Anal Bioanal Chem (2015) 407:4447–4457

DOI 10.1007/s00216-014-8449-5

RESEARCH PAPER

Determination of selected veterinary antimicrobials in poultry

excreta by UHPLC-MS/MS, for application in Salmonella control
programs
Brecht Gorissen & Tim Reyns & Mathias Devreese &
Patrick De Backer & Joris Van Loco & Siska Croubels

Received: 31 October 2014 / Revised: 20 December 2014 / Accepted: 22 December 2014 / Published online: 30 January 2015
# Springer-Verlag Berlin Heidelberg 2015

Abstract The most important source of Salmonella spp. in-
fection in humans is by the consumption of contaminated
poultry products. Due to the risk of resistance development
and its transfer from animals to humans, the Belgian Royal
Decree concerning the eradication of Salmonella (C-2007/
22784) prohibits treatment of poultry with antimicrobials
against zoonotic Salmonella spp. To uncover illicit use, an
analytical method using ultra-high performance liquid
chromatography-tandem mass spectrometry (UHPLC-MS/
MS) for the determination of antimicrobial residues in poultry
excreta was developed and validated for classes having an
active spectrum against Salmonella spp. in poultry: β-
lactams (amoxicillin and penicillin V), fluoroquinolones
(enrofloxacin, difloxacin, and flumequine), polymyxins (co-
listin), sulfonamides in combination with trimethoprim
(sulfachloropyridazine, sulfadiazine, and sulfaclozine), and
tetracyclines (chlortetracycline and doxycycline). A generic
and high-throughput sample preparation was developed. Ex-
traction of samples was performed by ultrasonication using a
combination of acetonitrile and McIlvaine buffer, followed by
centrifugation and filtration prior to analysis. The method was
validated according to Commission Decision 2002/657/EC
Published in the topical collection on Hormone and Veterinary Drug
Residue Analysis with guest editors Siska Croubels, Els Daeseleire,
Sarah De Saeger, Peter Van Eenoo, and Lynn Vanhaecke.
B. Gorissen (*) : T. Reyns : J. Van Loco
Department of Food, Medicines and Consumer Safety, Service of
Chemical Residues and Contaminants, Scientific Institute of Public
Health, Rue Juliette Wytsmanstraat 14, 1050 Brussels, Belgium
e-mail: Brecht.Gorissen@wiv-isp.be
B. Gorissen : M. Devreese : P. De Backer : S. Croubels
Department of Pharmacology, Toxicology and Biochemistry, Faculty
of Veterinary Medicine, Ghent University, Salisburylaan 133,
9820 Merelbeke, Belgium
for linearity, apparent recovery/trueness, repeatability, repro-
ducibility, limit of quantification, limit of detection, specifici-
ty, matrix effect, and storage stability in matrix. To demon-
strate the applicability of the method, an in vivo experiment
was conducted. For each antimicrobial class, one registered
drug was selected and administered in the drinking water to
two laying hens. Excreta samples were collected every 12 h
during and until 2 days after treatment and analyzed using the
developed method.
Keywords Salmonella spp. . Veterinary antimicrobials .
Residues . Poultry manure . Liquid chromatography/
electrospray ionization-tandem mass spectrometry
Introduction and aims
Salmonella spp. is a genus of rod-shaped, Gram-negative bac-
teria that can be found in the intestinal flora of various species.
It is a major cause of reported food poisoning worldwide,
leading to gastrointestinal disorders. Consumption of
undercooked and raw shell eggs is the most important source
of human salmonellosis [1].
Next to the risk of disease, a major concern is antimicrobial
resistance development and the transfer of resistant
Salmonella spp. bacteria to man. The antimicrobial resistance
profile of Salmonella spp. is allocated to a gene cluster carried
by chromosomal genomic island 1 (SGI1). To date, SGI1
variants have been identified carrying up to six resistance
genes, making the (mis)use of veterinary antimicrobials criti-
cal in conferring resistance to multiple antimicrobial families
[2, 3]. The European Food Safety Authority (EFSA) and the
European Centre for Disease Prevention and Control (ECDC)
4448
recently reported occurrence data of multidrug-resistant
(MDR) Salmonella spp. isolates from human cases at the
EU level. In general, multidrug resistance of the isolates was
high (28.9 %; N=13,496; country average 33.5 %), with the
highest levels reported from Italy (63.1 %; N=130) and Hun-
gary (55.8 %; N=588). Seven member states (Austria, France,
Hungary, Ireland, Lithuania, Spain, and the UK) even reported
a few isolates which were simultaneously resistant to ten an-
timicrobials [3].
Because of both health hazards, a Salmonella spp. erad-
ication program for poultry has been set up in Belgium
