Food Chemistry 190 (2016) 982989

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Development and validation of a modied QuEChERS protocol coupled

to LCMS/MS for simultaneous determination of multi-class antibiotic
residues in honey
Amr H. Shendy a, Medhat A. Al-Ghobashy b,c,, Sohair A. Gad Alla a, Hayam M. Lotfy b,d
a

Central Laboratory of Residue Analysis of Pesticides and Heavy Metals in Food, Agricultural Research Center, Ministry of Agriculture and Land Reclamation, Giza, Egypt
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
Bioanalysis Research Group, Faculty of Pharmacy, Cairo University, Cairo, Egypt
d
Pharmaceutical Chemistry Department, Faculty of Pharmaceutical Sciences and Pharmaceutical Industries, Future University, Cairo, Egypt
b
c

a r t i c l e

i n f o

Article history:
Received 1 July 2014
Received in revised form 8 April 2015
Accepted 17 June 2015
Available online 18 June 2015
Keywords:
LCMS/MS
Honey
Veterinary drug residues
Nitrofuran
Nitroimidazoles
Ronidazole
Dimetridazole
AHD
AOZ
AMOZ
SEM

a b s t r a c t
LCMS/MS assay was developed and validated according to EU guidelines for determination of nitrofuran
metabolites and nitroimidazole residues in honey. Crude samples were acid-treated to liberate
matrix-bound residues and a modied QuEChERS protocol was employed. Nitrofurantoin, furazolidone,
furaltadone and nitrofurazone were determined via analysis of their metabolites AHD, AOZ, AMOZ and
SEM, respectively while nitroimidazole residues; ronidazole (RNZ) and dimetridazole (DMZ) were determined directly. For all analytes, neat standard calibration curves, after correction for matrix effect were
successfully employed. Decision limit (CCa) and detection capability (CCb) were below the MRPL for
nitrofurans (1.00 lg kg1) and the recommended concentration for nitroimidazole (3.00 lg kg1), respectively. The CCa, CCb, percentage recovery and CV% ranges were 0.120.74 lg kg1, 0.211.27 lg kg1,
90.96104.80% and 2.6512.58%, respectively. This work is part of the national initiative for establishing
a national monitoring program for drug residues in Egyptian honey.
2015 Elsevier Ltd. All rights reserved.

1. Introduction
Illegal use of antibiotics as veterinary drugs is well documented
around the world, regardless of the socioeconomic status. The
presence of antibiotic residuals in food products constitutes an
important health risk and is associated with the increased microbial resistance to antibiotics (Barganska, Namiesnik, & Slebioda,
2011; Butaye, Devriese, & Haesebrouck, 2001; Venable, Haynes, &
Cook, 2014). Recently, WHO identied antimicrobial resistance as
one of the three greatest threats to global health (WHO:
Antimicrobial resistance: global report on surveillance, 2014).
Galarini et al. reported that during the last ve years 71% of the
notications issued by the Rapid Alert System for Food and Feed
(RASFF Portal), Directorate-General for Health and Consumers
involved the presence of antimicrobial residues in honey bee products. Alerts concerning nitrofurans (NF) and nitroimidazoles (NMZ)
Corresponding author at: Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt.
E-mail address: medhat.alghobashy@cu.edu.eg (M.A. Al-Ghobashy).
http://dx.doi.org/10.1016/j.foodchem.2015.06.048
0308-8146/ 2015 Elsevier Ltd. All rights reserved.

formed about 25% of these notications (Galarini, Saluti,

Giusepponi, Rossi, & Moretti, 2015).
NF are broad-spectrum antibiotics currently in use either as a
prophylactic measure or for treatment of diseases such as the
American foulbrood (Barganska et al., 2011; Cronly et al., 2010).
The most commonly used members of the NF are nitrofurantoin,
furazolidone, furaltadone and nitrofurazone (Lopez, Feldlaufer,
Williams, & Chu, 2007; OKeeffe et al., 2004). The presence of NF
residues in honey has been previously reported (Barganska et al.,
2011; Bottoni & Caroli, 2015; Kaufmann, Butcher, Maden,
Walker, & Widmer, 2015; Venable et al., 2014). Due to concerns
about the carcinogenicity and mutagenicity of NF and their
metabolites, they were banned from use in the EU (1995) and
the United States (2002) (Barganska et al., 2011; Kaufmann et al.,
2015). Such zero tolerance compounds were classied under group
A: Prohibited substances, according to the Council Directive
96/23/EC and Commission Regulation (EU) 37/2010. Owing to the
rapid metabolism of NF, recent reports (Barganska et al., 2011;
Lopez et al., 2007; Venable et al., 2014) focused on the detection
of NF metabolites;1-aminohydantoin (AHD, metabolite of

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A.H. Shendy et al. / Food Chemistry 190 (2016) 982989

