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Multi-residue determination of antibiotics in fish by

liquid chromatography-tandem mass spectrometry
a a a a a
M. Gbylik , A. Posyniak , K. Mitrowska , T. Bladek & J. Zmudzki
a
Pharmacology and Toxicology Department, National Veterinary Research Institute, Pulawy,
Poland
Accepted author version posted online: 28 Feb 2013.Published online: 10 Apr 2013.

To cite this article: M. Gbylik, A. Posyniak, K. Mitrowska, T. Bladek & J. Zmudzki (2013) Multi-residue determination of
antibiotics in fish by liquid chromatography-tandem mass spectrometry, Food Additives & Contaminants: Part A, 30:6,
940-948, DOI: 10.1080/19440049.2013.780210

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 Food Additives & Contaminants: Part A, 2013
Vol. 30, No. 6, 940–948, http://dx.doi.org/10.1080/19440049.2013.780210

Multi-residue determination of antibiotics in fish by liquid chromatography-tandem mass

spectrometry
M. Gbylik*, A. Posyniak, K. Mitrowska, T. Bladek and J. Zmudzki
Pharmacology and Toxicology Department, National Veterinary Research Institute, Pulawy, Poland
(Received 13 September 2012; final version received 17 February 2013)

A multi-residue method for the determination of 34 antibacterial drugs (three aminoglycosides, nine β-lactams, nine
fluoroquinolones, three macrolides, five sulfonamides, trimethoprim and four tetracyclines) in fish samples by LC-MS/
MS was developed. The procedure enables the isolation of residues based on double extraction of the fish sample with m-
phosphoric acid and heptafluorobutyric acid as an ion-pair agent and acetonitrile. All compounds were determined at the
Downloaded by [Moskow State Univ Bibliote] at 04:15 21 October 2013

same time on a C18 column with gradient elution. The method was validated according to the requirements of European
Decision 2002/657/EC. Specificity, recovery, repeatability, within-laboratory reproducibility, decision limit CCα and
detection capability CCβ were calculated. CCα ranged from 55.3 to 1085 µg kg−1 and CCβ ranged from 59.5 to
1141 µg kg−1. The overall recoveries were from 96% to 111% with respect to the internal standards.
Keywords: multi-residue; fish; antibiotics; LC-MS/MS

Introduction two separate analytical procedures: one sample is

Many different groups of antibacterial compounds such as
aminoglycosides, β-lactams, fluoroquinolones, macrolides,
sulfonamides, and tetracyclines were widely used in
human and veterinary practice. The physicochemical prop-
erties of these drugs may cause their easy transfer to
ground and surface water. Because of that fact, they can
accumulate in water organisms, especially fish, and cause
disturbances to the aquatic ecosystem. The occurrence of
antibiotics in fish can also be dangerous for consumers.
Antibiotic accumulation in aquaculture ecosystems can
lead to antibiotic resistance in microorganisms, which
causes an increase in antibiotic-resistant infections in
humans and potential adverse side-effects such as allergies
(Kasprzyk-Horden et al. 2007; Canada-Canada et al. 2009;
Smith et al. 2009; Chafer-Pericas et al. 2011). The
European Union sets MRLs for veterinary substances
whose residues can be dangerous for human health
(Commission Regulation (UE) 37/2010 of 22 December
2009). Therefore, it appears to be very important to set up
a simple, fast and reliable analytical method for the deter-
mination of antibiotics in fish. Many multi-residue meth-
ods for determination of antibacterial compounds in
animal muscles (Granelli & Branzell 2007; Hurtaud-
Pessel et al. 2011) have been described in the literature.
However, most of the methods do not cover the analysis of
aminoglycosides in the same analytical protocol (Li et al.
2006; Smith et al. 2009; Chafer-Pericas et al. 2011).
Several multi-class methods include aminoglycosides
(Hammel et al. 2008), but these methods are based on
extracted with acetonitrile or acetonitrile with water buf-
fer; the second sample is extracted with trichloroacetic
acid (Gaugain-Juhel et al. 2009; Hurtaud-Pessel et al.
2011). Some methods that utilise single sample prepara-
tion for the simultaneous analysis of highly polar amino-
glycosides with other components either require a double-
injection system with two different chromatographic col-
umns (Martos et al. 2010) or do not include cephalospor-
ins in the analytical scheme (Chiaochan et al. 2010). So
far there is no published multi-class method enabling the
simultaneous determination of aminoglycosides, cephalos-
porins, fluoroquinolones, macrolides, penicillins, sulfona-
mides, trimethoprim and tetracyclines in a single
analytical method. This paper describes the development
of a sensitive, efficient and rapid procedure to determine a
wide range of antibacterial residues in fish by LC-MS/MS.
Materials and methods
Reagents
All reagents used were of an analytical grade. m-Phosphoric
acid, acetonitrile, and methanol were obtained from J.T.
Baker (Deventer, The Netherlands). Ethylenediamine tetra-
acetic acid (EDTA) was from POCH (Gliwice, Poland),
trichloroacetic acid (TCA) was from Sigma-Aldrich (St.
Louis, MO, USA). Heptafluorobutyric acid (HFBA) was
from Fluka (Newport News, VA, USA). Water was deionised
(>18 MΩ cm−1) by the Millipore system. Amoxicillin
(AMOX), penicillin G (PEN G), cephapirin (CFPI), ceftiofur

