**3.1. Matrix-analyte**

**2. Honeybee diseases**

**Compounds Extraction/**

, 5 MACs, 6 SAs, 8 TCs (22)

dTRM, trimethoprim.

LIN, lincomycin.

3 AGs, LIN<sup>e</sup>

338 Honey Analysis

a

b

c

e

f

**clean-up**

Na<sup>4</sup>

Four subsequent LLE steps were carried out.

Water, 2 M, HCl in MeOH,

EDTA to pH 2.0/PSA (d-SPE)

NFPA, nonafluoropentanoic acid (ion-pairing reagent).

HFBA, heptafluorobutyric acid (ion-pairing reagent).

**Table 11.** Multiclass confirmatory methods.

foulbrood, European foulbrood and nosemosis [3].

prevent the rapid diffusion within a colony.

have been demonstrated to be an effective treatment.

Honeybees are affected by fungal, bacterial, viral (Thai Sac brood) and acarine (*Varroa*) diseases. Antibiotics are generally used to fight bacterial and fungal diseases such as American

**Separation Equipment CCβ or LOD** 

LC-MS/MS (ESI+)

**Column Mobile phase**

Gradient: 100 mM HFBA<sup>f</sup> /

water/ACN

CCβs for permitted antibiotics (lincomycin, MACs, QNs, SAs, TCs) were provided considering a hypothetical MRL of

Zorbax SB-C18 (100 × 2.1 mm, 3.5 μm)

100 or 200 μg/kg. For banned substances (NMZs), CCβs were in the range 1.2–2.6 μg/kg.

**(µg/kg)**

7–33 [98]

**References**

American foulbrood is by far the most virulent brood disease known in honeybees. The disease is caused by the spore-forming bacterium, *Paenibacillus larvae*. Larvae up to 3 days old become infected by ingesting spores that are present in their food. Spores germinate in the gut of the larva and the vegetative form of the bacteria begins to grow, taking its nourishment from the larva. Infected larvae normally die after their cell is sealed. The vegetative form of the bacterium, before to die, produces many millions of spores which are extremely resistant to desiccation and can remain viable for more than 40 years in honey and beekeeping equipment. Because of this persistence, in most countries official apiary inspectors are required to burn all infected colonies. Other countries (e.g. USA, Canada, and Argentina) allow the use of antibiotics, such as oxytetracycline and tylosin, to keep the disease in control. However, antibiotics are not a cure or a treatment of the infection since they affect only the vegetative stage of American foulbrood, inhibiting its development in the gut of the larvae. This may

European foulbrood is closely related to American foulbrood in symptomatology, and until 1906, these two diseases were not differentiated. The causative organism of European foulbrood is the bacterium *Melissococcus plutonius*, which does not produce spores, and therefore, this disease is considered less severe than American foulbrood. European foulbrood occurs primarily in spring when numbers of *M. plutonius* reach their peak. The bacterium is ingested by honey bee larvae and it replicates in mid-gut. If the bacteria out-compete the larva, the larva will die before the cell is capped. Alternatively, the bee may survive until adulthood if the larvae has sufficient food resources. Some antimicrobials, for example, oxytetracycline, Sample treatment is fundamental in the residue analysis of food, since the achievement of low detection limits (some parts per billions) and suitable selectivity involves extensive purification of generally complex food matrices. The sample preparation process consists of the extraction followed by one or more purification steps. Rarely, the purification step is omitted. To decide the sample treatment strategy, main aspects have to be considered: the characteristics of both sample matrix and he physico-chemical properties of analyte(s) have to be taken into account, together with, in addition, the already developed procedures (literature searching).

Because of the hydrophilic nature of honey, frequently, the extraction coincides with the sample dissolution in pure water or in acidified aqueous solutions or in buffers. After that, besides the traditional liquid-liquid extraction (LLE) and solid-phase extraction (SPE) purifications, more recent clean-up methodologies have been applied such as quick, easy, cheap, effective, rugged and safe (QuEChERS), molecularly imprinted polymers (MIPs) and multiwalled carbon nanotubes (MWCNs). These two latter are particular kinds of SPE, whereas QuEChERS methodology is a variation of LLE, followed by a dispersive solid-phase extraction (d-SPE) step. It is important to keep in mind that, despite the proliferation of dozens of new purification approaches with various acronyms, essentially all these fall into LLE or SPE techniques. Some additional examples are microextraction by packed sorbent (MEPS), stir bar sorption extraction (SBSE), dispersive liquid-liquid microextraction (DLLME) and phase separation-based magnetic-stirring salt-induced liquid-liquid microextraction (PS-MSLM). These recent methodologies give also evidence of the current trend towards "micro", that is, towards a lower consumption of reagents and materials during the sample treatment. Less common and expensive purification systems such as turbo-flow chromatography are not here considered.

