**1. Introduction**

Antimicrobials are used in food-producing animals to prevent and/or treat animal diseases. Although epidemiological data on the real magnitude of their adverse effects are very scarce, they indicate that the presence of antibiotic residues in food could be an important vehicle for the development of antibiotic-resistant bacterial strains. Because of these concerns, many countries have restricted the use of antibiotics in farm. The major honeybee diseases for which antibiotics are applied are American foulbrood, European foulbrood infections and nosemosis. Foulbrood infections are caused by bacteria, whereas *Nosema* disease is caused by a fungus. Currently, in the European Union the maximum residue limits (MRLs) for antibiotics in food are listed in Regulation (EU) No 37/2010 [1]. This regulation stipulates that each antibiotic

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

must have a MRL before it can be used on a food-producing animal. European Union does not allow the use of antibiotics for treatment of honeybees, and therefore, there are not MRLs in honey for these substances. The lack of harmonized rules with regard to acceptable control methods, limits of detection or sampling methods, results in different interpretations by European Member States. Some Member States and Switzerland have established action limits or tolerance levels [2, 3]. In the CRL Guidance Paper (2007) [4], the European Union References Laboratories (EURLs, ex CRLs) proposed recommended concentrations for analysis of macrolides, streptomycin, sulphonamides and tetracyclines in honey within the national residue control plans carried out in accordance with Council Directive 96/23/EC [5] (**Table 1**). These recommended concentrations, however, have no real legal basis. They are used as reference during method development since detection capability (CCβ) for screening methods or decision limit (CCα) for the confirmatory ones [6] should be lower than recommended concentrations. All the veterinary drugs just mentioned belong to the Group B of Annex I of Council Directive 96/23/EC [5], that is, they are permitted substances with fixed MRLs in several food commodities. On the other hand, in the case of banned substances (Group A, Annex I of Council Directive 96/23/EC) such as chloramphenicol (CAP) and nitrofurans (NFs), the European Union has set minimum required performance limits (MRPLs) of 0.3 and 1.0 μg/kg, respectively. MRPLs are foreseen in Article 4 of Commission Decision 2002/657/EC


a Maximum residue limits (MRLs) or tolerances (legal limits).

b Recommended concentrations (RCs) which only represent a reference point for analytical method performances. c Working residue level (WRL) below which there is considered to be undue risk to human health. dSum of tylosin A and B.

**Table 1.** Worldwide limits for antibiotics in honey.

[6] and they are reference point for action. They are intended to harmonize the analytical performance of methods ensuring the same level of consumer protection in the European Union. Among banned substances, also the use of nitroimidazoles has been documented in beekeeping practice. However, no MRPLs have been fixed for nitroimidazoles, the European Union has not fixed the relevant MRPLs, and during the development of analytical methods, the recommended concentration of 3 μg/kg (CRL Guidance Paper [4]) is taken into account.

must have a MRL before it can be used on a food-producing animal. European Union does not allow the use of antibiotics for treatment of honeybees, and therefore, there are not MRLs in honey for these substances. The lack of harmonized rules with regard to acceptable control methods, limits of detection or sampling methods, results in different interpretations by European Member States. Some Member States and Switzerland have established action limits or tolerance levels [2, 3]. In the CRL Guidance Paper (2007) [4], the European Union References Laboratories (EURLs, ex CRLs) proposed recommended concentrations for analysis of macrolides, streptomycin, sulphonamides and tetracyclines in honey within the national residue control plans carried out in accordance with Council Directive 96/23/EC [5] (**Table 1**). These recommended concentrations, however, have no real legal basis. They are used as reference during method development since detection capability (CCβ) for screening methods or decision limit (CCα) for the confirmatory ones [6] should be lower than recommended concentrations. All the veterinary drugs just mentioned belong to the Group B of Annex I of Council Directive 96/23/EC [5], that is, they are permitted substances with fixed MRLs in several food commodities. On the other hand, in the case of banned substances (Group A, Annex I of Council Directive 96/23/EC) such as chloramphenicol (CAP) and nitrofurans (NFs), the European Union has set minimum required performance limits (MRPLs) of 0.3 and 1.0 μg/kg, respectively. MRPLs are foreseen in Article 4 of Commission Decision 2002/657/EC

