Documenti di Didattica
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Journal of Chromatography A
journal homepage:www.elsevier.com/locate/chroma
Review article
Article history: Flavonoids have elicited significant attention as a result of their importance in plants, their influence on the properties of
Received 14 September 2015 Accepted natural-product derived commodities and especially as a consequence of their pur-ported health benefits. Research in all of
25 November 2015 Available online 28 these fields relies heavily on accurate analytical data, and in this LC–MS has come to play an influential role by allowing
November 2015 relatively fast tentative identification and accurate quantification of low levels of flavonoids in a variety of matrices. The field
has undergone rapid expansion in the last decade due to important developments in both HPLC and MS instrumentation,
Keywords: which nowadays allow much faster and more accurate analysis of flavonoids. This contribution aims to provide an overview
Comprehensive two-dimensional LC
of these developments and their application in flavonoid analysis since 2009. The discussion is focussed first on
(LC × LC)
methodologies which provide improved LC separation of flavonoids in terms of speed and/or resolution, including ultra high
Flavonoids
Liquid-chromatography–mass pressure LC (UHPLC), monolithic and superficially porous phases, high temperature LC (HTLC) and comprehensive two-
spectrometry (LC–MS) dimensional LC (LC × LC). The fun-damental background relevant to each of these will be briefly outlined, as well as the
MS/MS implications and promise of their hyphenation to MS. Secondly, the possibilities and limitations of a range of the latest MS
Superficially porous phases instruments available in combination with advanced LC analysis will be discussed, including ion trap, triple quadrupole, time-
Ultra high pressure liquid chromatography of-flight, Orbitrap, ion mobility and various hybrid instruments. Examples from the latest literature will be used to illustrate
(UHPLC) the performance gains achievable in flavonoid analysis by the hyphenation of advanced LC separation and high-end MS
instrumentation.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2. LC–MS analysis of flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.1. General aspects
of LC separation of flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
2.2. Detectors used in the LC analysis of flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.3. General aspects
(n)
of LC–MS analysis of flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.3.1. Flavonoid glycosides . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.3.2. Flavonoid
aglycones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3. Advances in HPLC separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.1. One-dimensional
HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.1.1. Ultra high pressure liquid
chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.1.2. Monolithic columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.1.3. Superficially porous stationary phases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.1.4. High temperature liquid chromatography (HTLC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 3.2.
Comprehensive multi-dimensional LC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Fundamental aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 4.
Advances in LC–MS analysis of flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
∗
Corresponding author.
E-mail address: ajdevill@sun.ac.za (A. de Villiers).
http://dx.doi.org/10.1016/j.chroma.2015.11.077
0021-9673/© 2015 Elsevier B.V. All rights reserved.
A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 17
Fig. 1. Structures of the main classes of flavonoids, with numbering of rings and carbons indicated. Note the difference in the numbering scheme for chalcones [5]. Substituents on the backbones are
mostly hydroxyl and methoxy groups, while additional mono- to oligosaccharides with varying degrees of acylation are also common.
NMR. Whereas NMR provides average compositional information on the current state-of-the-art in flavonoid analysis by LC–MS; in this work we
complex mixtures, MS addresses the composition of single molecules. will endeavour to fill this gap.
Developments in commercial mass analysers over the last decade and a half The emphasis of the current report will be on the most impor-tant
now routinely allow determination of molec-ular formulae and molecular advances in the HPLC separation and MS instrumentation and their
fragmentation using collision induced dissociation (CID) on single or multi- application in the LC–MS analysis of flavonoids in a variety of matrices.
stage MS systems, and are largely responsible for the fact that LC–MS has Considering the very large number of liter-ature reports appearing annually in
assumed its current position of eminence in the field of flavonoid analysis. this field, we will limit the scope of this review to the last 7 years (since
2009). We will fur-ther limit the scope to UHPLC separation (defined here as
The field of LC–MS has experienced exceptional growth during this period
HPLC separations performed on columns packed with sub-2 m phases and/or
due to the commercial availability of a range of high resolution and ◦
hyphenated MS detectors based on atmospheric pressure ionisa-tion (API) operated at pressures above 400 bar), the use of elevated temperatures (≥50
ionisation sources [41,42]. C), alternative stationary phase morphologies (including monoliths,
superficially porous phases), comprehensive two-dimensional LC separations
1
The combination of chromatographic resolution provided by HPLC and (LC × LC) and multi-stage and/or high resolution MS. The fundamental
n (n)
structural information provided by MS enables rou-tine separation and background of advanced HPLC methods and MS instrumentation and their
tentative identification of complex mixtures of flavonoids spanning several application to flavonoid analysis will be addressed briefly, and the benefits of
orders of magnitude in concentra-tion, something that was not possible 25 these developments will be highlighted on the hand of important literature
years ago. Furthermore, important advances in LC technology have been reports. We will attempt to highlight the most important contemporary trends
realised in the last decade. Significant developments include the use of ultra in the LC–MS analysis of flavonoids based on these reports.
high pressures and sub-2 m stationary phases (ultra high pressure liq-uid
chromatography, UHPLC), alternative stationary phases such as monoliths
and superficially porous phases, high temperature HPLC [43,44] and
multidimensional HPLC [45–49]. Note that sample preparation and extraction of flavonoids, although
essential in all analytical workflows for flavonoids and especially so in the
As a consequence of the intensive research activity involving flavonoids, case of LC–MS analysis to reduce matrix effects, will not be addressed in
a wealth of information may be obtained regarding important aspects such as detail in the current work. This aspect has been covered in detail by several
their analysis, biochemistry, health prop-erties and recently identified reviews [16,33,56–59,62,65,66,74,75] and developments in the field are less
compounds. Especially a number of comprehensive book series serve as dramatic than those involving the analysis step.
excellent reference works that provide detailed overviews of the field and are
updated periodi-cally [3,4,50–52]. More dedicated information on the analysis Finally, a caveat should be added: because of the very large number of
of flavonoids may be found in books such as [53,54], while landmark reports papers falling within the scope of this review, a com-prehensive list of all
provide comprehensive overviews of particular aspects such as the analytical relevant reports is not possible in this format; neither will we attempt to
chemistry of flavonoids in food [55,56], extraction and analysis of phenolics discuss each report in detail. Rather, tables will be used to summarise the
[57,58], the role of MS in the structural analysis of flavonoids [59,60], also in information, and the discus-sion will focus on important contributions and
biological samples trends deduced from these works.
[27] and wine chemistry [61], separation and detection methods for
flavonoids [62] and advanced separation methods for selected flavonoids 2. LC–MS analysis of flavonoids
[33]. In light of the spectacular developments in the field during the last 5–10
years, however, these seminal reference-works are somewhat outdated. 2.1. General aspects of LC separation of flavonoids
Several more recent review papers have addressed particular areas of the
HPLC or LC–MS analy-sis of flavonoids, such as developments in the HPLC Contemporary HPLC separation of flavonoids is almost exclu-sively
separation of phenolic compounds [63,64], analysis of food polyphenols by performed using reversed phase liquid chromatography
ultra high pressure LC (UHPLC) combined with MS [65], chromato-graphic
analysis of flavonoids and metabolites [66], developments in the analysis of
natural product [67] and food phenolics [68] and metabolites [69–73]. None
1 This of course implies that a large number of excellent research papers published in the last 7 years will not be
of these works comprehensively address the fundamental and instrumental
included due to the criteria defined for the scope of the current work. This was however important to limit the number of
aspects pertinent to
papers to manageable numbers.
A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 19
(RP-LC), with the notable exceptions being normal phase liq-uid according to molecular weight (MW), first reported in 1979 [76] (and based
chromatography (NP-LC) for oligomeric proanthocyanins [76], and the recent on previous findings on silica gel TLC [115]) and sub-sequently adapted to
increasing application of hydrophilic interaction chromatography (HILIC). HPLC format and refined by several groups [116,117]. This type of separation
Other modes of separation which have been applied to flavonoid separation is typically performed in adsorp-tive mode on bare silica stationary phases and
include mixed-mode ion-exchange-reversed phase [77–79] separation of mobile phases containing dichloromethane/methanol/acidified water
anthocyanins, size exclusion chromatography (SEC) analysis of flavonol [116,117], hexane/acetone or hexane/methanol/ethyl acetate [118]. Proan-
glycosides [80,81] and theaflavins and proanthocyanidins [81]. Because of the thocyanins elute in order of their degree of polymerisation (DP) under these
relatively sparse use of the latter modes, however, they will not be discussed conditions, with isomeric compounds of the same DP co-eluting.
in detail in the current work (except when used in LC × LC), which will focus
primarily on RP-LC in accordance with the scope and preponderance of this
mode in flavonoid literature. A more recent trend is the application of HILIC for flavonoid anal-ysis.
HILIC can be considered an aqueous variant of NP-LC, where retention is
RP-LC has demonstrated its suitability for the separation of flavonoids primarily governed by partitioning of polar compounds into an aqueous-rich
based on the nature of the aglycone (including the oxidation state, substitution layer immobilised on the surface of polar bonded phases [119,120]. The first
patterns and stereochemistry), the nature and degree of glycosylation as well application of HILIC to flavonoid analysis, and one of the first applications of
as the nature and degree of acylation. By far the majority of RP-LC this mode of separa-tion [121] (although the term and a description of its
separations are performed using C18 octadecyl-silica (ODS) phases, although separation mechanism was to follow only much later), was reported by Lea
C8 [82,83], C12 [84], phenyl or phenyl-hexyl [76,85–90], pentafluo-rophenyl [76], who used a pellicular polyamide phase and a mobile phase comprising
(PFP) [90–95] and polar embedded RP phases [88,96–98] as well as methanol, acetic acid and water for the separation of epicatechin and
polymeric RP-LC phases [99] have also found appli-cation in flavonoid phloridzin. Similar retention of proanthocyanins is observed in HILIC
analysis [100–102]. Mobile phases typically comprise aqueous/organic phases compared to NP-LC [122,123]. However, the more polar mobile phases used
containing methanol, acetoni-trile and less commonly tetrahydrofuran [103], in HILIC, typically comprising binary mixtures of acetonitrile and acidified
isopropanol [101] or ethanol [99] and acidic modifiers such as acetic acid, water, makes the technique more suitable for flavonoid analysis than NP-LC
formic acid, ammonium acetate or trifluoroacetic acid (TFA) [101] and improves ion-isation efficiency for these compounds compared to RP-LC
(phosphoric, citric or perchloric acids have also been used in combination due to higher organic content of the mobile phase [124]. Indeed, the appli-
with UV detection, although these are not suited to hyphenation with MS). cation of HILIC for the analysis of flavones and flavonols [125–127],
For anthocyanins, highly acidic mobile phases (>4–10% formic acid, 0.1– dihydrochalcones and flavanones [127], anthocyanins [128] and phlorotannins
0.6% TFA) [104–107] are used to ensure prevalence of the flavylium cationic [129] has been demonstrated recently. HILIC sta-tionary phases used in
species in solution and therefore improve chromatographic efficiency flavonoid analysis include silica [130], diol [122,131], polyethylene glycol
[33,100,101,105]. (PEG) [131], cyclodextrin (CD) [130], ZIC-HILIC [132] and amide [128,130]
phases. One of the major driv-ing forces behind the interest in HILIC for
flavonoid analysis is the alternative separation mechanism to RP-LC offered
Retention of flavonoids in RP-LC is based on hydrophobicity; some by this separa-tion mode [133], which typically results in class–type
selectivity differences may be observed based on the nature of the stationary separations of flavonoids according to the degree of glycosylation and/or acy-
phase, endcapping, carbon loading, polar embedded groups as well as the lation. As a consequence, HILIC has found increasing application in the LC ×
nature of the silica phase (e.g. organo-silanes) [108,109]. However, the LC separation of flavonoids in combination with RP-LC (see Table 2).
general elution order of flavonoids on RP-LC columns is mostly consistent in
the following sequence for flavonoid aglycones: flavanols < flavanones <
flavonols < flavones [4,59,110,111] (anthocyanins elute close to flavanols
using ‘generic’ (i.e. not highly acidic) mobile phases [111,112]). For each of
these classes, retention decreases with the number of hydroxyl groups on the
flavonoid backbone, and increases with the number of methoxy substituents. 2.2. Detectors used in the LC analysis of flavonoids
Glycosylation decreases RP-LC retention, whereas acylation of glycosyl
groups increases retention relative to the corresponding glycosylated A wide range of detectors can and have been used in combi-nation with
derivatives. Common sugars encountered in glycosylated flavonoids include HPLC separation to detect and/or identify flavonoids. These include
glucose, galac-tose, rhamnose, xylose and arabinose, with mannose, fructose electrochemical detection (ED) [134,135], fluores-cence (FL) [62,136], UV–
and glucoronic and galactoronic acids being rarer. The general elution order vis, diode array [101,137], NMR [38–40] and of course MS detectors
of glycosylated flavonoids in RP-LC is -galactosides < - glucosides < [59,60,62,110,138]. Of these, by far the most common nowadays are diode
-arabinosides < -xylosides < -rhamnosides [101]. Di-and higher oligomeric array and MS detectors, which will be discussed briefly in this and the next
saccharides are common in some prod-ucts, with the most common section, respectively.
disaccharides being rutinoside (rhamnosyl-( 1→6)-glucose) and neo- The conjugated aromatic character of flavonoids proved to be a significant
hesperidose (rhamnosyl-(-( 1→2)-glucose) [59]. The most common aliphatic benefit especially in early flavonoid research: absorption at relatively long
and aromatic acids involved in acylation include (in rough order of their RP- wavelengths increases the selectivity of qual-itative and quantitative
LC elution sequence) malic, acetic, malonic, succinic, gallic, proto-catechuic, spectrophotometric methods, while the characteristic spectra of different
hydroxybenzoic, vanillic, caffeic, syringic, p-coumaric, ferulic and synaptic classes of flavonoids allow dis-tinction between them. These characteristics
acids [101]. For more detailed informa-tion regarding the relative elution are equally useful when UV–vis detection is performed in combination with
orders of different classes of flavonoids in RP-LC, the reader is referred to HPLC separation.
[101].
Flavonoids display two UV–vis absorption maxima: an absorp-tion band
in the region of 240–285 nm (Band II) ascribed to the A-ring, and a second
band at 300–550 nm due to the B-ring (Band I). Of these, the latter is more
As a rule, NP-LC is not suited to flavonoid analysis due to the lim-ited useful because it provides more selective information, since all flavonoids
solubility of these compounds in typical NP-LC mobile phases, the exception absorb in the region of 240–285 nm. Flavanols exhibit only Band II
being apolar flavonoids such as polymethoxylated flavones [113] and absorption (269–279 nm) due to a lack of conjugation between A- and B-
acetylated flavonoids [114]. One notable appli-cation of NP-LC is however rings; the same applies to flavanones, dihydroflavonols and isoflavones.
the separation of proanthocyanins
20 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Fig. 2. Examples of UV–vis spectra of different classes of flavonoids: (A) anthocyanins, (B) flavanols, (C) flavanones, (D) flavones, (E) flavonols and (F) isoflavones.
Source: Reproduced with permission from [56].
Flavonols and flavones show Band I absorption between 300 and 380 nm, aromatic acids leads to increased absorbance in the UV region close to Band
whereas anthocyanidins are easily distinguished by their Band I absorption II (260–290 nm), whereas for hydroxycinnamic acids increased absorbance is
between 460 and 550 nm in the visible range. Typical examples of the UV–vis observed in the region 305–325 nm. In the interpretation of UV spectra
absorbance spectra of the principal classes of flavonoids are presented in Fig. obtained using on-line diode array detection, it is important to consider the
2. effect of the solvent (both the nature of mobile phase constituents and their
As a rule, increasing hydroxylation of A- and B-rings leads to composition at the point of elution) when comparing with reference spectra,
bathochromic shifts in the wavelength of maximum absorption, max, of Bands since this can have a significant effect on flavonoid spectra. For more detailed
II and I; this effect is somewhat offset by methylation of the hydroxyl groups. information on the UV–vis absorption characteristics of flavonoids, the reader
Glycosylation does not have a significant effect on the absorbance spectra of is referred a range of excellent dedicated texts available [101,110,140].
most flavonoids, and the nature of the sugar moiety has no effect. Exceptions
are glycosylation at C-3, which causes a hypsochromic shift of 15–20 nm in
absorption Band I for flavonols and flavones, C-glycosylation which results in While the structural analysis of flavonoids based on their UV–vis spectra
a bathochromic shift of Band II, and the absence of a shoul-der at 440 nm for has in recent years largely been surpassed by more pow-erful tools such as
anthocyanidin-3,5-di-O-glycosides compared to anthocyanidin-3-O- MS and NMR, useful information can easily be obtained based on UV–vis
diglycoside [101,139]. Acylation involving spectral properties, the most impor-tant being the chemical class of
flavonoids. For this reason UV–vis
A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 21
detection remains a very useful technique in combination with HPLC and MS on general ionisation and fragmentation processes. The contem-porary
for flavonoid analysis. Because UV–vis detection is non-destructive, the importance of LC–MS in flavonoid analysis is reflected by the large number
detector can be connected before other detec-tors such as MS and NMR, is of excellent review articles reported in the last decade on this topic
compatible with high mobile phase flow rates and provides extremely reliable [59,60,110], and the reader is referred to these for more in-depth information
quantification (provided that co-elution of compounds with similar on flavonoid identification by LC–MS. Furthermore, much more detailed
absorbance properties can be avoided) [137]. The sensitivity of UV detection infor-mation may be obtained regarding specific classes of flavonoids such as
depends on the flavonoid target and instrument used, but is generally in the dihydroflavonols [151], isoflavones [152], flavonoid-
region of 0.02–10 ng injected mass [94,95,103,141,142], which is sufficient
for most natural product and food applications. Further-more, UV–vis spectra [153] and flavone-di-C-glycosides [154], flavonoid aglycones [155,156],
can be used to distinguish between different classes of flavonoids as outlined flavonoid-O-glycosides [157,158] and flavonoid glyco-sides [108,159,160] in
above. While this may sound trivial, this information in many cases may dedicated research reports.
prove pivotal. For example, sev-eral glycosylated flavonoids exhibit identical The following discussion will be limited to the currently most relevant
MS behaviour due to identical molecular formulae and fragmentation, yet LC–MS ionisation sources, the API sources electrospray ionisation (ESI),
belong to dif-ferent flavonoid families [143]. As an example, even accurate atmospheric pressure chemical ionisation (APCI) and atmospheric pressure
mass MS cannot distinguish between the delphinidin and cyanidin gly-cosides photoionisation (APPI). Fortunately, the general ionisation and fragmentation
behaviour of flavonoids is largely independent of the ionisation source and
(anthocyanins) and quercetin and kaempferol glycosides (flavonols),
instrument used [60,62,161,162], although relative ion intensities may vary
respectively, unless high-collision energy CID exper-iments are performed.
some-what [60,153,163] and some differences have been noted between CID-
However, UV–vis spectra of anthocyanins and flavonols are clearly distinct 2 3
(Fig. 2) and therefore allow facile assignment of the chemical class without MS/MS and in-source CID spectra [164] and between MS and pseudo MS
additional experiments. Furthermore, UV absorbance at ∼500 nm can be used spectra on IT and Q-TOF instruments [162]. Never-theless, the following brief
for selec-tive detection and accurate quantification of anthocyanins, even in overview can be considered generally applicable to most instruments.
cases where co-elution occurs with flavonols. In light of the fact that UV
detectors are relatively cheap and commonly available, it is no surprise that
on-line hyphenation of HPLC separation with UV and MS detection in For all API sources, the composition of the mobile phase plays a critical
sequence has become the norm in flavonoid analysis, especially in profiling role in ionisation, since analytes should either be ionised in solution (ESI) or
methods where the aim is to obtain maximum information [144]. in the gas phase after mobile phase evaporation (APCI and APPI). In fact,
divergent conclusions have been drawn regarding the best mobile phase
composition for flavonoid analysis. For negative mode ESI ionisation, optimal
mobile phases reported in literature include 0.1% formic acid [108], 10 mM
ammonium acetate (pH 4) [165] or 0.5% acetic acid [156], whereas for posi-
Post-column addition of UV shift reagents has been used to study tive mode, 0.2–0.75% formic acid [108,165] is preferred; although
flavonoid substitution patterns, and post-column or pre-column derivatisation trifluoroacetic acid has been used, this additive has also been shown to reduce
(for example fluorescent labelling to exploit the benefits of this mode of ESI-MS sensitivity due to its ion-pairing prop-erties [108,165]. For
detection) has also been used in flavonoid analysis, although these are less anthocyanins, highly acidic mobile phases containing 5–10% formic acid are
commonly used, and since the anthocyanidins occur as flavylium cations in
common nowadays [145,146].
such mobile phases, positive ionisation is almost exclusively used [138]. The
Fluorescence detection is used much less often in the LC analysis of two most common organic modifiers used in the RP-LC anal-ysis of
flavonoids, a consequence of the fact that only few flavonoid classes exhibit flavonoids, methanol and acetonitrile, have little direct effect on the ionisation
fluorescent behaviour: isoflavones, methoxylated flavones and flavonoids of molecules, although nebulisation effi-ciency will be affected by the %
organic modifier in the mobile phase (generally low percentages of
with an OH substituent at carbon 3 (Fig. 1) [62]. Significantly, this includes
acetonitrile are detrimental)
flavanols such as (epi)catechin and the procyanidins, which makes
fluorescence detection extremely useful for the selective and sensitive
analysis of these compounds, and the technique has continued to find
[108].
application in research focussed on these classes [147–150].
