Chapter 1

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ANALYTICAL CHEMISTRY AND MICROCHEMISTRY

NEW TRENDS IN SAMPLE PREPARATION
TECHNIQUES FOR FOOD ANALYSIS

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ANALYTICAL CHEMISTRY
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ANALYTICAL CHEMISTRY AND MICROCHEMISTRY

NEW TRENDS IN SAMPLE PREPARATION
TECHNIQUES FOR FOOD ANALYSIS

OSCAR NÚÑEZ, PHD
AND

PAOLO LUCCI, PHD
EDITORS

New York


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Library of Congress Cataloging-in-Publication Data
Names: Núñez Burcio, Oscar, editor. | Lucci, Paolo, editor.
Title: New trends in sample preparation techniques for food analysis /
editors, Oscar Núñez and Paolo Lucci (Department of Analytical Chemistry, Faculty of
Chemistry, University of Barcelona, Barcelona, Spain, and others).
Description: Hauppauge, New York : Nova Science Publishers, Inc., [2016] |
Series: Analytical chemistry and microchemistry | Includes bibliographical references and index.
| Description based on print version record and CIP data provided by publisher; resource not viewed.
Identifiers: LCCN 2016019751 (print) | LCCN 2016012406 (ebook) | ISBN
9781634850896 (HERRN) | ISBN 9781634850728 (hardcover)
Subjects: LCSH: Food--Analysis. | Sample preparation (Chemistry)

Classification: LCC TX541 (print) | LCC TX541 .N534 2016 (ebook) | DDC
664/.07--dc23
LC record available at />
Published by Nova Science Publishers, Inc. † New York


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CONTENTS
Preface
Chapter 1

Chapter 2

vii
Novel Sorbent Materials for Off-Line and On-Line Solid-Phase
Extraction Applied to Food Analysis
Michele Balzano, Deborah Pacetti and Natale G. Frega
Application of Molecularly Imprinted Polymers
to Solid-Phase Extraction in Food Analysis
Paolo Lucci and Oscar Núñez

1

27

Chapter 3

Turbulent Flow Chromatography in Food Analysis
Marta Llorca and Marinella Farré

45

Chapter 4

QuEChERS Procedures in Food Sample Preparation
Oscar Núñez and Paolo Lucci

73

Chapter 5

Microextraction Methods in Food Sample Preparation
Anna Damascelli

127

Chapter 6

Ionic Liquids in Food Analysis Sample Preparation
Omar J. Portillo-Castillo, Marsela Garza-Tapia,
Abelardo Chávez-Montes and Rocío Castro-Ríos

175

Chapter 7

Supercritical Fluid Extraction (SFE) for Rapid and Efficient Sample
Preparation, with a Special Focus on Food Contaminants
Sabrina Moret and Lanfranco S. Conte

223

About the Editors

247

Index

249


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PREFACE
Nowadays, there is a growing need for applications in food control and safety analysis to
cope with the analysis of a large number of analytes in very complex matrix. New analytical
procedures are demanding sensitivity, robustness, effectiveness and high resolution within a
reduced analysis time. Most of these requirements may be met to a certain extend by the total
or partial automation of the conventional analytical methods, including sample preparation or
sample pre-treatment coupled on-line to an analytical system. Despite the advances in
chromatographic separations and mass spectrometry techniques, sample preparation is still
one of the most important parts in any analytical method development and an effective
sample preparation is essential for achieving good analytical results. Obviously, ideal sample
preparation methods should be fast, accurate, precise and must keep sample integrity. For this
reason, and over the last years, considerable efforts have been made to develop modern
approaches in sample treatment techniques that enable the reduction of the analysis time

without compromising the integrity of the extraction process. This book examines new trends
in sample preparation techniques for food analysis.
Chapter 1 - Since the end of the twentieth century, Solid-Phase Extraction (SPE) has been
considered as one of the most popular analytical extraction technique, due to its simplicity,
quickness and low solvent consumption. The SPE approach provides a powerful tool in the
field of food quality and safety control. Within this contest, the SPE can be applied on a
several complex liquid matrices (i.e., milk, drinkable water, wine, beer, aqueous beverages,
oils) likewise on solid matrices (i.e., plant tissues, fruits, vegetables, grains, meat, fish and
animal tissues) for many purposes, such as purification, trace enrichment, desalting, and class
fractionation.
Due to the high complexity of food matrices and the complex nature of food target
compounds, there is considerable interest in novel SPE materials with high selectivity or even
specificity towards such compounds concurrently applicable to a wider range of matrixes and
analytes – from extremely polar to hydrophobic species. Moreover, significant efforts have
been also devoted to development of new, advanced SPE sorbent materials with high sorption
capacity and enhanced chemical or physical mechanical stability. Finally, taking into account
the high attention paid to the development of on-line analytical techniques that combine SPE
sample preparation and separation plus detection in one fully automated analytical set up,
innovative material for on-line column pre-concentration and separation systems coupled with
chromatographic techniques (liquid chromatography–tandem mass spectrometry; gas
chromatography–tandem mass spectrometry) are recently investigated. In view of this, the


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viii

Oscar Núñez and Paolo Lucci

present book chapter describes the off-line and on-line SPE approaches and reviews the