*Corresponding author. Email: malgorzata.gbylik@piwet.pulawy.pl

© 2013 Taylor & Francis


(CFT), cefoperazone (CFPE), cephalexin (CFLE), cefqui-
nome (CFQ), cefazolin (CFZ), cefalonium (CFLO), dano-
floxacin (DAN), difloxacin (DIF), enrofloxacin (ENR),
ciprofloxacin (CIP), norfloxacin (NOR), flumequine (FLU),
sarafloxacin (SAR), oxolinic acid (OXO), nalidix acid
(NAL), chlortetracycline (CTC), tetracycline (TC), doxycy-
cline (DC), oxytetracycline (OTC), streptomycin (STRP),
dihydrostrepromycin (DISTRP), spectinomycin (SPEC),
neomycin (NEO), gentamycin (GEN), sulfamerazine (SME),
sulfamethazine (SMT), sulfamethoxazole (SMA), sulfamono-
methoxine (SMM), sulfadimethoxine (SDMX), trimethoprim
(TMP), tylosin (TYL), erythromycin (ERY), azithromycin
(AZY), sulfafenazole (SFF) – IS1, ciprofloxacin d-8 (CP-d8)
– IS2, and roxithromycin (ROX) – IS3 were all from Sigma-
Aldrich. Strata X-CW (60 mg, 3 ml) cartridges were obtained
from Phenomenex (Torrance, CA, USA); syringe filters
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0.45 μm PVDF were from Restek (Bellefonte, PA, USA).
Standard solutions
Individual stock standard solutions (1 mg ml–1) for macro-
lides (TYL, ERY, AZY), tetracyclines (TC, CTC, DC,
OTC), sulfonamides (SME, SMT, SMA, SMM, SDMX)
and diaminopyrimidine (TMP) were prepared in methanol,
whereas for fluoroquinolones (DAN, DIF, ENR, CIP, NOR,
FLU, SAR, OXO, NAL) they were prepared in methanol
with the addition of sodium hydroxide; for aminoglycosides
(STRP, DISTRP, SPEC, NEO, GEN) and β-lactams
(AMOX, PEN G, CFPI, CFT, CFPE, CFLE, CFQ, CFZ,
CFLO) they were prepared in deionised water in amber
volumetric flasks and stored at –18°C. Mixtures of working
standard solutions were prepared in deionised water in
plastic flasks and stored at 4°C. Individual stock internal
standard solutions (1 mg ml−1) for SFF (IS 1), CP-d8 (IS 2)
and ROX (IS 3) were prepared in deionised water in amber
volumetric flasks and stored at –18°C. Mixtures of working
internal standards (2 µg ml−1) were prepared in deionised
water in amber volumetric flask and stored at 4°C.
Fish samples
For method validation and the optimisation process, all
fish samples including common bream, roach, pike, zander
and catfish were collected from different commercial fish
farms and checked that they were free from antibiotic
residues. Before the experiment, muscles and skin in nat-
ural proportions were collected from different fish species.
Thereafter, all fish samples were minced, mixed and stored
at –18°C until analysis. Contrary to the matrix effect, the
samples from different fish species were prepared
separately.
Sample preparation
To the fish sample (2.0 ± 0.1 g), a 100 µl mixture of
internal standard solution (CP-d8, ROX, SFF) was added
Food Additives & Contaminants: Part A 941
before sample extraction and the sample was mixed and
left to incubate at 4°C in the dark for 30 min. The 0.1 M
EDTA (0.5 ml), 3% m-phosphoric acid pH 5.5 (6 ml),
0.02 M HFBA (2 ml) and 20% TCA (0.6 ml) were added;
the sample was homogenised with a vortex mixer for
2 min and mechanically shaken for 10 min and then
centrifuged at 4500 rpm for 10 min at 5°C. An aliquot
of the supernatant was loaded into a Strata X-CW column
preconditioned with 3 ml of methanol, water and 0.02 M
HFBA. The cartridge was dried under vacuum for 6 min
and eluted with 2% formic acid in methanol (twice 3 ml).
The eluate was evaporated to dryness under a stream of
nitrogen at 45 ± 5°C. The remaining fish pellet was re-
extracted with 6 ml acetonitrile, vortexed, mechanically
shaken for 10 min and centrifuged at 4500 rpm for 10 min
at 5°C. The supernatant was evaporated to dryness under a
stream of nitrogen at 45 ± 5°C. Both residues were dis-
solved in 0.025% HFBA (250 µl) and combined. The
coupled extracts were filtered through 0.45 µm PVDF
syringe filters into LC vials (Figure 1).
Liquid chromatography-mass spectrometry equipment
and conditions
The LC analysis was performed using an Agilent 1200
HPLC system (Agilent Technologies, Böblingen,
Germany) with an automatic degasser, a binary pump and
an autosampler. The chromatographic separation was carried
out with the use of a Luna C18 (2) 100A column
(50 × 4.6 mm, particle size 3 µm; Phenomenex) at 35°C.
The LC flow was maintained at 250 µl min−1. The mobile
phase A was acetonitrile; the mobile phase B was 0.025%
HFBA in water. A gradient elution programme started
with 5% of A, increased to 50% of A at 2 min and 90% of
A at 7 min, was kept at 90% of A for 4 min, returned to the
initial composition at 11.01 min and equilibrated for another
7 min before the next injection. The LC system was
connected to a PE Sciex API 4000 quadrupole mass spectro-
meter (PE Sciex, Framingham, MA, USA). An electrospray
source was used in positive-ion mode. Data acquisition was
in MRM mode. The ion transitions and mass parameters for
each analyte monitored are listed in Table 1. The following
parameters were used in the tune mode: resolution Q1 and
Q3 – unit; temperature – 500°C; nebuliser gas (gas 1) – 40;
curtain gas – 20; collision gas – 3; auxiliary gas – 50; and ion
spray voltage – 5500 V. Analyst 1.5 software controlled the
LC-MS/MS system and processed the data.
Method validation
The method was validated according to the requirements
of European Decision 2002/657/EC. For the validation
process mixed fish samples were used. Internal standard
mixture (100 µl) was added to each sample, and all sam-
ples were fortified with a 50, 100 and 150 µl mixture of
standard solution that corresponded to the following con-
centration levels of 0.5, 1.0 and 1.5 MRL/VL (validation
 942 M. Gbylik et al.