nitrofurantoin), 3-amino-2-oxazolidinone (AOZ, metabolite of

furazolidone),
3-amino-morpholinomethyl-2-oxazolidinone
(AMOZ, metabolite of furaltadone) and semicarbazide (SEM,
metabolite of nitrofurazone). The chemical structures of parent
NF compounds and their metabolites are shown in Fig. S1.
On the other hand, NMZ are antibiotics that are used to combat
anaerobic bacterial and parasitic infections (Hernandez-Mesa,
Garcia-Campana, & Cruces-Blanco, 2014; Mital, 2009).
Nitroimidazoles; most commonly ronidazole (RNZ) and dimetridazole (DMZ) have been in use to prevent and control Nosema apis in
hives that is one of the most common adult honey bee diseases
(Cronly et al., 2010). Both of RNZ and DMZ along with their
hydroxy metabolites are suspected of being genotoxic, carcinogenic and mutagenic (Hernandez-Mesa et al., 2014; Huet et al.,
2005). Similar to NFs, NMZ were detected in honey (Bottoni &
Caroli, 2015; Galarini et al., 2015; Venable et al., 2014) and are
classied as zero tolerance substances for all food producing species according to EU Commission Regulation 37/2010 and belong
to Group A (Prohibited substances). The chemical structures of
the most commonly used NMZ are shown in Fig. S1.
With the implementation of the stringent requirements CD
2002/657/EC and 2003/181/EC, the development of highly sensitive and specic analytical methods for the determination of drug
residues has become a challenging task. The EU has established a
harmonized minimum required performance limit (MRPL) for the
detection of NF residues at 1.00 lg kg1 and a recommended concentration for NMZ residues of 3.00 lg kg1 (CRL guidance paper
2007). Such ultra-low levels required sophisticated sample preparation and/or analysis techniques. Few methods have been
reported for the analysis of NF and/or NMZ in a variety of matrices
including honey (Bottoni & Caroli, 2015; Kaufmann et al., 2015;
Venable et al., 2014; Xia et al., 2008). A rapid multi-class,
multi-residue method for the determination of NMZ in honey
using LCMS/MS has been reported (Cronly et al., 2010; Lopez
et al., 2007). Recently, LCMS/MS has also been employed for the
determining NF metabolites in different matrices (Vass, Hruska,
& Franek, 2008). To the best of our knowledge, there are no reports
on the simultaneous determination of NF and NMZ in honey.
The aim of this study was to develop and validate a sensitive,
simple and multi-class LCMS/MS analysis protocol for the simultaneous determination of residues of NF metabolites (AHD, AOZ,
AMOZ and SEM) and NMZ (DMZ and RNZ) in honey.
Derivatisation of NF metabolites using 2-nitrobenzaldehyde
(2-NBA) will be carried out followed by extraction using a modied
QuEChERS sample preparation technique. The proposed protocol
was validated to the quality criteria of CD 2002/657/EC by measuring linearity, accuracy, repeatability, within-laboratory reproducibility, decision limit (CCa) and detection capability (CCb).
The applicability of the developed protocol for the routine monitoring of locally produced honey will be investigated, as part of
the national monitoring program for drug residues in Egyptian
honey. This should help various Egyptian honeybee products to
penetrate international markets.
2. Materials and methods
2.1. Chemicals, reagents and standard solutions
Reference standards for AHD, AOZ, AMOZ, SEM, RNZ and DMZ
with purity of at least 98% were obtained from Dr. Ehrenstorfer
GmbH (Germany). All other chemicals were of HPLC grade and
were
obtained
from
SigmaAldrich
(USA).
50 mM
2-nitrobenzaldehyde (NBA) solution was prepared by dissolving
189 mg of NBA powder into 25 mL methanol in an amber volumetric ask. Individual standard stock solutions were prepared by
accurately weighing 10 mg of each standard into a set of 100 mL

amber volumetric asks. The volume was completed to the mark

with methanol: acetonitrile (80:20 v/v) in case of NF metabolites
and methanol in case of NMZ to a nal concentration of
100.00 lg mL1. Stock solutions were stored at 20 2 C away
from direct light. Two working standard solutions of the four NF
metabolites (1.00 lg mL1 and 0.10 lg mL1) were prepared by
diluting suitable aliquots of each stock solution with methanol.
Similarly, two working standard solutions of NMZ standards
(1.00 lg mL1 and 0.10 lg mL1) were prepared in methanol.
Ultra-pure water was obtained using a MilliQ UF-Plus system
(Millipore, Germany) with a resistivity of at least 18.2 MO cm at
25 C and TOC below 5 ppb.
2.2. Instrumentation and conditions
Analysis was carried out using an Agilent 1200 HPLC system
(Agilent Technologies, USA) connected to an API 4000 QTrap tandem
mass
spectrometer
(Applied
Biosystems,
USA).
Chromatographic separations were accomplished using a
ZORBAX Eclipse XDB C-18 column (4.6  150 mm, 5 lm) that
was obtained from Agilent Technologies (USA). The temperature
of the column compartment and sample tray were maintained at
40 C and 48 C, respectively. A gradient elution at a ow rate of
0.3 mL min1 was employed. The mobile phase composition was:
(A) 5 mM ammonium formate buffer in methanol/water (1:9 v/v),
pH 3.0 0.05 and (B) methanol. The gradient was optimised in
order to achieve a run time with enough data points for each peak
over 14 min: 0.06.0 min (6095% B), 6.012.0 min (95% B) and
12.014.0 min (9560% B). The injection volume was 25 lL and
detection was achieved using ESI in positive ion mode. Nitrogen
was used as: nebuliser gas, curtain gas, heater gas and collision
gas according to manufacturers recommendations. Ion spray voltage was 5500 V and the temperature source was set at 400 C.
Acquisition was performed in MRM mode in which one MRM
was used for quantication (quantier peak) and the other was
used for conrmation (qualier peak). The MS/MS transitions and
optimal operational conditions used for analysis are summarized
in Table 1.
2.3. Sample preparation
2.3.1. Derivatisation
Organic honey samples were obtained from local market and
used as blank samples. Aliquots of 1.00 0.02 g were accurately
weighed into 50 mL polypropylene centrifuge tubes. Suitable aliquots of the NF metabolites and NMZ working standard solutions
Table 1
MS/MS transitions and optimal operational conditions used for analysis.
Compound(s)

Precursor
ion m/z

MRM
transitions
m/z

RT

DP
V

EP
V

CE
V

EXP
V

NP-AHD

249.09

6.85

NP-AOZ

236.10

NP-AMOZ

335.09

NP-SEM

209.14

RNZ

201.12

DMZ

142.13

134.00a
104.00b
134.00a
104.00b
291.10a
128.00b
165.90a
192.00b
140.10a
55.10b
96.10a
81.10b

71
71
41
41
66
66
61
61
26
26
56
56

10
10
10
10
10
10
10
10
10
10
10
10

19
33
19
33
17
33
15
17
17
35
23
35

6
6
6
6
16
6
8
10
8
8
16
6

6.96
7.40
7.03
5.55
6.21

RT, retention time (min); DP, declustering potential (volt); EP, entrance potential
(volt); CE, collision energy (volt); EXP, exit potential (volt).
a
Transitions for quantier peaks.
b
Transitions for qualier peaks.

