High-performance capillary electrophoresis for food quality evaluation

High-performance capillary electrophoresis for food quality evaluation

14

Adele Papetti, Raffaella Colombo University of Pavia, Department of Drug Sciences, Pavia, Italy

14.1

Introduction

The electrophoresis principle is based on the migration of ions in a buffer solution under an applied voltage and separation is determined by their mass to charge ratio. In 1937 the Swedish biochemist A. Tiselius was the first to introduce the electrophoretic technique applied to serum proteins by using a U-shaped cell [1]. From 1960 to 1980 horizontal polyacrylamide or agarose gel slabs became very diffused to analyze low or high molecular weight samples, respectively, thus developing so-called gel electrophoresis, a technique widely used to separate analytes (mainly peptides, proteins, and nucleic acids) based on their size. In the early 1980s, J.W. Jorgenson and K.D. Lukacs, and then S. Hjerte`n reported the first electrophoretic separations within open, fused-silica capillaries [2–4]; therefore electrophoresis principles have transferred from conventional slab gel electrophoresis to automated and fast

channel (10-200 mm I.D.)

polymer coating (12-20 mm thickness)

substrate (150-360 mm O.D.)

Amines Amino acids Carbohydrates Catecholamines Fatty acids Inorganic ions Nucleosides and nucleotides

Migration

Organic acids Peptide and proteins Polyphenols Vitamins detection point

high voltage power supply

sample buffer inlet

buffer outlet

Evaluation Technologies for Food Quality. https://doi.org/10.1016/B978-0-12-814217-2.00014-7 © 2019 Elsevier Inc. All rights reserved.

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capillary column systems, giving a basis to the modern high-performance capillary electrophoresis (HPCE), also known as capillary electrophoresis (CE). In addition, novelties in the semiconductor industry allowed a progression of electrophoresis from capillary to microchip, which has the advantages of integrating multiple analytical steps (such as sample pretreatment) and ensuring portability [5].

14.2

Basic principles

14.2.1 Electrophoresis, migration time, and mobilities Mobility represents the velocity of an ion/analyte through the capillary. Therefore separation by electrophoresis is based on differences in solute velocity in an applied electric field (E); the velocity of an ion can be given by the product of its electrophoretic mobility (μe) and E (v ¼ μeE). Mobility for a given ion and medium is a constant that is characteristic of that ion and is determined by the electric force (FE) that the molecule experiences, balanced by the frictional ones (FF) through the medium (steady-state electrophoresis) [6, 7]: FE ¼ FF

(14.1)

FE ¼ qE FF ¼ 6πηrv

(14.2)

where q ¼ ion charge, η ¼ viscosity of the background electrolyte (BGE) solution, r ¼ ion radius, and v ¼ ion velocity. Solving for velocity: v ¼ qE/6πηr and considering that v ¼ μeE: μ ¼ q=6πηr

(14.3)

From this equation it is evident that small, highly charged species have high mobilities, whereas large, minimally charged species have low mobilities. Analogously, the magnitude of electroosmotic flow (EOF) is expressed as a mobility term (μEOF or μ0) and is represented by the velocity of the bulk flow (vEOF) in an applied electric field (E): vEOF ¼ μEOF E

(14.4)

The apparent mobility (μapp) refers to the observed mobility of an analyte and to the parameters of electrophoretic separation, i.e., diffusion constant of the particle, applied E, time spent in the capillary, and resolution of components. When EOF is present, it is the combination of two mobility terms (apparent, μapp and effective, μp) that produces the observed migration of analytes: μapp ¼ μEOF + μp

(14.5)

In normal polarity and with injection at the anode pole, cations and neutral species migrate with EOF and anions migrate against it. In CE it is also possible to work in reverse polarity, where EOF is driven in the opposite direction, by introducing a positive charge on the capillary wall to mobilize an anionic double layer toward the anode [6, 7].

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From the measured migration time and other electrophoretic parameters, it is possible to calculate the mobility: μapp ¼ l=tE with E ¼ V=L

(14.6)

μapp ¼ lL=Vt

(14.7)

where l (effective length of the capillary, cm) and L (total length of the capillary, cm), V ¼ applied voltage (V), and t ¼ migration time (s).

14.2.2 Dispersion and efficiency The resolution of two migration zones is dependent on their length. Zone length is strongly correlated to the dispersive process, which should be controlled because it increases zone length and the mobility difference necessary to obtain the separation. For a Gaussian peak, dispersion corresponds to the baseline peak width wb ¼ 4σ (σ ¼ standard deviation of the peak). Under CE ideal conditions (for example, with small injection plug length and without analyte-wall interactions) the only contribution to solute-zone broadening is longitudinal diffusion along the capillary. Radial diffusion across the capillary is irrelevant, because of the plug flow profile, and also convective broadening cannot be considered thanks to capillary anticonvective properties [6, 7]. Thus the parameter of efficiency N (number of theoretical plates) can be related to the molecular diffusion coefficient of the solute (D): σ 2 ¼ 2Dt ¼ 2DlL=μV

(14.8)

N ¼ μVl=2DL ¼ μEl=2D

(14.9)

14.3

Procedures

14.3.1 Instrumentation In CE, separations are carried out in a bare fused silica capillary (30–100 cm long), which are circular in cross-section with an inner diameter (i.d.) of 10–200 μm (mainly 50–100 μm) and an outer diameter (o.d.) of 150–360 μm. Both capillary ends (inlet and outlet) must be immersed in buffer containers, into which the platinum electrodes are located. A high-voltage power supply is connected to electrodes and can apply constant voltage values between 10 and 30 kV. The electric field is simply the ratio between the applied voltage and the capillary length (V/cm). The detection system is online/real time near a capillary zone, called the revelation or detection window [6, 8]. CE instruments provide mechanisms to control the temperature of the capillary (15–60°C) with high-speed forced-air coolers or recirculating liquid coolant systems, reducing joule heating (Q) and zone broadening, generated by electrophoresis and high electric fields. A constant temperature is a crucial parameter; it is important to maintain buffer viscosity and by consequence to achieve reproducible migration times. It is also possible to set and control the temperature of samples (10–40/60°C) with an external water bath or a sample storage unit [7, 8].

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14.3.2 Capillaries The capillary is characterized by two lengths: the capillary length, also known as total length (L), on which the voltage is applied, and the effective capillary length to the detector (l), which is the distance between the injection site (inlet) and the detection window on-capillary. Capillaries are made up of a hard, high-temperature pure glass (e.g., fused silica and quartz), used for ultraviolet-visible (UV-vis) components, and they are covered with a layer of polyimide (10–20 μm thick), which is a copolymer with high heat resistance and flexibility, not transparent to UV light. So, at a certain distance from the outlet, which is independent of the CE instrument, it is necessary to remove polyimide to create a detection window transparent to UV [8]. Capillaries can be uncoated or coated. In an uncoated capillary the walls are made of silanol groups (Si-OH) on the glass surface and they become negatively charged (SO–x ) in basic solutions and buffers. When the voltage is applied to the circuit, one electrode becomes net positive and the other net negative. The wall silanol anions pair with mobile buffer cations, producing an electrical double layer along the wall. The remaining buffer cations are attracted to the negative electrode, giving foundation to the so-called EOF, which, for an uncoated capillary, is toward the negative electrode [9]. This phenomenon is also known as electroosmosis and exists in any electrophoretic system when the liquid near a charged surface is placed in an electrical field, resulting in the bulk movement of fluid near that surface. The electric potential near the wall is called ζ potential (V) and represents the potential at the interface of the compact and diffuse layers, where the EOF shear takes place. Because the surface volume ratio is very high inside a capillary, EOF becomes a significant factor in CE [8, 9]. The velocity of the EOF through a capillary is given by the Smoluchowski equation [8]: VEOF ¼ ðεζ=4πηÞE

(14.10)

where ε ¼ dielectric constant of the BGE solution, ζ ¼ potential, π ¼ constant, η ¼ viscosity of the BGE solution (P), and E ¼ applied potential (V/cm). Not all separations can be optimized using bare silica; in fact, in some applications, wall-analyte interactions, which produce an EOF mobility variation, cause great difficulty in obtaining reproducible analysis time. To avoid these problems, the modification of inner wall and the reduction/elimination of EOF represent important solutions, preventing adsorption of the analyte and also stabilizing the pH. A capillary inner wall can be chemically modified with covalent attachments of silanes (covalent coating) with neutral or hydrophilic substituents [9–12] or with the addition of polymeric modifiers (N,N-dimethylacrylamide, N,N-diethylacrylamide, poly(vinylpyrrolidone) [PVP], polybrene, poly(ethylene oxide) [PEO], and hydroxypropylmethylcellulose [HPMC]) in BGE (dynamic or adsorptive coating) [8, 9, 12]. An uncoated fused-silica capillary is prepared for its first use by rinsing it with 10–15 column volumes of NaOH or KOH (0.1–1 M), followed by 10–15 column volumes of the run buffer. For a coated capillary, NaOH is replaced with other solvents, such as ethanol or toluene. By using commercially coated capillaries, it is very important to follow the manufacturer’s instructions for cleaning and

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regeneration procedures, because coated capillaries are easily converted into bare silica capillaries [7–9].

14.3.3 Buffers Capillary ends must be immersed in and filled with a fresh buffer solution, called run buffer or BGE, responsible for conductivity when the voltage is applied. Buffers are compounds used to control the pH of a solution, which is responsible for reproducibility in CE. They are generally weak acids or bases that can accept or donate protons, reducing the change in pH that is caused by the introduction of additional acid or base. CE separation takes place in the run buffer, in which the differences in migration time and mobility can exist. Buffers can either be made or purchased [6, 7]. A suitable CE buffer should have high purity, high buffer capacity at the pH of interest (within one pH unit of the buffer pKa), low absorbance at the wavelength of interest, low mobility to minimize current generation, and low temperature coefficient (change in pH per °C). Preference should be given to BGEs with a high buffering capacity and low specific conductivity [13]. The choice of a buffer depends on the nature of the separation, and it is connected to the desired pH value, ionic strength, type of salts (inorganic or organic), and operating temperature. For instance, a change in pH can affect the current, a change in current can affect temperature, and a change in temperature can affect pH. The pH directly modulates the rate of dissociation of surface groups. Silanol groups on the surface behave as weak acids (pK 7) and remain protonated in acidic pH, but gradually dissociate to generate negative siloxy groups (SiO–) when the pH increases toward alkaline conditions. A capillary can be used in a pH range 2–9, because the fraction of negatively charged silanol groups becomes significant at a pH of about 2 and increases with pH to reach saturation around 9 [9]. The buffer capacity and the ionic strength are correlated: by increasing ionic strength, the buffer capacity increases. In addition, a high ionic strength reduces ζ potential and EOF [9, 13]. Concentration of the BGE is very important also in relation to peak shape and method sensitivity. In fact, by reducing the concentration/conductivity of the sample buffer relative to the BGE or by increasing the concentration/conductivity of the BGE relative to the sample buffer, it is possible to obtain the so-called “sample stacking.” This effect increases peak efficiency and method sensitivity, and it is recommended to keep samples at about one-tenth the concentration of the BGE to optimize it [6, 8]. Organic buffers have high buffer capacity compared to inorganic ones and have the advantage of having low mobility/conductivity and giving less joule heating, with the possibility of using higher voltages and increasing peak efficiency [13]. On the contrary, organic buffers have the disadvantage of absorbing UV-vis light, and for high-sensitivity experiments the background absorbance may become a very important issue. Buffer molecules exhibit a temperature coefficient and the pH of a buffered system changes with temperature. Although the capillary temperature can be fixed and monitored by efficient systems (liquid coolant), during electrophoresis the temperature

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inside the capillary cannot be completely controlled and measured, and it can be estimated only by calculating the joule heating (Q) [8, 13], as reported here: Q ¼ E2 Λc

(14.11)

where Q ¼ joule heating generated (Ω/cm3), E ¼ voltage gradient (V/cm), Λ ¼ molar conductivity of the BGE (cm2 mol–1 W–1), and c ¼ concentration of the BGE solution (mol/L). Capillary performance is optimal when it is dedicated only to a specific type of buffer species [8].

14.3.4 Rinse, injection, and separation A rinsing procedure (conditioning or flushing) is necessary to charge the surface of a new capillary and also for so-called “capillary regeneration.” This procedure consists of filling the capillary to create the same surface on its inner wall prior to every analytical run (conditioning inter-run). Positive pressure and vacuum are the most common methods of rinsing capillaries with typical rinse values of 20 and 10 psi, respectively, but positive pressures up to 100 psi can also be applied in all the latest generation of CE instruments [8]. Capillaries used in CE have a total volume of a few nanomicroliters and to avoid potential band broadening, only a small fraction of the capillary can contain sample. So, only a minute sample amount of 1–50 nL (injection plug, mm; or injection volume, nL/s) can be injected into the capillary. A long injection plug may cause wide bands and poor resolution. For injection by pressure and vacuum (hydrodynamic injection) it is possible to choose the desired value of pressure and time. Typical injection pressures and times are 0.5–1 psi and 3–10 s, respectively. Rinsing and injection pressures are supplied from an on-board air pump that applies pressures to the headspace of a buffer reservoir/vial. To calculate the volume of liquid injected by pressure, it is necessary to use the Poiseuille equation [8]: V ¼ ðΔPd4πtÞ=ð128ηLÞ

(14.12)

where P ¼ pressure drop down the length of the capillary (Pa), d ¼ capillary’s internal diameter (m), t ¼ time during which the pressure is applied (s), η ¼ viscosity of the BGE solution (Pa s), and L ¼ total length of the capillary (m). As an alternative to hydrodynamic injection, low-voltage values (electrokinetic or electrophoretic injection) can be applied for short times. This injection type is subjected to errors and low reproducibility because components that migrate more rapidly in the electrical field will be overrepresented in the sample compared to slowermoving components. After sample injection, the voltage is applied and the migration of components in the electrical field occurs. An electrophoretic separation can be affected by various factors: pH and viscosity of the BGE, temperature of the system, and hydrodynamic radius of the molecules. For example, temperature influences the electrical resistance and current, the viscosity, and finally the velocity of the molecules [6, 8].

