Research Article

Received: 22 February 2007 Revised: 29 February 2008 Accepted: 12 August 2008 Published online in Wiley Interscience: 7 October 2008

(www.interscience.com) DOI 10.1002/jsfa.3411

Residues of antibiotics and sulfonamides

in honeys from Basque Country (NE Spain)
J Serra Bonvehı́a∗ and A Lacalle Gutiérrezb

Abstract
BACKGROUND: The aim of this work was to ensure that Label Basque market honey is free of veterinary residues.

RESULTS: A total of 567 Basque honey samples were previously analyzed with the respective Charm II system – 68 samples were
presumptive positive for sulfonamides (SA-s), 24 samples for tetracyclines (TC-s), and no positive samples for chloramphenicol
(CAP) (<0.3 µg kg−1 ) residues. The residues were mostly confirmed by liquid chromatography fluorescence detection (LC-FD)
and tandem mass spectrometry (LC-MS/MS), according to the latest European Union criteria for the analyses of veterinary drug
residues (2002/657/EC). These techniques confirmed that 19 of the 68 samples, presumptive contaminated with SA-s, contained
sulfathiazole (STZ) residues at levels from 20 to 210 µg kg−1 , and the 24 samples presumptive contaminated with TC-s, were also
confirmed, showing tetracycline (TC) levels from 15 to 920 µg kg−1 . Linearity range, decision limit (CCα), detection capability
(CCβ), precision and reproducibility were also determined.

CONCLUSION: Residues of veterinary drugs were confirmed in a very limited number of honey samples: sulfathiazole (3.40%)
and tetracycline (4.22%). This work reports the advantages of the Charm II assay, but also its limitations, detecting SA-s in
most (87.7%) of the heather (Erica vagans) honey samples. The false positives detected in this honey were assumed to be of an
unknown compound that has not been confirmed as a drug residue. Until now, no studies have been performed to find out if
other heather honeys of different geographical origins give similar false positives for SA-s.

c 2008 Society of Chemical Industry

Keywords: sulfonamides; tetracyclines; raw honey; Charm II test; liquid chromatography/tandem mass spectrometry

INTRODUCTION Required Performance Limit (MRPL) of the analytical method of

Antibiotics are used in the prevention and treatment of European
foulbrood (EFB) (Melissococcus pluton) and American foulbrood
(AFB) (Paenibacillus larvae) in honeybees.1 Since the mid-1990s,
there has been an increase number of AFB cases in Europe.2 Drugs
known to be effective against these diseases are tetracyclines
(TCs) [mainly oxytetracycline hydrochloride (OTC), and tetracycline
(TC)], streptomycin and sulfathiazole (STZ). Powdered Terramycin
(oxytetracycline hydrochloride) is used in Spain (200 mg per hive).
The drug is administered in three applications of sugar syrup or
three dustings at 4- to 5-day intervals. Terramycin should be fed
early in the spring or fall and consumed by the honeybees before
the main honey flow begins, to avoid contamination of honey
production. The treatment has to be removed at least 6 weeks
before main honey flow. Apicicline was also used in veterinary
practice in Spain; it contains 0.4% oxytetracycline and 4% sul-
fathiazole as active compounds. However, the drugs do not kill
the P. larvae because of the presence of resistant bacteria.1,2 The
safety of sulfonamides (SA-s) to consumers has been questioned
because of their apparent toxicity.3 OTC and TC are also used in
treatment of human bacterial infections and, thus, this method of
control is not advisable. As a result, honey could become contam-
inated with these medical products. In Germany and several other
countries (Japan, Switzerland, Scandinavia, etc.), the use of such
antibiotics is not approved for the treatment of honeybees.4 In the
Council Regulation (EEC) 2377/90,5 no maximum residues (MRLs)
detection was established for chloramphenicol (0.3 µg kg−1 ).6 The
consumer does not expect any contamination with antibiotics in
a natural product such as honey, because honey itself has a bac-
teriostatic action.7 Switzerland has established a general MRLs
of 50 µg kg−1 for sulfonamides, 20 µg kg−1 for tetracyclines, and
20 µg kg−1 for streptomycin in honey.8 A procedure to decontam-
inate AFB-infected equipment and to control AFB is the utilization
of gamma-radiation.9 This practice showed good results in Aus-
tralia, the only country where gamma-radiation is allowed on a
commercial basis. Several screening kits (Tetrasensor, Elisa, Scin-
tillation detection)10 – 14 are available to detect the presence of
antibiotic residues in honey at low levels, but they show sev-
eral handicaps: (1) they do not identify analyte; (2) they display
interferences and false positive results; (3) presence of epimers
reduces the sensitivity; and (4) matrix effects are observed. Charm
II assay has been used (since about 2000) in the detection of
antibiotics in animal food and other matrices, including honey.
∗ Correspondence to: J Serra Bonvehı́, Research and Development of Nederland,
Co., P.O. Box 34, 08890 Viladecans (Barcelona), Spain.
E-mail: serrjosep@gmail.com
a Research and Development of Nederland, Co., P.O. Box 34, 08890 Viladecans
(Barcelona), Spain
b Neiker, Nekazal Ikerketa eta Garapenerako Euskal Erakundea, Berreaga Kalea
63

are fixed for antibiotics in honey; however in 2003, a Minimum 1, 48160 Derio (Bizkaia), Spain

