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Determination of Antibiotic Residues in Honey: - Zaneta Bargan Ska, Marek S Lebioda, Jacek Namies Nik
Determination of Antibiotic Residues in Honey: - Zaneta Bargan Ska, Marek S Lebioda, Jacek Namies Nik
7, 2011 Trends
Honey produced by honeybees is a valuable food product. The presence of xenobiotics in honey may harm its quality and
constitute a danger to human health. Antibiotics are commonly applied by beekeepers to eliminate disease among honeybees.
Moreover, ubiquitous administration of antibiotics may cause bacteria to become resistant to many drugs and spread antibiotic-
resistant strains of bacteria. Appropriate sample preparation and determination of antibiotics at very low concentrations in
foodstuffs are real analytical challenges. This article reviews analytical methods used for determination of residues of different
sorts of antibiotic in honey and other honeybee products.
ª 2011 Elsevier Ltd. All rights reserved.
Keywords: Antibiotic residue; Chromatographic determination; Gas chromatography; Honey; Liquid chromatography; Mass spectrometry; Multi-
class residue method; Sample preparation; Screening test; Tandem mass spectrometry
0165-9936/$ - see front matter ª 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2011.02.014 1035
Trends Trends in Analytical Chemistry, Vol. 30, No. 7, 2011
run, so the choice of sample-preparation technique is shows a general scheme of determination of antibiotics
very important. in honey.
Semi-quantitative immunoassay tests are character-
ized by high specificity, high sensitivity, simplicity and
2. Screening tests cost effectiveness, which make them particularly useful
in routine work. The main drawbacks of these methods
Screening methods can detect the presence of an analyte are high background absorption, a large number of
or group of analytes at the level of interest, and usually incubation and washing steps, and degradation of
provide semi-quantitative or quantitative results. enzyme during storage. Biosensor methods that contain
Screening tests should provide low rate of false compliant a biological-recognition element (e.g., enzymes, proteins,
samples, high throughput, ease of use, short analysis nucleic acids, cells, and tissues) coupled to a signal-
time, good selectivity, and low cost. Table 2 lists assays transduction element can operate fully automatically.
that are commonly used to determine the content of Instability of the biological-sensing component, which
antibiotic residues in honey. can lose its activity in hours or days, depending on the
Laboratories facing the analysis of large numbers of nature of the molecule and exposure to environmental
samples usually first apply screening methods covering stresses (e.g., pH, temperature or ionic strength), is the
families of antibiotics. Samples found to be non-compli- chief disadvantage of these types of test. Another limi-
ant are then analyzed by confirmatory methods. Fig. 1 tation is on the size of the physico-chemical transducers
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Trends in Analytical Chemistry, Vol. 30, No. 7, 2011 Trends
Table 2. Assays used to determine the contents of various antibiotic residues in honey
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Trends Trends in Analytical Chemistry, Vol. 30, No. 7, 2011
Table 3. Sample preparation and analytical techniques used to determine antibiotics in honey
Analytes Sample preparation Recovery [%] Analysis LOD [ppb] LOQ [ppb] Ref.
2
Sulfonamides Hydrolysis/LLE 44–73 LC-MS – – [27]
Hydrolysis/LLE/SPE 38–116 LC-FLD, post-column 1–2 2–5 [28,29]
derivatization
Dissolution/LLE/SPE 80–125 LC-MS2 7–12 [30]
Homogenization/LLE/SPE 65–86 LC-UV 0.3–0.4 1.5–2.2 [31]
Dissolution/SPE 74–94 LC-FLD 2–5 5–10 [32]
Hydrolysis 56–96 LC-FLD – 4–15 [33]
Hydrolysis/on-line – LC-MS2 0.5–2 – [9]
extraction/SPE
Tiamulin Homogenization/SPE 88–106 LC-UV and LC-MS 29–35 and 0.5–1.2 101–122 and 1.4–4 [48]
Abbreviations: CL, Chemiluminescence detector; dSPE, Dispersive solid-phase extraction; FLD, Fluorescence detector; LC, Liquid Chromatog-
raphy; LC-MS, Liquid chromatography-mass spectrometry; LC-MS2, Liquid chromatography-tandem mass spectrometry; LLE, Liquid-liquid
extraction; LOD, Limit of detection; LOQ, Limit of quantitation; MISPE, Molecular imprinted solid-phase extraction; OCLLE, On column
liquid-liquid extraction; RRS, Rayleigh resonance scattering detector; SPE, Solid-phase extraction; UV, Ultraviolet detector; QuEChERS, Quick,
easy, cheap, effective, rugged and safe extraction.