2 ± 0.01 g of fish + 100 µl IS

0.5 ml 0.1 M EDTA

6 ml 3% meta-phosphoric acid pH = 5.5
2 ml 0.02 M HFBA
0.6 ml 20% TCA

Vortex, mechanically shake

centifugation: 4500/10 min, 5°C

Remaining fish matrix Supernatant

SPE extration: Strata X-CW

6 ml ACN
conditioning:
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3ml: MeOH, ERY-H2O, 0.02M HFBA

Vortex, mechanically shake
centrifugation: 4500/10 min, 5°C Application of extract

Elution:
Supernatant evaporation: N2, 45°C 2 × 3 ml 2% formic acid in MeOH

Eluat evaporation: N2, 45°C

Dilution: 0.025% HFBA
Dilution: 0.025% HFBA

Combinating residues and

filtrating:
0.45 µm PVDF syringe filters

Figure 1. Sample preparation.

level). For analytes without established MRLs, the VL
was set at a concentration level of 50 µg kg−1 (Table 2).
Calibration and quantification
The matrix-matched calibration curve was prepared by fortify-
ing blank fish samples at six concentration levels (0.25, 0.5,
0.75, 1.0, 1.5 and 2.0 times the MRL/VL). The evaluation of
quantitative results was performed by comparing the analyte/
internal standard peak area ratio from a matrix-matched cali-
bration curve to the analyte/internal standard peak area ratio in
the analysed samples.
Selectivity
The possible presence of interfering substances around the
retention times of the compounds of interest was checked
by analysing 20 mixed blank samples of fish muscles and
skin from different sources.
Recovery, repeatability and within-laboratory
reproducibility
The repeatability was determined after fortifying of six
blank samples at each of the three concentration levels of
0.5, 1.0 and 1.5 times the MRL/VL for each analyte. The
samples were analysed on the same day with the same
instrument and the same operators and the CVs (%) of
the fortified samples were calculated. The within-laboratory
reproducibility was determined after fortifying another two
sets of blank samples at the same concentration levels of
analysed compounds as for the repeatability and analysing
on two different days with the same instrument and differ-
ent operators and the overall CVs of the fortified samples
were calculated. The percentage recovery (with reference to
the internal standard) was evaluated in the same experiment
as repeatability by comparing the mean measured concen-
tration with the fortified concentration of the samples.
Decision limit and detection capability
Decision limit (CCα) and detection capability (CCβ) were
determined by the matrix-matched calibration curve proce-
dure according to ISO 11843 (243). CCα was calculated
with a statistical certainty of 1 – α (α = 0.05), whereas CCβ
was calculated with a statistical certainty of 1 – β (β = 0.05).
Stability of the antibiotics in solution and matrix
The stability of each stock standard solution of all
antibiotics groups (aminoglycosides, β-lactams,
 Food Additives & Contaminants: Part A 943

Table 1. MS/MS ion transitions and parameters.

Analyte Ion transition 1 (m/z) Ion transition 2 (m/z) DP (V) CE (Ev) Dwell time (ms)
a
AMOX 366.1/349.1 366.1/208.0 45 14 30
PEN G 335.1/160.0a 335.1/176.1 60 17 30
SMT 279.2/156.0a 279.2/108.0 50 25 20
SME 265.0/156.0a 265.0/108.0 50 27 20
SDMX 311.0/156.0a 311.0/108.0 50 23 20
SMA 254.0/107.8a 254.0/155.9 40 24 20
SMM 281.0/156.0a 281.0/108.0 50 35 20
TMP 292.1/262.2a 292.1/231.3 52 36 20
DC 445.0/428.0a 445.0/154.0 50 23 20
OTC 461.0/426.0a 461.0/444.0 40 28 20
TC 445.0/410.0a 445.0/427.0 55 27 20
CTC 479.0/444.0a 479.0/462.0 60 29 20
ERY-H2O 716.5/558.5b 716.5/158.2 75 28 20
AZY 749.5/591.2b 749.5/158.4 89 40 20
TYL 916.0/174.0b 916.0/772.5 110 52 20
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STRP 582.0/263.0a 582.0/246.0 166 52 40

SPEC 351.1/333.2a 351.1/207.2 67 32 40
DISTRP 584.3/263.2a 584.3/246.2 150 42 40
CIP 332.0/314.0c 332.0/231.0 65 28 20
ENR 360.0/342.0c 360.0/286.0 100 33 20
FLU 262.0/244.0c 262.0/202.0 44 25 20
SAR 386.0/368.0c 386.0/348.0 50 31 20
NAL 233.0/215.0c 233.0/187.0 42 30 20
OXO 262.0/244.0c 262.0/216.0 52 25 20
DIF 400.5/382.1c 400.5/356.0 50 30 20
DAN 358.0/340.0c 358.0/255.0 60 33 20
NOR 320.0/302.0c 320.0/231.0 50 30 20
CFPI 424.0/152.0a 424.0/124.0 50 35 20
CFT 524.0/241.0a 524.0/125.0 50 25 20
CFQ 529.0/134.0a 529.0/125.0 50 25 20
CFLO 459.0/337.1a 459.0/152.0 46 16 20
CFZ 455.0/323.0a 455.0/156.0 50 15 20
CFLE 348.0/158.0a 348.0/106.0 50 10 20
CFPE 646.0/530.0a 646.0/530.0 60 17 20
Notes: aIon transition used for quantification with sulfaphenazole as IS 1.bIon transition used for quantification with roxithromycin as IS 2.
c
Ion transition used for quantification with ciprofloxacin d-8 as IS 3.