[4]. One of the main measures of this Belgian Royal
Decree C-2007/22784 is that a Salmonella spp.-positive
flock has to be culled. As culling leads to significant
economic losses for the breeder, they might treat the flock
with antimicrobials in order to obtain a negative analysis
for the presence of Salmonella spp. bacteria. To expose
this deception, the decree prohibits the use of antimicro-
bials against zoonotic Salmonella spp. in positive flocks,
and this is controlled by residue analysis of meat samples
originating from sacrificed animals.
In poultry, drinking water medication is the preferred meth-
od for administration of antimicrobials [5]. Significant
amounts of the used antimicrobials are eliminated in poultry
excreta as parent compound [6]. The actual amount of excre-
tion will vary with the type of antimicrobial, dosage level, as
well as the type and age of the treated animal [7]. For some
antimicrobials, such as colistin, over 90 % of the oral dose
passes through the animal unchanged [8, 9]. Therefore,
excreta might be a suited matrix to screen for illicit
antimicrobial use. A noninvasive method would certain-
ly be more feasible in terms of animal welfare and labor
efficiency.
As of today, the use of multiclass methods for the analysis
of antimicrobials in manure of poultry origin is lacking. Due
to the various classes of antimicrobials having quite different
physicochemical properties, it has mostly been limited to a
few analytes from the same class. However, a couple of
multiclass methods have also been reported which include
tetracyclines, sulfonamides (in combination with trimetho-
prim), and fluoroquinolones [10–13].
The aim of the present study was to develop and validate an
analytical method for the reliable determination of antimicro-
bial residues in poultry manure, based on ultra-high perfor-
mance liquid chromatography-tandem mass spectrometry
(UHPLC-MS/MS). All selected compounds were registered
antimicrobials with an active spectrum against Salmonella
spp. in poultry, namely β-lactams (amoxicillin and penicillin
V), fluoroquinolones (enrofloxacin, difloxacin, and
flumequine), polymyxins (colistin), sulfonamides in combina-
tion with trimethoprim (sulfachloropyridazine, sulfadiazine,
and sulfaclozine), and tetracyclines (chlortetracycline and
doxycycline). Finally, the validated method was applied to
B. Gorissen et al.
biological samples derived from laying hens treated with
one of the aforementioned antimicrobials.
Materials and methods
Reagents
Acetonitrile (ACN) and methanol were of UHPLC grade and
obtained from Biosolve BV (Valkenswaard, the Netherlands).
Formic acid, citric acid, and disodium hydrogen phosphate
dihydrate (Na2HPO4) were from Merck KgaA (Darmstadt,
Germany). HPLC grade water was produced by means of a
Milli-Q® Gradient A10 water purification system (Millipore,
Bedford, MA, USA).
The McIlvaine buffer (pH 3.6) used for sample extraction
was prepared by mixing aqueous solutions of Na2HPO4
(0.2 mol/L) and citric acid (0.1 mol/L) in suitable proportions
(32.2/67.8, v/v).
Standards and stock solutions
The reference standards chlortetracycline hydrochloride
(CTC), doxycycline hyclate (DOX), amoxicillin trihydrate
(AMO), penicillin V potassium salt (PEV), enrofloxacin
(ERF), difloxacin hydrochloride (DFX), flumequine (FEQ),
sulfachloropyridazine (SCP), sulfadiazine (SDZ), sulfaclozine
sodium (SCL), trimethoprim (TMP), and colistin sulfate salt
(COL) were all purchased from Sigma-Aldrich (Bornem,
Belgium).
The internal standards (demeclocycline hydrochloride
(DMC), ampicillin (AMP), norfloxacin (NFX), and
sulfaphenazole (SPZ)) were also purchased from Sigma-Al-
drich. Chemical structures for all selected antimicrobials and
internal standards are presented in Fig. 1.
Individual stock solutions (1 mg/mL) were prepared by
accurately weighing 10 mg of reference standard into a
10-mL volumetric flask, with correction for purity where nec-
essary. AMO, PEV, AMP, and COL were dissolved in purified
water; all other standards were dissolved in methanol. Stock
solutions were divided in Eppendorf® tubes in volumes of
300 μL and stored at −20 °C in the dark.
On each analysis day, cups were thawed and working so-
lutions of 0.5, 1, 1.5, 5, 10, 25, and 50 μg/mL were prepared
by diluting an appropriate amount of the stock solution with
0.1 % formic acid in water. In the same way, an internal stan-
dard working solution of 5 μg/mL was also prepared on a
daily basis. All working solutions were discarded after use.
Biological samples
Twelve 17-week-old brown laying hens were randomly dis-
tributed and housed into six groups of two birds per cage. One
Determination of selected veterinary antimicrobials 4449