During the determination of antibiotics in honey, (56–96%) of 13 sulfonamides with relative standard
sugars and other interfering substances (e.g., wax and deviations below 10% [6].
dyes) have to be removed from the sample at the prep- To meet the continuing challenges in the analytical
aration stage [21] and that is not always straightfor- determination of sulfonamides in honey, and various
ward. For example, hydrolysis of the N-glycoside bond to requirements of the Codex Alimentarius [22], sample-
convert sugar-bonded sulfonamides into unbound forms preparation techniques were employed for extraction
sometimes led to poor recoveries for almost all the [i.e. solid-phase extraction (SPE) and solvent extraction
sulfonamides, and it was necessary to modify the sample- (SE)]. One solution that can further eliminate these
preparation method. Methanol was used to break problems is the Quick, Easy, Cheap, Effective, Rugged,
the N-glycoside bond to give satisfactory recoveries and Safe approach (QuEChERS), which was developed in
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Trends in Analytical Chemistry, Vol. 30, No. 7, 2011 Trends
2003 [23] and became very popular among analysts in multiple-reaction monitoring (MRM) mode [54].
worldwide for determining pesticide residues, especially According to EU criteria (2002/657/EC), when an MS
in food, because of its rapidity, cheapness and the pos- detector is used to confirm the presence of veterinary
sibility of determining a broad spectrum of compounds. drugs in food, at least two MRM transitions have to be
The conventional QuEChERS approach is based on employed [55] to provide information enabling unam-
acetonitrile/water partitioning of the analytes followed biguous confirmation of the analytes. Honey is a com-
by salting out with sodium chloride and magnesium plex matrix that contains sugars, pigments and phenolic
sulfate. Afterwards, dispersive SPE (dSPE), which in- compounds that must be removed prior to LC-MS anal-
volves the addition of small amounts of a bulk sorbent to ysis [26]. Usually methods start with acidic hydrolysis,
the extracts, is usually applied. The QuEChERS method followed by liquid-liquid extraction to liberate sugar-
reduces the number of steps in the analytical procedure, bound sulfonamides and sample clean up is continued by
thereby minimizing potential sources of error [24]. The SPE. The hydrolysis time must be long enough to ensure
application of the QuEChERS method provides high that the reaction is complete. The final analysis is by
recovery rates for analytes over a wide polarity range, LC-MS2 or LC-FLD, usually with post-column derivati-
and allows the use of smaller amounts of organic sol- zation.
vents. Methods allowing determination 37 out of the 42
A modified QuEChERS technique was used to analyze monitored multi-class antimicrobals (tetracyclines,
honey for chloramfenicol. Clean up by primary-second- macrolides, aminoglycosides, b-lactams, amphenicols
ary amine (PSA) was performed after evaporation of the and sulfonamides) in honey samples are available [5].
acetonitrile phase to decrease the limit of quantification The authors performed a single LC-MS2 analysis using a
(LOQ) and to minimize problems with the LC column stacking injection method. Thus, 10 lL of each of four
[25]. honey extracts were successively taken, giving a final
A review on general methods of preparing honey injection volume of 40 lL. The gradient elution and MS2
samples for chromatographic determination of contam- acquisition was started after the injection of the last
inants appeared recently [26]. Table 3 shows how the honey extract. The order of extract injection was very
choice of sample-preparation techniques depends on the important and influenced the loss of b-lactam due to the
properties of the target antibiotics in honey. presence of acid in two of the extracts. Up to 12 honey
samples can be prepared within 5 h using this analytical
3.2. Liquid chromatography procedure.
HPLC is widely used for routine analyses, where target A procedure involving sample dissolution with EDTA
antibiotics are determined. HPLC with UV detection was under mildly acidic conditions (pH 4.0) followed by SPE
used in a study of the degradation of tetracycline and allowed determination of 17 compounds (macrolides,
oxytetracycline in honey [51], but the limit of detection tetracyclines, quinolones, and sulfonamides) in honey
(LOD) was quite high (about 1 ppm). HPLC applied to samples [37] by ultra-performance LC-MS2 (UPLC-MS2)
determination of antibiotics has some inherent draw- in less than 5 min.
backs: Summarizing, the development of a multi-residue MS
(1) each antibiotic class has to be tested separately; detection methods, especially coupled with UPLC, is
(2) confirmation of the target analytes is based mainly highly promising and reduces the time and the effort
on retention-time comparison to standards; and, necessary for sample preparation.
