Trends in Analytical Chemistry, Vol. 30, No.

7, 2011 Trends

Determination of antibiotic residues

in honey
_
Zaneta Bargańska, Marek Ślebioda, Jacek Namieśnik

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

1. Introduction diseases in honeybees [3]. Consequently,

_
Zaneta Bargańska*,
Jacek Namieśnik
Department of Analytical
Chemistry, Chemical Faculty,
Gdansk University of
Technology, Traugutta 11/12,
80-233 Gdańsk, Poland
Marek Ślebioda
Perlan Technologies Polska Sp.
z. o. o., Puławska 303, 02-785
Warszawa, Poland
*
Corresponding author.
Tel.: +48 58 3472110;
Fax: +48 58 3472694;
E-mail: zanjarz@gmail.com
Safety of food and feed is one of the main
objectives in consumer health policy.
Maintaining a high level of protection in
this area is vital not only for public health,
but also to preserve consumer confidence
in food. The regulation of the European
Parliament and of the Council (EC) No
178/2002 [1], laying down the general
principles and requirements of food law,
established the Rapid Alert System for
Food and Feed (RASFF). The principle of
RASFF action is based on the collection
and the rapid communication of informa-
tion on risks detected in relation to food or
feed, and the RASFF portal website con-
tains an online searchable database of
notifications [2].
The contamination of the environment
by xenobiotics is linked to industrialization
and intensive agriculture. Honeybees can
be used for environmental monitoring
because they are good biological indica-
tors due to two signals: their mortality and
the residues present in their bodies or
beehive products that may be detected by
laboratory analyses.
The use of antibiotics in apiculture has
been known since 1940. Sulfonamides,
tetracyclines, nitrofurans and macrolides
are used in farming to prevent and to
combat diseases of cultivated plants and
by beekeepers to prevent and to combat
antibiotic residues in honey may have got
there as a result of the treatment of dis-
eases [e.g., American foulbrood (AFB) or
European foulbrood (EFB)]. The treatment
of honeybees with antibiotics is prohibited
in the European Union (EU) and there
have been significant advances in EU
legislation concerning risk assessment.
European Commission (EC) Directive
2377/90 with annexes states that honeys
should be free of antibiotic contamination
[4], so honeys containing these substances
cannot be sold in most EU countries and
no maximum residue levels (MRL) of
antibiotic residues have been laid down. In
some countries (Switzerland, UK and
Belgium), MRLs have been set for each
class of antibiotics (10–50 ppb) [5,6].
Table 1 lists information on therapeutic
substances (including prohibited sub-
stances), which may be used for veterinary
purposes, and which are covered by the
program for monitoring chemical residues
in honey and other honeybee products.
Two main approaches are used to
determine the content of antibiotic resi-
dues in honey: screening tests and multi-
stage analytical methodologies. The simple
tests provide qualitative or quantitative
information, enabling determination of a
single target analyte. With multi-stage
methods, a fairly broad spectrum of ana-
lytes can be determined in one analytical

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Trends Trends in Analytical Chemistry, Vol. 30, No. 7, 2011

Table 1. Characteristics of antibiotics usually applied by beekeepers

Antibiotic Characteristics Ref.