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This screening procedure is based on a radio immuno assays de-
tection system for chemical families of drug residues with different
limits of detection.15 It uses class-specific receptors or antibodies
in imune/binding assay formats. Results are given in numerical
counts, exhibiting high sensitivity and good repeatibility for the
antibiotics tested. For the detection of any antimicrobial drug, two
reagents are required: a [14 C]- or [3 H]-labeled antimicrobial drug
and a binding reagent (microbial cell containing receptors for
the antimicrobial drugs). A variety of chromatographic methods
has been used to confirm and quantificate presumptive antibi-
otic residues in honey. In this paper, two analytical methods are
proposed for the quantitative determination of SA-s and TC-s
in honey by LC-FD, and the positive samples were also con-
firmed by liquid chromatography coupled to mass spectrometry
(LC-MS/MS). Several liquid chromatographic methods have been
reported for determination of SA-s and TC-s using ultraviolet (UV)
detection; however, these methods have insufficient sensitivity,
and interferences from the honey matrix do not allow quantifi-
cation by UV at the level 10 µg kg−1 .16 – 19 A rapid reverse-phase
high-performance liquid chromatography method with fluorimet-
ric detection is mainly applied, due to the low sensitivity of UV
detection and the high cost of the mass spectrometry instru-
ment. SA-s have bean determined by fluorescence detection with
precolumn derivatization with fluorescamine, using previous liq-
uid–liquid extraction.20,21 In addition, in the experiment reported
here, extract ion and cleanup procedure was applied to suc-
cessfully isolate TC-s, which combined postcolumn magnesium
acetate with fluorescence detection to quantify TC-s at this low-
level.22 Fortunately, the cleanup for honey extracts through Oasis
HLB cartridges considerably reduces the interferences and their
negative influences on the selectivity of the analytical signal and
maintenance of instruments, especially in LC-MS/MS analysis.8,23
To improve the precision and accuracy of the analytical procedure
utilized, the validation was established taking into account the
European Community Decision 2002/657/EC.24 For the analysis
of banned compounds (such as sulfonamides and tetracyclines
for which no MRLs have been set) by LC-MS/MS, a minimum of
four identification points (IP-s) are required. Pang et al.23 present
LC-MS/MS procedure with satisfactory results without the hy-
drolysis step required by other chromatographic methods with
different detection systems, including LC-MS analysis. However,
in the present study a hydrolysis step prior to extraction was
adopted to ensure that sugar-bound sulfonamides in honey were
included in the final result.8,20,25 In addition, the above-mentioned
data confirm that honey interactions between sugars and SA-s
decreases biological activity, making the product more available
under acidic conditions. In consequence, it could be transformed
into free active compound in the human digestive system.
The aim of the present work was to obtain an overview to ensure
that Label Basque marketed honey is free from drug residues.26
Moreover, all honey and syrup stored during medication must be
removed to avoid human consumption. In fact, honey purchasers
requests suppliers to certify that the offered honey is pure and free
of veterinary drugs.
MATERIALS AND METHODS
Materials
The study was carried out on 567 selected samples from different
floral origins. All samples were unheated, and stored in dark
and dry places at room temperature. The samples were taken
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directly from the containers that beekeepers use for the storage of
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c 2008 Society of Chemical Industry
J Serra Bonvehı́, A Lacalle Gutiérrez
Table 1. Detection and confirmation of presumptive positive
residues in unifloral and multifloral honeys
Charm II Test LC-FD
Botanical origin
(no. samples) SAs TCs CAP STZ TC
Erica spp. (65) 57 15 0 8 15
Eucalyptus sp. (37) 0 1 0 0 1
Castanea sativa (12) 0 0 0 0 0
Multifloral (453) 11 8 0 11 8
SA-s: sulfonamides; TC-s: tetracyclines; CAP: chloramphenicol
honey. For each sample, data about its handling by the producer,
extraction date, type of bee-hive and route of conveyance was
collected. Maturity and deterioration criteria indicated that the
quality of the honeys was good, and none of the samples showed
signs of fermentation. After this laboratory study, the samples
were reclassified as unifloral or multifloral honey, according
requirements defined in the Basque Label Quality.26 The unifloral
honeys (provided by Basque beekeepers and produced in the years
2003 and 2004) were defined as sweet chestnut (Castanea sativa),
heather (Erica vagans plus E. cinerea), and eucalyptus (Eucalyptus
sp.) (Table 1).
Chemicals
HPLC-grade dichloromethane, methanol, and acetonitrile were
from Scharlab (Barcelona, Spain). Analytical grade citric acid, suc-
cinic acid, boric acid, oxalic acid, magnesium acetate, sodium
hydroxide, trichloroacetic acid, disodium phosphate, phosphoric
acid, hydrochloric acid (37%), formic acid (98%), and ammo-
nia (25%) were supplied by Acros (Geel, Belgium), and flu-
orescamine was obtained from Merck (Darmstadt, Germany).
Sodium 1-decanesulfonate (for ion-pair chromatography) was
obtained from Sigma Chemical Co. (St Louis, MO, USA). Ultra-
pure water (Milli-Q, Millipore Corp., Bedford, MA) was prepared
for chromatographic use. Sulfonamides – sulfamethizole (SML),
sulfamethoxypyridazine (SMP), sulfadoxine (SDX), sulfathiazole
(STZ), sulfamerazine (SMR), sulfamethazine (SMZ), sulfachloropy-
ridazine (SCP), sulfadimethoxine (SDM), sulfamethoxazole (SOZ),
sulfisoxazole (SXZ), sulfaguanidine (SGD), sulfadiazine (SDZ), sul-
fanilamide (SNL), sulfacetamide (SAA), and sulfapyridine (SPY),
and the hydrochlorides of tetracycline (TC), oxytetracycline (OTC),
and chlortetracycline (CTC) – were from Sigma, sulfachlorpyrazine
(SPZ) (Ciba-Geigy Corporation, Ardsley, NY), 4-epitetracycline (4-
epiTC), 4-epioxytetracycline (4-epiOTC), and 4-epichlortetracycline
(4-epiCTC) were obtained from Acros (Fisher Scientific, Schwerte,
Germany).
Reagents, standards and buffers [tablet reagents (radioactive
material), MSU multi-antimicrobial concentrate standard, zero
control standard, MSU extraction buffer, and scintillation fluid
(Optifluor)] are included in the Charm II drug test for honey
(Charm Sciences, Inc., Lawrence, MA).
Instruments
Charm II system
The Charm II 7600 analyzer (Charm Sciences, Inc.) assay is a rapid
antibody assay that uses [3 H]- and [14 C]-tagged drug tracers with
broadly specific binding agents in a multipurpose single sample,
liquid scintillation counter (LSC) and luminometer.14,27 When the
J Sci Food Agric 2009; 89: 63–72
Antibiotic and sulfonamide residues in Basque Country honey
binding reagent is added to a sample with antimicrobial drugs, the
contaminating antimicrobial drug binds to receptors in the cell.
This prevents the [14 C] or [3 H] antimicrobial drug from binding to
these sites. Therefore, the more [14 C]- or [3 H]-labeled antimicrobial
drug bound, the less antimicrobial drug there is in the sample. The
detection reaction is stopped with a centrifugation step, where
unbound tracer is separated from bound tracer–binder complex,
and analyzed in a scintillation counter for 1 minute to give a
resulting count. The amount of [14 C] or [3 H] bound to the cells
is measured in counts per minute (cpm). The higher the count,
the less drug contamination in the sample, and vice versa. The
result is simplified to a present/absent result using a control
point. The control point is the cutoff number between a negative
and a positive result. For establishing a control point, six negative
controls (known negative honey samples) have to be run. Then the
results are averaged and 25% subtracted. This value indicates the
control point. Test results greater than the control point indicate
a negative sample, while results less than or equal to the control
point indicate the sample is presumptive positive and needs to be
retested.14,27
Liquid chromatography system
The liquid chromatography consisted of a Model Prostar 240
LC pumping unit; Model 410 Autosampler fitted with a 50 µL
loop; 330 photodiode array detector (PAD), and 363 fluorescence
detector (all from Varian Chromatography Division, Palo Alto,
USA). Chromatographic data from LC were processed on Star
Chromatography WorkStation software, version 6.20, for data
processing. LC-MS/MS experiments were performed using a
Finnigan MAT TSQ 70 triple quadrupole MS/MS system (Finnigan
MAT, San José, CA) equipped with Finnigan MAT thermospray
interface.
Sample preparation for analysis by Charm II test
The honey extract had active reagents added in sequential and
competitive assay formats at various incubation temperatures
optimized for drug detection. Extraction procedure was as
described in Operator’s manual for sulfa 011 and test for
antimicrobial drugs in honey (beta-lactams and tetracyclines),
including a new edition for chloramphenicol assay.27,28 The test
took 12–20 minutes for a multitude of antibiotics. Sulfonamides