-lactams

amoxicillin (AMO) penicillin V (PEV) ampicillin (AMP)

fluoroquinolones

enrofloxacin (ERF) difloxacin (DFX) flumequine (FEQ) norfloxacin (NFX)

sulfonamides/trimethoprim

sulfachloropyridazine (SCP) sulfaclozine (SCL) sulfadiazine (SDZ)

trimethoprim (TMP) sulfaphenazole (SPZ)

tetracyclines

chlortetracycline (CTC) doxycycline (DOX) demeclocycline (DMC)

polymyxins

colistin A (COL A)

Fig. 1 Chemical structures for all selected antimicrobials and internal standards

group served as control and did not receive any antimicrobial
treatment (blank). All other groups were administered medi-
cated drinking water, at a dose according to the summary of
product characteristics, with one representative of following
antimicrobial classes: tetracyclines, DOX (Doxylin® 50 %
WSP, Dopharma Research B.V., Raamsdonksveer,
The Netherlands); β-lactams, AMO (Octacillin®, Eurovet
A n i m a l H e a l t h B . V. , B l a d e l , T h e N e t h e r l a n d s ) ;
fluoroquinolones, DFX (Dicural®, Zoetis Belgium s.a., Lou-
vain-la-Neuve, Belgium); sulfonamides/trimethoprim, SCP/
4450
TMP (Cosumix® Plus, V.M.D. n.v., Arendonk, Belgium); and
polymyxins, COL (Coliplus®, Divasa-Farmavic s.a., Barcelo-
na, Spain).
During the 5-day treatment, medicated water was
refreshed every 12 h. Furthermore, during and until
2 days after the last treatment, bedding was refreshed
every 12 h, and fresh solid droppings (15 g on average)
were collected from multiple positions in the cage and
pooled per treatment group. All excreta samples were
homogenized with a stomacher and then stored at ≤
−80 °C until analysis. The animal experiment was ap-
proved by the Ethical Committee of the Faculty of Vet-
erinary Medicine of Ghent University (case number: EC
2013/111).
Sample extraction
Frozen (≤ −80 °C) biological samples were left to equil-
ibrate to room temperature before extraction. Aliquots
(1 g) were weighed into Falcon® 50-mL polypropylene
conical tubes (BD, Franklin Lakes, NJ, USA) and
spiked with internal standards by adding 100 μL of
working solution, followed by vortex-mixing. Then,
10 mL of the extraction solution, consisting of
acetonitrile/McIlvaine buffer (75/25, v/v), was added.
Samples were consequently vortex mixed (30 s), rotated
for 10 min, followed by ultrasonification (10 min) and
centrifugation (3,220×g, 10 min, 4 °C). After ultracen-
trifugation of 1-mL supernatant (10 min, 12,354×g), the
upper layer was filtered through a Whatman™ PVDF
0.2-μm syringe filter (GE Healthcare, UK) and trans-
ferred to an autosampler vial.
Matrix-matched calibration curves, prepared by spiking
negative poultry manure samples with working standard solu-
tions, were submitted to the full extraction procedure.
UHPLC instrumentation and chromatographic conditions
The UHPLC equipment consisted of an AcquityTM UPLC®
class system (sample and quaternary solvent manager) and an
Acquity UPLC® BEH C18 column (100 mm×2.1 mm i.d.,
1.7 μm), both from Waters (Millford, MA, USA). Mobile
phase A consisted of 0.1 % aqueous formic acid, while mobile
phase B was 0.1 % formic acid in ACN.
The column was maintained at 40 °C, and the injector
volume was set to 5 μL. The mobile phase flow rate was
0.4 mL/min, and the following gradient elution method was
used: 0–0.5 min: 95 % A/5 % B; 0.5–1 min: linear decrease to
50 % A/50 % B; 1–3 min: 50 % A/50 % B; 3–5 min: linear
decrease to 0 % A/100 % B; and 5–7 min: linear increase to
95 % A/5 % B.
B. Gorissen et al.
MS/MS instrumentation and parameters
Analysis was performed on a Xevo™ TQ-S triple quadrupole
mass spectrometer from Waters (Millford, MA, USA) with an
electrospray ionization (ESI) source operated in the positive
mode. The mass spectrometer parameters were manually op-
timized by direct infusion of individual standard solutions at a
concentration of 10 μg/mL. The following conditions were
utilized: capillary voltage of 3 kV, source temperature of
150 °C, desolvation temperature of 550 °C, cone and
desolvation gas flow rates of 150 and 1,000 L/h, respectively,
and a collision gas flow (argon) of 0.17 mL/min. The selected
quantification and confirmation traces with their respective
collision energies are listed in Table 1.
Validation criteria
European Commission Decision 2002/657/EC is extensively
used for validation purposes in food and environmental ma-
trices [14]. Though there is no agreement about whether it
should also be used for the analysis of animal manure, it was
applied in the validation of the presented method for the fol-
lowing set of parameters: linearity, apparent recovery/true-
ness, precision (repeatability and reproducibility), limit of
quantification (LOQ), limit of detection (LOD), specificity,
matrix effect (signal suppression/enhancement (SSE)), and
storage stability in matrix [14–18].
Linearity
Six-point calibration curves in a range of 50–5,000 ng/g were
prepared using spiked blank poultry manure samples. Peak
area ratios between the analytes of interest and their respective
internal standards were plotted against their concentration lev-
el, and a linear regression was performed. Linearity was
established with a correlation coefficient (r) of ≥0.99 and a
goodness-of-fit of ≤10 % [14–16].
Apparent recovery/trueness
Trueness was determined by analyzing six independently
spiked blank poultry manure samples at both 50, 100, and
200 ng/g. Matrix-matched calibration curves were used to
calculate apparent recoveries from these experiments. The
mean apparent recoveries had to be within 80 to 110 %
[14–17].
Precision
Repeatability or within-day precision was determined on the
same samples as used for the trueness evaluation. From the
analysis results, the coefficients of variation (CVr) were deter-
mined and had to fall within two thirds of the values calculated
Determination of selected veterinary antimicrobials 4451