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A.H. Shendy et al. / Food Chemistry 190 (2016) 982989

were added to nal concentration of 1.00 lg kg1 and 3.00 lg kg1,

respectively. Spiked blank samples were vortex mixed for 30 s and
left to stand away from light at room temperature for 10 min.
Deionised water (4.0 mL) was added to the samples followed by
0.5 mL of 1.0 M HCl and 150 lL NBA reagent solution. Samples
were vortex mixed until complete homogeneity was obtained
and then incubated at 55 C in an agitated water bath for 4 h away
from light. Neutralisation to pH 7.0 0.5 was carried out using
5.0 mL of 0.1 M di-potassium hydrogen phosphate followed by
300 lL of 1.0 M NaOH. Throughout this manuscript, nitrophenyl
(NP) derivatives of the NF metabolites will be abbreviated as
NP-AHD, NP-AOZ, NP-AMOZ and NP-SEM (Fig. S1).
2.3.2. Extraction
A modied QuEChERS protocol (Anastassiades, Lehotay,
Stajnbaher, & Schenck, 2003) using acetonitrile extraction, without
addition of primary secondary amine followed by evaporation
under vacuum was employed. NP derivatives of the NF metabolites
and NMZ were extracted using 10.0 mL acetonitrile followed by
agitation for 2 min at 700 rpm. The efciency of extraction was further enhanced by adding 4 g of MgSO4 and 1 g NaCl followed by
agitation for 1 min at the same speed. Samples were then centrifuged at 15,000g for 10 min using a cooling centrifuge. The
organic layer was carefully transferred into 50 mL ask and evaporated at 45 C using a rotatory evaporator. The residue was then
reconstituted into 1.0 mL mobile phase A, sonicated for 1 min then
ltered through a disposable 0.45 lm PTFE membrane lter into an
amber glass vial and injected into LCMS/MS.
2.4. Calibration and validation
A mixture of NF metabolite standards was derivatised with NBA
as described above. The nal preparation of NP-derivatives was
then mixed with NMZ to a nal concentration of (1 lg mL1 each).
A serial dilution of the obtained mixture was prepared using
mobile phase A (0.25, 0.50, 1.00, 2.00, 5.00 and 10.00 ng mL1)
and analysed using LCMSMS as described. The neat standard calibration curves were constructed by plotting concentration
(ng mL1) versus instrument response and correlation coefcients
were estimated. In order to determine the matrix effect; calibration curves were constructed using matrix matched standards
and results were compared to those of the neat calibration curves.
In-house validation with a model-dependent performance
parameters approach was performed according to the criteria
and recommendations of the European Commission Decision
(CD) 2002/657/EC implementing the Council Directive 96/23/EC.
The ResVal software (EC 2002, ver. 2.0) was used for data analysis
and calculations of decision limit (CCa), detection capability (CCb)
and measurement uncertainty (MU).
Blank honey samples were fortied at three levels, 0.5, 1.0 and
1.5  MRPL/recommended concentration. The nal concentrations
for NF metabolites were 0.5, 1.0 and 1.5 lg kg1 while for NMZ the
concentration levels were 1.5, 3.0 and 4.5 lg kg1. At each level the
analysis was performed on three different days using six replicates/day in order to verify the accuracy, repeatability and
within-laboratory reproducibility. Twenty blank honey samples
from different botanical and geographical origins were analysed
on the fourth day to distinguish possible interference in identication and/or quantication of the analytes. Results were expressed
as percentage recovery and coefcient of variation (CV%). The
CCa and CCb were obtained through the analysis of one blank sample and ve fortied samples at concentration levels (0.5, 1.0, 1.5,
2.0 and 5.0  MRPL/recommended concentration) in accordance to
ISO 11843. MU was then estimated by combining the
within-laboratory reproducibility and matrix effect variances in
agreement to SANCO/2004/2726-rev 4.

2.5. Application to real samples

One point matrix matched standard at the MRPL/recommended
concentration and an equivalent neat standard sample were prepared and analysed. Results were used to estimate the matrix
effect and correct for minor variability in test results. The optimised assay protocol was applied for the analysis of thirty commercial honey samples obtained from local market. Naturally
incurred sample, a prociency testing sample (PT sample) by our
laboratory for NF metabolites, round 02249 (2015) was tested. In
the case of the PT sample, results of the proposed assay were compared to those obtained using the currently employed assay with
modications (Verdon, Couedor, & Sanders, 2007). Briey, derivatisation was carried out using NBA, ethyl acetate was used for
extraction without further cleanup. Collected extracts were evaporated under N2 stream at 45 C and reconstituted in
methanol/1 mM ammonium formate (60/40 v/v) before analysis.
3. Results and discussion
3.1. Instrumentation and conditions
LC separation was optimised for all studied analytes (NP-AHD,
NP-AOZ, NP-AMOZ, NP-SEM, RNZ and DMZ). Different mobile
phases: 1, 5 and 10 mM ammonium formate in methanol/water
(1:9 v/v) with pH 3.04.0 were tested using ve LC columns of different dimensions and from different suppliers; Agilent Zorbax
Eclipse XDB C18 (4.6  150 mm, 5 lm), Phenomenex Kinetex C18
(4.6  150 mm, 2.6 lm), Waters Symmetry C18 (4.6  150 mm,
5 lm), Thermo scientic Hypersil C18 (4.6  150 mm, 5 lm) and
Sun Fire C8 (4.6  75 mm, 2.5 lm). The ow rate and gradient were
optimised as described above in order to obtain separation with
enough data points (at least 1012) for each component in a relatively short run time. Efcient separation, symmetric peaks along
with the highest possible signal intensity was successfully
achieved using the described conditions in a total run time of
14 min. The fragmentation conditions and collision energies were
optimised for each analyte by direct infusion of analyte standard
solutions into the mass spectrometer. For quantication and conrmation, the protonated parent ions [M+1]+ and two transition
products were monitored (Table 1). This yields four identication
points, 1 for the precursor ion and 1.5 for each product ion, in
agreement with CD 2002/657/EC for conrmatory methods.
Moreover, identication and quantication of all analytes was
based on four criteria: (1) chromatographic retention time stability, (2) matching of the retention time of analytes in spiked samples and standard solutions, (3) presence of the two relevant
transitions from the analyte molecular peak, a signal to noise ratio
of the ionic transitions (S/N > 3) and (4) stability of the ion ratio
between the quantier and qualier peaks in accordance with
the tolerances recommended by CD 2002/657/EC (point 2.3.3.2).
The ion ratio for each analyte was regularly monitored throughout
the study in order to ensure that they were within acceptable
ranges. The less intense signal (low transition) was used as the
qualier and the most intense one (high transition) was used as
the quantier (Fig. S2).
3.2. Sample preparation
It has been reported that NF metabolites could bind to proteins
or peptides and might form Schiffs base adducts with carbohydrates, aldehydes and/or ketones in honey (Lopez et al., 2007).
Moreover, Tolgyesi et al. reported that RNZ might bind to sugars
forming an N-glycoside bond (Tolgyesi et al., 2012). Thus, mild acid
treatment for honey samples prior to analysis was carried out in