Negative ionisation has been shown to be more sensitive than positive
NMR clearly remains the most important spectroscopic tech-nique for ionisation for all ionisation sources [165], with ESI being the most sensitive
flavonoid analysis, although the technique is still relatively rarely used on-line [62]. This is not so much due to ionisation effi-ciency, but is rather a
in combination with HPLC separation due to the challenges associated with consequence of increased chemical noise in positive ionisation mode
this hyphenation. In LC-NMR, two approaches are possible: direct coupling [108,165]. Positive ionisation generally results in more extensive
using a flow probe for the NMR, or stop-flow NMR measurements. In the fragmentation than negative ionisation [59,108,165], and remains popular due
former case, deuterated solvents are used in combination with pulse to the amount of struc-tural information that may be obtained using this mode
sequences aimed at suppressing background signals. Stop-flow NMR involves [59,62,110], especially for the analysis of flavonoid-C-glycosides [153].
the use of a valve triggered from an upstream detector to park the peak of
In first-order MS spectra of flavonoids, with low cone voltages, protonated
interest in the flow cell, and provides much higher sensitiv-ity as a + −
consequence. The power of LC-NMR for natural products has been [M+H] and de-protonated [M−H] ions are most com-mon, with little
fragmentation present. Depending on the mobile phase composition, however,
demonstrated admirably by Wolfender and co-workers; since the topic falls − − −
outside the scope of this work, readers are referred to several important adducts such as [M−H+AcONa] , [M+HSO4] , [M+AcO] and
−
overviews of the field for further information [38–40]. [M−H+AcONa+MeOH] may be detected, in addition to molecular
− + +
complexes such as [2M−H] , [2M+H] and [2M+Na−H] [110,153].
Complexation with metals and chelating ligands (added post-column in the
case of LC–MS) has also been used in the MS/MS study of flavonoids
(n) [37,60].
2.3. General aspects of LC–MS analysis of flavonoids
For most of the fragmentation pathways outlined below, tan-dem mass
In this section, a brief overview of the background infor-mation relevant spectrometers operated under conditions of relatively high collision energies
(10–47 eV) are used to generate second-order mass spectra. However, single
to the MS detection and MS/MS structural elucidation of flavonoids will be
stage mass spectrometers may
presented, with the emphasis
22 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
be used to obtain fragmentation information by adaptation of the cone voltage FAB-MS/MS following acetylation has been reported [173], although this
to attain in-source CID [110,166]. approach has found limited application since.
Flavonoid-di-O-glycosides, where two monosaccharides are linked to
±3 ±7
different aglycone hydroxyls, show Y 0, Y 0 fragments due to the cleavage
2.3.1. Flavonoid glycosides ±3 ±5
of one of the glycosidic bonds (for anthocyanins these will be Y 0, Y 0). In
The nomenclature proposed by Domon and Costello [167] has found
±3 ±7
widespread acceptance for the fragmentation of glycosylated flavonoids, and some cases Y 0Y 0 fragments corre-sponding to cleavage of both sugar
is illustrated for flavonoid O- and C-glycosides in Fig. 3A and B, respectively. linkages are also observed [110]. The susceptibility of the sugar–aglycone
(Note that the ionisation mode is spec-ified by the superscript + or − in this bond to cleavage varies according to position, with fragmentation
nomenclature [62]). When the charge is located on the aglycone fragment, preferentially occurring at the C-3 position compared to the C-7 position
k,l ± ± ± [174]. This allows assignment of glycan substitution for flavonoid-di-O-
ions are labelled X j , Y j and Z j , where the subscript j denotes the
glycosides containing sugars of different molar masses.
number of the interglycosydic bond cleaved (the aglycone–glycosidic bond is
numbered 0) and superscripts k and l indicate cleavages within the The relative stability of the C-glycosidic bond means that high collision
carbohydrate rings. When more than one glycosylation pos-itions are energies are required for fragmentation, which mainly involves cross-ring
involved, an additional superscript m is used to denote the position of cleavages of the saccharide residue and the loss of water molecules. Typical
k,l ±m ±m fragmentation patterns are pre-sented in Fig. 3B, with the main fragment ions
aglycone substitution, e.g. ions X j , Y j . For charged carbohydrate
k,l ± ± ± 0,2 ± 0,1 ± 0,4 ±
fragments, ions are designated A i , B i and C i , where the subscript i corresponding to X 0, X 0, and X 0, often coinciding with the loss
0,3 ±
(≥1) represents the number of the glycosidic bond cleaved, counting from the of water or CH2O fragments [175,176]. The X 0 ion is only present at
non-reducing end numbered 1. 0,3 ± 0,2 ±
noticeable levels for flavonoid-C-8-glycosides [110]. X 0, X 0, and
0,1 ±
X 0 fragment ions correspond to losses of 90, 120 and 150 amu,
Flavonoids commonly occur in nature as glycosidic species, with two respectively, for flavonoid-hexosides. The corresponding fragments for
flavonoid-pentosides result in losses of 60, 90 and 120 amu [59,110].
types distinguished based on the nature of the flavonoid–saccharide linkage.
Flavonoid-O-glycosides are more common (and exclusively occur in the case
of anthocyanins), sugars are attached to one or more hydroxyl groups through C-6 and C-8 positional isomers can be distinguished based on more
an acid-labile C O bond; hydroxyl groups at C-3 and C-7 are common 0,2 ± 0,3 +
extensive fragmentation of X 0 fragments for the former; the X 0 and
glycosylation sites (C-3 and C-5 for anthocyanidins). Flavonoid-C-glycosides +
[M+H−4H2O] ions have been proposed as being diagnostic for the
contain acid-resistant C C bonds linking the sugar directly to the flavonoid
differentiation of C-8 and C-6 isomers in posi-tive ESI-MS/MS [153]. Waridel
nucleus (Fig. 3B). C-glycosidic bonds occur virtually exclusively at carbons
et al. [162] compared Q-TOF and IT instruments in positive and negative
C-6 and C-8 of the aglycone [108]. mode APCI, and found loss of water to be characteristic for the C-6
0,2 +
glycosides, whereas MS/MS of the X ion in positive mode allowed
distinction if the iso-mers based on the losses of small molecules (and the
The labile nature of the O-glycosidic bond ensures that the bond is 1,3 +
cleaved under low collision energy conditions, lead-ing to the loss of neutral presence of a A ion observed in Q-TOF spectra only). A more complete set
sugar residues (loss of 162, 146 and 132 amu for hexosides, deoxyhexosides of guidelines has been reported in both positive and negative ionisation [177].
± Similar to flavonoid-di-O-glycosides, flavonoid-di-C-glycosides containing
and pentosides, respec-tively) and the corresponding Y 0 aglycone ion in the
case of monoglycosides [59,60,110]. The mechanism for this fragmenta-tion sugar residues of different mass can be assigned based on the more extensive
has been shown to entail rearrangement involving hydrogen migration [169]. fragmentation of the C-6-glycoside moiety [59]. More recently, an approach to
± iden-tify flavone-di-C-glycosides based on negative ionisation MS/MS spectra
In some cases the corresponding saccharide ions, B 1, are observed at m/z has been reported [154] (refer to Section 4.2.1 for further details).
163 or 147 for hexoses and pentoses, respectively [59,110]. In negative
• −
ionisation mode, a radical agly-cone ion, [Y 0−H] , resulting from the
homolytic cleavage of the glycosidic bond, may also be observed [59,170]. Flavonoid-O,C-diglycosides can contain the O-glycoside moiety linked
Flavonoid-O-diglycosides, where a disaccharide is attached to the aglycone, either to a hydroxyl group of the aglycone or to a hydroxyl group of the C-
± ±
show Y 1 and Y 0 fragments, with higher collision energies favouring the bound glycoside. Fragmentation of these compounds entails both cleavage of
± the O-glycosidic bond, which typically occurs at lower collision energies, and
formation of the Y 0 aglycone ion. An interesting exception is observed for
flavonol- and flavanone-O-rutinosides (rhamnosyl-( 1→6)-glucose) and cross-ring cleavage of the C-bound saccharide at higher collision energies
flavanone-7-O-neohesperidoses [59,110].
Differentiation of isomeric acylated flavonoid-glycosides is also possible
to some extent based on MS/MS spectra [178], although limited systematic
+ studies for this class of compounds have been reported.
(rhamnosyl-( 1→2)-glucose), where an Y * ion is observed. This ion results
+
from the loss of the inner glucose residue ([M+H−162] ) through hydrogen
migration from the C-5 hydroxyl of the agly-cone to the terminal rhamnose,
± 2.3.2. Flavonoid aglycones
followed by a rearrangement reaction [59,110,171]. The ratio of the Y 1and According to the nomenclature proposed by Ma et al. [168] (improved
±
Y 0 fragments in positive ionisation mode can be used to assign the class of from that developed by Mabry et al. [140]) for flavonoid aglycones, ions are
flavonoid-O-disaccharide, whereas negative or positive ionisation second designated i,jA± 0+ and i,jB± 0 for fragment ions containing intact A- and B-
order spectra can be used to distinguish between iso-meric flavonoid-O- rings, respectively. The superscripts i and j indicate the cleaved C-ring bonds,
neohesperidosides and -O-rutinosides, which differ in terms of their while the subscript 0 dis-tinguishes these ions from carbohydrate fragments
interglycosidic linkage (1 → 2 and 1 → 6, respectively) [157,158,168]. This k,l ±
A i and B± i (i ≥ 1). In general, the fragmentation pathways discussed below
approach can be extended to di- to penta-glycosylated flavonoids to allow are applicable to both positive and negative ionisation, although the former
characterisation of the interglycosidic linkage and to differentiate positional mode is more commonly used in the structural analysis of flavonoids
isomers based on negative ionisation MS/MS spectra [172]. Especially the (exclusively in the case of anthocyanidins). Nega-tive ionisation requires
− − − 7−
relative abundance of Y 1, Z 1, Y 0 and Y 0 ions are determina-tive in higher collision energies and may lack some diagnostic ions in product spectra
this regard. The stereochemical assignment of the terminal monosaccharide [59,62,179]. Fragment ions
units in flavonoid-O-glycosides using ESI and
A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 23
(n)
Fig. 3. General fragmentation scheme and illustration of nomenclature used for the MS analysis of (a) flavonoid-O-glycosides (b) flavonoid-C-glycosides and (c) flavonoid aglycones [167,168].
formed from cleavage of the C-ring bonds are very informative, as they 0,2 ±
RDA fission of ring C (in essence resulting in the A fragment of the
provide information on the number and nature of substituents on the A- and relevant proanthocyanidin moiety) [181–183]; this path-way is energetically
B-rings. The fragmentation pathways entail retro-Diels–Alder (RDA) favoured in the top unit of proanthocyanidins
reactions involving the C-ring, typically at bond positions 1/2, 0/2, 0/3, 0/4 or [184] and is commonly followed by the loss of an additional water molecule.
2/4 [56,59,62,110,155] (Fig. 3C). The second fragmentation pathway, designated het-erolytic ring fission (HRF)
The relative prevalence of each fragmentation pathway depends on the [184], involves loss of the A-ring, again primarily of the top unit (this
1,3 ± 1,4 ±
class and substitution pattern of the flavonoid. The A 0 ion, observed for corresponds to the B fragment). The loss of a 126 amu neutral fragment is
all flavonoid subclasses in positive and neg-ative modes, is most readily indicative of a 1,3,5-trihydroxybenzene structure of the A-ring. The third
formed and often constitutes the base peak ion in second-order mass spectra fragmentation pathway involves quinone methide (QM) cleavage of the inter-
[59,62]. For flavones and flavonols, cleavage of the 0/2 bonds is common, flavan bond [181–183]. This pathway is useful in the assignment of the
0,2 ± 0,2 ±
leading to the formation of the B 0 ion, while the corresponding A 0 position of A-type interflavan bonds in higher oligomers [181,184]. A fourth
ion is characteristic for flavonols [155]. Further losses of 18 amu ( H2O), 28 mechanism, benzofuran-forming (BFF) fission, follows a similar mechanism
to HRF to produce a C-ring benzofuran derivative [185]. Further loss of water
amu ( CO), 42 amu ( C2H2O) and in the case of nega-tive ionisation 34 amu
molecules is also common in MS/MS spectra of proanthocyanins, which may
( CO2) or successive losses of these groups are also commonly observed in further complicate the spectra of high molecular weight compounds. Li and
MS/MS spectra. In the case of O-methylated flavonoids the loss of 15 amu Deinzer
± ± •
( CH3) is prominent, with the [M H−CH3] radical ion generally
constituting the base peak. Direct cleavage of the bond between the B- and C- [185] proposed a set of rules that may be used to identify the composition of
ring can be observed for some ions in negative ionisation mode and antho- procyanidins based on MS/MS spectra and the four fragmentation pathways,
±
cyanidins in positive mode [180], resulting in an [M−B-ring] fragment demonstrating the applicability thereof by assigning molecules up to a DP of
[179]. six. A similar approach has also been used in the characterisation of A-type
proanthocyanins [186]. It is important to note though that assignment of
Chalcones and dihydrochalcones differ from the other flavonoid stereoisomers is not possible based on MS/MS data.
subclasses in that they do not possess a heterocyclic C-ring (Fig. 1). In the
case of dihydrochalcones, the saturated 3-carbon chain frag-ments easily at Finally, it is worth pointing out that a range of electronic databases
low collision energies in positive ionisation mode. Cleavage of the bond containing MS/MS and HR-MS(/MS) data for metabo-lites, phenolics or
between the A-ring and the carbon atom from the keto group, followed by flavonoids are nowadays available [54,187,188], or may be compiled in-house
+ +
loss of a ketone, yields the product ions [C9H9O2] and [C7H7O] , [189], and these are finding increasing application in the screening and
respectively. Other ions characteristic for the dihydrochalcones include identification of plant flavonoids [190–193].
+ +
[C8H9O4] and [C6H7O3] , produced from the rupture of the and carbons
adjacent to the carbonyl [110]. Chalcones on the other hand have been shown
to fragment according to two pathways, depending on the number and 3. Advances in HPLC separation
position of phenolic substituents on the A-ring. The first reaction, prevalent
for compounds with a C -2 hydroxyl, involves formation of the cor- Like all forms of chromatographic separation, the performance of HPLC
responding flavanone isomer, which then fragments according to a RDA methods is inherently constrained by the properties of the target analytes, the
mechanism as outlined above. For compounds without a C -2 hydroxyl, stationary and mobile phases as well as instru-mental limits. This also has
cleavage of the bonds on either side of the carbonyl group is common [143]. clear implications for the analysis of flavonoids by HPLC. The two main
driving forces behind improve-ments in HPLC in general apply equally in the
HPLC analysis of flavonoids: the need for decreased analysis times and
Proanthocyanidins show relatively distinct fragmentation reac-tions, increased resolution. The former applies mainly in routine environments,
illustrated schematically in Fig. 4 for positive ionisation. The first of these where large numbers of samples need to be analysed daily, but is
involves elimination of a B-ring as a result of
24 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
OH HO O -H O
2 HO O
HO O OH BFF m/z 287
OH RDA OH OH
OH HO O HO
OH OH -H2O O
OH OH
HO O OH OH
OH
OH HRF m/z 427
m/z 563 O
O
QM BFF
OH OH OH
-H2O
OH
HO O
HO O
OH
O O
OH m/z 297
OH
OH
OH RDA
OH O
m/z 273 m/z 437
OH
OH OH m/z 285
HO O
HO O
OH
OH
m/z 291 OH
Fig. 4. General fragmentation scheme for procyanidins illustrated for a procyanidin dimer in positive ionisation mode. The fragmentation pathways involving retro-Diels–Alder (RDA), heterolytic ring
fission (HRF), quinone methide (QM) and benzofuran-forming fission (BFF) are demonstrated.
Source: Adapted from [185].
also important from a cost-saving perspective and in cases where analytical peaks – in this case, the sample components approximate a ran-dom
data is required within a short time. The latter require-ment is important for distribution and Davis and Giddings’ findings emphasise the importance of
the analysis of complex samples, especially where numerous chemically improved chromatographic performance.
related species which cannot be dis-tinguished by the detector used occur in For gradient separations, the peak capacity, n c, can be deter-mined
the same sample. This is clearly the case for a wide range of flavonoid- according to [197]:
containing natu-ral products [4] and therefore explains the incentive for (1)
n =1+
c t g
improving HPLC separation in flavonoid analysis.
(1/n) 1w
n b
As a measure of the separation performance of chromatographic systems,
where tg refers to the gradient time (i.e. the separation window) and w b the
the concept of peak capacity has gained popularity as an alternative to more
baseline width of analyte peaks averaged for n peaks. Typical peak capacity
conventional measures such as efficiency and resolution, which are limited to
values for RP-LC vary between ∼50 for fast separations and in the region of a
isocratic analyses and selected peak pairs, respectively. The peak capacity of a
separation represents the number of compounds that can theoretically be few hundred for highly opti-mised separations. Taking cognisance of the
separated under defined experimental conditions (column, gradient, flow rate, findings of Davis and Giddings, and considering that many natural products
tem-perature, etc.) [194]. It is important to note though that no real-life contain in excess of 100 flavonoids, it is clear that continued efforts to
separation can resolve a number of components equal to the peak capacity of improve the performance of LC separations are of critical importance also in
that separation, a consequence of irregular distribu-tion of analyte peaks the field of flavonoid analysis.
across the separation window. Davis and Giddings [195] used the statistical
theory of component overlap to demonstrate that to improve the probability of
obtaining pure chromatographic peaks, the peak capacity should exceed the 3.1. One-dimensional HPLC
num-ber of components to be separated by a significant factor. To use an oft-
quoted example, to resolve with 98% certainty n randomly dis-tributed peaks, In the last 15 years, a number of significant developments in the field have
the peak capacity should be equal to 100 × n [196]. For relatively simple extended the performance limits of (uni)-dimensional HPLC. Several of these
samples containing few analytes of interest, the experienced chromatographer have gained widespread acceptance also in the area of flavonoid analysis,
can select experimental condi-tions so as to improve the likelihood of notably the use of UHPLC, alternative stationary phase morphologies such as
obtaining separation (for example through the careful selection of stationary monolithic and superficially porous phases, comprehensive multidimensional
phase, mobile phase composition and gradient, temperature, etc.). However, separations and to a lesser extent the used of elevated temperature. In the
for highly complex samples, this is no longer possible for all analyte following sections, the fundamental background for each of these approaches
will be outlined briefly prior to presenting an overview of their application in
the analysis of flavonoids during the last 7 years.
A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 25
where ε and εT are the external and total porosities, respectively. It is clear that a reduction in d p coincides
where u0 is the linear velocity and the viscosity of the mobile phase. K v0 is related
to the particle size via [200,201]:
u · ·L with a significant decrease in column permeability. The somewhat counter-intuitive result of this is that
Kv0 = 0
higher overall efficiencies are attained with an increase in particle size, a result of the relative importance
P of column per-meability and (current) instrumental pressure constraints.
2 3
dp · ε In this context, plate height curves such as the van Deemter provide limited information due to the fact
Kv0 = 2 that column perme-ability, and by extension the maximum column length, are not taken into account (H is
180 · (1 − ε) · εT
independent of L). A more accurate rep-resentation of the ultimate performance of column/instrument
combinations can be obtained using so-called kinetic plots, the origins of which can be traced to Giddings
[202], but which have been adapted by allied band broadening due to the establish-ment of a radial temperature
various authors [203–205]. In essence, these gradient inside the column [211,212]; this has also been confirmed
plots combine plate height data with experimentally [213]. This effect can be minimised by reducing the pressure
column permeability and pressure (i.e. the flow rate or column length), or more practically by reducing the
constraints to provide an accurate reflection internal diameter of the column (which coincides with a reduction in
of the performance of specific volumetric flow rate, thereby further reducing the effects of frictional
column/instrument combination as a heating). It is for this reason that most UHPLC separations are performed on
function of analy-sis time. The group of columns
Desmet developed a family of kinetic plots
[200,205–207] which allow facile
comparison of the performance of different
columns as a function of a range of
parameters; this approach has also been (2) of 2.1 mm internal diameter (i.d.) or lower. From the perspective of
extended to gradient separations, where hyphenation to MS, this is clearly beneficial. Optimal flow rates on these
systems can be compared in terms of peak columns (taking into account the plate height behaviour of these phases, cf.
