**5.1. Chloramphenicol (CAP)**

frequency-only quadrupole, fragments the analyte through interaction with a collision gas. The most used acquisition mode is multiple reaction monitoring (MRM). Increased selectivity, improved signal-to-noise ratio (S/N), lower limits of quantitation, wider linear range and improved accuracy are some of the benefits of this technique. LC-MS/MS instrumentation tends to give better quantitative precision and improved sensitivity than alternative configurations, making it a superior choice for routine analysis of specific targeted contaminants.

346 Honey Analysis

An alternative to LC-MS/MS system is the coupling of liquid chromatography with high-resolution mass spectrometry (LC-HRMS). At the beginning, these analysers were mainly used for research purposes, but after 2007 they started to be applied in routine analysis, too. With HRMS analysers, full-scan spectra are continuously obtained throughout the analytical run allowing for exhaustive qualitative post-acquisition analysis. There are two technologies of high-resolution mass spectrometry: time-of-flight (TOF) and orbitrap. However, single-stage high-resolution mass spectrometry demonstrated to not be suitable for the confirmation of residues at very low concentrations in highly complex matrices such as honey. More recently, hybrid platforms have been available at the bench level such as Q-TOF and Q-Orbitrap combining a quadrupole with an accurate mass analyser. These configurations provide exceptional selectivity and sensitivity over single-stage equipment, and they are increasingly applied in residues analysis of food.

With regard to the chromatographic separation, although the coupling between gas chromatography and mass spectrometry (GC-MS) has been realized before LC-MS, gas chromatography is rarely used for the determination of antibiotics, due to their polar nature, low volatility and thermal instability. Therefore, high-performance liquid chromatography (HPLC) is the technique of choice for antibiotic analysis. Since its introduction in 1970s, HPLC progressively improved mainly thanks to the evolution of packing materials used to carry out the separation. Columns packed with 10 and 5 μm fully porous particles dominated the field for nearly thirty years (1975– 2000). In 2004, a great advance in instrumentation and column technology was made achieving very significant increases in resolution, speed and sensitivity. Columns with smaller particles (sub 2-μm) and instrumentation able to deliver mobile phase at 15,000 psi (1000 bar) allowed the achievement of a new level of performance. This new step of HPLC is known as UHPLC technology. In 2007, LC columns with core-shell (superficially porous) particles were introduced. This new generation of microspheres provides the same high efficiency of sub 2-μm UHPLC totally porous particles, but with lower backpressures. The first commercially available coreshell sorbent was the Halo® from Advanced Material Technologies. Currently, the most applied core-shell columns are Kinetex® (Phenomenex), Poroshell® (Agilent), Accucore® (Thermo Fisher Scientific), Ascentis Express® (Supelco), Cortecs® (Waters) and Nucleoshell® (Macherey Nagel).

Many of these have been used to determine residues in honey (see **Tables 3**–**11**).

**5. Overview of methods for the determination of drug residues in honey**

In the following paragraphs for each compound or class of compounds, an overview of the published confirmatory methods for the determination of residues in honey is given in **Tables 3**–**11**. Although widely applied in routine laboratories as screening methods, procedures based on bioanalytical techniques such as immunoenzymatic or receptor tests are not considered.

Chloramphenicol is a potent, broad-spectrum antibiotic and a potential carcinogen and has been banned in the European Union since 1994 for use in food-producing animals. The United States and Canada, as well as many other countries, have completely banned its usage in the production of food, too. In January 2002, concerns regarding serious deficiencies of the Chinese residue control system and problems related to the use of banned substances in food-producing animals led the European Union to issue a suspension of imports of all products of animal origin from this country. Meanwhile, a growing number of rapid alert notifications related to the presence of CAP in imported honey have been issued. In beekeeping practice, this antibiotic is mainly used to fight the American foulbrood disease. In 2002, 31 cases out of 34 positive CAP honey detected by the RASFF system (**Table 2**) were from China. These findings were confirmed by Verzegnassi et al. [17] who in the same period analysed 176 raw honeys of various geographical origins, showing very extensive contamination in those of Chinese origin (29 positive samples out of 32). One year later (2003), the percentage of positive chloramphenicol honey from China fell down with only one notification. The import ban was lifted in July 2004.