Tetracycline Commonly applied in the treatment of many bacterial infections of the digestive system, the respiratory [7,8]
system and the skin. Also used as a growth stimulant in animals, hence its use in some countries as an
animal-feed additive. The use of tetracyclines as growth stimulants is prohibited in the European Union
(EU). The large-scale application of tetracyclines carries the risk of their residues appearing in food.
Tetracyclines are not registered in the EU for treating honeybees, so there are no established residue levels
for this type of antibiotic in honey. Oxytetracycline is currently the only antibiotic registered for use by
Canadian beekeepers to treat American foulbrood (AFB), a highly contagious bacterial disease of larvae,
difficult to eradicate, caused by the rod-shaped bacteria Paenibacillus larvae. Tetracyclines act on both
Gram-positive and Gram-negative bacteria.
Sulfonamides Used for 10–15 years to combat varroatosis, a disease of bees caused by the mite Varroa destructor, and in [7,9,10]
the treatment of AFB. In 1940, sodium sulfathiazole was registered in the USA for the control of AFB, but its
application was later prohibited, as residues of this antibiotic accumulated in honey.
Aminoglycosides This is a group of bactericidal antibiotics, of particular significance in combating dangerous Gram-negative [7]
bacterial infections. The best-known aminoglycosides are gentamycin, lincomycin, neomycin and
streptomycin. Their polar nature makes it difficult to isolate them from samples and to determine them
chromatographically.
b-lactams The most commonly used class of antibiotics in veterinary practice for treating bacterial infections in
domestic animals and dairy cattle. These antibiotics contain b-lactam bonds, which are broken by certain
enzymes, the so-called b-lactamases, produced by some bacteria. This is why these compounds tend to
decompose under the influence of heat and in the presence of alcohols.
Macrolides Commonly applied in veterinary practice to treat infections of the respiratory system and as growth
stimulants added to animal feeds. They are easily assimilable following oral administration and disperse to
a large extent into the tissues, especially lungs, liver and kidney.
Chloramphenicol A broad-spectrum antibiotic. Chloramphenicol acts on various pathogens affecting both humans and [7,11]
animals. It is one of the most toxic antibiotics, damaging bone marrow and causing anemia, leucopenia
and granulopenia.
Nitrofuran The nitrofuran derivatives furazolidone, nitrofurazone, nitrofurantoin, furaltadone are antibacterial drugs [7,12]
derivatives which were used in veterinary practice to treat infections of the urinary tract, digestive system and skin, and
were also used as food preservatives. The antibacterial action of nitrofurans covers a broad spectrum of
micro-organisms (Streptococcus, Staphylococcus, Gram-negative rods). Nitrofurans also have
antiprotozoal and fungicidal properties. Besides their pharmacological value, nitrofurans elicit numerous
side effects like mutagenicity, carcinogenicity, damage to the lungs and cardiac muscle. In animal
organisms nitrofurans are metabolized quite quickly, so their metabolites are used to monitor nitrofuran
residues as residue biomarkers because of their long half-life in animals (1.9–3.8 weeks). The respective
biomarkers of furazolidone, nitrofurantoin, nitrofurazone and furaltadone are the metabolites 3-amino-2-
oxazolidone, 1-aminohydantoin, semicarbazide, and 3-amino-5-morpholinomethyl-2-oxazolidone. The
administration of nitrofuran to animals destined to be human food has been prohibited in the EU since
1997, because the metabolites of this compound can accumulate in animal tissues and their products for a
long time, even after the conclusion of treatment.

run, so the choice of sample-preparation technique is
very important.
2. Screening tests
Screening methods can detect the presence of an analyte
or group of analytes at the level of interest, and usually
provide semi-quantitative or quantitative results.
Screening tests should provide low rate of false compliant
samples, high throughput, ease of use, short analysis
time, good selectivity, and low cost. Table 2 lists assays
that are commonly used to determine the content of
antibiotic residues in honey.
Laboratories facing the analysis of large numbers of
samples usually first apply screening methods covering
families of antibiotics. Samples found to be non-compli-
ant are then analyzed by confirmatory methods. Fig. 1
shows a general scheme of determination of antibiotics
in honey.
Semi-quantitative immunoassay tests are character-
ized by high specificity, high sensitivity, simplicity and
cost effectiveness, which make them particularly useful
in routine work. The main drawbacks of these methods
are high background absorption, a large number of
incubation and washing steps, and degradation of
enzyme during storage. Biosensor methods that contain
a biological-recognition element (e.g., enzymes, proteins,
nucleic acids, cells, and tissues) coupled to a signal-
transduction element can operate fully automatically.
Instability of the biological-sensing component, which
can lose its activity in hours or days, depending on the
nature of the molecule and exposure to environmental
stresses (e.g., pH, temperature or ionic strength), is the
chief disadvantage of these types of test. Another limi-
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

Assays Principle Remarks Ref.