Fig. 5a) are in the region of 0.2–0.5 mL/min and are therefore ideally
capacities obtainable as a function of compatible with ESI-MS. The much narrower peak
analysis time [208]. (3) widths typically obtained do however place severe demands on the acquisition
rate of MS detectors. While 4.6 mm i.d. UHPLC columns
Operation at
elevated
pressures is
associated with
the risk of
frictional
heating and
26 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
H (µm)
t R (s)
10 1000
5 100
0 10
0 1 2 3 4 5 6 7 10 100 1000
6 u0 (mm/s) 1000000 nc
tetrameric procyanidin, 25°C tetrameric procyanidin, 25°C
tetrameric procyanidin, 50°C 100000 tetrameric procyanidin, 50°C
catechin, 25°C c catechin, 25°C d
4
catechin, 50°C 10000 catechin, 50°C
(s)
1000
H (µm)
Rt
2
100
0 1 2 3 4 5 6 7 10 100 1000
u0 (mm/s) nc
Fig. 5. (a) Comparison of plate height curves obtained for the flavanol catechin on a 5 m porous phase ( Pmax 400 bar, blue diamonds), a 1.7 m porous phase ( Pmax 1000 bar, yellow circles) and a 2.6
m superficially porous phase ( Pmax 600 bar, brown squares). (b) shows the corresponding peak capacity (n c ) as a function of analysis time for catechin (as determined using the corresponding
◦
retention factors). (c) illustrates the plate height curves for catechin (MW 290) and a tetrameric procyanidin (MW 1155) at 25 and 50 C on a 1.7 m porous phase ( P max 1000 bar), and (d) illustrates
the corresponding retention time vs nc plot. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
are commercially available, and have also found extensive use in flavonoid validation results, which amply demonstrate the suitability of columns and
analysis, optimal flow rates on these columns (typically 1.2–2.5 mL/min, instrumentation for routine analysis. We will not attempt to address each of
depending on the analytes) require flow split-ting prior to MS detection. these, but rather to highlight some trends concerning the application of
Furthermore, frictional heating effects become a concern on such column UHPLC in this field.
dimensions (something which is not sufficiently recognised by many The principal application of UHPLC technology, in flavonoid analysis as
practitioners in flavonoid analysis). in HPLC in general, is to attain faster separation and higher throughput. This
makes sense, since 100 and 50 mm columns packed with sub-2 m particles
Finally, the impact of typical UHPLC column dimensions should be provide roughly equiv-alent efficiencies to 250 and 150 mm columns packed
considered in the context of extra-column band-broadening. For the short, 2.1 with 5 m particles, respectively. This performance is obtained in much shorter
mm i.d. columns which are the norm in UHPLC, peak volumes are especially analysis times for UHPLC columns because (i) the column is shorter and (ii)
small because of the reduction in flow rate and the high column efficiency. In the optimal mobile phase linear velocity is higher. The practical consequence
these circumstances, dispersion in extra-column volume can significantly is that similar separation is attained as for conventional HPLC, with
affect the measured peak width. For example, Gritti and Guiochon showed commensurate sensitivity, robustness etc., but at analysis times 2–9× shorter.
that even the most sophisticated modern instruments (excluding capillary and A typical example is the speeding up of routine methods on conventional
nano-systems) lead to losses of 15–25% of column efficiency in the case of HPLC systems to UHPLC columns and instrumentation [107]. Another
the latest generation high-efficiency columns in isocratic mode [214]. Fekete application area is in the second dimension of LC × LC separations, where
et al. [215] reached similar conclusions for sub-2 m porous, superficially sub-2 m phases show clear benefits for maximising performance in the
porous and monolithic columns. While the effect of extra-column band shortest possible analysis time – here the reduced C-term band broadening on
broadening is significantly reduced in gradient separations due to focusing on these phases is exploited on short columns operated at high flow rates.
the column, dispersion in connection tubing at the outlet of the column can
still have a pro-found effect on the measured peak width. This is often evident
from comparison of peak widths in UV and MS chromatograms obtained by
UHPLC-DAD-MS, where much broader peaks are observed in the latter. This A second approach is to use longer columns packed with sub-2 m phases,
may be ascribed in part to the size of most MS instru-ments, which requires in this case utilising the increased pressure capa-bilities of UHPLC columns
relatively long connection tubing between the exit of the UV cell and the and instrumentation to obtain higher resolution for complex flavonoid
ionisation chamber. However, small changes in tubing length and/or internal mixtures. Examples are found in the analysis of rooibos tea [216] and
diameter can produce much better MS chromatograms. strawberry [217] phenolics and red wine anthocyanins [218] on 200 mm
UHPLC columns. These columns provide isocratic efficiencies ∼2.4× higher
than standard 250 mm 5 m columns, which translates into an increase in reso-
lution by a factor of about 1.5.
UHPLC technology has matured rapidly in the last decade, and has found
widespread acceptance in the separation community – to the extent that the
technique has in many fields replaced conven-tional HPLC methods for 3.1.2. Monolithic columns
routine analysis. That this is also the case in flavonoid analysis is confirmed An alternative approach to overcome the
by the large number of applications reported in Tables 1, 2 and 4–7, also
evidenced by the observa-tion that many of the papers cited in this work permeability/pressure limitations of HPLC is to use monolithic
report method columns. Monolithic stationary phases are cast as single
continuous phases with interconnected skeletons and
through-pores. The higher external
A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 27
Table 1
Applications of advanced 1-dimensional LC methods without MS detection to flavonoid analysis (2009–2015).
Staghorn sumac fruit Quercetin-3-rhamnoside, UV C18 (100 mm × 2.1 mm, 1.7 m dp ), A: [309]
◦
(Rhus hirta L.) quercetin and peonidin-3- QIT FR = 0.35 mL/min, 30 C, 14 min MeOH/H2 O/FA,
O-galloyl-hexose (ESI+/−) 95:2:3 (v/v/v),
B:
MeOH/H2 O/FA,
C18 (100 mm × 2.1 mm, 1.7 m dp ), 2:95:3 (v/v/v)
Hops (Humulus lupulus Xanthohumol and UV A: 0.1% FA, B: [310]
◦
L.) isoxanthohu-mol FR = 0.25 mL/min, 30 C, 13 min ACN
‘Copaíba’, ‘copaiva’ or Quercitrin and afzelin UV C18 monolith (100 mm × 4.6 mm), A: H2 O, B: ACN [226]
‘paú-de-óleo’ FR = 1.0 mL/min, 60 min
(Copaifera Anthocyanins, flavonols, UV C18 (100 mm × 4.6 mm, 1.8 m dp ), A: 0.5% TFA, B: [107]
langsdorffii)
Juices of strawberry,
◦ d
American cranberry, flavanones, FL FR = 1.0 mL/min, 25 C, 40 min TFA /ACN/H2 O
dihydrochalcones and b (0.5/50/49.5,
bilberry, sour cherry, Q (ESI+)
black grape, orange and flavan-3-ols (48 flavonoids) v/v/v)
apple Apigenin, luteolin and UV C18 (150 mm × 2.1 mm, 1.7 m dp ), A: H2 O/MeOH [266]
Natural dyestuffs
◦
genistein FR = 0.2 mL/min, 40 C, 40 min (10/90, v/v), B:
MeOH C: 0.1%
Q (ESI+/−) PFP (50 mm × 2.1 mm, 1.7 m dp SP), FA
Chokeberry (Aronia Anthocyanins, flavonol A: 5% FA, B: [94]
melanocarpa, Aronia glycosides (7 flavonoids) FL FR = 0.2 mL/min, 18 min MeOH/H2 O
arbutifolia and Aronia UV (30/70, v/v)
prunifolia) Catechins (8 compounds) UV C18 monolith (100 mm × 4.6 mm), H2 O/ACN/MeOH [227]
Green, oolong and
black teas FR = 1.4 mL/min, 7 min (83/6/11, v/v/v,
C18 (50 mm × 2.1 mm, 1.7 m dp ) isocratic)
Red, white, black, and Flavan-3-ol derivatives and UV A: 1% FA, B: [150]
◦
green teas, cocoa quercitin (8 flavonoids) FL FR = 0.5 mL/min, 35 C, 2 min MeOH
LIT-
Orbitrap
(ESI+) C18 (50 mm × 2.1 mm, 1.8 m dp ),
Barringtonia racemosa Rutin, quercetin and UV A: 0.1% TFA, B: [142]
kaempferol FR = 0.6 mL/min, 13.4 min ACN
Standards Flavones, isoflavones, UV C18 (50 mm × 3.0 mm, 2.6 m dp SP), A: 0.05 mM [90]
flavanones, flavan-3-ols FR = 3.0 mL/min, 2 min ammonium
and flavonols (15 acetate, B: ACN
flavonoids) C18 (50 mm × 2.1 mm, 1.8 m dp ),
Chia seeds (Salvia Isoflavones (5 flavonoids) UV A: 2% AA, B: [141]
hispanica L.) FR = 0.4 mL/min, 18 min H2 O/ACN/AA
C18 monolith (50 mm × 2.0 mm), (68/30/2, v/v/v)
Herbal dietary Flavonol, chalcone and UV A: 0.05% TFA [311]
◦
supplements flavanone aglycones and LIT FR = 1.2–1.3 mL/min, 20 C, 5 min (UV) or 0.1% FA
glycosides and flavan-3-ols (MS), B: ACN
(14 flavonoids) Q (ESI−) C18 (150 mm × 4.6 mm, 2.6 m dp SP),
Zhi-Zi-Da-Huang Isoflavone and flavanone A: 0.1% FA, B: [312]
decoction (TCM )
a glycones and glycosides, FR = 0.8 mL/min (0.2 mL/min to MS), ACN
flavonol glycoside and ◦
35 C, 55 min
flava-3-ols (13 flavonoids) Q (ESI−) PFP (100 mm × 4.6 mm, 2.6 m dp SP),
Soy Isoflavones (20 A: 1% AA, B: [93]
◦
compounds) UV FR = 0.8 mL/min, 25 C, 37 min ACN
Forsythia flowers Rutin UV UV: C18 (75 mm × 4.6 mm, 2.7 m dp A: [313]
◦
QIT (ESI−) SP), FR = 1.4 mL/min, 30 C, 22 min H2 O/orthophosphoric
QIT: C18 (150 mm × 2.1 mm, 1.9 m acid (99.5:0.5,
◦
dp ), FR = 0.2 mL/min, 25 C, 68 min v/w), B: ACN
A:
H2 O/ACN/FA,
95:5:0.1
(v/v/v), B: 0.1%
C18 (100 mm × 3.0 mm, 2.6 m dp SP) FA in ACN
Grape pomaces Flavonols and flavan-3-ols UV A: 0.1% FA, B: [314]
◦
(8 flavonoids) FR = 0.8 mL/min, 35 C, 20 min ACN
Soy bean, black bean, Isoflavones (14 UV C18 (100 mm × 2.0 mm, 2 m dp ), A: 0.5% FA, B: [315]
compounds) ◦ ACN
red bean and three FR = 0.3 mL/min, 40 C, 25 min
soybean pastes
a
TCM, traditional Chinese medicine.
c
b LIT, linear ion trap; QIT, quadrupole ion trap; FL, fluorescence detection; Q, (single) quadrupole; Q-TOF, quadrupole-time-of-flight. FR, flow rate;
SP, superficially porous; PFP, pentafluorophenyl.
d
ACN, acetonitrile; FA, formic acid; AA, acetic acid; MeOH, methanol; EDTA, ethylenediaminetetraacetic acid; THF, tetrahudrofuran; TFA, trifluoroacetic acid.
A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 29
Table 2
Sample(s) Flavonoids Hyphenation Stationary phases (column Mobile phases Detector(s) Reference(s)
mode(s), dimensions), flow rates,
separation temperature, analysis times
modes,
sampling
time(s)
Apple and cocoa Procyanidins and Off-line, 1 1 d UV [147]
D: Diol (250 mm × 1 mm, ( D) A: 1% AA in
c d e
flavonol glycosides HILIC × RP-LC, 5 m dp ), FR = 0.05 mL/min, ACN , B: FL
1 b d e
(41 compounds) ts = 1 min 70 min MeOH /H2 O/AA Q-TOF (ESI−)
2 (94.05/4.95/1
D: C18 ((2×)
50 mm × 4.6 mm, 1.8 m (v/v/v))
◦ 2 d
dp ), FR = 1.8 mL/min, 50 C, ( D) A: 0.1% FA ,
4/30 min B: ACN
Mate extracts Flavonol glycosides On-line, 1 1 UV [367]
D: RP-Amide ( D) A: H2 O (pH
e
(3 compounds) RP-LC × RP-LC, (250 mm × 1.0 mm, 5 m 3), B: ACN (pH 3) IT -TOF
1 ◦ 2 (ESI+/−)
ts = 2 min dp ), FR = 0.01 mL/min, 25 C, ( D) A: H2 O (pH
70 min 3), B: ACN (pH 3)
2
D: C18 (30 mm × 4.6 mm,
c
2.7 m dp , SP ),
◦
FR = 4 mL/min, 45 C
1 c 1
Longdan Xiegan Flavone and On-line, D: ILC (150 mm × 4.6 mm), ( D) 10 mM UV [368]
a b e
Decoction (TCM ) isoflavone ILC × RP-LC, FR = 0.05 mL/min, 400 min ammonium APCI-Q-MS
aglycones and 1 * 2 acetate (pH 6.8, (−)
ts = 10 min D: C18 (150 mm × 4.6 mm),
glycosides, FR = 2.0 mL/min (0.8 mL/min isocratic)
flavanone to MS) 2
( D) A: 0.1% AA, B:
glycoside (8 ACN
flavonoids) 1 c
D: PEG (150 mm × 2.1 mm,
Standards Flavan-3-ols, On-line, 1 2 UV [357]
( D, D) A: 10 mM
flavones, flavanone RP-LC × RP-LC, 5 m dp ), FR = 0.25 mL/min, ammonium
1 ◦
aglycones and ts = 0.58 min 40 C, 35 min, acetate, B: ACN
glycosides, flavonol 2
D: C18 (30 mm × 3 mm,
aglycones and 2.7 m dp SP), FR = 4 mL/min
◦
glycosides, (0.2 mL/min to MS), 60 C
isoflavone (13
flavonoids) 1
D: C8 (250 mm × 4.6 mm,
Fructus aurantii (TCM) Polymethoxylated Off-line, 1 2 UV [374]
( D, D) A: water,
flavones (42 RP-LC × RP-LC, 5 m dp ), FR = 1 mL/min, B: ACN IT-MS (ESI+)
1
flavonoids) ts = 1 min 30 min
2
D: C18 (250 mm × 4.6 mm,
5 m dp ), FR = 1 mL/min,
30 min
Red wines Flavan-3-ols and On-line, 1 1 UV [344]
D: Phenyl (250 mm × 1 mm, ( D) A: H2 O (pH
flavonol glycosides RP-LC × RP-LC, 5 m dp ), FR = 0.01 mL/min, 3), B: ACN (pH 3)
1 ◦ 2
(7 flavonoids) ts = 2 min 25 C, 80 min ( D) A: H2 O (pH
2 3), B: ACN (pH 3)
D: C18 (30 mm × 4.6 mm,
2.7 m, dp SP),
FR = 4 mL/min (0.2 mL/min
◦
to MS), 45 C
Red wine Flavan-3-ols, On-line, 1 1 2 UV [355]
D: PEG (150 mm × 2.1 mm, ( D, D) A: 10 mM
flavones, flavonols, RP-LC × RP-LC, 5 m dp ) FR = 40, 70, and ammonium
flavonol glycosides, 1 240 L/min, 35, 110, and acetate, B: ACN
ts = 1.5 min
◦
flavanone 140 min, 40 C
aglycones and 2
D: C18 (50 mm × 3.0 mm,
glycosides, 2.7 m dp SP), FR = 3.3 and
isoflavones (14 4.8 mL/min, 0.42, 1.4,
◦
flavonoids) 2.0 min, 50 C
Green tea Flavan-3-ols, Off-line, 1 1 UV [126]
D: Diol (250 mm × 1 mm, ( D) A: 1% AA in
proanthocyanidins, HILIC × RP-LC, 5 m dp ), FR = 0.05 mL/min, ACN, B: FL
1
flavonol aglycones ts = 1 min 70 min MeOH/H2 O/AA Q-TOF (ESI−)
and glycosides and 2 (94.05/4.95/1
D: C18 (50 mm × 4.6 mm,
flavone glycosides 1.8 m dp ), FR = 0.8 mL/min, (v/v/v))
(48 flavonoids) ◦ 2
50 C, 30 min ( D) 0.1% FA, B:
ACN
Stevia rebaudiana Flavone and On-line, 1 c 1 UV [366]
D: PA -G ( D) A: ACN (pH
extracts flavonol glycosides HILIC × RP-LC, (250 mm × 1.0 mm, 5 m 3), B: H2 O (pH 3)
(5 flavonoids) 1 2
ts = 20 s dp ), FR = 0.02 mL/min, ( D) A: H2 O (pH
101 min 3), B: ACN (pH 3)
2
D: C18 (30 mm × 2.1 mm,
1.8 m dp ), FR = 3.4 mL/min,
◦
70 C
Citrus juices Flavanones and On-line, 1 1 2 UV [343]
D: PEG (250 mm × 2.1 mm, ( D, D) A: 0.1%
flavones (11 RP-LC × RP-LC, 5 m dp ), FR = 50 L/min, FA, B: H2 O/ACN/ Q-MS (ESI−)
1
flavonoids) ts = 1 min 80 min isopropanol/FA
2 (39.9:20:40:0.1
D: C18 (50 mm × 4.6 mm,
2.7 m dp SP), (v/v))
◦
FR = 3.0 mL/min, 40 C
30 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Table 2 (Continued )
Sample(s) Flavonoids Hyphenation Stationary phases (column Mobile phases Detector(s) Reference(s)
mode(s), dimensions), flow rates,
separation temperature, analysis
modes, times
sampling
time(s)
Scutellaria barbata D. Flavone and Off-line, HILIC × HILIC: HILIC × HILIC: Q-TOF (ESI+) [375]
Don (TCM) flavanone HILIC × HILIC, 1 1 2 UV
D: Silica ( D, D) A:
aglycones and HILIC × RP–LC, (250 mm × 4.6 mm, 5 m ACN/water (95:5
1
glycosides (22 ts = 1 min dp ), FR = 1.0 mL/min, 30 min (v/v)) with 5 mM
flavonoids) 2 ammomium
D: Amide
(150 mm × 4.6 mm, 5 m formate, B: 5 mM
dp ), FR = 1.0 mL/min, 30 min ammonium
HILIC × RP: formate
1
D: Amide HILIC × RP:
(150 mm × 4.6 mm, 5 m 1 2
( D, D) A: 0.1% FA,
dp ), FR = 1.0 mL/min, 30 min B: 0.1% FA in ACN
2
D: C18 (150 mm × 2.1 mm,
5 m dp ), FR = 0.2 mL/min,
50 min
Standards Flavan-3-ols, On-line, 1 1 UV [362]
D: DioEDMA, monolith ( D) A: ACN, B:
flavones, flavonol HILIC × RP-LC, (180 mm × 0.53 mm), 10 mM
1 ◦
and flavanone ts = 1.5 min FR = 0.01 mL/min, 40 C ammonium
2
aglycones and D: RP-Amide, C18 and acetate (pH 3.1)
glycosides, phenyl hexyl (50 and 2
( D) A: 10 mM
isoflavone (13 30 mm × 3.0 mm, 2.6 and ammonium
flavonoids) 2.7 m dp SP), acetate (pH 3.1),
◦
FR = 3–4 mL/min, 50 C, B: ACN
1.5 min
Green and black teas Flavonol- Off-line, 1 c 1 e [81]
D: SEC ( D) A: H2 O, B: ELSD
(Camellia sinenis) glycosides, SEC × RP-LC, (300 mm × 7.8 mm, ACN UV
flavone-glycosides, 1 3 2 e
ts = 1 min 5 × 10 Da size exclusion), ( D) A: 0.1% FA, B: QqQ (ESI−)
◦
flavan-3-ol FR = 1.0 mL/min, 60 C, MeOH
derivatives (54 60 min
flavonoids) 2
D: C18 (50 mm × 2.1 mm,
1.7 m dp ), FR = 1.0 mL/min
◦
(0.1 mL/min to MS), 80 C,
10 min
Rooibos tea (Aspalathus Dihydrochalcones, Off-line/on- 1 1 UV [127]
D: Diol (250 mm × 1 mm, ( D) A: 2% AA in
linearis) flavanones, line, 5 m dp ), FR = 0.05 mL/min, ACN, B: Q-TOF (ESI+/−)
flavones, and HILIC × RP-LC, 100 min MeOH/H2 O/AA
flavonols (20 1 2 (93.05/3.95/2
ts = 1/2 min D: C18 (50 mm × 4.6 mm,
flavonoids) (off-line/on- 1.8 m dp ), (v/v/v))
line) ◦ 2
FR = 1.0 mL/min, 48 C, ( D) A: 1% AA, B:
29 min ACN
Six TCMs Isoflavone, flavone Off-line, 1 c 1 Q-TOF (ESI+) [376]
D: Click CD D) A: H2 O, B:
and flavanone HILIC × RP-LC, (150 mm × 4.6 mm, 5 m ACN, C: 100 mM UV
1 *
aglycones and ts = 2 min dp ), FR = 1.0 mL/min, 30 min ammonium
2
glycosides (25 D: C18 (150 mm × 2.1 mm, formate
2
flavonoids) 5 m dp ), FR = 0.3 mL/min, ( D) A: 0.2% FA, B:
c 0.2% FA in ACN
15 min or Click OEG
(150 mm × 2.1 mm, 5 m
dp ), FR = 1 mL/min, 30 min TOF (ESI−)
Qingkailing (TCM) Baicalin and On-line, 1 1 [350]
D: SEC (20 mm × 2.1 mm), ( D) A: 10 mM
◦
wogonoside SEC × RP-LC, FR = 0.02 mL/min, 25 C ammonium
1 ** 2
ts = 5 min D: C18 (100 mm × 2.1 mm, formate (isocratic)
2
1.7 m dp ), ( D) A: 0.1% FA, B:
◦
FR = 0.7 mL/min, 80 C ACN
Standards Flavan-3-ols, Online, 1 1 2 UV [377]
D: PEG ( D, D) A: 10 mM
flavones, flavonols RP-LC × RP-LC, (150 mm × 2.1 mm, 5 m ammonium
and flavanones (14 1 acetate (pH 3.1),
ts = 1.5 min dp ), FR = 0.066 mL/min,
◦
flavonoids) 40 C, 75 min B: ACN
2 c
D: PFP (50 mm × 3.0 mm,
2.6 m dp SP),
◦
FR = 2.5 mL/min, 40 C
Standards Flavones, flavonols, On-line, 1 1 2 UV [231]
D: Zwitterionic monolith ( D, D) A: 0.01 M
flavanones, HILIC × RP-LC, (210 mm × 0.53 mm), FR = 2 ammonium
flavan-3-ols and 1 ◦ acetate, B: 0.01 M
ts = 1.5 min and 3 L/min, 60 C, 60,
isoflavones (15 100 and 140 min ammonium
flavonoids) 2 acetate in ACN
D: C18 (30 mm × 3.0 mm)
◦
FR = 4.5 mL/min, 50 C, 1.0
and 1.5 min
A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 31
Table 2 (Continued )
Sample(s) Flavonoids Hyphenation Stationary phases (column Mobile phases Detector(s) Reference(s)
mode(s), dimensions), flow rates,
separation temperature, analysis times