assay used a more complex acid hydrolysis and reverse phase
preparation to eliminate p-aminobenzoic acid (PABA), and convert
carbohydrate-SAs into free form (total time 1 h).
Sulfonamides
About 5 g of honey was transferred into a 50 mL conical centrifuge
tube and homogenized with 20 mL of 1 mol L−1 HCl. After ho-
mogenization, the solution was incubated at room temperature
for 1 h. Then, 2.3 mL of 7.5 mol L−1 NaOH were added to sample
solution, followed by the measurement of pH (1.0 to 5.0). The pH
was adjusted to 7.7–8.0 drop by drop with 0.075 mol L−1 NaOH.
The filtrate solution was applied to an Bond Elut C18 cartridge
(500 mg, 3 mL) (Varian, Harbour City, CA), preconditioned with
5 mL of methanol and 5 mL of distilled water (twice). After the
extract had passed through at a flow rate of 1–2 drops per sec-
ond, the cartridges were washed with 5 mL of distilled water. The
analytes were eluted with 1 ml of methanol, and the eluate was
evaporated to dryness under a nitrogen stream (40–45 ◦ C). The
dry residue was reconstituted in 5 mL of Zero Control Standard so-
lution, and stopped with cold ice for 10 min. Finally, the deuterated
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standard solution was added (tablet reagent dissolved in 300 µL
of distilled water), and incubated at 85 ◦ C for 3 min. The samples
were then homogenized and centrifuged (2000 × g, 10 min). The
supernatant was discarded and the residue redissolved in 300 µL of
distilled water and 3 mL scintillation fluid (Opti-fluor). Immediately,
the solution was measured on [3 H] channel of Charm analyzer in
counts per minute (cpm), and compared with control point.27
Tetracyclines
Sample preparation for tetracyclines was restricted to a simple
dilution step (1 : 10) by dissolving 1 g of honey in 9 mL of MSU
extraction buffer supplied with the test kit. The receptor tablet was
suspended in 300 µL of distilled water, 4 mL of the diluted honey
solution added, and the mixture incubated at 35 ◦ C for 5 min. The
samples were then homogenized, centrifuged (2000 × g, 10 min.),
and the supernatant discarded. Finally, the residue was dissolved
with 300 µL distilled water and 3 mL scintillation fluid (Opti-fluor).
Immediately, the solution was measured on [3 H] channel of the
Charm analyzer (cpm), and compared with control point.27
Chloramphenicol
The procedure is recommended for honey samples with a Pfund
colour index of 83–114 mm (e.g. heather honey). About 10 g
of honey was weighed in a 50 mL centrifuge tube, and 30 mL
of the supplied MSU extraction buffer added. The solution was
thoroughly mixed for 1 min., and adjusted the pH to 7.5 with
M2 buffer included in the test kits. Then 5 mL of the solution
was loaded onto a CH cartridge Varian (500 mg, 6 mL) that had
been previously conditioned with 5 mL of 100% methanol. The
samples were washed with 5 mL of 20% methanol, and the
analyte was extracted with 2 mL of 75% methanol. The receptor
white tablet was suspended in 300 µL of distilled water, 5 mL of
the diluted honey solution added, and the mixture incubated at
50 ◦ C for 3 min. The sample was homogenized and the test tube
returned to the incubator at 50 ◦ C for an additional 3 min. The
green tablet was added, the mixture homogenized and incubated
at 50 ◦ C for 3 min., then centrifuged at 5000 rpm for 5 min. The
supernatant was discarded. Finally, the residue was dissolved
with 300 µL distilled water and 3 mL scintillation fluid (Opti-fluor).
Immediately, the solution was measured on [3 H] channel of Charm
analyzer (cpm), and compared with control point.27,28
Sample preparation for LC-FD
Sulfonamides
The sulfonamides were extracted from 5 g of homogenized honey
with 5 mL of 0.1 g mL−1 trichloroacetic acid solution. The samples
were shaken for 10 min. at room temperature on a mechanical
shaker and heated for 1 hour at c. 64 ◦ C. The pH was adjusted to
6.5 with 1 mol L−1 Na2 HPO4 , pH 12 (c. 2.5 mL), and later 10 mL
of acetonitrile and 2.5 mL of dichloromethane were added. The
mixtures were shaken again for another 10 min. on a mechanical
shaker, and centrifuged at 6000 rpm for 10 min. The supernatant
was poured into a 25 mL volumetric flask. Re-extract twice and
made up to volume with acetonitrile. Of the organic extract, 10 mL
was evaporated to dryness under a nitrogen stream at 50 ◦ C.
The extract residue was reconstituted in 1 mL of solution buffer
for derivatization [acetonitrile/H3 PO4 pH 6.0 and citrate buffer
0.5 mol L−1 pH 3.0) in the ratio (6/4, v/v)], adding 20 µL of internal
standard (5 µg mL−1 of sulfanilamide in methanol), and 0.2 mL of
10 mg mL−1 fluorescamine solution (50 mg fluorescamine in 5 mL
of acetone). The solution was allowed to develop the reaction for
40 min. at room temperature.20,21
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Tetracyclines
The tetracyclines were extracted from 5 g of homogenized honey
with 25 mL of succinic buffer solution at pH 4 (5.9 g succinic
acid in 1 L distilled water adjusting pH 4 with 1 mol L−1 NaOH),
and this mixture was vortexed to complete dissolution, before
being centrifuged at 3500 rpm for 15 min. at room temperature.
The Oasis HLB solid phase extraction (200 mg per 6 mL) (Waters
Chromatography Division, Milford, MA) was conditioned with
3 mL acetonitrile and 3 mL distilled water. The supernatant of the
centrifuged extract was poured into the reservoir and the solution
passed through the SPE cartridge for 12–15 min. The samples
were washed with distilled water (3 mL) for 1–2 min., and the TC-s
were eluted with 5 mL of extraction solution [oxalic buffer solution
(0.01 mol L−1 oxalic acid adjusted to pH 2.25–2.5 with 1 mol L−1
NaOH), and acetonitrile in the ratio (30/8, v/v)].22,29
Sample preparation for LC/MS-MS confirmation
Sulfonamides and tetracyclines
About 10 g honey was dissolved in 20 mL of 2 mol L−1 HCl and the
samples were allowed to stand for 30 min. at room temperature.
After incubation, 40 mL of 0.3 mol L−1 citric acid was added. The pH
was adjusted to 3.4–4.5 with ammonia solution (7.14 mol L−1 ) in
20 mL honey filtrate. The solution was passed for 10–15 min. over
SPE cartridge (Waters Oasis HLB 200 mg per 6 mL) preactivated
with 3 mL of acetonitrile and 2 mL of distilled water. Later the
sample was washed with 3 mL of distilled water (three times), dried,
and the retained analytes were eluted with 3 mL of acetonitrile in
a graduated flask. The extract was then evaporated at 40 ◦ C under
nitrogen flow to a small volume, and diluted by adding 0.5 mL
of mobile phase A, and the graduate flask reweighed. Finally, the
solution was transferred without filtration into a vial.8
Chloramphenicol
About 1 g honey was dissolved in 1 mL of distilled water at 60 ◦ C
for 5 min. Then, 5 mL acetonitrile was added, and the mixture
shaken vigorously for 10 s, centrifuged for 3 min. at 3500 rpm, and
the supernatant was transferred to glass-stoppered tube. Then
1 mL distilled water and 5 mL acetonitrile were added, and the
mixture was shaken vigorously for 6 s, recentrifuged for 2 min. at
3500 rpm, and the upper layer (water) discarded. The organic layer
was evaporated to dryness under a nitrogen stream (60 ◦ C), and the
residue was dissolved in 100 µL of methanol, and 3 mL of distilled
water and 0.5 mL of buffer solution 0.5 mol L−1 Na2 HPO4 .2H2 O
(pH 6 adjusted with 0.85 g mL−1 phosphoric acid) added.30,31
Instrumental analysis
LC-FD detection
Sulfonamides. The separation was obtained with a Nucleosil
100-5 C18 AB (250 × 4.6 mm i.d., 5 µm particle size) column
provided with a Nucleosil 100-5 C18 AB guard column (8×4.6 mm)
(Varian, Middelburg, The Netherlands). The mobile-phases were
(A) 0.02 mol L−1 H3 PO4 and (B) CH3 OH/Acetonitrile (1 : 1, v/v). The
column was eluted by using a linear gradient: initial time, 40%
B; 0–20 min., 40% B; 20–25 min., 50% B; 25–39 min., 50% B;
39–40 min., 55% B; 40–50 min., 55% B; 50–51 min., 40% B;
51–58 min., 40% B. The mobile phase flow rate was of 0.6 mL
min .−1 , injection volume, 50 µL. Use of photodiode array detector
coupled on line with fluorescence detection. The settings of the
fluorescence detection were λexc 405 nm, λem 496 nm, sensitivity
0.3, and slit 7 nm (Model 363 ProStar); the maximum UV absorption
of the SA-s were found to be in the range of 260–290 nm.20,21
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Tetracyclines. LC was performed with a Chromsep 5 C8 (100 ×
4.6 mm i.d.; 5 µm particle size) column and a guard column
Chromsep 5 C8 (4.0 × 2 mm i.d.) (Varian). The mobile-phases were
(A) 1.22 g sodium 1-decanesulfonate dissolved in 1 L of 0.01 N
oxalic buffer solution adjust to pH 2.25–2.5 with 1 N NaOH
and (B) 1.22 g sodium 1-decanesulfonate dissolved in 1 L of 0.01
N oxalic buffer solution/acetonitrile/methanol (2 : 1:1, v/v). The
column was eluted by using a linear gradient: initial time, 32% B;
0–15 min., 100% B; 15–17 min., 32% B; 17–25 min., 32% B. The
mobile-phase flow rate was of 0.8 mL min−1 ; Injection volume,
50 µL. Fluorescence detection was carried out after post-column
addition of magnesium acetate [(6 g L−1 ) in 0.1 mol L−1 boric acid
buffer pH 9], and the derivation reagent was delivered at a flow
of 0.3 ml min .−1 . The settings of the fluorescence detection were:
λexc 385 nm, λem 500 nm, sensitivity 0.3, slit 7 nm [Model 363