Dissociation constants (pK<sup>a</sup> s) and lipophilicity are key parameters to understand the behaviour of drugs, and therefore, to perform appropriate extraction and purification strategies, physico-chemical properties of a drug molecule are described by its pK<sup>a</sup> (s) and its polarity pK<sup>a</sup> (dissociation constant) is a measure of the strength of an acid or a base. It determines the charge on a molecule at any given pH. The lipophilicity polarity is measured by the partition coefficient, P, or better, by the distribution coefficient, D, which are the key parameters to understand the behaviour of molecules, and therefore, to design appropriate purification strategies during the method development, P is the ratio of the concentration of a compound in octanol to its concentration in water P (Eq. (1)):

$$\mathbf{P} = \frac{\left[\text{drug}\right]\_{\text{actual}}}{\left[\text{drug}\right]\_{\text{water}}} \tag{1}$$

P is generally expressed as logarithm of the log P. Log P is a constant for the molecules under its neutral form, and its value is a measure of lipophilicity or hydrophobicity. On the other hand, the distribution coefficient (D, or better, its logarithm, log D) takes into account all neutral and charged forms of the molecule. Therefore, for ionizable solutes, such as drugs, the pH-dependant lipophilicity descriptor, that is, the distribution coefficient (D), is more appropriate. D is the ratio of the sum of the concentrations of all forms of the compound (ionized plus un-ionized) in each of the two phases, octanol and water, (Eq. (2)): <sup>D</sup><sup>=</sup> [ drug  molecule] octano<sup>l</sup> \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ [ drug  molecule] wate<sup>r</sup> <sup>+</sup> [ drug  ion] wate<sup>r</sup>

$$\mathbf{D} = \underbrace{\left[\text{drug molecule}\right]\_{\text{actual}}}\_{\left[\text{drug molecule}\right]\_{\text{water}} + \left[\text{drug ion}\right]\_{\text{water}}} \tag{2}$$

Roughly, when log D < 0, the molecule is polar (hydrophilic) and vice versa. Because the charged forms hardly enter the octanol phase, this distribution varies with pH. In the pH region where the molecule is mostly unionized, log D = log P. Acids are neutral when protonated and negatively charged (ionized) when deprotonated. Bases are neutral when deprotonated and positively charged (ionized) when protonated. Therefore, the log D of a compound is strongly influenced by its acid-base dissociation constant(s), pK<sup>a</sup> . However, log D values cannot furnish precise information about the ionization status of the compound mainly because frequently more than one acidic or basic centre can be present in its structure. Only the knowledge of the pK<sup>a</sup> s allows the understanding of the predominant forms at the various pH values. In **Figures 1**–**3**, the plots of log D versus pH of one representative compound per class are shown. These plots were obtained applying the MoKa® package (Molecular Discovery Ltd.) [13]. This software package is able to predict also the pK<sup>a</sup> s. Ranges of pHs increasing log D (lipophilicity) can favour RP-SPE and LLE purification strategies, which are based on the analyte transfer from a more polar medium (honey solution) to a less polar one. On the other hand, selective purifications such as ion-exchange SPE are enabled when the analytes are in their ionized form and, therefore, in pH intervals where log D values are lower (higher hydrophilicity).

#### **3.2. Purification**

Liquid-liquid extraction (LLE) is one of the first sample preparation approaches and continues to be widely used. LLE is based on the transfer of an analyte from the aqueous sample to a water-immiscible solvent based on its distribution coefficient, D. The water-immiscible solvents can be ethyl acetate, dichloromethane and chloroform. Nevertheless, some shortcomings, such as emulsion formation, the use of relatively large sample volumes and toxic organic solvents, make the traditional LLE (relatively) expensive and environmentally harmful. To avoid emulsion formation, supported liquid extraction (SLE) can be applied.