applications of innovative SPE sorbent (i.e., molecular recognition sorbents, graphene,
nanostructured materials and mixed mode polymeric sorbents) in the identification and
quantification of target food compounds, especially contaminants.
Chapter 2 - In spite of the huge development of analytical instrumentation during last two
decades, sample preparation remains a crucial part of the whole analytical process since
effective sample preparation is still essential for achieving satisfactory analytical results.
Therefore, great efforts have also been conducted to develop sample treatment procedures
able to increase the efficiency and, especially, the selectivity of the extraction technique for
obtaining cleaner chromatographic traces, and more sensitive, precise, and accurate analytical
methods. Within this context, molecularly imprinted polymers (MIPs), which are synthetic
polymeric materials with an artificially generated three-dimensional network able to
specifically rebind a target analyte, thus reducing co-extraction of matrix interferences, has
recently emerged as a promising and selective sorbent for the clean-up and preconcentration
of several target compounds from food, biological, pharmaceutical and environmental
samples.
In the present chapter, the basic principles involved in the synthesis of molecular
imprinted polymers, as well as the use of MIPs as solid-phase extraction in food analysis, will
be described by means of relevant and recent application examples.
Chapter 3 - Food innovation, food quality standards and food safety need for rapid,
sensitive and robust analytical methods. In addition, the quantity and variety of toxic
contaminants in food is continuously increasing as a consequence of industrial development,
new agricultural practices and environmental pollution. During recent years an important
research has been paid on to the improvement on sample preparation methods for food
matrices, in which the new tendencies on greener methodologies, the use of online clean up
systems, the development of new materials and new mass spectrometry analysers have played
an important role.
This chapter presents a general overview on the use of Turbulent Flow Chromatography
(TFC), a relatively new technique for sample preparation that has shown a great potential for
on-line sample pre-treatment in food analysis, in particular for very complex matrices.
The technique fundamental, materials employed and different examples of application

will be presented and discussed with special emphasis on recent application to trace
determinations and emerging food contaminants.
Chapter 4 - The requirements for a simple, rapid, cost-effective and multiresidue method
able to provide high quality of analytical results led Anastassiades et al. to develop in the
years 2001 and 2002 a new sample treatment method called “QuEChERS”. Although initially
this methodology was developed for the analysis of veterinary drugs (anthelmintics and
thyreostats) in animal tissues, its great potential in the extraction of polar and particularly
basic compounds make it ideal on pesticide residue analysis in plant materials where today is
widely recognized as a multiresidue sample treatment. However the application of this sample
procedure method has widely spread to other applications being very effective in the
determination of other groups of compounds such as pharmaceuticals and mycotoxins in a
wide variety of complex matrices. In this chapter, the principles of QuEChERS method and
its application in food analysis will be addressed. Coverage of all kind of applications is
beyond the scope of the present contribution, so it will focus on the most relevant applications
published in the last years.


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Preface

ix

Chapter 5 - The new trends in food analysis are microextraction techniques due to their
potential as fast, simple and inexpensive sample preparation, high enrichment factor,
adaptability to field sampling and possibility of automation. Moreover, they use a low amount
of solvents or are solventless contrary to traditional extractions with environmental
advantages. The emerging techniques in this area are solid-phase microextraction (SPME),
stir bar sorptive extraction (SBSE) and liquid-phase microextraction (LMPE). SPME is nonexhaustive and solventless technique and is based on absorption–adsorption of analyte into a
fiber coating and subsequent desorption. SPME was rapidly accepted as a simple,

miniaturized and green technique because it combines sampling, extraction, concentration,
clean up and sample introduction in a single step. Based on its advantages, SPME quickly
became one of the most widely used techniques in various fields of analytical chemistry.
SBSE is a method based on a coated stir bar, which can be added to the sample for stirring
and extraction (direct SBSE) or can be exposed to the headspace (HS-SBSE). The extraction
mechanisms and advantages are similar to those of SPME, but the extraction efficiency is
improved compared to SPME because of the greater amount of coating in SBSE. However,
extraction and desorption time of SBSE is longer than SPME. Liquid-phase microextraction
is liquid-liquid extraction (LLE) with a minimized solvent volume (acceptor phase – water
immiscible solvent) used to extract analytes from aqueous solution (donor phase). It can be
divided into three main categories: (a) single drop microextraction (SDME), (b) hollow fiberbased liquid-phase microextraction (HF-LPME) and (c) dispersive liquid–liquid
microextraction (DLLME). SDME usually employs a drop of 1–3 µL of organic solvent at the
tip of a microsyringe needle. Then the drop can be withdrawn inside the barrel after the
extraction process and can be injected into the instrument. The main disadvantage is related to
the instability of solvent droplet at the tip of the microsyringe, especially during stirring at
high speeds. To overcome the fragility of solvent drop in SDME, hollow fiber-based liquidphase microextraction is applied to stabilize the extracting phase. For extraction of analytes
from aqueous solutions porous hollow fiber is first immersed in organic solvent immiscible
with water to immobilize it in the pores of hollow fiber (HF). Then the lumen of HF is filled
either with the same organic solvent (two-phase systems) or with aqueous acceptor phase
(three-phase systems). Finally, in DLLME a mixture of the extraction phase (low solubility in
water and high density) and the dispenser solvent (miscible with extraction solvent and water)
is rapidly injected into the aqueous sample. Tiny droplets are formed in the aqueous sample,
which provides a vast interphase contact and accelerates the mass transfer of analytes. The
advantages of DLLME are simplicity of operation, rapidity, low cost, high recovery and high
enrichment factor. In conclusion, microextraction techniques may prove invaluable
approaches as analytes, which cannot easily be extracted with LLE, now can be isolated
simply and at low cost.
Chapter 6 - Ionic liquids (ILs) are organic salts with melting points below 100 ºC. These
compounds are formed by the combination of a bulky organic cation and an anion, which is
usually inorganic but can be organic; the asymmetry of the molecule lowers the lattice energy

and is responsible for the low melting point. Their ionic nature gives ILs unique properties
such as negligible volatility, high electric and thermal conductivity, higher viscosity and
density than molecular solvents, and a multitude of varying solvation interactions. Due to
their low volatility, ILs are considered “green solvents”, safer than conventional organic
solvents for both the analyst and the environment. Both cation and anion have a significant
effect in defining the physical and chemical properties of ILs, so by changing the cation-anion


