Separation & Purification Reviews

ISSN: 1542-2119 (Print) 1542-2127 (Online) Journal homepage: http://www.tandfonline.com/loi/lspr20

Analysis of Polyphenols in Honey: Extraction,

Separation and Quantification Procedures

Ana Pascual-Maté, Sandra M. Osés, Miguel A. Fernández-Muiño & M. Teresa

Sancho

To cite this article: Ana Pascual-Maté, Sandra M. Osés, Miguel A. Fernández-Muiño

& M. Teresa Sancho (2018) Analysis of Polyphenols in Honey: Extraction, Separation
and Quantification Procedures, Separation & Purification Reviews, 47:2, 142-158, DOI:
10.1080/15422119.2017.1354025

To link to this article: https://doi.org/10.1080/15422119.2017.1354025

Accepted author version posted online: 13

Jul 2017.
Published online: 16 Aug 2017.

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Separation & Purification Reviews, 47: 142–158, 2018
Copyright © Taylor & Francis Group, LLC
ISSN: 1542-2119 print / 1542-2127 online
DOI: https://doi.org/10.1080/15422119.2017.1354025

Analysis of Polyphenols in Honey: Extraction,

Separation and Quantification Procedures
Ana Pascual-Maté, Sandra M. Osés, Miguel A. Fernández-Muiño, and M. Teresa Sancho
Nutrition and Bromatology Division, Department of Biotechnology and Food Science, Faculty of
Sciences, University of Burgos, Burgos, Spain

Polyphenols are secondary plant metabolites playing a major role as potentially functional
components. They can also be used for honey authentication. This review gathers the recent
literature references about honey extraction procedures, as well as instrumental analysis of
phenolic compounds found in honey. Liquid-Liquid extraction is widely used for both
extraction and purification purposes, with adequate recovery percentages. However, the use
of high solvent volumes is a major disadvantage. More environmentally friendly methods
include accelerated solvent extraction, and dispersive and inverse dispersive liquid-liquid
microextraction. Solid phase extraction is the most common method for honey polyphenols’
isolation. Polyphenol isolation by a combination of liquid-liquid and solid phase extraction
allows good recoveries for a variety of different compounds. High-performance liquid chro-
matography with ultraviolet or mass spectrometry detectors is by far, the most commonly
employed instrumental procedure to separate and quantify polyphenols in honey although
capillary electrophoresis has been also successfully used for these purposes. The use of new
sorbents, the optimization of current procedures and the development of other simple and
rapid analytical techniques are challenges for future analysis of polyphenols found in honey.

Keywords: Honey, phenolic compounds, flavonoids, extraction, analysis

INTRODUCTION flavonoids are the flavanones, flavones and flavonols, which

Polyphenols, also known as phenolic compounds, are nat-
ural phytochemical products of secondary plant metabolism.
They are responsible for most of the antioxidant activity of
honey. The phenolic acids and flavonoids are the two the
most important classes of secondary metabolites (1).
Phenolic acids found in honey can be benzoic acid deriva-
tives, such as gallic and ellagic acids, or cinnamic acid
derivatives, such as caffeic, sinapic, ferulic and coumaric
acids (2). Flavonoids consist of phenolic hydroxyl functions
attached to ring structures (1). These compounds can be
subdivided into different families, according to structural
variations within the rings. In honey, the most important
Received 19 September 2016, Accepted 1 July 2017.
Address correspondence to Miguel A. Fernández-Muiño; M. Teresa
Sancho, Nutrition and Bromatology Division, Department of
Biotechnology and Food Science, Faculty of Sciences, University of
Burgos, Plaza Misael Bañuelos García s/n, 09001 Burgos, Spain.
E-mail: mafernan@ubu.es; mtsancho@ubu.es
originate from propolis, nectar and pollen (3). Flavonoid
aglycones are the main flower-derived honey flavonoids
(4), although flavonoid glycosides have been found in
honey as well (5, 6).
The identification of individual phenolic compounds, as
well as the whole polyphenol profile can help to differenti-
ate and to authenticate honeys of different botanical and
geographical origins (1). For the same botanical origin,
polyphenol profile differences can be likely due to the
different regions of honey collection. Several international
bodies, such as the Association of Official Analytical
Chemists and the International Honey Commission, propose
official and/or standardized procedures for honey analysis.
Nevertheless, despite their huge importance, no method
regarding honey polyphenol determination has been offi-
cially recommended so far. The reasons might be related
to the difficulties that emerge when trying to put together
simplicity, rapidity, low cost, environmental sustainability,
as well as good precision, accuracy and sensitivity. It is also

necessary to consider cis isomerization and other possible
structural changes that might occur throughout the analysis
(7). Furthermore, the comparison of different methods
aimed to choose the most suitable one is very difficult
because of the different conditions for sample preparation
and the variety of procedures for phenolic isolation and
quantification. Moreover, for the same method, details
regarding different solvents and solvent proportions, differ-
ent columns, different chromatographic conditions, different
detectors and different improvements for particular detec-
tors, can be found in the literature. This review deals with
the current methods of analysis of polyphenols in honey,
both as a group and individually, describing the advantages
and disadvantages of the main steps for extraction, separa-
tion, identification and quantification of these important
compounds, thereby hoping to help establish a standardized
procedure for their determination in the near future.
ANALYSIS OF POLYPHENOLS IN HONEY AS A
GROUP
Total phenolic compound (TPC) and total flavonoid content
(TFC) of honey have been widely determined by spectro-
photometric methods that are simple, rapid and cost-effective.
These methods are not selective, likely providing overesti-
mated values, in particular, when using crude honey. The
most common method for TPC estimation is based on a
modification of the Folin-Ciocalteu procedure (8), which is
very unspecific. It actually determines the total reducing
capacity of polyphenols, sugars, aminoacids, vitamins and
other compounds (9–11). Some researchers determined TPC
on crude honey. Interferences were minimized using a blank
of honey and polyvinylpolypyrrolidone (12), or using a blank
FIGURE 1 Polyphenols extraction and quantification in honeys.
POLYPHENOLS IN HONEY 143
of an artificial honey (13). To obtain more reliable results of
honey’s TPC, it is highly recommended to remove interfering
compounds, as described below in the paragraph “isolation of
phenolic compounds” (10, 11, 14).
TFC is usually determined by methods based on the
formation of aluminum-flavonoid complexes. With regard
to honey, the most commonly employed procedures are
carried out in neutral and alkaline media. These two assays
do not actually quantify the TFC, because each of them
shows different specificity for various flavonoids (15). The
assay in neutral medium jointly quantifies quercetin, morin,
kaempferol, rutin (flavonols) and the flavone luteolin,
whereas the assay in alkaline medium jointly quantifies
rutin, luteolin, catechin, as well as some phenolic acids,
such as chlorogenic acid (15). Sugars interfere (16), so
that TFC must be determined from honey extracts instead
of from whole honey, subtracting, where appropriate, the
interferences due to the color of extracts (17).
POLYPHENOLS PROFILE DETERMINATION
The following paragraphs will focus on the procedures of
polyphenol separation and quantitation (Figure 1). Tables 1
and 2 summarize the highlights of polyphenol isolation as
well as high-performance liquid chromatograpy (HPLC) and
capillary electrophoresis (CE) techniques, respectively, with
the corresponding literature references (2–6, 11, 18–107).
Isolation of Phenolic Compounds
The isolation of phenolic compounds from the complex
honey matrix is a critical step. The final honey extract
must be concentrated with all phenolic compounds of the
 TABLE 1
Isolation and high-performance liquid chromatography (HPLC) procedures for the analysis of honey’s polyphenols.
144

Extraction method Extraction solvent Final solvent Column Mobile phase Detection Ref.

No extraction step − Acidified Atlantis C18 (50 × 2.1 mm ID; 2 mM Formic acid (A) and methanol (B) ESI(-)(+)-MS and APCI(-) (18)
deionized particle size 3 μm) (+)-MS (QTRAP, MRM
water mode)
No extraction step − Ultrapure water Gemini C18 (150 × 4.6 mm ID; 0.2 M Phosphoric acid (A) and acetonitrile DAD (280 nm, 360 nm) and (19)
particle size 3 μm) (B) (DAD)/Water (A) and methanol (B) ESI(-)(+)-MS/MS (triple
(MS) quadrupole, MRM mode)
No extraction step (Bath − Ultrapure water Hypersil gold C18 (250 × Potassium dihydrogen phosphate buffer pH UV (280 nm, 320 nm) (20)
Sonication) 4.6 mm ID; particle size 5 2.92 (A) and methanol (B)
μm) and same guard column
A. PASCUAL-MATÉ ET AL.