**Figure 1.** Log D versus pH for chloramphenicol (CAP), fumagillin, lincomycin and tylosin A (MAC).

charge on a molecule at any given pH. The lipophilicity polarity is measured by the partition coefficient, P, or better, by the distribution coefficient, D, which are the key parameters to understand the behaviour of molecules, and therefore, to design appropriate purification strategies during the method development, P is the ratio of the concentration of a compound

[ drug ] wate<sup>r</sup>

P is generally expressed as logarithm of the log P. Log P is a constant for the molecules under its neutral form, and its value is a measure of lipophilicity or hydrophobicity. On the other hand, the distribution coefficient (D, or better, its logarithm, log D) takes into account all neutral and charged forms of the molecule. Therefore, for ionizable solutes, such as drugs, the pH-dependant lipophilicity descriptor, that is, the distribution coefficient (D), is more appropriate. D is the ratio of the sum of the concentrations of all forms of the compound (ionized

Roughly, when log D < 0, the molecule is polar (hydrophilic) and vice versa. Because the charged forms hardly enter the octanol phase, this distribution varies with pH. In the pH region where the molecule is mostly unionized, log D = log P. Acids are neutral when protonated and negatively charged (ionized) when deprotonated. Bases are neutral when deprotonated and positively charged (ionized) when protonated. Therefore, the log D of a compound is strongly

precise information about the ionization status of the compound mainly because frequently more than one acidic or basic centre can be present in its structure. Only the knowledge of the

can favour RP-SPE and LLE purification strategies, which are based on the analyte transfer from a more polar medium (honey solution) to a less polar one. On the other hand, selective purifications such as ion-exchange SPE are enabled when the analytes are in their ionized form

Liquid-liquid extraction (LLE) is one of the first sample preparation approaches and continues to be widely used. LLE is based on the transfer of an analyte from the aqueous sample to a water-immiscible solvent based on its distribution coefficient, D. The water-immiscible solvents can be ethyl acetate, dichloromethane and chloroform. Nevertheless, some shortcomings, such as emulsion formation, the use of relatively large sample volumes and toxic organic solvents, make the traditional LLE (relatively) expensive and environmentally harmful. To avoid emulsion formation, supported liquid extraction (SLE) can be applied.

and, therefore, in pH intervals where log D values are lower (higher hydrophilicity).

s allows the understanding of the predominant forms at the various pH values. In **Figures 1**–**3**, the plots of log D versus pH of one representative compound per class are shown. These plots were obtained applying the MoKa® package (Molecular Discovery Ltd.) [13]. This soft-

(1)

(2)

. However, log D values cannot furnish

s. Ranges of pHs increasing log D (lipophilicity)

in octanol to its concentration in water P (Eq. (1)):

<sup>P</sup><sup>=</sup> [ drug ] \_\_\_\_\_\_\_\_\_ octano<sup>l</sup>

influenced by its acid-base dissociation constant(s), pK<sup>a</sup>

ware package is able to predict also the pK<sup>a</sup>

pK<sup>a</sup>

340 Honey Analysis

**3.2. Purification**

plus un-ionized) in each of the two phases, octanol and water, (Eq. (2)): <sup>D</sup><sup>=</sup> [ drug  molecule] octano<sup>l</sup> \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ [ drug  molecule] wate<sup>r</sup> <sup>+</sup> [ drug  ion] wate<sup>r</sup>

**Figure 2.** Log D versus pH for AOZ, derivatized AOZ (NBAOZ), metronidazole (NMZ) and enrofloxacin (QN).

**Figure 3.** Log D versus pH for flumequine (QN), sulfathiazole (SA), streptomycin (STR) and tetracycline (TC).

Its principle is simple: a chemically inert, high surface area support, highly purified, graded diatomaceous earth (Extrelut®, Hydromatrix®, Celite®, etc.) serves as a stationary vehicle for the aqueous phase of the liquid-liquid extraction experiment. The aqueous-based sample (e.g. diluted honey) is added to the dry sorbent and allowed to wet the diatomaceous earth. A small volume of immiscible organic extraction solvent is then added and allowed to percolate by gravity through the supported aqueous phase. Because the aqueous sample has been widely dispersed throughout the solid support, the organic solvent has intimate contact with the thin film of aqueous phase and rapid extraction (equilibration) occurs.