ProStar].22,29
LC-MS/MS system
Sulfonamides and tetracyclines. Chromatographic separation was
performed on a column Nucleosil 100-5, C18 HD (50 × 2 mm i.d.,
5 µm particle size) equipped with 5 µm Nucleosil 100, C18 HD
guard column (4.0 mm × 2 mm i.d.). Column temperature set at
25 ◦ C. The mobile-phases were (A) 50 mL acetonitrile and 3 mL
formic acid (98%) l L distilled water, and (B) 3 mL formic acid
in 1000 mL graduated flask and adjusted with acetonitrile. The
column was eluted by using a linear gradient: 0–10 min., 30% B;
10–12 min., 30% B; 12–12.1 min., 0% B; 12.1–19 min., 0% B. The
solvent flow rate was 0.2 mL min .−1 ; injection volume, 10 µL. A
tandem mass spectrometer equipped with electrospray ionization
(ESI) was used for detection. The ESI source operated in the positive
ionization mode at 90 ◦ C, and desolvation was performed at 250 ◦ C
with a desolvation gas flow of 560 L h−1 and a cone gas flow of
50 L h−1 . For desolvation and nebulization, high-purity nitrogen
was used, and argon (99.998%) served as the collision gas. MS/MS
was performed in both the daughter scan and neutral loss scan
modes, applying multi-reaction monitoring (MRM) using the two
most intense and specific fragment ions.8
Chloramphenicol. Chromatographic separation was performed
on a column Hypersil-Keystone Beta Basic C18 (150 × 2.1 mm i.d.,
5 µm particle size) (Thermo Electron, Madison, WI). The mobile-
phases were (A) water and (B) methanol. The column was eluted
by using a linear gradient: initial time, 30% B; 0–3 min., 95%
B; 3–5 min., 95% B; 5–5.1 min., 30% B. The solvent flow rate
was 0.25 mL min .−1 ; injection volume, 20 µL. MS/MS conditions:
Ionization mode polarity: ESI, negative; source temperature,
350 ◦ C; scan event (m/z 152–256) and collision energy (m/z
157–272).30,31
Standards solutions
Sulfonamides
Stock solutions of sulfonamides and sulfanilamide (SA) as internal
standard at the concentration of 1 mg mL−1 were prepared in
methanol, and stored protected from light at 4 ◦ C. A stock standard
mixture (10 µg mL−1 ), was prepared in distilled water. Working
standard solutions with concentrations in the expected ranges
were prepared from this stock standard solution at 10, 20, 50, 100
and 200 ηg mL−1 .
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Antibiotic and sulfonamide residues in Basque Country honey
Tetracyclines
The tetracyclines were dissolved in 100 mL of methanol (21.6 mg
tetracycline hydrochloride, 22.2 mg chlortetracycline hydrochlo-
ride and 21.6 mg oxytetracycline hydrochloride) for a final con-
centration of 200 µg mL−1 . The stock solution was diluted to
appropriate concentration (50 µg mL−1 ) with methanol. A second
standard solution (2.5 µg mL−1 ) was prepared daily by dilution
of standard solutions with extraction solution [oxalic buffer solu-
tion/acetonitrile (30/8, v/v)]. A calibration curve in the range 10 to
300 ηg mL−1 was prepared daily. Standard solution of the 3-epi
metabolites was also prepared, but they were only analyzed for
identification.
Chloramphenicol (CAP)
A stock solution of CAP was prepared by dissolving 25 mg of
chloramphenicol in 25 mL of methanol (stable for at least 3 months
at 4 ◦ C). The stock solution was diluted with appropriate volumes
of methanol/water (3/7, v/v) to create working standards for
calibration curve (0.2, 0.5, 1, 2, 5, and 10 ηg mL−1 ).
Method validation
Validation of the LC method for sulfonamides and tetracyclines
determination
Method validation was carried out according to the criteria
described in the Council Directive 96/23/EC.24 The parameters
taken into account were the following: response linearity, decision
limit, detection capability, trueness, and precision. Usually the
quantification of drug residues is performed using a matrix-
matched calibration curve made from fortified blank samples
prepared in the same matrix as the real samples. A calibration curve
was established by measuring sulfonamides and tetracyclines
peak areas over a five-fold range of concentrations in fortified
blank honeys. The slope, intercept, and correlation coefficient
were calculated by linear regression analysis. Standard calibration
curves (four replicates each) for the analytes were plotted. These
calibration curves were prepared repeatedly during the study with
highly reproducible results. In order to improve the accuracy, the
internal standard quantification was applied to detect sulfonamide
residues at low levels. Internal standard (IS) was a difficult
process, as there were no regions of the chromatogram that were
completely free of extract-born interferences. The sulfanilamide
(SLN) was selected based on the relative lack of interferences,
appropriate retention time, low cost, and well-characterized purity,
and is the compound most frequently referred to as suitable for
this purpose.20,21 Furthermore, among the samples analyzed, none
was found for SLN. However, the selection of a possible internal
standard has to be done with great care and, if is possible, an
adequate stable isotope labeled internal sulfonamide is preferred.
The rations of the peak area (A) of each SA-s to that of the internal
standard (IS) were plotted against concentration described in
calibration standard solutions. The precision study was evaluated
with the SA-s (STZ, SML, SMZ, and SLN as internal standard), and
TC-s (TC, OTC, and CTC) most used in veterinary practice in Spain.
The recovery and repeatability of the procedures were evaluated
by the analysis of six spiked samples with these SA-s and TC-s
at 25, 50, 100, and 150 µg kg−1 based on the linearity range,
on three different days (n = 24). Repeatability was evaluated
by applying the whole extraction procedure six times to the
same sample. Reproducibility was determined by analyzing each
sample of honey fortified on five different days over about a
month. Precision and accuracy were determined using spiked
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c 2008 Society of Chemical Industry
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samples with the SA-s and TC-s described at three concentrations
ranging from 50 to 150 µg kg−1 (n = 5 for each concentration).
The following equations were used:
Accuracy (%) = [(measured concn − actual concn)/
actual concn] × 100
Precision (%) = (standard deviation/mean concn) × 100
According to the guidelines, limit of detection (LOD) and limit of
quantification (LOQ) are expressed as the decision limit (CCα) and
detection capability (CCβ), respectively. The decision limit is the
lowest concentration level of the analyte that can be detected
in a sample with a an error probability of 1% that a sample is
noncompliant. The detection capability is the lowest concentration
at which a method is able to detect truly contaminated samples
with an error probability of β (β = 5% for banned compounds).
Blank material was fortified at 5 different concentrations (n = 20)
and the standard error of the y intercept was calculated.
The decision limits (CCα = 2.33 × standard error of the y
intercept) and the detection capabilities [CCβ = CCβ + (1.64 ×
standard deviation of 20 spikes at CCα)] were determined.24
Charm test
The validation method for each antibiotic and sulfonamide is
incorporated as an appendix to the Cham II manuals for honey. The
decision limit was obtained using negative honey with established
control point, spiked with the sulfonamides, tetracyclines and
chloramphenicol at different concentrations for obtaining results
less or equal to this control point.
Melissopalynological analysis
The analysis of floral origin was carried out in accordance with the
methods of the International Commission of Bee Botany (ICBB)
of the International Union of Biological Sciences (IUBS),32 which
involved 1200 pollen grains per sample, as well as honeydew
indicators.33 The microscope preparations were made without
acetolysis to preserve all the components in the sediments
extracted. Pollen was observed at ×400–1200 magnifications.
RESULTS AND DISCUSSION
Charm test
The results of drug contamination in autochthonous honey
samples (Table 1) indicated a low incidence of SA-s and TC-s
residues in honey from Basque Country market. Of a total of 567
samples, 68 samples (12%) were positive for sulfonamides, and
24 samples (4%) for tetracyclines. The CCα was about 4 µg kg−1
(SOZ), 5 µg kg−1 (SDM, SMR, SDZ, SPZ, and SMP), 6 µg kg−1 (SXZ),
8 µg kg−1 (SDX), 9 µg kg−1 (STZ), 10 µg kg−1 (SMZ), 15 µg kg−1
(SML), and 50 µg kg−1 (SAA). For tetracyclines, the CCα was
5 µg kg−1 (TC), 10 µg kg−1 (OTC and CTC), and 0.3 µg kg−1 for
CAP. In general, the frequency of residues of antibiotics in honey
from local beekeepers was low.15,20,34,35 No residues of CAP were
found. However, positive results were obtained in the majority of
the samples of heather (Erica vagans) honey with the Charm II test
for sulfonamides, but could not be confirmed by chromatographic
procedures. Only 19 of 57 samples presumptive contaminated
with SA-s were confirmed. This divergence has been induced by
the presence of a natural compound detected in this honey, which
67
was not confirmed as residue by LC-MS/MS detection, but at the
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 www.soci.org J Serra Bonvehı́, A Lacalle Gutiérrez