(10 × 4 mm)
No extraction step (Bath − Ultrapure water Zorbax RP SB-C18 (150 × 4% Acetic acid (A) and methanol (B) ECD (potential 1.0 V, oxidative (21)
Sonication) 4.5 mm ID; particle size 5 mode) and DAD (288 nm,
μm) and same guard column 320 nm)
(20 × 4.0 mm ID)
No extraction step (Bath − 40% methanol: Kinetex C18 (100 × 2.1 mm ID, 8 mM Formic acid pH 2.8 (A) and UV and ESI(-)-MS/MS (22)
Sonication/MAE) acidified water particle size 2.6 μm) and acetonitrile (B) (QTRAP, MRM and SIM
(pH 2, HCl) guard column modes)
No extraction step − 80% Methanol BEH C8 (150 × 2.1 mm ID; Methanol (A) and 7.5 mM formic acid (B) DAD and ESI-MS (triple (23)
(Centrifugation and acid particle size 1.7 μm) quadrupole, MRM mode)
hydrolysis (heat and HCl))
LLE Ethanol Ethanol Lichrosorb RP-18 (200 × 3 mm, Acidified distilled water pH 2.6 with DAD (280 nm, 310 nm) (24)
particle size 7 µm) and C18 orthophosphoric acid (A) and acetonitrile (B)
guard column (10 × 2.1 mm,
particle size 30–40 µm)
LLE Ethyl acetate Methanol Chromspher RP-C18 (200 × Acidified water (A) and acetonitrile (B) DAD (280 nm) (2)
3 mm ID; particle size 5 μm)
LLE Ethyl acetate Methanol Eclipse XDB RP-C18 (150 × 2% Acetic acid (A) and acetonitrile (B) UV (254 nm, 280 nm) (25)
4.5 mm ID; particle size 5
μm)
LLE Ethyl acetate Methanol Eclipse XDB RP-C18 (150 × 2% Acetic acid (A) and acetonitrile (B) UV (280 nm, 330 nm) (26)
4.5 mm ID; particle size 5
μm)
LLE Ethyl acetate Methanol Lichrosorb RP-18 (250 × 4.5 mm) 2.5 g/100 mL Acetic acid solution (A) and UV (280 nm, 330 nm) (27)
acetonitrile (B)
−2
LLE Ethyl acetate Methanol:10 N Spherisorb RP ODS2 (250 × Methanol:10−2 N sulfuric acid (10:90) DAD (292 nm), EI-MS and (28)
sulfuric acid 4.6 mm ID; particle size 5 NMR
(10:90) μm)
LLE Ethyl acetate Methanol Acquity UPLCTM BEH C18 0.5% Acetic acid (A) and 0.5% acetic acid in ESI(-)-MS/MS (29)
(100 × 2.1 mm ID; particle acetonitrile (B)
size 1.7 μm)
LLE Ethyl acetate Methanol:water Hypersil gold C18 (50 × 2.1 mm 0.1% Formic acid (A) and 0.1% formic acid HESI(-)-MS/MS (LTQ- (30)
(3:2) ID; particle size 1.9 μm) in acetonitrile (B) OrbiTrap)
LLE Ethyl acetate Methanol:water Acquity UPLCTM BEH C18 (100 2% Acetic acid (A) and methanol (B) ESI(-)-MS (Q-TOF) (31)
(3:2) × 2.1 mm ID; particle size 1.7
μm)
LLE Methanol followed by Methanol Zorbax Eclipse RP XDB-C18 80% Acetonitrile in methanol (A) and 2% UV (280 nm, 315 nm) (32)
diethyl ether:ethyl acetate (150 × 4.6 mm ID; particle acetic acid (B)
(1:1) size 5 μm)
LLE Butanol followed by ethyl 50% Methanol Hyperclone ODS C18 (200 × Methanol (A) and 0.3% formic acid (B) DAD (260 nm, 320 nm) (33)
acetate 2.0 mm ID; particle size 5
μm)
LLE followed by SPE Amberlite Hexane (LLE1), methanol Methanol Phenomenex RP-C18 column Water:acetic acid (99:1) (A) and UV (290 nm) (34)
XAD-2 resin column, (SPE) and diethyl ether (250 × 4.6 mm ID; particle methanol (B)
followed by LLE (LLE2) size 5 μm)
LLE followed by SPE Isolute Ethyl acetate (LLE) and 50% 50% Methanol C18 column (250 × 4.6 mm ID; Methanol (A) and 5% formic acid (B) DAD (280 nm) (35)
C18 cartridges methanol (SPE) particle size 5 μm)
ASE followed by LLE Acidified water (ASE) and Methanol:water Xterra RP-18 (150 × 4.6 mm ID; 1% Acetic acid (A) and acetonitrile (B) UV (280 nm, 330 nm) (36)
diethyl ether (LLE) (50:50) particle size 5 μm)
IDLLME Acetonitrile (disperser) and 1-Octanol GmbH C18 (250 × 4.6 mm ID; Methanol:0.3% phosphoric acid (58:42) UV (370 nm) (37)
1-octanol (extractant) particle size 10 μm) Isocratic elution
DLLME Acetonitrile (disperser) and Acetonitrile Discovery RP HS PEG (150 × Acetonitrile: 0.1% formic acid DAD (280 nm,370 nm) and (38)
chloroform (extractant) 4.6 mm ID; particle size 5 ESI(-)-MS (ToFMS)
μm)
DLLME/SPE Amberlite XAD-2 Acetone (disperser) and Methanol:water Chromolith FastGradient RP- 0.1% Formic acid (A) and 0.1% formic acid DAD (265 nm, 290 nm, 330 (39)
resin column followed by LLE chloroform (extractant) (1:4) 18e ((50 × 2)×2 mm ID) in methanol (B) nm, 370 nm) and HESI
(DLLME)/ Methanol (-)-HRMS (LTQ-OrbiTrap,
(SPE) and diethyl ether CID)
(LLE)
SPE Amberlite XAD-2 resin Methanol Methanol Shim-pack RP CLC-ODS (250 2% Acetic acid (A) and acetonitrile:methanol DAD (40)
column × 4.6 mm; particle size 5 μm) (2:1) (B) (phenolic acids)/1% Acetic acid
(A) and methanol (B) (flavonoids)
SPE Amberlite XAD-2 resin Methanol Methanol:water Gemini 5u RP-C18 (250 × 0.01 M Phosphate buffer pH 2.5 (A) and DAD (214 nm, 280 nm) (41)
column (50:50) 4.6 mm ID; particle size 5 methanol (B)
μm)
SPE Amberlite XAD-2 resin Methanol Water Ascentis Express RPAmide (100 0.14% Formic acid (A) and 0.14% formic DAD (255 nm, 275 nm, 300 (42)
column × 2.1 mm ID; particle size 2.7 acid in acetonitrile (B) nm, 330 nm) and ESI(-)-MS/
μm) and same guard column MS (Q-TOF)
(5 × 2.1 mm)
SPE Amberlite XAD-2 resin Methanol Methanol Zorbax RP SB-C18 column (250 1% Acetic acid (A) and methanol (B) ECD (potential 0.9 V, oxidative (43)
column × 4.6 mm ID; particle size 5 mode)
μm),
SPE Amberlite XAD-2 resin Methanol Methanol Zorbax RP SB-C18 column (250 Methanol (A) and 1% acetic acid (B) ECD (potential 0.9 V, DC (44)
column × 4.6 mm ID; particle size 5 mode) and DAD (254 nm,
μm) 280 nm, 290 nm, 324 nm)
SPE Amberlite XAD-2 resin Methanol Methanol C18 (250 × 4.6 mm ID; particle 0.1% Formic acid (A) and methanol (B) ECD (potential 0.9 V, oxidative (45)
column size 5 μm) mode) and DAD (280 nm,
290 nm)
SPE Amberlite XAD-2 resin Methanol (SPE), Methanol:0.02 Nucleodur Sphinx RP (150 × Methanol:0.02M phosphate buffer (20:80) at ECD (CEAD) (six electrodes, (46)
column + bath sonication/ASE methanol:0.02 M M phosphate 4.6 mm ID; particle size 5 pH 3.2 with 1 M sodium hydroxide (A) potentials 0.3–0.8 V) and
+ bath sonication phosphate buffer (1:1) buffer (1:1) μm) and Phenomenex C18 and same (80:20) (B) (CEAD)/0.5% ESI(-)(+)-MS (ion trap)
(bath sonication) and ethyl guard column (4 × 3 mm ID; Acetic acid in methanol:water (20:80) (A)
acetate or ethanol:water particle size 5 μm) and same (80:20) (B) (MS)
(80:20) (ASE)
SPE Amberlite XAD-2 resin Methanol Methanol Lichrocart RP-18 (100 × 4 mm Methanol:water:formic acid (50:47:3). UV (340 nm) (47)
column followed by Sephadex ID; particle size 5 μm) Isocratic elution
LH-20 column
SPE Amberlite XAD-2 resin Methanol Methanol Lichrocart RP-18 (100 × 4 mm 5% Formic acid (A) and methanol (B) DAD (340 nm) (3, 48)
column followed by Sephadex ID; particle size 5 μm)
LH-20 column
SPE Amberlite XAD-2 resin Methanol Methanol Lichrocart RP-18 (125 × 5 mm 5% Formic acid (A) and acetonitrile (B)/ DAD (340 nm) (49)
column followed by Sephadex ID; particle size 5 μm)/ Water:formic acid (95:5) (A) and methanol
LH-20 column Spherisorb ODS-2 (250 × (B)/ Methanol:THF: 5% formic acid in
POLYPHENOLS IN HONEY

