onion),

herbs

OH

HO

OH

Cyanidin
Anthocyanidins

Blackberries, strawberry

OH

+
O

HO

O-CH3
OH

OH

Malvidin

+
O

HO

HO

Black grape, wine

O-CH3
OH

ed

HO

Luteolin

Flavones

OH

O-CH3

pt

Flavanone

OH

OH
HO

Apigenin

Naringenin

ce

Ac

O-CH3

Herbs (oregano), olive oil

OH
HO

OH

Citrus juices, fruits, tomatoes,

herbs (thyme)

HO

OH
O

HO
HO

OH

HO

Herbs (thyme)

OH
O

HO

Taxifolin

OH

Herbs (oregano)

OH
HO

HO

Genistein

Soy and soy products

HO

Isoflavones

OH

HO

Daidzein

Soy and soy products

OH

OH

Catechin

OH
O

HO

Grapes, cocoa, tea, wine

OH

Flavanols

OH

OH

Epigallocatechin

HO

HO

OH

Tea

OH
HO

OH

Condensed tannins

HO
HO

Proanthocyanidins

OH

Cocoa, fruit (apple)

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

Fruits (grape), tea

O
O

OO

HO OH
HO

OH

Figure 1

FLAVONOIDS

+
O

HO

Eriodictyol

Flavanonols

Herbs (sage, thyme, oregano),

vegetables (artichoke), olive oil

OH
+
O

HO

Page 37 of 42

Fruits (raspberry, blackberry)

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

Catechin and epicatechin

M
an

100

Catechin and epicatechin

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