In **Figure 4**, the sample preparation protocols proposed by the authors of the nine selected analytical methods listed in **Table 3** are summarized [17–25]. Using the CAF as "case study", the figure generalizes the sample purification concept, which is a modular process composed of one or more LLE and SPE steps. Generally, honey is dissolved in water or in acidic solutions due to better solubility of CAF in organic solvents at these pHs (**Figure 1**), thus enabling subsequent RP-SPE or LLE purification. Only Alechaga et al. [23] solubilize honey in an aqueous basic solution (1% NH3 ), to favour the adsorption on the stationary phase (Oasis HLB) of florfenicol amine which was included in the same procedure. As explained by its name, florfenicol amine (the main metabolite of florfenicol) is a basic drug non-ionized at pHs exceeding 9. The solubilized honey is then purified with one or two clean-up steps: (a) SPE [20, 21, 23];

**Figure 4.** Sample treatment strategies for the determination of **chloramphenicol** residues in honey (**Table 3**): (a) [20, 21, 23]; (b) [18, 19, 22, 24]; (c) [17]; (d) [25].

(b) LLE [18, 19, 22, 24]; (c) SPE and LLE [17]; (d) LLE and SPE [25]. The same scheme could be realized for all the other antibiotic methods summarized in **Tables 4**–**11**. A complete overview of the sample preparation issues is available in "Analysis of Antibiotic Residues in Food" [26].

#### **5.2. Fumagillin**

Fumagillin is a potent amoebicidal agent with properties known since 1950s. This compound is used by apiarists to protect bees from *Nosema apis*. A few articles have reported methods for its determination. The first procedure using LC-MS technique (single quadrupole) has been developed by Nozal et al. in 2008 [27]. In 2011 and in 2015, respectively, Kanda et al. [28] and van den Heever et al. [29] published methods based on LC-MS/MS (triple quadrupole). Nozal et al. [27] and van den Heever et al. [29] applied a quite similar purification approach, solubilizing honey in water and purifying it with polymeric RP-SPE cartridge. They also reached similar LODs ranging from 1 to 4 μg/kg, depending on the honey type (botanical origin). Surprisingly, Kanda et al. [28] reported LODs of two orders of magnitude lower (0.02–0.03 μg/kg), applying QuEChERS extraction with 0.1% FA in acetonitrile followed by non-retentive WAX-SPE. These authors estimate LOD by means of the standard deviation (SD) observed in replicate experiments carried out at a low spiking level, that is, 1 μg/kg (LOD = 3 × SD). However, following the analytical chemistry detection theory, to obtain a reliable estimation of LOD, the spiking level should be close to the found LOD. Clearly, the spiking level reported by Kanda et al. [16] is not suitable, being two orders of magnitude higher than the estimated LOD. This example demonstrates the well-known issues in the estimation of method limits, which can prevent correct comparison among method performances. On the other hand, most of the authors do not report how the LODs are obtained, simply declaring that they are calculated according to signalto-noise (S/N) ratio approach (LOD = 3 × S/N). Finally, it is worthy of note that among the multiclass procedures, only Lopez et al. [30] have included fumagillin within the determined analytes (**Table 11**).

#### **5.3. Macrolides (MACs) and lincomycin**

As a result of the development of resistance to oxytetracycline, in the last 15 years two macrolide antibiotics, erythromycin and tylosin, have been widely used for the prevention and treatment of apiculture diseases. Since 1970s, some studies report that tylosin was superior to sulphathiazole in the control of American foulbrood in field colonies of honeybees. In 2005 and in 2013, the US Food and Drug Administration (FDA) and Canada authorities, respectively, approved the use of tylosin in honeybees. In addition, Canada authorities fixed an MRL in honey equal to 200 μg/kg as sum of tylosin A and B (**Table 1**). The most significantly published procedures are summarized in **Table 4** [31–37]. Lincomycin belongs to the group of lincosamides, and its activity against *Paenibacillus larvae* strains has been reported. In 2012, lincomycin was approved by FDA to control tetracycline-resistant American foulbrood disease. Its structure is similar to that of macrolides, and some analytical methods determine simultaneously these substances [31, 33]. Because macrolides are unstable in acidic solution, that is, pH <4, sample extraction is generally carried out in water or in basic buffers (pH 8.0–10.5). Due to their basic nature, at these pHs the reversed-phase solid-phase extraction approach is favoured (**Figure 1**), and all procedures listed in **Table 4** purify the honey extract using silica C18 or polymeric cartridge (Oasis HLB and Strata-X).

## **5.4. Nitrofurans (NFs)**

(b) LLE [18, 19, 22, 24]; (c) SPE and LLE [17]; (d) LLE and SPE [25]. The same scheme could be realized for all the other antibiotic methods summarized in **Tables 4**–**11**. A complete overview of the sample preparation issues is available in "Analysis of Antibiotic Residues in Food" [26].