4 mm ID, particle size 3 μm) water (25:15:60) (A) and methanol (B)

(Continued )
145

Extraction method
SPE Amberlite XAD-2 resin
column followed by Sephadex
LH-20 column
SPE Amberlite XAD-2 resin
column followed by Sephadex
LH-20 column
SPE Amberlite XAD-2 resin
column followed by Sephadex
LH-20 column
SPE Amberlite XAD-2 resin
column followed by Sephadex
LH-20 column or LLE
SPE Amberlite XAD-2 resin
column, followed by
Sephadex LH-20 column or
by Maxi-Clean RP-C18
cartridges or LLE
SPE Amberlite XAD-2 resin
column followed by LLE
SPE Amberlite XAD-2 resin
column followed by LLE
SPE Amberlite XAD-2 resin
column followed by LLE
SPE Amberlite XAD-2 resin
column followed by LLE
SPE Amberlite XAD-2 resin
column followed by LLE
SPE Amberlite XAD-2 resin
column followed by LLE
SPE Amberlite XAD-2 resin
column followed by LLE
SPE Amberlite XAD-2 resin
column followed by LLE
SPE Amberlite XAD-2 resin
column followed by LLE
SPE Amberlite XAD-2 resin
column followed by LLE
SPE Amberlite XAD-2 resin
column followed by LLE
SPE Amberlite XAD-2 resin
column followed by LLE
SPE Amberlite XAD-2 resin
column followed by LLE
TABLE 1 146
(Continued)
Extraction solvent Final solvent Column Mobile phase Detection Ref.
Methanol Methanol Lichrocart RP-18 (125 × 4 mm Water:formic acid (19:1) (A) and methanol (B) DAD (340 nm) (50)
ID; particle size 5 μm)
Methanol Methanol Lichrocart RP-18 (125 × 4 mm Water:formic acid (19:1) (A) and methanol DAD (290 nm, 340 nm) (4)
ID; particle size 5 μm) (B)
Methanol Methanol Merck Lichrospher 100 RP-18 Water:formic acid (19:1) (A) and methanol DAD (280 nm, 320 nm) (51)
(125 × 3 mm ID; particle size (B)
A. PASCUAL-MATÉ ET AL.
5 μm)
Methanol (SPE) and diethyl Methanol Lichrocart RP-18 (125 × 4 cm Water:formic acid (19:1) (A) and methanol DAD (290 nm, 340 nm) (52)
ether (LLE) ID; particle size 5 μm) (B)
Methanol (Amberlite and Methanol Lichrocart RP-18 (125 × 4 mm Water:formic acid (19:1) (A) and methanol DAD (290 nm, 340 nm) (53)
Sephadex columns), 80% ID; particle size 5 μm) (B)
methanol (Maxi-Clean
RP-C18 cartridges) and
diethyl ether (LLE)
Methanol (SPE) and diethyl Methanol Lichrocart C18 (125 × 3 mm ID) Phosphoric acid at pH 2.5 (A) and methanol DAD (280 nm) (54)
ether (LLE) (B)
Methanol (SPE) and diethyl Methanol Lichrocart RP-18 (125 × 4 mm Water:formic acid (19:1) (A) and methanol DAD (340 nm) (55, 58, 59)
ether (LLE) ID; particle size 5 μm) (B)
Methanol (SPE) and diethyl Methanol Lichrocart RP-18 (125 × 4 mm Water:formic acid (19:1) (A) and methanol DAD (350 nm) (56)
ether (LLE) ID; particle size 5 μm) (B). Isocratic elution (65% A:35% B)
Methanol (SPE) and diethyl Methanol Zorbax Eclipse XDB-C18 (150 × Water:formic acid (99.5:0.5) (A) and DAD (280 nm, 340 nm) (57)
ether (LLE) 4.6 mm ID; particle size 5 μm) methanol: acetonitrile (50:50)(B)
Methanol (SPE) and diethyl Methanol Lichrocart RP-18 (125 × 4 mm 5% Formic acid (A) and methanol (B) DAD (290 nm, 340 nm) (60)
ether (LLE) ID; particle size 5 μm)
Methanol (SPE) and diethyl Methanol C18 column (150 × 4.6 mm ID; Water:formic acid (95:5) (A) and methanol DAD (290 nm, 340 nm, 370 (61)
ether (LLE) particle size 5 μm) (B) nm)
Methanol (SPE) and diethyl Methanol Sunfire TM analytical C18 (150 Water:formic acid (95:5) (A) and methanol DAD (290 nm, 340 nm, 370 (62)
ether (LLE) × 4.6 mm ID; 5 μm) (B) nm)
Methanol (SPE) and diethyl Methanol Xterra RP-18 (150 × 3.9 mm ID; 0.05% Formic acid (A) and methanol (B) DAD (285 nm, 340 nm) (63)
ether (LLE) 5 μm) (DAD)/Discovery C18 (XTerra)/DAD (285 nm, 340
(150 × 2.1 mm ID; particle nm) and ESI(+)-MS
size 5 μm) (ESI-MS) (Discovery)
Methanol (SPE) and diethyl Methanol Synergi MAX-RP packed with 10% Methanol:1% acetic acid (A) and UV (254 nm) and ESI(-)(+)-MS (64)
ether (LLE) Luna C18 stationary phase methanol (B)
(250 × 4.6 mm ID; particle
size 4 μm)
Methanol (SPE) and diethyl Methanol Lichrocart RP-18e C18 (250 × Water:formic acid (99:1)(A) and methanol: DAD (290 nm, 360 nm) and (65)
ether (LLE) 4 mm ID; particle size 5 μm) isopropanol (90:10) (B) ESI(-)-MS/MS (ion trap)
Methanol (SPE) and diethyl Methanol Zorbax C18 (150 × 4.6 mm ID; 0.5% Formic acid (A) and methanol (B) DAD (280 nm, 350 nm) and (66)
ether (LLE) particle size 5 μm) and ESI(-)-MS/MS (triple
Agilent C18 guard column quadrupole)
Methanol (SPE) and diethyl Methanol Lichrocart RP-18 (125 × 4 mm Water:formic acid (19:1) (A) and methanol DAD (290, 340 nm), EI-MS (67)
ether (LLE) ID; particle size 5 μm) (B) and 1H NMR
Methanol (SPE) and diethyl Methanol:water Eclipse XDB RP-C18 (250 × 1% Acetic acid (A) and acetonitrile (B) DAD (280 nm, 330 nm) and (68)
ether (LLE) (50:50) 3.0 mm ID; particle size 5 ESI(-)(+)-MS (single
μm) and guard column quadrupole)
SPE Amberlite XAD-2 resin
column followed by LLE
SPE Amberlite XAD-2 resin
column followed by LLE
SPE Amberlite XAD-2 resin Methanol (Amberlite), ethyl
column followed by LLE and acetate (LLE) and
SPE Sephadex LH-20 column
SPE Amberlite XAD-2 resin Methanol
column followed by C18
cartridges
SPE Amberlite XAD-4 resin Methanol (SPE) and diethyl
column followed by LLE
SPE Amberlite XAD-4 resin
column followed by LLE
SPE Amberlite XAD-4 resin
column followed by LLE
SPE C18 cartridges
SPE C18 cartridges
SPE Bond Elut C18 cartridges
SPE Bond Elut C18 cartridges
SPE Bond Elut C18 cartridges
SPE Chromabond C18 column
SPE Supelclean LC-18 cartridges 90% Methanol
SPE RESPREP C18 cartridges
SPE Sep-Pak RP C18 cartridges
SPE Sep-Pak RP C18 cartridges
Methanol (SPE) and ethyl Methanol Betasil C18 (250 × 4.5 mm ID; 1% Acetic acid (A) and 10% acetonitrile in
acetate (LLE) particle size 5 μm)
Methanol (SPE) and ethyl Ethyl acetate Shimadzu LC-18 (250 × 4.6 mm 5% Formic acid (A) and methanol (B)
acetate:water (LLE) ID), Rexchrom LC-18
column (150 × 4.6 mm ID)
and C18 guard column
Methanol Shim-pack RP CLC-ODS (250 5% Formic acid (A) and methanol (B)
× 4.6 mm ID; particle size 5 (phenolic acids)/1% formic acid (A) and
methanol (Sephadex μm)
LH-20)
Methanol LiChrosorb Hibar RP C18 (250
× 2.4 mm ID; particle size 5
μm) and C18 guard column
Methanol Lichrocart 250–4 Lichrospher
ether (LLE) 100 RP-18 C18 (250 × 4 mm
ID; particle size 5 μm)
Methanol (SPE) and diethyl Methanol Zorbax Eclipse Pus RP C18
ether (LLE) (150 × 4.6 mm ID; particle
size 1.8 μm)
Methanol (SPE) and ethyl Unknown Luna C18 (250 × 4.6 mm ID;
acetate (LLE) particle size 10 μm) and
Phenomenex ODS C18 guard
column
90% Methanol 90% Methanol Luna C18 100A (150 × 4.60 mm
ID; 5 μm)
Methanol Methanol Zorbax SB-C18 (250 × 4.6 mm
ID; particle size 5 μm)
Acetonitrile: THF (1:1) Methanol:water Spherisorb RP ODS-2 (250 ×
(1:1) 4.6 mm ID; particle size 5
μm) and Perisorb RP18 guard
column (30 × 4.6 mm ID,
particle size 30–40 μm)
90% Methanol Methanol Purospher Star RP-18e (150 ×
4.6 mm ID; particle size 5
μm) and Merck guard column
RP-18e
Methanol Methanol Zorbax Eclipse RP XDB-C18
(150 × 4.6 mm ID; particle
size 5 μm)
Methanol Methanol Spherisorb ODS2 (250 ×
4.6 mm ID; particle size 5
μm)
90% Methanol Lichrocart RP-18 C18 (150 ×
4.0 mm ID; particle size 5
µm)
Methanol Methanol Waters C18 (250 × 4.6 mm ID;
particle size 10 μm)
Methanol Methanol Lichrocart RP-18 C18 (250 ×
4 mm ID; 5 μm) and
Lichrocart C18 guard column
(4 × 4 mm)
Methanol Methanol Luna C18 (250 × 4.6 mm ID; 5
DAD (280 nm, 320 nm) (69)
methanol (B)
DAD (254 nm, 290 nm, (70)
(phenolic acids)/1% Formic acid (A) and 320 nm)
methanol (B) (flavonoids)
DAD (290 nm, 320 nm), ESI- (71)
MS and NMR
methanol (B) (flavonoids)
5% Formic acid (A) and methanol (B) DAD (280 nm) (11)
Methanol (A) and water:formic acid (19:1) UV (340 nm) (72)
(B)
0.5% Acetic acid (A) and methanol (B) ESI(-)-MS (Q-TOF) (73)
Mixture 0.2% Acetic acid:methanol: DAD (280 nm, 340 nm) and (74)
acetonitrile Isocratic elution ESI(-)-MS/MS
0.1% Phosphoric acid (A) and 0.1% UV (265 nm) (75)
phosphoric acid in methanol (B)
Water:formic acid (99.5:0.5) (A) and DAD (290 nm, 340 nm) and (76)
methanol (B) ESI(-)-MS/MS
20 mM Potassium dihydrogen phosphate UV (220 nm, 280 nm, 310 nm) (77)
buffer pH 2.92 (A) and methanol (B)
0.1% Phosphoric acid (A) and 0.1% DAD (220 nm) (78)
phosphoric acid in methanol (B)
0.25% Formic acid: 2% methanol (A) and DAD (290 nm, 340 nm) (79)
methanol (B)
Water:formic acid (19:1) (A) and methanol DAD (280 nm, 320 nm, 350 (80)
(B) nm)
0.1% Phosphoric acid (A) 0.1% phosphoric UV (265 nm) (81)
acid in methanol (B)
Water:acetonitrile: THF:glacial acetic acid UV (269 nm) (82)
(54:36:5:5) Isocratic elution
Water:formic acid (99:1) (A) and methanol DAD (290 nm, 320 nm, 340 (6)
(B) nm, 360 nm) and ESI(-)-MS/
MS (ion trap)
Milli-Q water (A) and methanol (B) DAD (270 nm, 330 nm) and (83)
POLYPHENOLS IN HONEY