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14.3.5 Detection systems Most CE detection systems are done on-capillary and the short path length in CE detection and its low concentration sensitivity represent the main problem that hinders the widespread application of this technique. Absorbance detectors are the most commonly encountered types of detector in CE instrument systems. The simplest absorbance detector (UV detector) uses only a portion of the available energy, while another type of absorbance detector, called the photodiode array detector (PDA), delivers the entire spectrum of light available from the source lamp. A PDA detector is very useful for confirming the identity of an analyte and for estimating its peak purity, and nowadays every commercial CE instrumentation is equipped with a PDA [6]. Absorbance detection is sufficient for many biological analytes, but in some cases the analyte has a weak chromophore and does not appreciably absorb the UV wavelengths. For example, inorganic ions do not absorb in UV and carbohydrates and some acids do not exhibit strong UV absorbance. To overcome this problem, indirect UV detection can be used. Indirect detection uses a wavelength-absorbing substance in a BGE, which also has a mobility close to that of the analytes. When analytes migrate in the BGE, they displace the absorbing substance, giving origin to a decrease in the absorbance and a negative peak [6, 8, 14]. Laser-induced fluorescence (LIF) detection systems for CE are also available. They are fluorescent detectors in which lasers are used as the source of the excitation energy and analytes must be fluorescent. Its application is not very diffused, as only a limited number of molecules contain a natural fluorophore and so a derivatization either on- or off-capillary is necessary [6, 8]. The CE-LIF method is applied to many sample matrices, from plasma to food samples (analysis of amino acids, carbohydrates, DNA, biogenic amines, vitamins, etc.) [14]. Chemiluminescence (CL) is a method based on the production of an electronically excited species derived from chemical reactions without the presence of an external source. CL systems in food analysis include the luminol reaction, the peroxyoxalate reaction, and the tris(2,20 -bipyridine)ruthenium(II) system, and are applied, for example, for derivatized amino acids, carbohydrates, and pesticides [14]. Electrochemical detection (ED) represents a powerful approach to the analysis of food samples. This consists of voltammetric or amperometric mode detection (VD or AD), which is able to measure voltage or current, respectively. This detection is based on oxidation/reduction reactions, which occur when an analyte interacts with an electrode at the outlet of the CE capillary and it can be used for inorganic ions, amino acids, carbohydrates, and biogenic amines. This type of detection is independent from path length and by consequence it has better sensitivity than a UV detector, but it is not very diffused because of the difficulty in aligning the capillary with the electrodes and the necessity to separate the high electric field applied for separation (kV potentials) from that used for detection (mV potentials) [8, 14]. It is also possible to couple CE to detectors that are outside of the capillary, although this requires a specialized interface, as in the case of CE-mass spectrometry (MS) with an electrospray ionization (ESI) interface, in which the outlet end of the CE capillary is inserted. MS adds the additional data of molecular weight, and MS/MS systems (MS2) also provide structural information. The design of these systems also

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Table 14.1 Capillary electrophoresis detectors (type and principle) and relative detection limits Detection type

Detection principle

Detection limit (M)

Spectrophotometric UV Spectrophotometric indirect-UV Spectrophotometric LIF Chemiluminescence Electrochemical Mass spectrometry

Absorption Absorption Fluorescence Electromagnetic Amperometric Mass to charge ratio

10–5–10–7 – 10–14–10–16 10–9–10–12 10–10–10–11 10–8–10–9

allows the use of UV or other detectors prior to the MS interface and the coupling of CE-MS has important advantages thanks to the speed and the resolving capacity of CE and the selectivity and sensitivity of MS [8]. CE-MS has many important applications in food quality and safety and in foodomics. For example, among food safety analysis, CE-MS has been mostly applied to the analysis of traces of contaminants and residues in different samples (water, milk, etc.). In addition, CE-MS is very useful for food metabolomics (metabolic profiling or metabolic fingerprinting) to study smallmolecule metabolites, mechanisms in food production processes and transgenic foods, and searching for new biomarkers of quality and authenticity [14–17]. See Table 14.1 for a comparison of detection systems and their relative detection limit.

14.3.6 CE separation modes Until now, we have discussed CE, which refers to capillary zone electrophoresis (CZE) also called free-solution capillary electrophoresis. CZE is the most widely used, but CE versatility is due not only to its wide number of applications, but also to its numerous different separation modes. In fact, it is sufficient to modify the medium into the capillary or the capillary internal surface to obtain different techniques and applications. The different CE modes achieve different selectivities because of their different separation mechanisms [6]. In Table 14.2 CE modes with principles and type of analyte and buffer modifiers are summarized. The CE analytical methodologies used in food analysis and mentioned here are: CZE, micellar electrokinetic chromatography (MEKC), capillary electrochromatography (CEC), capillary gel electrophoresis (CGE), capillary isotachophoresis (cITP), chiral capillary electrophoresis (CCE), and nonaqueous CE (NACE) [14, 18]. In food analysis, CZE represents the most diffused technique to analyze, for example, amino acids, biogenic amines, and contaminants [14, 18, 19]. The name CZE is misleading because MEKC and CGE are also zonal techniques (see later). CZE is applied only to charged analytes, while MEKC is a mode of CE that allows neutral molecule separation, adding surfactants to the BGE in a concentration sufficiently high to allow the formation of micelles (critical micelle concentration). These detergents, which can be anionic (sodium dodecyl sulfate, SDS), cationic (dodecyltrimethylammonium bromide; cetyltrimethylammonium bromide), nonionic (Triton X-100), or zwitterionic (3-(3-cholamidopropyl)dimethylammonio)1-propanesulfonate), arrange in dynamic micelles with a hydrophobic core and a

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Table 14.2 Capillary electrophoresis (CE) separation modes CE mode

Basic principles

Analyte

Medium

CZE

Mobility

BGE

MEKC

Hydrophobic/ionic interaction Mobility, hydrophobic/ionic interaction Molecular size

Charged molecules (ions, proteins) Neutral and charged molecules Charged/neutral molecules

CEC

CGE cITP cIEF CCE NACE

Moving boundary Isoelectric point Formation of diastereomeric entities Homo- and heteroconjugation ion pairing

Proteins, DNA Proteins, peptides Closely related proteins Chiral analytes Low-soluble analytes

BGE + surfactant BGE (organic, volatile) Gel (polymer sieving matrix) Two buffers Ampholytes BGE + chiral selector BGE + organic solvents

hydrophilic outer surface. The analytes can interact with micelles with hydrophobic and/or ionic interactions and move at their velocity. When they do not interact with micelles, they migrate with the EOF, if it is present. This technique is called “chromatography” because micelles constitute a pseudophase and for neutral species only the partition coefficient drives the distribution/separation of the analytes. Hydrophobic compounds interact more strongly with micelles and have longer migration time, so to decrease these interactions and to accelerate the chromatographic kinetics, the addition of organic modifiers (methanol, acetonitrile [ACN], and 2-propanol) up to 50% v/v is recommended. In addition, buffers with a basic pH are recommended to maintain EOF and to decrease undesired interactions between the capillary inner walls and the surfactant/solute [20, 21]. MEKC is used often in the analysis of flavonoids, vitamins, amino acids, and also racemic amino acids and different types of contaminants in food samples and beverages [18, 19, 22]. In CEC, capillaries are packed with a stationary phase as high performance liquid chromatography (HPLC) columns, giving foundation to a hybrid technique between LC and CE. Therefore separation is carried out thanks to two mechanisms: partition and mobility (chromatographic retention and electromigration separation mode, respectively) [8, 23]. This technique is widely used to analyze aromatic compounds such as polyaromatic hydrocarbons and aromatic carboxylic acids [23]. In food analysis, open tubular (OT)-CEC systems find applications for nitrites and nitrates [15], vitamins, nucleosides and nucleotides, contaminants [14], and also in enantiomeric determinations of organic acids [24]. CGE derives directly from slab gel electrophoresis with the great advantage of applying higher voltages (10–100 times) without the joule heating effect and with rapid analysis time. The term “gel” in CGE is ambiguous; in fact, solid matrices are not necessary, but polymers, which can be covalently cross-linked (bis-polyacrylamide) and

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hydrogen bonded (agarose), or linear polymer solutions (polyacrylamide or methylcellulose), are used. The polymer concentration necessary is inversely proportional to the size of the analyte. Linear polymers lead to more stable gels in respect of those generated from cross-linked ones. Resolution and efficiency in CZE and CGE are identical, and also for these modes it is possible to modulate the selectivity using different types of reagents (ion-pairing solvents, chiral selectors, complexing agents), which can be added to the BGE or covalently bound to the gel. CGE was created to study proteins, but nowadays detection of genetically modified organisms (GMOs), food-borne pathogens, and authenticity testing are the prevalent CGE applications for DNA analysis in foods [14, 15, 18]. For example, CGE-LIF is very diffused mainly to analyze DNA markers in varieties and GMO-derived DNA sequences, and to identify food-borne pathogens with a sensitivity and a rapidity comparable to real-time polymerase chain reaction (PCR) [15]. In cITP, a combination of two buffer systems (leading and terminating electrolytes) is used to create a state in which the separated zones move at the same velocity; in fact, cITP is considered a “moving boundary” technique. For example, for anionic analytes the buffer must be selected so that the leading electrolyte contains an anion with an effective mobility that is higher than that of the solutes. Similarly, the terminating electrolyte must contain an anion with a lower mobility than that of the solutes. Since the leading anion has the highest mobility, it moves fastest, followed by the anion with the next highest mobility, and so on. The individual anions migrate in discrete zones, but all move at the same velocity, as defined by the velocity of the leading anion. Since cITP is usually performed in constant current mode, a constant ratio must exist between the concentration and the mobility of the ions in each zone. Zones that are less (or more) concentrated than the leading electrolyte are sharpened (or broadened) to adapt to the proper concentration. This solute-concentrating principle is a sort of preconcentration step (stacking effect), which enhances efficiency and selectivity [25, 26]. This technique is particularly used in online combinations with CZE-UV to enhance CZE sensitivity, and some interesting applications in quality food control are present in the literature [18, 27, 28]. In a few old studies, cITP is applied alone, for example, to the analysis of ions or ionizable sweeteners of different food matrices [15, 29]. In capillary isoelectric focusing (cIEF), the capillary is filled with ampholytes, which are mixtures of buffers with a range of pKa able to create a pH gradient within the capillary. Analytes will migrate to the point where the pH of the gradient equals their isoelectric point (the pH at which a molecule, although charged, has a neutral behavior but behaves as if it was neutral). At this pH value, the analyte, which has no net charge, stops its migration. When voltage is applied, the samples and the ampholytes migrate to their appropriate positions in the gradient. When focusing is complete, the mixture is distributed throughout the length of the capillary, and to detect each component it is necessary to mobilize them (pressure or chemical mobilization). This CE mode is almost exclusively used for the separation of closely related protein species in the characterization of isoforms, resolving the broad bands that characterize these samples in CZE or CGE [8]. cIEF experiments are most frequently performed in coated capillaries or with dynamic coatings with very few applications in the food field [30].

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A CE mode, called CCE, is specific for chiral analysis [31]. CCE, with the simple addition of chiral selectors to the BGE and without any need of a chiral stationary phase, represents a powerful technique for the separation of racemic amino acids also in food analysis [15]. A great number of chiral selectors are available, but cyclodextrins and different types of antibiotics are often used and offer versatile applications [32]. NACE is a CE mode with purely nonaqueous BGE, which can be used as an alternative to MEKC. NACE analyzes substances with very low solubility in aqueous media and improves selectivity by using organic solvents (mainly methanol and ACN) [8, 18, 33]. MEKC [22, 34, 35], CEC [24], and NACE [34] applications, in few cases, allow enantioselective procedures also for food characterization without additives.

14.4

Advantages and limitations

The small i.d. of the capillary and the high surface area-to-volume ratio allow an anticonvective system with a controlled joule heating effect. In addition, the geometry of the capillary, in which the velocity of liquid is nearly uniform, results in a “plug flow.” This condition reduces the band broadening in contrast to the laminar/parabolic flow typical of the HPLC technique. CE analyses are usually very fast (10–20 min) and efficient, with a very low consumption of sample and reagents (solvents and buffers) and require minimum sample preparation, even in complex matrices [6–8]. On-capillary detection eliminates the problems of coupling the capillary to flow cells or other devices, removing the problem of dead volumes, but the effective length of the light path through the capillary is very small (for example, a capillary with a 50 μm i.d. has an effective path length of 32 μm) and by consequence the absorbance signal obtained from a CE system is low (low sensitivity). To overcome this limitation and increase sensitivity, capillaries with particular cells (low-volume flow cell or bubble cell) with a modified path length are also available [8], and many online and/or offline preconcentration methods in different CE modes and formats have been proposed [36]. The great number of available parameters (capillary type and length, buffer pH, type and concentration, injection type and parameters, voltage, temperature) represents a potential analytical resource in a method set up to resolve an analytical problem [8]. Quantitative analysis by CE can be a critical point; it depends on many parameters (temperature variations, sample adsorption, precise injection of small sample plugs) and some factors can be directly affected by the operator, while a few are completely instrument dependent. In addition, peak area results from different migration velocities of the solutes, so solutes of low mobility remain in the detection window for a longer time (overestimation of peak area) than those of higher mobility (underestimation of peak area). However, this phenomenon can be corrected simply by dividing integrated peak area by migration time (area normalization) [6, 7]. In addition, CE has demonstrated great potential for a wide range of applications, from small to large molecules (ions, drugs, proteins, natural products) [8, 18, 37]. In particular, regarding food analysis, CE was often used for a large variety of

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Table 14.3 Capillary electrophoresis (CE) separation modes with specific advantages and limitations CE mode

Advantages

Limitations

CZE MEKC

Simplicity Neutral molecules Surfactants enhance analyte solubility Velocity High efficiency Ideal for MS detectors Velocity

Only for charged molecules Interaction surfactant/hydrophobic solute and inner walls Not for proteins Accurate optimization of medium composition Need of high voltages No multilane capacity of SDS-PAGE Cross-linked polymers cause rapid gel polymerization, bubble formation, and gel rigidity Hydrodynamic injection cannot be applied Zone sharpening with samples rich in salts

CEC

CGE

cITP

cIEF CCE

NACE

High sensitivity Online coupling with CZE Ideal for conductance and MS detectors Velocity High resolution Simplicity Efficiency Small amounts of selectors Solvents enhance analyte solubility Selectivity High efficiency Velocity

Protein wall adsorption Dynamic coating instability More accurate selection of separative conditions Solvents induce changes of pKa values and mobility

food-related complex molecules, including amino acids, peptides, proteins, phenols, polyphenols, lipids, carbohydrates, DNAs, vitamins, additives, and contaminants, as well as small organic and inorganic compounds. In fact, CE versatility makes this technique an important analytical tool in food quality and safety, in food processing and stability, and also in foodomics [14, 15, 18]. In Table 14.3 supplementary advantages and limitations of each CE separation mode are reported.

14.5

Recent technology development

Miniaturized CE systems (microchip-CE devices) offer simple, rapid, and sensitive methods; for example, they are used both for monitoring analytes (such as proteins) and for detecting frauds or contaminations. This approach consists of a unique chip platform, which includes sample pretreatment, solution distribution/mixing, separation, and detection [36, 38–40].

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Novel immunoassays are based on electrophoretic principles (immunoaffinity CE); it combines CE advantages (high speed, efficiency, and small sample consumption) with selectivity of antibodies as binding agents. These systems are very sensitive and useful for enrichment and quantification of low abundant analytes or contaminants from complex matrices with important applications in allergen detections [41–43]. New coating polymers for CEC and development of pressurized (p)-CEC coupling: The development of molecular imprinted polymers (MIPs), which are novel materials with high specificity and versatility, or OT-CEC coating materials as stationary phases significantly increased CEC application fields [44, 45]. In addition, p-CEC systems with new columns/materials/mechanisms have been set up. In p-CEC, a micro-HPLC pump is connected to the inlet end of the capillary column to minimize the typical CEC problem of bubble formation [46–48]. Advances in coupling CE with electrochemical detectors: CE contactless coupled detection (CE-CCD) and CE capacitively coupled contactless conductivity detection (CE-C4D). In particular, CE-C4D is a sensitive technique that found interesting applications in pharmaceutical, biological, and food fields for the analysis of cations, inorganic/organic anions, biogenic amines, and free fatty acids [49]. Recent advances in combining CE with MS with the development and improvement in CE-MS interfaces, such as ESI, matrix-assisted desorption/ionization, and inductively coupled plasma (ICP), allow versatile applications in food analysis and foodomics, with interesting results in comparison with the conventional LC-MS methods [16, 17, 50–52]. Optimization of NACE-MS coupling allowed important applications for trace analysis in food samples [53]. Concentration procedures. Developments in preconcentration techniques coupled with CE: solid-phase extraction (SPE)-CE. In inline and online systems the SPE columns are integrated with the CE instrumentation, allowing simple, rapid, and economic analysis with minimal sample loss. The development of new SPE materials and on-chip SPE-CE allow the analysis of low-abundance analytes in different matrices with high rapidity and sensitivity [54].

14.6

Recent application progress in different types of foods

This section reports and discusses the most important CE applications present in the literature of the last decade. It is organized into six main sections: Solid food, Beverages and liquid food, Other foods (honey, food supplements, and baby foods), Additives, Contaminants, and Foodomics. For solid and liquid foods, a further classification based on vegetal or animal origin has been applied. Regarding Sections 14.10 and 14.11, CE techniques and applications are reported focusing on the type of detected molecules/compounds in all the mentioned foods. Analysis of nucleosides and nucleotides, as components of nucleic acids and as bioactive metabolites, is reported for every different food sample, while a specific section is dedicated to foodomics.

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CE modes of analysis are reported with general comments, and for liquid foods, food supplements, additives, and contaminants a table of applications is presented to simplify the widely available literature. Sample pretreatments are discussed when particular procedures are necessary due to the use of CE techniques. For every detail regarding sample preparation and CE methods, please see the cited references.