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x

Oscar Núñez and Paolo Lucci

combination or modifying the cation structure, ILs with different physicochemical properties
can be obtained, providing a wide range of potential solvents. These features make ILs a
potentially attractive replacement for volatile organic compounds as solvents in various
chemical processes. Sample preparation has a significant influence on the analysis and is still
considered a bottleneck in laboratory processes; as a consequence, much work has been
devoted to this issue. In recent years, publications on the use of these compounds as
extracting media have increased substantially. The usefulness of ILs has been demonstrated in
liquid-liquid microextraction, microwave-assisted extraction, dispersive liquid-liquid
microextraction, single-drop microextraction, and solvent bar microextraction, among other
methods. In this chapter, recent applications of ILs in sample preparation steps for the
determination of organic compounds and metals in different food matrices will be discussed
in depth.
Chapter 7 - Supercritical fluid extraction (SFE) is a well-established environmentallyfriendly extraction technique which can be used as an alternative to classical solvent
extraction, both on industrial scale (extraction of oil from seeds, extraction of essential oil and
aroma compounds, decaffeination, etc.) and on analytical scale. In the latter case, it is mainly
used off-line as a sample preparation step prior to the analytical determination, but it can be

easily coupled on-line to chromatographic instruments, other analytical apparatus or directly
to a detection system. SFE exploits the unique properties of a supercritical fluid, which,
thanks to its high density (supercritical fluids have a density and hence a solvating power
similar to that of a liquid), low viscosity and high diffusivity (which resembles that of a gas),
can easily and rapidly penetrate deep into the sample matrix, enabling fast and efficient
extractions.
Due to its low toxicity, low cost and convenient critical temperature and critical pressure,
carbon dioxide, sometimes in combination with a polar modifier, is the most common
substance used in SFE. It has also the advantages to be a gas at ambient temperature and
pressure, so it spontaneously evaporates at the end of the extraction process, without leaving
residue in the sample extract.
The nature of the matrix and the analyte, as well that of the supercritical fluid, can greatly
influence the extraction yields, so that the optimization of a number of parameters, such as
presence of a modifier, extraction temperature and pressure, extraction flow, extraction mode
(static or dynamic) and sample collection, is required to achieve optimal yields, selectivity
and fast extraction.
A wide range of applications is described in the literature for the extraction of lipids,
bioactive compounds, volatile aroma compounds and a number of different contaminants
from both environmental and food matrices. Particularly, some interesting applications, which
will be reviewed in this chapter, have been proposed in the field of food contaminants. Most
concerns pesticides, polycyclic aromatic hydrocarbons (PAHs), polychloro biphenyls (PCB)
and other priority organic pollutants (POP), residue of veterinary drugs, mycotoxins,
nitrosamines and metals.


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In: New Trends in Sample Preparation Techniques
Editors: Oscar Núñez and Paolo Lucci

ISBN: 
© 2016 Nova Science Publishers, Inc.

Chapter 1

NOVEL SORBENT MATERIALS FOR OFF-LINE
AND ON-LINE SOLID-PHASE EXTRACTION APPLIED
TO FOOD ANALYSIS
Michele Balzano, Deborah Pacetti and Natale G. Frega
Department of Agricultural, Food and Enviromental Sciences,
Polytechinic University of Marche, Ancona, Italy

ABSTRACT
Since the end of the twentieth century, Solid-Phase Extraction (SPE) has been
considered as one of the most popular analytical extraction technique, due to its
simplicity, quickness and low solvent consumption. The SPE approach provides a
powerful tool in the field of food quality and safety control. Within this contest, the SPE
can be applied on a several complex liquid matrices (i.e., milk, drinkable water, wine,
beer, aqueous beverages, oils) likewise on solid matrices (i.e., plant tissues, fruits,
vegetables, grains, meat, fish and animal tissues) for many purposes, such as purification,
trace enrichment, desalting, and class fractionation. Due to the high complexity of food
matrices and the complex nature of food target compounds, there is considerable interest
in novel SPE materials with high selectivity or even specificity towards such compounds
concurrently applicable to a wider range of matrixes and analytes – from extremely polar
to hydrophobic species. Moreover, significant efforts have been also devoted to
development of new, advanced SPE sorbent materials with high sorption capacity and
enhanced chemical or physical mechanical stability. Finally, taking into account the high
attention paid to the development of on-line analytical techniques that combine SPE
sample preparation and separation plus detection in one fully automated analytical set up,
innovative material for on-line column pre-concentration and separation systems coupled

with chromatographic techniques (liquid chromatography–tandem mass spectrometry;
gas chromatography–tandem mass spectrometry) are recently investigated. In view of
this, the present book chapter describes the off-line and on-line SPE approaches and
reviews the applications of innovative SPE sorbent (i.e., molecular recognition sorbents,



Corresponding author: Email:


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2

Michele Balzano, Deborah Pacetti and Natale G. Frega
graphene, nanostructured materials and mixed mode polymeric sorbents) in the
identification and quantification of target food compounds, especially contaminants.

Keywords: SPE, off-line/on-line SPE, mixed mode polymeric sorbents, molecular
recognition sorbents, immunosorbents, aptamers, nanostructured materials

1. INTRODUCTION
The SPE technique involves partitioning between a liquid (sample matrix or solvent with
analytes) and a solid sorbent phase. Unlike the liquid – liquid extraction (LLE) technique,
SPE can be considered more affordable due to its efficiency and yield quantitative
extractions. SPE is easy to perform, rapid and it can also be automated [1, 2].
The main objectives of SPE procedure are removal of interfering matrix components and
selective concentration and isolation of the analytes. Often the enrichment step is necessary to
reach the instrumental limit of detection for the analytes of interest in quali-quantitative food
analysis. Several factors can reduce the extraction efficiency, sample recovery and