μm) and Phenomenex C18 ESI(-)-MS/MS (ion trap)

guard column
147

(Continued )

Extraction method
SPE Sep-Pak RP C18 cartridges
SPE Sep-Pak RP C18 cartridges
SPE Sep-Pak RP C18 cartridges/ Methanol (SPE) and diethyl
SPE Amberlite XAD-2
column + LLE
Bath Sonication + SPE
Amberlite XAD-2 column
Bath Sonication followed by SPE Methanol
DSC-C18 cartridges
Bath Sonication followed by SPE Acetonitrile
Strata C18-E cartridges
Bath Sonication followed by SPE Acetonitrile
Strata C18-E cartridges
Bath Sonication followed by SPE Acetonitrile
Strata C18-E cartridges
SPE Chromabond HR-X
cartridges
SPE GL-Pak PLS-2 cartridges
SPE SDB-L cartridges followed
by SAX cartridges
SPE Strata X cartridges
SPE Strata X cartridges
148
TABLE 1
(Continued)
Extraction solvent Final solvent Column Mobile phase Detection Ref.
Methanol Methanol Lichrocart RP-18.5 (250 × 4 mm 1% Formic acid (A) and methanol (B) DAD (280 nm, 320 nm, 360 (84)
ID; 5 μm) and Lichrocart nm) and ESI(-)-MS/MS (ion
guard column (4 × 4 mm) trap, CID)
Methanol:0.1% formic acid Methanol:0.1% Gemini C18 110 Å (150 × 2 mm 0.1% Formic acid (A) and methanol (B) DAD (210 nm, 254 nm, 280 (85)
(90:10) formic acid ID; 5 μm) and Ultra cartridges nm) and ESI(-)(+)-MS/MS
(90:10) C18 Peptide guard column (ion trap, NMR)
Methanol Lichrocart RP-18 C18 (250 × Water:formic acid (99:1) (A) and methanol DAD (290 nm, 320 nm, 340 (5)
A. PASCUAL-MATÉ ET AL.
ether (LLE) 4 mm ID; particle size 5 μm) (B) nm) and ESI(-)-MS/MS (ion
and Lichrocart C18 guard trap)
column (4 × 4 mm)
Methanol Methanol Gemini RP C18 (250 × 4.6 mm 0.01 M phosphate buffer pH 2.5 (A) and DAD (214 nm, 280 nm) (86)
ID; particle size 5 μm) Methanol (B)
Methanol Whatman ODS-2 column (250 × Water:trifluoroacetic acid:acetonitrile UV (280 nm, 360 nm) (87)
4.6 mm ID; particle size 5 (87:3:10) (A) and same (40:50:10) (B)
μm)
Acetonitrile Syncronis C18 (100 × 2.1 mm 0.1% Formic acid (A) and same in HESI(-)-MS (LTQ-OrbiTrap (88)
ID; particle size 1.7 mm) acetonitrile (B) (HESI-MS/MS)/0.1% (identification), and DAD
Formic acid (A) and acetonitrile (B) (254 nm, 280 nm) and HESI
(DAD-HESI-MS/MS) (-)-MS/MS (triple
quadrupole, CID)
(quantification)
Acetonitrile Hypersil gold C18 (50 × 2.1 mm 1% Formic acid (A) and acetonitrile (B) HESI(-)-MS/MS (LTQ- (89)
ID; particle size 1.9 μm) OrbiTrap, CID)
Acetonitrile Hypersil gold C18 (100 × 0.1% Formic acid (A) and 0.1% formic acid HESI(-)-MS/MS (LTQ- (90)
2.1 mm ID; particle size 1.9 in acetonitrile(B) OrbiTrap, CID)
μm)
75% Methanol 75% Methanol Nucleodur C18 Pyramid (100 × 0.1% Formic acid (A) and 0.05% formic acid DAD (254 nm, 272 nm, 300 (91)
2.1 mm ID; particle size 1.8 in methanol (B) nm) and ESI(-)(+)-MS/MS
μm) (SRM mode) and NMR
Methanol Methanol Discovery RP Amide C16 (150 0.5% Acetic acid (A) and 0.5% acetic acid in ECD (CEAD) (seven (92)
× 4.5 mm ID; particle size 5 methanol (B) electrodes, potentials 0.05–
μm) (ECD) and Senshu Pak 0.6 V), ESR and ESI(-)-MS
(2.0 × 150 mm) (MS-ESI)
Methanol: acetonitrile (2:1) Methanol: Luna C18 (150 × 4.6 mm ID; Water:formic acid (19:1) (A) and methanol DAD (290 nm, 340 nm) and (93)
(SDB-L) and 1N sulfuric acetonitrile particle size 5 μm) (DAD) (B) (DAD)/0.1% acetic acid (A) and 0.1% ESI(-)-MS/MS (triple
acid (SAX) (2:1) (SDB-L) and X-Terra MS-C18 (150 × acetic acid in acetonitrile (B) (MS) quadrupole, MRM mode)
and 1N 4.6 mm ID; particle size 5
sulfuric acid μm) (MS)
(SAX)
Methanol: acetonitrile (2:1) Methanol: Luna RP-C18 (150 × 2 mm ID; 1% Formic acid (A) and acetonitrile (B) DAD (maximum UV) and ESI (94)
acetonitrile particle size 3 μm) and (-)-MS (triple quadrupole)
(2:1) diluted Gemini C18 guard column (4
twofold with × 2 mm)
10 mM
sulfuric acid
Methanol Methanol Kinetex C18 (100 × 2.1 mm ID; 8 mmol/L Formic acid solution pH 2.8 (A) ESI(-)-MS/MS (QTRAP-triple (95)
particle size 2.6 μm) and and acetonitrile (B) quadrupole linear trap, SRM
guard column mode)
SPE Oasis HLB cartridges Methanol 0.1% Formic Zorbax SB-C18 column (100 × 0.1% Formic acid (A) and methanol (B) DAD (270 nm, 360 nm) and (96)
acid: 2.1 mm ID; particle size 1.8 ESI(-)-MS/MS (triple
acetonitrile μm) quadrupole, MRM mode)
(70:30)
SPE cartridges (Oasis HLB/ Methanol Methanol Ascentis C18 (150 × 4.6 mm ID; 2 mmol/L Formic acid pH 2.7 (A) and DAD (254 nm)-FLD/UV-ESI (97)
Strata X/Bond Elut C18)/SPE 5 μm) (DAD/FLD) and methanol (B) (-)-MS (QTRAP, MRM
Amberlite XAD-2 resin Atlantis C18 (50 × 2.1 mm mode)
column ID, 3 μm) (UV-ESI(-)-MS)
SPE RP cartridges (Strata X/ Formic acid:methanol (1:9) 2% Acetic acid Gemini RP C18 (150 × 4.6 mm 2% Acetic acid (A) and 2% acetic acid in DAD (280 nm)-ESI(-)(+)-MS (98)
Oasis HLB/WondaSep in methanol ID; 5 μm) methanol (B) (Q-TOF)
Pharma/BRP)/SPE RP-AE
cartridges (Strata X-A/P-SAX,
Oasis MAX, WondaSep
MAX)
SPE Al2O3/SiO2 cartridges/SPE Methanol:water (4:1) pH 1.8 Methanol:water ODS column (150 × 4.6 mm ID; 0.2% Phosphoric acid (42%) and methanol DAD (360 nm) (99)
with H3PO4 (4:1) pH 1.8 5 μm) (58%)
with H3PO4
SPE Sil-CBM-C30 cartidges/ Methanol:water (4:1) Methanol:water ODS column (150 × 4.6 mm ID; 0.2% Phosphoric acid (A) and methanol (B) DAD (360 nm) (100)
SPE Welchrom C18 cartridges (4:1) 5 μm)
Fe3O4@SiO2@ ImC18 Methanol or 0.3% H3PO4 in Methanol or ODS column (150 × 4.6 mm ID; 0.2% Phosphoric acid (42%) and methanol DAD (360 nm) (101)
methanol: water (6:1) 0.3% H3PO4 5 μm) (58%)
in methanol:
water (6:1)
SPE Timesnano MWCNTs Methanol (SPE) and diethyl Methanol Betasil RP C18 (150 × 4.6 mm 1% Formic acid (A) and methanol (B) DAD (270 nm) (102, 103)
followed by LLE ether (LLE) ID; 3 μm)
SPE Timesnano MWCNTs Methanol (SPE) and diethyl Methanol Acquity UHPLC BEH C18 (150 0.1% Formic acid (A) and methanol (B) ESI(-)-MS (triple quadrulope, (104)
followed by LLE ether (LLE) × 2.1 mm ID; 1.7 μm) SIR mode)
POLYPHENOLS IN HONEY
149