(3) some analytes have to be derivatized to obtain an
appropriate LOD. 3.3. Gas chromatography
Sulfonamides are derivatized with fluorescamine (kex GC is rarely used for the determination of antibiotics in
403 nm and kem 492 nm, LOQ 10 ppb) [20] prior to honey, due to the polar nature, low volatility and ther-
separation by reversed-phase (RP)-HPLC for fluorimetric mal instability of these drugs. Derivatization of these
detection (FLD) (LOD 5 ppb, LOQ 10–15 ppb). polar compounds is advisable to improve peak shape and
Streptomycin was analyzed using post-column derivati- sensitivity of the method, acetylation being most widely
zation with 5-amino-6-hydroxynaphthalene-2-sulfonic used because the reaction can be carried directly in
acid with an LOD of about 5 ppb, and an LOQ of 10 ppb aqueous phase. Most applications require an extraction
[52]. step, for both sample clean up and preconcentration
Since 1990, LC-MS2, in combination with electrospray before analysis [7]. Table 4 summarizes the GC methods
ionization (ESI) or atmospheric pressure chemical ioni- used for determination of antibiotic residues in honey.
zation (APCI), has become widely used in the quantita- The use of atomic emission detection (AED) as a GC
tive analysis of residues of veterinary drugs in food [53]. detection method of N-methylated sulfonamides has the
A triple-quadrupole analyzer is usually used for the noticeable advantage of combining qualitative identifi-
quantitative analysis of different classes of antibiotics in cation of the analyte by evaluating the ratio of the ele-
food because of the high selectivity of the measurement ments forming the target compound with quantitative
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Trends Trends in Analytical Chemistry, Vol. 30, No. 7, 2011
Table 4. Gas chromatography methods used for determination of antibiotic residues in honey
Abbreviations: AED, Atomic emission detector; CCa, Decision limit; CCb, Detection capability; ID, Internal diameter; MS2, Tandem mass
spectrometry.
determination. Application of this technique extends given to sample preparation and extract clean up before
significantly the linear dynamic range and lowers the the final determination.
LOD [58]. Once again, MS detection offers the major
advantage of the qualitative identification of the analytes References
by their mass spectrum. However, the quantitative [1] European Commission, Council Directive 178/2002, Off. J. Eur.
determination of antibiotics is hampered by their non- Commun. L031 (2002) 1.
linear response, with deviations depending on the com- [2] European Commission (http://ec.europa.eu/food/food/rapidalert/
index_en.htm).
pound being determined.
[3] K. Kümmerer, Chemosphere 75 (2009) 417.
In conclusion, there is a downward trend in utilizing [4] European Commission, Council Regulation 2377/90, Off. J. Eur.
GC for the determination of antibiotics in honey, due to Commun. L224 (1990) 1..
the long analysis time and the additional step of deriv- [5] Y.-A. Hammel, R. Mohamed, E. Gremaud, M.-H. Le Breton, A.G.
atization, as demonstrated by a very small number of Philippe, J. Chromatogr., A 1177 (2008) 58.
[6] J. Bernal, M.J. Nozal, J. J Jiménez, M.T. Martı́n, E. Sanz, J.
recent publications on this method of determination. The
Chromatogr., A 1216 (2009) 7275.
only recommended GC-MS method that meets the cri- [7] M.C. Moreno-Bondi, M.D. Marazuela, S. Herranz, E. Rodriguez,
teria of Directive 2003/181/EC [20] is for the determi- Anal. Bioanal. Chem. 395 (2009) 921.
nation of chloramphenicol. [8] N. Moeller, E. Mueller-Seitz, O. Scholz, W. Hillen, A. Bergwerff, M.
Petz, Eur. Food Res. Technol. 224 (2007) 285.
[9] T.S. Thompson, D.K. Noot, Anal. Chim. Acta 551 (2005) 168.