**Figure 4.** Sample treatment strategies for the determination of **chloramphenicol** residues in honey (**Table 3**): (a) [20, 21,

Fumagillin is a potent amoebicidal agent with properties known since 1950s. This compound is used by apiarists to protect bees from *Nosema apis*. A few articles have reported methods for its determination. The first procedure using LC-MS technique (single quadrupole) has been developed by Nozal et al. in 2008 [27]. In 2011 and in 2015, respectively, Kanda et al. [28] and van den Heever et al. [29] published methods based on LC-MS/MS (triple quadrupole). Nozal et al. [27] and van den Heever et al. [29] applied a quite similar purification approach, solubilizing honey in water and purifying it with polymeric RP-SPE cartridge. They also reached similar LODs ranging from 1 to 4 μg/kg, depending on the honey type (botanical origin). Surprisingly, Kanda et al. [28] reported LODs of two orders of magnitude lower (0.02–0.03 μg/kg), applying QuEChERS extraction with 0.1% FA in acetonitrile followed by non-retentive WAX-SPE. These authors estimate LOD by means of the standard deviation (SD) observed in replicate experiments carried out at a low spiking level, that is, 1 μg/kg (LOD = 3 × SD). However, following the analytical chemistry detection theory, to obtain a reliable estimation of LOD, the spiking level should be close to the found LOD. Clearly, the spiking level reported by Kanda et al. [16] is not suitable, being two orders of magnitude higher than the estimated LOD. This example demonstrates the well-known issues in the estimation of method limits, which can prevent correct compari-

**5.2. Fumagillin**

348 Honey Analysis

23]; (b) [18, 19, 22, 24]; (c) [17]; (d) [25].

Nitrofurans have been used for long time in veterinary practice as antibacterial agents for treating infections caused by bacteria and protozoa. At present in Europe and other several countries, these substances are explicitly prohibited or not authorized for all food-producing animals because of their potentially carcinogenic and mutagenic effects on human health. Several studies have showed that animals rapidly metabolize nitrofurans and the in vivo stability of parent drugs is no longer than a few hours. Consequently, the detection of parent drugs in animal tissues is impractical [38]. The covalent binding of NFs with protein tissues has been proven applying the 14C technique to furazolidone drug. After this observation, analytical methods able to liberate the covalently bound drugs were developed. An acidic hydrolysis followed by a derivatization step with 2-nitro-benzaldehyde (NBA) and subsequent neutralization demonstrated to be the more suitable procedure for NF residue determination. The acid hydrolysis does not release the intact drug, but a structural unit of the parent molecule. 3-Amino-2-oxazolidinone (AOZ), 5-methyl-morpholino-3-amino-2-oxazolidinone (AMOZ), semicarbazide (SEM) and 1-aminohydantoin (AHD) are the released metabolites of furazolidone, furaltadone, nitrofurazone and nitrofurantoin, respectively. It must be underlined that the derivatization with NBA of the cleaved drug metabolites is essential, since AOZ, AMOZ, SEM and AHD are very polar compounds scarcely retained on RP columns and with poor ionization properties in the electrospray interface of MS analysers. It was thanks to the application of the hydrolysis and derivatization procedure together with the use of LC-MS/MS technique that, in the early 2000s, a large number of contaminated food samples were discovered (**Table 2**). Currently, all the methods are based on this treatment. The analysis of commercialized honey samples demonstrated that furazolidone (AOZ) is the main nitrofuran antibiotic used in apiculture [12]. Inevitably, all methods in **Table 5** apply the LC-MS techniques [39–45]. The first procedure for the determination of metabolites in honey was published by Khong et al. in 2004, using isotopic dilution [39]. Most of the procedures perform the honey solubilization directly in the derivatization mixture (usually an HCl aqueous solution with NBA) [39, 42–44], then purifying the less polar derivatized metabolites (NBAOZ, NBAMOZ, NBSEM and NBAHD). Since after derivatization the solution is neutralized (pH about 7), the LLE and RP-SPE approaches work well (log D about 1 for NBAOZ: **Figure 2**). On the other hand, a limited number of methods perform the derivatization after the first purification step [40, 41]. Tribalat et al. [40] solubilize honey in a 100 mM HCl solution and then carry out a non-retentive RP-SPE (Oasis HLB) since the non-derivatized metabolites are very polar with scarce affinity for non-polar sorbents. As shown in **Figure 2**, at pH < 2 the log D of AOZ is lower than −2. After derivatization, a second (retentive) RP-SPE to isolate NBAOZ, NBAMOZ, NBSEM and NBAHD is carried out. Analogously, Lopez et al. [41] solubilize honey in a 10% NaCl solution, and after a non-retentive RP-SPE (Oasis HLB), they derivatize the metabolites and carry out a LLE using ethyl acetate. For the first time, in 2015, Kaufmann et al. [43] applied an LC-HRMS/MS platform (LC-Q-Exactive) to identify and quantify NFs and CAP, demonstrating acceptable performances for all the four metabolites, except for SEM with CCα and CCβ higher than the fixed MRPL (1 μg/kg).