fluoroquinolones, macrolides, sulfonamides, trimethoprim,
tetracyclines) was determined in the following order: 1
day and 1, 3, 6 and 10 months. All individual stock
standard solutions were stored at two different tempera-
tures: 4°C and –18°C.
The stability of antibiotics in fish muscles was evalu-
ated by analysing fortified blank fish samples at the 1
MRL/VL concentration level. Prepared samples were
stored at –18°C and analysed after 1 day and 1, 2, 3 and
6 months.
Robustness
The robustness of the method was estimated according to
the Youden approach for each of eight fortified blank fish
samples at the 1 MRL/VL concentration level (Plackett &
Burman 1946; Van der Heyden et al. 2001). The four
different possible factors that could influence the method
results were checked (the amount of ACN used for extrac-
tion, the amount of solvent used for SPE conditioning and
the elution part, and the temperature of the evaporation of
the extracts).
Matrix effect
The matrix effect was checked by analysing five different
fish matrices of 1 MRL/VL concentration level for all
analytes and was calculated using equation (1), which
was proposed by Kasprzyk-Horden et al. (2007). The
matrix effect was evaluated as a percentage of the signal
intensity of a sample extract fortified after extraction (IM)
in relation to the signal intensity of water fortified (IW) at
the same concentration level:
 
IM
Matrix effectð%Þ ¼  100 (1)
IW
 944 M. Gbylik et al.

Table 2. Performance data of the LC-MS/MS multi-residue method for mixed fish samples.

Within-laboratory
Analyte MRL/VL (µg kg–1) Repeatability, CV (%) reproducibility, CV (%) CCα (µg kg–1) CCβ (µg kg–1) Recovery (%)
AMOX 50 4.2 12.5 65.5 79.0 111
PEN G 50 18.0 18.3 65.9 80.8 96
SMT 100 6.9 11.4 115.3 133.3 105
SME 100 4.1 8.8 110.2 126.9 100
SDMX 100 4.4 4.3 109.4 119.4 99
SMA 100 5.2 4.8 106.7 114.0 103
SMM 100 4.4 6.7 107.39 119.1 97
TMP 100 3.0 5.7 119.8 127.17 103
DC 100 9.9 8.7 124.8 160.3 102
OTC 100 5.6 7.9 118.4 138.3 106
TC 100 3.9 6.5 109.8 121.8 100
CTC 100 4.7 5.3 106.0 114.2 99
ERY-H2O 200 3.3 3.8 215.5 229.7 99
AZY (50)a 11.2 12.1 56.9 67.9 102
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TYL 100 7.0 10.2 111.4 127.0 97

STRP 500 5.8 6.5 589.8 714.9 107
SPEC 300 3.1 7.5 378.0 444.5 106
DISTRP 500 2.6 4.4 529.4 571.0 101
CIP 100 2.8 4.1 108.8 121.4 100
ENR 100 12.6 15.0 121.8 146.7 96
DIF 300 3.3 4.7 331.4 362.6 99
DAN 100 2.0 5.3 106.4 115.6 98
NOR (50)a 7.5 7.9 59.0 65.6 105
FLU 600 4.7 6.3 692.3 806.4 98
SAR 30 8.1 11.5 39.6 48.3 103
NAL (50)a 5.4 7.8 63.8 78.9 100
OXO 100 2.9 5.1 116.2 134.9 99
CFPI (50)a 2.6 6.5 57.6 62.8 103
CFT 1000 2.3 4.9 1085 1141 99
CFQ (50)a 3.8 6.3 55.3 59.5 102
CFLO (50)a 9.2 13.6 61.8 68.5 104
CFZ (50)a 7.9 9.4 60.5 66.6 105
CFLE (50)a 3.4 7.0 56.7 61.8 103
CFPE (50)a 4.0 7.8 57.1 63.1 101
Note: aValidation level (VL) for analytes without the MRL based on the compound characteristics or MRL of the specific analytes in other matrices.

Results and discussion
Method development
The development of multi-class methods for the determi-
nation of antibiotics in biological matrices is difficult as
regards many analytical and chemical aspects. The chemi-
cal structure of these analytes is disparate, so that a sample
preparation procedure should be versatile to ensure the
extraction of all compounds. The double-step extraction
that was applied in the procedure and combining both
dissolved residues in one vial before LC-MS/MS analysis
allowed only a single LC-MS/MS run to be performed. In
the majority of the published methods for the determina-
tion of aminoglycosides, it appears necessary to use an
ion-pair agent as a mobile phase in gradient mode
(Gaugain-Juhel et al. 2009; Turnipseed et al. 2009;
Hurtaud-Pessel et al. 2011). In order to find the best
composition of the mobile phase to produce good peak
shapes and to minimise matrix effects for all analytes,
some experiments were performed. Three different ion-
pair reagents (heptafluorobutyric acid, trifluoroacetic acid
and pentafluoropionic acid) mixed with organic solvents
(methanol and acetonitrile) were tested. Good results were
obtained with all of them, but the mixture of a water
solution of HFBA with acetonitrile gave the most satisfac-
tory results such as the highest ionisation, the best peak
shapes, good recoveries and a short elution time for all
groups of tested analytes. On the basis of the literature and
the authors’ experience, two columns – Luna C8 and Luna
C18 (Canada-Canada et al. 2009; Gaugain-Juhel et al.
2009; Hurtaud-Pessel et al. 2011) – were chosen for further
experiments. It was found that the Luna C18 column, in
comparison with the Luna C8, gave a better peak shape,
higher intensity and shorter retention times. In order to
minimise the retention time of the most retained analytes
within the shortest total analysis, a 50 mm Luna C18
column was tested and a gradient elution applied. Due to
 Food Additives & Contaminants: Part A 945