14.7

Solid food

14.7.1 Fruits and vegetables 14.7.1.1 Adrenergic amines and catecholamines In the last few years the use of Citrus aurantium (bitter orange) fruit extracts in food supplements for hypocaloric diets dramatically increased due to the significant content of adrenergic amines such as synephrine, octopamine, and tyramine, which could increase thermogenesis and induce lipolysis in the human body. A maximum dose of 30 mg/day was suggested to avoid undesirable effects, including muscular fasciculation, arrhythmia, and tachycardia. Therefore it is important to quantify these compounds for quality control purposes and for the detection of food adulteration in commercial formulations. A CZE method was developed for the simultaneous analysis of synephrine, octopamine, and tyramine; it was validated, obtaining satisfactory values of precision and extraction yield, and used for the analyses of water extracts of C. aurantium dried whole fruits or fruit parts (endocarp, mesocarp, and exocarp) or of commercial formulations [55]. Other substances that could regulate many physiological processes in humans are catecholamines, i.e., dopamine, epinephrine, and norepinephrine. Banana fruit is a clear example of food containing all these compounds, and microchip electrophoresis was demonstrated as a suitable technique to quantify them in food. Because these substances exhibited native fluorescence, a microchip-CE method with LIF detection was set up for the analysis of Cavendish dessert banana extracts. Catecholamine and its precursors tyrosine and tryptophan were quantified and their chemical structures were confirmed by microchip-CE-MS (single quadrupole with a nanoelectrospray). The only disadvantage registered was analyte ion suppression due to the high signal of mono- and disaccharides present in the food matrix [56].

14.7.1.2 Amino acids, peptides, and proteins In foods, amino acids are responsible for nutritional and organoleptic properties. Their natural composition could be modified by a technological process (for example, fermentation, aging, and distillation could affect amino acids modifying their concentration or also generating new amino acids). Thus amino acids and their relative concentration in a food matrix can also be considered as an important marker of authenticity, quality, and origin. Many researches described methods based on protein patterns to detect food adulteration. Therefore the importance of having high-throughput, sensitive, and not so expensive analytical techniques to detect amino acids, but also their aggregates, i.e., peptides and proteins, is extremely important.

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CE may be used as an alternative method to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and chromatographic methods because of its many general and sometimes specific advantages. CZE-UV methods are the most applied for the detection of amino acids and proteins in fruits and vegetables. Some examples are listed later. As regards amino acids, Cebolla-Cornejo [57] reported the application of this technique for the analysis of glutamic acid in muskmelon, winter squash, and orange (using the same method, carboxylic acids and sugars were also detected). CZE-UV was also used for the analysis of methanol-soluble proteins of sweet cherry, performed after a simple direct evaporation with nitrogen of the solvent. The results provided information on the most important physicochemical parameters related to the sensorial quality of the fruit and therefore this method could be used routinely for quality control [58]. This technique is also the choice for discriminate and/or fingerprint proteins for food authentication purposes as in the case of the investigation of adulteration of Spanish smoked paprika “Pimento´n de La Vera” with foreign paprika of an inferior quality [59]. In this work, the most important aim was to develop a simple procedure for protein extraction based on temperature-induced phase partition with Triton X-114 that allowed high sensitivity in the determination of smoked paprika adulteration. The same procedure was adopted for discriminating autochthonous varieties of peppers by the protein profile produced by different applied drying processes [60] and as a good alternative to DNA-based analysis methods or to the morphological analysis of plants in the differentiation of lentil cultivars from false lentil species (i.e., single-flour vetch and common vetch) [61]. Over the last decade, the market for basic and dairy-like soybean products has increased day by day, therefore there is a need for good alternatives to milk products for people with allergies or intolerance to animal milk proteins. Most of the CZE methods reported in the literature are dated in the 1990s and generally require only a sample dissolution in an appropriate separation medium [62]. The only improvement could be to consider the method proposed by Kanning [63] in 1993 for the characterization of β-conglycinin (7S) and glycinin (11S) soybean protein fractions. This has been performed by using a hydrophilically coated capillary and a polymeric hydrophilic additive to the BGE. CZE methods applied to the quantitative analysis of soybean proteins in basic and dairy-like soybean commercial products showed similar results for precision, accuracy, and robustness to those obtained by reverse-phase (RP)-HPLC methods. A CE-method was developed for the separation and quantification of soybean proteins in powdered and liquid soybean milks, and in soybean infant formulas derived from soybean protein isolates (SPI), but the distinction between different types of products within each SPI was not possible, differently by HPLC. The same occurred for soybean flour, textured soybean, and liquid soybean milks derived from whole soybean seeds [64]. Conversely, the results obtained by the CZE method were similar to those obtained by chromatographic methods in the detection of undeclared additions of bovine whey proteins in powdered soybean milk samples [65].

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In 2006, a satisfactory method was set up for the detection and quantification of soybean proteins in gluten-free bakery products in the presence of possible interferences such as egg and milk proteins; limit of detection (LOD) and limit of quantification (LOQ) values were one order of magnitude higher than those registered for an HPLC method [66]. More recently, a 2D microchip-CE device has been developed to assess the adulteration of soybean proteins in dairy products. This system integrated different CE modes: an IEF on a microchip and an ITP/CZE in the embedded capillary. The advantage of this technique was the isolation of specific fragments of proteins by on-chip IEF that can remove most milk proteins in a short time rather than conventional sample pretreatment procedures. Further analysis of this protein fragment by ITP/CZE in an embedded capillary allowed the detection of a low percentage of soybean proteins (0.1%) in total dairy proteins. This microchip-CE device provided a more sensitive technique, for example, to discover the adulteration of soybean proteins in dairy products (see Fig. 14.1A) [67], representing a very promising alternative to CE in the rapid detection of food frauds.

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Fig. 14.1 Capillary electrophoresis protein analysis in food frauds: (A) discrimination of milk (peak 1) and soybean (peaks 2–9) proteins in adulterated bovine milk products by a 2D microchip-capillary electrophoresis-ultraviolet device [67]; (B) protein fingerprint (P1–P11 peaks) of different white wine variety compositions by capillary zone electrophoresisultraviolet. ACS Reprinted with permission from D. Chabreyrie, S. Chauvet, F. Guyon, M.H. Salagoity, J.-F. Antinelli, B. Medina. Characterization and quantification of grape variety by means of shikimic acid concentration and protein fingerprint in still white wines, J. Agric. Food Chem. 56 (2008) 6785–6790. Copyright (2008) American Chemical Society.

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A CGE-UV method has been developed for the first time by Montealegre [68] to differentiate olive varieties depending on the protein profile. The sample preparation consisted of a homogenization of stone and pulp (separately), followed by treatment with a chloroform/methanol mixture for removing lipid components, and of protein precipitation with cold acetone. This technique over commonly used SDS-PAGE has the advantages of full automation, online detection, small sample amount, and high resolution. Therefore it was possible to identify a number of protein fractions not detected by using SDS-PAGE. In addition, by applying discriminant analysis a classification of olive varieties according to their geographical origin was possible. An interesting application concerned the use of carbosilane dendrimers (symmetrical macromolecules with 3D structures) as a nanoadditive to improve the separation of soybean and olive seed proteins by MEKC-UV. These dendrimers have interior carbon-silicon bonds and were negatively charged in the surface with carboxylic acid as functional groups. In particular, the dendrimer with 32 surface carboxylate groups allowed improvement in protein profiles (six peaks instead of two and five peaks instead of two were registered by adding dendrimers in the BGE for olive seed and soybean proteins, respectively). This represents a useful tool to obtain the specific fingerprinting of protein for the differentiation and classification of varieties. Sample preparation required a simple extraction with a mixture of water:ACN and then Tris-HCl added with SDS and dithiothreitol for soybean and olive seed proteins, respectively. An advantage deriving from the use of dendrimers in the separation buffer in CE originated from their uniform and versatile structure whose skeletons and surfaces may be modified changing their cationic or anionic nature, concentration, and structure to adjust themselves to a specific application and to improve the separation selectivity [69]. Finally, CZE-UV was also used to analyze nonprotein amino acids, such as N-oxalyl-L-α-diaminopropionic acid (β-ODAP), and homoarginine. β-ODAP is present in Lathyrus species legumes, an alternative protein source for human and animal nutrition. When consumed at high doses it could lead to lathyrism. A CZE-UV method was used to quantify β-ODAP and homoarginine (another nonprotein amino acid present in Lathyrus spp. with interesting implications for human and animal nutrition) in two different Lathyrus species seeds, comparing the performance of two extraction methods: a simple extraction of ground seeds with an ethanol:water mixture for 24 h and a homogenization of ground seeds with the same solvent mixture. In both cases, the supernatants obtained were evaporated before the resuspension in buffer prior to analysis. The second sample preparation method was more sensitive [70].

14.7.1.3 Organic acids CZE-UV is the preferred CE technique used in the analysis of organic acids present in foods. In fact, citric and malic acids were detected in muskmelon [71], and together with oxalic acid in other fruit and vegetable crops using a method that improved the resolution of malate/citrate by the optimization of BGE parameters and rinsing procedures [57]. The effect of the commercial supply chain on citric and malic acids concentrations in different accessions of rocket salad has been investigated by Bell [72].

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14.7.1.4 Carbohydrates (mainly oligosaccharides) Glucose, fructose, and sucrose are key components in the taste intensity of fruits and vegetables or of fruit-derived beverages such as juices or wines. Their detection by CZE-UV is common and in the literature many applications are reported: muskmelon [71], grape [18, 73], salad [72], and other fruits and vegetables [57].

14.7.1.5 Secondary metabolites (polyphenols, glucosinolates) Consumption of foods containing high amounts of bioactive natural products is increasing day by day and therefore these compounds are more and more extensively studied for their beneficial effects. Among the substances synthetized and accumulated in plant organs, secondary metabolites such as polyphenols are the target of the highest number of studies. RP-HPLC coupled with UV or MS detectors is a widely used technique; however, in the last decade the use of CE has become more frequent for developing rapid, accurate, and simple methods of analysis. Grape berry polyphenolic composition was extensively studied and one interesting application dealt with the evaluation of the effects of UV-B radiation, which upregulates some genes of the phenylpropanoid and flavonoid biosynthetic pathways, on grape berry skin extracts without a sample cleanup procedure [74]. A more complex sample preparation was used for the CZE analysis of phenolic acids in exotic fruit; in fact, a preconcentration step of the phenolic fraction after different liquid-liquid extractions and an alkaline hydrolysis step for the release of esterified phenolic acids were set up. A 32 factorial design was successfully applied to the electrolyte optimization and the resulting method proved to be suitable for the analysis of free and bound phenolic acids [75]. Another method was specifically set up for about 20 phenolic acids carrying out a univariate optimization of time of analysis, selectivity, and peak shape (only one parameter changes keeping the others constant); after validation, the method was applied to the analyses of the simple methanolic extract obtained from different avocado varieties harvesting at two ripening degrees (see Fig. 14.2A) [76]. Another group of fruits extensively studied are red fruits. CZE-UV was applied to define the polyphenolic profile of cranberries, blueberries, grapes, and raisins. The collected data treated with principal component analysis showed that samples were mainly clustered according to the fruit origin, confirming the results obtained by the HPLC method [78]. Two different CE-MS approaches, i.e., nontargeted and targeted (full scan or a multiple reaction monitoring method, respectively), were developed for the study of the profile of polar metabolites in complex samples such as avocado fruit. Thus it was possible to follow the quantitative evolution of different classes of metabolites detected in methanolic extracts, including not only phenolic acids and flavonoids, but also a carbohydrate, an organic acid, a vitamin, and a phytohormone, which change their levels during ripening [79].

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Fig. 14.2 Identification and quantification of (A) 21 phenolic acids in avocado samples [76] and of (B) 17 phenolic compounds in extra virgin olive oil samples [77] by validated capillary zone electrophoresis-ultraviolet methods.

Brassica vegetables are rich in phenolic acids and glucosinolates. A simple and rapid CZE method to quantify the phenolic acid contents was described by Lee [80] who used an SPE to replace the solvent extraction step in the isolation of the analytes, thus simplifying part of the extraction procedure. The same research group

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Evaluation Technologies for Food Quality

set up a CZE method involving an online large volume sample stacking procedure to preconcentrate kaempferol and quercetin, generally present in broccoli at low concentrations, below the limits of quantification of CZE-UV. This stacking procedure enabled sensitive detection and reproducible quantification of low concentrations of flavonoids, avoiding the need for other expensive detectors [81]. A method for the simultaneous separation and quantification of flavonoids and phenolic acids in tomato was set up using MEKC-UV. An accurate optimization of BGE composition (borax, ACN, methanol, and SDS concentrations) was performed. The repeatability, LODs, and recoveries in tomato samples are similar to the results of other authors working with similar or more sensitive techniques [82]. Glucosinolates are biologically inactive precursors of bioactive substances, i.e., isothiocyanates (ITCs), that could be activated by the action of enzymes (myrosinase) accumulated in a separate compartment after tissue damage. Gonda and coworkers [83] set up a fast, robust, and simple MEKC method for the simultaneous detection of glucosinolates, myrosinase enzyme activity, and ITC conversion rates in Brussels sprouts, radish, watercress, and horseradish. MEKC parameters have been optimized, followed by optimization of a myrosinase-compatible derivatization procedure for ITCs. The method was suitable not only for the screening of glucosinolates and allyl ITC, but also as a higher specificity myrosinase assay that also allows quantification of online generated ITCs.

14.7.1.6 Glycerophospholipids Glycerophospholipids are polar lipids, which have a glycerol backbone esterified with fatty acids in positions sn-1 and sn-2, and with a phosphate group in sn-3. Analysis of this class of compounds generally involves a number of problems due to the high variety of fatty acids that can be present in each phospholipid class, low abundance with respect to the nonpolar triglycerides, and lack of chromophores and the consequent low UV absorption. A NACE method with ESI-MS detection (NACE-ESI-MS) was developed. The results obtained for the qualitative/quantitative composition of olive fruits (and also olive oils) indicated the presence of phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, lysophosphatidic acid, and phosphatidylglycerol, and a correlation between their relative abundance and the botanical and geographical origin was pointed out. Moreover, interesting differences in the glycerophospholipid compositions for olive stone and pulp were shown [84].

14.7.1.7 Phytohormones (auxins) Interesting applications of CE deal with the detection and quantification of phytohormones produced by plants, a group of substances displaying vital roles in a plant’s lifecycle. Today, these substances are applied exogenously to regulate plant growth, and sometimes their concentration in vegetables and fruits is so high that it could be

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considered to have potential side effects for humans and animals (carcinogenicity, neurotoxicity, impaired reproduction). The simultaneous determination of gibberellic acid, indole-3-acetic acid, abscisic acid, jasmonic acid, indole butyric acid, 1-naphthalene acetic acid, and 2,4-dichlorophenoxy acetic acid (phytohormones containing a carboxyl group) was possible in banana using an efficient and sensitive CZE method with LIF detection based on the derivatization with 6-oxy-(acetypiperazine) fluorescein [85]. A CZE method proposed for the detection of auxins was based on the addition of chitosan-modified silica nanoparticles into the running buffer solution as a pseudostationary phase in capillary electrophoresis. The optimization of nanoparticle concentration, pH, and running buffer solution concentration led to the separation of indole-3-acetic acid, indole butyric acid, 2,4-dichlorophenoxyacetic acid, and 1-naphthaleneacetic acid extracted by octyl alcohol from bean sprout, radish, garlic bolt, cherry tomato, and kiwifruit. High separation efficiency, very short time of analysis, and simplicity in operation, in addition to high recovery, demonstrated applicability to real sample analysis [86].