 **(µg/kg) RCsb (µg/kg) Source**

(MRLs) for Veterinary Drugs in

Agricultural Chemical Residues

1.4.2—Schedule 20) [10]

CRL Guidance Paper [4] Tetracyclines – <sup>20</sup>

Regulations—Title 21 [8] Oxytetracycline – –

EU Streptomycin – 40 European Regulation 37/2010 [1]

Canada Fumagillin 25 – List of Maximum Residue Limits

Foods [9] Oxytetracycline 300 –

Australia/New Zealand Oxytetracycline 300 – Food Standards Code (standard

in Foods [11] Amoxicillin <sup>8</sup> –

Japan Oxytetracycline 300 – Positive List System for

Recommended concentrations (RCs) which only represent a reference point for analytical method performances.

Sulphonamides – 50 Erythromycin Tylosin – 20

Tylosin – –

Erythromycin – 30<sup>c</sup> Tylosin 200d –

Ampicillin 9 –

Working residue level (WRL) below which there is considered to be undue risk to human health.

Maximum residue limits (MRLs) or tolerances (legal limits).

**Table 1.** Worldwide limits for antibiotics in honey.

USA Lincomycin – – CFR—Code of Federal

**Country Approved substance MRLsa**

a

326 Honey Analysis

b

c

dSum of tylosin A and B.

The worldwide standard, the Codex Alimentarius, has not fixed any MRL for antibiotics in honey [7]. The Codex Alimentarius or "Food Code" was established by FAO and the World Health Organization in 1963 to develop harmonized international food standards, which protect consumer health and promote fair practices in food trade. Similarly, in United States, no tolerances for antibiotics in honey have been established, although oxytetracycline is approved for longtime in beekeeping practice to control American foulbrood. At present, lincomycin and tylosin are authorized, too. The MRLs (or tolerances) for residues of antibiotics in food are set by the US Food and Drug Administration (USFDA) and listed in the Code of Federal Regulations, Title 21 [8]. Conversely, Canada, Australia, New Zealand and Japan have established MRLs for oxytetracycline (300 μg/kg) [9–11]. In addition, also MRLs for fumagillin, oxytetracycline and tylosin are provided by Canadian authorities (**Table 1**). For erythromycin, a working residue level is provided, below which no risk to human health is considered.

Sulphathiazole and oxytetracycline are probably the first antibiotics used to fight honeybee diseases. Starting from 1980s, analytical methods have been developed for these two drugs in honey at trace levels mainly based on liquid chromatography coupled to UV-Vis (LC-UV-Vis) and fluorescence detectors (LC-FLD). In the early 2000s, the availability of liquid chromatography systems coupled to mass spectrometric analysers (LC-MS) at bench level involved the progressive development of procedures using this technique which allows a more selective and universal detection than the traditional detectors based on UV absorption (quite universal, but not selective) or fluorescence (selective, but not universal). Therefore, existing methods have been progressively converted using LC-MS improving performances and sample throughput, and new challenging analytical problems have been solved thanks to this technique equipments (e.g. the detection of nitrofurans metabolites in food).

From a toxicological point of view, in the European Union law system the distinction is between permitted drugs (aminoglycosides, lincomycin, macrolides, quinolones, sulphonamides and tetracyclines) and banned drugs (chloramphenicol, nitrofurans and nitroimidazoles) belonging to substances of group B and A, respectively (Annex I of Directive 96/23 [5]). As discussed before, there are not MRLs for antibiotics in honey (**Table 1**). Hence, in this context, "permitted drugs" are drugs with an MRL in food commodities other than honey (meat, milk, liver, etc), whereas the banned ones (chloramphenicol, nitrofurans and nitroimidazoles) cannot be used in any food-producing species generally not only in European Union, but also in several other countries. This distinction is also fundamental to choose the analytical technique to develop confirmatory methods, which are procedures fulfilling Commission Decision 2002/657/EC criteria [6]. For the banned substances, the use of mass spectrometric detectors is mandatory, whereas, for the permitted ones, traditional detectors, UV-Vis or FLD, are suitable, too. In addition, for banned drugs, the required method limits are in the range from 0.1 to 1 μg/kg; for permitted drugs, limits of about one order of magnitude greater can be acceptable (**Table 1**).