Charm II (Charm Target analytes bind to receptors and the level of
Sciences, Inc.) radioactivity of H3 or C14 is determined.
ELISA (enzyme-linked An immunological test used for the quantitative
immunosorbent assay) determination of antibiotics by means of antibodies.
Tetrasensor The test uses two elements: a reagent containing an
(Unisensor) amount of labeled receptor and a dipstick consisting
of a set of two capture membrane lines. The first line
captures the remaining active receptor and the second
line takes the excess of reagent that has passed
through the first line.
Sulfasensor (CAP This test employs an antibody capable of detecting 10
Transia, Biacore) key sulfonamides. The protocol includes a rapid acid-
hydrolysis step to liberate the bound sulfonamide
residues from the sugar component.
Used in screening tests for sulfonamides, [13,14]
tetracyclines, b-lactams, macrolides, amphenicols
and streptomycin amino-glycosides.
The method is frequently applicable only in [14]
preliminary quantitative analysis. It enables
detection of an antibiotic in honey in range above
10–50 ppb, depending on its chemical properties.
Used as a screening test for the quick [15]
determination of tetracyclines present in honey
even at 10 ppb levels.
Used in screening test for sulfonamides at the [16]
target detection level of 25 ppb.

Figure 1. Flowchart for screening of antibiotics in honey.

used in biosensors. The performance of biosensor tech- 3. Analytical procedures

nology and immunoassay applied to testing honey was
found to be in good agreement to confirmatory high-
performance liquid chromatography (HPLC) methods,
although some false positives were observed for tissue
samples [17].
The possibility of using diluted samples from screening
for confirmation by gas or liquid chromatography coup-
led to tandem mass spectrometry (GC-MS2 or LC-MS2)
makes the complete procedure extremely convenient to
use [18]. However, most chromatographic methods are
limited to one class or related classes of compounds and
only a few studies have described simultaneous analysis
of unrelated compound classes [19].
3.1. Sample preparation
In EU countries, there are no MRL values for antibacte-
rial substances in honey. Only chloramphenicol and
nitrofuran metabolites have MRLs of 0.3 ppb and 1 ppb,
respectively [20]. Analytical methods intended for
detection of residues of antimicrobial drugs in honey
at trace levels are therefore necessary, and advanced
sample-preparation techniques have to be applied in
order to enable determination of trace and ultra-trace
quantities of antibiotic analytes because of the complex
composition of the honey matrix. The main focus has been
to decrease the LOD of the target compounds (<5 ppb).

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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

Multiclass Dissolution/on-line – LC-MS2 0.5–10 – [34]

antibiotics extraction
Dissolution/LLE – LC-MS2 27–81 – [5]
Dissolution/SPE 65–104 LC-MS2 <2 <7 [35,36]
Dissolution/SPE > 50 LC-MS2 0.1–1 <4 [37]

Macrolides Dissolution/SPE 42–136 LC-MS2 – – [38]

Tetracyclines Dissolution/on-line 84–121 LC-UV 5–12 17–40 [39]

extraction/SPE
MISPE 95–103 LC-MS2 0.1–0.3 0.2–1.1 [40]
Dissolution/SPE 72–98 LC-MS2, LC-UV 0.02–1.1 – [41]
Dissolution/SPE 95–105 LC-RRS 0.4–1 – [42]
Dissolution/OCLLE LC-CL, LC-UV 0.02–0.1 – [43]

Aminoglycosides Dissolution/SPE 81–84 LC-MS2 – 2 [44]

Chloramphenicol Dissolution/LLE/SPE 64–74 LC-MS2 0.2 0.5 [45]

Dissolution/QuEChERS/dSPE 78–92 LC-MS 0.002 0.006 [25]

Chloramphenicol Homogenization/SPE 45–85 LC-MS2 0.2–5 0.6–15 [46]

and sulfonamides

Chloramphenicol, LLE/SPE 75–107 LC-MS2 0.02–0.2 0.1–20 [47]

Florphenicol and
Tiamphenicol

Tiamulin Homogenization/SPE 88–106 LC-UV and LC-MS 29–35 and 0.5–1.2 101–122 and 1.4–4 [48]

Streptomycin Dissolution/SPE 94–109 LC-MS2 3–5 – [49]

Tylosin Dissolution/LLE/SPE 94–113 LC-MS2 – – [50]