modes,
sampling
time(s)
Apple Procyanidins, On-line, 1 1 IT-MS (ESI−) [369]
D: Diol (150 mm × 1.0 mm, ( D) A: ACN/AA
dihydrochalcones HILIC × RP-LC, 5 m dp ), FR = 21 L/min, (98/2 (v/v)), B: UV
1
and flavonols (64 ts = 78 s 50 min MeOH/H2 O/AA
2
flavonoids) D: C18 (50 mm × 4.6 mm, (95/3/2 (v/v/v))
2
2.7 m dp SP), ( D) A: 0.1% FA, B:
FR = 3.0 mL/min ACN and A: 0.1%
FA, B: ACN/MeOH
1 (50/50 (v/v))
D: Diol (250 mm × 1 mm,
Cocoa Procyanidins (38 On-line, 1 UV [340,342]
( D) A: 1% AA in
compounds) off-line, 5 m dp ), FR = 0.025 mL/min ACN, B: FL
stop-flow, (on-line) and 0.05 mL/min MeOH/H2 O/AA Q-TOF (ESI−)
HILIC × RP-LC, (stop-flow and off-line), (94.05/4.95/1
1
ts = 1 min 50 min (stop-flow and (v/v/v))
(off-line and off-line) and 100 min 2
( D) A: 0.1% FA, B:
stop flow), (on-line) ACN
1 2
ts = 2/3 min D: C18 column
(on-line) (50 mm × 4.6 mm, 1.8 m
◦
dp ), FR = 1.5 mL/min, 50 C,
12.6 min Q-TOF (ESI−)
Grape seed Procyanidins (78 On-line, 1 1 [148]
D: Diol (250 mm × 1 mm, ( D) A: 1% AA in
single peaks, HILIC × RP-LC, 5 m dp ), FR = 0.025 mL/min, ACN, B: FL
2
species of DP up to ts = 2 min 100 min MeOH/H2 O/AA
16 detected) 2 (94.05/4.95/1
D: C18 (50 mm × 4.6 mm,
2.6 m dp ), FR = 1.5 mL/min, (v/v/v))
◦ 2
50 C, 2 min ( D) 0.1% FA, B:
1 ACN
D: Diol (150 mm × 1.0 mm,
Grape seeds Procyanidins (43 On-line, 1 UV [230]
( D) A: 2% AA in
flavonoids) HILIC × RP-LC, 5 m dp ), FR = 0.015 mL/min, ACN, B: IT-MS (ESI−)
1
ts = 1.3 min 85 min MeOH/H2 O/AA
2
D: C18 (50 mm × 4.6 mm, (95/3/2 (v/v/v))
2
2.7 m dp SP), FR = 3 mL/min ( D) A: 0.1% FA, B:
(0.6 mL/min to MS), 1.3 min ACN/MeOH (50/50
2 (v/v))
D: C18 monolith
(100 mm × 4.6 mm), 1.3 min 2
( D) A:0.1% FA, B:
MeOH or
ACN/MeOH (50/50
1 (v/v))
D Diol (250 mm × 1 mm,
Cocoa and red grape Proanthocyanidins Off-line/on- 1 UV [363]
( D) A: 1% AA in
seeds (42 compounds) line, 5 m dp ), ACN, B: Q-TOF (ESI−)
e
HILIC × RP-LC, FR = 0.05–0.025 mL/min MeOH/H2 O/AA ABTS assay
1 2 (94.05/4.95/1
ts = 1 min D C18 (50 mm × 4.6 mm,
(off-line), 2 min 2.6 m dp SP), (v/v/v))
(on-line) ◦ 2
FR = 1.5 mL/min, 50 C, ( D) A: 0.1% FA, B:
15/2 min (off-line/on-line) ACN
Fenugreek seeds Flavone glycosides Off-line 1 1 d UV [378]
D: C18/propylphenyl ( D) A: 0.1% TFA ,
(15 flavonoids) (heart-cutting) (100 mm × 1 mm, 5 m dp ), B: MeOH/H2 O/TFA Q-MS (ESI+/−)
◦
and on-line, FR = 0.017 mL/min, 20 C (50/50/0.1 (v/v/v))
2 2
RP-LC × RP-LC, D: C18 (100 mm × 2.1 mm, ( D) A: 0.1% TFA,
1
ts = 1.5–4 min 2.6 m dp SP) (off-line) or B: ACN/H2 O/TFA
* C18 (75 mm × 2.10 mm, (33/4/8/0.05
(on-line)
3 m dp ) (on-line), (v/v/v))
FR = 2.3 mL/min (1:1 split
◦
before MS), 20 C
Blueberries, black Anthocyanins (73 Off-line, 1 1 UV [248]
D: Amide ( D) A: 0.4% TFA in
beans, red radish, red compounds) HILIC × RP-LC, (150 mm × 4.6 mm, 2.5 m ACN, B: 0.4% TFA Q-TOF (ESI+)
grape skins, red 1 ◦ 2
ts = 1 min dp ), FR = 0.2 mL/min, 50 C, ( D) A: 7.5% FA, B:
cabbage. 79–113 min 7.5% FA in ACN
2
D: C18 (50 mm × 4.6 mm,
2.6 m dp SP),
◦
FR = 0.5 mL/min, 50 C,
40 min
Ge-Gen (kudzu root, Isoflavones (19 Heart-cutting, 1 c 1 UV [364]
D: CSH C18 ( D) A: 0.1% FA, B:
TCM) flavonoids) on-line, (100 mm × 2.1 mm, 1.7 m MeOH IT-MS (ESI−)
◦ 2
RP-LC × RP-LC, dp ), FR = 0.1 mL/min, 50 C, ( D) A: 0.1% FA, B:
1
ts = 0.5 min 35 min ACN
2
D: Phenyl-hexyl
(50 mm × 3.0 mm, 2.7 m dp
SP), FR = 2.5 mL/min
(0.25 mL/min to MS), 50 ◦ C
32 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Table 2 (Continued )
Sample(s) Flavonoids Hyphenation Stationary phases (column Mobile phases Detector(s) Reference(s)
mode(s), dimensions), flow rates,
separation temperature, analysis
modes, times
sampling
time(s)
Sugarcane (Saccharum Flavones (15 On-line, 1 c 1 UV [356]
D: CN (250 mm × 1 mm, ( D) A: 0.1% AA, B:
d
spp.) flavonoids) RP-LC × RP-LC, 5 m dp ), FR = 0.02 mL/min, 0.1% AA in EtOH Q-MS (ESI+/−)
1 2
ts = 2 min 172 min ( D) A: 0.1% AA, B:
2 0.1% AA in MeOH
D: C18 (30 mm × 4.6 mm,
2.7 m dp SP),
FR = 3 mL/min (0.4 mL/min
to MS)
Radix Astragali (TCM) Isoflavones (34 Off-line, 1 1 UV [370]
D: SEC ( D) A: H2 O, B:
flavonoids) SEC × RP-LC, (300 mm × 4.6 mm), ACN Orbitrap (ESI+)
1 2
ts = 2 min FR = 1 mL/min, 30 min ( D) A: 0.1% AA, B: IT-MS (ESI+)
2 0.1% AA in ACN
D: C18 (250 mm × 4.6 mm,
3 m dp ), FR = 0.8 mL/min
(0.2 mL/min to MS)
Licorice (Glycyrrhiza Flavanone On-line, 1 c 1 UV [379]
D: CSH C18 ( D) A: 0.1% FA, B:
uralensis) aglycones and RP-LC × RP-LC, (100 mm × 2.1 mm, 1.7 m MeOH IT-MS (ESI−)
glycosides, 1 ◦ 2
ts = 0.5 min dp ), FR = 0.1 mL/min, 50 C, ( D) A: 0.1% FA, B:
chalcone 40 min ACN
glycosides, 2
D: Phenyl-hexyl
isoflavones, (50 mm × 3.0 mm, 2.7 m
flavonols and dp SP), FR = 2 mL/min
◦
flavones (39 (0.2 mL/min to MS), 50 C
flavonoids) 1
D: CN (150 mm × 2.0 mm,
Hedyotis diffusa (TCM) Flavonol glycosides On-line, 1 UV [365]
( D) A: H2 O, B:
(10 flavonoids) RP-LC × RP-LC, 3 m dp ), MeOH Q-TOF (ESI+)
1 ◦ 2
ts = 1 min FR = 0.025 mL/min, 50 C, ( D) A: H2 O, B:
150 min 0.05% FA in ACN
2
D: C18 (50 mm × 3.0 mm,
2.6 m dp SP),
FR = 2.0 mL/min
◦
(0.25 mL/min to MS), 50 C
a TCM, traditional Chinese medicine.
1
b ts , sampling time; ILC, immobilised liposome chromatography.
c
FR, flow rate; ILC, immobilised liposome chromatography; PEG, polyethylene glycol; PA, polyamine; SEC, size exclusion chromatography; CD, cyclodextrin; OEG, oligo(ethylene glycol); PFP,
pentafluorophenyl; SP, superficially porous; CN, cyanopropyl; CSH, charged surface hybrid.
d
AA, acetic acid; ACN, acetonitrile; FA, formic acid; MeOH, methanol; EDTA, ethylenediaminetetraacetic acid; TFA, trifluoroacetic acid; EtOH, ethanol.
e
ABTS, 2,2 -azino-bis(3-ethylbenzothiazoline)-6 sulphonic acid; ELSD, evaporative light scattering detector; FL, fluorescence detector; Q, (single) quadrupole; IT, ion trap; TOF, time-of-flight;
QqQ triple quadrupole.
* **
Not truly comprehensive due to the use of excessive sampling times. Not truly
comprehensive due to fraction transfer protocol.
porosity of these phases, resulting from the large through-pores, is responsible Comparison of the kinetic performance of monolithic columns with
for the higher permeability of monolithic columns (cf. Eq. (3), where ε values porous silica phases is challenging due to the wide range of monolithic phases
are ∼0.5–7 for monoliths compared to 0.4 for silica packings). of different properties available. Neverthe-less, kinetic studies on silica
monoliths, which are mostly used in flavonoid analysis, generally show the
Two types of monolithic columns can be distinguished, namely polymeric benefit of these phases to primarily lie in the high-efficiency regime, with
and silica monoliths [219]. The former are produced by in situ polymerisation conventional phases outperforming monoliths for fast analyses [200,222]. A
using a suitable monomer (such as polymethacrylates, polystyrene- ®
second generation of commercial silica monoliths (Chromolith High-
divinylbenzene, etc.), free radical initiator and porogenic solvent [220]. In Resolution) with macropores of 1.2 m and mesopores of 15 nm was
contrast, silica monoliths are produced using sol–gel processes involving introduced in 2011. These columns have closed the gap to porous phases in
polycondensation of tetra-alkyloxysilanes with polyethylene glycol (PEG) as terms of minimum plate heights of ∼7 m [225]. However, this performance
poro-gen, followed by derivatisation of the phase [221]. Control of the silica comes at the cost of lower perme-ability; the net result being that their optimal
skeleton and macro- and meso-pore sizes is possible through the selection of kinetic performance shifts to lower efficiencies and faster analysis, yet these
the silane starting material and PEG concentration [222]. columns are still outperformed by sub-2 m and superficially porous phases in
this region [44]. Increasing the maximum pressure above the cur-rent 200 bar
dictated by the column cladding might improve the competitiveness of
The increased permeability of monolithic phases may be exploited in one monolithic columns [44].
of two ways. First, very high flow rates can be used, which in combination
with the favourable mass transfer proper-ties of monoliths allows for fast
analyses. This is the tactic most often used in flavonoid analysis. Fast Monoliths have found some application in flavonoid analysis, primarily in
analyses using monolithic columns have also been used in the second two areas. The first is for the fast separation of a limited number of target
dimension of LC × LC separations [223]. Alternatively, high-permeability flavonoids, where typically commercial (Chromolith ® ) C18 silica monoliths
silica mono-liths can be used as very long columns to provide high efficiency of 100 mm length (or two of these coupled in series [186,226]) are used at
[224]; this approach has not been utilised in flavonoid analysis to date. high flow rates to increase analysis speed. For simple mixtures, isocratic
elution is often used [149,227–229]. The second main application
area of
A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 33
monoliths is in comprehensive 2-dimensional LC analyses (refer to Section catechin, compared to 4.24 m and 1.27 mm/s for a fully porous 1.7 m phase
3.2), where the high-flow compatibility of these phases are beneficial for fast [199]. In fact, this column outperforms fully porous phases of various particle
second dimension analyses [230], or alterna-tive retention mechanisms of sizes across the entire range of practical efficiencies (Fig. 5b).
polymeric monoliths are exploited in the first dimension [231].
Since 2006 a range of superficially porous columns of different (mostly
Considering that monolithic columns have been around since the late RP-LC) chemistries, column dimensions and particle sizes varying between
1980s [232,233], they have found relatively limited appli-cation in the LC– 1.3 and 5 m have become commercially avail-able from several manufacturers
MS analysis of flavonoids, especially compared to UHPLC (only 5% of the [257]. HILIC superficially porous phases are nowadays also available [251],
applications listed in the tables, refer to Fig. 7). One reason for this may lie in although to the best of our knowledge these have not to date been applied in
the fact that the very high flow rates typically used for fast analyses on the LC–MS analysis of flavonoids.
commercially available monolithic column dimensions of i.d.’s 4.6 or 3 mm
are a serious drawback from the perspective of solvent consumption and Mirroring the contemporary situation in HPLC, application of these phases
hyphenation to MS. For example, in typical studies utilising mono-liths, flow has grown rapidly in both one- and multi-dimensional HPLC analysis of
rates between 2.5 and 4 mL/min [149,229,234] were used, with flow splitting flavonoids during the past seven years (Tables 1, 2 and 4–7). These phases are
required prior to MS detection [186]. Cer-tainly the familiarity and experience increasingly being used as alternatives to sub-2 m porous phases, since they
with conventional phases of most practitioners, coupled with the range of gen-erally provide similar performance at lower operating pressures.
high-performance silica-based columns available nowadays, also plays a role Accordingly, superficially porous phases are also utilised to achieve two
in this regard, reflected also in the relatively sparse use of monoliths in principal goals: to obtain faster separations, and, to a lesser extent, to
general. maximise resolution for complex mixtures. The major-ity of applications
therefore use relatively short columns to benefit from speed gains, although
column lengths up to 150 mm have been used to provide maximum resolution
[96,124,258–263] – these provide much higher resolution than conventional
3.1.3. Superficially porous stationary phases columns of 150 or 250 mm length and packed with 5 m phases.
Superficially porous (also referred to as core–shell, shell or fused-core)
phases were originally introduced in the 1960s [235,236], but it has only been
with the introduction of second gen-eration superficially phases since 2006 Several studies have compared the performance of core–shell and fully
[237,238] that an upsurge in interest in these phases has occurred. This is a porous phases. For example, Ruiz et al. [260] compared
consequence of the remarkable performance of these columns: in contrast to a conventional (250 mm × 4.6 mm, 5 m), a superficially porous (150 mm ×
conventional porous silica phases, minimum plate heights (H min) as low as 4.6 mm, 2.6 m) and 2.2 m (150 mm × 2.0 mm) C18 phases for the analysis of
glycosylated flavonols, and found the superficially porous phase to be
1.1–1.8dp have been measured for superficially porous phases, where for
superior for this application. For the analysis of polyphenols in lentil seed
years Hmin = 2dp was considered a practical limit for HPLC columns. Initially coats, two types of core–shell 2.6 m columns (Kinetex C18 and PFP) where
marketed as providing reduced diffusion distances due to the smaller porous compared to a fully porous 4 m phase. The core–shell columns outperformed
layer surrounding a solid core, and therefore a reduced stationary phase mass the fully porous particles, with the PFP phase providing better separation of
transfer term, exten-sive subsequent fundamental research has demonstrated flavone and flavonol glycosides than the C18 core–shell col-umn [91]. This
that the improved performance of these phases is mostly due to reduced A- phase has also been found to be ideally suited for the analysis of hydrophobic
term [239–242] (ascribed to a narrower particle size distribu-tion [238,239]
isoflavones [92,93] and some antho-cyanins [95]. Manchón et al. similarly
and/or better packing efficiency [243]) and B-term [239,242,244–247]
reported better performance for a 2.6 m superficially porous C18 phase
contributions to plate height, in addition to a lower C-term [242,247]. The
compared to 3.5 m fully porous C18 phase and a monolithic column for the
latter advantage in terms of the C-term behaviour of superficially porous
phases is particularly pronounced for high MW compounds due to the slow separa-tion of isoflavones [264]. Zhang et al. [265] found a 150 mm 1.6 m
diffusion of these com-pounds [238,241,242]. From the perspective of core–shell column to provide better peak capacity and symmetry than two 100
flavonoid analysis, this is pertinent in that it implies particular advantages of mm sub-2 m phases for the separation of rhubarb constituents. An example of
these phases for the analysis of oligomeric proanthocyanidins [199] and the performance obtained on this col-umn in gradient mode is presented in
highly glycosylated and acylated flavonoids such as anthocyanins [128,248]. Fig. 6. Note that this column should, based on a minimum plate height of
1.8dp, provide an iso-cratic efficiency of 52,000 plates – the increased
resolving power is clearly evident from Fig. 6. This performance is only
attainable on UHPLC instrumentation though. It should be noted that
compari-son between column performance in gradient mode for complex
The primary benefit of superficially porous phases is therefore that samples is far from straightforward due to the effect of differ-ent particle
performance similar to fully porous phases of smaller d p can be obtained, sizes, column dimensions and selectivity differences between phases. For
however at lower pressures due to the larger particles (cf. Eqs. (2) and (3)) example, Serrano et al. found a polar embed-ded 1.7 m fully porous phase to
[247] – the same applies for larger particle size superficially porous phases be better suited than superficially porous phases for the separation of natural
[201]. This reduces the need for ultra high pressure operation and the risk of flavonoid dyes [266]. In line with the characteristics of superficially porous
frictional heating (super-ficially porous phases have also been shown to be phases, they have also found growing application in LC × LC separations (see
characterised by higher heat conductivity, further reducing this risk [249]). Section 3.2).
The only potential drawback of these phases is reduced loadability asso-ciated
with the increase in the phase ratio [250], although this remains a point of
discussion and is more relevant in the case of ionisable analytes [251–253]
than for flavonoids. Several recent reviews on superficially porous phases can
be consulted for details on the theoretical aspects, applications, stationary It is worth pointing out that although the reduced pressure-drop for
phase manufac-turing and available chemistries [254–257]. superficially porous columns allows their utilisation on con-ventional HPLC
instruments – a benefit which is widely heralded by manufacturers and in
literature – special care should be taken regarding extra-column band
Fig. 5a and b illustrates the advantages of superficially porous phases for broadening on such instrumentation. Most conventional HPLC instruments
flavanols: for a 2.6 m superficially porous phase, H min and uopt values of 3.9 are characterised by large delay- and extra-column volumes, and even
m and 1.2 mm/s were measured for allowing for careful
34 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Fig. 6. Representative RP-LC–TOF-MS chromatogram illustrating 104 common peaks identified in rhubarb using a 150 mm × 2.1 mm 1.6 m superficially porous phase.
◦
Analysis conditions: 0.1% formic acid/ACN gradient; 0.3 mL/min; 40 C; detection: ±ESI-Q-TOF-MS.
Source: Reproduced with permission from [265].