The use of liquid chromatography coupled to mass spectrometry equipments and the worldwide improvement of law systems probably explains the decrease in the incidence of veterinary drug residues in honey and honeybee products (royal jelly and propolis). The number of cases per year in 2003 was 40, whereas in 2015 only six notifications have been recorded by the European Rapid Alert System for Food and Feed (RASSF) as shown in **Table 2** [12]. In place since 1979, RASSF enables information to be shared efficiently between its members [national food safety authorities of EU Member States, the EU Commission, the European Food Safety Authority (EFSA), Norway, Liechtenstein, Iceland and Switzerland]. It provides an efficient service to ensure that urgent notifications are sent, received and responded to in the shortest time possible. Thanks to RASSF, many food safety risks had been averted before any harm to European consumers was caused.


**Table 2.** RASFF notifications in the period 2002–1015 (hazard category "residues of veterinary medicinal products"; product category "honey and royal jelly").

In Section 4, an extensive overview of the main published analytical methods for the determination of residues of the antibiotics (legally or illegally) used in apiculture is carried out. The analytical steps of each selected method (sample treatment, analytical technique and detection limits) are summarized in **Tables 3**–**11**.


a FF, florfenicol; FFA, florfenicol amine; TAP, thiamphenicol.

The use of liquid chromatography coupled to mass spectrometry equipments and the worldwide improvement of law systems probably explains the decrease in the incidence of veterinary drug residues in honey and honeybee products (royal jelly and propolis). The number of cases per year in 2003 was 40, whereas in 2015 only six notifications have been recorded by the European Rapid Alert System for Food and Feed (RASSF) as shown in **Table 2** [12]. In place since 1979, RASSF enables information to be shared efficiently between its members [national food safety authorities of EU Member States, the EU Commission, the European Food Safety Authority (EFSA), Norway, Liechtenstein, Iceland and Switzerland]. It provides an efficient service to ensure that urgent notifications are sent, received and responded to in the shortest time possible. Thanks to RASSF, many food safety risks had been averted before any harm to

European consumers was caused.

328 Honey Analysis

**Year No of notifications No of found substancesa Number (substance)**

2002 45 57 34 (CAP), 13 (STR/DSTR), 7 (SAs), 3 (TCs)

2006 16 17 7 (CAP), 6 (SAs), 2 (STR), 1 (NFs), 1 (TCs)

2004 25 27 10 (SAs), 7 (CAP), 5 (NFs), 5 (STR)

2009 10 10 4 (TCs), 3 (NFs), 2 (STR), 1 (SA)

2011 6 6 3 (SAs), 2 (NMZs), 1 (lincomycin)

2012 6 7 5 (SAs), 2 (NFs),

In the same sample more than one residue could be present.

product category "honey and royal jelly").

a

2013 4 4 2 (SAs), 1 (NFs), 1 (TCs),

2014 1 1 1 (SA—sulphamethoxazole)

2003 40 53 20 (SAs), 17 (CAP), 11 (STR), 3 (TCs), 2 (NFs)

2005 41 49 25 (CAP), 8 (STR), 6 (SAs), 5 (TCs), 4 (NFs), 1 (MAC)

2007 20 41 24 (SAs), 6 (QNs), 6 (TCs), 2 (MACs), 1 (CAP), 1 (STR),

2008 27 29 9 (MACs), 7 (TCs), 5 (SAs), 3 (CAP), 2 (QNs), 2 (STR),

2010 8 9 3 (lincomycin), 2 (STR), 1 (TC), 1 (NMZ), 1 (MAC),

2015 6 10 3 (CAP), 3 (STR/DSTR), 2 (TCs), 1 (NMZ), 1 (SA)

**Table 2.** RASFF notifications in the period 2002–1015 (hazard category "residues of veterinary medicinal products";

1 (NFs)

1 (NFs)

1 (QN),

b An enzymatic hydrolysis was carried out to deconjugate CAF in the muscle sample included in the method scope.

c Extrelut (diatomaceous earth) was used to help the liquid-liquid extraction process.

dLMA-MAA-EDMA: poly(lauryl methacrylate-co-methacrylic acid-co-ethylene glycol dimethacrylate).