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,
sugars and other interfering substances (e.g., wax and
dyes) have to be removed from the sample at the prep-
aration stage [21] and that is not always straightfor-
ward. For example, hydrolysis of the N-glycoside bond to
convert sugar-bonded sulfonamides into unbound forms
sometimes led to poor recoveries for almost all the
sulfonamides, and it was necessary to modify the sample-
preparation method. Methanol was used to break
the N-glycoside bond to give satisfactory recoveries
(56–96%) of 13 sulfonamides with relative standard
deviations below 10% [6].
To meet the continuing challenges in the analytical
determination of sulfonamides in honey, and various
requirements of the Codex Alimentarius [22], sample-
preparation techniques were employed for extraction
[i.e. solid-phase extraction (SPE) and solvent extraction
(SE)]. One solution that can further eliminate these
problems is the Quick, Easy, Cheap, Effective, Rugged,
and Safe approach (QuEChERS), which was developed in

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Trends in Analytical Chemistry, Vol. 30, No. 7, 2011
2003 [23] and became very popular among analysts
worldwide for determining pesticide residues, especially
in food, because of its rapidity, cheapness and the pos-
sibility of determining a broad spectrum of compounds.
The conventional QuEChERS approach is based on
acetonitrile/water partitioning of the analytes followed
by salting out with sodium chloride and magnesium
sulfate. Afterwards, dispersive SPE (dSPE), which in-
volves the addition of small amounts of a bulk sorbent to
the extracts, is usually applied. The QuEChERS method
reduces the number of steps in the analytical procedure,
thereby minimizing potential sources of error [24]. The
application of the QuEChERS method provides high
recovery rates for analytes over a wide polarity range,
and allows the use of smaller amounts of organic sol-
vents.
A modified QuEChERS technique was used to analyze
honey for chloramfenicol. Clean up by primary-second-
ary amine (PSA) was performed after evaporation of the
acetonitrile phase to decrease the limit of quantification
(LOQ) and to minimize problems with the LC column
[25].
A review on general methods of preparing honey
samples for chromatographic determination of contam-
inants appeared recently [26]. Table 3 shows how the
choice of sample-preparation techniques depends on the
properties of the target antibiotics in honey.
3.2. Liquid chromatography
HPLC is widely used for routine analyses, where target
antibiotics are determined. HPLC with UV detection was
used in a study of the degradation of tetracycline and
oxytetracycline in honey [51], but the limit of detection
(LOD) was quite high (about 1 ppm). HPLC applied to
determination of antibiotics has some inherent draw-
backs:
(1) each antibiotic class has to be tested separately;
(2) confirmation of the target analytes is based mainly
on retention-time comparison to standards; and,
(3) some analytes have to be derivatized to obtain an
appropriate LOD.
Sulfonamides are derivatized with fluorescamine (kex
403 nm and kem 492 nm, LOQ  10 ppb) [20] prior to
separation by reversed-phase (RP)-HPLC for fluorimetric
detection (FLD) (LOD  5 ppb, LOQ  10–15 ppb).
Streptomycin was analyzed using post-column derivati-
zation with 5-amino-6-hydroxynaphthalene-2-sulfonic
acid with an LOD of about 5 ppb, and an LOQ of 10 ppb
[52].
Since 1990, LC-MS2, in combination with electrospray
ionization (ESI) or atmospheric pressure chemical ioni-
zation (APCI), has become widely used in the quantita-
tive analysis of residues of veterinary drugs in food [53].
A triple-quadrupole analyzer is usually used for the
quantitative analysis of different classes of antibiotics in
food because of the high selectivity of the measurement
Trends
in multiple-reaction monitoring (MRM) mode [54].
According to EU criteria (2002/657/EC), when an MS
detector is used to confirm the presence of veterinary
drugs in food, at least two MRM transitions have to be
employed [55] to provide information enabling unam-
biguous confirmation of the analytes. Honey is a com-
plex matrix that contains sugars, pigments and phenolic
compounds that must be removed prior to LC-MS anal-
ysis [26]. Usually methods start with acidic hydrolysis,
followed by liquid-liquid extraction to liberate sugar-
bound sulfonamides and sample clean up is continued by
SPE. The hydrolysis time must be long enough to ensure
that the reaction is complete. The final analysis is by
LC-MS2 or LC-FLD, usually with post-column derivati-
zation.
Methods allowing determination 37 out of the 42
monitored multi-class antimicrobals (tetracyclines,
macrolides, aminoglycosides, b-lactams, amphenicols
and sulfonamides) in honey samples are available [5].
The authors performed a single LC-MS2 analysis using a
stacking injection method. Thus, 10 lL of each of four
honey extracts were successively taken, giving a final
injection volume of 40 lL. The gradient elution and MS2
acquisition was started after the injection of the last
honey extract. The order of extract injection was very
important and influenced the loss of b-lactam due to the
presence of acid in two of the extracts. Up to 12 honey
samples can be prepared within 5 h using this analytical
procedure.
A procedure involving sample dissolution with EDTA
under mildly acidic conditions (pH 4.0) followed by SPE
allowed determination of 17 compounds (macrolides,
tetracyclines, quinolones, and sulfonamides) in honey
samples [37] by ultra-performance LC-MS2 (UPLC-MS2)
in less than 5 min.
Summarizing, the development of a multi-residue MS
detection methods, especially coupled with UPLC, is
highly promising and reduces the time and the effort
necessary for sample preparation.
3.3. Gas chromatography
GC is rarely used for the determination of antibiotics in
honey, due to the polar nature, low volatility and ther-
mal instability of these drugs. Derivatization of these
polar compounds is advisable to improve peak shape and
sensitivity of the method, acetylation being most widely
used because the reaction can be carried directly in
aqueous phase. Most applications require an extraction
step, for both sample clean up and preconcentration
before analysis [7]. Table 4 summarizes the GC methods
used for determination of antibiotic residues in honey.
The use of atomic emission detection (AED) as a GC
detection method of N-methylated sulfonamides has the
noticeable advantage of combining qualitative identifi-
cation of the analyte by evaluating the ratio of the ele-
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