Table 1 SRM transitions for all selected compounds and internal standards

Compound Precursor ion (m/z) Product ion(s) (m/z) CE (eV) Retention time (min)

amoxicillin 366 114e 15 2.01

207.9 10
chlortetracycline 479.1 370.8 20 2.44
443.8e 30
colistin A 390.8 379.2 10 2.86
– –
difloxacin 400.8 299.8 20 2.39
356.9e 30
doxycycline 445.3 320.9 20 2.49
427.9e 40
enrofloxacin 360.4 244.9 20 2.35
316e 30
flumequine 261.6 201.9 20 3.00
243.9e 40
penicillin V 351 114 20 2.98
160e 20
sulfachloropyridazine 285.1 107.8 20 2.55
155.8e 30
sulfaclozine 285.7 92 20 2.71
130e 20
sulfadiazine 250.3 91.8 10 2.23
155.8e 20
trimethoprim 291 123 25 2.31
230e 25
ampicillina 350.2 106.1 20 2.27
demeclocyclineb 465.1 448.1 20 2.40
norfloxacinc 320.1 302.1 20 2.46
sulfaphenazoled 315.1 158 25 2.67

m/z mass-to-charge ratio, CE collision energy

a
Internal standard (IS) for β-lactams
b
IS for tetracyclines
c
IS for fluoroquinolones
d
IS for sulfonamides and trimethoprim
e
The quantifier ion