**Table 3.** Confirmatory methods for chloramphenicol (CAP).


a AIVT, acetylisovaleryltylosin (tylvalosin); AZI, azithromycin; CLA, clarithromycin; ERY, erythromycin; JOS, josamycin; LIN, lincomycin; NEO, neospiramycin; OLE, oleandomycin; SPI, spiramycin I; TILM, tilmicosin; TYL, tylosin A; TYLB, tylosin B; TYLC, tylosin C; TYLD, tylosin D.

b TRIS, tris(hydroxymethyl)aminomethane.

**Table 4.** Confirmatory methods for lincomycin and macrolides (MACs).



a AHD, nitrofurantoin metabolite; AMG, nitrovin metabolite; AMOZ, furaltadone metabolite; AOZ, furazolidone metabolite; DMZ, dimetridazole; DNSH, nifursol metabolite; Ft, furaltadone; Fz, furazolidone; Nt, nitrofurantoin; Nz, nitrofurazone; NPIR, nifurpirinol; PSH, nifuroxazid metabolite; RNZ, ronidazole; SEM, nitrofurazone metabolite.

b Derivatization with 2-NBA in HCl solution with subsequent neutralization.

c Derivatization with 2-NBA and HCl after the indicated purification step.

**Table 5.** Confirmatory methods for nitrofurans (NFs).

**Compoundsa Extraction/**

ERY, TYL 100 mM

**Compoundsa Extraction/**

LIN, TYL Na2

ERY, OLE, SPI, TILM, TYL

330 Honey Analysis

ERY, LIN, JOS, SPI, TILM, TYL

TYL, TYLB, TYLC,

ERY, NEO, OLE, SPI, TILM, TYL,

AIVT, AZI, CLA, ERY, JOS, SPI, TILM, TYL

TYLD

TYLB

a

b

**clean-up**

CO3 – NaHCO3 buffer (pH 9.0)/ C18-SPE

100 mM NaH2 PO<sup>4</sup> buffer at pH 8.0/Oasis HLB-SPE

TRIS<sup>b</sup>

100 mM Na2 CO3 – NaHCO3 buffer (pH 9.0)/ Strata-X-SPE

100 mM NaH2 PO<sup>4</sup> buffer (pH 8.0)/ Oasis HLB-SPE

Na3 PO<sup>4</sup> (pH 8.0)/C18-SPE

Water/Oasis HLB-SPE

 buffer (pH 10.5)/Oasis HLB-SPE

AHD, AMOZ, AOZ, SEM

AHD, AMOZ, AOZ, SEM, Ft, Fz, Nt, Nz

**clean-up**

tylosin B; TYLC, tylosin C; TYLD, tylosin D.

TRIS, tris(hydroxymethyl)aminomethane.

100 mM HCl/ Oasis HLB-SPE<sup>c</sup>

, AF buffer to pH 6–7, Oasis HLB-SPE

b /EtAc, Lichrolut EN-SPE

**Separation Equipment CCβ or LOD** 

**Separation Equipment CCβ or LOD** 

(a) LC-MS (ESI+) (b) LC-MS/MS (ESI+)

LC-MS/MS (ESI+)

(a) LC-MS (ESI+) (b) LC-DAD

(a) LC-MS/MS (ESI+) (b) UHPLC-HRMS/MS (Q-TOF) (ESI+)

LC-MS/MS (ESI+)

LC-MS/MS (ESI+)

Gradient: 0.1% TFA/0.1% TFA in ACN/MeOH

Gradient: 1% FA/water/ACN

Gradient: 10 mM NH<sup>4</sup>

Gradient: 1% FA/ACN/ MeOH

(a) Gradient:10 mM NH<sup>4</sup>

ACN (b) Gradient: Water/1% FA/ ACN

Isocratic: water/ACN (30:70, v/v)

AIVT, acetylisovaleryltylosin (tylvalosin); AZI, azithromycin; CLA, clarithromycin; ERY, erythromycin; JOS, josamycin; LIN, lincomycin; NEO, neospiramycin; OLE, oleandomycin; SPI, spiramycin I; TILM, tilmicosin; TYL, tylosin A; TYLB,

