Professional Documents
Culture Documents
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
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.
983
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.
984
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
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
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
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
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
987
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
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
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
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.