14.7.1.8 Acrylamide Proteins or amines can react with reducing compounds to give foundation to Maillard reaction products, which play an important role in the formation of flavors and colors in foods during processing and storage. Among them, acrylamide has been classified as “probably carcinogenic to humans” by the International Agency for Research on Cancer. It is easily formed in cooked carbohydrate-rich foodstuffs at elevated temperatures and thus its determination using sensitive and selective analytical techniques is necessary. A CZE-MS2 method applying an inline preconcentration injection mode (field amplified sample injection [FASI]) in reversed polarity was developed, validated, and used for quantifying acrylamide in potato crisps, biscuits, crisp bread, breakfast cereals, and coffee. A particular sample preparation step was set up; it consisted of a defatting process followed by purification by Strata-X-C SPE cartridges prior to the derivatization step of amino acid with 2-mercaptobenzoic acid to obtain an ionizable compound suitable for CE analysis [87]. More recently, a microchip-CE method based on a five-step online multiplepreconcentration process was developed for the analysis of acrylamide in potato chips and French fries. This technique combined prolonged field-amplified sample stacking (FASS) and reversed-field stacking to extend the FASS time and remove the vacant sample matrix. After optimization, a LOD value about 700-fold lower than the ones previously reported for CE methods without the concentration process was obtained [88].

14.7.1.9 Nucleosides and nucleotides Dietary nucleosides and nucleotides play an important role in the maintenance of functions of bone marrow hematopoietic cells, intestinal mucosa, and brain. Therefore the quantification of these compounds in food is very important too.

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As an example, details of a CZE-UV method can be found in the paper of Lignou [71].

14.7.1.10 Inorganic cations A method for the direct injection from fruit and vegetable tissues (zucchini, apple, and mushroom) without any sample pretreatment has been developed by Kalsoom, allowing the determination of different cations, as shown in Fig. 14.3A [89]. A small piece of the tissue was simply placed into a capillary electrophoresis vial followed by application of an electrokinetic injection. The addition of HPMC to BGE allowed for an accurate electrokinetic injection of sodium, potassium, calcium, and magnesium ions from the plant material. The main disadvantage of this approach was the limitation of the applicability to analytes that can be charged on application of voltage; however, the main advantage was the simplicity of the approach.

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Fig. 14.3 Determination of different ions: (A) cations (1. K+, 2. Ca2+, 3. Na+, 4. Mg2+) in fruits and vegetables by direct injection in a CE-indirect UV system [89]; (B) cations in tap and mineral waters by a validated CZE-indirect UV method [90]; (C) anions (Cl– and F–) in original (Y) and adultered (Y1–Y5) whiskey samples by a high-throughput microchip-CE-C4D device [40].

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14.7.2 Cereals 14.7.2.1 Amino acids, peptides, and proteins Another interesting application concerned the detection of contamination of durum wheat flour with less expensive wheat by the detection of gliadins and albumins [91]. The separation of albumins by CZE-UV may encounter difficulties; in fact, this protein fraction has a high tendency to bind to the inner walls of fused-silica capillaries for the presence of high amounts of basic amino acids. A good resolution was the use of acidic buffers, such as phosphate/L-alanine buffer, that provided good resolution and repeatability [92]. The simultaneous separation and identification of triticale high molecular weight glutenin and secalin subunits by CZE is an efficient alternative to SDS-PAGE and should facilitate the breeding of valuable cultivars. A method using hydrophilic polymers, such as PVP and HPMC mixtures, for dynamic coating of the capillary inner wall and a low-concentration solution of PEO for the isoelectric separation buffer, was set up. In this case, the sample preparation procedure was quite complex; in fact, after having removed albumins, globulins, and gliadins using a saline solution and organic solvents in sequence, glutenins and secalins were extracted with Tris-HCl buffer. A final step of selective preparation of high molecular weight glutenin and secalin subunits was required. In comparison with previous CZE methods, the presented method offered significant improvements in terms of both run-to-run reproducibility and separation efficiency of faster-migrating subunits; moreover, the method also allowed the quantification of individual subunits [93, 94]. High molecular weight glutenin subunits in common wheat are a class of heterogeneous proteins and are generally difficult to separate and characterize; a cIEF method was developed and applied to 16 different cultivars of wheat with good results [30].

14.7.2.2 Carbohydrates A CZE-UV method was set up for the analysis of carbohydrates in breakfast cereals. This method did not require derivatization and presented many advantages compared to traditional reducing sugar and glucose-specific methods because it was possible to quantify more than one sugar in a single run even when present in low concentrations. In comparison with chromatographic methods, CZE did not require complex sample preparation, but only a grinding of the sample (500–1000 μm particle size) and its dilution in water [95].

14.7.3 Cocoa and coffee beans CZE-UV was used to analyze a nonprotein amino acid N-phenylpropenoyl-L-amino acid (NPA). It was quantified in raw and roasted cocoa beans and the results obtained by the CE method lacked sensitivity when compared with those obtained by ultrahighperformance liquid chromatography (UHPLC); however, CE needed a very simple sample preparation with respect to UHPLC requiring an SPE cleanup procedure.

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Evaluation Technologies for Food Quality

Therefore the CZE-UV method is suitable for cocoa samples with high NPA contents compared to the UHPLC method recommended for low concentrations [96].

14.7.3.1 Carbohydrates (mainly oligosaccharides) Today, the accidental or more frequently fraudulent adulteration of coffee is one of the problems affecting the economy of coffee producers. A method based on home-made CE-C4D equipment was developed, validated, and applied to the analysis of instant coffee powder adulterated with corn and coffee husks. This method was based on the controlled acid hydrolysis of xylan and starch present in adulterants, followed by the analysis of the corresponding resulting monosaccharides, i.e., xylose and glucose, respectively [97].

14.7.4 Dairy products and eggs 14.7.4.1 Fatty acids Nowadays, determination of the level of cis-trans fatty acids in food is an important quality parameter since their intake is a key dietary factor, especially for people affected by hypertension, heart disease, and obesity. CZE and indirect UV could be considered a good alternative method to monitor total trans-fatty acids in raw material such as hydrogenated vegetable fat, which is widely used as a dairy cream substitute in the manufacture of spreadable processed cheese and in final products, as demonstrated by de Castro [98, 99]. No derivatization or extraction procedures for sample preparation were needed, but only a simple saponification step and appropriate sample dilution. Therefore the method had a high analytical throughput. An alternative method with direct UV detection was proposed by Porto [100] and applied for the analysis of butter toffee, a mix for Brazilian cheese bread, and also cake mix, stuffed wafers, and chocolate. The main advantages of this method were represented by the use of an aqueous tetraborate buffer without cyclodextrin that made analysis simpler and cheaper, and by the use of a polyimide capillary that is stable when exposed to BGE solution and can be used for a larger number of analyses. Considering that fatty acids are a complex class of molecules being constituted by a high number of isomers and homologs, in addition to their low solubility in aqueous media and low molar absorptivity, a multivariate approach (23 central composite design) could be useful to optimize CE variables [101]. A suitable CZE-UV method for the quantification of cis-trans long chain fatty acids (stearic [C18:0], elaidic [C18:1t], oleic [C18:1c], palmitic [C16:0], linoleic [C18:2cc], and linolenic [C18:3ccc]) in butter, margarine, and filled cookies (and also in olive oil, soy oil, and hydrogenated vegetable fat) was developed. The obtained results were not statistically different from those obtained applying the AOAC official gas chromatography (GC) method. Much research has been focused on the particular healthy properties of omega-3 fatty acids; for example, a qualitative differentiation between natural and enriched

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chicken eggs was successfully proposed by the evaluation of omega-3 fatty acid profiles using a simple and rapid CZE-UV method [102]. The detection of saturated fatty acids is generally performed by GC-MS or LC-MS methods, which have two main disadvantages: the need to derivatize the sample and/ or the need to detect fatty acid types having specific chain lengths. Conversely, a sensitive and selective CZE-MS method was developed by using dicationic ion-pairing reagents (IPRs) forming singly charged complexes with anionic fatty acids. The best results were obtained using 1,5-pentanediyl-bis(1-butylpyrrolidinium) difluoride as an ion pair reagent, and the use of a postcolumn IPR infusion method gave the most efficient sensitivity. The developed CZE-paired ion ESI-MS method was applied for the quantification of fatty acids in coffee powder, cheese, and also in coffee extracts (see Section 14.8 [103]).

14.7.4.2 Peptides and proteins The nutritional value attributed to goat milk increased the consumption of goat milk cheeses, and thus the investigations on changes in microbiological and chemical properties of fresh goat milk cheese during storage have attracted researchers. A CZE-UV method was used to monitor proteolysis in goat milk cheese stored in different conditions. By CZE, the degradation of the main casein fractions and the formation of new peptides was followed [104]. The same analytical methodology was applied to compare the effect of different proteases on the protein composition of Prato cheese [105] and also to characterize the peptide profile of the insoluble protein fraction in acidic medium (pH 4.6) of 10 different brands of Prato cheese. This last work concluded that commercial Prato cheeses, even if produced with different raw materials and under different processing conditions, showed very similar peptide profiles when assessed at the molecular level [106]. Another very similar application consisted of the analysis of the water-insoluble protein fraction of Estonian hard cheese Old Saare; the different products generated by primary proteolysis (especially casein degradation) during ripening were detected [107].

14.7.5 Fish and meat 14.7.5.1 Peptides and proteins Recently, CZE-UV was used in parallel with 1H nuclear magnetic resonance (NMR) to study the effect of an in vitro-simulated digestion process on the bioactive dipeptide carnosine in two commercial samples of the Italian cured beef meat bresaola. Sample preparation involving raw material was simple and consisted of a homogenization step, acidification at pH 2.5, and analysis of the supernatant. The results obtained using the two applied analytical techniques were in agreement. Considering the digested samples, CZE analysis was still performed for gastric digested samples because the pH value used in this step was the optimum for CE analysis (pH 2.5), whereas NMR spectroscopy was performed for intestinal digested samples at pH 7.

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Applying this procedure, it was possible to evidence the difference between the total carnosine content, measured by CZE, and its free diffusible fraction detected by NMR; this difference represented the not accessible carnosine for intestinal absorption (it was adsorbed to the food matrix dispersed in the digestion fluid) [108].

14.7.5.2 Biogenic amines Biogenic amines are molecules widespread in nature deriving from the enzymatic decarboxylation of natural amino acids; they are especially present in protein-rich food, such as fish and meat. Their excessive consumption could generate human disorders and tumor development; therefore it is important to set up detection and quantification methods. The analysis of histamine, spermidine, cadaverine, putrescine, phenylethylamine, and spermine in oriental crucian carps was performed by a new MEKC method based on multiphoton excitation fluorescence (MPEF) detection after derivatization with fluorescein isothiocyanate (FITC), using a home-built CE-MPEF system. Results indicated a higher resolution in comparison to MEKC with single photon excitation fluorescence detection using an attoliter detection volume [109]. A nonionic MEKC method coupled to LIF detection was developed for the quantitative determination of FITC-derivatized biogenic amines in two Turkish traditionally processed fish products: brined Atlantic bonito and dry-salted sardine. The salting process can enhance the production of biogenic amines, especially if the salt contains nitrates or nitrites, and therefore their quantification in processed foods is an important public health issue. Use of the CZE-LIF technique had the great advantage of being able to accurately analyze food matrices extremely rich in salt that generally creates an important matrix effect. In fact, very low analyte concentrations can be usefully employed in CZE-LIF, thus making it possible to create an appropriate dilution of the sample and avoid the matrix effect generated by high salt concentration. Other main advantage of this technique consisted of the use of an additive in a BGE separation electrolyte that enhanced the fluorescent intensity of FITC-labeled biogenic amines considerably, thus shortening the separation time; furthermore, the additive could adjust the selectivity of amines without any change in current, avoiding the joule heating problem. This method is highly sensitive and could be easily used for the rapid analysis of biogenic amines even present in low concentrations [110]. A selective and rapid CZE-UV method was proposed for the quantification of histamine in tuna fish samples. Experimental design proved to be an important tool in choosing the appropriate BGE components and separation conditions without the need for experiments; thus it was possible to set up and validate a method free of interferences in a complex food matrix [111]. In 2015, the concentrations of putrescine, histamine, tyramine, phenylethylamine, and spermidine were evaluated in oyster samples under different storage conditions by the CZE-electrochemiluminescence (ECL) method [112]. A cITP method was set up and validated for the quantification of the content of spermine, spermidine, putrescine, cadaverine, histamine, tyramine, tryptamine, and 2-phenylethylamine in fresh white and red meat samples and also in samples fortified

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with additives and stored for 4 days at 4°C. The aim of this study was to develop a suitable method for the estimation of the impact of different additives on the formation of biogenic amines in meat stored at refrigerated temperatures [113]. Gizzerosine, or 2-amino-9-(4-imidazolyl)-7-azanonanoic acid, is a biogenic amine formed following the reaction between the amino group of lysine and the imidazole ring of histidine during an overheated fish meal manufacturing process. A sensitive detection method based on microchip-CE with LIF detection was developed. Gizzerosine was derivatized with FITC prior to CE analysis. The proposed method was advantageous in terms of reagent and sample consumption, analysis time, assay sensitivity, and applicability to complex samples such as fish meals, and trace levels could be quantified without any preenrichment [114].

14.7.5.3 Fatty acids For the analysis and separation of 15 n-3 and n-6 fatty acids in complex food matrices, such as grass-fed and grain-fed beef samples, a direct and sensitive CE-UV method using aqueous borate buffer containing beta cyclodextrin, SDS, urea, and ACN was used [115].

14.8

Beverages and liquid food

14.8.1 Water The analysis of water samples represents an important issue in food quality and safety, to monitor different types of analytes and contaminants in environmental, tap, or mineral waters. Concentration of analytes in the aquatic environment is usually low and this, in addition to CE low sensitivity, makes a sample preparation step necessary before instrumental analysis. To overcome this problem, which represents the most critical step in the analysis of water samples, pretreatments consisting of simple filtration or neutralization and many offline and/or online preconcentration methods were set-up and are continuously suggested in the literature [36, 116, 117]. Because of its sample preparation issue, this section deals with an overview of the particular preconcentration steps that must precede CE analysis, while for a detailed list of applications in the analysis of specific water contaminants (pesticides, toxins, and others), see Section 14.11. Water samples can be treated by using the common SPE procedure [54, 118–120] or the most innovative solid-phase microextraction (SPME) [121] and molecularly imprinted SPE (MI-SPE) techniques [117]. SPME is solventless and requires the use of fibers coated with a liquid (polymer) and/or a solid (sorbent). It is more rapid than SPE, ensuring good linearity and sensitivity, but fibers have higher costs and a shorter lifetime [121]. The MI-SPE technique is based on the MIPs mechanism and many examples of the use of MIPs to preconcentrate phenolic compounds in water samples are present in the literature [117]. Among offline pretreatments, offline liquid phase microextraction (LPME), by using, for example, supported liquid membranes, has great potential to clean up

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and preconcentrate analytes with low costs (low consumption of solvents) and a great facility to couple with analytical systems. In LPME, analytes are extracted from aqueous samples across or into volumes of organic solvents, which are water immiscible. New interesting developments in LPME concern so-called electromembrane extraction and microelectromembrane extraction (μ-EME) across free liquid membranes (FLMs), which exhibit the highest preconcentration capability among sample pretreatment techniques. In this method, an electric field moves analytes across the FLMs [122–125]. Many authors have focused their attention on online sample preconcentration techniques in CE, in which the enrichment process has been mainly performed in the separation capillary. Principal online sample preconcentration techniques applied in food analysis include field-amplified stacking and transient isotachophoresis (tITP) [126]. Among them, electrokinetic supercharging (EKS) and FASI are valid procedures with important applications in water sample analysis. They are different methods, in which analytes are introduced electrokinetically, and FASI is particularly suitable for large amounts of samples [116, 126, 127]. Also, the combinations of preconcentration methods represent an important solution in CE applications. For example, FLMs online combined with EKS show a very highly efficient preconcentration technique, in comparison with EKS or FASI alone, and it is very useful for hydrophilic contaminants [116]. Other examples are represented by a particular LPME technique called dispersive liquid-liquid microextraction (DLLME), which can be directly coupled with CZE for the determination of mercury and its compounds [128], and by using the stacking effect coupled with microemulsion electrokinetic chromatography, in which BGE is a microemulsion, useful for the analysis of hydrophobic analytes (phenols and chlorophenols) [129]. For the detection of aldehydes, which can originate during water disinfection, a precapillary derivatization step is necessary and only a few methods are available [130, 131]. For more details, see Section 14.11. A second option to detect analytes in traces in water samples and soft drinks is the use of sensitive detectors, such as electrochemical detectors (AD or C4D) [122, 124, 125, 132, 133] or LIF [120], but also in these cases a preconcentration step is recommended. In addition, for the detection of cations and anions in water samples, indirect UV detection [90] can be used as an alternative to complex interfaces (direct connection between sampling and separation capillary) coupled with C4D [134]. Ultrasensitive methods, such as CE-ICP-MS, have been applied in the analysis of mercury compounds to directly determine ultratraces without any preconcentration step in water and fish samples [135]. In detail, CE applications for water samples (environmental, tap, mineral) or soft drinks concern the detection of water ions (cations and anions) [90, 125, 134] and contaminants (pesticides, toxins, drugs, and other pollutants, for which see Section 14.11). As regards the analysis of mono- and divalent cations (Na+, K+, Li+, Ca2+, Mg2+ ions) in tap and mineral water, dynamic coating-CZE-indirect UV (see Fig. 14.3B) [90] and flow-gating interface CZE-C4D [134] are the two most recent applications. For the analysis of anions (ClO–4, Cl–, NO–3, and SO–2 4 ) in tap water, μ-EME across FLMs and CZE-C4D [125] were proposed.