Even today, probably, solid-phase extraction (SPE) is the most used sample purification tool in trace analysis. This technique was developed in the mid-1970s as an alternative to LLE. The degree of selectivity of SPE technique can be very different, depending on the attractive forces between the analytes and the functional groups on the sorbent surface. SPE sorbents are most commonly categorized by the nature of their primary interaction or retention mechanism with the analyte(s) of interest. The sorbent can interact with analytes by hydrophobic (non-polar/ non-polar), hydrophilic (polar-polar, hydrogen bonding, dipole-dipole, π-π interactions) and cationic-anionic interactions. The most common SPE sorbents packing can be classified into non-polar phases (reversed phases—RP), polar phases (normal phases—NP), ion-exchange and immunoaffinity adsorbents.

Non-polar sorbents are used under RP chromatography conditions and are suitable for the extraction of hydrophobic or polar organic analytes from aqueous matrices. Accordingly, reversed phase is the most used SPE sorbent type to purify honey, which is a water-soluble matrix. These sorbents comprise alkyl silica and polymer-based materials. Alkyl silica sorbents are manufactured by bonding alkyl or aryl functional groups, such as octyl (C8), octadecyl (C18) and phenyl (Ph) to the silica surface. It should be noted that in SPE, the interactions described above are not found in pure form, but in combination. For example, C18 silica-based sorbents are non-polar sorbent, but it still possess free silanol groups, which can produce hydrophilic secondary interactions. The retention of analytes under RP conditions is due primarily to the van der Waals attractive forces between the carbon-hydrogen bonds in the analytes and the functional groups on the silica surface. The elution of adsorbed compounds is generally made by using a non-polar solvent (compared to water) to disrupt the forces that bind the compound to the sorbent. However, silica-based bond phases contain non-uncapped silanols, which can cause the strongly binding of some group of compounds (i.e. tetracyclines), and in addition, they can be used only in a limited pH range (2–8). Currently, silica materials have been more and more replaced by polymeric sorbents. The macroporous wettable hydrophilic-lipophilic balance (HLB) polymeric sorbent (divinylbenzene-N-vinylpyrrolidone) was at first introduced by Waters Company (Oasis HLB). Later, other manufacturers commercialized similar reversed-phase proprietary polymeric sorbents such as Strata-X (surface-modified styrene-divinylbenzene; Phenomenex), LiChrolut EN (highly cross-linked polystyrene-divinylbenzene; Merck, Darmstadt, Germany) and Evolute ABN (cross-linked polystyrene-divinylbenzene functionalized with oligomeric hydroxyl groups; Biotage). These cartridges have been widely applied in honey purification of almost all antibiotic classes.

The intrinsic honey characteristics undoubtedly favour the wide application of RP-SPE purification approaches since NP-SPE is more suitable to isolate a polar analyte in a mid- to nonpolar matrix (acetone, chlorinated solvents, hexane, etc). The most common polar stationary phases are silica, alumina and florisil. Retention of an analyte under NP conditions is primarily due to interactions between polar functional groups of the analyte and polar groups on the sorbent surface (hydrogen bonding and π-π interactions, among others). The passing of a solvent that disrupts the binding mechanism, usually a solvent that is more polar than the sample matrix, allows the elution of the adsorbed compounds. To the best of our knowledge, examples of NP-SPE purification applied to determination of veterinary drug residues in honey are limited to nitroimidazole family (**Table 6**). This is probably why nitroimidazoles are very polar compounds. The application of this kind of sorbents generally involves a preliminary liquidliquid extraction step to transfer the analytes from the aqueous phase (solubilized honey) to an organic phase (non-polar matrix) which is then loaded onto the cartridge.

Its principle is simple: a chemically inert, high surface area support, highly purified, graded diatomaceous earth (Extrelut®, Hydromatrix®, Celite®, etc.) serves as a stationary vehicle for the aqueous phase of the liquid-liquid extraction experiment. The aqueous-based sample (e.g. diluted honey) is added to the dry sorbent and allowed to wet the diatomaceous earth. A small volume of immiscible organic extraction solvent is then added and allowed to percolate by gravity through the supported aqueous phase. Because the aqueous sample has been widely dispersed throughout the solid support, the organic solvent has intimate contact with the thin film of aqueous phase and rapid extraction (equilibration) occurs.