according to the Horwitz equation: CV (%)=2(1−0.5×log c), Limit of detection

where c equals the mass fraction expressed as a power of 10
(e.g., 1 mg/g=10−3) [14].
Reproducibility or between-day precision (CVR) was eval-
uated using the same spike levels, with six replicates prepared
for each concentration and on each of three analysis days. The
CVR had to be below the calculated CV using the Horwitz
equation [14].
Limit of quantification
The LOQ was defined as the lowest concentration that fell
within the abovementioned acceptance criteria for trueness
and precision during the method validation [14]. This concen-
tration was also established as the lowest point of the calibra-
tion curve.
The LOD is defined as the lowest measured concentration
from which it is possible to deduce the presence of the analyte
with reasonable statistical certainty. These values were calcu-
lated from samples spiked at the LOQ level, using a signal-to-
noise (S/N) ratio of 3/1 [14, 16].
Specificity
Specificity is defined as the power of discrimination between
the analyte of interest and closely related substances which
may be present in the sample matrix. Blank poultry manure
samples were extracted and analyzed with the proposed meth-
od, and the presence of interferences was determined around
the retention times of the analytes of interest [14, 16].
4452
Matrix effect (signal suppression/enhancement)
The presence of SSE was determined by comparing the slopes
of calibration curves prepared using standard solutions with
calibration curves prepared from spiking blank matrix after
extraction (n=3), by means of a statistical t test [18].
Storage stability
Long-term storage stability of the compounds in matrix during
storage at ≤ −80 °C was investigated by spiking 18 blank
poultry manure samples at 1 μg/g. Three samples were ana-
lyzed immediately after preparation, whereas the other sam-
ples were stored at ≤ −80 °C until analysis (n=3) after a period
of 1 week, 2 weeks, 1 month, 3 months, and 5 months. True-
ness should fall within the range of −20 to +10 % [14].
Colistin
For COL A, a qualitative validation procedure was performed,
also according to the European Commission Decision
2002/657/EC [14]. The concentration at which there were
≤5 % false compliant results was determined by analysis of
20 samples of blank poultry manure spiked at a concentration
of 50 ng/g and 20 samples of blank poultry manure. Blank
manure samples originated from different animals that did not
receive any antimicrobial treatment.
Results and discussion
UHPLC-MS/MS method development
The liquid chromatography conditions were optimized by
injecting mixed standard solutions (100 ng/mL) of all analytes
on an Acquity UPLC® BEH C18 column. Mobile phase se-
lection was based on reversed phase LC-MS methods in re-
cent literature that shared compounds of interest with the pre-
sented method [12, 19–32].
The addition of 0.1 % formic acid to water and ACN was
found to give best separation and enhance mass spectrometry
intensity. The addition of formic acid to aqueous and organic
mobile phases was also frequently reported in literature for the
chromatographic separation of the different COL constituents
[20, 28–30] and for multiresidue methods including veterinary
antimicrobials [13, 27, 31, 32].
A range of isocratic and gradient elution profiles was stud-
ied for the separation of the selected components. To achieve
adequate retention of high polarity compounds, the gradient
was started with only 5 % of ACN. The content of organic
solvent was raised to 100 % during the gradient profile to
prevent carryover of analytes and column contamination due
B. Gorissen et al.
to matrix compounds between two consecutive injections.
Each run ended with a return to initial conditions in order to
prepare the column for a subsequent sample. The final select-
ed gradient (see section “UHPLC instrumentation and chro-
matographic conditions”) allowed the elution of all analytes
within a short run time of 7 min, including preconditioning
and rinsing steps.
Sample extraction
Poultry manure has a highly complex chemical composition
with various metabolized organic substances (e.g., proteins,
fat, fiber) and lots of inorganic elements. Moreover, it presents
a heterogenous matrix consisting of manure, feed residues,
and bedding, so several analyte–matrix interactions and pos-
sible impurities must be considered [33]. The above makes the
development of an extraction procedure a challenging step,
and to date, the use of multiclass methods for the analysis of
antimicrobials in poultry manure has not been extensively
applied. Due to the different physicochemical properties of
selected families of antimicrobials, reported methods have
been limited to a few analytes from the same or, at most, three
different classes of antimicrobials [10–13].
Some of these reported “multiclass” methods still require
different chromatographic and/or extraction conditions for dif-
ferent classes of antimicrobials [12, 13, 31, 34]. Moreover,
polymyxins and/or β-lactams were not included in previously
reported methods for analysis of veterinary antimicrobials in
poultry manure. Furthermore, for COL (polymyxin antibiot-
ic), no analytical method for its determination in biological
matrices has been reported so far. Its complex chemistry leads
to several difficulties to accurately quantify this compound.
COL is a mixture of at least 30 closely related polypeptides,
with COL A (polymyxin E1) and COL B (polymyxin E2)
constituting around 85 % of total COL. Both main compo-
nents are lipodecapeptides and present five free amines which
are able to protonate separately from one another [35]. At
physiological pH, the cationic polypeptide molecules interact
noncovalently with anionic lipopolysaccharides in the bacte-
rial membrane. This causes loss of integrity of the cell mem-
brane and increases permeability of the cell envelope, leading
to cell lysis and cell death [36, 37]. These nonspecific interac-
tions make it more difficult for bacteria to develop resistance
against COL, which has recently led to an increased interest in
a world where MDR bacteria are emerging [20, 38]. Because
it has a very low oral bioavailability, the withdrawal period is
very short (only 1 day) [39]. This makes it a highly interesting
compound for inclusion in official control programs for the
detection of illicit antimicrobial use.
Extraction of pharmaceuticals from biological matrices is
usually based on ultrasonication [10, 12, 13, 31, 34, 40] and
liquid–liquid extraction (LLE) [32]. Microwave-assisted ex-
traction [27] and accelerated solvent extraction [11] were also
Determination of selected veterinary antimicrobials 4453