reproducibility of SPE procedure such as an inappropriate cartridge conditioning, too strong
loading and wash solvent, too large volume or mass of loaded and too small volume of
elution mobile phase [2]. In particular, the success of SPE depends on the knowledge about
the properties of target analytes and the kind of the samples. The ability of a SPE phase to
discriminate between the analyte and other sample components become of fundamental
importance when the development of SPE method is planned. The selectivity of SPE phase
depends on the chemical structure of the analyte, the properties of the adsorbent, the
composition of the sample matrix and the eluent used. As such, in order to select a proper
sorbent material, all of these factors should be taken into account (Table 1).
For the absorption of the analytes of interest on sorbet material, you can take advantages
of a variety of absorptive forces: the weak (non polar-non polar, van der Waals), hydrophilic
(polar-polar, hydrogen bonding, dipole-dipole and dipole-induced dipole), strong
hydrophobic interactions, the ion exchange process and the chemical modification of the
analytes by on-cartridge derivation reaction (e.g., immunoaffinity reaction). As result, a
variety of sorbents is available today, each offering a different selectivity.
The traditional SPE sorbent materials can be grouped into three main categories:
reversed, normal and ion exchange phases.
The material used as reversed phase include bonded silica, carbon-based, polymer-based
and polymer-coated media. They are usually selected to investigate, for instance, organic
acids in beverages. The strongly and moderately hydrophobic silica-based bonded sorbent
(i.e., octadecyl – C18 and octyl – C8 silica endcapped) are generally used to adsorb analytes
of even weak hydrophobicity from aqueous solution (i.e., water samples, wine, fruit juice).
These sorbents are synthesized by reacting on organosilane (silicon atom bonded to an
organic functional group) with the silica surface. The carbon-based media include graphitized
carbon black (GCB) and porous graphitic carbon (PGC). They have a high attraction for
organic polar and non-polar compounds from both polar and non-polar matrices. Retention of
analytes is based primarily on the analyte’s structure, rather than on interactions of functional
groups on the analyte with the sorbent surface. The drawback of the carbon-based sorbents is
that they have excessive retention (some analytes can even be irreversibly adsorbed).

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Novel Sorbent Materials for Off-Line and On-Line Solid-Phase Extraction …

3

Table 1. Key proprieties of the sample matrix, analyte and eluent considered when
the SPE phase selection is performed
Sample
Matrix
Aqueous

Analyte Solubility

Aqueous

Organic Soluble

Organic

Organic Soluble

Water Soluble

Eluent
recommended
Aqueous

Organic

(water miscible)
Organic

Organic
(water miscible)

Analyte Polarity
Non Polar
Moderately Polar
Polar
Cationic
Anionic
Non Polar and
Cationic
Non Polar and
Anionic
Non Polar
Polar
Moderately Polar
Cationic
Anionic
Non Polar and
Cationic
Non Polar and
Anionic

Phase
Recommended
Reversed
Reversed

Reversed
Ion Exchange
Ion Exchange
Reversed and Ion
Exchange

Reversed
Normal
Normal
Ion Exchange
Ion Exchange
Reversed and Ion
Exchange

Porous polymeric sorbents overcome some of the disadvantages. The most widely used
polymeric sorbent are styrene/divinylbenzene (SDVB) materials. It can be used for retaining
hydrophobic compounds whose contain some hydrophilic functionality, especially aromatic
ones. The higher potential of PS-DVB resins, such as Amberlite XAD-type, over C18 silicas
for trapping polar compounds was largely demonstrated [3, 4]. Taking into account the
loading capacity comparing polymer-based SPE and traditional silica-based SPE, the
Polymer-Based SPE is more suitable for its lower solvent consumption, blow-down time, and
sample variation. Another advantage is represented by its great pH stability from 1 to 14.
The normal phase includes polar adsorption media such as non-derivatized silica material
(SPE-Si), modified silica (SPE-CN, SPE-NH2, SPE-Diol) magnesium silicate (SPE-Florisil)
and aluminium oxide materials (SPE-alumina). They are suitable to adsorb polar compounds
from non-polar matrices. In particular, the magnesium silicate sorbent (Florisil) is particularly
suited to clean up extracts from fatty foods because it retains some lipids preferentially. Florisil is very good for cleaning up extracts containing non-polar pesticides, such as the
chlorinated hydrocarbons. Recently, a dual layer SPE cartridge (EZ-POP NP) has been
applied as a novel tool to extract the polynuclear aromatic hydrocarbons (PAHs) from olive
oil [5, 6]. The SPE cartridge was packed with Florisil® as the top sorbent bed and a mixture of

Z-Sep/C18 as the bottom bed. It allowed to retain preferentially lipid matter, while the PAHs
were eluted using acetonitrile. As result, the dual layer SPE shown higher recovery and
reproducibility than traditional silica SPE cartridge.


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4

Michele Balzano, Deborah Pacetti and Natale G. Frega

The ion exchange phase consists of silica based, hydrophilic, strong ion-exchanger with
large pore size sorbent material. Ion exchange sorbents containing fixed ion exchange sites
are used to isolate ionic compounds (acids and bases) from aqueous solutions. To achieve
optimum extraction conditions, the ion-exchanger sorbent and the analyte should be
oppositely charged. The cation exchanger material could contain ammines or inorganic
cations (e.g., calcium, sodium, magnesium, etc.) whereas the anionic exchanger material
could contain carboxylic and sulphonic acids, phosphates and similar groups. The –SO3group is a strong anion exchanger (SCX) for the extraction of basic analytes from solution.
The –N+(CH3)3 group is a strong anion exchanger (SAX) for the adsorption of acids.
The main applications of SPE procedures using traditional phases in food contaminants
analysis are summarized in Table 2.
Table 2. Applications of traditional SPE phases in food analysis
Analyte
Aflatoxins

Herbicides

Food Matrix
cereal, nuts, peanut butter
corn flour, liver

fish
honey
water
vegetable oil
water
UHT milk and water
citrus fruit
fish
water
water

Insecticides
Metals

water
water

Mycotoxins

cereal, nuts, peanut butter
corn flour, maize, milk
cereal, foodstuff
homogenized milk, plants and
meat, water
animal fat
oils
vegetables
adipose tissue
animal fat
water

meat
homogenized milk, maize, water
soy beans
cereals, foodstuff
water

Antibiotics
Aromatic hydrocarbons
Atrazine
Bisphenol A
Chromium(VI)
Fungicides

Ochratoxin A
Organochlorine
pesticides
PAH
PCB + pesticides
Phthalates
Sulfonamides
Triazines
Zearalenone
Zinc

SPE Mechanism
normal phase
reversed phase
normal phase
reversed phase
reversed phase

reversed phase
reversed phase
normal phase
normal phase
normal phase
reversed phase
normal phase
reversed phase
ion exchange
reversed phase
normal phase
reversed phase
normal phase
reversed phase
normal phase
reversed phase

SPE phase
Silica (SiOH)
C18
Silica (SiOH)
C18
C18
Silica-Diol
PSDVB
Silica-NH2
Silica-Diol
Silica (SiOH)
C18
Silica-CN