#### **5.5. Nitroimidazoles (NMZs)**

Metronidazole (MNZ), dimetridazole (DMZ), ronidazole (RNZ) and ipronidazole (IPZ) are all nitroimidazole drugs with antibiotic and antiprotozoal activity. NMZs have been traditionally used for treatment and prevention of histomoniasis and coccidiosis in poultry, trichomoniasis in cattle and dysentery in swine. Due to their mutagenicity, genotoxicity and carcinogenicity, in 1990s NMZs have been classified in Europe as prohibited substances for all food-producing species (Group A6 of Annex I of Directive 96/23 [5]). NMZs can prevent and control *Nosema apis*, and in China, these drugs have been used as a cheap alternative to fumagillin. The presence of NMZ residues in honey has been reported only in the last few years [46]. CRL Guidance Paper (2007) [4] requires methods to reach 3 μg/kg. The main published methods based on LC-MS/MS technique are listed in **Table 6** [47–51]. The 5-nitroimidazoles are known to be rapidly metabolized in animals forming the relevant hydroxy metabolites which are generally determined together with the parent drugs because they may have similar mutagenic potential. The first confirmatory procedure in honey has been published by Cronly et al. [47] in 2010, following the detection of metronidazole residues in imported honey from China and from other non-EU countries [12]. Since at pH lower than 2.5 the NMZs are ionized, the solubilization of honey in water or in buffered solution at pH 6–7 favours RP-SPE or LLE purifications (**Figure 2**). On the other hand, some authors have taken advantage of NMZ ionization in strong acidic solutions performing effective cationicexchange purifications (SCX).

#### **5.6. Quinolones (QNs)**

thanks to the application of the hydrolysis and derivatization procedure together with the use of LC-MS/MS technique that, in the early 2000s, a large number of contaminated food samples were discovered (**Table 2**). Currently, all the methods are based on this treatment. The analysis of commercialized honey samples demonstrated that furazolidone (AOZ) is the main nitrofuran antibiotic used in apiculture [12]. Inevitably, all methods in **Table 5** apply the LC-MS techniques [39–45]. The first procedure for the determination of metabolites in honey was published by Khong et al. in 2004, using isotopic dilution [39]. Most of the procedures perform the honey solubilization directly in the derivatization mixture (usually an HCl aqueous solution with NBA) [39, 42–44], then purifying the less polar derivatized metabolites (NBAOZ, NBAMOZ, NBSEM and NBAHD). Since after derivatization the solution is neutralized (pH about 7), the LLE and RP-SPE approaches work well (log D about 1 for NBAOZ: **Figure 2**). On the other hand, a limited number of methods perform the derivatization after the first purification step [40, 41]. Tribalat et al. [40] solubilize honey in a 100 mM HCl solution and then carry out a non-retentive RP-SPE (Oasis HLB) since the non-derivatized metabolites are very polar with scarce affinity for non-polar sorbents. As shown in **Figure 2**, at pH < 2 the log D of AOZ is lower than −2. After derivatization, a second (retentive) RP-SPE to isolate NBAOZ, NBAMOZ, NBSEM and NBAHD is carried out. Analogously, Lopez et al. [41] solubilize honey in a 10% NaCl solution, and after a non-retentive RP-SPE (Oasis HLB), they derivatize the metabolites and carry out a LLE using ethyl acetate. For the first time, in 2015, Kaufmann et al. [43] applied an LC-HRMS/MS platform (LC-Q-Exactive) to identify and quantify NFs and CAP, demonstrating acceptable performances for all the four metabo-

lites, except for SEM with CCα and CCβ higher than the fixed MRPL (1 μg/kg).

Metronidazole (MNZ), dimetridazole (DMZ), ronidazole (RNZ) and ipronidazole (IPZ) are all nitroimidazole drugs with antibiotic and antiprotozoal activity. NMZs have been traditionally used for treatment and prevention of histomoniasis and coccidiosis in poultry, trichomoniasis in cattle and dysentery in swine. Due to their mutagenicity, genotoxicity and carcinogenicity, in 1990s NMZs have been classified in Europe as prohibited substances for all food-producing species (Group A6 of Annex I of Directive 96/23 [5]). NMZs can prevent and control *Nosema apis*, and in China, these drugs have been used as a cheap alternative to fumagillin. The presence of NMZ residues in honey has been reported only in the last few years [46]. CRL Guidance Paper (2007) [4] requires methods to reach 3 μg/kg. The main published methods based on LC-MS/MS technique are listed in **Table 6** [47–51]. The 5-nitroimidazoles are known to be rapidly metabolized in animals forming the relevant hydroxy metabolites which are generally determined together with the parent drugs because they may have similar mutagenic potential. The first confirmatory procedure in honey has been published by Cronly et al. [47] in 2010, following the detection of metronidazole residues in imported honey from China and from other non-EU countries [12]. Since at pH lower than 2.5 the NMZs are ionized, the solubilization of honey in water or in buffered solution at pH 6–7 favours RP-SPE or LLE purifications (**Figure 2**). On the other hand, some authors have taken advantage of NMZ ionization in strong acidic solutions performing effective cationic-