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14.8.2 Milk 14.8.2.1 Bovine milk In the analysis of milk, when the target is protein content/degradation, milk samples are only defatted by centrifugation (for 10–30 min at 4°C) and filtrated to remove the fat layer [136, 137]. Conversely, to analyze nonprotein analytes or contaminants, due to the high concentrations of lipids, carbohydrates, and proteins present in milk products, previous protein precipitation and preconcentration steps are required. For milk preconcentration, many methods were proposed: from simple SPE [138] or DLLME procedures [139] to MIPs [140] or combined techniques [141–143]. Milk proteins are classified into caseins (αS1, αS2, β-casein, and κ-casein) and whey proteins (mainly α-lactalbumin and β-lactoglobulin, considered as small whey proteins), and are prone to degradation by native and bacterial enzymes. Milk protein composition affects both industrial milk processing and its nutritional value, and the monitoring of protein profiles becomes very important to ensure milk quality and also to investigate the correlation between protein profile and casein genetic variants [136, 137]. The concentration of caseins and the concentration and conformation of β-lactoglobulin affect many different milk products, mainly fermented products or milk powders. CZE monitors the change in protein profile/degradation, which can also depend on different bacterial strains, obtaining the percentage of degradation in relation to a sterile control milk [136], and also identifying protein genetic variants, as in the case of β-casein [137]. Adding a sample preconcentration step (for example, t-ITP), the CZE-matrix-assisted laser desorption/ionization (MALDI)-MS technique obtains a very sensitive method (LOD  2.1 nM) for both whey proteins [42]. Monitoring whey protein content has an important application in the control of adulteration of expensive caprine or ovine milk with bovine milk; CZE-ESI-MS offers a rapid and accurate approach to quantify whey proteins of bovine milk in “nonbovine” ones [144]. A recent work proposed a CZE-UV method as an alternative to conventional techniques (GC) to detect another type of milk adulteration, i.e., the addition of whey, which can be detected by monitoring the fatty acids profile [145]. Proteins or amines can react with reducing compounds to give origin to Maillard reaction products, which play an important role in the formation of flavors and colors in foods during processing and storage. In milk products, furosine concentration increases with increasing processing time/temperature conditions; it can be detected by CZE-UV or CZE-MS2 [146, 147]. Studies on other nonprotein amino acids are present in the literature, concerning bovine and human breast milk [147, 148]. A very important aspect in food safety regards the abuse or illegal use of drugs (nonsteroidal antiinflammatory drugs [NSAIDs], antibiotics, synthetic estrogens), in particular in animal food. Their wide use in veterinary medicine and the presence of drug residues in food products represent a potential risk for human health. For the analysis of these substances, preconcentration steps [141–143] or ECL detectors [149] are necessary. For the detection of NSAIDs, Alshana set up a rapid competitive DLLME-FASS-CZE-method, compared with conventional techniques (mainly

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SPE-LC-UV and SPE-LC-MS) [141]. Many methods have been proposed for antibiotics: β-lactams [142], tetracyclines [149], fluoroquinolones [138, 140], and 5-nitroimidazoles [150]. Moreover, CZE-UV methods able to simultaneously separate and quantify different antibiotics (β-lactams, tetracyclines, quinolones, amphenicols, and sulfonamides) are present in the literature [143]. For estrogens, different CE modes, such as MEKC or CEC coupled with MS or AD detectors, have been suggested to obtain simple and sensitive analyses [46, 139]. In the literature, many CE applications have been reported for the rapid detection of melamine in human foods, as an alternative to the most diffused LC methods. This is a very important issue for health safety because this molecule is a trimer of cyanamide, used as a fertilizer, derived from plastic materials and resins manufacturers and insecticide metabolism. It is a very important food and food-contact materials contaminant, but it is also used to adulterate milk products and animal feed, because it is able to increase the apparent protein content. Simple CZE-UV methods [151–153] are available for this application. Some CZE-UV methods that rapidly and effectively detect polycyclic aromatic hydrocarbons (important environmental pollutants contaminating animal feed) in milk are also present in the literature [154]. For a list of specific applications in the detection of drugs, melamine, and other pollutants in milk and derivatives, see Section 14.11.

14.8.2.2 Goat milk Goat milk has a particular composition and it is a high-quality product, mainly for infants and elderly people. It is more digestible in comparison to bovine milk. A CZE-UV method has been proposed to simply recognize milk from goats receiving organic products or commercial feed by monitoring the presence of organic acids (end-products of carbohydrate metabolism in lactic acid bacteria or additives) and quantifying hippuric acid content [155].

14.8.2.3 Human breast milk As regards human milk samples, many CZE applications dealing with the analysis of carbohydrate content are present in the literature. MEKC-UV, CZE-LIF, and CZE-UV have been demonstrated to be very useful for analyzing and quantifying neutral or acidic human milk oligosaccharides, which are very important dietary factors [156]. The main CE mode is CZE with UV [157] and C4D detection systems (for oligosaccharides determination, see other sections: energy drinks, juices, wine, honey) [158]. In addition, hyphenated CZE-LIF-ESI-MS methods rapidly obtain an oligosaccharide profiling with a characterization of about 50 molecules [159]. Another important application for human breast milk concerns the presence of drugs, but for these analytes, see Section 14.11. Detailed CE applications are reported in Table 14.4.

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Table 14.4 Determination of different analytes in milk by capillary electrophoresis (CE) Analyte

Food matrix

CE technique

References

αS1, αS2, β-casein, κ-casein, α-lactalbumin, and β-lactoglobulin Protein genetic variants Small-molecular weight whey proteins Bovine milk proteins Organic acids Oligosaccharides

UHT and degraded bovine milk

CZE-UV

[136]

Bovine milk

CZE-UV

[137]

UHT milk and skimmed milk powder Ovine or caprine milk Goat milk Human breast milk Human breast milk Human breast milk Human breast milk

t-ITP, followed by immunoaffinity CZEUV and CZE-MALDI-MS CZE-ESI-MS

[42]

Nonprotein amino acids

Fatty acids

Furosine Nonsteroidal antiinflammatory drugs β-Lactam antibiotics Tetracyclines Fluoroquinolones 5-Nitroimidazoles β-Lactams, tetracyclines, quinolones, amphenicols, and sulfonamides Estrogens Melamine

Bovine and human breast milk

Adulterated bovine milk (with whey) Bovine milk Bovine milk

Fortified bovine milk Bovine milk Bovine milk Bovine milk Bovine milk Bovine milk

Bovine milk Bovine milk Milk powder, bovine milk Milk powder Bovine milk

[144]

CZE-UV CZE-LIF-ESI-MS CZE-UV CZE-C4D MEKC-UV, CZE-LIF and CZE-UV CZE-UV

[155] [159] [157] [158] [156]

CZE-LIF, CZE-C4D, and MEKC-LIF CZE-UV

[147] [145]

CZE-UV and SPE-CZE-UV CZE-MS2 (FASS)-CZE-UV

[146] [147] [141]

SPE-LVSS-CZE-UV

[142]

CZE-ECL SPE-CZE-UV MSPE-HPCE-UV LLE-SPE-CEC-UV CZE-UV

[149] [138] [140] [150] [143]

SPE-p-CEC-AD DLLME-MEKC-ESI-MS2 SPE-CZE-UV

[46] [139] [151]

CZE-UV SPE-CZE-UV

[152] [153]

[148]

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14.8.2.4 Soy milk and drinks A very few CE publications are present in the literature for soy milk and drinks, e.g., the detection of aspartic acid enantiomers in soy milk (and beer) [160], of furosine, a Maillard reaction product in soy beverages [161], and of isoflavones, the main representative flavonoid compounds in soy beverages [162]. For pesticides analysis, see Section 14.11.

14.8.3 Olive oil Studies of olive oil mainly concern the detection of adulterations. In fact, mixing olive oil with seed oils of lower economic value is a common fraudulent practice. This represents an important issue for human health, because seed oils (hazelnut, peanut, sesame, soybean) may be allergenic or toxic. Many CZE methods are present in the literature, in particular those that detect phenolic compounds (simple phenols, secoiridoids, lignans, flavonoids, and phenolic acids), fatty acids profile, chlorophylls, betaines, and tocopherols, allowing assurance of quality and botanical origin of olive oil. The content of phenolic compounds depends on olive variety, environmental conditions (fly attack included), and extraction procedure, and it represents a marker of olive oil quality. Their extraction is very simple and is usually carried out with liquid liquid extraction (LLE) or SPE; on the contrary, microextraction techniques for olive samples are seldom explored [163]. Phenols can be monitored by CZE-UV [164] and CZE-MS, often preceded by sample derivatization [165, 166] or by advanced CZE, which is able to provide complete phenolic profiles (see Fig. 14.2B) [77] and different CE modes (NACE or CEC) [167, 168], which obtain a more complete phenolic profile. Fast and sensitive microchip-CE-AD techniques are also available [169]. An interesting CZE-UV method is able to quantify secoiridoids (oleocanthal and oleacein), a particular class of phenolic compounds with antiinflammatory properties [170]. The analysis of fatty acids (mainly oleic and palmitic acids and in less concentration linoleic acid) and their derivatives is both a measure of olive oil acidity and stability, and the concentration of these compounds is also correlated to botanical origin. It is usually carried out by GC approaches, but CE can overcome GC’s necessity to have volatile compounds [163]. Among CE modes, CZE with indirect UV detection is the suggested method [171]; in fact, only older publications on other CE modes are available [172]. Monitoring of fatty acids cannot detect when olive oil is adulterated with hazelnut oil. In addition, other oil species, such as corn and sunflower, have been measured only in small quantities. As an alternative, a very sensitive approach of a combined PCR-CE system has been proposed to analyze olive oil adulteration (with a sensitivity to detect fraud as low as 5%) with different oil types (soybean, palm, rapeseed, sunflower, sesame, cottonseed, and peanut). The obtained barcode profile gives the direct percentage of each plant oil [173]. This approach has been used in the past for the first time to discriminate olive oil botanical origin [174]. See also Section 14.12.

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In 2010–11, the first CZE-UV and CZE-MS methods to detect the pyridine betaine trigonelline were set up. Trigonelline is an alkaloid with many positive bioactivities (it shows hypoglycemic, hypocholesterolemic, osmoregulatory, and antitumoral effects). These analytical methods became very useful to discriminate the adulteration of olive oil with sunflower and soy oils. In fact, trigonelline is present in many vegetables and derived products, including sunflower and soy oils, but it is not detected in olive oil; so, it represents a marker of olive oil quality [166, 175]. Chlorophylls are important markers of authenticity, quality, and product stability and therefore their monitoring is often used as a quality control parameter. Thanks to chlorophyll fluorescence, CZE-LIF is the ideal technique allowing to discriminate natural pigments from synthetic ones that are often added, as in the case of refined olive oil [176]. Tocopherols (vitamin E) content represents an important issue to reveal sophisticated adulteration of olive oil (for example, with hazelnut oil) too. These compounds have important antioxidant properties, increasing the product shelf life. Because of their apolarity, CEC and NACE modes have been suggested [177, 178]. The analysis of proteins in olive oil by CE methods is relatively recent because proteins are minor components, and by consequence minor markers of olive oil quality; however, monitoring protein content could be considered as a control of the refining process, which is the main cause of protein loss [179]. On the contrary, nonprotein amino acids, such as ornithine, β-alanine, γ-aminobutyric acid, alloisoleucine, citrulline, and pyroglutamic acid, which can be monitored by CZE-UV and CZE-MS2, are considered new markers of adulteration [180]. For the analysis of aldehydes, representing toxic pollutants with remarkably unpleasant pungent odors in vegetable oils (and in wine), see Section 14.11. Detailed CE applications are reported in Table 14.5.

14.8.4 Coffee, tea infusion, and energy drinks 14.8.4.1 Coffee beverage/extract Concerning the quantification of alkaloids in coffee, tea, and soft drinks, an interesting publication of Li showed the set-up of a CZE method with the introduction of a nanoinjector and a particular electrical system suitable to decrease potential current instability, thus increasing precision [181]. Simple CZE methods can also be applied for detecting inorganic and organic anions, but even these methods are only reported in relatively older publications [182]. Fatty acids, important molecules for human health because they contribute to increased cholesterol level, are responsible for coffee acidity. Different CE methods reported in the literature in the last decade showed the separation of different types of fatty acids [103, 183]. A CZE-MS method proposed by Lee allowed the separation of a wide range of fatty acids without derivatization compared to GC-MS and LC-MS techniques. The same validated method offers potentialities also for trace anions detection in food samples [103].

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Table 14.5 Determination of different analytes in oils by capillary electrophoresis (CE) Analyte

Food matrix

CE technique

References

Phenolic compounds

Olive oil

CZE-UV and LIF, CZEESI-MS, SPE-NACE-UV, and LIF LLE-CZE-UV

[163]

Secoiridoids (oleocanthal and oleacein) Fatty acids

Protein

Nonprotein amino acids Trigonelline

Betaines Chlorophylls Tocopherols

Extra virgin olive oil Olive oil Olive oil, seeds oil Extra virgin olive oil Olive oil Olive oil Olive oil Olive oil

CZE-ESI-MS CZE-ESI-MS2 CZE-UV

[165] [166] [77]

SPE-NACE-ESI-MS LLE-CEC-UV Microchip-CE-AD LLE-CZE-UV

[167] [168] [169] [170]

Olive Olive Olive Olive Olive Olive Olive

CZE-UV, CZE-LIF CZE-indirect UV MEKC-UV PCR-CZE-fluorescence PCR-CZE-fluorescence FASI-CZE-UV CZE-UV and CZE-ESI-MS2

[163] [171] [172] [173] [174] [179] [181]

CZE-ESI-MS2 or CZE-UV CZE-UV

[147] [175]

CZE-ESI-MS2

[166]

SPE-CZE-LIF CEC-UV, NACE-LIF LLE-CEC-UV SPE-NACE-LIF

[176] [163] [177] [178]

oil oil oil oil oil oil oil

Olive oil Sunflower, soy, and extra virgin olive oils Extra virgin olive and seed oils Olive oil Olive oil Vegetable oils Vegetable oils

[164]

In coffee quality control, the problem relative to the presence of adulterants is very important and suitable analytical methods are needed. In particular, methods able to monitor the different carbohydrates composition could recognize a specific adulteration, as, for example, for xylose; its quantification is a useful indicator of husk and twigs addition to coffee. Another example is represented by the high level of glucose, which is a marker of adulteration with maltodextrin, caramel, or cereals, while high fructose levels indicate the addition of chicory. A very recent CZE-MS method was set up to obtain a complete profile of monosaccharides (fucose, galactose, arabinose, glucose, sucrose, rhamnose, xylose, mannose, fructose, and ribose) composition, as an index of coffee adulteration with soybean and corn [184].