same time present affinity by a microbial cell containing a specific

Table 2. Absolute recoveries of sulfonamides and tetracyclines from
receptor incorporated in the Charm test for sulfonamide (and spiked samples (ηg g−1 )
not antibodies). Also honey with very high hydroxymethylfurfural
content can lead to false positive results. Component Component Rec.
Component added found (%)

LC-FD method for sulfonamides STZ 25.1 18.6 74.1

51.4 37.6 73.2
Numerous analytical methods have been applied for the identifi-
101.1 77.2 76.4
cation and quantification of SA-s in honey, including colorimetric
150.7 122.5 81.3
procedures, derivative spectrophotometry, and chromatographic
SMZ 25.4 18.1 71.3
methods (TLC/LC), after a simple extraction and clean-up.16,17,20,36
52.4 37.1 70.8
In our laboratory, the LC-FD method has been developed to verify
103.1 74.3 72.1
and quantify trace drugs in this biological matrix, using pre-column
149.7 110.6 73.9
derivatization with fluorescamine at pH 3. This derivatization tech-
SML 25.2 17.9 71
nique is preferred because the reagents are easily prepared, the
50.3 36.0 71.6
reaction occurs at room temperature, and the fluorescamine and
102.1 74.9 73.4
reaction byproducts are not fluorescent. The disadvantage of this
151.2 120.2 79.5
method is that the derivatives are not stable and they must be
OTC 25.1 22.2 88.4
prepared and analyzed one at a time. The derivatization was es-
50.4 44.4 88.1
tablished in 40 min. at room temperature (25 ◦ C). The CCα for STZ
101.7 91.8 90.3
and SMZ was 10 µg kg−1 , and 12 µg kg−1 for SML. The CCβ was
150.4 137.9 91.7
15 µg kg−1 for STZ and SMZ, and 17 µg kg−1 for SML. When IS was
TC 25.3 22.7 89.7
incorporated in standard calibration and sample determination,
50.7 45.3 89.4
calculated peak response ratios, ranges of the recoveries, and
100.3 91.4 91.1
coefficients were highly improved compared to those mentioned
151.1 139.9 92.6
in the literature.20,21 Recover rates obtained were 73.2–81.3% for
CTC 25.4 21.9 86.2
STZ, 70.8–73.9% for SMZ, and 71–79.5% for SML (Table 2). The
51.1 44.2 86.5
precision of the method was determined as repeatability (RSDr )
101.4 89.8 88.6
and reproducibility (RSDR ) at three concentrations for STZ, SMZ,
150.2 135.3 90.1
and SML (Table 3). The RSDr and RSDR obtained were lower than
2.10%, with a mean of 1.92%, and 2.54% with a mean of 2.38%, STZ: sulfathiazole; SMZ sulfamethazine; SML sulfamethizole; OTC:
respectively. The results were analyzed according to an outlier oxytetracycline; TC: tetracycline; CTC: chlortetracycline.
n = 24 determinations.
test (Cochran and Grubbs test). As expected, the SA-s gave linear
response from 0 to 200 µg kg−1 . The correlation coefficient should
be >0.995, and linear regression of the amount injected vs peak
area/height gave a correlation coefficients of 0.9957 for STZ, 0.9971
for SMZ, and 0.9997 for SML. These results indicate that the method
had satisfactory accuracy.37 Our studies showed the need to adjust
the initial gradient conditions in order to elute all SA-s, providing
a satisfactory capacity factor for the first eluting SA-s (STZ) (Fig. 1).
The retention time (RT) of the SA-s referenced ranged from 10 to
30 min. on the basis of five parallel determinations over 5 days,
the precision relative standard deviation (SD) of the RT was 2.7%
for STZ, 3.1% for SMZ, and 4.12% for SML, whereas the precision
of peak area values were 3.57%, 3.83%, and 4.12% for STZ, SMZ,
and SML, respectively. Of the 68 presumptive positive samples,
19 were confirmed as such, with sulfathiazole residues at levels
from 20 to 210 µg kg−1 . Of the 57 presumptive positive samples of
heather honey, residues of sulfathiazole were confirmed in only 8 Figure 1. LC chromatograms of blank honey and spiked samples with the
(Table 1), showing the detection in these honeys of a natural com- sulfonamides most used in Spain (0.050 µg mL−1 ). Peaks: sulfanilamide
(intern standard) (1); sulfathiazole (2); sulfamethizole (3); sulfamethazine
pound not confirmed as residue. Honey samples fortified with STZ (4).
(2000 µg kg−1 ) were reduced in 6 weeks to values of 80 µg kg−1 ,
whereas if the honey was fortified with 1000 µg kg−1 , and stored
at 4 ◦ C, the residue content was still 85% of the initial amount after
solvents, and SPE elution volumes for TC-s. When phenyl columns
11 months; this is in agreement with Belliardo.38
were used, some interference could be observed in the RT of the
TC-s. Under the experimental conditions reported here, the SPE
LC-FD method for tetracyclines was applied through Waters Oasis HLB (200 mg per 6 mL). This
The TC-s were separated on a reverse-phase C8 column and polymeric adsorbent does not contain a silanol backbone, without
reacted post-column with magnesium acetate in boric acid buffer the interference of TC-s interacting too strongly with the silanols
(pH 9), at room temperature. To obtain optimum conditions for of a silica-based cartridge.29 Experimental results indicated that
SPE clean-up, several factors were investigated, including the it was not necessary to do a second cleanup through a COOH
68

following: type of clean-up cartridge, washing solvents, eluent solid-phase extraction with this methodology, as adequate values