Gradient: 0.2% FA/0.2% FA in ACN

Ac/

Ac (pH 3.5)/ACN

**Column Mobile phase**

Zorbax C8 (150 × 2.1 mm, 5.0 μm)

YMC ODS-AQ S-3 120 Å 50 × 2.0 mm

Synergi Hydro-RP (150 × 2.0 mm, 4.0 μm)

Luna C18(2) 100 Å (150 × 4.6 mm, 5.0 μm)

(a): YMC ODS-AQ S-3 120 Å (50 × 2.0 mm) (b): Acquity BEH C18 (100 × 2.1 mm, 1.7 μm)

Gemini C18 110 Å (50 × 2.0 mm, 5.0 μm)

C18HCE (100 × 2.1 mm, 5.0 μm) (home-made)

**(µg/kg)**

LC-MS (APCI+) 7–10 [31]

(a) <1 (b) 0.01–0.07

(a) 2–3 (b) 49–57

(a) 0.01–0.5 (b) 0.2–1.0

5.0–5.2 [36]

0.01–0.5 [37]

0.24–2.10 [33]

**References**

[32]

[34]

[35]

LC-MS/MS (ESI+)

LC-MS/MS (ESI+)

**Column Mobile phase**

Gradient: 0.025% AcOH/ ACN

Gradient: 20 mM AF buffer (pH 3.8)/ACN

Symmetry Shield C18 (150 × 2.1 mm, 3.5 μm)

**Table 4.** Confirmatory methods for lincomycin and macrolides (MACs).

Inertsil ODS3 (150 × 2.0 mm, 3.0 μm)

**(µg/kg)**

0.12–0.56 [39]

0.15–2.1 [40]

**References**



a DMZ, dimetridazole; HMMNI, 2-hydroxymethyl-1-methyl-5-nitroimidazole; IPZ, ipronidazole; IPZ-OH, ipronidazole metabolite; MNZ, metronidazole; MNZ-OH, metronidazole metabolite; RNZ, ronidazole; TRZ, ternidazole; CAP, chloramphenicol.

b Other less common NMZs are included in the method scope.

c Styrene-divynilbenzene copolymer (RP-SPE).

**Table 6.** Confirmatory methods for nitroimidazoles (NMZs).



a CIPRO, ciprofloxacin; DANO, danofloxacin; DIFLO, difloxacin; ENRO, enrofloxacin; FLUME, flumequine; MARBO, marbofloxacin; NALI, nalidixic acid; NOR, norfloxacin; OXO, oxolinic acid; SARA, sarafloxacin.

b Other less common QNs are included in the method scope.

**Compoundsa Extraction/**

DMZ, HMMNI, IPZ, IPZ-OH, MNZ, MNZ-OH, RNZ, SCZ, TRZ<sup>b</sup>

332 Honey Analysis

chloramphenicol.

(a) CIPRO, DANO, ENRO, MARBO, NOR, SARA<sup>b</sup> (b) FLUME, NALI, OXO

CIPRO, DANO, DIFLO, ENRO, FLUME, MARBO, NALI, NOR, OXO, SARA

CIP, DAN, DIFLO, ENRO, NOR, SARA<sup>b</sup>

CIPRO, DANO, DIFLO, ENRO, MARBO, SARA<sup>b</sup>

CIPRO, DANO, DIFLO, ENRO, FLUME, MARBO,

CIP, ENR, NOR Water, H2

OXO

a

b

c

**clean-up**

10 mM NH<sup>4</sup>

Styrene-divynilbenzene copolymer (RP-SPE).

**clean-up**

pH 11.0, SAX-SPE<sup>c</sup>

NaH2 PO<sup>4</sup> /

MacIlvaine buffer (pH 4.0)

Na2 HPO<sup>4</sup> buffer (pH 6.3)/ hexane, Oasis HLB-SPE

(Na2 EDTA)/ Oasis HLB-SPE, MCAC-SPE

30 mM NaH2 PO<sup>4</sup> buffer (pH 7.0)/ QuEChERS (5% FA in ACN)

to pH 1.0/ PS-MSLMd

(a) 2% AcOH in ACN, SCX-SPE (b) ACN, 50 mM Na2

HPO<sup>4</sup>

**Compoundsa Extraction/**

(pH 6.0)/MIP

Ac

Other less common NMZs are included in the method scope.