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Concerning nonprotein amino acids [148] and Maillard reaction products (melanoidins and acrylamide) [87, 146], CZE is the common CE mode used, often with an online preconcentration step.

14.8.4.2 Tea infusion In the literature, only a few CE works are presented on the determination of phenolic content in tea, but the selectivity of this technique represents an important advantage in the separation of closely related phenolic compounds and gives results comparable to those obtained by HPLC [185]. In particular, the detection and quantification of catechins and/or methylxanthines are very important for their biological positive effects and for tea quality and stability. In fact, catechins are responsible for the bitter taste of tea and caffeine for tea flavor. For both classes of compounds, MEKC is the most commonly used technique [35, 186, 187]. A limited number of methods have been developed for the analysis of amino acids [188] and nonprotein amino acids [147, 148] in tea samples. A publication concerning nucleosides and nucleotides analysis in tea samples has also been reported [189].

14.8.4.3 Energy drinks A very few publications are present on energy drinks. In addition to the monitoring of oligosaccharides [190] and vitamins [191], a very important issue concerned the determination of taurine. Taurine is a nonprotein amino acid added to energy drinks, mainly to stimulate the brain. This substance has a positive effect also on the liver and cardiovascular system, but it also has potential negative effects if consumed at high doses, especially in relation to caffeine content; therefore its monitoring is very important [147, 192]. The main applications for coffee, tea, and energy drinks are reported in Table 14.6.

14.8.5 Fruits and vegetables juices Fruit juices are among the most popular beverages consumed around the world and their economic value makes this product easily disposed to adulteration. The most common fruit juice adulteration practices are dilution with water, addition of sugars or pulp wash, and blending with cheaper fruit juices. To overcome this problem in 2010 the Association of the Industry of Juices and Nectars provided guidelines for general fruit authenticity and quality criteria [193]. Regarding this, the main CE application concerns the detection of organic acids, which are responsible for juice acidity and are frequently substituted with cheaper material in frauds. The issue since the 2000s has been to find methods able to analyze these compounds in different juice matrices because they are markers of quality that distinguish a pure juice from juice mixtures. The main CE techniques reported in the literature concern CEC-indirect UV [194, 195] and CZE-MS [196]. The enantiomeric separation of DL-malic, DL-tartaric, and DL-isocitric acids has been performed by CCE-UV [197] or OT-CEC-UV [24]. Analysis of carbohydrates profile by CE provides a simple way to classify juices from different fruits and to evaluate adulterated juice mixtures. The analysis of

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Table 14.6 Determination of different analytes in coffee, tea, and energy drinks by capillary electrophoresis (CE) Analyte

Food matrix

CE technique

References

Methylxanthines

Tea, coffee, Coca-Cola Tea Energy drink Tea

CZE-UV

[181]

Chiral-MEKC-UV Short capillary MEKC-C4D/UV CZE-UV

[35] [192] [185]

Polyphenolic compounds

Fatty acids Anions

Oligosaccharides Amino acids Nonprotein amino acids

Melanoidins Acrylamide Vitamins (water soluble) Nucleosides and nucleotides

Tea Tea Coffee Coffee Coffee beverage Coffee Coffee Energy drinks Tea Coffee, tea

MEKC-UV chiral-MEKC-UV CZE-PIESI-MS2 MEKC-UV CZE-indirect UV

[186] [187] [103] [183] [182]

CZE-PIESI-MS2 CZE-ESI-MS2 Short capillary CE format-C4D OT-CEC-UV CZE-UV, MEKC-UV

[103] [184] [190] [188] [148]

Tea, energy drink Energy drink Energy drink Coffee Coffee Coffee Energy drinks

CZE-LIF and micro-CE-LIF, or MEKC-C4D Flow-gating interface CE-C4D Short capillary MEKC-C4D/UV CZE-UV FASI-CZE FASI-CZE-MS2 MEKC-UV

[147] [134] [192] [146] [146] [87] [191]

Tea

CZE-UV

[189]

oligosaccharides has been generally made by CZE-indirect UV [198, 199] and CZE-C4D [158]. Different CE modes (CZE, MEKC, CCE) can be applied for amino acids, nonprotein amino acids, and Maillard reaction products. In more detail, MEKCUV has been adopted for amino acids [200], MEKC-LIF, CCE-MS [148], CZE-UV, and CZE-C4D [146, 147] for nonprotein amino acids, and finally MEKC-UV for hydroxymethylfurfural [146]. With a preconcentration step, based on magnetic nanoparticles, it is possible also to control the presence of metals, such as Co, Zn, Cu, Ni, and Cd. The simple addition of a chelating agent and the consequent formation of complexes with metals overcome the conventional problem in the determination of transition metal ions by CE. In fact, they have similar mobilities because they have similar size and identical charge [201]. For more details, see Section 14.11.

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14.8.6 Sauces In sauces samples, a specific CE application concerns the monitoring of taste enhancers, such as glutamic acid, a compound correlated to a number of toxic processes (allergy and obesity). CZE-C4D and in-capillary derivatization-CZE-UV methods have been developed for its detection in soy, fish, and chili sauces [202, 203]. These methods provide a different strategy to obtaining a stacking effect offline or in-capillary. A few other applications in soy sauce have been reported: a CEC-indirect UV method for the analysis of organic acids in soy sauce [194] and a MEKC-LIF method for the quantification of biogenic amines in soy sauce [204]. CE techniques have also been applied to detect benzoic and sorbic acids, which are added to different sauces (soy, fish, and chili sauces) as preservatives; for more details, see Section 14.10.

14.8.7 Alcoholic beverages 14.8.7.1 Wine Regarding wine samples, the first CE methods were set up to analyze mainly polyphenols and protein contents as reported in the review by Coelho [205]. Wines present a large amount and variety of phenolic compounds that could be easily oxidized; therefore the detection of these compounds is a key factor not only for their biological effects but also because polyphenols (especially phenolic acids, catechins, proanthocyanidins, and some flavonoids) play an important role in wine quality (especially in red wines), contributing to flavor and color properties. In addition, in white wine the profile and quantification of some polyphenols is correlated to grape varieties [206]. Simple CZE methods with UV [207, 208] or AD [209, 210] can obtain rapid separation without the need of other sample treatments less than a simple filtration. In addition, phenol oxidation and the subsequent formation of phenol-protein complexes, which represent a key parameter for food manufacturing control and wine stability, could be monitored by CE, as proposed by Trombley on a model protein [211]. Amino acids, which are due to enzymatic degradation of the grape proteins and to autolysis of dead yeast cells, are markers of wine nutritional properties and are known to influence the aroma of wine and the foam characteristics of sparkling wines. Protein fingerprint and amino acid content can be monitored by CZE-UV [212], as shown in Fig. 14.1B for different white wines, or by CZE-LIF [213], and the determination of chiral amino acids is possible by CCE-UV with a simple precapillary derivatization procedure [214]. The determination of biogenic amines in wine samples represents a toxicological issue. They derive from the degradation of amino acids and their content is correlated to grape variety and environmental factors. In addition, they represent an indicator of alteration and potential health risk, so the search for pretreatment procedures and advanced analytical methods able to detect these compounds is very important.

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The main techniques used are MEKC-LIF and MEKC-UV [215], but also automatized online combination of cITP-CZE coupled with UV detector [216] and CZE-MS2 are available [217]. Organic acids, i.e., tartaric acid, malic acid, citric acid, succinic acid, acetic acid, and lactic acid are the main acids responsible for wine taste, and CE is considered a conventional method for their detection and quantification in wine, as demonstrated by the most recent CZE-indirect UV [218] and MEKC-indirect UV [219] applications. These methods have been developed for the analysis of white and red wine samples and rice wine, respectively. Among the analysis of organic acids, the enantioseparation of tartaric acid is important, because D-tartaric acid, which is not a natural product, can be added to beverages, but it has to be declared. This detection can be carried out with a particular chiral separation mode by applying a CE-CCD method [220]. Other markers of authenticity and quality of food products are carbohydrates. Recently, CE methods were also developed for their quantification in wine; a particular coated CZE-UV method has been set up for the quantification of oligosaccharides in red wines, similar to apple juice [221]. The search for melatonin and its isomers in grape-related foodstuffs, mainly wine, is relatively recent. This molecule has not only positive physiological functions, but it is also an index of alcohol fermentation and its content depends on grape products. Melatonin content is commonly determined by LC, and the first CZE and CEC methods to detect melatonin in wine were set up in 2010 [222, 223]. Also, the presence of carbonates, such as monoalkyl carbonate, which is correlated with the type of vinification, can be monitored by CE. Some examples perform the analysis of monoethyl carbonate in sparkling wine and other alcoholic carbonated beverages, such as beer and soft drinks, by CZE-C4D [224]. In conclusion, CZE remains the main mode for wine analysis [206]. Particular mention should be given to the development of microchip-CE, able to detect different wine compounds (polyphenols, amines, organic acids, sugars, and contaminants) with important applications in wine quality and authentication, and to obtain wine fingerprinting useful for wine characterization and classification [225]. Conversely, capillary and chip-based systems are mainly applied for monitoring wine-making processes (i.e., fermentation and cell culture processes) [226]. The detection of pesticides, mainly fungicides, in wine represents an important issue in relation to the lack of uniformity in the maximum residue limits (MRLs). In spite of its poor sensitivity, offline or online preconcentration steps can help CE to become competitive with conventional LC or GC techniques [227]. A more detailed description of the most used CE applications for the detection of pesticides and other substances in wine samples and derivatives is reported in Section 14.11. Other applications are listed in Table 14.7.

14.8.7.2 Beer Food quality control of beer samples is generally performed by GC techniques for sensory and chemical evaluation of beer aroma and analysis of pesticide residues, or by LC methods applied for the detection of amino acids, gluten peptides, phenolic acids,

High-performance capillary electrophoresis for food quality evaluation

339

Table 14.7 Determination of different analytes in alcoholic beverages by capillary electrophoresis (CE) Analyte

Food matrix

CE technique

References

Polyphenols

White wine White, ros
e, and red wines Red wine White wine White wine Wine Wines Red wine

CZE-UV CZE-UV

[206] [207]

CZE-UV CZE-AD CZE-AD Microchip-CE-UV CZE-UV CZE-UV

[208] [209] [210] [225] [208] [211]

Whiskey

CZE-UV

[228]

White wine White and red wines Wine Beer Beer Vinegar

CZE-UV CZE-LIF CCE-UV CZE-UV CZE-UV MEKC-LIF

[212] [213] [214] [229] [230] [148]

Wine

MEKC-UV/LIF, microchip CE-fluorescence MEKC-LIF cITP-CZE-UV CZE-ESI-MS2 CZE-CD CCE-direct UV/indirect UV CZE-indirect UV MEKC-indirect UV CE-CCD

[215]

[218] [219] [220]

Microchip-CE CZE-UV CZE-C4D Microchip-CE-UV CZE-UV, CEC-UV CZE-UV, CEC-UV CZE-ESI-MS CZE-UV or CZE-ESI-MS

[225] [221] [158] [225] [222] [223] [233] [189]

CZE-C4D

[224]

Microchip-CE-UV/C4D

[40]

Polyphenol– protein complexes Phenolic aldehydes Amino acids

Nonprotein amino acid Biogenic amines

Wine Red wines Wine and beer Beer Beer Organic acids

Carbohydrates

Melatonin Iso-α-acids Nucleoside and nucleotide Carbonate Anions

White and red wines Rice wine and beer White and red wines, wine grapes Wine Red wines Wine Wine White and red wines White and red wines Beer Beer Wine, beer, and drinks Whiskey

[204] [216] [217] [231] [232]

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Evaluation Technologies for Food Quality

aldehydes, and contaminants [230]. The application of CE still remains limited. It has been proposed for the detection and quantification of amino acids playing a significant role in beer fermentation, flavor, and quality, but because their content is low, derivatization was necessary to overcome amino acid sensitivity limits. CE methods with in-capillary derivatization have been suggested [229]. A known method of coordinating interaction between amino acids and copper ions has been proposed to obtain direct monitoring of amino acids content without derivatization with an online preconcentration step followed by CZE-UV [230]. As previously reported, in wine, beer, and in all fermented products, biogenic amines represent important food toxics. Direct rapid and sensitive methods without derivatizations and using CZE-UV [231] or CCE-UV [232] are present in the literature. In addition, CE can also be applied for the detection of α-acids and β-acids in hops and iso-α-acids in beer. Hops are prone to oxidation and deterioration, and storage conditions are the main factors affecting these processes, thus creating these acids, which cause changes in beer flavor [233]. The development of CE methods suitable for the rapid detection of organic acids (oxalic, tartaric, formic, citric, malic, lactic, succinic, acetic), important compounds for taste, flavor, and aroma of beverages (see also wine), as well as for monitoring the fermentation process needs the addition of a chromophore to BGE [219]. Other applications are listed in Table 14.7.

14.8.7.3 Whiskey The definition of chemical composition and markers is useful to certify whiskey quality and authenticity to protect consumers from adulterated and/or falsified products. For this purpose, GC and LC techniques are available to monitor different substances (phenolics, furans, sugars, alcohols). CE represents an interesting alternative to the analysis of the content in phenolic aldehydes (vanillin, syringaldehyde, coniferaldehyde, and sinapaldehyde), which are known to be markers of authenticity. With simple stacking procedures before CE analysis, the results were comparable to those obtained by LC-MS2 methodologies [228]. Chips with C4D detection have also been proposed for a rapid and high-throughput analysis of anions (Cl– and F–) to discriminate authentic whiskey from diluted samples (see Fig. 14.3C) [40].

14.9

Other foods

14.9.1 Honey The mineral profile of honey gives an important indication of the geographic origin. Inorganic anions are related to the conductivity of honeys, and this parameter is regulated in the quality control process of honey samples. A CZE-indirect UV method has been set up for the detection of cations, mainly Na+, K+, Ca2+, Mg2+, and Mn2 + [234, 235], and anions, i.e., chloride, nitrate, sulfate, and phosphate [236].

High-performance capillary electrophoresis for food quality evaluation

341

Among the functional ingredients present in this food, the most widely studied group is the family of antioxidants, i.e., flavonoids/phenolic compounds or phenolic derivatives, which could be considered markers of healthy properties or of toxicity, respectively. MEKC-UV was one of the first CE modes set up to detect flavonoids and phenolic acids in honey samples [237]; by using trace amounts of poly-βcyclodextrin wrapping carbon nanotubes for microextraction (microSPE) before the application of CZE-LIF, better results have been obtained [238]. A programmed nebulizing-gas pressure mode for quantitative CZE-ESI-MS was used to quantify phenolic derivatives [239]. According to Codex Alimentarius a minimum of 60% (w/w) for monosaccharides and a maximum of 5% (w/w) for sucrose has been established for honey. Therefore suitable methods, including CE methods, for the determination of monooligosaccharides (fructose, glucose, and sucrose) has been set up to describe the quality and authenticity of honey. As an example of CE applications, noteworthy methods could include CZE-C4D [158], MEKC-UV [240], and microchip CE-AD/ED [241]. In addition, the proline content can be considered not only a quality marker, but also a marker of honey maturity, and it is very useful to detect sugar adulteration, as demonstrated by Dominguez [242] who developed and validated an MEKC-indirect UV method for the simultaneous quantification of oligosaccharides and proline. As previously reported for other food matrices, the quantification of organic acids could be performed by CZE-UV methods [243]. Honey sample preparation before CE analysis is generally simple; it consists of dissolution in an appropriate solvent/buffer before a dilution step or a simple filtration procedure [234, 236]. For phenolic compounds and flavonoids analysis, LLE or SPE steps are generally added [237–239].