HTLC ovens have only relatively recently become commercially available. anthocyanin analysis and LC × LC separations, increasing aware-ness of the
benefits of elevated temperature and the means to assess the risk of analyte
In the case of flavonoid analysis, the well-known thermal instability of degradation, coupled to the commercial avail-ability of growing numbers of
these molecules raises concern about the risk of on-column degradation columns and instruments suitable for HTLC operation may see a concomitant
occurring, and is likely the main reason for the restricted application of HTLC increase in the number of reports utilising modest increases in temperature (in
in the field. However, to assess the risk of on-column degradation it is ◦
the range 30–80 C) for improved flavonoid analysis in the future.
important to consider the resi-dence time of the analyte in the heated column
relative to the rate of its thermal degradation at this temperature [268,272]. Guillarme et al. [43] provided an informative comparison of the kinetic
Simply put, if the analyte spends less time in the heated column than is
benefits of UHPLC, monolithic columns, superficially porous phases, HTLC,
required for its thermal degradation, HTLC at that temperature is a viable
and combinations of these, compared to conventional HPLC for the flavonol
option. For example, anthocyanins are notoriously thermally labile [280,281].
rutin (MW 610). Fig. 8 presents a graphical overview of their findings, where
◦
Yet, it has been demonstrated that operation at tempera-tures up to 80 C are the x-axis shows the highest pos-sible peak capacity (P in this figure) for a
feasible for anthocyanin analysis provided that analysis times are kept to less gradient time of 3 h, and the y-axis the time required for a peak capacity of
than an hour [105,282]. Grata et al. 100. The benefits of these technologies for both high throughput and high
resolution separations are evident, as is the complementary nature of UHPLC
[283] similarly reported that no thermal degradation was observed for and HTLC approaches.
metabolites, including flavonoids, in Aridopsis thaliana based on a detailed
◦
analysis of TOF-MS data for separations performed at 30 and 90 C. Table 1 lists the applications of UHPLC, monolithic and superfi-cially
porous columns without MS detection to flavonoid analysis during the last 7
From Tables 1, 2 and 4–7, it is clear that the use of temperatures above 40 years (applications with MS detection are summ-arised in Tables 4–7 and will
◦ be discussed below).
C in flavonoid analysis is relatively rare. Nevertheless, a number of studies
◦ ◦
employed column temperatures of 50 C [98,143,180,199,218,284–293], 60
3.2. Comprehensive multi-dimensional LC
◦ ◦ ◦
C [80,217,294], 70 C [282,295], 80 C [81,296] or 90 C [283] in one- and
two-dimensional analy-ses. Qi et al. [297] implemented a temperature Fundamental aspects
◦
gradient between 22 and 35 C for the analysis of flavones and flavonols in Multidimensional liquid chromatography (MD-LC) provides a powerful
traditional Chinese medicine (TCM) in order to limit pressure restraints on a means to significantly improve the performance of LC separations. This is
1.8 m RP column operated on conventional instrumentation. achieved by combining the separation power and selectivity of multiple 1-
Two particular applications of HTLC are worth mentioning. Reichelt et al. dimensional HPLC methods. MD-LC approaches can be divided in heart-
◦ cutting methods, where only selected part(s) of a sample are subjected to
[99] used a polymeric PRP-1 column operated at 120 C for the fractionation
of natural products prior to the sen-sory evaluation of the fractions (an multiple separations, and comprehensive separations, where all constituents of
approach termed LC Taste). The mobile phase contained only water and the sam-ple are analysed in several dimensions. In this report, we will limit
ethanol, and fractions could be directly tasted after dilution with a basic our attention to comprehensive MD-LC separations, as these are more generic
tastant solution. High temperature operation is essential to accommodate the in nature and provide much higher resolving power. (As an aside, although
high vis-cosity of the ethanol-based mobile phase. The authors confirmed the comprehensive systems where the number of separation dimensions exceeds
flavour modulating activities of several flavonoids using this approach [99]. two have been demonstrated [316], this has not yet been achieved for
flavonoid analysis). The subsequent discussion will therefore focus on
comprehensive 2-dimensional liquid chromatography (LC × LC) only. For
High temperature operation also provides a particular benefit in instances detailed overviews of the field, the reader is referred to several dedicated LC
where on-column conversion between multiple species of an analyte occurs × LC reviews [45–49,317–320], as well as more specific reviews covering
[298,299]. A pertinent example is the case of anthocyanins, where several hyphenation of LC × LC with MS [321], column selectiv-ity in LC × LC
different chemical forms may occur in the mobile phase. It has been shown [322] and multidimensional chromatography in food analysis [323].
[105] that the inter-conversion reaction between the flavylium cation and
carbinol pseudoba-sic species is directly responsible for additional band
broadening in the separation of these compounds under conventional con-
ditions (flow rate and temperature). An increase in the analysis temperature The incentive behind the growing interest in LC × LC lies in the
results in faster kinetics of the relevant equilibria, and therefore a reduction in exceptional performance gains that may be realised when combin-ing two
band broadening [105]. Operation at elevated temperature and optimal flow HPLC separations under ideal conditions: the peak capacity of such a
rates result in significant increases in chromatographic performance for separation is theoretically the product of peak capacities of both 1-
anthocyanin analy-sis, without the risk of analyte degradation [105,218]. dimensional separations [324–328]. LC × LC is therefore capable of providing
separation performance an order of magnitude higher than obtainable by even
highly optimised 1-dimensional HPLC methods. Attaining such performance
◦ is however more chal-lenging in practice. Two particular aspects should be
Even a modest increase in temperature to 80 C can have a large impact
on mobile phase viscosity and therefore chromatographic behaviour. For pure addressed to meet the criteria for methods to be considered comprehensive
water for example, the viscosity will decrease by about 60% compared to
room temperature (the correspond-ing values are about 40% and 35% for [329] and exploit the potential of these separations.
methanol and acetonitrile, respectively). This implies that the benefits of The first pertains to the requirement that different separa-tion mechanisms
elevated temperature operation can be exploited with even a modest increase be used in both dimensions. This is essential to effectively utilise the two-
in tem-perature in the range where commercial column ovens operate. It has dimensional separation space pro-vided by LC × LC. The degree to which two
to be stressed though that for high-flow operation (for example 4.6 mm i.d. separations provide complementary information is referred to as their
columns and above), efficient mobile phase pre-heating is critical even if the ‘orthogonality’. A combination of highly orthogonal separations will result in
column can effectively be thermostatted at these temperatures. the spread of analytes across the two-dimensional space, and therefore
maximise resolution, whereas weakly orthogonal separations (i.e. where
retention of sample constituents between the two separa-tions are correlated to
While the use of reasonably high temperatures in flavonoid analysis will a significant degree) provide limited benefit.
likely remain limited to certain niche fields such as
A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Fig. 8. Comparison of the performance achievable (x-axis: peak capacity, P, for a gradient time of 3 h, y-axis: gradient time required for a peak capacity of 100) by HPLC, UHPLC, monolithic
phases, HTLC and combinations of these. Data obtained for the flavonol rutin.
Source: Reproduced with permission from [43].
1 2
where nc and nc are the peak capacities of the first- and second-dimension
separations, respectively, and nc,2D the practical peak capacity of the LC × LC
separation. From this equation it is clear that the potential performance gains of
LC × LC are heavily reliant on both the degree of orthogonality and under-
sampling.
In practice, LC × LC can be performed in one of three ways. The first, and
1
most simple, is the off-line approach, where fractions elu-ting from the D
column are collected (manually or automatically). These can then be
concentrated, dissolved in a suitable solvent or directly injected onto the
second dimension column. Off-line LC × LC can therefore be performed on a
single HPLC instrument, since fractions can be injected after their collection.
n
c
them next to each other in a matrix format which can be viewed as a surface
plot or more commonly as a contour plot (see for example Figs. 11–13).
Quantification can also be performed in LC × LC by combining the
2
that the D analyses must be very fast to meet first-
dimension sampling criteria. Alternative
configurations are also possible, such as for example
using trapping columns and an added make-up flow
to trap analytes eluting from the first dimension. This
approach has the benefit of essentially removing all
or the majority of the first-dimension mobile phase
prior to injection on the second dimension, and can
be used to minimise dilution (see below) and remove
unwanted mobile phase components such as salts (the
approach is often used in ion exchange (IEC) × RP-
2
LC-MS for this reason). Furthermore, multiple D
columns can be used [335,336] to effectively provide
longer second dimension analysis times and therefore
increase the overall resolution, at the cost of
increased instrumental and method complexity
[337,338].
Fig. 9. Schematic representations of instrumental configurations used for on-line LC × LC using (a) empty storage loops, (b) trapping columns and (c) multiple 2D columns; and (d) for stop-flow LC
× LC.
Source: Reproduced with permission from [48].
38 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
2 1 1
contribution of each individual D ‘slice’ of a D chromatographic the effect of effectively de-coupling fraction volumes from the D
peak, or by using more advanced algorithms [343,344]. Advanced flow rate. In off-line LC × LC, this limitation can be circumvented
data analysis techniques can also be applied to LC × LC data [345], by injection of a portion of each fraction, diluting fractions (both
although multivariate data analysis is necessarily more compli- resulting in lower sensitivity) or by evaporating the solvent fol-
cated than in 1-dimensional separations since alignment of data lowed by dissolution in a weaker injection solvent.
in two dimensions present several challenges [346]. Second dimension column stationary phases are selected based
Compared to one-dimensional HPLC, method development and on performance for fast separations – technologies such as UHPLC
optimisation is much more complex in LC × LC [347–349]. This topic [360], superficially porous phases [357] and HTLC [147] have there-
is much too complex to be addressed fully in the context of this con- fore all found application in the second dimension of LC × LC
tribution, and we will therefore focus on providing some general systems. For the same reason, relatively short columns are gen-
guidelines for parameter selection in LC and the implications for erally used, typically of i.d. 2.1–4.6 mm to increase capacity and
performance and hyphenation to MS. The first choice involved in minimise injection problems. While monolithic columns are ide-
LC × LC method development is the selection of separation modes, ally suited for fast analyses at high flow rates, the fact that splitting
where orthogonality, performance (speed and efficiency) and com- is required prior to MS detection represents a significant drawback.
patibility with the detection strategy to be used are important Finally, the dilution of analytes occurring in both dimensions
criteria. Considering the chemical properties of flavonoids, LC × LC should be considered. For passive modulation (i.e. where analytes
methods for their analysis have almost exclusively involved RP-LC 1
are not trapped and focussed after the D separation), such as
and/or HILIC (with a few notable exceptions [81,296,350]). RP-LC mostly used in LC × LC, total dilution is generally much higher than
is most often used in the second dimension because of compatibil- is the case for 1-D HPLC. Dilution in each dimension, and the total
ity with MS and the fact that good performance is obtained in this dilution in LC × LC, can be determined according to [347,361]:
mode for fast separations [351], although HILIC is in principle also √ 1
× F
1
2 1 (6)
suited to fast D analysis [352,353] prior to MS detection and has DF 2 1
been used for this purpose in proteomics research. The combination = Vinj
of HILIC and RP-LC promises higher orthogonality than RP-LC × RP- √ 2
F×
2
× SR
2
LC, although hyphenation of the former modes is more complicated DF 2
(7)
due to the relative elution strengths of the mobile phase used in = 1 1
F × ts
each dimension (see below). The orthogonality of RP-LC × RP-LC 2D 1 2
systems can be improved through judicious selection of the mobile DF = DF × DF (8)
phase composition (typically the organic modifier used, since pH in
the range used for flavonoid analysis has little effect on the selec- where DF is the dilution factor, the standard deviation in time
tivity) and especially the stationary phase chemistries used in both units and F the flow rate for each dimension, as specified by the
dimensions. Furthermore, in the case where gradient separation is 1 1
superscript, Vinj is the D injection volume and SR is the ‘split ratio’
performed in both dimensions – the most common approach which to accommodate cases where only a portion of each fraction may
2
provides this highest overall performance [347] – the D gradi- be injected. Eqs. (6)–(8) clearly illustrate that analytes are diluted
1 to a far greater extent following passage through two columns than
ent can be varied throughout the D separation to maximise the
utilisation of the 2-dimensional space [354–356]. This may require is the case for 1-dimensional HPLC; this has clear implications for
2 the detector requirements in LC × LC.
correction of D retention times when multiple slices of the same
compound elute in consecutive gradients [357]. LC × LC has found considerable and growing application in
The choice of hyphenation mode, assuming availability of suf- flavonoid analysis, especially during the last five years (Table 2).
ficient instrumentation, depends primarily on the performance An overview of the applications listed in this table is pre-
requirements and time considerations: for maximum resolution sented in Fig. 10. Several different combinations of separation
off-line or stop-flow approaches should be used, whereas on-line modes have been reported, including RP-LC × RP-LC, HILIC × RP-LC,
is preferable for faster separations. Regarding column dimensions, HILIC × HILIC and SEC × RP-LC. HILIC × RP-LC has found the most
the selection is governed largely by mobile phase considerations. widespread application in the LC × LC analysis of flavonoids (47%
RP-LC and HILIC mobile phases are perfectly miscible, but the of applications listed in Table 2), followed by RP-LC × RP-LC (38%)
solvent composition of most fractions generally implies high (Fig. 10a). Compared to LC × LC in general, where according to Li
elution strength in the second dimension. In HILIC × RP-LC (or RP- et al. [351] RP-LC × RP-LC is the most common combination of
LC × HILIC) this is because of the disparate percentages of organic modes (32% vs. 22% for NP-LC/HILIC × RP-LC), this relative prefer-
modifier used in each of these modes, whereas in RP-LC × RP-LC ence for HILIC in flavonoid analysis is likely associated with the
1 unique and complementary retention order obtained in this mode
this follows from the fact that compounds are eluted from the D
column in a solvent composition where their retentivity is low. (It is compared to RP-LC. Noteworthy examples include glycosylated
for this reason that RP-LC columns with lower retention are mostly flavonoids, where compounds are separated according to their
1
used in D for RP-LC × RP-LC). The practical implication of this is degree of glycosylation and/or acylation [126,248], and proantho-
2 cyanidins, where HILIC separation according to size is extensively
that the injection volume on the D column should be minimised
to avoid injection band broadening. The volume of each 1st dimen- used to study this family [147]. Most reports (two thirds during
1 1
sion fraction (Vfrac) is related to the D flow rate, F, and sampling the last 7 years, Fig. 10b) use on-line LC × LC, followed by off-line
1 LC × LC (31%); stop-flow HILIC × RP-LC has also only been used in
time, ts, according to:
1 1
Vfrac = ts × F two reports for the separation of procyanidins [340,342]. For high
(5) speed separations in the second dimension, it is noteworthy that
the use of superficially porous phases exceeds that of fully porous UHPLC
The most common approach to minimise injection effects in on-line LC × phases; monoliths have found limited application in LC × LC separation of
1
LC is therefore to reduce the i.d. of the D column, leading to a concomitant flavonoids (with some exceptions [230,231,362]). Furthermore, the elevated
1 temperature has found extensive use to facilitate fast separations in the second
reduction in F. Other options to address this lim-itation include the use of
trapping columns [358] or incorporating a split between the two dimensions dimension, with temper-atures of 50 ◦ C [147,231,248,340,342,362–365], 60 ◦
[342,359]. The latter approach is simpler, but reduces overall sensitivity, C [357], 70 ◦ C [366] and 80 ◦ C [81,350] being used.
whereas the former has
A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 39
Fig. 10. Overview of the LC × LC analysis of flavonoids (2009–2015) summarising the different separation modes (a), hyphenation modes (b), column formats (c) and mass spectrometers (d) used in
the reports listed in Table 2.
Virtually all classes of flavonoids, including flavonols [344,367], surprisingly good size-based separations in the first dimension, with tetra-,
flavanols and procyanidins [81,147,344], chalcones [127], (iso)flavones tri-, di- and monoglycosylated flavonoids eluting in the order of decreasing
[357,368], flavanones [357,368] and anthocyanins size. The separation mechanism is not clearly defined, however, with isomeric
[248] have been separated successfully in LC × LC. Furthermore, the flavonol glycosides also being separated to some extent in this dimension. In
◦
suitability of LC × LC-UV for quantitative analysis of flavonoids has been the sec-ond dimension, a conventional RP-LC column operated at 60 C
demonstrated [343,344]. The authors noted that LC × LC provided higher
limits of detection than 1-dimensional HPLC, a consequence of greater ◦
[80] or a UHPLC column at 80 C [81] was used. The SEC × RP-LC
dilution occurring in two dimensions (cf. Eqs. (6)–(8)), but that in some cases combination showed good orthogonality, and by using an off-line hyphenation
improved chromatographic resolution provided more accurate results. strategy involving fraction concentration, good sensi-tivity was obtained.
Thirty six flavonoid-glycosides were identified in the heart-cutting 2D LC
Hyphenation of LC × LC separation with MS is clearly beneficial from analysis of Maytenus ilicifolia, whereas 54 flavonoids were identified in green
the perspective of compound identification and selectivity. The improved and black teas using the off-line comprehensive approach with detection
separation offered by LC × LC separation is also ben-eficial from the point of performed by means of a triple quadrupole MS. An example of the contour
view of MS, since reduced co-elution minimises the number of potential plot obtained for the off-line SEC × RP-LC analysis of a black tea is presented
interfering ions and thereby matrix effects [321]. However, especially on-line in Fig. 12.
LC × LC also places strict demands on the speed of MS detection due to the
2 Alternative separation modes have also found application in the 2-
very nar-row peak widths commonly obtained for fast D separations. This is
reflected in the fact that most (48%) of LC × LC–MS methods for flavonoid dimensional analysis of flavonoids, for example the combination of CPC
analysis during the review period were performed using TOF (or Q-TOF) [371] or CCC [372] with RP-LC and RP-LC with micellar elec-trokinetic
instruments (Table 2). IT detectors have also been used [230,369], while chromatography (MEKC) [87], although these fall outside the scope of the
Zhang et al. [370] recently used an Orbitrap instrument operated at a current report.
resolving power of 50,000 and scan speed of 2 Hz in combination with off- While many of the applications of LC × LC report similar num-bers of
line SEC × RP-LC analysis. compounds than are nowadays also identified using advanced LC–MS
methods (Section 4), the benefits of improved chromatographic resolution
Qiao et al. [364] reported an interesting heart-cutting/RP-LC × RP-LC cannot be overstated. Especially in qualitative analyses of flavonoids, the
approach for the analysis minor compounds in herbal medicines. Major main application area of LC × LC to date, the quality of mass spectral data is
1 greatly improved as a consequence of less co-elution provided by the
compounds were removed following D separa-tion using a time-activated
heart-cutting method, whereas minor compounds were separated in combination of complementary separation modes [321].
comprehensive mode using shifted gradients in the second dimension to
improve the orthogonality of the RP-LC × RP-LC system. The instrumental A field where LC × LC demonstrates clear benefits is in the analysis of
configuration and an example contour plot obtained in this work are presented proanthocyanidins. The complexity of these com-pounds, with the number of
in Fig. 11. Separated compounds were identified based on MS/MS spectra isomeric structures increasing exponentially with DP, means that they cannot
obtained using IT-MS. be resolved using any available HPLC method [373]. Furthermore, because of
the large number of isomeric structures, high resolution MS and MS/MS data
SEC and RP-LC have also been combined for flavonoid analysis, first in are also of limited utility. LC × LC operation com-bining HILIC separation
heart-cutting mode by de Souza et al. [80] for the separation of flavonol according to MW and RP-LC separation according to isomeric composition
glycosides. In a follow-up report, the same group used an off-line SEC × RP- has been shown to offer a powerful approach for the elucidation of
LC method for the analysis of green and black tea flavonoids [81]. The SEC procyanidin content in a variety of natural products, especially when
mobile phase was collected automatically and evaporated between the two hyphenated to
dimensions. The authors reported
40 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Fig. 11. (A) Instrumental configuration used for the on-line heart-cutting/RP-LC × RP-LC analysis of Puerarialobata herbal medicine. Valve 1 was used to selectively cut the major constituents
(underlined peak numbers in (B)), and valve 2 for the comprehensive LC × LC analysis of the remainder of the constituents. (B) shows the UV contour plot obtained for this analysis, with a zoom of
the separation area in the red block on the right. Numbered compounds except for 12, 13 and 19 are isoflavones. Analysis conditions:
◦ 2 ◦
1D: 100 mm × 2.1 mm CSH C18 column, 50 C, 0.1 mL/min 0.1% formic acid/methanol gradient; D: 50 mm × 3.0 mm 2.7 m superficially porous C18 phase, shifted gradients, 50 C, 2.5 mL/min
(split 1:9 before ESI-IT-MS detection).
Source: Reproduced with permission from [364].