150 A. PASCUAL-MATÉ ET AL.
sample, after having removed sugars and other substances
that interfere in the analysis (108). Then, the extracts can be
used for polyphenol analyses as a whole (commented
above), or for flavonoid and phenolic acid identification,
usually by liquid chromatography.
Several researchers only filter the sample before HPLC,
thus omitting the isolation step, because it is time-consum-
ing and could lead to the loss of some trace analytes. Using
acidified samples, Biesaga and Pyrczynska (18) identified
nine phenolic acids and six flavonoids. Neither sugar inter-
ferences nor contamination of the MS ion source due to
sugar caramelization were observed. On the other hand,
using samples simply diluted with ultrapure water,
Tuberoso et al. (19) only determined gallic and homogenti-
sic acids. More recently, Kováčik et al. (23) analyzed honey
polyphenols by diluting the sample with 80% aqueous
methanol, followed by centrifugation to remove insoluble
particles. These authors employed acid hydrolysis (heat and
HCl), for the analysis of phenols bound to glycosides,
determining fourteen phenolic acids and five flavonoids.
With a minimum sample preparation, honey phenolics
and phenolic acids in particular can be efficiently extracted
by using ultrasonic bath homogenation (bath sonication),
wrongly named as ultrasonic extraction, despite it not
being an extraction procedure (1). Some researchers (20,
21) carried out bath sonication for 10 minutes at 25°C on
aqueous honey samples. Perna et al. (20) achieved recov-
eries higher than 98.5% for six phenolic acids (gallic,
chlorogenic, caffeic, p-coumaric, benzoic and ferulic
acids), and seven flavonoids (epicatechin, catechin, galloca-
techin, rutin, myricetin, quercetin and hesperetin). Biesaga
and Pyrzynksa (22) compared application of bath sonication
and microwave-assisted extraction (MAE), on samples
diluted with 40% methanol/acidified water. Bath sonication
was applied for 5 minutes, accomplishing recoveries of
about 90% for standard phenolic compounds (flavonoids
aglycones and benzoic acid derivatives). The phenolic
extraction yield as well as stability decreased only slightly
with time. MAE was applied for 1 minute at 90 W, obtain-
ing lower recoveries (70–80%) compared to bath sonication.
The phenolic recoveries decreased when a microwave irra-
diation of 160 W was used.
In comparison with solvent extraction, both bath sonica-
tion and MAE increase the extraction speed, simplifying the
process and reducing the consumption of solvents. They
provide similar extraction yields for flavonoid glycosides
than solvent extraction, but lower yield for flavonoid agly-
cones (quercetin, kaempferol, rhamnetin, myricetin and
luteolin), which are almost completely decomposed because
of their instability during irradiation. Therefore, the control
of irradiation times is of paramount importance, since longer
radiations could decrease the percentage of extracted com-
ponents due to degradation and oxidation processes (108),
especially when MAE is used since it works at temperatures
higher than those of bath sonication.
Liquid-Liquid Extraction (LLE)
Liquid-liquid extraction or solvent extraction is a separation
process where a solute present in a solution is transferred to
another immiscible liquid. LLE can be carried out with
different solvents.For honeys, the most common solvent
used is ethyl acetate (2, 25–31). After LLE, the extracts
are concentrated by evaporating the solvent r, and finally,
the residue is usually re-dissolved in methanol and filtered.
Applying this procedure, on a vacuum rotary evaporator,
Karabagias et al. (25) reported recoveries higher than 95%
for five phenolic compounds (quercetin, myricetin, kaemp-
ferol, chrysin and syringic acid). Several researchers set up
serial solvent extractions, such as methanol followed by
diethyl ether/ethyl acetate (32), or butanol followed by
ethyl acetate (33). Spilioti et al. (33) used ethyl acetate as
second solvent extractant, because of the higher recoveries
obtained for phenolic acids in comparison with diethyl
ether. In addition, compared to other extracts, ethyl acetate
extracts were likely to possess higher antibiotic and anti-
inflammatory activities.
The main drawbacks of LLE are emulsion formation,
which makes the complete recovery of some phenolic com-
pounds difficult (53), and requires a high volume of sol-
vents. Furthermore, LLE is often used as a purification step
after SPE, with diethyl ether being the most employed
solvent in this case (Table 1).
Accelerated Solvent Extraction (ASE) is a recent auto-
mated LLE procedure with a single extraction cell that
combines high temperature and pressure, thus reducing
time and solvent consumption. The procedure is expensive
and the high temperature employed could lead to the degra-
dation of a significant amount of phenolic compounds.
Petrus et al. (46) optimized the ASE operation conditions
(100°C, 5 minutes) for the analysis of eight flavonoids
(myricetin, quercetin, naringenin, luteolin, hesperetin,
kaempferol, isorhamnetin and galangin), testing different
solvents and eventually obtaining the best recoveries with
ethanol/water 80:20 v/v (>84%) and ethyl acetate (>82%).
The use of the 100°C was likely to cause the degradation of
some polyphenols as demonstrated by Habib et al. (36),
minimizing compound degradation by decreasing the ASE
temperature to 25°C, thus completing the extraction by
LLE.
An improvement of LLE is the Dispersive Liquid-Liquid
Microextraction (DLLME) and Inverse Dispersive Liquid-
Liquid Microextraction (IDLLME) methods. Both extraction
techniques are simpler, cheaper, faster and more environmen-
tally friendly than the conventional LLE method (38, 39). They
are based on the complete dispersion of a high-density extracting