**5.5. Nitroimidazoles (NMZs)**

350 Honey Analysis

exchange purifications (SCX).

QNs are widely used in veterinary practice because of their rapid effect and broad-spectrum antibacterial activity. Despite the lack of scientific data demonstrating efficacy, the application of these antibiotics in apiculture, especially in Asia, as a prophylaxis for bee diseases increased during the last few years. The first RASFF notifications for the presence of QNs in honey were reported in 2007 in Chinese products. Their use was confirmed by the frequent detection of QN residues in honey also by other control authorities, such as the US Department of Agriculture (USDA) and the Canadian Food Inspection Agency (CCFIA) [14]. To date, the only compounds found in bee products are enrofloxacin, ciprofloxacin and norfloxacin. The native fluorescence of quinolone ring has been extensively exploited to determine these antibiotics in biological fluids and food. Thanks to the high sensitivity of fluorescence detection and the lower cost of equipment compared to LC-MS, this technique is still used to detect and confirm quinolone residues in food. In **Table 7**, the most significant methods are listed [52–58]. Generally, the solubilized honey is purified by reversed-phase SPE [53, 54] or by LLE [55–57]. SPE sorbents, other than reversed-phase types, are reported in the papers published in 1998 by Rose et al. [52] and in 2011 by Yatsukawa et al. [54]. Rose et al. describe two parallel sample treatment protocols using ion-exchange solid-phase extraction: one for nine amphoteric QNs (ciprofloxacin, danofloxacin, enoxacin, enrofloxacin, lomefloxacin, marbofloxacin, norfloxacin, ofloxacin and sarafloxacin) and another for three acidic ones (flumequine, nalidixic acid and oxolinic acid). Amphoteric QNs bear both an acidic group (carboxylic acid) and a basic group (piperazinyl group), and therefore, they are positively ionized at acidic pH, enabling isolation with strong cation-exchange mechanism (SCX-SPE). On the other hand, acidic quinolones can only be neutral, or at basic pHs, they are negatively charged enabling anion-exchange purification. Yatsukawa et al. apply the classical RP-SPE (Oasis HLB) followed by metal chelate affinity chromatography (MCAC). This particular type of SPE acts via the specific chelation of quinolones with ferric ions previously bound to the stationary phase (sepharose fast flow resin). The elution is performed with a buffer (pH 4) containing Na2 EDTA. This is probably the only published application of MCAC to quinolone purification, exploiting their chelating properties. The achievable selectivity allows an efficient removal of interferences also in dark-coloured honey samples such as manuka and buckwheat [54]. On the other hand, MCAC is a well-known stationary phase to purify tetracycline antibiotics using copper (Cu2+) as metal ion (see Section 5.9). Finally, in 2014, Tayeb-Cherif et al. [58] proposed a cheap and simple procedure without any sample purification (**Table 7**): the solubilized honey was just injected in the LC-FLD system. As a result, high detection capabilities (CCβ) are observed (10–100 μg/kg)