MS [126,147,148,230,340,342,363,369]. As an example, Fig. 13 instruments [383]. By the middle of the next decade [59,62] ESI and APCI
demonstrates the information obtained for the on-line HILIC × RP-LC-FL- had become the norm in flavonoid analysis, while time-of-flight (TOF) and
ESI-TOFMS analysis of grape seed procyanidins [148]. Mass spectral data quadrupole-time-of-flight (Q-TOF) instruments were only just finding
allowed extraction of information for particular classes of compounds, as application in flavonoid analysis [384].
illustrated in Fig. 13D for the mono-galloylated procyanidins. While this As will be shown in the following sections, the contemporary sit-uation is
methodology provides much more detailed molecular information than could significantly different. In addition to very significant gains in the sensitivity
be obtained using 1-dimensional LC–MS, the complexity of the high MW and speed of MS systems which have been pro-gressively attained with newer
fraction (Fig. 13C) also serves to emphasise the challenges associated with generation instruments, a range of new high resolution analysers and tandem
proanthocyanidin analysis. For such applications, LC × LC is expected to MS configurations have also found application in flavonoid analysis. We will
play an increasingly important role in the future. not attempt to address in any detail instrumental developments or the
operation of all instruments, but rather to highlight the potential benefits of the
different types of mass analysers and the types of analyses for which they are
4. Advances in LC–MS analysis of flavonoids suited on the hand of recent applications.
From the perspective of LC–MS, technological advances in MS From the perspective of hyphenation of MS to high-performance
instrumentation over the last fifteen years have largely over-shadowed those separations, several pertinent aspects should be considered. First, the fact that
in HPLC. Since the beginning of the century, the commercial availability of optimal ESI performance is attained at flow rates of 0.2–0.4 mL/min is
more robust and more advanced LC–MS instruments, coupled to the entirely compatible with the trend in UHPLC of a reduction in column
reduction in the price of these instru-ments, has certainly contributed to the diameter from conventional 4.6–2.1 mm i.d. columns dictated by frictional
much more widespread use and utility of the technique in flavonoid analysis. heating considerations [199,213]. On the other hand, commercial monolithic
The extent of these improvements, as well as the most important columns are mostly used in 4.6 mm i.d. formats, which, coupled to the higher
developments, are clearly highlighted when comparing current practice with flow rates used for fast analysis on these columns, implies flow splitting prior
the state of affairs at the time of several important reviews. By the end of the to MS detection. Superficially porous phases, due to their relatively high
previous century, the majority of LC–MS applications utilised fast atom permeability and favourable thermal conductivity properties
bombardment (FAB) and thermospray (TSP); ESI and APCI were recent
additions to the ion sources used for flavonoid analysis [55,380–382]. [249] are viable to use in both 4.6 and 2.1 mm formats, although clearly the
Furthermore, most analyses were performed using quadrupole (single stage or latter is preferred from the perspective of hyphenation to MS. Indeed, of the
triple quadrupole, QqQ) or ion trap applications listed in Tables 4–7, the majority employ columns of internal
diameter 2.1 mm.
A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 41
Fig. 12. Contour plot (325 nm UV detection) obtained for the off-line SEC × RP-LC separation of a black tea hydro-alcoholic extract (note that the first dimension SEC separation is on the y-axis).
1 ◦ 2
Compounds 24–63 are flavonoids, as well as 5, 9–10, 12, 15–18 and 21 and 22. Analysis conditions: D: 300 mm × 7.8 mm SEC column, 60 C, 1 mL/min water/ACN gradient; D: 50 mm × 2.1
◦
mm 1.7 m porous C18 phase, 80 C, 1 mL/min.
Source: Reproduced with permission from [81].
Lower column volumes however also place stricter demands on [293]; the rest without exception used ESI. Compared to the global picture for
instrumentation in terms of extra-column volume. For example, Wolfender LC–MS reported in 2012 [41] (82% ESI, 16% APCI, 2% APPI), the higher
and co-workers [283] showed that peak capacities of UHPLC separations are prevalence of ESI is a consequence of the fact that most flavonoid classes are
typically reduced by between 15% and 30% upon connection to MS more efficiently ionised using this mode. ESI was used in negative ionisation
detection, even if care is taken to reduce the volume of the connection tubing. mode in 48% of all applications, and both positive and negative ionisation
Indeed, comparison of UV and MS chromatograms in many literature reports modes in 33% of applica-tion during this time (refer to Section 2.3 for a
visually reveals the effect of this phenomenon. Finally, the scan speed is of general discussion of the MS behaviour of flavonoids).
obvious importance for hyphenation to very fast separations – this aspect will
be addressed in more detail for the different mass spectrome-ters below. APCI is generally considered an alternative to ESI for relatively non-polar
compounds which are less efficiently ionised in ESI. While APCI shows
comparable to better sensitivity compared to ESI for flavonoid aglycones
[161], and therefore is well suited to the analysis of hydrolysed samples, the
4.1. Ionisation modes latter typically outperforms the former for natural glycosylated species
[165,407,408]. Two poten-tial advantages of APCI over ESI are better
Early ionisation techniques applied for the direct analysis of flavonoid compatibility with high flow rates, and lower susceptibility to matrix effects
analysis include chemical ionisation (CI) [385] and electron impact (EI) [386] during the ioni-sation process [383]. APCI continues to find application in
(these also in combination with GC sep-aration), field desorption [387], flavonoid analysis, and indeed has been used in recent years in combination
plasma-desorption-MS (PD-MS) [388,389], FAB [169,390–392] and matrix- with UHPLC-(HR)–MS [163,407] and LC × LC [368], especially in the
assisted laser desorp-tion/ionisation (MALDI) [393,394]. analysis of more apolar isoflavonoids and isoflavones. Nevertheless, the better
performance of ESI for a wide range of flavonoid species is likely an
However, it is only with the development of ionisation sources that important contributing factor to the dominance of this form of ionisation in
allowed coupling of HPLC with MS that the technique found the pre- contemporary flavonoid analysis.
eminence it enjoys today in flavonoid analysis. Initial ionisation techniques
used for this purpose such as TSP [395,396], moving belt (MB) [397,398], APPI presents an alternative API source which is complementary to ESI or
continuous-flow FAB [399] have now largely been replaced by API APCI, especially for the analysis of non-polar compounds. Similar to APCI,
techniques such as ESI [400–403], APCI [404–406] and to a lesser extent the liquid sample is vaporised by a modified heated nebuliser, but instead of
APPI. using a corona discharge to establish ioni-sation, the process relies on charge
Of these, ESI is undoubtedly the most important. Indeed, in the advanced transfer from dopant or solvent molecules ionised by 10 eV photons produced
LC–MS analysis of flavonoids during the period cov-ered by this review, only by a discharge lamp [409]. Rauha et al. [165] compared ESI, APCI and APPI
three reports utilised APCI [163,368,407], mostly involving analysis of with a vari-ety of mobile phases in positive and negative ionisation modes for
isoflavones, and only one used APPI
42 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Fig. 13. Total ion chromatogram (TIC) (A) and fluorescence (B) contour plots for the HILIC × RP-LC–FL–ESI-MS separation of a grape seed extract. (C) A detailed section of the TIC and (D) the
1
extracted ion contour plot for monogalloylated procyanidins. Analysis conditions: D: 250 mm × 1 mm Diol column, 100 min gradient at 25 L/min; sampling time 2 min (split 1:24 between two
2 ◦
dimensions); D: 50 mm × 4.6 mm 1.8 m porous C18 phase, 50 C, 1.5 mL/min (split 1:1.2 before ESI-QTOF-MS detection). Peak labels: PC X GY refers to a procyanidin of degree of polymerisation
X and galloylation Y. F denotes the z-axis scale.
Source: Adapted from [148].
the analysis of five selected flavonoids, and concluded that nega-tive mode instrument, leads to an additional 30% loss in peak capacity for highly
ESI with ammonium acetate containing mobile phases provided the lowest efficient separations [283]. Therefore this option should be employed with
limits of detection (LODs). While this was espe-cially true for flavonoid- care, especially for ultra-fast separations.
glycosides, similar LODs were obtained between all three modes for It is relevant to note that in addition to the extensive use of MALDI, a
flavonoid aglycones in both positive and negative ionisation, although range of ionisation sources suitable for the direct analysis of flavonoids from
different optimal mobile phase compositions were used for each ionisation liquid or solid samples have been developed and found application in
mode [165]. APPI has also more recently been used in flavonoid analysis. flavonoid analysis. The most important of these are desorption electrospray
Riffault et al. chose this ionisation method for the phytochemical characterisa- ionisation (DESI) [40] and direct anal-ysis in real time (DART) [410–412].
tion of rose flowers, although the choice of ionisation mode was primarily Since these ionisation sources are not applicable in LC–MS, they will not be
based on its performance for more apolar compounds such as fatty acids and discussed here.
sesquiterpenoids [293].
4.2. Mass analysers
The ability to perform swift polarity switching between posi-tive and
negative ionisation modes has proven beneficial in several research fields due Mass analysers can broadly be divided into two main classes, namely high
to the complementary information that may be gained for compound classes and low resolution (or nominal mass) instruments, based on their ability to
preferentially ionised in either mode. In the case of flavonoid analysis, this is distinguish ions with small mass-to-charge (m/z) differences. The critical
however less important since most flavonoids are sufficiently ionised in either parameter here is the resolving power, defined as the ratio of an ions’ mass to
mode (the exception being anthocyanins, where mobile phase considerations the width of its peak at half height (full width at half maximum, FWHM). The
for efficient separation in any case preclude the use of nega-tive ionisation). resolving power is normally specified with the mass at which it is determined,
Nevertheless, when flavonoids are determined together with other classes of as resolving power decreases with ion mass. High resolution mass
compounds, such as for example in metabolomic research or studies aimed at spectrometers (HR-MS) are generally considered to provide resolving power
comprehensive char-acterisation of natural product constituents, polarity greater than 10,000, while for ultrahigh resolving power instruments this value
switching becomes a valuable tool. A common example is where complete is above 100,000 [41]. The former class includes TOF analysers, while the
phenolic analysis is the goal and flavonoids are analysed together with Fourier transform (FT) instruments Orbitrap and ion cyclotron resonance
phenolic acids. However, Wolfender and co-workers showed that polarity- (ICR) fall in the latter class. Mass accuracy is the relative difference between
switching, using a switch time of 0.3 s on a TOF measured and theoretical m/z values, reported in parts per million
A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 43
Table 3
Overview of the specifications of various mass analysers used in the LC–MS analysis of flavonoids. The table is based on the work of Holcapekˇ et al. [41], updated using the latest specifications for
QqQ, Q-LIT and Orbitrap instruments.
a 3 Mass range m/z (upper limit) Mass accuracy (ppm) b Dynamic range
Mass analyser Resolving power (FWHM) [×10 ] Scanning speed (Hz)
QqQ – 2000–3000 – 10–30 6
10
a 6
IT – 2048–6000 – 10–60 10
a 4 5
TOF 10–60 100,000 <2–5 10–100 10 –10
4
ICR 750–2500 4000–10,000 <0.25–1 2 10
a 100–500 2000–6000 <1–3 1–20 4000–5000
Orbitrap
a Includes hybrid configurations.
b
Calculated for a mass range of m/z 1000.
(ppm) units. Sufficient mass accuracy provides the key benefit of allowing whereas for untargeted or screening analyses the goal is to iden-tify as many
determination of elemental composition [413]. The mass accuracy required compounds as possible, often only qualitatively. The nature of the analysis
for this however depends on the mass of the ion, with mass accuracy of <5 being performed will determine the best suited mass analyser for the given
ppm typically sufficient for compounds of m/z < 300; for higher MW ions, application.
this degree of mass accuracy is no longer sufficient because of the much In the case of targeted analyses, selectivity and quantifica-tion
larger number of potential combinations of elements [41]. specifications (linearity, dynamic range, sensitivity) are of paramount
importance. Currently the most widely used approach for targeted analysis
HR-MS instruments such as double-focusing sector and modi-fied relies on the high selectivity inherent to tan-dem MS operation, most
magnetic sector instruments used in the early LC–MS analysis of flavonoids commonly exploited by utilising QqQ instruments operated in multiple
[108,171] have nowadays largely been replaced by TOF, Orbitrap and to a reaction monitoring (MRM) mode. While IT instruments are also capable of
lesser extent FT-ICR systems. this measurement mode, the quantitative performance of these detectors is
A second classification of MS instruments is based on whether they are generally inferior to QqQ. An alternative strategy involves exploiting the ben-
n efits of HR-MS data to increase selectivity, with the added benefit that full-
single or multistage systems. Multistage instruments allow MS (where n > 1)
operation either in time, as in ion trap (IT) instruments, or by combining scan data are also available. Especially Orbitrap instru-ments are increasingly
multiple mass analysers in space. Especially in combination with LC–MS being used for this purpose, where the high resolving power of these
using soft ionisation tech-niques such as ESI, where limited fragmentation instruments is beneficial.
n
information is obtained, tandem MS (MS/MS) or MS capabilities are
For untargeted analysis, resolving power is critical. In combi-nation with
essential for structural elucidation purposes. An array of very powerful hybrid
high efficiency or fast separations, however, the duty cycle of the mass
instruments is nowadays commercially available, including QqQ, quadrupole-
linear ion trap (Q-LIT), Q-TOF [414,415], ion-trap-TOF (IT-TOF) [416–418], spectrometer is equally important. Currently, TOF-MS instruments still
quadrupole- and linear ion trap-Orbitrap [419] and quadrupole- and linear ion provide the best compromise between these parameters, although
trap-ICR instruments. With the exception of the ICR instruments, all of these developments in Orbitrap systems show promise to obtain higher resolving
have found increasing application in flavonoid analysis during the last decade. power for similar scan times in the future. Secondly, tandem MS
measurements are highly beneficial, especially so in the case of ESI-MS
From the perspective of hyphenation to advanced LC separa-tions such as analysis of flavonoids due to the amount of information regarding substitution
UHPLC and LC × LC, a critical requirement of any mass analyser is a patterns of isomeric compounds that may be obtained (cf. Section 2.3). The
sufficiently high acquisition speed [420]. Peak widths in the order of a few combination of both MS/MS and high resolving power is ideal, and this
seconds are often encountered, which implies that fast acquisition rates, in the explains the contemporary popularity of Q-TOF systems for the non-targeted
order of 5–10 Hz and higher, are required to obtain the requisite ∼15 data analysis of flavonoids. On the other hand, the latest generation of Orbitrap
points across each peak. The latest generation of quadrupole and ion trap instruments are capable of providing the same information at high resolving
based analysers are mostly capable of meeting this requirement, while TOF power (although lower scan speeds), and have gained more widespread
systems provide the best scanning speeds of all analysers. acceptance in the last few years. In the following sections, the applications of
advanced LC separation with each of the different types of mass
Another important characteristic of mass analysers is the mass range they spectrometers for flavonoid analysis during the last seven years will be
are capable of covering. This value is relatively limited for quadrupole, ion outlined.
trap and Orbitrap instruments, with TOF sys-tems providing the highest mass
range of all systems.
An overview of the performance characteristics of mass anal-ysers 4.2.1. Nominal mass instruments: ion trap (IT) and triple
currently used in LC–MS analysis of flavonoids is presented in Table 3. It quadrupole (QqQ)
should be noted that these are generalised specifica-tions, with some variation Ion trap instruments use oscillating electric fields to trap and store ions in
occurring between instruments and the mode in which each instrument is two or three dimensions, based on which 3D (QIT or IT, also called Paul
operated. For example, the scan speed is inversely related to the resolving traps) and 2D (linear (quadrupole) ion trap (LQIT or LIT) systems can be
power in FT instruments (Orbitrap and ICR) and the mass range in scanning distinguished. The latter have higher trapping efficiencies and are less
instruments (Q and IT) as well as the sensitivity of all analysers to varying susceptible to space-charge effects, pro-viding higher sensitivity and an
increased dynamic range [413]. Accordingly, most recent applications in
degrees. For a relatively recent overview of the performance of the differ-ent
flavonoid analysis utilise LIT instruments (Table 4). The main advantage of
instruments, the reader is referred to the excellent reviews by Holcapekˇ et al.
n
[41] and Forcisi et al. [42], while more dedicated reviews may be consulted ion trap analysers is that they enable the true MS operation by allowing
for details on particular instruments such as Q-TOF [415], Orbitrap [421,422] selective trapping and fragmentation of parent and/or daughter ions as a
and FT-ICR [423] systems. function of time [161]. The primary application of these instru-ments has
therefore been in qualitative analysis.
Flavonoid analyses may broadly be divided into targeted and untargeted
applications. In the case of the former, a finite number of known target Ion trap instruments are extremely versatile and played a cru-cial role in
molecules are determined, mostly quantitatively, the early years of flavonoid analysis by LC–MS, primarily in terms of
n
structural elucidation by MS experiments. Nowadays,
44 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Table 4
Applications of advanced LC hyphenated to ion trap MS detectors to flavonoid analysis (2009–2015).
Sample Flavonoids Ionisation Detector(s) Stationary phase (column Mobile phase(s) References
source dimensions), flow rate,
temperature, analysis time
Licorice (Glycyrrhiza Prenylated and ESI+/− LIT
a C18 (150 mm × 2.1 mm, c
A: 0.1% AA , B: 0.1% [428]
c
glabra) glycosylated flavanones, UV 1.7 m dp ), AA in ACN
chalcones, isoflav-3-enes, b
FR = 0.3 mL/min, 25 min
isoflavones and
isoflavanes (18
flavonoids) ESI− C18 (150 mm × 2.1 mm,
Peanut skins A- and B-type LIT A: 0.1% AA, B: 0.1% [286]
proanthocyanidins (83 UV 1.7 m dp ), FR = 0.6 mL/min AA in ACN
◦
flavonoids) (0.3 mL/min to MS), 50 C,
27 min
Berry skins (Vitis Anthocyanins (18 ESI+ a C18 (250 mm × 4.6 mm, c [284]
QIT A: 2% FA , B: 2% FA
vinifera L.) flavonoids) 5 m dp ), FR = 1.0 mL/min, in ACN
◦
ESI− 50 C, 45 min
Black currant juice Flavone, flavonol and a C18 (50 mm × 2.1 mm, A: 0.1% FA, B: 0.1% [430]
Q-LIT
flavanone aglycones and 1.8 m dp ), FA in ACN
glycosides (13 flavonoids) ESI− FR = 0.3 mL/min, 45 min
Rooibos (Aspalathus Flavonol and flavone QIT C18 (150 mm × 4.6 mm, A: 1% FA in [424]
linearis) aglycones and glycosides, a
TOF 1.8 m dp ), FR = 0.5 mL/min H2 O/ACN (90/10,
◦
flavanone and chalcone (0.2 mL/min to MS), 25 C, v/v), B: ACN
glycosides (26 flavonoids) ESI+/− 35 min
Asian plant dyes Flavone aglycones LIT C18 (150 mm × 2.1 mm, c [431]
A: 0.03% TFA , B:
(Arthraxon hispidus glycosides (9 flavonoids) UV 1.8 m dp ), 0.024% TFA in ACN
and Miscanthus FR = 0.1 mL/min, 40 min
tinctorius) ESI+/− C18 (50 mm × 2.0 mm,
Pomegranate (Punica Flavan-3-ols, flavonols, LIT A: 0.1% FA, B: 0.1% [429]
granatum L.) juice flavanones, 1.8 m dp ), FA in ACN
◦
dihydrochalcones, and FR = 0.2 mL/min, 30 C,
flavones (14 flavonoids) ESI− 20 min
Eucalyptus wood Flavan-3-ols, flavonol QIT C18 (50 mm × 2.1 mm, A: H2 O/ACN (99:1, [432]
aglycones and glycosides, UV 1.9 m dp ), v/v) containing
◦
flavanones and FR = 0.6 mL/min, 25 C, 0.1% FA, B: 0.1% FA
flavanonols (18 37 min in ACN
flavonoids) ESI+/− C18 (50 mm × 2.0 mm,
Pomegranate (Punica Flavonol glycosides, LIT A: 0.1% FA, B: 0.1% [433]
granatum L.) juice anthocyanins, taxifolin 1.8 m dp ), FA in ACN
◦
hexoside and naringenin FR = 0.2 mL/min, 30 C,
(11 flavonoids) 20 min
Rat serum Isoflavones (4 flavonoids) ESI+ Q-LIT C18 (50 mm × 4.6 mm, A: 0.1% FA, B: 0.1% [92]
b FA in ACN
2.6 m dp SP ),
ESI− FR = 0.5 mL/min, 9 min
Halimione portulacoides Flavonol and flavone QIT C18 (50 mm × 2.1 mm, A: H2 O:ACN (99:1) [434]
glycosides (13 flavonoids) UV 1.9 m dp ), containing 0.1% FA,
◦
FR = 0.6 mL/min, 25 C, B: 0.1% FA in ACN
ESI+/− 37 min
Eastern Teaberry Flavan-3-ols, QIT C18 (150 mm × 2.1 mm, A: H2 O/ACN/FA [435]
(Gaultheria procyanidins and flavonol UV 1.7 m dp ), (95:5:0.1, v/v), B:
◦
procumbens L.) aglycones and glycosides FR = 0.3 mL/min, 25 C, 0.1% FA in ACN
(22 flavonoids) ESI+/− 80 min
Viola yedoensis Makino Flavone glycosides (35 LIT C18 (50 mm × 2.1 mm, A: H2 O, B: ACN [154]
flavonoids) UV 1.9 m dp ),
◦
FR = 0.25 mL/min, 30 C,
ESI− 33 min
Green tea, oolong tea Flavonol glycosides (18 QIT C18 (100 mm × 2.1 mm, A: 2% AA, B: ACN [436]
and black tea flavonoids) UV 1.8 m dp ),
◦
FR = 0.4 mL/min, 20 C,
ESI− 20 min
Forsythia flowers Rutin QIT C18 (150 mm × 2.1 mm, A: H2 O/ACN/FA [313]
UV 1.9 m dp ), (95:5:0.1, v/v/v), B:
◦
FR = 0.2 mL/min, 25 C, 0.1% FA in ACN
68 min
Dietary supplements Flavonol and flavanones ESI+ QIT C18 (100 mm × 0.075 mm, Isocratic: [437]
and food matrices (5 flavonoids) UV c
<2 m dp ), FR = 450 nL/min, ACN/MeOH /H2 O/FA
10 min (32.5:10:57:0.5,
ESI+/− C18 (150 mm × 1.0 mm, v/v/v/v).