solvent (chlorinated solvents) into an aqueous honey solution
with the help of a water-miscible and polar disperser solvent,
forming a cloudy solution that increases the sample-extractant
contact surface (37, 39).
After centrifugation, the extraction product is settled at the
bottom of the conical test tube (DLLME) or at the top
(IDLLME). The difference between both methods is that in
IDLLME the extractant has lower density than water. Ranjbari
et al. (37) optimized IDLLME for quercetin extraction in
honey with recoveries higher than 97%, by using acetonitrile
as disperser solvent, 1-octanol as extracting solvent and an
optimal sample pH of 4.5. Campone et al. (39) optimized
DLLME for five phenolic acids’ and ten flavonoids’ extrac-
tion, with recoveries higher than 70%, by using acetone as
disperser solvent, chloroform as extracting solvent and an
optimal sample pH of 2. The main handicap of this method
was the poor extraction of the most polar compounds, such as
glycosides (39). Campillo et al. (38) used acetonitrile as dis-
perser solvent for the analysis of eight flavonoid aglycone
compounds, adjusting sample pH to 3 and eventually obtain-
ing recoveries higher than 80%. Future investigations aimed to
improve the extraction of polar compounds should focus on
the research of other more efficient solvents.
Solid-Phase Extraction (SPE) with Amberlite Resin
Amberlite XAD-2 is a non-ionic polymeric resin (pore size 9
nm, particle size 0.3–1.2 mm) used for semi-polar compound
extraction. The Amberlite extraction method enabled recovery
values of 80–90% for flavonoids (108), allowing a more
effective elimination of sugars, acids, pigments and other
interfering compounds than reversed-phase cartridges (46).
The disadvantages of such extraction methods are the low
affinity of some phenolic compounds and polar glycoside
flavonoids and the need for high amounts of honey samples
and organic solvents. This is a time-consuming procedure (97).
Filtration through the Amberlite XAD-2 resin was applied to
honey for the first time in 1991 by Ferreres et al. (47). This is
today the most used method for polyphenol extraction
(Table 1). In the method, the sample is dissolved in acidified
water (pH 2). The use of acidic water is recommended because
it limits phenolic oxidation allowing for flavonoid aglycone
recovery higher than 95% (53). The sample is usually filtered
through cotton, paper, or membrane filters and the filtrate is
passed through the column containing the resin. Phenolic
compounds are adsorbed by the resin and remain in the col-
umn, while sugars and other polar compounds are eluted with
an aqueous solvent. Next, the phenolic fraction is eluted with
methanol. Many researchers reported the use of Amberlite
XAD-2 for honey phenolic extraction, sometimes with slight
modifications (47), such as adjusting the quantity of sample,
the acidified water volume used for polar compounds elution,
the methanol volume used for phenolic compounds elution
and/or the size of the glass column in which amberlite is
packed (3–5, 48–53, 55, 56, 58, 60, 67, 105, 106).
POLYPHENOLS IN HONEY 151
The most relevant optimization of Amberlite SPE extrac-
tion was the mixture of the sample with Amberlite XAD-2
particles before packing the chromatographic column, fol-
lowed by stirring at room temperature for 10 minutes. This
procedure resulted in higher exposed surface area of the
resin, increasing the capacity of polyphenol adsorption (5,
34, 41, 46, 54, 59, 60, 68–71, 107). Another modification
was the substitution of Amberlite XAD-2 with Amberlite
XAD-4, due to its higher surface area and thereby higher
efficiency (72–74).
After Amberlite extraction, a further clean-up step, is
highly recommended because potentially remaining sugars
could contaminate the phenolic fraction. Eventually,
cleaned-up phenolic compounds must be concentrated,
either by removing the solvent under reduced pressure or
by flushing with nitrogen. Right after this step, the extract is
re-dissolved mainly in methanol (Table 1). Nevertheless,
some authors avoid the clean-up step by just concentrating
the methanolic extract and then re-dissolving it, usually in
methanol or methanol/water mixtures (40–45).
The clean-up step was first performed with a size exclu-
sion column of hydroxypropylated dextran gel (Sepahadex
LH-20), after re-dissolving the methanolic concentrated
extract in methanol (4, 47–53). As this type of column
was rather complex and expensive, another clean-up proce-
dure was proposed. It consisted in re-dissolving the concen-
trated methanolic extract in water and extracting the
phenolic compounds with diethyl ether, as described in the
previous “LLE” section (53). This is by far the most com-
monly employed clean-up procedure (Table 1). However,
Ferreres et al. (52) realized that the extraction with diethyl
ether did not allow the proper determination of heather
honey flavonoids, probably due to their dark color.
Considering the small content of flavonoids and the high
amount of phenolic acid derivatives found in these honeys,
it is compulsory to use the filtration step through Sephadex
LH-20 (4, 51).
Different researchers have compared the SPE Amberlite
XAD-2 and LLE methods. Campone et al. (39) found simi-
lar recoveries for the majority of flavonoids between SPE
Amberlite XAD-2 followed by LLE and DLLME methods,
although the extraction efficiency was different for some
compounds depending on the procedure. In comparison
with the combined procedure of SPE Amberlite and LLE,
DLLME showed higher extraction efficiency for the flavo-
noids myricitin and quercetin, but lower recoveries for the
phenolic acids caffeic, coumaric, ferulic and vanillic acids.
Petrus et al. (46) found similar recoveries for ASE and non-
polar resins.
SPE with Commercial Cartridges
Reversed-phase (RP) SPE procedures with commercial car-
tridges packed with different sorbents and followed by vacuum
manifold system seem to be the simplest and most effective
152 A. PASCUAL-MATÉ ET AL.
means for phenolic compound extraction. RP-SPE cartridges
separate compounds according to their polarity, adsorbing non-
polar hydrophobic analytes. The use of commercial cartridges is
faster and less expensive than all procedures with Amberlite
XAD-2 resin. Commercial cartridges need small quantities of
sample and low volumes of organic solvents, thus being more
appropriate for flavonoid glycosides extraction (5, 94). The
main drawback is the initial investment, because vacuum mani-
fold and vacuum pumps might be expensive for the analysis of a
reduced number of samples. Truchado et al. (5) used Amberlite
XAD-2 for the extraction of flavonoid aglycones and propolis-
derived phenolics and SPE C18 for assessing the presence of
flavonoid glycosides from nectar. The main steps for phenolic
extraction by using SPE commercial cartridges are sorbent
conditioning (normally with methanol, and acidified or deio-
nized water), sample addition (adsorption), sorbent washing to
eliminate sample interferences (normally with acidified water),
and, finally, analyte elution with a solvent (usually methanol).
The sample preparation involves the dilution of honey in deio-
nized acidified water (pH 2), followed, if necessary, by sample
centrifugation. The most commonly used SPE cartridges are
hydrophobic reverse silica-based bonded phase octadecyl car-
tridges (C18) of different brands (5, 6, 75–85). The C18 reten-
tion mechanism depends on Van der Waals forces, hydrogen
bonds, or dipole–dipole interactions (97). C18 sorbents have a
strong hydrophobicity, ideal to extract non-polar compounds
from aqueous samples. Dimitrova et al. (77) tested C18 sorbents
with different carbon loads and pore sizes, choosing Bond Elut
C18 cartridges due to their high carbon load and small pore size
that provided better recoveries and stronger polyphenol
retention.
Polymeric sorbents are more pH resistant and possess
higher surface area than C18, with a mechanism of retention
based on π-π interaction for analytes containing aromatic
rings (97). The most common copolymer cartridges are
styrene-divinylbenzene, as well as N-vinylpyrrolidone-divi-
nylbenzene. Compared to conventional C18 cartridges, styr-
ene-divinylbenzene cartridges (91–95) show excellent
analyte separation due to their high surface area and higher
recovery rate for most phenolic compounds. With regard to
N-vinylpyrrolidone-divinylbenzene cartridges (96, 97), the
two monomers of their chemical structure confer superior
lipophilic and hydrophilic retention capacities.
Michalkiewicz et al. (97) compared Amberlite XAD-2
resin and different cartridges. The best recoveries for phe-
nolic acids were obtained using N-vinylpyrrolidone-divinyl-
benzene (Oasis HLB) SPE cartridges, both for aqueous
standard solutions and spiked honey samples. For flavonols,
the best recoveries were obtained with Bond Elut C18
cartridges (18.12% C, 60 Å), but only in case of standard
solutions. Sun et al. (98) found out that among RP-SPE
cartridges, Oasis HLB were the most efficient. These
researchers also proposed the use of RP anion exchange
(RP-AE) cartridges, since RP-AE sorbents possess a
quaternary amine group that adsorbs phenolic acids more
easily. They verified that RP-AE Strata X-A cartridges
achieved better results for polyphenols (mainly flavonoids)
than RP-SPE cartridges.
Liu and coworkers (99–101) observed that flavonoids could
not be efficiently extracted with commercial silica C18 sor-
bents, because these sorbents do not have polar groups (100),
so that they synthetized and characterized new sorbents for
honey flavonoid extraction. Nano-Al2O3-coated mesoporous
silica cartridges (Al2O3/SiO2) (99), and carbamate-embedded
triacontyl-modified silica (Sil-CBM-C30) (100) provided bet-
ter results than C18 sorbents for flavonoid extraction. Liu et al.
used 1-octadecylimidazolium ionic liquid-modified magnetic
nanoparticles (Fe3O4@SiO2@ImC18) with high surface area,
achieving fast and complete solid-liquid separation using an
external magnetic field allowing for easy recovery of the
loaded nanoparticles. Myricetin, quercetin and luteolin were
all detected and quantified, accomplishing excellent recoveries
(between 87% and 95%) (101).
Combination of Extraction Procedures
Honey polyphenols are also extracted by procedures based on
combinations of two techniques, such as Amberlite XAD-2
followed by either a purification (clean-up) step (“Amberlite
XAD-2” section), or by an extraction step with SPE C18
cartridges (11, 53). These two steps were used after LLE
(35) or ultrasonic bath homogenation (86–90). ASE was
followed by LLE (36) and multi-walled carbon nanotube
(MWCNT) extraction was also followed by LLE (102–
104). MWCNT extraction is a relatively new and inexpensive
technique for polyphenol extraction (102). Using this
method, Badjah-Hadj-Ahmed et al. (102) achieved recov-
eries between 89% and 96% for ten phenolic acids, nine
flavonoids and six phenols. The high surface area and the
tubular structure provided good adsorption capacity and high
stability, forming a wide range of interactions with the com-
pounds. The MWCNT extracts were added to an acidified
solution of water, and subsequent stirring helped for phenolic
compound adsorption. The MWCNT extracts were separated
by filtration and washed to remove polar constituents.
Polyphenols retained on the surface of MWCNT extracts
were eluted with methanol and concentrated under reduced
pressure in a rotary evaporator at about 40°C. For final
purification, the residue was re-dissolved in water, extracted
with diethyl ether, concentrated to dryness under nitrogen
and re-dissolved in methanol (102–104).
INSTRUMENTAL ANALYSIS
HPLC is the most commonly employed procedure for honey
polyphenol analyses. A simple external standard quantifica-
tion is sometimes followed by a CE control. Gas