#### **5.7. Streptomycin and dihydrostreptomycin (STR/DSTR)**

Streptomycin and its derivative, dihydrostreptomycin, are aminoglycoside (AGs) antibiotics used in apiculture to protect bees against a variety of brood diseases. They are polybasic cations consisting of two or more sugars, attached to an aminocyclitol ring with glycoside linkage. Despite the fact that streptomycin is not authorized in most countries in beekeeping practice, its use is often suggested in bee forums and in beekeeping handbooks. Residues of streptomycin and dihydrostreptomycin have been frequently detected in honey and honeybee products by the EU RASSF system (**Table 2**). Due to the lack of chromophore or fluorophore groups, the traditional absorbance or fluorescence detectors cannot be directly applied to AG determination, as shown in **Table 8** [59–68]. Fortunately, the primary amine groups in the aminoglycoside structure react with a number of derivatizing agents. Therefore, especially in the past when mass spectrometry detectors were not commonly available, methods for this antibiotic family were mainly based on liquid chromatography coupled to FLD after post-column derivatization with *o*-phthalaldehyde (OPA) or β-naphthoquinone-sulphonate (NQS). Since aminoglycosides are in polyionic form in aqueous solutions, both their extraction and preconcentration are difficult, and like the sugars of the honey, silica-based C18 sorbents are unable to retain them. The coating of silica C18 sorbents with an ion-pairing reagent such as 1-heptanesulphonic acid (AHS) was experienced to produce a temporary cation exchanger [59, 61, 62], favouring the analyte retention. In contrast, Bohm et al. [64] purify honey extracts with RP-SPE without any addition of ion-pairing reagents, probably thanks to the use of a polymeric sorbent (Oasis HLB), instead of the silica-based C18 stationary phases. Three procedures [60, 63, 66] applied weak cation-exchange extraction (WCX) to clean-up honey. In 2013, Ji et al. [65] synthesized a molecular imprinted polymer (MIP) by polymerization of methacrylic acid and ethylene glycol dimethacrylate in the presence of streptomycin as template molecule. The observed recoveries for four model compounds in honey (streptomycin, gentamicin, spectinomycin and dihydrostreptomycin) ranged from 90 to 110%. Currently, this developed MIP sorbent is commercially available and Moreno-Gonzales et al. applied it to determine aminoglycosides in honey using capillary zone electrophoresis coupled to an ion trap mass analyser [68]. Finally Wang et al. developed a home-made hydrophilic stationary phase (polyvinyl alcohol onto silica gel, PVA-Sil), which demonstrated satisfactory performances to pre-concentrate aminoglycosides in honey extracts [67].

With regard to chromatographic issues, because of their high polarity, the underivatized aminoglycosides are not sufficiently retained on standard reversed-phase HPLC columns. Therefore, there are two possible choices: (i) the addition of ion-pairing reagents such as alkyl sulphonates (e.g. sodium 1-heptansulphonic acid, AHS) or fluoropropionic acids (e.g. heptafluorobutyric acid, HFBA; pentafluoropropionic acid, PFPA) in the mobile phase and (ii) the application of HILIC (hydrophilic interaction chromatography) analytical columns, which are more compatible with MS detection since ion-pairing reagents cause strong ion suppression. HILIC is a variant of normal-phase chromatography that uses water as a strong eluent and water-miscible organic solvents like acetonitrile as organic components of the mobile phase. In **Table 8**, examples applying derivatization [59, 61], ion-pairing reagents [60, 62] and HILIC chromatography [63–67] are reported.

#### **5.8. Sulphonamides (SAs)**

As early as 1940s, sodium sulphathiazole was registered for the control of American foulbrood in United States, but its use was later banned because residues of the drug continued to be found many months after its administration. Residues of sulphadiazine, sulphadimethoxine, sulphamerazine, sulphamethazine and sulphamethoxazole have been also detected in honey [12, 14]. Sulphonamides have good UV absorption with maxima in the range of 260–275 nm, and since the 1980s, confirmatory methods have been developed using HPLC coupled to UV detection. Moreover, after derivatization with fluorescamine, sulphonamides give fluorescence and some procedures apply LC-FLD (with pre- or post-column derivatization), reaching limits of detections (LOD/CCβ) comparable to those of LC-MS methods. In **Table 9**, some example of these applications are listed [69–78]. Since considerable amounts of SAs are bound to honey sugars, in 2000 Schwaiger and Schuch [79] demonstrated the need of an acidic hydrolysis prior to the residue analysis. This step avoids the underestimation of the actual sample contamination.

The solubility of sulphonamides in acids and alkali is conditioned by their amphoteric properties, due to the presence of an anilino amino group (pKa1: 2–2.5) and of an amidic group, which contains a labile hydrogen atom with acidic properties (pKa2: 6–9). Thus, sulphonamides are positively charged in acidic medium at pH <2, neutral at pH 3–6 and negatively charged at pH >6. Therefore, at one hand, exploiting their basic moiety, some procedures use strong cation exchange (SCX-SPE) to isolate sulphonamides from the acidic honey extracts [71–73, 77]. On the other hand, to successfully apply RP-SPE or LLE clean-up, some researchers buffered honey extract in the pH range about 4–6 in which the neutral form of sulphonamides prevails [70, 74–76, 78]. In this interval, the distribution coefficients (D) reach their maximum and the compound lipophilicity is enhanced, as shown in **Figure 3** for sulphathiazole.