**Table 6.** Confirmatory methods for nitroimidazoles (NMZs).

**Separation Equipment CCβ or** 

**Separation Equipment CCβ or LOD** 

LC-MS/MS (ESI+)

UHPLC-MS/ MS (ESI+)

(a) Isocratic: 10 mM Phosphate buffer (pH 3.0)/ ACN (b) Isocratic: 10 mM OA/ ACN/MeOH (60:30:10, v/v/v)

Gradient: 1% FA/ACN

Isocratic: 1 mM SDS, 20 mM citrate buffer (pH 3.1)/ACN (70:30, *v/v*)

Isocratic: 1% FA/MeOH (71:29, v/v)

Gradient: 0.02% FA/ACN

Isocratic: MeOH/ ACN/0.34% PA, 0.6% TEA<sup>e</sup> (15:5:80, v/v)

LC-MS/MS (ESI+)

Gradient: 0.1% FA/0.1% FA in ACN

DMZ, dimetridazole; HMMNI, 2-hydroxymethyl-1-methyl-5-nitroimidazole; IPZ, ipronidazole; IPZ-OH, ipronidazole metabolite; MNZ, metronidazole; MNZ-OH, metronidazole metabolite; RNZ, ronidazole; TRZ, ternidazole; CAP,

**Column Mobile phase**

**Column Mobile phase**

(a) Zorbax RX C8 (250 × 4.6 mm, 5.0 μm) (b) Kromasil C8 (250 × 3.2 mm, 5.0 μm)

XBridge MS C18 (100 × 2.1 mm, 3.5 μm)

Inertsil ODS-4 (150 × 4.6 mm, 3.0 μm)

(150 × 4.6 mm, 5.0 μm)

Zorbax Eclipse Plus HHRD (50 × 2.1 mm, 1.8 μm)

Zorbax Eclipse XDB-C18 (150 × 4.6 mm, 5.0 μm)

ACN/hexane WondaSil C18

SO<sup>4</sup>

Kinetex XB C18 (150 × 2.1 mm, 2.6

μm)

**LOD (μg/kg)**

**(µg/kg)**

0.07–0.66 [53]

0.2–1.7 [56]

LC-FLD 5–50 [52]

LC-FLD 0.33–4.4 [54]

LC-DAD 0.4–19 [55]

LC-FLD 0.067–0.35 [57]

0.18–0.51 [51]

**References**

**References**

c SCX: strong cation exchange and SAX: strong anion exchange.

dPS-MSLM: phase separation-based magnetic-stirring salt-induced liquid-liquid microextraction method (LLE). e Triethylamine.

**Table 7.** Confirmatory methods for quinolones (QNs).



a AMI, amikacin; APR, apamycin; GEN, four isomers of gentamicin; HYG, hygromycin; KAN, kanamycin; NEO, neomycin; PAR, paromomycin; SIS, sisomycin; SPC, spectinomycin; TOB, tobramycin.

b AHS, sodium 1-heptanesulphonic acid (ion-pairing reagent).

c HFBA, heptafluorobutyric acid (ion-pairing reagent).

dPFPA, pentafluoropropionic acid (ion-pairing reagent).

e CZE-IT-MS: capillary zone electrophoresis coupled to ion trap mass spectrometry.

f PVA-Sil: polyvinyl alcohol-Silica.

g TE: thiol-ene.

**Table 8.** Confirmatory methods for streptomycin/dihydrostreptomycin (STR/DSTR).



a DAP, dapsone; SCP, sulphachloropyridazine; SDA, sulphadiazine; SDT, sulphadimethoxine; SDX, sulphadoxine; SGN, sulphaguanidine; SMM, sulphamonomethoxine; SMR, sulphamerazine; SMP, sulphamethoxypyridazine; SMX, sulphamethoxazole; SMZ, sulphamethazine; SNL, sulphanilamide; SPD, sulphapyridine; SQX, sulphaquinoxaline; STZ, sulphathiazole; TRM, trimethoprim.

b And less common SAs are included in the method scope. c AHS, sodium 1-heptanesulphonic acid (ion-pairing reagent).