4. Conclusion [10] J. Kisała, M. D_zugan, Zeszyty Naukowe 11 (2009) 115.
[11] H. Chen, J. Ying, H. Chen, J. Huang, L. Liao, Chromatographia 68
(2008) 629.
The presence of xenobiotics in food is particularly dan- [12] K. Cooper, G. Kennedy, Analyst (Cambridge, UK) 130 (2005)
gerous for human health, so it is essential to monitor 446.
their residues in food. EU requirements mean that it is [13] M. Morlot, P. Beaune, Apiacta 38 (2003) 226.
necessary to determine antibiotic residues in different [14] S. Bogdanov, Apiacta 38 (2003) 190.
[15] W. Reybroeck, S. Ooghe, H. de Brabander, E. Daseleire, J. Agric.
compartments of the environment and in food. The EU Food Chem. 55 (2007) 8359.
relies on MS detection for unequivocal identification of [16] N. Moeller, Eur. Food Res. Technol. 224 (2007) 285.
chemical residues in foodstuffs. EC Decision (2002/657/ [17] J.P. Ferguson, G.A. Baxter, J.D.G. McEvoy, S. Stead, E. Rawlings,
EC) states that ‘‘methods based only on chromatographic M. Sharman, Analyst (Cambridge, UK) 127 (2002) 951.
analysis without the use of molecular spectrometric [18] G. Alfredsson, C. Branzell, K. Granelli, A. Lundstrom, Anal. Chim.
Acta 529 (2005) 47.
detection are not suitable for use as confirmatory [19] R.J.B. Peters, A.A.M. Stolker, J.G.J. Mol, A. Lommen, E. Lyris, Y.
methodsÕÕ. Samples found to be non-compliant with Angelis, A. Vonaparti, M. Stamou, C. Georgakopoulos, M.W.F.
bioassay techniques therefore usually have to be con- Nielen, Trends Anal. Chem 29 (2010) 1250.
firmed with separation techniques utilizing specific [20] European Commission, Commission Decision 2002/657/EC, Off. J.
detectors (GC-MS or LC-MS). Eur. Commun. L71 (2003) 17.
[21] S.R. Rissato, M.S. Galhiane, M.V. de Almeida, M. Gerenutti, B.M.
The determination of antibiotics in honey is based Apon, Food Chem. 101 (2007) 1719.
mainly on multi-class residue methods enabling the [22] Establishment of Regulatory Programmes, Codex Alimentarius 3
simultaneous determination of a wide spectrum of target (1995) 66.
analytes in a single sample. Because the drugs are [23] M. Anastassiades, S.J. Lehotay, D. Stajnbaher, F.J. Schenck, J.
present in honey at low concentrations, priority is being AOAC Int. 86 (2003) 412.
1040 http://www.elsevier.com/locate/trac
Trends in Analytical Chemistry, Vol. 30, No. 7, 2011 Trends
[24] A. Wilkowska, M. Biziuk, Ford Chem. 125 (2011) 803. [42] L.-F. Wang, J.-D. Peng, L.-M. Liu, Anal. Chim. Acta 630 (2008)
[25] C. Pan, H. Zhang, S. Chen, Y. Xu, S. Jiang, Acta Chromatogr. 17 101.
(2006) 320. [43] G.-H. Wan, H. Cui, H.-S. Zheng, J. Zhou, L.-J. Liu, X.-F. Yu, J.
[26] M.W. Kujawski, J. Namieśnik, Trends Anal. Chem. 27 (2008) Chromatogr., B 824 (2005) 57.
785. [44] M. van Bruijnsvoort, S.J.M. Ottink, K.M. Jonker, E. de Boer, J.
[27] L. Verzegnassi, M.-C. Savoy-Perroud, R.H. Stadler, J. Chromatogr., Chromatogr., A 1058 (2004) 137.
A 977 (2002) 77. [45] D. Ortelli, P. Edder, C. Corvi, Chromatographia 59 (2004) 61.
[28] K.E. Maudens, G.-F. Zhang, W.E. Lambert, J. Chromatogr., A [46] R. Sheridan, B. Policastro, S. Thomas, D. Rice, J. Agric. Food
1047 (2004) 85. Chem. 56 (2008) 3509.