A.H. Shendy et al. / Food Chemistry 190 (2016) 982989

order to liberate matrix-bound compounds. Moreover, acidic conditions provided a suitable environment for the derivatisation
reaction using NBA (Verdon et al., 2007), as will be discussed in
detail.
3.2.1. Derivatisation
In order to improve the sensitivity of detection, NF metabolites
are commonly derivatised prior to determination using LCMS or
LCMS/MS (Chu & Lopez, 2007; Lopez et al., 2007). In this study,
spiked honey samples were prepared using all analytes at the
MRPL of NF metabolites and recommended concentration for
NMZ (CRL guidance paper, 2007), as described above.
Derivatisation of NF metabolites using NBA and extraction of NP
derivatives using ethyl acetate was carried out in accordance to
the method of Verdon et al. that was previously reported for the
analysis of NF metabolite residues in poultry muscle tissue
(Verdon et al., 2007). The effect of duration of incubation step
(0.54 h) has been investigated at 55 C in an agitated water bath.
Results were calculated relative to a control sample of equivalent
concentration prepared in solvent and corrected for matrix effect
(Table S1).
In agreement to previously reported results (Verdon et al.,
2007), the reaction between NBA and NF metabolites was complete
after 3 h. Thus, in all determinations NP derivatives were analysed
after incubation at 55 C for up to 4 h (Table S1). Lack of signicant
difference in the percentage recovery of NMZ conrmed their stability under the studied experimental conditions. The derivatisation experiment was also carried out at a higher temperature
(80 C) in order to investigate whether increasing the reaction temperature would help reduce the reaction time. Results showed that
at 80 C, maximum reaction yield for all NF metabolites was
reached within 0.5 h. However, a gradual decrease in the percentage recovery was noted upon incubation for longer period of time.
Results raised a concern about the stability of NP derivatives at
high temperature. Moreover, percentage recovery obtained upon
incubation at 80 C for 0.5 h was not signicantly higher than that
obtained at 55 C for 4 h, for all analytes. Neutralization of the reaction mixture was then carried out to pH 7.00 0.05 in order to
allow extraction using organic solvents as described.
3.2.2. Extraction
Owing to the complexity of honey matrix, that contains sugars,
enzymes, proteins as well as other minor components such as
lipids and waxes, sample clean-up is generally employed.
QuEChERS method (Anastassiades et al., 2003; Barganska et al.,
2011) has been in use for sample preparation prior to analysis of
multi-class residues in honey (Wang & Leung, 2012) and veterinary
drugs in honey and milk (Aguilera-Luiz, Vidal, Romero-Gonzalez, &
Frenich, 2008). Originally, QuEChERS method involved a single
extraction step, sample clean up via dispersive solid phase extraction using primary secondary amine and direct injection of large
extract volumes (Anastassiades et al., 2003).
In this study, a modied QuEChERS extraction protocol without
sample clean-up followed by evaporation was optimised and
employed. Initially, the effect of extraction solvent composition;
ethyl acetate, acetonitrile, dichloromethane and mixtures of acetonitrile dichloromethane (1:1, 2:1, 3:1 and 4:1 v/v) was investigated. Results were evaluated on the basis of extraction efciency
for all components, cost and safety of employed solvent. Although
extraction efciency was relatively higher in case of acetonitrile
dichloromethane mixtures, acetonitrile was chosen in order to
achieve efcient extraction of all studied analytes at approximately
the same percentage recovery (Fig. 1). In this study, salting out and
complete phase separation was achieved via addition of 4 g MgSO4
and 1 g NaCl in accordance to previously published protocol
(Anastassiades et al., 2003). It should be noted that salting out