**Compoundsa Extraction/**

DSTR, GEN SPC, STR

334 Honey Analysis

AMI, APR, DSTR, GEN, HYG, KAN, NEO, PAR, SIS, SPC, STR, TOB

DSTR, GEN, SPC, STR

APR, DSTR, GEN, NEO, PAR, SPC, STR

a

b

c

e

f

g

TE: thiol-ene.

SCP, SDT, SDX, SDZ, SMP, SMR SMX, SMZ, SPD,

SCP, SDT, SMP, SMR, SMX, SPD<sup>b</sup>

STZ

**clean-up**

20 mM K2 HPO<sup>4</sup>

(pH 7.4)/ MIP-SPE

Water/10 mM NH<sup>4</sup>

0.4 mM Na2 EDTA, 0.5% NaCl, 5% TCA, 1–10 M NaOH, WCX-SPE

5 mM K2

50 Mm KH2 PO<sup>4</sup> buffer

(pH 7.0)/ MIP-SPE

PVA-Sil: polyvinyl alcohol-Silica.

**Compoundsa Extraction/**

STZ Acetone/1 M

**clean-up**

HCl, diethyl ether

10% TCA, 1 M

0.1% PA (pH 2.0)/SCX-SPE, AHS<sup>c</sup>

, PA to pH 6.0, Oasis HLB-SPE

Na2 HPO<sup>4</sup> to pH 6.5/ACN, DCM

buffer (pH 11.0)/PVA-Sil-SPE<sup>f</sup> (home-made sorbent)

buffer

Ac,

HPO<sup>4</sup>

AHS, sodium 1-heptanesulphonic acid (ion-pairing reagent).

HFBA, heptafluorobutyric acid (ion-pairing reagent). dPFPA, pentafluoropropionic acid (ion-pairing reagent).

TEg

**Separation Equipment CCβ or LOD** 

**Column Mobile phase**

Isocratic: 20 mM AF (pH 3.0)/ACN (40:60, v/v)

Gradient: 1% FA/1% FA in ACN

30 mM AF/ FA (99/1, v/v)/ ACN/water/FA (80:19:1, v/v/v)

200 mM FA/7 mM NH3

AMI, amikacin; APR, apamycin; GEN, four isomers of gentamicin; HYG, hygromycin; KAN, kanamycin; NEO,

**Separation Equipment CCβ or LOD** 

LC-MS/MS (ESI+)

LC-FLD (with derivatization)

XAmide HILIC (150 × 4.6 mm, 5.0 μm)

Obelisc R 100 Å, (100 × 2.1 mm, 5.0 μm)


Bare fusedsilica capillary (90 cm × 50 μm × 375 μm)

neomycin; PAR, paromomycin; SIS, sisomycin; SPC, spectinomycin; TOB, tobramycin.

CZE-IT-MS: capillary zone electrophoresis coupled to ion trap mass spectrometry.

**Table 8.** Confirmatory methods for streptomycin/dihydrostreptomycin (STR/DSTR).

μBondapack phenyl (300 × 3.9 mm)

Nucleosil C18 HD (50 × 2.0 mm, 3.0 μm)

Symmetry Shield C18 (150 × 3.9 mm)

**Column Mobile phase**

Isocratic: KH2

Gradient: 0.3% FA in water:ACN (95:5, v/v)/0.3% FA in ACN

ACN

KH2 PO<sup>4</sup> (pH 3.5–4.0)/ACN (73:27, v/v)

PO4 buffer (pH 3.0) in 10%

Isocratic: 10 mM

**(µg/kg)**

LC-MS/MS (ESI+) 1.8–6.0 [65]

LC-MS/MS (ESI+) 1–12 [66]

LC-MS/MS (ESI+) 2.3–6.1 [67]

CZE-IT-MS<sup>e</sup> 6–103 [68]

**(µg/kg)**

<10 [70]

2–5 [71]

LC-UV 60 [69]

**References**

**References**

**Table 9.** Confirmatory methods for sulphonamides (SAs).


b PLS-2, polystyrene-divinylbenzene polymer (RP-SPE).