C18
SCX
C18
Silica-NH2
C18
Silica (SiOH)
C18
Silica (SiOH)
C18 or C8

ion exchange
normal phase
normal phase
ion exchange
reversed phase
reversed phase
ion exchange
reversed phase
ion exchange
normal phase
reversed phase

SCX
Florisil®
Silica (SiOH)
SCX
C18
C18
SCX
C18

SCX
Silica (SiOH)
C18


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Novel Sorbent Materials for Off-Line and On-Line Solid-Phase Extraction …

5

In order to overcome many of limitation of traditional SPE phase (instability at extreme
pH, low recovery in the extraction of analytes, low selectivity), novel SPE material sorbents
have been developed. Especially, innovative sorbent material for on-line column preconcentration and separation systems coupled with chromatographic techniques (liquid
chromatography–tandem mass spectrometry; gas chromatography–tandem mass
spectrometry) are recently investigated.
In this context, the present book chapter describes the off-line and on-line SPE
approaches and reports the applications of innovative SPE sorbent in the identification and
quantification of food compounds, especially contaminants.

2. OFF-LINE AND ON-LINE SPE APPROACHES
The SPE procedure consists in five steps process: selection of SPE stationary phase,
conditioning, sample addition, washing and elution.
At the beginning of each SPE protocol, it is necessary to select the most appropriate solid
phase. The selection of sorbent material plays a fundamental role because it controls
parameters of primary importance such as selectivity, affinity and capacity [7]. The choice
depends strongly on the nature of the analytes and their physical and chemical properties,
which should define the interaction with the chosen sorbent material.
After the phase selection, the sorbent material is conditioned using an affordable solvent
(conditioning step) and the sample is added to the solid sorbent (sample addition step).

Afterward, the purification of the compounds of interest can be performed, during the
washing and/or elution steps, in three ways: selective extraction, selective washing, selective
elution.
In the selective extraction, the SPE phase exclusively bind selected components (analytes
or the sample impurities) when the sample passes through the SPE tube. Thus, either collect
the retained compounds through elution, or discard the tube containing the extracted
impurities. In the selective washing, both target analytes and impurities are retained when the
samples are percolated through the SPE packing. Thus, the impurities are rinsed through with
wash solutions that are able to remove them, but no the analytes. Differently, in the selective
elution the adsorbed analytes are eluted in a solvent that leaves the strongly retained
impurities behind.
Taking into account the possibility to combine sample preparation and separation plus
detection in one fully automated analytical set-up, the SPE procedure can be coupled on-line
with instrumental analytical techniques. Thus, the SPE methodology can be performed
following manual (off-line SPE) or automated (on-line SPE) procedure. In both approaches,
the method development is carried out taking into account pH, ionic strength, polarity, flow
rate of elution solvent and physic-chemical characteristics of the sorbent bed.
The off-line SPE procedure is economical and needs simple equipment, moreover it is
fully applicable to on-site sampling. The off-line SPE system is composed by a sorbent
material, which can be supported by different packaging (syringe barrels, microtubes-tips and
discs), a solvent system and a vacuum manifold. The most popular packaging format for offline SPE is a syringe barrels which are easy to handle by using vacuum or positive pressure


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manifold. In any case, the flow-rate is difficult to control, and care should be taken to prevent

the column from drying out prior to sample application.
Several factors can reduce the extraction efficiency, sample recovery and reproducibility
of off-line SPE procedure such as an inappropriate cartridge conditioning, too strong loading
and wash solvent, too large volume or mass of loaded and too small volume of elution mobile
phase [2]. The off-line SPE procedure leads also some critical points such as time consuming,
request of large amount of the organic solvent for the elution, which can cause a possible loss
of analytes during the evaporation steps. Furthermore, off-line SPE involves a large
manipulation of the samples followed by a possible contamination, reduction of accuracy and
precision of the analysis. However, in spite of these disadvantages, the off-line SPE approach
remains useful for analyzing complex samples, due to its greater flexibility, whenever elution
solvent is not compatible with the subsequent method of analysis.
The on-line SPE approach consists of an automatic procedure wherein the manual
preparation steps and the risk of human error are intensely reduced. As such, on-line SPE
methodology guarantees sensitive, precise and selective bioanalysis. Additionally, it strongly
improves sensitivity for microliter-scale injections in capillary and nano liquid
chromatography analysis [7]. On-line SPE approaches are suggested when the amount of
sample is limited, or when very high sensitivity is required for the analysis. The automated
on-line instrument is simple to use, but it requires experienced personnel for method
development and eventual trouble-shooting. The comparison between on-line and off-line
SPE approaches features is summarized in Table 3.
Table 3. On-line and off-line SPE approaches: comparison between their features
On-line SPE
Small sample volumes are required to obtain
enough sensitivity
Matrix effects, ionic suppression or
enhancement in MS spectrometry
Reusable cartridges
Less flexibility, most systems do not allow the
combined use of different cartridge
Automatization and minimal sample handling

leads better precision
Direct and fast elution of sample after preconcentration.
Minimal degradation
Minimal consumption of organic solvent
No loss of analytes due to evaporation
Absence of extract for further analysis
Short analysis time
Limited portability
Expensive equipment

Off-line SPE
High sample volumes are necessary
Less matrix effects in MS spectrometry
Disposable cartridges
Sequential extraction and possibility of using
different combinations of cartridges connected in
series
Manipulation of sample lead contamination and
less precision
Risk of degradation of compound

High consumption of organic solvent for elution
Possible loss of analytes during evaporation step
Several measures can be carried on with the same
extract
Long analysis time
Portable SPE system
Cheap equipment

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Figure 1. On-line SPE-HPLC-MS/MS configuration, with guard cartridges after SPE and HPLC pumps.
The A-port switching valve configuration shown corresponds to the time period when the sample is
loaded onto the SPE cartridge.