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Antibiotic and sulfonamide residues in Basque Country honey www.soci.org

Table 3. Analytical Relative Standard Deviation (RSD) of sulfonamides

STZ STZ SMZ SMZ SML SML
added found RSD added found RSD added found RSD
(ηg g−1 ) (ηg g−1 ) (%) (ηg g−1 ) (ηg g−1 ) (%) (ηg g−1 ) (ηg g−1 ) (%)

Intraday
60.1 43.9 ± 0.81 1.85 59.4 41.8 ± 0.87 2.08 60.3 42.9 ± 0.81 1.89
59.7 45.5 ± 0.83 1.82 60.5 43.6 ± 0.91 2.09 59.7 43.7 ± 0.83 1.90
60.4 49 ± 0.87 1.78 59.9 43.7 ± 0.87 1.99 60.6 47.8 ± 0.91 1.90
Interday
60.1 44 ± 0.97 2.21 59.4 41.7 ± 1.04 2.49 60.3 42.8 ± 1.03 2.41
59.7 45.4 ± 1.01 2.22 60.5 43.4 ± 1.09 2.51 59.7 43.8 ± 1.09 2.49
60.4 48.9 ± 1.07 2.19 59.9 43.9 ± 1.11 2.53 60.6 48.1 ± 1.12 2.33

n = 18 determinations.

Table 4. Analytical Relative Standard Deviation (RSD) of tetracyclines

OTC OTC TC TC CTC CTC
added found RSD added found RSD added found RSD
(ηg g−1 ) (ηg g−1 ) (%) (ηg g−1 ) (ηg g−1 ) (%) (ηg g−1 ) (ηg g−1 ) (%)

Intraday
56.4 49.6 ± 0.71 1.43 53.7 47.9 ± 0.79 1.65 59.7 51.7 ± 0.83 1.61
55.7 50.2 ± 0.68 1.36 54.3 49.4 ± 0.71 1.44 58.4 51.6 ± 0.78 1.51
56.9 51.9 ± 0.75 1.45 52.6 48.6 ± 0.75 1.54 60.3 54.4 ± 0.86 1.58
Interday
56.4 49.7 ± 1.03 2.07 53.7 48 ± 0.98 2.04 59.7 51.5 ± 1.07 2.08
55.7 50.1 ± 0.98 1.96 54.3 49.2 ± 1.05 2.13 58.4 51.5 ± 1.11 2.16
56.9 52 ± 1.08 2.08 52.6 48.5 ± 1.09 2.25 60.3 53.9 ± 1.15 2.13

n = 18 determinations.

of recovery and repeatability were achieved, without the retention
of TC-s on the COOH column.18,39 Thus, the TC-s were eluted on
Oasis cartridge with oxalic buffer (pH 2.25–2.5) and acetonitrile
in ratio 30 : 8 (v/v). The eluent was also assayed at 3, 4, 5, and
6 mL. The results show that 3 mL was not enough volume to
elute all TC-s (OTC 50%, TC 71%, and CTC 50.2%), indicating
that the volume recommended in the literature does not have
good selectivity. However, with 5 mL, all TC-s were eluted (all
>85%), and no improvement was observed in a further elution
with 6 mL. Recoveries for the different TC-s were 88.1–91.7% (for
OTC), 89.4–92.6% (for TC), and 86.2–90.1% (for CTC) (Table 3).
Several mobile-phases were tested for efficient elution of TC-s: the
best results were obtained with gradient program with excellent
elutions for all analytes – the peak co-eluting with tetracycline
disappeared and the interference was decreased significantly.
Under these conditions, the TC-s identified were successfully
separated within 16 min., as shown in chromatogram (Fig. 2). The
RSDr , RSDR , and recoveries of spiked samples were determined at
three concentrations for TC, OTC, and CTC (Table 4). The RSDr and
RSDR obtained were lower than 1.66% and 2.26%, respectively. The
correlation coefficient should be >0.995: linear regression of the
amount injected vs peak area/height gave a correlation coefficient
of 0.9954 for OTC, 0.9981 for TC, and 0.9984 for CTC. The CCα
values were 2 µg kg−1 for CTC, 4 µg kg−1 for TC, and 5 µg kg−1
for OTC. The CCβ values were 5 µg kg−1 for CTC, 9 µg kg−1 for
TC, and 12 µg kg−1 for OTC. Method precision and accuracy were
4.90% and 29.7% for SA-s, and 4.54% and 13.9% for TC-s (Table 5).
The results demonstrate that the applied analytical procedure
gives enough guarantees for quality control.29,37 Contamination
with tetracycline was detected in all 24 presumptive positive
samples at levels from 15 to 920 µg kg−1 (Table 1). A further
complication in the determination of TC-s, in particular CTC, is the
fact that it can rapidly isomerizes, even under mild conditions,
to form 4-epimers.39,40 This paper also reviews the stability of
the main tetracyclines utilized in Spain. At 4 ◦ C and 20 ◦ C, OTC
standard solutions were very stable after 11 weeks, with only 4%
of epimeric forms. In contrast, CTC and TC epimerized rapidly
after 11 weeks at 20 ◦ C, and low levels have been reported after
11 weeks at 4 ◦ C, which may lead to false quantification of TC-s, by
summing the areas of TC-s and their respective epimers without
appropriate correction factors.39 Thus, TC-s epimer values were not
determined. Similar results were obtained with standard solutions
stored in the dark.
LC-MS/MS confirmation
For the identification of positive samples, according to the
European Community Decision 2002/657/EC,24 the follow criteria
were used: (a) the signal-to-noise ratio (S/N) of the characteristic
ions selected must be ≥3; (b) the differentiation of the retention
time of analyte and corresponding standard should be within
±2.5%; (c) the allowable deviation of the relative abundance of the
characteristic ions of the target matter and that of the characteristic
ions of the corresponding standard should be within ±20% to
±50%, depending on the relative ion intensities. The decision limit
(CCα) varies between 0.8 and 12 µg kg−1 for the 16 sulfonamides
referenced in European Union (SML, SMP, and SDX, 0.8 µg kg−1 ;
STZ, SMR, SMZ, SCP, SDM, SOZ, and SXZ, 1 µg kg−1 ; SGD and
69

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 www.soci.org J Serra Bonvehı́, A Lacalle Gutiérrez