**Figure 3.** Log D versus pH for flumequine (QN), sulfathiazole (SA), streptomycin (STR) and tetracycline (TC).

Even today, probably, solid-phase extraction (SPE) is the most used sample purification tool in trace analysis. This technique was developed in the mid-1970s as an alternative to LLE. The degree of selectivity of SPE technique can be very different, depending on the attractive forces between the analytes and the functional groups on the sorbent surface. SPE sorbents are most commonly categorized by the nature of their primary interaction or retention mechanism with the analyte(s) of interest. The sorbent can interact with analytes by hydrophobic (non-polar/ non-polar), hydrophilic (polar-polar, hydrogen bonding, dipole-dipole, π-π interactions) and cationic-anionic interactions. The most common SPE sorbents packing can be classified into non-polar phases (reversed phases—RP), polar phases (normal phases—NP), ion-exchange

and immunoaffinity adsorbents.

342 Honey Analysis

Due to their selectivity, ion-exchange SPE sorbents can be generally used only in single-residue or single-class procedures. These sorbents are very efficient for extraction of charged analytes, such as acidic and basic compounds, from aqueous or non-polar organic samples. Ion-exchange phases are comprised of positively (aliphatic quaternary amine, aminopropyl) or negatively (aliphatic sulphonic acid, aliphatic carboxylic acid) charged groups. Porous polymer, ion-exchange resins have a higher exchange capacity and a wider pH operating range than silica-based materials. Ion-exchange sorbents are usually classified as weak or strong, depending on the identity of the ionic group and whether its charge is independent of the sample pH (strong ion exchanger) or can be manipulated by changing pH (weak ion exchanger). Antibiotic substances have frequently basic functional groups, and therefore, the application of both strong cation exchange and weak cation exchange has been reported also in honey, mainly for the determination of streptomycin/dihydrostreptomycin (**Table 8**) and sulphonamides (**Table 9**). Finally, the immunoaffinity chromatography is a SPE technique, based on very selective antigen-antibody interactions (immunosorbents); examples of its application to purify honey have been reported, too.

In some cases, both liquid-liquid extraction and solid-phase extraction can be used in an "opposite manner", that is, solubilizing or retaining the interfering substances rather than the analytes. An important example in antibiotic analysis is the so-called defatting to purify food extracts in water-miscible solvents: the added water-immiscible solvent (generally hexane) does not solubilize the analytes of interest, but the highly lipophilic interfering substances (fats), and therefore, it is discarded. Analogously, in the "non-retentive" SPE the sorbent has no affinity for the analytes, but for the sample contaminants. The solid phase is simply used to "filter" the sample: analyte passes through the column without being retained, while (part of) the contaminants are retained. This kind of extraction is generally applied when the analyte is highly soluble in the sample matrix (or in the dilution solvent), and therefore, it cannot be partitioned out onto a solid sorbent (retentive SPE) or an immiscible solvent (LLE).

Among the relatively modern purification approaches, it may be worthwhile to describe the QuEChERS, molecularly imprinted polymers (MIPs) and multi-walled carbon nanotubes (MWCNs) methods. The QuEChERS approach has become particularly popular for the multiresidue analysis of pesticides in various food matrices, and it generally consists of two steps: first, the homogenized sample is extracted and partitioned using an acetonitrile and salt solution (MgSO<sup>4</sup> and NaCl), and then, an aliquot of the supernatant is cleaned using a dispersive solid-phase extraction (d-SPE) technique. Dispersive SPE is a "non-retentive" SPE, because the matrix co-extractives are adsorbed onto the sorbent, while leaving analytes of interest in the solvent. In some applications of QuEChERS, the second step (d-SPE purification) can be omitted. MIP sorbents are highly cross-linked polymers with a predetermined selectivity towards a single analyte or group of structurally related analytes. This selectivity is obtained during the synthesis of the polymer by using a template molecule to form cavities with specific shape. The process usually involves initiating the polymerization of monomers in the presence of the template molecule that is extracted afterwards, thus leaving complementary cavities behind. Due to the high selectivity of these sorbents, they generally allow for lower detection limits. In recent years, multi-walled carbon nanotubes, a new kind of carbon material, have attracted much interest that is directed towards the development of solid-phase extraction adsorbents. The MWCNs were promising sorbents because of the larger specific area and the dramatic hydrophobic characteristic of the surface. The adsorption mechanisms involve weak interactions (mainly π-π stacking, van der Waals and electrostatic forces), facilitating the adsorption of analytes in a selective and reproducible manner.