14.9.2 Food supplements The consumption of food supplements increases day by day, as well as the number of commercially available products. Directive 2002/46/CE [244] strictly regulates vitamins and minerals levels that could be added to food supplements, including in Annex II the list of permitted vitamins and minerals sources. Furthermore, the European Commission prepared a report on the use of substances other than vitamins and minerals to be used in food supplement formulations. Therefore to protect consumers against potential health risks following the consumption of adulterated food supplements, suitable analytical methods had to be developed. CE analysis of vitamins, sugars, or amino acids in different types of supplements has been reported in the literature [245–247]. A lot of brand of supplements containing carnitine are on the market; carnitine is a nonprotein amino acid and an essential nutrient, in particular for infants and for patients with certain diseases (renal, cardiovascular, and Alzheimer’s diseases). The formulations generally contain only L-carnitine isomer or the racemic mixture. Whereas L-carnitine is highly effective, D-carnitine displays serious side effects, therefore it is essential to apply suitable detection methods for carnitine [248].

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Evaluation Technologies for Food Quality

A similar issue involves lipoic acid, whose natural form is R-lipoic acid, but synthetic lipoic acid is racemic and the potency of S-lipoic acid is poorly clarified. Electrophoretic methods (CZE and CCE) are useful alternatives to the conventional LC and GC approaches in enantiomeric separation. They could determine the content of the enantiomers, which is very important to monitor the chemical process and purity of L-carnitine [249, 250] or R-lipoic acid preparations [251]. The main problem with food supplements is adulteration with an indiscriminate and illegal addition of active principles, which should be absent. Food supplements for weight control represent one of the classes too often adulterated with caffeine or drugs. To detect these substances in the formulations, CZE-UV and CZE-MS2 are the most used CE methods [55, 252–254]. A more recent issue regards the addition of metallic nanoparticles (such as gold, platinum, and palladium) during the manufacturing process to improve the performance of dietary supplements. Electrophoretic methods (mainly CZE-ICP-MS) allow rapid and high-resolution monitoring and characterization [255]. Other details of the methods are listed in Table 14.8.

14.9.3 Baby foods α-Lactalbumin, immunogloublin G, and lactoferrin are commonly used as additional components in infant formula with the aim of promoting defense from infections. Since their beneficial effects are strictly dependent on manufacturing and storage Table 14.8 Determination of different analytes in food supplements by capillary electrophoresis (CE) Analyte

CE technique

References

Vitamins (ascorbic acid, thiamine, riboflavin, nicotinic acid, and nicotinamide) Glucosamine Amino acids (leucine, isoleucine, valine)

MEKC-UV

[245]

CZE-CCD CZE-UV or indirect UV CZE-C4D CCE-MS CCE-UV CZE-UV CZE-UV CZE-UV CZE-ESI-MS2 CZE-ESI-MS2

[246] [247] [249] [250] [251] [252] [252] [55] [253] [254]

CZE-ICP-MS

[255]

Carnitine Lipoic acid Caffeine Furosemide, norephedrine, and ephedrine Adrenergic amines Amphetamines Furosemide, trichlormethiazide, hydrochlorothiazide, triamterene, spironolactone, acetazolamide, dioctyl sulfosuccinate, bisacodyl, sennoside A, sennoside B, picosulfate, phenolphthalein, phentermine, sibutramine, N-didemethylsibutramine, fenfluramine, N-nitrosofenfluramine, mazindol, fluoxetine, diazepam Metallic nanoparticles

High-performance capillary electrophoresis for food quality evaluation

343

conditions, their detection and quantification, frequently together, are important key points. In addition, the developed CE methods could often be used for the simultaneous detection of other milk proteins, such as β-lactoglobulin and bovine serum albumin, with CE results in good agreement with those obtained by LC methods. One of the first CE methods applied for the detection of α-lactalbumin, β-lactoglobulin, and bovine serum albumin in infant formula was a CZE-LIF method reported in 2005 by Veledo [256]. More recently, a microchip-CE-UV was used for quantifying α-lactalbumin, β-lactoglobulin, immunoglobulin G, lactoferrin [257], and a simple CZE-UV for the detection of lactoferrin [258]. In addition, a validated CGE method with UV detection was proposed to give a complete protein profile, including high-molecular weight (> 50 kDa) whey proteins (immunoglobulins, bovine serum albumin, lactoferrin) in different infant formulas. Applying this method, it is possible to quantify the ratio of whey to casein in infant products, produced with different whey ingredients [259]. CZE-UV can be useful also in the monitoring of nitrate and nitrite (mainly coming from vegetables) in baby foods. Nitrate is not toxic, but under low pH condition or by bacteria action it can be reduced to nitrite, which can lead to methemoglobinemia, also called blue baby syndrome [260]. In hypoallergenic infant formulas, the detection of peptides deriving from complex bovine milk protein hydrolysates to prevent allergy is very important. Peptides, which exhibit different bioactivities (immunostimulating, antimicrobial, opioid, angiotensin converting enzyme inhibition, mineral binding, antithrombotic, allergenic), can be derived from enzymatic reactions or can be a consequence of particular food processing systems. For this purpose, CE-MS (mainly CZE-MS) applied after an SPE purification step provides further rapid methods in comparison to LC-MS [261]. Other analytes detected by CE methods in baby food, are: l

l

l

Anions (nitrite, nitrate) by CZE-UV [260]; Nonprotein amino acids (carnitine) in baby food supplements by CCE-MS2 [262]; Nucleosides and nucleotides in infant formula and baby foods by CZE-MS [263, 264].

14.10

Additives

14.10.1 Dyes Among additives, dyes represent one of the class of substances often illegally added to foodstuff, as in the case of Sudan dyes and tartrazine (E102), most used to enhance the red/orange color of food. Sudan dyes have been classified by the International Agency for Research on Cancer as category 3 (they induce liver and bladder cancer in animals [265]); nowadays, it is still frequently used because of its low cost and wide availability. Tartrazine can cause hyperactivity in children, allergy, and asthma, and the acceptable daily intake has been fixed at 7.5 mg kg–1 body weight. Sudan dyes are used often in sauces, and tartrazine in different types of foods (milk, soft drinks, juices, candies, cakes, cereals, and soups). Different CE modes are available to monitor dyes [266–268]. Fig. 14.4A shows the determination of four Sudan dyes in a chilli tomato sauce by MEKC-UV [267].

344

Evaluation Technologies for Food Quality

0.0008

0.0006

(a) 0.0004 AU

I.S. S II

0.0002 SI

S III S IV

0.0000

(b) –0.0002 3.0

4.0

5.0

(A)

6.0

7.0 Minutes

8.0

9.0

16 SA

BA

(a) ∗

Absorbance (mV)

(b) (c) 8

(d) (e) ∗

(f) (g)

0 3.5

(B)

4.5 Time (min)

5.5

Fig. 14.4 Determination of different additives by micellar electrokinetic chromatographyultraviolet (MEKC-UV): (A) chilli tomato sauce (a) and chilli tomato sauce spiked with Sudan dyes (I–IV) (b) [267], (B) preservatives (benzoic acid, BA and sorbic acid, SA) in different samples (juice—(b); soft drinks—(c) and (d); soy sauces—(e) and (f ); wine—(g)) [269]. ACS Reprinted with permission from T.S. Fukuji, M. Castro-Puyana, M.F. Tavares, A. Cifuentes, Fast determination of sudan dyes in chilli tomato sauces using partial filling micellar electrokinetic chromatography, J. Agric. Food Chem. 59 (2011) 11903–11909. Copyright (2011) American Chemical Society.

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345

14.10.2 Preservatives Benzoic acid and sorbic acid are used as preservatives to inhibit bacterial and antifungal development, respectively, but their use can be set under control. In fact, benzoic acid is nontoxic, but it can give foundation to carcinogenic benzene, and sorbic acid can produce mutagenic products. Sodium benzoate and potassium sorbate remain the most used preservatives because of their good solubility in water. CE food applications in the monitoring of preservatives are mainly focused on sauce samples (soy, tomato, fish, and chili sauces) [269–271] and beverages, such as wine and soft drinks (see Fig. 14.4B) [269], and also allow simultaneous and rapid determination of many different types of preservatives [272]. Of interest is an electrokinetic flow analysis system equipped with an electroosmotic pump, five solenoid valves, and one online home-made SPE for cleaning up and concentrating samples. These systems combined with CZE were set up for the analysis of benzoic and sorbic acids and their relative salts in fruit jams (and also in milk beverages and soy sauce). An ionpairing reagent (tetrabutylammonium bromide) was added to sample solutions to enhance the breakthrough content of the preservatives on the SPE column [273].

14.10.3 Sweeteners Sweeteners are used often in sugar-free soft drinks, juices, jellies, and chocolate, and their potential toxicity often remains controversial. C4D is mainly proposed as the CE application and the main advantage, in comparison with standard LC techniques, is the short analysis time. Also, cITP can be applied for this application allowing the simultaneous determination of different sweeteners [29]. Several papers have demonstrated the utility of CE for the analysis of different artificial sweeteners, from the most diffused aspartame and saccharin [274] to new substances, such as stevia [275] and neotame [276]. Stevia, a natural sweetener extracted from plants belonging to the Stevia rebaudiana family, is a natural substitute for saccharose, and its use has recently been increased; its sweetness is mainly due to its content in glycoside derivatives (rebaudiosides, stevioside, steviolbioside, and dulcosides). Moreover, it has no calories and does not interfere with insulin. CE methods to detect sugar alcohols or polyols (erythritol, maltitol, xylitol, and sorbitol), which are low-calorie sweeteners particularly adapted for diabetics, are also available [277].

14.10.4 Synthetic antioxidants Only a few CE publications are present in the literature for the determination of synthetic antioxidants, such as alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, and tert-butylhydroquinone [278].

14.10.5 Other additives The detection and quantification of benzoyl peroxide, a specific additive used for wheat flour, commonly known as “flourbrightener,” is extremely important for the toxic effect of its metabolite benzoic acid in humans when present in high concentrations. A simple

346

Evaluation Technologies for Food Quality

Table 14.9 Determination of different additives in food by capillary electrophoresis (CE) Analyte

Food matrix

CE technique

References

Sudan dyes

Sauces Sauces Milk, soft drinks, juices, candies, cakes, cereals, and soups Milk, soft drinks, juices, candies, cakes, cereals, and soups Soy sauce and beverages Sauces Wine and soft drinks Wine and soft drinks Milk beverages, soy sauces, and fruit jams Sauces

CEC-AD MEKC-UV CZE-UV

[266] [267] [268]

MEKC-UV

[268]

FASI-CZEC4D MEKC-UV MEKC-UV MEKC-UV EFA-SPECZE-UV CZE-UV

[270] [269] [269] [272] [273]

Soft drinks

CZE-C4D

[274]

Fruit beverages Nonalcoholic beverages Chocolate

CZE-C4D CZE-UV CZE-C4D

[275] [276] [277]

Food supplement

MEEKC-UV

[278]

Wheat flour

CZE-UV

[279]

Tartrazine

Sorbic and benzoic acids

Sorbic and benzoic acids, monosodium glutamate Aspartame, cyclamate, saccharine, and acesulfame-K Stevia Neotame Erythritol, maltitol, xylitol, and sorbitol Alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, tert-butylhydroquinone Benzoyl peroxide

[271]

method using CE was developed and benzoyl peroxide was determined as benzoic acid after a reduction step by potassium iodide added to the suspension of flour in methanol. The precision, accuracy, and LOD and LOQ values were in agreement with those using HPLC methods and thus the set-up CE methods could be considered a good alternative for routine monitoring in wheat flour [279]. Other details of CE in additives analysis are reported in Table 14.9.

14.11

Contaminants

14.11.1 Pesticides, herbicides, and fungicides The Food and Agriculture Organization/World Health Organization (WHO) and Codex Alimentarius Commission fixed the MRL for pesticides and contaminants in foods, and subsequently the European Union set different directives for MRL levels

High-performance capillary electrophoresis for food quality evaluation

347

for various fruits and vegetables [280]. Therefore it is fundamental to develop analytical methods suitable for the detection of pesticide residues aimed at food quality control. The main disadvantage of CE in analyzing pesticide residues and other contaminants is inadequate sensitivity of UV detection. Among analytical techniques, CE-MS is certainly the ideal technique for trace contaminant analysis in food [281], but recent developments in sample pretreatment represent a valid alternative. In the last few years, CE low sensitivity was mainly overcome with sample enrichments by SPE and SPME, mainly used for liquid samples [282]. Conversely, LLE, LPME, and DLLME techniques [283–285] and particular SPE modes (i.e., matrix solid-phase dispersion or stir-bar sorptive extraction) [286–288] have been demonstrated to be very efficient for solid samples, such as vegetables and fruits. Also, MIPs technologies were applied for the selective recognition of pesticide residues [288–290]. A list of the most recent CE applications in the detection and quantification of pesticides, herbicides, and fungicides in different food matrices can be found in Table 14.10 [47, 227, 236, 291–295]. Table 14.10 Determination of contaminants in food by capillary electrophoresis (CE) Analyte

Food matrix

CE technique

References

Triazines Bipyridylium compounds

Fruits and vegetables Water

MI-MSPD-MEKC-UV SPE-CZE-UV

[288] [118]

Water

[116]

Water Water and grape Cereals Water

FASI-CZE-UV EKS-CZE-UV FLMs-EKS-CZE-UV SPE-LVSS-CZE-UV LVSS-MEKC-UV EME-CZE-UV

Soy milk and drinks

CZE-ESI-MS

[282]

Fruits and vegetables

SPE-MEKC-UV SBSE-MEKC-UV

[286]

Fruits and vegetables

pCEC-indirect AD

[47]

MEKC-LIF Microchip-CE-CCD

[291] [292]

MSPD-CZE-ECL DLLME-NACE-UV

[287] [284]

Sulfonylureas Benzimidazole derivatives Triazolopyrimidine sulfoanilide compounds Acrinathrin, bitertanol, cyproconazole, fludioxonil, flutriafol, myclobutanil, pyriproxyfen Organophosphate derivatives Sodium monofluoroacetate Phenylureas Imazalil, prochloraz, and thiabendazole

Fruits and vegetable juices Vegetables and rice Fruit juices and vegetables

[116] [119] [285] [123]

Continued

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Evaluation Technologies for Food Quality

Table 14.10 Continued Analyte

Food matrix

CE technique

References

Multiclass pesticides Dithiocarbamate

Wine Vegetables Vegetables Fruits Fruits and vegetables Honey

SPME-MEKC-UV CZE-UV CZE-ICP-MS MIPs-CEC-UV CZE-indirect LIF CZE-indirect UV PCR-CGE-UV PCR-CGE-UV Microchip-CEfluorescence PCR-CZE-LIF CZE-UV MEKC-UV CZE-MS PCR-MEKC-UV

[227] [293] [294] [290] [295] [236] [296] [297] [298]

PCR-MEKC-UV

[304]

PCR-MEKC-UV

[305]

[306] [120] [307]

Water Seafood Water Beverages Water, soft drinks Milk, dairy products

CPE-CZE-UV SPE-CZE-LIF tITP-CZE-UV tITP-CZE-C4D Online FASI-CZE-AD CZE-ICP-MS SPME-CZE-UV cITP-CZE-UV CZE-UV, CZE-MS DLLME-FASS-CZE-UV

Milk, yogurt Milk, yogurt Meat Bovine milk Bovine milk Feedstuff Bovine milk Honey

DLLME-MEKC-MS/MS p-CEC-AD SPE-CZE-MS2 SPE-CZE-UV MIPs-CZE-UV CZE-UV CZE-ECL CZE-UV

[139] [46] [309] [138] [140] [310] [149] [311]

Thiabendazole Strobilurin Formic acid Food-borne pathogens

Mycotoxins

Bacterial endotoxins Shellfish toxins

Acidic drugs Antimalarials Nonsteroidal antiinflammatory drugs Estrogens Quinolones Fluoroquinolones Tetracycline

Apple juice Apple juice Fruit juices, cereals Nuts, fruits, cooked meat, dry fermented sausage, cured cheese Cooked ham, dry fermented sausage, peach Fruits, nuts, cereals, spices, dry-ripened foods Cereal Water Mussel

[299] [300] [301] [302] [303]

[133] [308] [121] [27] [34] [141]

High-performance capillary electrophoresis for food quality evaluation

349

Table 14.10 Continued Analyte

Food matrix

CE technique

References

Macrolide antibiotics, tetracycline β-Lactams 5-Nitroimidazole Sulfonamide

Bovine milk

LLE-SPE-CZE-UV

[143]

Bovine milk Bovine milk Meat Meat Meat Dairy products, chicken eggs, honey Meat

SPE-LVSS-CZE-UV CCE-UV CEC-MS Microchip-CE-LIF MEKC-UV FASS-CZE-UV

[142] [150] [312] [313] [314] [315]

SPE-CZE-CL

[316]

Meat Pork meat Human breast milk

NACE-MS CSEI-sweep-MEKC-UV LLE-CZE-UV

[317] [318] [319]

Rice Fish Fish Water Water Water Water Treated water Water Water Water Soft drinks Soft drinks

CZE-ICP-MS CZE-ICP-MS CZE-ICP-MS DLLME-CZE CZE-ICP-MS EME-CZE-C4D FASI-CZE-C4D μSPE-MEKC-UV LPME-CZE-AD MISPE-CZE-UV DLLME-MEEKC-UV FASI-MEKC-UV EME-CZE-C4D

[320] [321] [322] [128] [135] [122] [132] [130] [131] [117] [129] [127] [124]

Sulfamethoxazole and trimethoprim Sulfadimidine, sulfadiazine, and sulfathiazole β-Agonists Tricyclic antidepressants Heavy metals

Metals Aldehydes Phenolic compounds Chlorophenols Bisphenols

14.11.2 Intracellular food-borne pathogens To detect microorganisms and their toxic products is a challenging issue because of many different formats and combinations of ingredients. In addition, microorganisms and toxins are not usually equally distributed in foods and an aliquot tested may not necessarily be representative of the overall sample. Many CE modes (CZE, MEKC, CGE) are applied to detect different microbial contaminants in different types of food (milk, juice, wine, corn, fruits, meat, fish, baby foods) [323]. Some food-borne pathogens represent important food contaminants able to provoke serious injury and death, mainly in subjects at risk such as pregnant women, infants, and elderly people [298]. During the last few years an increased development of PCR-CE systems, coupled with amplification reactions or preenrichment steps, allowed the detection of many

350

Evaluation Technologies for Food Quality

different food-borne pathogens in a single run [296, 297, 299, 324]. Also, microchipCE applications are present in the literature, ensuring more rapid and reproducible methods in comparison to PCR-CE [298].