985

was particularly important in the case of extraction of NMZ compounds using acetonitrile (Fig. 1). Extracts were then centrifuged
at 15,000g using a cooling centrifuge. This step enabled further
sample clean-up via removal of solidied lipids and waxes in the
sample. The resulting acetonitrile extracts were evaporated under
vacuum at 45 C till complete dryness using a rotary evaporator.
Residue obtained was then reconstituted with mobile phase A
and analysed directly by LCMS/MS.
It should be noted that vacuum evaporation resulted in acceptable percentage recovery for all studied analytes that were comparable to those obtained using nitrogen stream evaporation
technique. Evaporation under vacuum was considered superior to
the commonly employed evaporation using nitrogen stream with
respect to both duration of time and cost. Results showing a comparison between the two techniques relative to an equivalent control sample prepared in solvent are summarized in Table S2.
The obtained results were in agreement to the previously
reported recovery ranges for NF metabolites and NMZ in honey
at 0.502.00 lg kg1 (Lopez et al., 2007) and at 10.00
100.00 lg kg1 (Zhou et al., 2007), respectively. Slightly lower percentage recoveries for NMZ have also been reported when compared to the above results (Galarini et al., 2015; Tolgyesi et al.,
2012). The optimised sample preparation protocol enabled high
percentage recoveries for NMZ which could be attributed to integration of the effects of both acid hydrolysis and salting out.
3.3. Calibration and quantication
When a multi-residue, multi-class assay is developed, it would
be difcult to obtain a radio labelled internal standard for each
compound. In many cases, a representative internal standard is
used in order to overcome cost and availability limitations of internal standards (Nunez, Moyano, & Galceran, 2005). Matrix matched
calibration; the most commonly adopted approach is used to compensate for signal suppression/enhancement experienced during
MS/MS analysis. In the current study, internal standard was not
employed and the quantication was accomplished using a set of
neat standard calibration curves using external standardisation
approach. Results were corrected using one point matrix matched
standard at the MRPL of NF metabolites (1.00 lg kg1) and recommended concentration of NMZ (3.00 lg kg1). Assay validation and
application to spiked honey samples, commercial samples as well
as a PT sample was then carried out in order to verify the applicability of this approach.
3.3.1. Linearity and working range
A mixture of NF metabolite standards was prepared and derivatised as described and NMZ standards were then added. A serial
dilution of the standard mixture was prepared (0.25
10.00 ng mL1) and analysed using the optimised assay conditions.
Results were used to construct the neat standard calibration curves
for the studied compounds. The matrix effect on instrument
response was then investigated in order to reveal possible signal
suppression or enhancement as previously recommended
(Gosetti, Mazzucco, Zampieri, & Gennaro, 2010). Blank honey samples were extracted and fortied with appropriate aliquots of
NP-derivatives of NF metabolites as well as NMZ (0.25
10.00 ng mL1). Matrix matched calibration curves were constructed and regression equation parameters were compared to
those obtained using the neat standard calibration curves
(Table 2). Results showed that both curves were linear with acceptable correlation coefcients and random distribution of the residuals. Two spiked honey samples were prepared at 2.00 and
10.00 lg kg1 and their concentrations were determined using
both calibration curves. Acceptable percentage recoveries were
obtained when the matrix matched calibration was employed.

986

A.H. Shendy et al. / Food Chemistry 190 (2016) 982989

Fig. 1. The effect of extracting solvents on the percentage recoveries of NP-AHD, NP-AOZ, NP-AMOZ, NP-SEM, RNZ and DMZ from honey samples. Error bars of the average
percentage recovery are indicated.

Table 2
Regression equation parameters and differences obtained for both neat standard and matrix matched calibration curves.
Compound(s)

NP-AHD
NP-AOZ
NP-AMOZ
NP-SEM
RNZ
DMZ

Neat standard calibration curves

Matrix matched calibration curves

2

Slope

Intercept

19148.72
241928.21
150331.28
10666.46
88894.36
21390.77

1058.97
20097.44
10478.97
971.23
5200.51
944.62

0.999
0.998
1.000
0.994
0.999
1.000

Slope difference (%)

2

Slope

Intercept

14296.00
127845.10
104011.30
11205.95
17272.82
13607.18

2516.00
9004.10
4901.03
477.64
7270.26
8609.74

0.996
0.995
0.999
1.000
0.982
0.984

25
47
31
+5
81
36

Linear regression equation, y = ax + b; a, slope; b, intercept; y, response (cps); x, concentration (ng mL1).

On the other hand, when the neat standard calibration curves were
used, the percentage recoveries were signicantly lower than those
obtained using the matrix matched calibration curve, as shown in
ANOVA results (Table S3). Such results along with slope differences
shown in Table 2 indicated signicant matrix effect.
3.3.2. Matrix effect and quantication
For in depth evaluation of the matrix effect, the neat standard
calibration curves and matrix matched calibration curves were
compared. Slope differences calculated for each component indicated a signicant matrix suppression ranging from 25% up to
81%, as shown in Table 2. Such differences were out of the
10% limit that has been previously suggested (Matuszewski,
Constanzer, & Chavez-Eng, 2003) which indicated matrix
suppression.
In order to estimate a correction factor for the matrix effect, a
set of extracted blank honey samples were fortied at 0.50, 1.00,
1.50, 2.00 and 5.00  MRPL/recommended concentration of the
studied compounds. Analysis was carried out as described and
mean percentage recoveries and CV% were calculated using the
neat standard calibration curve for each compound (Table 3).
Results indicated a homogenous matrix effect throughout the linear concentration range for each of the studied compounds.
Based on the obtained results, a correction factor was proposed
for each compound at the validation level. For each batch of honey
samples, one point matrix matched standard was prepared at the
MRPL/recommended concentration of each compound. Results of
the one point matrix matched standard were used to estimate
the correction factor for each compound. The following mathematical equation can be then applied for the determination of analyte
concentration in samples after correction for the matrix effect:

Table 3
Statistical analysis for the results of the matrix matched standards determined using
neat standard calibration curves showing the matrix effect.
Compound(s)

NP-AHD
NP-AOZ
NP-AMOZ
NP-SEM
RNZ
DMZ


Cs Ci

V ext V f
V evp W

Descriptive statistical analysis for percentage recoveries

(N = 15)
Mean
percentage
recovery

Median
percentage
recovery

CV%

Condence
interval (95.0%)

66.96
51.85
66.56
101.93
16.00
63.20

68.07
51.98
67.51
102.06
16.14
64.67

5.02
2.42
4.55
4.25
1.49
3.77

6.23
3.00
5.65
6.77
1.85
4.68



C mtxlabelled
C mtxfound

Cs, analyte concentration in sample (lg kg1)