LC optimisation, a total run time which included column
equilibration and re-equilibration time was 18 min.
The MS settings were optimised with a direct infusion
of working standard solutions. According to European
Union criteria, two MRM transitions were monitored.
For each compound, the two most abundant product ions
were chosen as diagnostic ions. The characteristic MS/MS
parameters – collision energy (CE), declustering potential
(DP) and dwell time – were optimised separately for each
analyte (Table 3). The analyses were performed in positive
ionisation mode.
Optimisation of sample preparation procedure
The most important part of the developed multi-class
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aminoglycosides, which are strongly polar and stable in
the presence of acids, only high ionic-strength extraction
solvents are recommended (Zhu et al. 2008; Gaugain-
Juhel et al. 2009; CiaoChan et al. 2010; Martos et al.
2010; Hurtaud-Pessel et al. 2011). For that reason, most
of the multi-residue methods are limited to the analysis of
only β-lactams, macrolides, fluoroquinolones, sulfona-
mides and tetracyclines, except aminoglycosides. In
order to overcome the problem and to be able to use the
same sample preparation protocol for all these antibiotic
groups, a two-step extraction of the same sample was
applied. At the beginning of the experiment, acetonitrile
and methanol were tested for the organic extraction step.
The best recoveries for all compounds excluding amino-
glycosides was achieved with acetonitrile. Trichloroacetic
acid, m-phosphoric acid, formic acid and citric acid were

method was the choice of the extraction mixture to give

an acceptable recovery for all analytes characterised by
distinct physicochemical properties. Additionally, it is
very important to utilise the clean-up technique which is
efficient enough to eliminate interfering compounds. In
the study, a few experiments with the application of dif-
ferent types of extractions, extraction solvents and clean-
up processes were carried out. It is mentioned in the
literature that for the analysis of β-lactams, macrolides,
sulfonamides and fluoroquinolones, the use of organic
solvents such as acetonitrile or methanol as an extraction
solvent gave the best results (Posyniak et al. 2005;
Carretero et al. 2008; Kaufmann et al. 2008; Canada-
Canada et al. 2009; Smith et al. 2009; Chafer-Pericas
et al. 2011; Gajda et al. 2012), whereas for
checked under acidic extraction; the best result was
achieved with m-phosphoric acid. During the second part
of the experiment, two extraction steps were linked
together for the isolation of one sample. It was observed
that the order of the extraction steps is very important as
the use of acetonitrile in the first extraction step and an
acidic solvent in the second step cause the loss of amino-
glycosides. Therefore, it was necessary to apply the acidic
extraction in the first step and the organic extraction in the
second step to obtain the best aminoglycoside extraction.
Afterwards, several SPE sorbents were tested for sample
clean-up (Table 3). Because of the problematically struc-
tured aminoglycosides, it was necessary to use an ion-pair
reagent in the SPE extraction step, which facilitated

Table 3. Optimisation of the extraction, re-extraction step and SPE parameters.