[29] A. Posyniak, K. Ja_zd_zewski, K. Pietruszka, K. Mitrowska, A. Gajda, [47] L. Zhao, C.H. Ball, Agilent Technologies Appl. Note 5990-
Bull. Vet. Inst. Pulawy 52 (2008) 87. 3615EN, 2009 (http://www.chem.agilent.com/Library/applica-
[30] V. Tamoši unas, A. Padarauskas, Chromatographia 67 (2008) tions/5990-3615EN.pdf).
783. [48] M.J. Nozal, J.L. Bernal, M.T. Martı́n, J.J. Jiménez, J. Bernal, M.
[31] H. Sun, L. Ai, F. Wang, Chromatographia 66 (2007) Higes, J. Chromatogr., A 1116 (2006) 102.
333. [49] R.H.M.M. Granja, A.M.M. Niño, R.A.M. Zucchetti, R.E.M. Niño, R.
[32] G.-F. Pang, Y.-Z. Cao, C.-L. Fan, J.-J. Zhang, X.-M. Li, Z.-Y. Li, G.- Patel, A.G. Salerno, Anal. Chim. Acta 637 (2009) 64.
Q. Jia, Anal. Bioanal. Chem. 376 (2003) 534. [50] C. Benetti, N. Dainese, G. Biancotto, R. Piro, F. Mutinelli, Anal.
[33] J. Bernal, M.J. Nozal, J.J. Jiménez, M.T. Martı́n, E. Sanz, J. Chim. Acta 520 (2004) 87.
Chromatogr., A 1216 (2009) 7275. [51] S. Bogdanov, Trakia J. Sci. 1 (2003) 19.
[34] C. Lafontaine, Y. Shi, F.A. Espourteille, Thermo Scientific Appl. [52] P. Edder, A. Cominoli, C. Corvi, Mitt. Geb. Lebensmittelunters.
Note, 2009 (http://www.thermo.com/eThermo/CMA/PDFs/Arti- Hyg. 89 (1998) 369.
cles/articlesFile_51570.pdf). [53] H.F. De Brabander, H. Noppe, K. Verheyden, J.V. Bussche, K.
[35] D. Debayle, G. Dessalces, M. Florence, G. Loustalot, Anal. Bioanal. Wille, L. Okerman, L. Vanhaecke, W. Reybroeck, S. Ooghe, S.
Chem. 391 (2008) 1011. Croubels, J. Chromatogr., A 1216 (2009) 7964.
[36] M.I. Lopez, J.S. Pettis, I. Bartonand, P.S. Chu, J. Agric. Food Chem. [54] S.J. Lehotay, K. Mastovska, A. Amirav, A.B. Fialkov, T. Alon, P.A.
56 (2008) 1553. Martos, A. de Kok, A.R. Fernández-Alba, Trends Anal. Chem. 27
[37] J.L. Martı́nez-Vidal, M.M. Aguilera-Luiz, R. Romero-González, A. (2008) 1070.
Garrido-Frenich, J. Agric. Food Chem. 57 (2009) 1760. [55] European Commission, Commission Decision 2002/657/EC, Off. J.
[38] J. Wang, J. Agric. Food Chem. 52 (2004) 171. Eur. Commun. L221 (2002) 8.
[39] J. Li, L. Chen, X. Wang, H. Jin, L. Ding, K. Zhang, H. Hang, [56] N. Campillo, R. Peñalver, M. Hernández-Córdoba, J. Chromatogr.,
Talanta 75 (2008) 1245. A 1125 (2006) 31.
[40] T. Jing, X.-D. Gao, P. Wang, Y. Wang, Y.F. Lin, X.-Z. Hu, Q.-L. [57] S. Impens, W. Reybroeck, J. Vercammen, D. Courtheyn, S. Ooghe,
Hao, Y. -K Zhou, S.-R. Mei, Anal. Bioanal. Chem. 393 (2009) K. De Wasch, W. Smedts, H. De Brabander, Anal. Chim. Acta 483
2009. (2003) 153.
[41] A.C. Pancorbo, S.C. Terrones, A.S. Carretero, A.F. Gutiérrez, J. [58] B. Chiavarino, M.E. Crestoni, A. Di Marzio, S. Fornarini, J.
Chromatogr., A 1195 (2008) 107. Chromatogr., B 706 (1998) 269.
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