Ci, found analyte concentration determined from calibration
curve (lg kg1)
Vext, total volume of extraction solvent (mL)
Vevp, aliquot volume taken for evaporation (mL)
Vf, nal volume after reconstitution of evaporated aliquot (mL)
W, sample weight (g)
Cmtx (labelled), labelled concentration of one point matrix
matched standard (lg kg1)
Cmtx (found), found concentration of one point matrix matched
standard (lg kg1)
Cmtx (labelled)/Cmtx (found), matrix effect correction factor

A.H. Shendy et al. / Food Chemistry 190 (2016) 982989

In order to verify the applicability of the proposed method of

calculation, two spiked honey samples (2.00 and 10.00 lg kg1 of
each compound) were prepared and analysed as described above.
The concentration of each compound was determined using the
corresponding neat standard calibration curve. Results were corrected using the proposed one point matrix matched method of
calculation. Statistical comparison was then carried out to those
obtained directly from the matrix matched calibration curves,
using one-way ANOVA. Signicant difference between the results
obtained using the neat standard calibration curve and the matrix
matched calibration curve conrmed the profound matrix effect as

987

explained above. On the other hand, no signicant difference

between the results corrected for matrix effect and those obtained
using the conventional matrix matched calibration conrmed the
applicability of our approach. A summary of the results of the statistical analysis are summarized in Table S3.
3.4. Validation procedure
Validation was carried out in accordance with the procedures
outlined in CD 2002/657/EC covering specicity and recovery
(trueness/accuracy),
precision
(repeatability
and

Fig. 2. Typical chromatograms for NP-AHD, NP-AOZ, NP-AMOZ, NP-SEM, RNZ and DMZ in fortied samples at the respective MRPL/recommended concentration level in
comparison to the corresponding blank samples, fortication level; 1.00 lg kg1 for NP-AHD, NP-AOZ, NP-AMOZ and NP-SEM and 3.00 lg kg1 for RNZ and DMZ.

988

A.H. Shendy et al. / Food Chemistry 190 (2016) 982989

within-laboratory reproducibility), decision limits (CCa), detection

capability (CCb) and measurement uncertainty (MU). The ruggedness of the assay was demonstrated on an ongoing basis through
its use for routine analysis of honey samples.
3.4.1. Specicity
A specicity study was conducted in order to verify the absence
of potential interfering compounds at the retention time of the
studied analytes. The assay was applied to twenty blank honey
samples of different origins/matrix composition (viscosity, pigment content, pollen grain contents. . .). Representative honey
samples
were
fortied
with
all
analytes
at
the
MRPL/recommended concentration and analysis was carried out
as described. No interfering peaks were detected in the region of
interest for all analytes as shown in the chromatograms of blank
honey samples (Fig. 2).

0.53 lg kg1 while for NMZ values were 0.530.74 lg kg1 and
0.911.27 lg kg1, respectively. The obtained results conrmed
the high sensitivity of the developed assay. The calculated critical
concentrations for CCa and CCb for the studied analytes are summarized in Table 4.
3.4.4. Measurement uncertainty (MU)
Measurement uncertainty has not been explicitly mentioned in
CD 2002/657/EC. However, it can be determined correctly by systematically taking into account all relevant inuencing factors possibly affecting the measurement results. According to
SANCO/2004/2726-rev 4, the within-laboratory reproducibility
can be regarded as a good estimator for the combined MU of individual methods. In this work, MU% was calculated for all studied
analytes (1121%) as summarized in Table 4.
3.5. Application to commercial honey samples

3.4.2. Accuracy and precision

The accuracy and precision (repeatability and within-laboratory
reproducibility) of the assay were measured at 0.5, 1.0 and
1.5  MRPL/recommended concentration for each analyte. Results
indicated acceptable performance of the assay for all analytes over
the studied validation levels. The percentage recoveries were in the
range of 90.96104.80% with CV% of 1.355.10% and 2.6512.58%
for repeatability and within-laboratory reproducibility, respectively. These results were in agreement to the requirements of
CD 2002/657/EC regarding the CV% for repeated analysis of spiked
or incurred material. Results are summarized in Table 4.
3.4.3. Decision limit and detection capability
The CD 2002/657/EC recommended analytical limits: (1) decision limit (CCa); the critical concentration at risk alpha (also called
the decision limit) and (2) the detection capability (CCb); the critical concentration at risk beta (also called the detection capability).
In this study, CCa and CCb were calculated using the calibration
curve procedure in accordance with ISO 11843. Blank honey samples were analysed along with fortied samples with the studied
NF metabolites (0.50, 1.00, and 1.50 lg kg1) and NMZ (1.50,
3.00, and 4.50 lg kg1). Responses were plotted against the added
concentration, and CCa and CCb were determined. The CCa and
CCb for the NF metabolites were 0.120.31 lg kg1 and 0.21

In order to demonstrate the applicability of the optimised assay

protocol as well as the correction factor calculation, thirty commercial honey samples of different botanical origin were obtained
from local market. The validated assay protocol was then applied
for the detection and determination of the studied analytes.
Results showed that AHD, AOZ, AMOZ, SEM, RNZ and DMZ were
not present in amounts above the detection capability or decision
limit of the employed assay. Samples were claimed appropriate for
human use according to the CRL guidance paper 2007. In all determinations, 1 g of honey sample was employed in order to reduce
the time and cost of sample preparation and the amounts of the
residual matrix components.
3.6. Application to previously analysed PT sample
Further verication of assay performance and calculation
approach was carried out through analysis of a PT sample (round
02249), as part of the Food Analysis Performance Assessment
Scheme (FAPAS). An incurred honey claimed to contain NF metabolites was analysed as described and results were calculated using
the proposed calculation method. Matrix-corrected results were
compared to those obtained using the currently adopted method
in our laboratory (Verdon et al., 2007) with modications

Table 4
Accuracy, precision values, decision limit (CCa), detection capability (CCb) and measurement uncertainty (MU) obtained for the studied NF metabolites and NMZ residues in
honey.
Compound(s)