reported. Given the need for specialized and expensive equip-
ment for the latter, it was chosen to develop a method based on
ultrasonication with solvent extraction.
Most recently reported methods for the analysis of an-
timicrobials in animal manure used combinations of an
organic solvent (methanol, ACN) with acids and/or buffers
as solvent for ultrasonication to extract antimicrobials from
biosolids [10, 12, 13, 27, 31, 34]. After evaluating several
different combinations, a mixture of ACN and McIlvaine
buffer gave overall best results and was optimized to ob-
tain sufficiently high recoveries for all analytes (see sec-
tion “Validation criteria”). In literature, cleanup of manure
extracts proved to be essential in most instances due to
significant matrix effects. Solid-phase extraction (SPE) or
LLE was used to ensure accuracy and reliability of the
method [10–13, 27, 32–34].
In the developed method, the problems of matrix interfer-
ences were reduced by using a small volume of sample with a
large volume of extraction solvent, in combination with more
sensitive and selective instruments. Also, matrix-matched cal-
ibration curves together with the addition of structurally relat-
ed internal standards for each antimicrobial class were used to
overcome these problems and compensate for possible losses
in sample preparation. Referenced methods did not report the
use of internal standards at all or add one single standard for
multiple classes [11]. Ho et al. (2012) did report the use of

a 366 > 114

b 366 > 114
amoxicillin 100 5.16e4 100 2.69e3

% %

479.1 > 443.8 479.1 > 443.8

chlortetracycline 100 1.03e5 100 1.40e 3

% %

390.8 > 379.2 390.8 > 379.2

colistin A 100 1.09e5 100 3.90e 3

% %

400.8 > 356.9 400.8 > 356.9

difloxacin 100 6.14e4 100 2.15e3

% %

445.3 > 427.9 445.3 > 427.9

doxycycline 100 2.89e5 100 2.50e 3

% %

360.4 > 316 360.4 > 316

enrofloxacin 100 9.13e5 100 5.45e 3

% %

261.6 > 243.9 261.6 > 243.9

flumequine 100 7.41e 5 100 4.91e 3

% %

351 > 160 351 > 160

penicillin V 100 9.90e 4 100 4.42e 3

% %

285.1 > 155.8 285.1 > 155.8

sulfachloropyridazine 100 4.63e 5 100 8.61e 3

% %

285.7 > 130 285.7 > 130

sulfaclozine 100 1.09e 5 100 6.06e 3

% %

250.3 > 155.8 250.3 > 155.8

sulfadiazine 100 3.60e 4 100 1.09e3

% %

291 > 230 291 > 230

trimethoprim 100 2.49e 5 100 6.39e 3

% %

1.6 2.5 3.4 1.6 2.5 3.4

time (min) time (min)
Fig. 2 MS/MS chromatograms for A a blank poultry manure sample spiked at 50 ng/g (limit of quantification) and B a blank poultry manure sample
illustrating that no interferences from matrix-related components were present around the retention times of the selected compounds
4454 B. Gorissen et al.

multiple internal standards in their method, which were all
stable isotope-labelled (SIL) analogues of the analytes of in-
terest [10].
The use of SIL analogues is in fact the best way to correct
for possible matrix effects [41]. However, given that these are
usually difficult to obtain and quite expensive, this approach is
less feasible when a high number of target compounds have to
be included [10]. In addition, structural analogues that were
used as internal standards in the developed method are not
registered for use in poultry and provided good relative recov-
ery results.
In Fig. 2, representative MS/MS chromatograms for a
blank poultry manure sample spiked at 50 ng/g (LOQ) for
all analytes and a blank poultry manure sample are shown.
This illustrates that no interferences from matrix-related com-
ponents were present around the retention times of the select-
ed compounds. An impurity in the elution time window of
sulfaclozine could be argued but only showed an average S/

Table 2 Results of the quantitative method validation for selected parameters

Compound Linearitya Conc. (ng/g) Trueness (%) Precision LODd (ng/g)