Briefly, the on-line SPE approach involves in the automated injection of the sample onto
the SPE cartridge, which retains the target molecules while the potentially interfering
compounds are wash out. Afterward, the retained analytes are eluted on-line, via a switching
valve, onto the series connected analytical column. Concurrently with the analytical
separation, an exchange or reconditioning of the cartridge can take place [8].
The SPE technique can be easily coupled on-line to high performance liquid
chromatography (HPLC) and gas-chromatography (GC) systems.
The combination SPE - HPLC is extensively applied to food and drinking water analysis,
especially to determine polar compounds in water solution. Different configurations of online SPE/HPLC system are available. The most widespread approach involves the
implementation of a small SPE column within the injection loop of a six-port rotary valve
(Figure 1). After conditioning, the sample is loaded in SPE column, then the valve is switched
in order to elute analytes out of the sorbent by the liquid chromatography mobile phase and
transfer them into the analytical column [9-11].
It is possible to reuse the SPE column. However, the reusability of the SPE cartridges
cause a progressive deterioration of the pre-column material and thus, lead to a change in
their selectivity and capacity. Another disadvantage deriving from the cartridge reusability is
the risk of sample cross-contamination if the sample compounds or matrix components are
not completely removed during the wash and elution steps, especially when complex or
highly polluted samples are analyzed.
Since the development of Ultra Performance Liquid Chromatography (UPLC)/mass

spectrometry (MS) system, UPLC/MS can be considered the perfect instrument for
combination with on-line SPE. The on-line SPE-UHPLC/MS allows complete separation of
high number of analytes via a single chromatographic run that takes few minutes.
Besides to HPLC or UPLC system, the SPE procedures can also be combined on-line
with a GC system. Anyway, the strong differences among the SPE principle and the gas
chromatographic analysis made the on-line combination of SPE and GC more complicated
[12]. In a first step, the procedure involves in activation and conditioning of SPE cartridge
using a proper eluent. Then, the analytes from the sample are enriched. Finally, after drying


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the cartridge, the analytes are desorbed and directly injected into the liner of the GC system.
A weak point in on-line SPE-GC is the sample introduction step. The main problem being
that the injection volumes are limited to some 1 to 5 µL. Since, after suitable sample pretreatment, the volume of the final SPE extract typically is between 50 and 500 µL, this
implies that in the last step prior to the GC analysis, around 95-99% of the collected analytes
is discarded. Furthermore, the introduction of water into GC should be avoided, since it
hydrolyses the siloxane bonds in GC columns, causing deterioration of the column
performance. For this purpose, the SPE column is dried with a gas flow after trapping and
before elution of the analytes. In this way, volatile analytes can be lost. Alternatively, the SPE
extract is dried with a separate drying column packed with copper sulphate or silica and
placed after the SPE column. It is also possible to heat the column during the drying process,
but this increases the risk of losing volatile analytes. The elution of the analytes is performed
using a suitable solvent for the GC injector system.

3. APPLICATION OF THE SPE PHASE IN FOOD ANALYSIS

In order to overcome the main limitations of traditional SPE phase, the novel SPE
materials with higher selectivity, specificity, sorptive capacity and enhanced chemical or
physical mechanical stability, are continually developed. Among them, molecular recognition
sorbents, graphene, nanostructured materials and mixed mode polymeric sorbents have been
widely applied to the analysis food compounds, especially food contaminants.

3.1. Molecular Recognition Sorbents
Molecular recognition sorbent depend on mediation of a wide range of highly targetspecific biomolecules, whose chemical structure is such that they can selectively bind to the
substances and trigger the desired reactions or transfer mass through cellular interfaces [13].
Those biomolecules include monoclonal and polyclonal antibodies, RNA and DNA [14].
Molecular characterization processes can be recognized highly specific and can be replicated
in vitro for several applications, including being the basis for specific or highly selective
media for SPE. Molecular recognition sorbents include molecularly-imprinted materials (see
chapter 2), immune sorbents and aptamer-modified sorbents.

3.1.1. Immunosorbents
The interaction between antibody and antigen is highly specific and it can be utilized as
the basis for the development of highly selective SPE immunosorbents [15, 16]. The antibody
can be covalently immobilized on a suitable support solid or gel (e.g., Sepharose, silica, or
cellulose) and the material obtained is packed into a cartridge or a column for on-line
extraction/separation/detection.
The immuno-SPE technique has been widely utilized in the detection of mycotoxin in
foodstuff matrices. The steps process in immuno-SPE clean up of the sample are showed in
Figure 2.


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Figure 2. Representation of immunoaffinity clean up. Red points represent the target compound.

Taking into account the agriculturally relevant mycotoxin to animal and human health, Li
et al. [17] utilized the immunoaffinity clean-up approach in order to determine
deoxynivalenol (DON; 12,13-epoxy-3-alpha, 7-alpha, 15-trihydroxy-9-trichothecen-8-one) in
cereals as wheat, rice, and millet. The DON is one of the type B trichothecenes produced by
fungi in taxonomically unrelated genera, such as Fusarium, Myrothecium and Stachybotrys.
The method was supported by coupling an immunoaffinity column, prepared using a new
deoxynivalenol monoclonal antibody, UPLC-MS/MS analysis. The column capacity was 2.86
μg DON per mL of gel, and the limit of detection (LOD) and limit of quantification (LOQ)
were 0.3 and 0.8 μg/kg, respectively.
Other applications of immune-SPE included the determination of methandrostenolone in
the animal tissue and foodstuff [18]. Since the immunoSPE technique resulted comparable to
the conventional extraction protocols [19-21] as regard the determination of mycotoxin,
Wilcox et al. [22] coupled in tandem two immunoaffinity columns (IACs), providing
selective clean-up, based on targeted mycotoxins known to co-occur in specific matrices. An
IAC for aflatoxins + ochratoxin A + fumonisins (AOF) was combined with an IAC for
deoxynivalenol + zearalenone + T-2/HT-2 toxins (DZT); an IAC for ochratoxin A (O) was
combined with a DZT column; and an aflatoxin + ochratoxin (AO) column was combined
with a DZT column. Taking into account the European food safety regulation for mycotoxin
limit, samples of rye flour, maize, breakfast cereal and whole meal bread were analyzed.
After IAC clean-up extracts were analyzed by LC-MS/MS. The experimentation
demonstrated the accuracy of the multi-mycotoxin IAC methods. As such, the limit of
detection in food matrices investigated were much lower than EU regulatory limits.
Stroka and Seindler [23] developed a new immunoaffinity clean-up procedure for the
isolation of mycotoxins from maize extracts without organic solvents from IAC after
isolation. This approach replace organic solvents with water at 70°C as alternative and the
resulting SPE eluate is suitable for direct and complete injection onto a reversed phase liquid

chromatography column. Moreover, it is interesting underline that evaporation, reconstitution
or dilution are not required. This procedure was validated for a variety of mycotoxins, as:
DON, zearalenone, T-2 and HT-2 toxins, aflatoxins and ochratoxin A in an array of different
matrices.