Fortication level
(lg kg1)

Accuracy

Repeatability

Within-lab reproducibility

Percentage
recovery

Mean concentration
(lg kg1)

CV%

Mean concentration
(lg kg1)

R2

CCa
(lg kg1)

CCb
(lg kg1)

MU%

CV%

NP-AHD

0.5
1.0
1.5

95.60
96.40
97.33

0.49
1.00
1.50

3.76
4.74
3.66

0.48
0.96
1.46

9.83
9.18
6.45

0.998

0.12

0.21

18

NP-AOZ

0.5
1
1.5

99.80
99.60
97.73

0.51
1.01
1.48

2.24
2.93
2.66

0.50
1.00
1.47

2.96
5.16
4.78

0.994

0.22

0.38

11

NP-AMOZ

0.5
1
1.5

95.00
104.80
104.00

0.49
1.04
1.50

1.35
5.10
3.37

0.48
1.05
1.56

4.71
8.92
8.32

0.999

0.2

0.34

21

NP-SEM

0.5
1
1.5

98.20
95.60
100.93

0.50
0.99
1.50

4.42
2.47
1.36

0.49
0.96
1.51

7.27
5.63
2.65

0.999

0.31

0.53

21

RNZ

1.5
3
4.5

96.20
94.03
95.67

1.51
2.89
4.47

3.74
1.51
2.83

1.44
2.82
4.31

5.44
6.81
5.06

0.999

0.53

0.91

13

DMZ

1.5
3
4.5

93.33
97.00
90.96

1.51
3.03
4.40

4.12
1.81
5.04

1.40
2.91
4.09

12.58
7.57
9.62

0.990

0.74

1.27

17

A.H. Shendy et al. / Food Chemistry 190 (2016) 982989

described above (Table S4). Results of the optimised assay conrmed the presence of AMOZ from the NF metabolite family.
These results were in agreement to those obtained using the currently adopted method and published in the results report (round
02249). The z-score was calculated for the obtained results and
was found within the acceptable range |z| < 2 (Table S4).
Satisfactory z-scores indicated the applicability of the proposed
assay protocol for the determination of the studied compounds
in honey samples.
4. Conclusion
An accurate and sensitive LCMS/MS assay was developed and
validated for the simultaneous determination of six banned antibiotics from two different classes in honey. Analytes were extracted
using modied QuEChERS protocol, without sample clean-up. Neat
standard calibration curves were successfully employed in conjunction with correction for matrix effect. Assay validation to the
quality criteria and requirements of CD 2002/657/EC was carried
out. The applicability of the method for the determination of the
studied compounds was veried using spiked honey samples as
well as PT samples. The assay was deemed suitable for the regulatory monitoring of NF metabolites and NMZ residues in locally produced honey. This should help develop an efcient national
monitoring plan for Egyptian honeybee products.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2015.
06.048.
References
Aguilera-Luiz, M. M., Vidal, J. L. M., Romero-Gonzalez, R., & Frenich, A. G. (2008).
Multi-residue determination of veterinary drugs in milk by ultra-high-pressure
liquid chromatographytandem mass spectrometry. Journal of Chromatography
A, 1205(1), 1016.
Anastassiades, M., Lehotay, S. J., Stajnbaher, D., & Schenck, F. J. (2003). Fast and easy
multiresidue method employing acetonitrile extraction/partitioning and
dispersive solid-phase extraction for the determination of pesticide residues
in produce. Journal of AOAC International, 86(2), 412431.
Barganska, Z., Namiesnik, J., & Slebioda, M. (2011). Determination of antibiotic
residues in honey. Trends in Analytical Chemistry, 30(7), 10351041.
Bottoni, P., & Caroli, S. (2015). Detection and quantication of residues and
metabolites of medicinal products in environmental compartments, food
commodities and workplaces. A review. Journal of Pharmaceutical and
Biomedical Analysis, 106, 324.
Butaye, P., Devriese, L. A., & Haesebrouck, F. (2001). Differences in antibiotic
resistance patterns of enterococcus faecalis and enterococcus faecium strains
isolated from farm and pet animals. Antimicrobial Agents and Chemotherapy,
45(5), 13741378.
Chu, P.-S., & Lopez, M. I. (2007). Determination of nitrofuran residues in milk of
dairy cows using liquid chromatographytandem mass spectrometry. Journal of
Agriculture and Food Chemistry, 55(6), 21292135.
CRL guidance paper (2007). In: <http://www.crl.fougeres.anses.fr/publicdoc/2013/
EURL_Guidance_Concentrations_Minimales_Recommend%C3%A9es_Methodes_
Analytiques.pdf>.
Cronly, M., Behan, P., Foley, B., Malone, E., Martin, S., Doyle, M., & Regan, L. (2010).
Rapid multi-class multi-residue method for the conrmation of
chloramphenicol and eleven nitroimidazoles in milk and honey by liquid
chromatography-tandem mass spectrometry. Food Additives and Contaminants:
Part A, 27(9), 12331246.
European Commission (1996). Commission Directive 96/23/EC. Ofcial Journal of
the European Commission, L125, 1032.
European Commission. (2002). Commission Decision 2002/657/EC of August 12,
2002: Implementing Council Directive 96/23/EC (2002) Concerning the
Performance of Analytical Methods and the Interpretation of Results. Ofcial
Journal of the European Commission, L221, 836. http://ec.europa.
eu/food/food/chemicalsafety/residues/lab_analysis_en.htm.
European Commission. (2003). Commission Decision 2003/181/EC of 13 March
2003 amending Decision 2002/657/EC as regards the setting of minimum