Figure 2. LC chromatograms of blank honey and spiked samples with Figure 3. LC chromatograms of unknown peak solely detected in heather
oxytetracycline (OTC), tetracycline (TC), and chlortetracycline (CTC) honey. Peak: unknown (1).
(0.050 µg mL−1 ). Peaks: oxytetracycline (1); tetracycline (2); chlortetra-
cycline (3).
the appropriate control point), 57 samples of a total of 65 samples
(87.7%) were classified as honeys presumptive contaminated
Table 5. Precision and accuracy for determination of sulfonamides with sulfonamides. However, the chromatography (LC-FD and
and tetracyclines LC-MS/MS) showed clearly the presence of a compound not
confirmed as a veterinary drug residue (Fig. 3), with significant
Component Component Error Precision
Component added (ηg g−1 ) found (ηg g−1 ) (%) (%) interactions for individual Charm receptor assay.
To investigate this anomaly, the LC-MS/MS conditions were
STZ 50.7 37.1 ± 1.37 26.8 3.69 optimized to corroborate possible contamination or the presence
100.3 76.4 ± 2.09 23.5 2.74 of a natural compound in heather honey. Sulfonamides are known
149.7 121.3 ± 3.41 18.9 2.81 to possess amphoteric character due to the presence of nitrogen
SMZ 51.1 35.9 ± 1.47 29.7 4.09 functions in their molecules, which because of their positions in
102.1 73.8 ± 2.74 27.7 3.71 the structure can protonate/deprotonate depending on the pH of
150.7 110.2 ± 4.17 26.9 3.78 the medium.8 Usually, the separation of SA-s is performed by LC
SML 50.3 35.9 ± 1.76 28.6 4.90 in a reversed-phase mode under acidic conditions. Mass spectra
101.1 74.4 ± 3.07 26.4 4.12 of all the sulfonamides and tetracyclines were acquired in a full
150.3 110.9 ± 4.39 26.2 3.96 scan mode with cone voltage of 25 V, using positive electrospray
OTC 50.3 44.4 ± 1.67 11.7 3.76 ionization. Cone voltages were optimized for maximum signal
100.7 90.8 ± 2.23 9.8 2.46 intensity of typical ions during an injection of single compounds
149.8 136.9 ± 3.44 8.6 2.51 into the mass spectrometer. The detection of the compounds was
TC 50.1 44.7 ± 1.53 10.8 3.42 divided into reaction time windows with dwell times for single
101.2 92.4 ± 2.83 8.7 3.12 reaction monitoring (SRM), which were set to 0.05 and 0.20 s (per
150.3 138.4 ± 4.37 7.9 3.16 SMR), depending on the intensity of the products ions for the
CTC 50.4 43.4 ± 1.97 13.9 4.54 transition. The most intense fragments in the positive ion mode
100.7 89 ± 2.87 11.6 3.22 were detected at m/z 156, 108, and 92, although the abundance
150.4 135.1 ± 4.57 10.2 3.38 was different depending on the compounds. For confirmatory
n = 15 determinations. purposes, transitions (product ions), cone and collision energy
voltages applied for the analytes are summarized in Table 6 for
each sulfonamide and tetracycline.
As expected, considerable difference was detected between
SDZ, 2 µg kg−1 ; SNL and SAA, 5 µg kg−1 ; SPZ, 9 µg kg−1 ; and SPY, this unknown component and the 16 SA-s according MS/MS
12 µg kg−1 ), and between 1 and 5 µg kg−1 for the tetracyclines conditions, retention times, and by comparison with spectra
(CTC, 1 µg kg−1 ; OTC and TC, 5 µg kg−1 ). While detection capability in Wiley6N and NIST98 libraries. Additionally, when applying
(CCβ) varies between 1.2 and 16.5 µg kg−1 for the sulfonamides the same chromatographical techniques to other multifloral
(SML, SMP, and SDX, 1.2 µg kg−1 ; STZ, SMR, SMZ, SCP, SDM, SOZ, and unifloral honeys (e.g. eucalyptus, sweet chestnut) free from
and SXZ, 1.7 µg kg−1 ; SGD and SDZ, 3.5 µg kg−1 ; SLN and SAA, residues, no peak was detected. For this reason, five ecological
7 µg kg−1 ; SPZ, 12.6 µg kg−1 ; SPY, 16.5 µg kg−1 ), and between 1.6 heather honey lots free of veterinary treatments and collected
and 7.5 µg kg−1 for the tetracyclines (CTC, 1.6 µg kg−1 ; OTC and in the same area of flowering with an official biocontrol label
TC, 7.5 µg kg−1 ). CCα was 0.08 µg kg−1 , and CCβ was 0.1 µg kg−1 were evaluated and submitted to additional assays. The same
for chloramphenicol. A strong matrix effect was found in honeys response and confirmation were observed in all the batches
of selected unifloral origin during the detection of SA-s by Charm of the ecological heather honey analyzed, characterized by the
assay. It is important to note that the interference was restricted presence of the same unknown peak. The same peak has also
and localized only in heather honeys (dominance required for recently been identified in the nectar extracted of Erica vagans
such classification: >45% pollen Erica vagans). Thus, floral origin (data not published). Apparently, this unknown peak could be
of the samples of this honey were confirmed, all characterized assigned as a natural compound characteristic of this kind of
for the strong presence of Erica vagans (pollen 67–84%; mean honey. This indicates a difference in response between the Charm
74 ± 5%). As mentioned previously, using the Charm II test (with
70

assay and the chromatographical procedures applied on this

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Antibiotic and sulfonamide residues in Basque Country honey www.soci.org

contain tetracycline. Finally, CAP was not detected in any of the

Table 6. MRM conditions for detecting sulfonamides and tetracy-
clines by tandem MS samples analyzed.

MRM Cone Collision

transitions voltage energy
Component (m/z) (V) (eV)
CONCLUSIONS
Residues of veterinary drugs were found in a very limited number
Sulfacetamide 215 > 156 17 20 of honey samples. No residues of chloramphenicol were found.
215 > 108 17 33 The Charm test was highly acceptable for detecting tetracyclines
Sulfapyridine 250 > 156 26 24 in honeys. However, it needs correction for the detection of
250 > 108 26 35 sulfonamides in heather (Erica vagans) honeys. The applied
Sulfadiazine 251 > 156 24 22 LC-FD and LC-MS/MS procedures allowed good resolution of
251 > 92 24 38 sulfonamide and tetracycline residues. The precision and accuracy
Sulfathiazole 256 > 156 25 22 of the system and sample preparation validation were acceptable,
256 > 108 25 34 with a high degree of reproducibility. It is therefore concluded
Sulfamerazine 265 > 156 28 25 that either of the two chromatographic methods presented here
265 > 172 28 28 could be used to determine drug residues in honey with a high
Sulfamethoxazole 254 > 156 20 18 degree of reliability. Also both procedures detected an unknown
254 > 108 25 35 natural compound in heather honey with a high affinity by the
Sulfamethizole 271 > 156 20 21 Charm analyzer.
271 > 108 20 35
Sulfamethazine 279 > 186 31 28
279 > 204 31 24 REFERENCES
Sulfadimethoxine 311 > 156 31 29 1 Hansen H and Brodsgaard CJ, American foulbrood: a review of its
311 > 218 31 28 biology. Bee World 80:5–23 (1999).
Sulfanilamide 173 > 156 20 9 2 Otten Ch, A general overview on AFB and EFB pathogen, way of
infection, multiplication, clinical symptoms and outbreak. Apiacta
173 > 92 20 30 38:106–113 (2003).
Sulfaguanidine 215 > 156 20 15 3 Sáenz Z, Zarazaga M, Brinas L, Lantero M, Ruiz-Larrea F and Torres C,
215 > 108 24 18 Antibiotic resistance in Escherichia coli isolates obtained from
Sulfachlorpyrazine 265 > 156 20 22 animals, foods and humans in Spain. Int J Antimicrob Agents
18:353–358 (2001).
285 > 126 20 36
4 Wallner K, Sulfonamide residues in German honey – The actual
Sulfamethoxipyridazine 281 > 156 20 26 situation. Apidologie 34:489 (2003).
281 > 126 20 31 5 EEC, Establishment of Maximum Residue Levels of veterinary medical
Sulfachloropyridazine 285 > 156 20 22 products in foodstuffs of animal origin, Council Regulation (EEC)
285 > 108 20 36 No. 2377/90, Off J Eur Commun L224/1 (1990).
6 EEC, Commision decision 2003/181/EC, March 13, 2003, amending
Sulfadoxine 311 > 156 20 29 decision 2002/657/EC as regards the setting of minimum required
311 > 218 20 28 performance limits (MRPLs) for certain residues in food animal
Sulfisoxazole 268 > 156 15 21 origin. Off J Eur Communities L71, pp. 17–18 (2003).
268 > 113 20 25 7 Serra Bonvehı́ J, Soliva Torrentó M and Muntané Raich J, Invertase
activity in fresh and processed honeys. J Sci Food Agric 80:507–512
Tetracycline 445 > 410 18 12
(2000).
445 > 427 18 18 8 Kaufmann A, Roth S, Ryser B and Widmer M, Quantitative LC/MS-MS
Oxytretracycline 461 > 426 25 17 determination of sulfonamides and some other antibiotics in honey.
461 > 443 25 10 J AOAC Int 85:853–860 (2002).
Chlortetracycline 479 > 444 23 18 9 Hornitzky MAZ, Commercial use of gamma radiation in the
beekeeping industry. Bee World 75:135–142 (1994).
479 > 462 23 16 10 Tetrasensor, Rapid test. Assay for a panel of tetracycline residues in honey.
Unisensor, S.A. (info@unsensor.be), Liège, Belgium (2003).
11 Shet HB and Sporns P, Enzyme immmunoassay for screening of
sulfathiazole in honey. J AOAC 73:871–874 (1990).
12 Masher A, Lavagnoli S and Curatolo M, Determination of residual
honey. Comparative data on biological activities (antibacterial, oxytetracycline in honey with an immunoassay kit. Apidologie
antioxidant, etc.), and full characterization of this compound are 27:229–233 (1996).
required. 13 Heering W, Usleber E, Dietrich R and Märtlbauer E, Immunochemical
As a first step, appropriate correction response has been screening for antimicrobial drug residues in commercial honey.
Analyst 123:2759–2762 (1998).
established in this honey for detecting sulfadrug residues by 14 Salter R, Charm II system – comprehensive residues analysis system
Charm II system. These differences are mainly due to the plant for honey. Apiacta 38:198–206 (2003).
source and to the different chemistry of the honey. This result 15 Edder P and Corvi Cl, Utilisation du Charm II test pour le dépistage
emphasizes that the floral origin and habitats are an important des résidus d’antibiotiques dans les denrées alimentaires d’origine
animal. Mitt Lebensm Hyg 1:218–228 (2001).
factor to be taken into account for the appropriate detection of 16 Barry CP and MacEachern GM, Reverse phase liquid chromatographic
trace residues by screening procedures. Until now, no studies determination of sulfathiazole residues in honey. J AOAC Int 66:4–7
have been performed to find out if other heather honeys of (1983).
different geographical origin would give similar results. Only in 17 Horie M, Saito K, Nose N and Nakazawa H, Simultaneous
determination of sulfonamides in honey by liquid chromatography.
8 of the samples presumptive contaminated with sulfonamides J AOAC Int 75:786–789 (1992).
was the presence of sulfathiazole confirmed, while all 24 samples 18 Oka H and Patterson J, Chemical analysis of tetracyclines. in Chemical
71