#### **5.9. Tetracyclines (TCs)**

practice, its use is often suggested in bee forums and in beekeeping handbooks. Residues of streptomycin and dihydrostreptomycin have been frequently detected in honey and honeybee products by the EU RASSF system (**Table 2**). Due to the lack of chromophore or fluorophore groups, the traditional absorbance or fluorescence detectors cannot be directly applied to AG determination, as shown in **Table 8** [59–68]. Fortunately, the primary amine groups in the aminoglycoside structure react with a number of derivatizing agents. Therefore, especially in the past when mass spectrometry detectors were not commonly available, methods for this antibiotic family were mainly based on liquid chromatography coupled to FLD after post-column derivatization with *o*-phthalaldehyde (OPA) or β-naphthoquinone-sulphonate (NQS). Since aminoglycosides are in polyionic form in aqueous solutions, both their extraction and preconcentration are difficult, and like the sugars of the honey, silica-based C18 sorbents are unable to retain them. The coating of silica C18 sorbents with an ion-pairing reagent such as 1-heptanesulphonic acid (AHS) was experienced to produce a temporary cation exchanger [59, 61, 62], favouring the analyte retention. In contrast, Bohm et al. [64] purify honey extracts with RP-SPE without any addition of ion-pairing reagents, probably thanks to the use of a polymeric sorbent (Oasis HLB), instead of the silica-based C18 stationary phases. Three procedures [60, 63, 66] applied weak cation-exchange extraction (WCX) to clean-up honey. In 2013, Ji et al. [65] synthesized a molecular imprinted polymer (MIP) by polymerization of methacrylic acid and ethylene glycol dimethacrylate in the presence of streptomycin as template molecule. The observed recoveries for four model compounds in honey (streptomycin, gentamicin, spectinomycin and dihydrostreptomycin) ranged from 90 to 110%. Currently, this developed MIP sorbent is commercially available and Moreno-Gonzales et al. applied it to determine aminoglycosides in honey using capillary zone electrophoresis coupled to an ion trap mass analyser [68]. Finally Wang et al. developed a home-made hydrophilic stationary phase (polyvinyl alcohol onto silica gel, PVA-Sil), which demonstrated satisfactory perfor-

mances to pre-concentrate aminoglycosides in honey extracts [67].

chromatography [63–67] are reported.

**5.8. Sulphonamides (SAs)**

352 Honey Analysis

With regard to chromatographic issues, because of their high polarity, the underivatized aminoglycosides are not sufficiently retained on standard reversed-phase HPLC columns. Therefore, there are two possible choices: (i) the addition of ion-pairing reagents such as alkyl sulphonates (e.g. sodium 1-heptansulphonic acid, AHS) or fluoropropionic acids (e.g. heptafluorobutyric acid, HFBA; pentafluoropropionic acid, PFPA) in the mobile phase and (ii) the application of HILIC (hydrophilic interaction chromatography) analytical columns, which are more compatible with MS detection since ion-pairing reagents cause strong ion suppression. HILIC is a variant of normal-phase chromatography that uses water as a strong eluent and water-miscible organic solvents like acetonitrile as organic components of the mobile phase. In **Table 8**, examples applying derivatization [59, 61], ion-pairing reagents [60, 62] and HILIC

As early as 1940s, sodium sulphathiazole was registered for the control of American foulbrood in United States, but its use was later banned because residues of the drug continued to be found many months after its administration. Residues of sulphadiazine, sulphadimethoxine, sulphamerazine, sulphamethazine and sulphamethoxazole have been also detected in honey The efficacy of the oxytetracycline for control of European foulbrood has been widely demonstrated as early as 1950s. In honey, beyond oxytetracycline (brand name: Terramycin®), tetracycline and chlortetracycline residues have been detected, too [12, 14]. Because of their polar nature, tetracyclines have the ability to strongly bind to proteins as well as to chelate with divalent metal ions. Therefore, most extractions incorporate acidic solvents with the addition of metal chelating agents. Frequently, the extraction approaches use Na2 EDTA-McIlvaine buffer (pH = 4). Known as the "universal tetracycline extractant", McIlvaine buffer consists of citric acid and disodium hydrogen phosphate. Other common buffers used for tetracyclines extraction are oxalic acid, succinic acid and citric acid. Another challenge in tetracycline determination is their epimerization. In mildly acidic conditions (pH 2–6), epimerization occurs at position C-4. Accordingly, European Union MRLs in food are established as sum of tetracycline and its epimer, that is, tetracycline and epi-tetracycline, oxytetracycline and epi-oxytetracycline, chlortetracycline and epi-chlortetracycline [1].