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3.1.2. Aptamers
Aptamers are a special class of high-affinity molecules derived from ribonucleic (RNA)
or deoxyribonucleic (DNA) acids. They are short (up to 110 base pairs), single stranded,
synthetic oligonucleotides, that can fold in characteristic shapes able to bind with high
specificity the target molecules; recognition arises from hydrogen bonding, van der Waals
forces, and dipole and stacking interactions [24]. As molecular recognition probes, aptamers
have binding affinities and specificities that are comparable to, and in some cases even
surpass those of monoclonal antibodies. Aptamers are discovered using an in vitro process
called Systematic Evolution of Ligands by EXponential enrichment (SELEX), a procedure
where target-binding oligonucleotides are selected from a random pool of sequences through
iterative cycles of affinity separation and amplification by PCR [24]. As results, the process is
automatable and reasonable amounts of highly-specific aptamers for the desired target
analytes – which can range from small molecules with molecular weights from 100 D to large
biomolecules and even whole cells and viruses - can be obtained.
The ability of these synthetic oligonucleotides to bind target species with high specificity
was first applied to the purification of a target molecule by Romig et al. [25], who isolated a
protein on chromatographic columns packed with an aptamer based sorbent phase.
Nowadays, different aptamer-based techniques were developed and improved specially for

the application in food safety testing. As such, there are different aptamer-based technology
as aptamer-nanoparticle colorimetry [17, 18], surface plasmon resonance (SPR) biosensor
[26-28], dynamic light scattering (DLS) [29], fluorescent biosensor [30-32], electrochemical
biosensor [33], and solid-phase extraction (SPE) column [34, 35].
When compared to the more conventional high-affinity reagents such as antibodies or
enzymes, the aptamers present several attractive advantages:









Wide range of target molecules;
High specificity and strong affinity featuring aptamers compared with antibody and
other kinds of ligands [36];
High sensibility;
Easy preparation and convenient modification [37];
Small molecular weight than antibody, allows to synthetize necessary DNA
sequences in vitro rapidly and flexibly with the chemical synthesis method;
Low cost;
Easy to repeat;
Safety and reliability;

As such, the aptamer-based solid phase extraction technique provides a suitable approach
for the detection of biotoxins in foodstuff and beverage. Lee et al. [38] developed an aptamersandwich based carbon nanotube sensor able to detect Bisphenol A at very low concentrations
in food matrices.
Several authors [34, 39] applied aptamer-SPE for the detection and quantification (as

parts per billion) of the ocratoxina A in wheat samples. A DNA aptamer with high affinity
and specificity to ochratoxin A was conjugated to a coupling gel (diaminodipropylamideagarose resin) and used as sorbent for the preparation of SPE columns. In the 2011, ChapuisHugon et al. [40] developed a new solid phase extraction method based on aptamers for the


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determination of ochratoxin A in red wine. In this case, two solid supports were chosen to
immobilize OTA aptamer by covalent binding (cyanogen bromide-activated sepharose) or
noncovalent binding (streptavidin-activated agarose). Although the two solid supports
showed a similar behavior as regards satisfactory capacity and binding efficiency, the
immobilization by covalent bonding appeared more reliable for the determination of OTA in
the wine [41].

3.2. Graphene
Graphene is considered the basic building block of all graphitic forms (i.e., carbon
nanotubes, graphite and fullerene) material. This material possesses a single layer of carbon
atoms in a closely packed honeycomb two-dimensional lattice. The large delocalized-electron
system of graphene can form strong-stacking interaction with the benzene ring, which might
make graphene a good choice for the extraction of benzenoid form compounds. This novel
carbon nanomaterial revealed many exceptional properties (e.g., large surface area, strong
fracture strength, and excellent adsorption performance) [42]. As such, graphene shows good
performance with excellent absorbability, easy preparation process, and stable chemical
properties [43, 44]. The exceptional properties of graphene make it a superior candidate as a
good SPE adsorbent in different sample preparation methods.
Graphene can be easily modified with functional groups, especially via graphene oxide.
Graphene oxide/polypyrrole (GO/Ppy) has been synthesized by mixing graphene oxide and

polypyrrole in a specific proportion. This material was characterized by a unique structure
similar to that of foam. Wang et al. [45] used GO/Ppy as the adsorbent phase of Pipette TipSPE for determining three auxins (indole-3-propionic acid, indole-3-butyric acid, and 1naphthaleneacetic acid) present in trace amounts in papaya juice. The work highlighted how
this material has a high affinity and adsorption capacity for all the three auxins investigated.
Han et al. [46] used graphene-based SPE cartridges for the analysis of the
organophosphorus pesticides (OPPs) in apple juice. The results showed that effective clean up
of interferences and a high enrichment factor can be obtained through graphene-based SPE
cartridges. Following the optimization of SPE procedures, the analytical performance of the
method showed that the proposed method was sensitive, simple, and cost saving. Good
linearities were obtained for all the OPPs (i.e., dichlorvos, dimethoate malathion). The LODs
and LOQs were in the range of 0.04-0.35 and 0.15-1.18 ng/mL, respectively. The LODs of
this work was lower than or nearly to the previously reported methods, showing the high
sensitivity of the proposed method.

3.3. Nanostructured Materials
The recent advancements in materials research, especially as regards the development of
nano-particle size, determined at the nanometer (nm) scale, had contributed to improve SPE
techniques. The application of nanomaterials as SPE sorbents, such as electrospun polymer
nanofibers (NFs) and carbon nanotubes (CNTs) was investigated in novel works.