Correlation coefficient (r) Goodness-of-fit (%) CVrb (%) CVRc (%)

amoxicillin 0.996 4.11 50 107.6 2.8 8.7 7.50

100 95.7 3.7 5.4
200 100.1 6.5 9.1
chlortetracycline 0.995 5.34 50 89.2 6.6 11.5 3.17
100 89.9 6.2 7.1
200 93.9 3.5 3.7
difloxacin 0.996 5.83 50 89.2 3 4.1 9.05
100 100.7 7.8 8.5
200 106.5 4.6 5.8
doxycycline 0.999 2.21 50 101.8 2.9 3.3 2.19
100 99.8 4.9 7.1
200 100 3.5 3.9
enrofloxacin 0.995 7.24 50 90.3 4.1 5.7 7.76
100 105 5.1 6.8
200 99.5 5 8.2
flumequine 0.998 6.96 50 102.8 1.9 4.9 1.84
100 90.1 2.7 3.1
200 107.8 4.2 7.8
penicillin V 0.998 7.07 50 96.7 1.5 4.5 8.50
100 104.4 3.3 4.2
200 102.4 5.3 11.5
sulfachloropyridazine 0.998 3.77 50 99.3 4.6 9.6 9.22
100 89.8 2.7 8.3
200 90.3 3.1 6.6
sulfaclozine 0.995 7.76 50 92.4 5.3 7.2 8.39
100 100.7 3.3 6
200 89.9 6.2 6.5
sulfadiazine 0.996 4.57 50 91.8 5.3 7.4 7.93
100 90.9 4.9 8.3
200 89.5 3.1 6.4
trimethoprim 0.996 5.96 50 93.1 3.7 6.1 2.47
100 96.7 6.2 8
200 92.6 3.9 6.5

The limit of quantification was set at 50 ng/g for all compounds

LOQ limit of quantification, CV coefficient of variation
a
Linearity range, 50–5,000 ng/g
b
Coefficient of variation for within-day repeatability (n=6)
c
CV for between-day reproducibility (n=6)
d
Limit of detection (n=6)
Determination of selected veterinary antimicrobials 4455

N ratio of 1.63 and thus was not considered to interfere with
the accurate quantification of this compound.
The advantage of the present method was the exclusion of a
time-consuming cleanup step, resulting in a quick and simple
extraction protocol. Extracts were simply filtered before injec-
tion, without additional treatments. The achieved sensitivity
was sufficient to determine the analytes at the concentration
levels of interest.
Method validation
The results for all analytes, except COL A, are summarized in
Table 2. For the calibration curves, linearity was observed up
to 5,000 ng/g for all components with correlation coefficients
(r) all >0.99. The trueness fell within the acceptance criteria of
−20 to +10 %, with apparent recoveries ranging from 89.2 to
107.8 % for the three spiked levels. Repeatability fell within
the maximum CV values; reproducibility also fell within the
ranges specified.
High sensitivity was reached, as shown by low LOD values
(ranging from 1.84 to 9.22 ng/g) (Table 2). The 50-ng/g level
could be quantified fulfilling the criteria for trueness and pre-
cision and was therefore set as LOQ of the method. Lower
LOQs have already been reported in multiclass methods for
CTC (2.94 ng/g), DOX (14 ng/g), SDZ (3 ng/g), SCP
(3.66 ng/g), TMP (1.2), ERF (14 ng/g), and FEQ (6 ng/g)
[10, 12, 27].
However, these methods all require time-consuming sam-
ple cleanup procedures making them less suitable for high-
throughput analysis in official control programs. Also, Hu

Fig. 3 A Results of an excretion 45000

study showing mean 40000
concentrations of selected
35000
antimicrobials in excreta of laying
concentration (ng/g)

hens (n=2/antimicrobial), during 30000

a 5-day oral administration and 2- AMO
25000
day post-administration of either DOX
difloxacin (DFX, 10 mg/kg BW/ 20000
SCP
day), sulfachloropyridazine/ 15000 TMP
trimethoprim combination 5/1
10000 DFX
(SCP/TMP, 24 mg/kg BW/day),
doxycycline (DOX, 25 mg/kg 5000
BW/day), or amoxicillin (AMO, 0
16 mg/kg BW/day) in the 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7
drinking water. B Representative time (days)
MS/MS chromatograms for
amoxicillin, colistin A,
difloxacin, doxycycline, and 100 366 > 114
amoxicillin
sulfachloropyridazine/ 1.23e6
%
trimethoprim in incurred samples
obtained on the third day of
administration during the 100 390.8 > 379.2
colistin A
excretion study 1.17e6
%