As shown in **Table 10** [80–88], besides the classical reversed-phase solid-phase extraction cartridges (phenyl, Oasis HLB, Strata-X and C18), tetracyclines can be selectively purified applying a particular type of solid-phase extraction, that is, metal chelate affinity chromatography (MCAC) [82, 86]. As mentioned before for quinolones (Section 5.6), MCAC exploits tetracycline metal complexing properties to allow for additional clean-up. The sorbent (sepharose resin) is treated with aqueous copper (II) sulphate. The sample extract is then loaded onto the column and TCs are retained. The copper ions give visualization of the clean-up process: the analytes are found where the blue copper ions appear. Initially, the tetracyclines are bound to the blue copper ions on the column until disruption by an EDTA containing buffer and elution of the copper ions, EDTA and tetracyclines.

#### **5.10. Multiclass methods**

In efforts to increase the cost-effectiveness of antimicrobial residue enforcement programmes, the development of analytical methods able to detect as many contaminant compounds as possible is highly preferred. However, it is well known that one of the difficulties in the development of these procedures is the incompatibility of selective sample treatments with acceptable accuracies for a wide range of analytes. Therefore, only a generic purification protocol such as liquid-liquid extraction or reversed-phase solid-phase extraction is achievable (**Table 11**). Since generally reversed-phase sorbents provide the least selective retention mechanism when compared to normal phase or ion exchange ones, they allow the most universal solid-phase extraction approach retaining most molecules with any hydrophobic character.

There are some considerations to do before to take on multiclass methods for antibiotics: (i) the extraction of nitrofuran metabolites requires acid hydrolysis and derivatization steps that would be destructive to other analytes of interest. Therefore, this class should be extracted apart from a multiclass method to obtain satisfactory recovery and avoid degradation of acidlabile compounds; (ii) as mentioned before, highly polar compounds, such as aminoglycosides, do not perform well in multiclass methods as they are relatively insoluble in organic solvents and exhibit little or no affinity for non-polar stationary phases used in RP-LC.LC. For this reason, in **Table 11** only two papers include aminoglycosides among the determined classes adding an ion-pairing reagent in the mobile phases; (iii) in addition, in honey, the determination of sulphonamides in honey requires a preliminary hydrolysis step to measure residues bound to sugars, and therefore, also in this case, acid-sensitive antibiotics can be destroyed.

In this context, "multiclass" are procedures involving the determination of more than two drug classes. Probably, the first multiclass method in honey has been published in 2004 by Kaufmann et al. [89], reporting the determination of three antibiotic families, including sixteen sulphonamides together with three tetracyclines and flumequine, a quinolone antibiotic for which until now there is no evidence of use in apiculture. In 2008, Hammel et al. [90] developed an LC-MS/MS protocol for 42 substances including five tetracyclines, seven macrolides, three aminoglycosides, eight beta-lactams, two amphenicols and seventeen sulphonamides. Four subsequent liquid-liquid extraction steps were necessary to adequately extract all the analytes. After this paper, many confirmatory multiclass methods have been published mainly applying triple quadrupole platforms [29, 91–98]. This is in accordance with the general trend in analysis of residues in food started in the late 2000s. Although triple quadrupoles have been introduced in the mid-to late-1990s, only in recent years these equipments have improved their electronics enabling the possibility of acquiring dozens of compounds in the same chromatographic run.

## **6. Conclusions**

The performances of an analytical method are mainly determined by the applied sample preparation and instrumental technique. The coupling honey-antibiotic (matrix-analyte) can be a "case study" to discuss the general strategies of developing methods for trace analysis in food. It must be kept in mind that the sample preparation protocol has to start from the knowledge of the matrix composition and analyte properties (MW, pK<sup>a</sup> , log D, etc). Moreover, the choice of the more suitable clean-up also involves the knowledge of the available methodologies, but in most of the cases the selection is limited to the SPE stationary phases. In the last years, new sorbent materials are more and more produced, enabling new possibilities for more efficient, rapid and cheap protocols. Undoubtedly, aminoglycosides and, to a lesser extent, tetracyclines are the more difficult classes to analyse. Obviously, when multiresidue or multiclass procedures are optimized, the challenge is the achievement of the best compromise among the different properties of each single-class challenging. The current trends in honey sample preparation and, more generally in food, 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; and the development of multiclass procedures. All these strategies aim at the reduction in the employed reagents/materials and at the increase in the analysis throughput. The choice of the analytical equipment is less free. Today, LC-QqQ systems (triple quadrupoles) are able to solve almost each analytical problem. With regard to the analyte separation, except for aminoglycosides, reversed-phase stationary phases are generally used. Various column types (traditional, sub 2-μm and core-shell) and manufacturers have been reported in literature to determine the same analyte or class of analytes (**Tables 3**–**11**), but frequently the applied selection criteria are not explained or compared.