Compounds Gas chromatography LOD or LOQ or Ref.

CCa [ppb] CCb [ppb]
Column Detection
Stationary phase Length [m] I.D [mm]
(film thickness [lm])
Chloramphenicol 5% diphenyl 95% dimethyl 30 0.32 AED 0.1–2.4 – [56]
derivatized with acetic polysiloxane (0.25)
anhydride
Chloramphenicol 5% phenyl-polysilphenylene- 30 0.25 MS2 0.1 0.25 [57]
siloxane (0.25)
Sulfonamides 5% phenylmethylsilicone (0.33) 12.5 0.22 AED – – [58]
derivatized via
N-methylation

Abbreviations: AED, Atomic emission detector; CCa, Decision limit; CCb, Detection capability; ID, Internal diameter; MS2, Tandem mass
spectrometry.

determination. Application of this technique extends
significantly the linear dynamic range and lowers the
LOD [58]. Once again, MS detection offers the major
advantage of the qualitative identification of the analytes
by their mass spectrum. However, the quantitative
determination of antibiotics is hampered by their non-
linear response, with deviations depending on the com-
pound being determined.
In conclusion, there is a downward trend in utilizing
GC for the determination of antibiotics in honey, due to
the long analysis time and the additional step of deriv-
atization, as demonstrated by a very small number of
recent publications on this method of determination. The
only recommended GC-MS method that meets the cri-
teria of Directive 2003/181/EC [20] is for the determi-
nation of chloramphenicol.
4. Conclusion
The presence of xenobiotics in food is particularly dan-
gerous for human health, so it is essential to monitor
their residues in food. EU requirements mean that it is
necessary to determine antibiotic residues in different
compartments of the environment and in food. The EU
relies on MS detection for unequivocal identification of
chemical residues in foodstuffs. EC Decision (2002/657/
EC) states that ‘‘methods based only on chromatographic
analysis without the use of molecular spectrometric
detection are not suitable for use as confirmatory
methodsÕÕ. Samples found to be non-compliant with
bioassay techniques therefore usually have to be con-
firmed with separation techniques utilizing specific
detectors (GC-MS or LC-MS).
The determination of antibiotics in honey is based
mainly on multi-class residue methods enabling the
simultaneous determination of a wide spectrum of target
analytes in a single sample. Because the drugs are
present in honey at low concentrations, priority is being
given to sample preparation and extract clean up before
the final determination.
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