presumptive contaminated with antibiotics were confirmed to Analysis for Antibiotics used in Agriculture, ed. by Oka H, Nakazawa N,

J Sci Food Agric 2009; 89: 63–72

c 2008 Society of Chemical Industry www.interscience.wiley.com/jsfa
 www.soci.org
Harada K and McNeil J-D. AOAC Publ, Gaithersburg, MD, pp.
333–405 (1995).
19 Vinas P, Balsalobre N and Hernández-Córdoba LE, Liquid determi-
nation with ultraviolet absorbance detection for the analysis of
tetracycline residues in honey. J Chromatogr A 75:786–789 (1992).
20 Diserens JM and Savoy-Perroud MC, Determination of sulfonamide
residues in honey. Quality & Safety Assurance Department, Nestlé
Research Center, Lausanne, Switzerland (2002).
21 Edder P, Cominoli A and Corvi C, Dosage de résidus de sulfamides
dans des denrées alimantaires d’origine animale (foies, rognoms,
viandes, poissons, oeufs, lait) par HPLC avec prédérivatisation et
détection fluorimétrique. Trav Chim Aliment Hyg 88:554–569 (1997).
22 Kaufmann A, Pacciarelli B, Prijic A, Ryser B and Roth S, Bestimmung
von rückständen von tetracyclinen in lebensmitteln. Mitt Lebensm
Hyg 90:167–176 (1999).
23 Pang GF, Cao YZ, Zhang JJ, Jia GQ, Fan CH, Li XM, et al, Determination
of 16 sulfonamides in honey by liquid chromatography/tandem
mass spectrometry. J AOAC Int 88:1304–1311 (2005).
24 EC, Implementing Council Directive 96/23/EC concerning the
performance of analytical methods and the interpretation of results,
EC Decision 2002/657, Off J Eur Commun L221/8 (2002).
25 Schwaiger I and Schuch R, Bound sulfathiazole residues in
honey – need of a hydrolysis step for the analytical determination
of total sulfathiazole content in honey. Dtsch Lebensm Rundsch
96:93–98 (2000).
26 B.O.P.V. (Boletı́n Oficial del Paı́s Vasco) N◦ 207 ZK/1993 (28 October),
Label Vasco de Calidad Alimentaria de la Miel, Dpto. Agricultura y
Pesca, Vitoria, pp. 9592–9606 (1993).
27 Charm Sciences, Charm II 6600/7600 Analyzer Operating Instructions.
Version 2.13. Charm Sciences, Inc., St Lawrence Mass, USA (2001).
28 McMullen SE, Lansden JA and Schenck FJ, Modifications and
adaptions of the Charm II rapid antibody assay for chloramphenicol
in honey. J Food Protect 67:1533–1536 (2004).
29 Pena A, Pelantova N, Lino CM, Silveira MIN and Solich P, Validation of
an analytical methodology for determination of oxytetracycline and
tetracycline residues in honey by HPLC with fluorescence detection.
J Agric Food Chem 53:3784–3788 (2005).
72
www.interscience.wiley.com/jsfa

c 2008 Society of Chemical Industry
J Serra Bonvehı́, A Lacalle Gutiérrez
30 Hormazábal V and Yndestad M, Simultaneous determination of
chloramphenicol and ketoprofen in meat and milk and
chloramphenicol in egg, honey, and urine using liquid
chromatography-mass spectyrometry. J Liq Chrom Rel Technol
24:2477–2486 (2001).
31 Yuan J, Jin ZX, Chong YS, Tao D, Hui LC, Bin W, et al, Determination of
chloramphenicol in royal jelly by liquid chromatography/tandem
mass spectrometry. J AOAC Int 89:1432–1436 (2006).
32 Louveaux J, Maurizio A and Vorwhol G, Methods for melissopalynol-
ogy. Bee World 59:139–157 (1979).
33 Vergeron Ph, Interprétation statistique des résultats à matière
d’analyse. Ann Abeille 7:384–394 (1964).
34 Reybroeck W, Residues of antibiotics and sulphonamides in honey on
the Belgian market. Apiacta 38:23–30 (2003).
35 Morlot M and Beaune P, An experience with Charm II system. Apiacta
38:226–234 (2003).
36 Nagaraja P, Yathirajan HS, Sunitha KR and Vasantha RA, A new,
sensitive, and rapid spectrophotometric method for the
determination of sulfa drugs. J AOAC Int 85:869–874 (2002).
37 Cuadros Rodrı́guez C, Garcı́a Campaña AM, Alés Romero F, Jiménez
Linares C and Román Ceba M, Validation of an analytical
instrumental method by standard addition methodology. J AOAC
Int 78:471–476 (1995).
38 Belliardo F, Determination of sulphonamide residues in honey by
high-pressure liquid chromatography. J Apic Res 20:44–48 (1991).
39 Bogialli S, Curini R, Corcia A, Laganà A and Rizzuti G, A rapid
confirmatory method for analyzing tetracycline antibiotics in
bovine, swine, and poultry muscle tissues: matrix solid-phase
dispersion with heated water as extractant followed by liquid
chromatography-tandem mass spectrometry. J Agric Food Chem
54:1564–1570 (2006).
40 Khong SP, Hammel YA and Guy PhA, Analysis of tetracyclines in
honey by high-performance liquid chromatography/tandem mass
spectrometry. Rapid Commun. Mass Spectrom 19:493–502 (2005).
J Sci Food Agric 2009; 89: 63–72