chromatography can only be used for volatile and semivo-
latile compounds.
HPLC
The modern trend seen in polyphenol analyses is a slow
replacement of HPLC by ultra-high performance liquid
chromatography (UHPLC), initially introduced by the
Waters® company with the trade name UPLC (Table 1);
UHPLC is the modern version of HPLC operating in shorter
run times with a considerably better efficiency obtained
operating under higher pressure conditions with miniatur-
ized equipment (columns, detector, injector) (30, 31, 39,
88–91, 104).
Most HPLC or UHPLC columns used for honey poly-
phenol separation are reversed-phase (RP) C18 columns,
sometimes protected by a precolumn or security guard col-
umn (Table 1). Wabaidur et al. (104) tested different lengths
of BEH C18 RP columns, observing the best results with
the longest column (15 cm). Nevertheless, Tomás-Barberán
et al. (48) verified that the high resolution obtained with the
longest column disappeared after few analyses. Liang et al.
(21) tested various commercial RP columns, with Zorbax
SB-C18 providing the best results. However, Badjah-Hadj-
Ahmed et al. (103) accomplished better results with Betasil
C18. Biesaga and Pyrzynska (18) observed that the sensi-
tivity, resolution and efficiency were higher using a Kinetex
C18 column (2.6 µm particle size), than using the conven-
tional Atlantis C-18 column (3 µm particle size), due to the
smaller particle size, the narrower size distribution and the
shorter diffusion path.
With regard to elution, the most common procedure is
gradient elution because isocratic mode leads to a co-elution
of phenolic compounds, resulting in a loss of selectivity
(46). Phenolic compounds are usually separated by binary
gradient elution, using diverse mobile phases (Table 1). As
for the mobile phases, mobile phase A is usually composed
by an aqueous solution of formic acid, acetic acid, or
phosphoric acid. Acidic mobile phases accomplish proper
polyphenol separation and peaks with sharp shapes, which
is particularly important for phenolic acids. Mobile phase B
is usually composed of methanol, acetonitrile, or different
concentrations of formic, acetic, or phosphoric acids in
methanol or acetonitrile. In general, acetonitrile is more
efficient than methanol, because the sensitivity achieved
with the former is better and the peaks sharper.
Conversely, for flavonoids separation, methanol is the
proper choice because of better resolution (96). Moreover,
the addition of modifiers to the mobile phase (formic, acetic,
or phosphoric acids) in concentrations lower than 0.05%
improves ion efficiency and diminishes peak tailing, thus
increasing the retention of flavonoids (96). Some authors
carried out optimizations of the chromatographic conditions,
testing different mixtures as mobile phases (21, 38, 48, 82,
96, 97, 102–104). Liang et al. (21) assayed different acetic
POLYPHENOLS IN HONEY 153
acid aqueous phases (0–6%), observing that the more acidic
the conditions, the better the UV detector response and
chromatographic resolution were. Zhou et al. (96) observed
that the combination of methanol and 0.1% formic acid at a
flow rate of 0.2 mL/minute was the optimum mobile phase
for the identification and quantification of flavonoids.
Badjah-Hadj-Ahmed et al. (102) assayed several mixtures
of water, acetonitrile, methanol and mixtures of 1% formic
acid with methanol or acetonitrile in isocratic and gradient
elution modes at different flow rates. The best and fastest
separation was accomplished with a binary mobile phase of
1% formic acid aqueous solution (A) and methanol (B) at
0.2 mL/minute. The same research group improved this
method using 0.1% instead of 1% v/v formic acid aqueous
solution (103, 104).
HPLC-UV
In respect of detection, the most common method used to
quantify honey phenolic compounds is HPLC coupled to
ultraviolet (UV) detectors (Table 1), such as photodiode
array detector (DAD or PDA). Phenolic compounds exhibit
an absorbance maximum between 260 and 370 nm (96).
Identification is possible by comparison with standards
using both the UV spectra and the retention times. The
main drawbacks of UV detection are first, the poor sensi-
tivity for compounds that are present in low quantities;
second, the difficulties in setting reliable detection limits
for substances with low UV-sensitivity; and finally, the
fact that identification can be difficult for components show-
ing similar UV spectra or co-eluting at the same time (21,
94). For these reasons, other detectors, such mass spectro-
metry detector (MS) and/or nuclear magnetic resonance
detector (MS or NMR) are commonly connected in series
as a complementary technique for identification purposes
(Table 1).
HPLC-MS
HPLC mass spectrometry (HPLC-MS) is also widely
employed for polyphenol quantification purposes
(Table 1). This method enables high selectivity and sensi-
tivity for the analysis of polyphenol in complex matrices
such as honey, providing precise structural information of
the compounds. It imposes the use of volatile mobile
phases, e.g. phosphoric acid must be banned. MS system
can be used in single-mode detection or in tandem mode
(MS-MS), thus increasing the sensitivity and selectivity of
the procedure and obtaining additional structural informa-
tion. Biesaga and Pyrzynska (18) showed that phenolic
compounds could be determined by HPLC-MS omitting
the isolation step. Using HPLC-MS, co-elution is not a
problem because reliable detection is carried out according
to molecular weights and m/z values (94). Phenolic com-
pounds are identified according to the corresponding spec-
tral characteristics: mass spectra, accurate mass,
154 A. PASCUAL-MATÉ ET AL.
characteristic fragmentation and retention time. The quanti-
fication is carried out using calibration curves of standard
compounds in different scan modes such as multiple reac-
tion monitoring (MRM) (Table 1), selected reaction mon-
itoring mode (SRM) (91, 95), selected ion monitoring (SIM)
(22), or selected ion reaction monitoring (SIR) (104).
The most commonly employed ionization technique for
compound fragmentation is electrospray ionization (ESI). It
is commonly used in negative ionization mode (ESI(-)),
increasing the sensitivity and selectivity toward flavonoids in
respect of the positive mode due to better ionization (18, 96,
104). Sometimes, both negative and positive modes are
employed (ESI(-)(+)) (Table 1). The positive mode provides
extra certainty in the molecular mass determination (68).
Instead of ESI, electron impact ionization (EI) (28, 67), head
electrospray ionization (HESI) (30, 39, 88–90) and atmo-
spheric pressure chemical ionization (APCI) (18) have been
used. HESI provides better ionization and therefore higher
sensitivity. Biesaga and Pyrzynska (18) observed that ESI
was a gentler ionization method than APCI, being less likely
to cause the analytes’ fragmentation. ESI is generally used for
a wide range of polar compounds that can be ionized in
solution, whereas APCI is used for less polar molecules that
can undergo acid-based reactions in the gas phase. Although
the linear range in APCI mode is usually wider compared to
ESI, quantification by ESI might be advantageous when work-
ing with small concentrations (18). Together with ESI and
HESI, collision-induced dissociation (CID) was employed
for fragmentation studies (39, 84, 88–90).
Some literature references describe the use of mass-detec-
tors with a single analyzer such as linear ion trap (5, 6, 46, 65,
83–85) and single quadrupole (68). However, the combination
of more than one analyzer increases the accuracy of mass
measurements and provides a considerably better sensitivity.
These multiple analyzer systems (Table 1) include triple quad-
rupole, linear trap quadrupole (LTQ) OrbiTrap, quadrupole-
ion trap (QTRAP) and quadrupole/time-of-flight (Q-TOF). A
triple quadrupole mass analyzer is neither suitable for full scan
measurements, nor good enough for the analysis of com-
pounds, whose ion transition has not been predefined with
this detector (30). On the basis of their molecular masses and
fragmentation patterns, honey phenolic acids, their derivatives
and flavonoid aglycones can be accurately detected with LTQ
OrbiTrap MS, because it provides advantages in comparison
with triple quadrupole MS and quadrupole/time-of-flight MS
(30). The main advantages of LTQ OrbiTrap are the possibility
of simultaneous qualitative and quantitative analyses, MSn
determination on the basis of high-resolution exact mass mea-
surement and data-dependent observations (30).
Other HPLC detectors
For phenolic quantification in honeys, the use of electro-
chemical detectors, such as the coulometric electrode array
detector (CEAD) provides better selectivity and sensitivity
than the DAD (21, 43, 44). The CEAD allows for the detection
of oxidative-reductive compounds like polyphenols (46, 92),
which sometimes cannot be detected by UV due to very low
concentration or a weak UV absorption (21). The CEAD can
also provide more information about the analyte than other
detectors (43). ESI-MS (46, 92), DAD (21, 44, 45) and elec-
tron spin resonance (ERS) (92) have been coupled to electro-
chemical detectors for identification purposes. Fluorescence
detector (FD) increases the sensitivity and selectivity of the
HPLC method, allowing the quantification of several fluores-
cent compounds without modifying the gradient elution.
Michalkiewicz et al. (97) quantified honey fluorescent pheno-
lic compounds with acceptable recoveries and precision using
FD together with DAD as a confirmation method.
Unfortunately very few compounds are fluorescent.
Capillary Electrophoresis
CE with UV or MS detection has the advantage of high
separation efficiency and short analysis time in comparison
to HPLC (50). However, it is difficult to separate uncharged
flavonoids in one run, for it is necessary to wash the fused-
silica capillary tube after each analysis in order to obtain a
better reproducibility (46). With regard to CE, micellar
electrokinetic capillary chromatography (50, 105) and capil-
lary zone electrophoresis (106, 107) have been successfully
used. Different aqueous solutions have been employed as
running buffer (Table 2). In order to improve the method
sensitivity, Arráez-Román et al. (107) researched different
types of sheathflow liquids, obtaining the best results by
using 2-propanol/water (60:40, v/v) with 0.1% (v/v) tri-
methylamine. They employed a mass detector, making
CE-MS a quick and efficient method with high selectivity
and sensitivity. The main drawback was a prolonged analy-
sis time due to the fact that long capillary lengths were
needed to couple the MS detector.
CONCLUSION
The most suitable procedures for the determination of
honey polyphenol profile include a first SPE step to
isolate the phenolic compounds. Nowadays, the resin
Amberlite is being replaced by considerably faster meth-
ods with commercial ready-to-use extraction cartridges.
The cartridge sorbents show better affinity for phenolic
compounds than Amberlite, helping to reduce the amount
of solvents used. The best results have been obtained with
RP C18 (18.12% C, 60 Å), N-vinylpyrrolidone-divinyl-
benzene and anion exchange cartridges. Other promising
extraction techniques involve MWCNTs and more
recently silica-based magnetic nanoparticles such as
Fe3O4@SiO2@ImC18. These solids are easily separated
from the bulk honey solution.
 POLYPHENOLS IN HONEY 155