Rosé wines Flavonols, flavan-3-ols, QIT A: 1% FA, B: 1% FA [438]
dihydrochalcones and ESI+/− QqQ 1.8 m dp ), in MeOH
◦
anthocyanins (113 UV FR = 0.08 mL/min, 40 C,
flavonoids) 46 min
a b
LIT, linear ion trap; Q-LIT, Quadrupole-linear ion trap; QIT, quadrupole ion trap; TOF, time of flight; QqQ, triple quadrupole. FR, flow rate;
SP, superficially porous.
c
AA, acetic acid; ACN, acetonitrile; FA, formic acid; TFA, trifluoroacetic acid; MeOH, methanol.
A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 45
however, IT instruments are less commonly used on their own, but are often MRM scanning was used to detect the presence of selected agly-cones
combined with HR analysers in powerful hybrid configu-rations such as IT- following in-source fragmentation of glycosylated flavonoids using a high
TOF and IT-Orbitrap instruments, which have largely superseded declustering potential. This was followed by a second full scan detection
conventional IT-MS in flavonoid analysis. In such instruments, the ion storage mode in the same analysis to allow tenta-tive assignment of the molecular
and sequential fragmentation capabilities of IT instruments are highly weight of the suspected target analytes. Finally, a second LC analysis was
beneficial, particularly in structural elucidation studies. Alternatively, IT performed with the detector used in product ion scan mode (with the third
multi-stage frag-mentation is often used in combination with separate LC– quadrupole used as a linear ion trap) for the identification of flavonoid
HR-MS analysis for structural elucidation [424–426]. From the perspec-tive derivatives.
of hyphenation to ultra-fast and high resolution separations, several latest
Within the scope of this review, we will limit our atten-tion to triple
generation IT and especially LIT instruments provide sufficient acquisition
quadrupole (QqQ) instruments. These combine two quadrupoles with a RF
speeds of up to ∼60 Hz (Table 3).
collision chamber in-between to allow tandem MS operation in space. Since
Ion trap instruments are generally considered inferior to quadrupole or both quadrupoles may be operated in selected ion monitoring (SIM) or scan
triple quadrupole instruments for quantitation. This is because of the need to modes, a range of different modes can be used on QqQ instruments. Scan
account for variable trapping times and also the lower dynamic range of these mode, where either of the quadrupoles is scanned, is less commonly used in
instruments (the latter limited by the capacity and need to avoid space-charge flavonoid analysis, in part because better performance in terms of sensitivity
effects). Nevertheless, Stanoeva et al. [427] have demonstrated the quanti- and accurate mass information can be obtained on other instruments. For
2 structural studies, product ion scan (Q1 in SIM, Q3 in scan), neutral loss (Q1
tative analysis of flavonoids in full-scan MS and MS modes using an IT
instrument, providing similar results but better sensitivity than UV detection. and Q3 in SIM mode at a fixed off-set) and precursor ion scan (Q1 scan, Q3
Gavina et al. [92] also utilised an IT instrument in MRM mode for the SIM) modes may be used [439]. QqQ instruments are however mostly utilised
quantitative analysis of isoflavones in rat serum, reporting a dynamic range of as selective and sensitive detectors in selected or multiple reaction monitoring
3 orders of magnitude and LOD’s of 0.01–3 pg on-column. An overview of mode (SRM or MRM), where Q1 is used in SIM mode to isolate par-ent ions,
the application of IT instruments in combination with advanced separation and Q3 in SIM to monitor selected daughter ions. For such targeted analysis,
methods is presented in Table 4; some noteworthy reports are discussed QqQ instruments provide very high sensitivity and selectivity as a
briefly below. consequence of the combination of two mass filters in SIM mode.
Table 5
Applications of advanced LC hyphenated to triple quadrupole MS detectors to flavonoid analysis (2009–2015).
Vitaflavan
®
and cocoa Flavanol ESI− b
QqQ (MRM )
b 1.7–692 g C18 (100 mm × 2.1 mm, d
A: 2% AA , B: [452]
d
nibs monomers and (not specified) 1.8 m dp ), ACN
procyanidins (4 c
FR = 0.4 mL/min, room
flavonoids) temp, 13 min
Milk-based food Anthocyanins and ESI+ QqQ (MRM) 3–3000 pg C18 (50 mm × 2.1 mm, d [408]
A: 10% FA , B:
products flavonols (26 on-column 1.7 m dp ), ACN
flavonoids) (10 L) FR = 0.4 mL/min, room
ESI− temp, 13 min
Cocoa Procyanidins (10 QqQ (MRM) 1–30 pg/ L C18 (100 mm × 2.1 mm, A: 0.2% AA, B: [453]
flavonoids) 1.8 m dp ), ACN
◦
FR = 0.4 mL/min, 30 C,
12.5 min
GegenQinlian Puerarin, ESI+ b 12.7–51 pg C18 (100 mm × 2.1 mm, A: 0.1% FA and [454]
QqQ (SIM )
a
decoction (TCM ) daidzein, baicalin, on-column 1.7 m dp ), 5 mM AA, B:
wogonoside and (5 L) ◦ d
FR = 0.2 mL/min, 30 C, MeOH
liquiritin ESI− 6 min
Black tea Proanthocyanins QqQ (MRM) LOD’s not C18 (100 mm × 2.1 mm, A: 0.1% FA, B: [88]
(8 flavonoids) UV specified 1.7 m dp ), 0.1% FA in ACN
◦
FR = 0.5 mL/min, 30 C,
ESI+/− 7 min
Rat plasma Procyanidins and QqQ (MRM) 1.5–606 g C18 (100 mm × 2.1 mm, A: 0.2% AA [455]
anthocyanins (21 (not specified) 1.8 m dp ), (procyanidins),
flavonoids) FR = 0.4 mL/min, 10% AA
12.5 min (anthocyanins),
ESI+/− C18 (100 mm × 2.1 mm, B: ACN
Tea Catechins (8 QqQ (MRM) LOD’s A: 0.05% AA [447]
compounds) 4.5–7.2 pg 1.7 m dp ), (+mode) or
◦
(+mode) and FR = 0.45 mL/min, 35 C, 0.01% FA
45–72 pg 5.5 min (−mode), B:
(−mode) MeOH
on-column
ESI+/− (1.5 L) C18 (100 mm × 2.1 mm,
Chamomile flowers Flavones and QqQ (MRM) LOD’s A: 0.1% FA, B: [456]
and Chamomile tea flavonols (9 2.6–12.9 pg 1.7 m dp ), MeOH
◦
extracts flavonoids) (−mode), FR = 0.45 mL/min, 30 C
4.9–408 pg
(+mode)
on-column
ESI− (3 L) C18 (50 mm × 2.1 mm,
Radix puerariae Isoflavones QqQ (MRM) A: 0.2% FA, B: [457]
aglycones and c MeOH
2.6 m dp SP ),
◦
glycosides (13 FR = 0.5 mL/min, 35 C,
flavonoids) 12 min
Red wine Anthocyanins ESI+ QqQ (Neutral Qualitative C18 (100 mm × 2.1 mm, A: 7.5% FA, B: [180]
(121 flavonoids) loss, survey 1.7 m dp ), ACN
◦
scan) FR = 0.1 mL/min, 50 C,
30 min
Mung bean (Vigna Flavanones, ESI+ QqQ (MRM) LOD’s C8 (150 mm × 2.1 mm, A: 10 mM FA, [83]
radiate) flavones and UV 0.04–3173 pg 1.7 m dp ), B: MeOH
◦
isoflavones (24 on-column FR = 0.2 mL/min, 40 C,
flavonoids) ESI+/− (2 L) 17 min
Apple, berries, green Dihydrochalcones, QqQ (MRM) LOD’s C18 (100 mm × 2.1 mm, A: 0.1% FA, B: [442]
tea and red wine isoflavones, 0.5–50 pg 1.8 m dp ), 0.1% FA in ACN
◦
flavones, on-column FR = 0.4 mL/min, 40 C,
flavanones, (2 L) 15 min
flavan-3-ols and
flavonols (63
flavonoids) C18 (150 mm × 2.1 mm,
Sangiovese wines Pyranoanthocyanins ESI QqQ (MRM) LOD’s 2–860 pg A: 5% FA, B: 5% [458]
and anthocyanin UV on-column 1.7 m dp ), FA in ACN
◦
flavanol adducts (2 L) FR = 0.4 mL/min, 40 C,
(92 flavonoids) ESI− 18 min
Cherry tomatoes, Flavonols and QqQ (MRM, LOD’s C18 (50 mm × 2.1 mm, A: 0.1% FA, B: [445]
tomato sauce and flavanones (3 product ion 100–1540 pg 1.7 m dp ), 0.1% FA in ACN
◦
juice flavonoids) and neutral on-column FR = 0.4 mL/min, 30 C,
ESI− loss scan) (10 L) 3 min
Cocoa beans Procyanidins (3 QqQ (MRM, Semi- C18 (150 mm × 2.1 mm, A: 0.1% FA, B: [446]
flavonoids) product ion quantitative 1.7 m dp ), 0.1% FA in ACN
◦
scan) FR = 0.3 mL/min, 30 C,
UV 35 min
A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 47
Table 5 (Continued )
Nevertheless, neutral loss and precursor ion scans are use-ful in group- to identify compounds of this class. Neutral loss mode (monitor-ing losses of
type identification of flavonoids, for example by studying glycosylated and/or 146, 162, 176, 308 and 324 for pentosides, hexosides, glucoronides,
n rutinosides, di-hexosides and hexosyl-glucoronides, respectively) was used to
acylated flavonoid derivatives, prior to HR-MS identification of compounds
[180,448]. As an exam-ple, Alberts et al. [180] applied UHPLC-QqQ in confirm putative compounds of interest. The authors reported that the latter
neutral loss and ‘survey scan’ (neutral loss-triggered product ion scan mode) operation mode was less sen-sitive and relied on slower scan times compared
for the analysis of red wine anthocyanins (Fig. 15). In neu-tral loss mode, to precursor ion scan mode, such that closely eluting isobaric compounds were
class-type detection of anthocyanidin-glycosides, -acetylglycosides, not resolved. Further confirmation of the tentatively identified com-pounds
-coumaroylglycosides, -caffeoyglycosides and -di-glycosides was performed. was obtained using high resolution MS and MS/MS data acquired on a Q-TOF
Compound identity was then con-firmed based on characteristic high-energy instrument [449].
CID spectra obtained for the anthocyanidin aglycones in survey scan mode.
Significantly, the characteristic product ion spectra showed consistent Obtaining a general sense of the sensitivity of QqQ detection in MRM
behaviour also for the derived anthocyanins encountered in wine, which mode is complicated by the fact that sensitivity varies between instruments
allowed the authors to identify 121 compounds in red wine using this and as a function of the number and nature of target analytes, ionisation mode
approach. and chromatographic conditions. Nevertheless, reported limits of detection
(LODs) fall in the range of 0.02 pg–3 ng on-column (Table 5), with higher
values reported for aglycones [83,408]. Usual LOD values for glycosylated
De Rosso et al. [449] used UHPLC-QqQ in various modes for the detailed species are in the region of 0.1–10 pg on-column, and typical linear ranges are
analysis of red wine flavonols. First of all, precursor ion analysis of the six in the order of 2–5 orders of magnitude [408,442,450], which
known wine flavonol aglycones was used
A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 49
◦
Fig. 14. Overlay of MRM traces acquired for the UHPLC–ESI-QqQ-MS analysis of 152 phenolics in rosé wine. Analysis conditions: 100 mm × 1 mm 1.7 m porous C18 phase, 40 C, 0.17 mL/min.
Fig. 15. (A) Chromatogram obtained for the UHPLC–MS/MS analysis of red wine anthocyanins on a QqQ instrument in neutral loss mode for the detection of anthocyanin-glycosides (neutral loss of
m/z 162). The insert shows the fragmentation pattern at low collision energies (25 V). (B) The characteristic product ion spectrum of malvidin-3-O-glucoside obtained in ‘survey scan’ mode (neutral
loss-triggered product ion scan) at 50 V collision energy. The insert shows the fragmentation scheme for the aglycone following the loss of the neutral sugar moiety. Analysis conditions: 100 mm × 2.1
◦
mm 1.7 m porous C18 phase, 50 C, 0.1 mL/min.
Source: Adapted from [180].
50 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
explains the widespread use of these detectors for targeted quan-titative ions for flavonoid-C-glycosides). Further putative identification of the parent
analysis. It is relevant to note that matrix effects remain a concern for such ions was then obtained based on extracted ion chro-matograms (using a 10
applications using ESI. This can be minimised by using standard addition mDa window) of the characteristic ion and an automated screening approach
quantification – the increased number of injections is then offset by the where known ‘adducts’ such as hexosides, pentosides, etc. are searched for in
shorter analysis times offered by UHPLC separation [440,451]. the full scan spectral data acquired. Sodium adducts were used as added
confirmation of the proposed structure (based on the observation that fragment
ions do not form sodium adducts).
4.2.2. Time-of-flight (TOF) instruments
Time-of-flight mass spectrometers [470] work on the principle that lighter A trend in the last few years has been increasing use of hybrid TOF
ions travel faster than heavier ions following an initial acceleration by an instruments, with single stage TOF analysers increasingly being replaced by
electric field. All ions acquire the same kinetic energy during this initial especially Q-TOF systems in flavonoid analyses. While in-source CID can be
acceleration period, and are separated in the field-free flight tube according to used to obtain fragmentation information needed to identify compounds
their different velocities. The physical property that is measured is flight time, [164,193,297,478], hybrid systems are much more flexible in allowing dual
3
which is directly related to the mass-to-charge ratio of the ion. Due to this scan modes, true MS/MS and also pseudo MS operation by utilising in-
mode of operation, TOF instruments offer very high mass ranges (without source CID [479,480]. The most used configuration for flavonoid analysis is
compromising sensitivity, as is the case in scanning mass spectro-meters), the Q-TOF (two-thirds of the papers listed in Table 6 used this instrument). In
very high acquisition rates and for the latest generation of instruments, addition to the tandem MS capabilities of Q-TOF systems, alterna-tive
relatively high resolving power and good sensitivity (although the latter acquisition modes are also possible, such as recording spectra at both high and
E
decreases with high acquisition rates) (Table 3). low collision energy settings within a single analy-sis (referred to as MS by
one vendor). An example of the application of this approach for the
Several important developments have improved the per-formance of TOF identification of phenolic compounds in apple products by UHPLC–Q-TOF-
instruments [471]; from the perspective of hyphenation to LC with ionisation MS analysis is presented in Fig. 16 [481]. Fig. 16B shows the extracted ion
sources such as ESI or APCI the introduction of orthogonal acceleration to chromatograms for two partially co-eluting flavonoids, with the corresponding
E
transform continuous ion beams into a pulsed source is especially relevant MS spec-tra obtained using a collision energy ramp of 10–40 eV presented in
[472,473]. Fur-thermore, reflectron instruments improve the resolving power Fig. 16C. While this approach is useful for the fast screening of unknown
and mass accuracy of TOF instruments through the use of an electro-static flavonoids, providing as it does useful information for tentative compound
reflector (ion mirror) to correct for kinetic energy dispersion of ions leaving identification based on the molecular ion and major fragments in a single
the ion source [474], although this comes at the cost of somewhat reduced analysis, it also lacks the selectivity offered by the MS/MS mode. This is
E
sensitivity and dynamic range [42]. evident from Fig. 16C where MS spectra contain fragment ions from co-
eluting compounds, which may hamper structural elucidation of co-eluting
In addition, several hybrid TOF instruments are nowadays com-mercially flavonoids of the same class.
available, which provides the option of tandem MS operation with high
resolving power acquisition. The most com-mon of these, the quadrupole-
TOF (Q-TOF) [414,415] has found extensive use in flavonoid analysis (Table
6). The Q-TOF configu-ration allows collimation of the ion beam as well as UHPLC-Q-TOF has also been used in the detailed analysis of the MS/MS
the option of mass selection in Q1 and fragmentation in a RF only behaviour of flavonoids. For example, Guo et al. [177] reported a systematic
quadrupole. A further variant of the Q-TOF system equipped with an ion approach to differentiate between isomeric 6-C and 8-C flavone glycosides
mobility cell between the quadrupole and the TOF analyser is available (see based on negative and positive ionisation MS/MS spectra of the deprotonated
below for a discussion on ion mobility) [475]. In another hybrid instrument, and protonated molecular ions, with the latter proving to be more
the ion trap TOF (IT-TOF) [416], the IT is used to store ions from a discriminative. Ma et al. [482] reported a MS/MS method on a Q-TOF
continuous source such as ESI, from where they are ejected via a dc pulse to instrument to allow differentiation of regioisomers of methylated flavonols
the TOF, delivering enhanced sensitivity through the storage of ions in the IT. +
based on the position-specific [M+H−CH4] ion and methylated RDA frag-
n
This configuration can also be used to perform MS experiments prior to high ments, as well as complementary information obtained from the RP-LC
resolution TOF detection. elution order.
Table 6
Applications of advanced LC hyphenated to time-of-flight MS detectors to flavonoid analysis (2009–2015).
Fig. 16. (A) UV chromatogram obtained at 280 nm for the UHPLC–Q-TOF-MS analysis of an apple extract. (B) The extracted ion chromatograms for phloretin-2 -O-xylosyl-glucoside (m/z 569) and
E
quercetin-3-O-xyloside (m/z 435) and (C) the corresponding MS spectra for the compounds obtained using the simultaneous (non-selective) acquisition of spectral data using a collision energy ramp
◦
of 10–40 eV. Analysis conditions: 100 mm × 2.1 mm 1.7 m porous C18 phase, 40 C, 0.35 mL/min; ESI+ detection at 10 Hz.
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical to determine the most active spectrum using FT [421]. In the first commercial configuration (LTQ-
antioxidants. This methodology proved to be more sensitive than the Orbitrap), ions are initially stored in a linear ion trap, from where they are co-
conventional on-line DPPH assay. axially ejected into a bent quadrupole or ‘C-trap’ to focus and quickly inject
Q-TOF-MS has also been hyphenated with HILIC [128], with an amide them into the Orbitrap. The resolving power of these instruments can be as
◦ high as 500,000, depending on the particular instrument, scan mode and scan
column operated at 50 C being used for the analysis of diverse anthocyanins
in blueberries, grape skins, black beans, red cabbage and red radish. time.
Various forms of bench-top Orbitrap mass spectrometers have been
The widespread use of TOF, and especially Q-TOF detectors in TM
utilised in the analysis of flavonoids. In the LTQ-Orbitrap XL a collision
contemporary LC–MS analysis of flavonoids makes sense in light of the high cell is added after the C-Trap to perform CID for MS/MS analyses; the LTQ-
resolution mass data and high acquisition rates offered by these detectors and Orbitrap Velos shows increased MS/MS sensi-tivity due a more effective ion
their relative cost compared to higher resolv-ing power instruments. The mass TM
guide. The Orbitrap Elite uses a smaller Orbitrap with an increased electric
accuracy of the latest generation TOF detectors are sufficient to allow field, increasing the resolving power of the instrument to 240,000 at m/z =
determination of molecular formulae for flavonoids of MW up to ∼1200 400. A mod-ified version of this instrument utilising a compact analyser has
[128] and to distinguish between isobaric compounds based on this data. been shown to attain a resolving power of 960,000 at m/z 400 for a 3 s
Furthermore, the MS/MS capabilities of Q-TOF systems are critical in TM
detection time [552]. In the Orbitrap Exactive , the ion source is directly
tentative struc-tural elucidation of flavonoids. Wojakowska et al. [480] found connected to the ‘C Trap’ followed by a collision cell before the Orbitrap
a resolving power of higher than 15,000 sufficient for molecular for-mula analyser. Ions that underwent fragmentation in the collision cell are
determined for flavonoids of MW up to 700 in first order spectra, but transferred back into the ‘C-Trap’ before being injected into the Orbitrap for
TM
estimated that a resolving power of 40,000 would be needed to attain the mass analysis. The latest edi-tion is the non-hybrid Q-Exactive , which has
same level of mass accuracy of MS/MS spec-tra. This is within the reach of a lower resolving power (140,000 at m/z 200) but provides higher scan speeds
several commercial instruments. While the mass accuracy of Orbitrap and than previous versions (12 Hz at a resolving power of 17,500), making it
especially FT-ICR sys-tems are certainly much better than TOF systems, and compatible with high speed separations.
therefore hold clear benefits for the separation of high MW compounds such
as proanthocyanins, the performance of TOF systems for ultra-fast separations
currently remains unmatched between the high reso-lution instruments. The utility of high resolution MS and MS/MS data obtained through
utilisation of Orbitrap instruments in infusion mode has been demonstrated for
the identification of polyphenols [553,554], while a number of reports
utilising conventional HPLC with Orbit-rap detectors have also been reported
4.2.3. Fourier transform ion cyclotron resonance (FT-ICR) MS FT-ICR [150,192,555–560]. For example, Lamuela-Raventós and co-workers used
[547,548] is the mass spectrometric technique with the conventional HPLC methods in combination with an LTQ-Orbitrap Velos to
6
highest mass resolution (up to greater than 10 ) and sub-femtomol sensitivity. study the phenolic composition of tomatoes [448], beer [561], walnut
The technique works on the principle of exciting all ions in the cyclotron
[562] and sofrito [563], while Cahill et al. [564] developed and validated an
simultaneously and measuring the ion image resulting from their combined
HPLC-Orbitrap method for quantifying isoflavones in waste water following
trajectories, which is amplified and translated to the frequency domain using
APCI ionisation. In the following discussion pertaining to Table 7, the focus
FT [423]. The res-olution depends on the observation time, and as a
consequence FT-ICR instruments are too slow to allow on-line hyphenation to will however be on selected appli-cations of advanced LC methods
UHPLC separation, and are generally used only in infusion mode for hyphenated to Orbitrap detection for flavonoid analysis.
flavonoid analysis [507]. An alternative is to use at-line or off-line coupling to
The majority of these have to date been performed using the LTQ-Orbitrap
HPLC separations, which essentially involves collection of fractions from the
TM
chromatographic system and subsequent infu-sion into the FT-ICR. While XL instrument. Li et al. [565] used this instru-ment coupled to a UHPLC
time consuming and labour intensive, this is currently the most practical way separation to identify 15 flavonoids in the traditional Chinese medicine
to exploit the high resolving power of FT-ICR in combination with HPLC (TCM) Sarcandra glabra, including glycosylated flavanones, flavanonols and
n
separation, and shows promise for example in metabolomic analysis [42]. By a flavanol, based on neg-ative ESI MS spectra and accurate mass
2006 FT-ICR had not been used in the LC–MS analysis of flavonoids [145], information. Analysis of Serbian honeys [566–568] was achieved on a 1.9 m
and to the best of our knowledge the only application of LC–FTICR-MS to dp column, while Orbitrap MS/MS was used to identify up to 31 flavonols,
flavonoid analysis since then was reported by Suzuki et al. [549]. These flavanonols, flavanones and flavones. An example of the benefit of high
authors used conventional HPLC (5 m ODS phase) with posi-tive mode ESI resolution MS data is demonstrated for these samples in Fig. 17, where four
to characterise flavonoids in Lotus japonicas. The mass spectrometer was peaks are observed in the EIC obtained at 269.06 (Fig. 17(b), 200 ppm mass
operated with a resolving power of 100,000 (at m/z 400), although no precision). Extraction of masses 269.0459 and 269.0822 at 1 ppm mass
information was provided on the scan time and the TIC was not presented. precision (Fig. 17(c) and (d), respec-tively) shows the peak pairs observed for
On-going developments aimed at extending the resolving power and reducing closely eluting apigenin and galangin (m/z 269.0459) and alpinetin and
the time domain tran-sient length of FT-ICR instruments may in future allow pinostrobin (m/z 269.0822), respectively, as confirmed by data-dependent
hyphenation of the detector to fast HPLC separations [42]. MS/MS operation [566].