To conclude, the current trends in food sample preparation involve the following issues: the miniaturization of the equipment for sample preparation (micro techniques); the decrease in the amount of sample to be analysed; the reduction in the use of organic solvents; the development of multiclass procedures; and the development of automated methods for the preconcentration. All these strategies aim at the reduction in the employed reagents/materials and at the increase in the analysis throughput.

strong, depending on the identity of the ionic group and whether its charge is independent of the sample pH (strong ion exchanger) or can be manipulated by changing pH (weak ion exchanger). Antibiotic substances have frequently basic functional groups, and therefore, the application of both strong cation exchange and weak cation exchange has been reported also in honey, mainly for the determination of streptomycin/dihydrostreptomycin (**Table 8**) and sulphonamides (**Table 9**). Finally, the immunoaffinity chromatography is a SPE technique, based on very selective antigen-antibody interactions (immunosorbents); examples of its

In some cases, both liquid-liquid extraction and solid-phase extraction can be used in an "opposite manner", that is, solubilizing or retaining the interfering substances rather than the analytes. An important example in antibiotic analysis is the so-called defatting to purify food extracts in water-miscible solvents: the added water-immiscible solvent (generally hexane) does not solubilize the analytes of interest, but the highly lipophilic interfering substances (fats), and therefore, it is discarded. Analogously, in the "non-retentive" SPE the sorbent has no affinity for the analytes, but for the sample contaminants. The solid phase is simply used to "filter" the sample: analyte passes through the column without being retained, while (part of) the contaminants are retained. This kind of extraction is generally applied when the analyte is highly soluble in the sample matrix (or in the dilution solvent), and therefore, it cannot be

partitioned out onto a solid sorbent (retentive SPE) or an immiscible solvent (LLE).

tating the adsorption of analytes in a selective and reproducible manner.

Among the relatively modern purification approaches, it may be worthwhile to describe the QuEChERS, molecularly imprinted polymers (MIPs) and multi-walled carbon nanotubes (MWCNs) methods. The QuEChERS approach has become particularly popular for the multiresidue analysis of pesticides in various food matrices, and it generally consists of two steps: first, the homogenized sample is extracted and partitioned using an acetonitrile and salt solu-

solid-phase extraction (d-SPE) technique. Dispersive SPE is a "non-retentive" SPE, because the matrix co-extractives are adsorbed onto the sorbent, while leaving analytes of interest in the solvent. In some applications of QuEChERS, the second step (d-SPE purification) can be omitted. MIP sorbents are highly cross-linked polymers with a predetermined selectivity towards a single analyte or group of structurally related analytes. This selectivity is obtained during the synthesis of the polymer by using a template molecule to form cavities with specific shape. The process usually involves initiating the polymerization of monomers in the presence of the template molecule that is extracted afterwards, thus leaving complementary cavities behind. Due to the high selectivity of these sorbents, they generally allow for lower detection limits. In recent years, multi-walled carbon nanotubes, a new kind of carbon material, have attracted much interest that is directed towards the development of solid-phase extraction adsorbents. The MWCNs were promising sorbents because of the larger specific area and the dramatic hydrophobic characteristic of the surface. The adsorption mechanisms involve weak interactions (mainly π-π stacking, van der Waals and electrostatic forces), facili-

To conclude, the current trends in food sample preparation involve the following issues: the miniaturization of the equipment for sample preparation (micro techniques); the decrease in the amount of sample to be analysed; the reduction in the use of organic solvents; the

and NaCl), and then, an aliquot of the supernatant is cleaned using a dispersive

application to purify honey have been reported, too.

tion (MgSO<sup>4</sup>

344 Honey Analysis