Result
1 Extraction step 2 Extraction step SPE + –
Acetonitrile βLs, FQs, SAs, AGs
DPs, MLs, TCs
m-Phosphoric acid TCs, AGs βLs, FQs, MLs, SAs, DPs
m-Phosphoric acid Strata X TCs, AGs βLs, FQs, SAs, DPs, MLs
Strata X-C βLs, FQs, SAs, DPs, MLs TCs, AGs
Strata X-CW TCs, AGs βLs, FQs, SAs, DPs, MLs
Oasis HLB βLs, FQs, SAs, DPs, MLs TCs, AGs
m-Phosphoric Strata X-CW TCs, AGs βLs, FQs, SAs, DPs, MLs
acid + HFBA Strata X TCs, AGs βLs, FQs, SAs, DPs, MLs
m-Phosphoric Strata X-CW TCs, AGs βLs, FQs, SAs, DPs, MLs
acid + HFBA + EDTA
m-Phosphoric acid + HFB- Strata X-CW TCs, AGs βLs, FQs, SAs, DPs, MLs
A + EDTA + TCA
Acetonitrile m-Phosphoric acid + Strata X-CW βLs, TCs, FQs, AGs
HFBA + EDTA + TCA MLs, SAs, DPs
m-Phosphoric acid + HFB- Acetonitrile Strata X-CW βLs, TCs, FQs,
A + EDTA + TCA MLs, SAs, DPs,
AGs
Note: (+), Detected analytes; (–), non-detected analytes; βLs, β-lactams (AMOX, PEN G, CFPI, CFT, CFPE, CFLE, CFQ, CFZ, CFLO); TCs, tetracyclines
(TC, CTC, DC, OTC); FQs, fluoroquinolones (DAN, DIF, ENR, CIP, NOR, FLU, OXO, NAL, SAR); MLs, macrolides (TYL, ERY, AZY); SAs,
sulfonamides (SME, SMT, SMA, SMM, SDMX); DPs, diaminopyrimidines (TMP); and AGs, aminoglycosides (STRP, DISTRP, SPEC).
 946 M. Gbylik et al.
elution of the aminoglycosides from the SPE cartridge.
The selection of the ion-pair reagent was performed by
laboratory experiment. Heptafluorobutyric acid, trifluoroa-
cetic acid and pentafluoropionic acid were tested, but only
m-phosphoric acid with heptafluorobutyric acid gave the
optimal results. Since β-lactams and macrolide compounds
are very sensitive in the presence of acid (Becker et al.
2004; Berrarda et al. 2007; Msagati & Nindi 2007), it is
very important to adjust the pH values for acidic and
neutral compounds. It was found that 3% m-phosphoric
acid pH 5.5 with 0.02 M HFBA in water gave the optimal
recoveries for all compounds tested. To improve protein
precipitation, it was decided to add 0.6 ml of 20% TCA.
Another issue is the presence of metal ions in muscle
which may cause the bonding of several groups of anti-
biotics, especially tetracyclines (Cherlet et al. 2003; Tong
Downloaded by [Moskow State Univ Bibliote] at 04:15 21 October 2013
et al. 2009). Therefore, EDTA was included in the extrac-
tion solution. Consequently, the recoveries of tetracyclines
were better without changing the recoveries of the other
compounds. Finally, the extraction and clean-up process
was evaluated. In order to improve the purification of the
samples, different final extract filters (PVDF, NYLON and
Nanosep MF Centrifugal Devices) were tested (Gaugain-
Juhel et al. 2009; Hurtaud-Pessel et al. 2011). PVDF
syringe filters gave the best purity of the extract without
any influence on the analytes recoveries.
Method validation
The result provided in Table 2 summarises the validation
parameters such as precision – repeatability and within –
laboratory reproducibility, decision limits and detection
capability. The concentrations of the analytes were derived
directly from the matrix calibration curve with the use of
internal standards. The satisfactory percentage recoveries
were calculated in terms of the internal standards: SFF for
aminoglycosides, tetracyclines, sulphonamides, trimetho-
prim and β-lactams, CP-d8 for fluoroquinolones, and ROX
for macrolides. The matrix-matched curves showed good
linearity (r2 > 0.99) for all the analytes. As can be
observed, an overall CV for all compounds was in the
range of 2.0–18.0% for repeatability and 3.8–18.3% for