The most common instrumental method for the separation

0.2 M sodium borate (pH 8.0):50 mM sodium dodecyl sulfate:10% UV (280 nm) (105)

DAD (280 nm) (106)

(107)
Ref.

DAD (340 nm) (50)

and quantification of honey phenolic compounds is HPLC. After
considering the published research, a suitable HPLC procedure

100 mM ammonium acetate at pH 10 with 10% 2-propanol. Sheath UV (214 nm)

Detection
could consist of a column of Betasil C18 or Kinetex C18 (2.6 µm

and ESI
(-)-MS
particle size), with a binary mobile phase of an acidified (volatile
formic or acetic acid) aqueous solution (solvent A), and acetoni-
trile or methanol (solvent B), as well as a LTQ OrbiTrap ESI(-)
MS/MS detector. HPLC with an electrochemical detector or a
combination of UV and ESI-MS/MS detectors would also pro-

liquid: 2-propanol:water (60:40) with 0.1% trimethylamine

200 mM boric acid: 50 mM sodium dodecyl sulfate pH 8.5
vide good results.
In the coming future, the study of new sorbents will be
key to improve the extraction of honey polyphenols, in
100 mM sodium borate (pH 9.5):20% methanol

particular polyphenol glycosides, since they can decompose

in acidic extraction conditions and/or show low affinity for
Isolation and capillary electrophoresis (CE) procedures for the analysis of honey’s polyphenols.

Running buffer

the solvents or stationary phases used for extraction.

Investigation and improvements of other fast analytical
techniques, such as CE, will allow better efficiencies, thus
saving time and reducing costs.

FUNDING

The authors thank the PIRTU program of ‘Junta de Castilla

methanol

y León’ (Spain) and the European Social Fund for the

predoctoral study grant to Ana Pascual-Maté.
Fused-silica capillary column

Fused-silica capillary column

Fused-silica capillary column

TABLE 2

Bare fused-silica capillary

ORCID
(75 cm×75 μm ID)

(70 cm×50 μm ID)

(50 cm×50 μm ID)

Column

M. Teresa Sancho http://orcid.org/0000-0002-9128-9422

(50 μm ID)

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