**Table 10.** Confirmatory methods for tetracyclines (TCs).


**Compoundsa Extraction/**

CTC, OTC, TC MacIlvaine

CTC, OTC, TC Citrate buffer, (Na2 EDTA)/ PLS-2-SPE<sup>b</sup>

CTC, OTC, TC ACN/SPE

CTC, OTC, TC 50 mM NH<sup>4</sup>

methacycline; TC, tetracycline.

buffer (Na2

MCAC-SPE

5% HCl/ MIP-SPE

(home-made sorbent)

buffer (pH 5.5)/ MCAC-SPE, Oasis HLB-SPE

MacIlvaine buffer (Na2 EDTA) (pH 4.0)/ Strata-X-SPE

Water/chitosanmodified graphitized MWCN

PLS-2, polystyrene-divinylbenzene polymer (RP-SPE).

**Table 10.** Confirmatory methods for tetracyclines (TCs).

Ac

CTC, DC, MINO, MTC, OTC, TC

336 Honey Analysis

CTC, DC, OTC,

CTC, DC, OTC,

CTC, DC, OTC,

CTC, DC, MTC, OTC, TC

TC

a

b

TC

TC

**clean-up**

MacIlvaine buffer (Na2 EDTA) (pH 4.0)/ phenyl-SPE

50 mM oxalate buffer (pH 4.0)/ Oasis HLB-SPE

EDTA) (pH 4.0)/hexane, PLS-2-SPE<sup>b</sup>

,

**Separation Equipment CCβ or LOD** 

LC-MS/MS (ESI+)

LC-MS/MS (ESI+)

LC-MS/MS (ESI+)

LC-MS/MS (ESI+)

LC-MS/MS (ESI+)

LC-HRMS (Q-TOF) (ESI+)

**Column Mobile phase**

Gradient: 0.09% OA (pH 3.0)/ACN

Gradient: 1% FA/ ACN:MeOH (50:50, v/v)

Isocratic: 1 M Imidazole buffer/MeOH (82:18, v/v)

Isocratic: 1 M Imidazole buffer/MeOH (75:25, v/v)

Isocratic: 100 mM OA/ ACN/MeOH (70:20:10, v/v/v)

Isocratic: 0.05% TFA/ACN (60:40, v/v)

Gradient: 0.1% FA/0.1% FA in ACN:MeOH (50:50, v/v)

Gradient: 0.05% AcOH/0.05% AcOH in ACN

Gradient: 0.1% FA/MeOH

CTC, chlortetracycline; DC, doxycycline; DMC, demeclocycline; OTC, oxytetracycline; MINO, minocycline; MTC,

Discovery RP-Amide C16 (5.0 μm)

Atlantis dC18 (150 × 2.1 mm, 3 μm)

Hydrospher C18 HS-301–3 (100 × 4.6 mm, 3.0 μm)

Tsk-gel ODS-80Ts (150 × 4.6 mm)

Restek C18 (150 × 2.1 mm, 5.0 μm)

ShodexRSpak DE-613 (150 × 6.0 mm)

Waters Phenyl (100 × 2.1 mm, 3.5 μm)

Symmetry C18 (150 × 2.1 mm, 3.5 μm)

SB-C18 (50 × 4.6 mm, 5 μm) **(µg/kg)**

3.3 [81]

0.1–0.3 [84]

3–20 [85]

7.2–7.7 [86]

5.5–9.2 [87]

0.61–10 [88]

LC-DAD 15–30 [80]

LC-FLD 5–9 [82]

LC-DAD 10–20 [83]

**References**


a Four subsequent LLE steps were carried out.

b NFPA, nonafluoropentanoic acid (ion-pairing reagent).

c CCβs for permitted antibiotics (lincomycin, MACs, QNs, SAs, TCs) were provided considering a hypothetical MRL of 100 or 200 μg/kg. For banned substances (NMZs), CCβs were in the range 1.2–2.6 μg/kg.

dTRM, trimethoprim.

e LIN, lincomycin.

f HFBA, heptafluorobutyric acid (ion-pairing reagent).

**Table 11.** Multiclass confirmatory methods.