within-laboratory reproducibility. Such results are accep-
table and in agreement with the criteria of European
Decision 2002/657/EC. Selectivity was determined and
none of the interfering peaks that could interact with the
analysing compounds retention times was detected. CCα
and CCβ were in accordance with MRLs required by
European regulation. The robustness of the method was
evaluated for the following parameters: the amount of
ACN used for extraction (6 and 8 ml), the amount of
solvent used for SPE conditioning (2.8–4 ml), the volume
of the eluate solvent in SPE (2 × 2.5–3.5 ml), and the
temperature of extract evaporation (40–50°C). The robust-
ness test proved that there was no significant influence of
the chosen factors on the results of the analysis. The
signal-to-noise (S/N) results for all samples fortified on
CCα concentration levels meant the signal-to-noise ratio
for each diagnostic ion was (S/N > 3:1). The minimum
recovery value was 96% for PEN G; the maximum recov-
ery value was 111% for AMOX. ERY cannot be detected
in its original form; in acidic conditions it was determined
only by its degradation product ERY-H2O and all perfor-
mance data of this procedure were determined for ERY-
H2O. The most difficult analytes to determine were GEN
and NEO. The recoveries for GEN and NEO were <60%
with respect to the internal standard and were not repea-
table (CVs > 30%), therefore this method cannot be used
for a quantitative evaluation of these two antibiotics, pos-
sibly only for a qualitative evaluation.
Stability of the antibiotics
The stability of proven individual stock standard solution
for fluoroquinolones and tetracyclines stored at –18°C
remains stable for at least 6 months. The individual stock
standard solutions of sulfonamides, cephalosporins and
macrolides stored under the same conditions are stable for
10 months. The stability of penicillins (PEN G and AMOX)
was 3 months at least because of the considerable decrease
of concentration levels. All standard stock solution stored at
4°C were stable for not more than 3 months. The tested
antibiotics remained stable in fish muscles stored at –18°C
at least for 6 months, with the exception of the penicillins.
For this group of compounds, a significant reduction in
concentrations were observed after 2 months.
Matrix effect
The negative matrix effect of ion suppression or ion
enhancement constitutes a common problem that occurs
during the analysis of antibiotics in biological samples by
LC-MS/MS with an ESI source and can be explained by
the process of ionisation of the sample at the entrance of the
MS. This is one of the disadvantages of LS-MS/MS and it
is very difficult to eliminate. The matrix effect was calcu-
lated for all analytes in different fish species (common
bream, roach, pike, zander and catfish). It was decided to
present the matrix effect as average results because of slight
differences between fish species (Table 4). The tested ana-
lytes gave average signal suppression in the range from
1.6% to 15.3%. The lowest average signal enhancement
was observed for ERY-H2O and AZY (4.0% and 4.5%,
respectively). DC and CFQ are characterised by the highest
average signal enhancement (17.8% and 17.5%, respec-
tively), but it is still not affected by the determination of
the analytes. The highest signal enhancement was observed
for DC (18.2%), OTC (16.9%) and CFQ (18.3%) in bream
samples. Roach and pike are characterised by the lowest
signal enhancement for all analytes (1.1–11.3%). The
 Food Additives & Contaminants: Part A 947