TM
Lebel et al. [290] used a LTQ-Orbitrap XL instrument for the
quantitative analysis of erectile dysfunction ingredients, including 9 natural
4.2.4. Orbitrap instruments flavonoids. The authors used full scan mode for deter-mination of accurate
The Orbitrap mass analyser, first described by Makarov [550,551], was mass and data-dependent MS/MS of targeted precursors. Limits of detection
introduced on the market in 2005. This system relies on the orbital trapping of for the flavonoids varied between 3 and 23 pg on-column.
ions around a spindle-like central elec-trode and an outer flared barrel-like
electrode using electrostatic fields. The frequency of the harmonic ion Tchoumtchoua et al. [163] reported a comprehensive structure-oriented
oscillations, which are inversely proportional to the square root of the m/z approach based on UHPLC–ESI-MS/MS using an LTQ-Orbitrap
TM
ratios, is mea-sured as an image current transient which is converted to Discovery instrument for the characterisation of isoflavonoids (isoflavones,
frequency dihydroisoflavones and their cor-responding glycosides) in natural products.
The proposed
60 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
Table 7
Applications of advanced LC hyphenated to Orbitrap and ion mobility mass spectrometry to flavonoid analysis (2009–2015).
Fig. 17. UHPLC–ESI-Orbitrap analysis of an ethyl acetate extract of Serbian honey. (a) depicts the TIC obtained in negative ionisation mode, (b) the extracted ion chromatogram for m/z 269.09 at 200
ppm mass precision, and (c) m/z 269.0459 and (d) 269.0822 at 1 ppm mass precision, respectively. Apigenin and galangin (c) and pinetin and pinostrobin (d) were identified based on MS/MS spectra.
methodology was illustrated for the determination of isoflavones in an quantification of these compounds is impossible using UV detection only due
Amphimas pterocarpoides crude extract. Chromatographic, UV and MS to extensive co-elution and the lack of suitable commercial standards.
information was combined to prioritise potential com-pounds of the target
class, which were further characterised based on accurate mass fragmentation In another interesting paper, the group investigated the use of negative ESI
information. The structures of selected tentatively identified compounds were behaviour of anthocyanins [287]. These compounds are normally detected in
further confirmed by NMR, and investigated using APCI and ESI MS/MS positive ionisation mode due to the preva-lence of the flavylium cationic
following infusion of the isolated compounds. For methylated isoflavones, species in solution, but may be hard to distinguish from flavonol-O-glycosides
positive mode APCI was found to provide useful information for structural due to identical nominal masses and (low-energy) fragmentation patterns. The
elucidation purposes. motivation here was to use negative ionisation to discriminate between these
flavonoid classes. Indeed, unusual [M−2H] − and [M−2H+H2O]− ions were
n
The utility of UHPLC–DAD-ESI-HR-MS in the profiling of flavonoids observed for the anthocyanins, for which the authors pro-posed a possible
mechanism of formation. However, a more likely explanation is that these
in foods is amply illustrated by a series of papers from the group at the US
TM ions are due to the deprotonated quinoidal and carbinol pseudobasic forms of
Department of Agriculture. These authors used an LTQ-Orbitrap XL anthocyanins, which are present in the column due to the low acidity of the
(operated at a resolving power of 15,000) with positive and negative ESI and mobile phase (0.1% formic acid) – these species are not separated
a 1.9 m RP-LC column to screen a range of phenolics in various sam-ples. chromatographically due to their relatively fast inter-conversion [105]. In our
For example, 30 anthocyanins and 105 flavonol glycosides were identified in opinion negative ionisation of anthocyanins makes little sense in light of the
microgreens [569], 67 anthocyanins and 102 flavonol glycosides in red fact that these compounds can normally be distinguished from other classes
mustard greens [189], 19 flavonoids in strawberries [217], 46 flavonol
based on UV characteristics, and failing that based on distinct high collision
glycosides and acylated gly-cosides in Rorippa indica [570] and 247
energy CID spectra of anthocyanidins [180]. Furthermore, analysis of
proanthocyanidins in seven different food materials [571]. In the latter report,
the authors derived relative molar response factors for ion counts in SIM containing only 0.1% formic acid
anthocyanins using mobile phases
operation from UV data, and used these to quantify (sepa-rated) leads to significant and evident band-broadening for these
proanthocyanidins in grape seeds. Despite the assumptions inherent to this
approach, the methodology represents a signif-icant contribution in the field
compounds, thereby exacerbating co-elution and providing an
of proanthocyanin analysis, since additional criterion to distinguish them from flavonols, if
needed.
A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 63
The Q-Exactive
TM
Orbitrap has also found application in flavonoid a real alternative to TOF analysers for such analyses in the future.
analysis. Palermo et al. [557] used this instrument in combination with HPLC
separation to identify 40 flavonoids in arti-chokes. The resolving power of the
instrument was set to 25,000 FWHM for a scan time of 1 s. Yang et al. used 4.2.5. Ion mobility spectrometry
this instrument in combination with an HPLC method for the qualitative Ion mobility spectrometry (IMS) measures the drift-time of ions through a
analysis of the TCM Huang-Lian-Jie-Du decoction (HLJDD). 24 flavonoids buffer gas in the presence of an electric field. The drift-time of an ion depends
were identified, some of which were then used as markers for quality control on its size/charge ratio – ions can therefore be separated based on differences
and quantified in selected samples using a UHPLC–QqQ-MS method utilising in their net charge or collision cross-sections, or by interactions with the gas.
polarity switching [444]. Tarr et al. [572] used a superficially porous phase The technique has found extensive application in the detection of explo-sives,
TM drugs and chemical warfare agents. IMS is ideally compatible with mass
and Q-Exactive Orbitrap MS in a metabolomics study to investigate the
effect of terroir and variety on Vitis vinifera L. grape juices. The metabolic spectrometry and provides complementary informa-tion, which explains the
signatures in negative and positive ionisation showed clustering according to growth in popularity of ion mobility-mass spectrometry (IM-MS) [586]. IM-
varietal and terroir properties, respectively, while flavonoid profiles obtained MS is especially used in the sep-aration of isobaric compounds and isomers,
from identification of 466 compounds using a flavonoid database were found conformers and in the measurement of ion size. Interestingly, because of the
to be good indicators of varietal character indepen-dent of terroir. Garrido rela-tive timeframes of chromatographic separations (s), IMS (ms) and MS
Frenich and co-workers reported UHPLC–MS methods based on the Q- ( s), the technique may be considered a form of comprehen-sive 3D
TM separation, since the separation obtained in each previous ‘dimension’ is
Exactive Orbitrap for the qualitative and quantitative analysis of
flavonoids in green tea [573] and soy based nutraceutical products [574]. MS essentially maintained. A range of different IMS methods may be used in IM-
data were acquired in full MS (no fragmentation, resolving power 25,000 MS, including traditional drift-time ion mobility (DTIMS), aspiration ion
FWHM; scan time = 0.25 s) and all-ion fragment (collision energy 30 eV, mobility (AIMS), field-asymmetric waveform ion mobility (FAIMS) and
10,000 FWHM; scan time = 0.10 s) modes in both positive and negative travelling wave ion mobility (TWIMS). A diverse range of IM-MS
ionisation. The overall scan rate for the four acquisition functions was 0.56 instrumentation is furthermore available, with IMS having been hyphenated to
Hz. LOD values varying between 1 and 50 g/L were reported for a range of magnetic sector, TOF, quadrupole, IT and FT-ICR instruments. For further
different flavonoids. Mullen et al. [263] described an HPLC method details on the various instrumental configurations available, the reader is
TM referred to [587].
employing a superficially porous phase and an Exactive Orbitrap mass
spectrometer for the (semi)-quantitative analysis of procyani-dins, flavonols
and hydroxycinnamic acids in whole coffee fruits, beans and husks from
China, India, and Mexico. The instrument resolving power was set to 60,000,
although the scan speed was not specified. IM-MS has found relatively sparse application in flavonoid anal-ysis to
date (Table 7), with most work done in infusion mode (i.e. without
chromatographic pre-separations). For example, Clowers et al. [588]
described a method using a dual gate IM-LIT mass spectrometer to separate
cation adducts of flavonoid-diglycosides. This type of mass spectrometer can
It should be noted that although the Orbitrap is a true high resolution operate either in a dual gate scanning (DGS) or single mobility monitoring
instrument (providing for example resolving powers of 60,000–100,000 (SMM) mode. In DGS mode a complete mobility spectrum was obtained.
FWHM for flow injection analysis [190,553,554]), this performance is highly Once the drift-time(s) were obtained using DGS, ions can be selectively
dependent on the scan time and mode. In LC–MS applications where fast or transmitted to accumulate a specific ion population to improve efficiency.
high-resolution separation is performed, the instrument is commonly used at a Troc´ et al. [589] demonstrated the (travelling wave) IM-MS separation of the
resolution set-ting of between 7500 and 30,000 to acquire a sufficient number epimers (+)-catechin and (−)-epicatechin by complexation with chiral
of spectra across each peak, and lower values for MS/MS acquisition [422]. modifiers and transition metals.
(In fact, depending on the number of scan modes utilised, scan speeds are
often insufficient for high-speed separations on older Orbitrap instruments). In contrast, flavone-glycosides were separated under ambi-ent pressures
In practice therefore Orbitrap instru-ments provide little gain in resolving using a differential ion-mobility spectrometer [462]. This method included a
power compared to the latest generation TOF systems for such applications. chromatographic separation using a PFP column. For the identification of
On the other hand, the compromise between sensitivity and scan time is lower diosmetin-7-O-glycoside and diosmetin-3-O-glycoside an IMS coupled to a
than for other HR mass analysers [422]. Q-LIT analyser was used.
More recently, IM-MS has also been used in combination with UHPLC
The compromises involved in parameter selection for quanti-tative separation for flavonoid applications. Yassin et al. [585] described a method
UHPLC-Orbitrap analysis have been elegantly addressed by De Paepe et al. employing UHPLC coupled to a Q-IM-TOF mass spectrometer to separate
[112]. These authors found a scan speed of 2 Hz, obtained at a resolving isomeric flavanols unresolved by chromatographic separation. The authors
power of 50,000 (WFHM at m/z 200) in MS mode, to be optimal using an showed that isomeric structures of B-type prodelphinidins and theasinensins
TM as well as the isobaric compound rutin could be resolved based on their
Exactive instrument (for an anal-ysis time of 23 min; for fast gradients
resolution would need to be adapted to increase the scan speed). Furthermore, mobility drift times in the negative ion mode. Gonzales et al. [584] used the
mass accuracy performance is also related to the dynamic range, which in turn additional selectivity offered by ion mobility separation to obtain cleaner mass
is related to the scan range. It is therefore clear that careful opti-misation of spectra to assist in the identification of acyl-ated and non-acylated flavonol-
MS (and MS/MS) parameters is required for optimal performance of Orbitrap glycosides. In both of these works, a commercial instrument equipped with a
instruments. TWIMS cell [475] was used.
Fig. 18. Methodology for assignment of daughter ions based on two separate ion mobility-MS experiments and classical least squares data modelling. Data shown for isovitexin (apigenin-6-C-
glucoside).
Source: Reproduced with permission from [590].
allow parent ions to enter the IMS cell (low collision energy in trap cell) and 4.2.6. Overview of mass spectrometers used in flavonoid analysis Fig. 19
fragment the separated ions before transfer to the TOF (high transfer cell summarises graphically the relative proportion of appli-
collision energy) [585]. Garmón-Lobato et al. which utilised different MS
cations listed in Tables 1, 2 and 4–7
[590] reported an interesting chemometric method for using IMS-MS detectors (single quadrupole applications are not included). It
measurements to assign daughter ions formed in the transfer cell (i.e. after
IMS separation) to specific pre-cursor ions. Since CID fragmentation is also should be pointed out that this is not an accurate reflection of
performed in the trapping cell, this approach provides equivalent information the global picture in flavonoid analysis because of the focus in
3
to MS experiments, but for all precursor ions simultaneously. The procedure the cur-rent work on advanced LC separations (and inevitable
is illustrated schematically in Fig. 18.
bias based on the incomplete data set). Nevertheless, for
applications falling within the scope of this work, the relative
popularity of TOF sys-tems is clear (50% of all applications)
and not unexpected based on
A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 65
Advances in LC–MS analysis of flavonoids largely mirror, and to a degree The extent of the advances in LC–MS and their implications in terms of
build on those in fields such as proteomics and metabolomics, which are the quality of analytical data obtainable are often not fully appreciated. Fig. 20
underpinned by significant developments in both HPLC and MS presents an attempt to highlight the evolution of flavonoid analysis, using
instrumentation. Most influential have been technological advances in MS, anthocyanins as an example. Compar-ison of the contemporary situation with
and translation of these to commer-cial instruments which are becoming more the first HPLC separation of wine anthocyanins in 1978 shows a clear increase
pervasive in routine laboratories. Notable are the performance of the latest in separation performance coinciding with improvements in column
genera-tion of QqQ instruments, which makes these systems ideal for the technology and LC × LC operation. Concurrent progress in hyphenation to
MS
Fig. 20. Evolution of the HPLC analysis of anthocyanins. (A) One of the first applications of RP-LC-UV to the analysis of grape anthocyanins. Conditions: C18 column (250 mm × 4.6 mm, 5 m dp ),
10% formic acid/methanol gradient at 2.5 mL/min, UV detection at 520 nm. Reproduced with permission from [29]© 1978 American Society for Enology and Viticulture. AJEV 29:42–49. (B)
UHPLC–ESI-QqQ-MS analysis of rosé wine showing the MRM transitions for 152 phenolic compounds. Conditions: C18 column (150 mm × 1 mm, 1.8 m d p ), 1% formic acid/methanol gradient at
0.17 mL/min, 40 ◦ C. Reproduced from [438]. (C) depicts the high efficiency UHPLC–ESI-Q-TOF-MS analysis of red wine anthocyanins. Conditions: C18 column (200 mm × 2.1 mm, 1.7 m d p ),
7.5% formic acid/acetonitrile gradient at 0.06 mL/min, 50 ◦ C. Reproduced with permission from [218]. (D) presents a UV contour plot obtained for the off-line HILIC × RP-LC analysis of grape
anthocyanins. Conditions: 1 D: amide column (150 mm × 4.6 mm, 2.5 m d p ), 0.4% TFA/acetonitrile gradient at 0.2 mL/min, 50 ◦ C, 1 ts = 0.5 min; 2 D, superficially porous C18 column (50 mm × 4.6
mm, 2.6 m dp ), 7.5% formic acid/acetonitrile gradient at 0.5 mL/min, 50 ◦ C, ESI-Q-TOF-MS and UV detection at 500 nm. Reproduced with permission from [248].
66 A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78
detection now allows selective analysis of large numbers of target DESI desorption electrospray ionisation
compounds by tandem MS, whereas HR-MS hyphenated to high- DGS dual gate scanning
resolution separations enables identification of large numbers of DP degree of polymerisation
unknown compounds. In fact, comparison of the reports cited in DPPH 1,1-diphenyl-2-picrylhydrazyl radical
this work with literature from only ten years ago clearly highlights DTIMS drift-time ion mobility spectrometry
the performance gains in terms of both analysis time reduction and ED electrochemical detection
the number of compounds that can be separated and tentatively EI electron (impact) ionisation
identified allowed by the hyphenation of advanced separation and ELSD evaporative light scattering detector
MS technologies. Considering the pace of developments in HPLC ESI electrospray ionisation
and MS, any prediction of future progress is necessarily fraught FAB fast atom bombardment
with uncertainty; it is however clear that the growing application FAIMS field-asymmetric waveform ion mobility spectrometry
of advanced LC–MS methods to flavonoid analysis will increasingly FL fluorescence
play an important role in support of flavonoid research. FR flow rate
FT Fourier transform
Symbols and abbreviations GC gas chromatography
HILIC hydrophilic interaction chromatography
HPLC high performance liquid chromatography
Symbols HRF heterolytic ring fission
ˇ under-sampling correction factor HR-MS high resolution mass spectrometry
xD xth dimension HTLC high temperature liquid chromatography
Dm diffusion coefficient of the analyte in the mobile phase i.d. internal diameter
x dilution factor in the xth dimension ICR ion cyclotron resonance
DF
P pressure drop IEC ion exchange chromatography
Pmax maximum pressure IM-MS ion mobility-mass spectrometry
dp particle size IMS ion mobility spectrometry
ε external porosity IT ion trap
εT total porosity LC liquid chromatography
fc fractional surface coverage LC–MS liquid chromatography–mass spectrometry
xF volumetric flow rate in the xth dimension LC × LC comprehensive two-dimensional liquid
H height equivalent of a theoretical plate (typically in m) chromatography
H
min minimum height equivalent of a theoretical plate LOD limit of detection
Kvo 2 LIT linear ion trap
column permeability (typically in m )
L column length MALDI matrix-assisted laser desorption ionisation
max wavelength of maximum absorbance MB moving belt
N number of theoretical plates (dimensionless) MD-LC multidimensional liquid chromatography
mobile phase viscosity MRM multiple reaction monitoring
n c,2D (practical) peak capacity of a two-dimensional separation MS mass spectrometry
x
nc peak capacity of separation in the xth dimension MS/MS tandem mass spectrometry
x n
standard deviation in dimension x MS multistage mass spectrometry (where n ≥ 2)
1
ts first dimension sampling time MW molecular weight
x gradient time in the xth dimension NMR nuclear magnetic resonance
tg
uopt optimal mobile phase linear velocity (typically in mm/s) NP-LC normal phase liquid chromatography
u0 mobile phase linear velocity (determined using unre- ODS octadecyl-silica
tained marker) OEG oligo-ethylene glycol
V
frac fraction volume PD plasma-desorption
x injection volume in the xth dimension PEG polyethylene glycol
Vinj
wb peak width at baseline PFP pentafluorophenyl
ppm parts per million
Abbreviations Q quadrupole
1-D one-dimensional QM quinone methide fragmentation pathway
2-D two-dimensional QqQ triple quadrupole
ABTS 2,2 -azino-bis(3-ethylbenzothiazoline)-6 sulfonic acid RDA retro-Diels–Alder
AIMS aspiration ion mobility spectrometry RP-LC reversed phase liquid chromatography
amu atomic mass units SEC size exclusion chromatography
APCI atmospheric pressure chemical ionization SIM selected ion monitoring
API atmospheric pressure ionization SMM single mobility monitoring
APPI atmospheric pressure photoionisation SR split ratio
BFF benzofuran-forming fission SRM selected reaction monitoring
CCC countercurrent chromatography TCM traditional Chinese medicine
CD cyclodextrin TFA trifluoroacetic acid
CE capillary electrophoresis TLC thin layer chromatography
CI chemical ionisation TOF time-of-flight
CID collision induced dissociation TSP thermospray
CPC centrifugal partition chromatography TWIMS travelling wave ion mobility spectrometry
DAD diode array detection UHPLC ultra high pressure liquid chromatography (pressures
DART direct analysis in real time >400 bar)
A. de Villiers et al. / J. Chromatogr. A 1430 (2016) 16–78 67
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