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3.3.1. Electrospun Polymer Nanofibers
Electrospun polymer nanofibers (NFs) can be obtained by electrospinning process [47].
Continuous NFs are characterized by diameters in the nm range, very large surface areas,
strength and flexibility. Morphology, physical and chemical properties of electrospun NFs

and NF mats can be selected. Due to these features coupled the to the high sorptive capacity
and analyte selectivity, electrospun NFs are an extremely interesting materials for SPE.
In addition, electrospun nanofibers possess a large specific surface area that makes them
suitable candidates to miniaturize solid phase extraction devices by reducing the sorbent
mass.
Maddah et al. (2015) developed a method based on SPE for the pre-concentration and
determination of diazinon and fenitrothion. As such, diazinon and fenitrothion are insecticide
which can pollute tap waters [48]. The two pollutants were pre-concentrated on electrospun
nanofibers as a adsorbent and then monitored by HPLC with diode array detector system. The
authors optimized some relevant parameters such as fiber packing amount, elution solvent,
pH, and ionic strength of the sample solution, in order to make the new method suitable and
useful for the analysis of diazinon and fenitrothion in tap water with high precision and
accuracy.
Qi et al. (2008) developed and tested three kinds of nanofibers [poly(styrene-comethacrylic acid), poly(styrene-co-p-styrene sulfonate), polystyrene] as SPE sorbents with the
aim to extract six compounds (nitrobenzene, 2-naphthol, benzene, n-butyl phydroxybenzoate, naphthalene, p-dichlorobenzene) in environmental water by highperformance liquid chromatography [49]. The detection limit ranged from 0.01 to 0.15
ng/mL. The method was tested to four real water samples. As results, it was possible to
underline the importance of functional groups, the polarity of nanofibers in controlling
sorption of target compounds.
Qi et al. aimed to remove three estrogens as diethylstilbestrol (DES), dienestrol (DS), and
hexestrol (HEX) from aqueous solution, utilized Nylon 6 electrospun nanofibers mat [50]. By
considering the adsorption equilibrium, maximum adsorption capacity values in the range of
97.71 to 208.95 mg/g were obtained. This range was comparable with other sorbents already
known in the literatures as carbon nanomaterials, multi-walled carbon nanotube,
carbonaceous adsorbents and activated carbon fibers. By considering these features, Nylon 6
electrospun nanofibers mat has great potential as a novel sorbent material for estrogens
removal.
Sun et al. (2013) investigated on electrospun polymer nanofibers as a solid-phase
extraction sorbent for the determination of benzimidazoles in pork meat [51]. The target
compound was then monitored by a high performance liquid chromatography with ultraviolet
detector (HPLC-UV) system.

3.3.2. Carbon Nanotubes
Carbon nanotubes (CNTs) approach concerns a large number of application fields as
electronics, medicine, optics, nanotechnology, etc. Nowadays analytical chemistry is
becoming the main sector for this novel technology approach. In particular, it will focalized
the use of CNTs as solid-phase extraction.
CNTs are allotropic forms of carbon comprising tubular structures formed by a single
rolled graphite lamella in a cylinder, i.e., single-walled CNTs (SWCNTs), or by several


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concentrically arranged CNTs arranged around a common axis as multi-walled CNTs
(MWCNTs) [52]. SWCNTs can have diameters up to 3 nm whereas MWCNTs up to 100 nm.
By considering the main features of solid-based sorbents, CNTs techniques can be
applied for different purposes depending on the physicochemical properties of analyte and
stationary phases and then on the extraction mechanism principle. As such, analyte
enrichment or storage, sample clean up or fractioning, still support for derivatization
reactions.
SPE represents the sorbent technique in which CNTs have found the most numerous
applications. As such, due to the possibility of extracting both organic and inorganic analytes.
Lately, there was a large widespread use of SPE technique based on CNTs as collected by
Socas-Rodríguez et al. [53]. In Tables 4 and 5 are reported the main applications of CNTs as
sorbents for the analysis of inorganic and organic analytes in different foods.

3.4. Mixed Mode Polymeric Sorbents
Mixed – mode sorbents also called dual mode sorbents consist of materials that are able

to be at the same time non – selective or highly selective towards specifics analytes,
meanwhile carry on high sorptive capacity using a single extraction step [104]. Nowadays
polymer based materials are the popular ones, they can operate as ion exchangers or as
conventional reverse-phase.
Mixed mode polymeric phases can be divided into cationic (SPE-MCX) or anionic (SPEMAX) and as weak or strong ion exchange, depending on the ionic group linked to the resin.
As for the retention mechanism, a cationic exchange mixed-mode sorbent retains neutral or
basic species by a reversed-phase mechanism and acid species by ionic exchange.
This sorbent material allowed the isolation of polar and ionic species from highlycomplex matrices. In fact, there is no silica support and no surface silanol groups which can
lead to unwanted secondary interactions, this is particularly favorable when analyzing
compounds which are disposed to secondary interactions with the silica surface.
He and Giusti (2011) set up a method using Oasis® MCX sorbent for isolation of
anthocianines from fruits and vegetables [105]. The new approach considered the combined
mechanisms of strong cation-exchange and reversed phase adsorption. As such, the crude
aqueous extract of chokeberry and purple were applied to an MCX SPE cartridge (6 mL, 1 g
sorbent; Waters Corp, Milford, MA). After washing with 0.1% TFA (trifluoroacetic acid), the
other-phenols fraction was collected by elution with 2 vol of methanol (0.1% TFA).
Subsequently anthocyanins were eluted with 1 vol of methanol and 1 vol of water/methanol
(40:60, v/v), both containing 1% NH4OH. The combined alkaline eluate was immediately
mixed with an aliquot of formic acid (99%) to lower the pH to <2. Both the other-phenols and
anthocyanin fractions were obtained for HPLC analysis.
The Oasis MCX® cartridge has been also applied to determine triazione erbicides,
lorfenicol ammine and tricaine in vegetable and animal food products [106-108]. Differently,
the SEP-SAX has been widely applied for the removal of fatty and other organic acids and
sugars in food matrix [109].