989

required performance limits MRPLs for certain residues in food of animal origin.
Ofcial Journal of the European Union, L71, 1718. https://www.fsai.ie/
uploadedFiles/Legislation/Food_Legisation_Links/Veterinary_Medicines, _Control_
of_Illegal_Substances_and_Po/Commission_Decision_2003_181_EC.pdf.
European Commission. (2010). Commission Regulation 37/2010 of 22 December
2009: on pharmacologically active substances and their classication regarding
maximum residue limits in foodstuffs of animal origin. Ofcial Journal of the
European Union, L15, 172. http://ec.europa.eu/health/les/eudralex/vol-5/reg_
2010_37/reg_2010_37_en.pdf.
Galarini, R., Saluti, G., Giusepponi, D., Rossi, R., & Moretti, S. (2015). Multiclass
determination of 27 antibiotics in honey. Food Control, 48, 1224.
Gosetti, F., Mazzucco, E., Zampieri, D., & Gennaro, M. C. (2010). Signal suppression/
enhancement in high-performance liquid chromatography tandem mass
spectrometry. Journal of Chromatography A, 1217(25), 39293937.
Hernandez-Mesa, M., Garcia-Campana, A. M., & Cruces-Blanco, C. (2014). Novel
solid phase extraction method for the analysis of 5-nitroimidazoles and
metabolites in milk samples by capillary electrophoresis. Food Chemistry, 145,
161167.
Huet, A.-C., Mortier, L., Daeseleire, E., Fodey, T., Elliott, C., & Delahaut, P. (2005).
Development of an ELISA screening test for nitroimidazoles in egg and chicken
muscle. Analytica Chimica Acta, 534(1), 157162.
International Organization for Standardization (1997). ISO 11843:1. Capability of
detection - Part 1: terms and denitions, Part 2: Methodology in the Linear
Calibration case ISO 11843:1 (2000), ISO 11843:2. Statgraphics-Plus V. 5.1
(2002) Experimental Design, Appendix C, Manugistics, Rockville, MD. In:
<https://www.iso.org/obp/ui/#iso:std:iso:11843:-6:ed-1:v1:en>.
Kaufmann, A., Butcher, P., Maden, K., Walker, S., & Widmer, M. (2015).
Determination of nitrofuran and chloramphenicol residues by high resolution
mass spectrometry versus tandem quadrupole mass spectrometry. Analytica
Chimica Acta, 826, 4152.
Lopez, M. I., Feldlaufer, M. F., Williams, A. D., & Chu, P.-S. (2007). Determination and
conrmation of nitrofuran residues in honey using LCMS/MS. Journal of
Agriculture and Food Chemistry, 55(4), 11031108.
Matuszewski, B. K., Constanzer, M. L., & Chavez-Eng, C. M. (2003). Strategies for the
assessment of matrix effect in quantitative bioanalytical methods based on
HPLCMS/MS. Analytical Chemistry, 75(13), 30193030.
Mital, A. (2009). Synthetic nitroimidazoles: biological activities and mutagenicity
relationships. Scientia Pharmaceutica, 77(3), 497520.
Nunez, O., Moyano, E., & Galceran, M. T. (2005). LC-MS/MS analysis of organic toxics
in food. Trends in Analytical Chemistry, 24(7), 683703.
OKeeffe, M., Conneely, A., Cooper, K. M., Kennedy, D. G., Kovacsics, L., Fodor, A., et al.
(2004). Nitrofuran antibiotic residues in pork: the FoodBRAND retail survey.
Analytica Chimica Acta, 520(1), 125131.
RASSF Portal. In: <https://webgate.ec.europa.eu/rasff-window/portal/?event=
SearchForm&cleanSearch=1>.
SANCO/2004/2726-rev 4, Guidelines for the implementation of decision 2002/657/
EC (2008). In: http://crl.fougeres.anses.fr/publicdoc/Guidelines-Consolidated_
2002-657_2004-2726rev4_en.pdf.
Tolgyesi, A., Sharma, V. K., Fekete, S., Fekete, J., Simon, A., & Farkas, S. (2012).
Development of a rapid method for the determination and conrmation
of nitroimidazoles in six matrices by fast liquid chromatography-tandem
mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis, 64,
4048.
Vass, M., Hruska, K., & Franek, M. (2008). Nitrofuran antibiotics: A review on the
application, prohibition and residual analysis. Veterinarni Medicina, 53(9),
469500.
Venable, R., Haynes, C., & Cook, J. M. (2014). Reported prevalence and quantitative
LCMS methods for the analysis of veterinary drug residues in honey: A review.
Food Additives and Contaminants: Part A, 31(4), 621640.
Verdon, E., Couedor, P., & Sanders, P. (2007). Multi-residue monitoring for the
simultaneous determination of ve nitrofurans (furazolidone, furaltadone,
nitrofurazone, nitrofurantoine, nifursol) in poultry muscle tissue through the
detection of their ve major metabolites (AOZ, AMOZ, SEM, AHD, DNSAH) by
liquid chromatography coupled to electrospray tandem mass spectrometry: Inhouse validation in line with Commission Decision 657/2002/EC. Analytica
Chimica Acta, 586(1), 336347.
Wang, J., & Leung, D. (2012). The challenges of developing a generic extraction
procedure to analyze multi-class veterinary drug residues in milk and honey
using ultra-high pressure liquid chromatography quadrupole time-of-ight
mass spectrometry. Drug Testing and Analysis, 4(S1), 103111.
WHO: Antimicrobial resistance: global report on surveillance (2014). In: <http://
apps.who.int/iris/bitstream/10665/112642/1/9789241564748_eng.pdf?ua=1>.
Xia, X., Li, X., Zhang, S., Ding, S., Jiang, H., Li, J., et al. (2008). Simultaneous
determination of 5-nitroimidazoles and nitrofurans in pork by highperformance liquid chromatography-tandem mass spectrometry. Journal of
Chromatography A, 1208(1), 101108.
Zhou, J., Shen, J., Xue, X., Zhao, J., Li, Y., Zhang, J., et al. (2007). Simultaneous
determination of nitroimidazole residues in honey samples by highperformance liquid chromatography with ultraviolet detection. Journal of
AOAC International, 90(3), 872878.