14.11.3 Toxins Mycotoxins, including more than 100 compounds such as aflatoxins, ochratoxins, patulin, ergot alkaloids, and verrucosidin, are natural, toxic, secondary metabolites of filamentous fungi; they are considered the major contaminants of agricultural products and foods (cereal products, fruits, vegetables, juices, and meats) and represent important toxicological agents for human health. In fact, they have teratogenic, carcinogenic, and/or mutagenic effects, and can cause autoimmune diseases or allergies. Because a single filamentous fungus can produce many different mycotoxins, analytical methods able to identify and quantify in a single sample more than one mycotoxin are needed [325–327]. The maximum levels of mycotoxins in foodstuffs have been specified in Commission Regulation (European Commission, EC) No. 1881/2006 as amended by Commission Regulation (European Union, EU) No. 165/2010. Provisions for sample preparation and analytical methods for the official control of mycotoxins are laid down in Commission Regulation (EC) No. 401/2006 as amended by Commission Regulation (EU) No. 178/2010. Therefore a modern issue requires methods able to analyze multiple mycotoxins in a single run and for this purpose CE-MS hyphenation and PCR-MEKC techniques represent ideal strategies [302–305]. Potential contaminations of an aqueous environment and/or seafood, which require accurate and ultrasensitive determinations, regard endotoxins [120] and paralytic shellfish toxins, which are strong inflammatory agents and neurotoxins, respectively [133, 307, 308]. For these and other applications [300, 301, 306], see Table 14.10.

14.11.4 Drugs Today, the control of antibiotic residues in edible animal tissues is mandatory; in fact, EU Directive 96/23/CE [328] has been established to control special substances and their residues, potentially toxic to the consumer, in food of animal origin, setting also the MRLs in Directive 2377/90/EEC [329]. This is a consequence of the misuse of antibiotics not only in humans but also in food-producing animals that leads to the transfer of antibiotic-resistant bacteria to humans. Another class of drugs commonly used in veterinary medicine, whose maximum residue levels have been established by the EU Council Regulation, is quinolones [328, 329]. A particular inline SPE concentration system was used in the development of the CZE-MS2 method for the quantification of danofloxacin, sarafloxacin, ciprofloxacin, marbofloxacin, enrofloxacin, difloxacin, oxolinic acid, and flumequine in chicken muscle samples. A detailed study of kind of sorbent, sample pH, volume, elution plug composition, and design of the system was performed. Moreover, a pressurized liquid extraction (PLE) method was also developed and the resulting combination of inline SPE-CE-MS2 with PLE contributed to the validation of a suitable method for

High-performance capillary electrophoresis for food quality evaluation

351

mAU 8 6 4 2 0

(A)

2.5

5

7.5

10

12.5 min*

mAU IS

8 6 4 2

TIL

TC OTC DOC TYL

0

(B)

2.5

5

7.5

10

12.5 min*

Fig. 14.5 Determination of different drugs (macrolides and tetracycline antibiotics) in feedstuffs by capillary zone electrophoresis: (B) simultaneous separation of tilmicosin—TIL, tetracycline—TC, oxytetracycline—OTC, doxycycline—DOC, and tylosin—TYL, in comparison with blank sample (A) [310].

the simultaneous detection of a high number of quinolones in complex matrices [309]. For fluoroquinolones, see Table 14.10 [138, 140]. Also, macrolide and tetracycline antibiotics could be detected and analyzed by CE [143, 149, 311]. A real application was that reported by Tong (Fig. 14.5) [310] who simultaneously quantified tetracycline, oxytetracycline, doxycycline, tilmicosin, and tylosin in feedstuff by a CE method taking advantage of simplicity, speed, and economic cost. Most of the CE applications reported in the literature in the last decade concern sulfonamides. Nine of these antiobiotics were separated and quantified by CEC-MS; in particular, a series of poly(divinylbenzene-alkyl methacrylate) monolithic stationary phases prepared in situ by polymerization of divinylbenzene and various alkyl methacrylates were developed as separation columns. Better resolution was obtained using the poly(divinylbenzene-octyl methacrylate) monolith, and the crosssectional roughness of the monolithic column end, which was used to couple to the ESI interface, strongly influenced the electrospray stability in CEC-MS. Furthermore, a simple polishing on the end of the monolithic column increased mass signal reproducibility. A sample cleanup procedure for meat samples was performed using SPE cartridges [312].

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A method leading to the separation of sulfamethazine, sulfamethoxazole, sulfaquinoxaline, and sulfanilamide in chicken samples (muscles) within 1 min was set up by Wang [313]. Microchip electrophoresis with LIF detection was used and the plastic microfluidic chips used were cheap and disposable. The analytes were extracted from samples with ACN after a homogenization step and then derivatized with fluorescamine solution in acetone at 80°C for 20 min before an appropriate dilution for analysis. An MEKC-UV method was set up for the simultaneous determination of seven sulfonamides (sulfamethazine, sulfamerazine, sulfathiazole, sulfachloropyridazine, sulfamethoxazole, sulfacarbamide, and sulfaguanidine) and three amphenicol-type antibiotics (chloramphenicol, thiamphenicol, and florfenicol) in 20 commercial muscle, liver, and skin with fat poultry samples. A simple SPE cleanup step was required for the extraction of analytes from tissues. The application of this method resulted in a selective determination of each analyte without interference; therefore it could be successfully adopted for routine screening of foodstuffs instead of LC-MS methods because it achieved a sufficient sensitivity to detect and quantify residues at levels lower than the established EU MLR values [314]. A new feasible online preconcentration step in combination with CZE-UV was developed for the detection of sulfamethoxazole and trimethoprim (an antibacterial agent used to treat bacterial infections commonly used in veterinary medicine in combination with sulfamethoxazole) in dairy products, chicken egg, and honey. Combining micelle to solvent stacking (MSS) with FASS an highly improved sensitivity and reduced LODs by more than 100-fold occur. In fact, by the optimization of MSS, different migration velocities of the analytes either being complexed by the pseudostationary phase or eliminated from the pseudostationary phase could be applied, and by using FASS the velocity of the analytes between the sample matrix and BGS could be optimized [315]. Another interesting application for the quantification of sulfadimidine, sulfadiazine, and sulfathiazole was based on CZE with online CL detection. This method took advantage of the inhibitory effect of the analytes on Ag(III) complex anions used for luminol oxidation, thus reducing the generation of CL signals. Cleanup and enrichment of analytes from pork and chicken meat samples were performed by strong cation exchange SPE columns. The system was reliable, selective, and sensitive and therefore useful for the determination of veterinary drug residuals in animal-derived food [316]. NACE-MS was used for trace analyses of clenbuterol, salbutamol, and terbutaline, β-agonists misused to increase meat production in pork meat. A preconcentration step of the analytes was necessary and an SPE procedure using mixed mode reversed phase/cation exchange cartridges was set for improving LOD values up to 0.3 ppb. A combination of hydrodynamic and electrokinetic injection enhanced the sensitivity of the method. This methodology could be used as a good alternative to HPLC-MS2 [317]. Cation-selective exhaustive injection sweeping micellar electrokinetic chromatography (CSEI-sweep-MEKC), an online stacking capillary electrophoresis method, was developed by Wang [318]. Fractional factorial design and response surface

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methodology were used as tools of the chemometrics experimental design to optimize all parameters. The optimized method was applied for the quantification of ractopamine and dehydroxyractopamine in porcine meat and the results obtained agreed with those obtained by MS techniques or by the use of commercial testing kits. The main advantage of the CSEI-sweep-MEKC method with respect to a CZE method was the higher sensitivity (about 900-fold), enabling nanogram/gram levels in the analysis. For the detection of tricyclic depressants and aminoglycosides [319] and for all detailed applications in the detection of drugs, see Table 14.10.

14.11.5 Heavy metals 14.11.5.1 Cereals Exposure to inorganic arsenic has long been a concern of both public health agencies and scientists. Cereals, and in particular rice and its products, contain different forms of inorganic and organic arsenic compounds, thus accumulating higher concentrations than other crops. In 2014, WHO proposed a draft maximum level of 0.2 mg/kg for inorganic arsenic in polished rice. The toxicity and bioavailability of arsenic highly depends on its chemical form; in fact, inorganic arsenic compounds As(III) and As(V) are considered to be class I human carcinogens, while organic forms, such as dimethylarsinic acid (DMA) and monomethylarsonic acid (MMA), can be considered much less toxic. In 2015, an interesting capillary electrophoresis coupled with inductively coupled plasma mass spectrometry (CZE  ICP-MS) method was developed to quantify the common arsenic species in rice and rice cereal. This method was successfully applied to different commercially available rice samples for the quantification of arsenic species. A sample preparation consisting of an enzyme (i.e., α-amylase)-assisted water-phase microwave extraction was necessary to reduce the sample viscosity, which subsequently increased the injection volume and enhanced the signal response. The method can be considered an excellent alternative to disadvantages of HPLC methods consisting both of column deterioration because of carbohydrates in the sample (leading to poor resolution and precision as the number of injections increases) and of the presence of unknown arsenic species that could interfere with the determination of As(III) [320].

14.11.5.2 Fish An interesting application for the detection and quantification of four arsenic species (As(III), As(V), MMA, and DMA) in Mya arenaria Linnaeus and shrimp samples was reported by Yang [321]. The novelty of this application consisted of the use of an improved sheath-flow interface for coupling CZE with ICP-MS that contributed to transport analyte solution to ICP-MS easily and more efficiently, to avoid laminar flow in the CE capillary caused by suction from ICP-MS, making electric supply more stable in CE. Furthermore, two different quantitative analysis modes were possible:

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continuous sample-introduction mode, working as a normal sheath-flow interface in which CE eluent was continuously transported into ICP-MS suitable for samples containing high concentrations of analytes, and collective sample-introduction mode, in which a pump ensured that one analyte at a time after its complete separation and elution out of the CE capillary was transported to ICP-MS for determination, and stopped working until the second analyte was completely separated and eluted out of the CE capillary. This technique had the advantages of reducing dead volume, avoiding sample dilution, and giving much lower LOD and better electrophoretic resolution. Using the sample environmentally friendly microwave-assisted extraction procedure, it was possible to completely extract organic and inorganic lead from marine animal samples, and using the same instrumentation it was possible to quantify traces of inorganic lead, trimethyl lead chloride, and triethyl lead chloride [322]. Details of CE methods used to detect each contaminant considered in this section in the different food matrices are reported in Table 14.10.

14.12

Foodomics

Foodomics is a relatively new approach to the study of food and nutrients with the application of genomics, proteomics, peptidomics, and metabolomics to investigate food safety, quality, traceability, storage, nutritional value, and bioactivity [15]. Genomic studies are the basic approach for the authentication of species, the identification of botanical origins, and the detection of allergen species [173]. For these studies, DNA-based methods (analysis of DNA length polymorphism) are less complicated compared to proteomics and metabolomics, and several publications are present in the literature by using very fast PCR-CGE-LIF assays, as, for example, for olive oil [173, 174] and tea authenticity [330] or for the analysis of GMOs (yeasts) in wine [331]. PCR is often combined with laboratory-on-a-chip CGE technology; as an example, see a study on spelt flour adulteration with soft wheat [332]. CE-MS represents a very powerful tool, not only for proteomics, peptidomics, and metabolomics [17, 51, 333], but also for genomics. The contribution of these hyphenated techniques in the study of GMO characterization and traceability has become essential [334]. In addition, also for the identification and quantification of intracellular food-borne pathogens and toxins the development of specific, rapid, and sensitive high-throughput foodomics methodologies, among which is the CE-MS approach, represents a new alternative to the most conventional PCRCGE-LIF procedure (see also Section 14.11) [335]. The use of CE-MS in metabolomics is still relatively new but is rapidly increasing, thanks also to technological developments (i.e., new interfaces) and online preconcentration strategies (i.e., SPE coupled to CE-MS) able to improve the sensitivity of CE-MS-based systems [52, 333]. Also, microchip-CE systems have been proposed for metabolite profiling [336]. Global profiling of metabolites is important to determine key compounds and metabolic pathways associated with food quality and stability. In addition, knowledge of metabolite compositions of plants ensures the possibility of controlling environmental and manufacturing conditions and

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modifying plant growth, and by consequence the nutritional value and/or the qualitative aspects (aroma, taste) of products [337]. Concerning GMOs, which can represent a very important health risk, results of the detection of transgenic DNA provided by PCR-CGE and of protein profiling obtained by CZE-UV [338] can be implemented by CE-MS techniques [334]. In fact, profiling technologies provide rapid information with important applications in the labeling and traceability of approved GMOs and in the control of nonauthorized GMOs [334, 336, 339]. Some examples of CE-MS applications in different types of foods are: l

l

l

l

l

l

Metabolite profiling of lettuce leaves [337]; Analysis of protein fraction of transgenic cultivars (soybeans) [339]; Analysis of metabolite profiling of transgenic cultivars (maize, soybean) [336]; Metabolite profiling of meat [340]; Metabolite profiling of wine and juice samples [341]; Metabolite profiling of Japanese sake [342].

14.13

Summary and outlook

This chapter offered a comprehensive overview of both principles and applications of HPCE techniques. CE represents a powerful analytical tool that is widely applied in food quality and safety, and is also a novel interesting approach in foodomics. Starting with a brief introduction on its evolution from gel electrophoresis to microchip-CE devices, basic principles, detailed general procedures, and detection systems were described. Different CE separation modes were also summarized to show the wide range of applications of this technique in food analysis. Advantages and limitations of each CE mode and new technical improvements were also reported. After the general section, recent application progress in different types of foods were considered. In the first part, applications were divided into solid and liquid foods (vegetable and animal origin) and other foods (honey, food supplements, and baby foods). In the second part, the analysis of certain food additives and contaminants was discussed. The last section of this chapter introduced foodomics applications. The chapter provided the most up-to-date information of the last decade in food quality and safety evaluation by CE.

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