Table 4. Matrix effects of analytes at MRL/VL levels.

Analyte Matrix effect (%) Analyte Matrix effect (%)

AMOX −8.9 ± 0.7 DISTRP 8.6 ± 0.5
PEN G −7.2 ± 0.3 CIP −4.7 ± 0.4
SMT 8.8 ± 0.6 ENR −14.8 ± 0.4
SME −1.6 ± 0.2 DIF −15.3 ± 0.4
SDMX −14.6 ± 0.3 DAN −13.2 ± 0.4
SMA −12.3 ± 0.2 NOR −16.0 ± 0.3
SMM −9.3 ± 0.5 FLU −13.8 ± 0.8
TMP −4.4 ± 0.4 SAR −14.7 ± 0.6
DC 17.8 ± 0.8 NAL −9.5 ± 0.9
OTC 16.0 ± 0.6 OXO −13.3 ± 0.5
TC 13.2 ± 0.4 CFPI 17.0 ± 0.5
CTC 14.3 ± 0.3 CFT 13.2 ± 0.9
ERY-H2O 4.0 ± 0.5 CFQ 17.5 ± 0.9
AZY 4.5 ± 0.3 CFLO 8.3 ± 1.0
TYL 10.9 ± 0.6 CFZ 7.9 ± 1.3
Downloaded by [Moskow State Univ Bibliote] at 04:15 21 October 2013

STRP 6.9 ± 0.4 CFLE 8.4 ± 0.9

SPEC −6.2 ± 0.5 CFPE 10.0 ± 0.6

results of the studies show that fish spaces do not have a
significant influence on the matrix effect. The matrix effect
was minimised by selective extraction, an effective sample
clean-up method, and the use of matrix-matched calibration
curves and internal standard.
Conclusions
To conclude, a specific, fast and reliable multi-class
method for the determination of 34 antibiotics in fish
samples was successfully developed. The sample prepara-
tion allows a rapid screening analysis of fish samples. The
method gives the quantitative results for all antibiotics. It
has been successfully validated according to Commission
Decision 2002/657/EC and it proved to be highly specific
and sensitive. Data obtained show satisfactory precision
and accuracy. The method can be applied during the
routine analysis conducted by laboratories involved in
official residue control analyses.
Acknowledgements
This work was financially supported by the Ministry of Science
and Higher Education (NCBIR Project No. NR12-0127-10).
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