100 400.8 > 356.9

difloxacin
5.18e6
%

doxycycline 100 445.3 > 427.9

1.87e7
%

sulfachloropyridazine 100 285.1 > 155.8

8.37e6
%

100 291 > 230

trimethoprim
5.56e7
%

1.6 2.5 3.4

time (min)
4456
et al. (2010) used microwave-assisted extraction in their meth-
od, which requires specialized and expensive equipment [27].
With regard to specificity, no interfering peaks were ob-
served in the analysis of blank samples. The presence of
SSE due to matrix interferences was observed for CTC,
DOX, ERF, FEQ, AMO, PEV, SCP, SDZ, and TMP and
ranged between 55.61 % (CTC) and 69.47 % (SCP). These
effects were circumvented by preparation of matrix-matched
calibration curves for the quantification of biological samples
derived from the animal experiment. The selected antimicro-
bials were found stable in samples that were stored for a period
up to 3 months at ≤ −80 °C, with trueness values ranging
between 86.7 and 107.5 %.
For COL, the results of method validation analysis were
insufficient for the compound to be quantitatively determined.
However, its nonspecific mode of action and limited resis-
tance have recently led to an increased use of this antibiotic,
making it a highly interesting compound for inclusion in the
proposed method [39]. Therefore, a qualitative validation for
the COL A compound was established including the demon-
stration of a concentration (50 ng/g) at which COL A was
detected with less than 5 % false compliant results, with a
mean S/N ratio of 6.82 (±0.78). The analysis of blank samples
showed no interfering signals around the retention time of the
COL A peak.
Application to biological samples
An excretion study was performed in laying hens, after oral
administration of either DFX, SCP/TMP combination, DOX,
or AMO. The developed method was applied for analysis of
biological samples derived from this animal experiment (see
section “Biological samples”).
Six-point matrix-matched calibration curves were used to
calculate the residual concentrations of the analytes by inter-
polation. Samples showing a response outside the linear range
of the calibration curve were diluted with blank matrix prior to
extraction using a factor based on the extrapolated result and
subsequently reanalyzed.
The excretion profile of DFX, SCP/TMP, DOX, and AMO
in poultry manure samples is presented in Fig. 3A. Figure 3B
shows a representative MS/MS chromatogram for DFX, SCP/
TMP, DOX, AMO, and COL A in incurred samples, obtained
on the third day of administration.
More or less equal trends were observed for all studied
compounds. Steady state was reached during the administra-
tion period, followed by a rapid decrease in concentration
below the LOD within 48 h after the treatment. Concentra-
tions of up to 41.8 μg/g (DFX), 22.2 μg/g (DOX), 19.5 μg/g
(SCP), 3.3 μg/g (TMP), and 2.2 μg/g (AMO) were deter-
mined in these samples. Though no quantitative results could
be obtained, COL A was detected until 36 h after the
treatment.
B. Gorissen et al.
Previous methods also reported antimicrobial residues at
very high concentrations in poultry manure, such as 24 μg/g
CTC, 78 μg/g DOX, 51 μg/g SDZ, 35 μg/g SCP, 17 μg/g
TMP, 1,421 μg/g ERF, 12 μg/g DFX, and 52 μg/g FEQ
[10–13, 31–34]. These high levels indicate that most of these
antimicrobials were excreted as parent compounds.
Conclusions
This paper is the first—to our knowledge—to describe an
UHPLC-MS/MS method for the determination of 12 antimi-
crobials in poultry excreta, in combination with a simple and
efficient sample preparation procedure (ultrasonication with
solvent extraction). It allows simultaneous analysis of all
analytes within a short run time of 7 min and is validated
according to Commission Decision 2002/657/EC for the lin-
earity, apparent recovery/trueness, precision (repeatability and
reproducibility), limit of quantification, limit of detection,
specificity, signal suppression/enhancement, and storage sta-
bility in matrix. Furthermore, the method was successfully
applied in an in vivo experiment using laying hens where high
concentrations were determined in incurred manure samples.
Also, to the best of our knowledge, polymyxin and/or β-
lactam antimicrobials were not included in any of the previ-
ously reported methods for analysis of veterinary antimicro-
bials in poultry manure. The presented method can therefore
be applied in Salmonella control programs, as it offers the
advantages of a generic and simple sample preparation meth-
od and the specificity of UHPLC-MS/MS. Moreover, the non-
invasive sample collection of excreta is highly desired in the
field in terms of animal welfare and labor intensity.
Acknowledgments This study was supported by the Federal Public
Service of Health, Food Chain Safety and Environment (contract RT
12/06 AMPOULDETECT).
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