Advances in Clinical Toxicology

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Assessment of Tetracyclines Residues Contamination in Chicken

Meat Samples of Algiers Slaughterhouses Level
Zamoum R1*, Baroudi F2, Djerroud D2, Khouni B2, Alamir B1
Research Article
1
Pharmacy Department, Pharmacy Faculty of Algiers-University, Algeria
Volume 8 Issue 4
2
National Toxicology Centre, Algeria
Received Date: May 04, 2023
Published Date: October 20, 2023
*Corresponding author: Zamoum Radia, Pharmacy department, Pharmacy Faculty of Algiers-
DOI: 10.23880/act-16000280
University 1, 8 Rue Lieutenant Mohamed Benarfa, El Biar, Algeria, Tel: (+213)554759994;
Email: r.zamoum@univ-alger.dz

Abstract
Food contamination with antimicrobial residues may occur, entailing assessment and monitoring of the contamination level
in edible tissue. In that way, 151 chicken meat samples were randomly collected from Algiers slaughterhouses. Otherwise,
two analytical methods are validated for the simultaneous determination of oxytetracycline, tetracycline, doxycycline and
chlortetracycline residues in edible chicken tissues, by liquid chromatography combined with mass spectrometry detection.
Beforehand, an extraction step is performed. Indeed, acetonitrile is used as an extraction solvent for the qualitative method,
and then the quantitative method is carried out after a preliminary extraction with an acidified EDTA-McIlvaine buffer, followed
by a solid-phase extraction cleanup. Validation qualitative method results were satisfactory in terms of specificity (100%),
sensivity, detection capability (CCß) and detection limit (LOD). Furthermore, validation results of the quantitative method was
pertinent to the EU commission criteria. Thus, determination coefficient (r2) values were between de 0.91 to 0.98%, where
the lowest values were observed for doxycycline. Trueness was between 85.5% and 104.8, expect for tetracycline (76.6%
at 150µg/kg) and doxycycline (134.7% at 100µg/kg). As for intraday and interday precision, values respectively ranged
between 12.5 and 26% and between 14 and 35%, except doxycycline for which precision values were higher. The methods
have been successfully used for the identification, confirmation and quantification of tetracyclines in chicken muscle samples.
Consequently, 25 samples were suspects in regard tetracyclines residues. The comparison of these samples with the QC at
MRL (100µg/kg) revealed five samples needed a quantification. Thus, oxytetracycline, its epimer and doxycycline identified
in samples were quantified. Indeed, all samples contained oxytetracycline and its epimer, doxycycline or both showed levels
less than the decision limit (CCα).

Keywords: Tetracyclines; Residue; Algiers; meat; LC

Abbreviations: HFBA: Heptafluorobutyric Acid, PFPA: lincosamides, and sulphonamides Mungroo NA, et al. [1] for
Pentafluoropropionic Acid, TFA: TrifluoroaceticAcid; CTC : both human and veterian use. Global antibiotic consumption
Chlortetracycline; DC: Doxycycline; OTC: Oxytetracycline; increased by 65% between 2000 and 2015, from 21.1
TC: Tetracycline; Epi: Epimer ; SC: Calibration Standard ; billion daily doses determined in 2000 to 34.8 billion in
LMR: Maximal Residue Limit. 2015 in 76 countries around the world [2]. The family of
tetracyclines alone represents 36.5% of the tonnage sales.
Introduction Critical antibiotics i.e. latest generation cephalosporins and
fluoroquinolones represent nearly 1.0% of the tonnage
Several families of antibiotics exist such as beta-lactams of active ingredient sold. Over the years 2014 and 2015,
i.e. pencillins and cephalosporins, tetracyclines, macrolides, the average total sales volume is close to 650 tonnes of

Assessment of Tetracyclines Residues Contamination in Chicken Meat Samples of Algiers Slaughterhouses Level Adv Clin Toxicol
2 Advances in Clinical Toxicology

antibiotics per year [3]. The problem of antibiotic residues, from Mevet and Oxytetracycline Hydrochloride from Fluka.
particularly in meats, is widely discussed by both scientific Sulfaphenazole was obtained from Dr Ehrenstorfer GmbH.
and media communities. Standards were provided with their certificate of analysis,
and stored according to supplier’s recommendations.
Indeed, this problem could have a health impact involving
not only public health but also economics, thus limiting Acetonitrile for high-performance liquid
international trade. Therefore, multidrug resistance to chromatography (HPLC) and liquid chromatography/mass
antibiotics is a global threat because the powerful antibiotics spectrometry (LC/MS), and Methanol were purchased
available in human clinics are becoming extremely rare [4]. from Panreac Applichem; Ethylenediaminetetraacetic
Besides, a Canadian study showed a strong relationship acid disodium dihydrate (Na2EDTA dihydrate) from Merck
between the commensal E. coli from retail chicken and human Sigma-Aldrich and Trichloroacetic acid (TCA) was obtained
infections and the pathogen Salmonella enterica serovar from Panreacquimica SA. Ammonium acetate, Suprapur
Heidelberg. These results exemplify the fast propagation of formic acid were purchased from Merck. Citric acid and
resistant bacteria [5]. Trifluoroacetic acid (TFA) were purchased from TCI
Chemicals; and ammonium formate from VWR Chemicals.
Moreover, despite the ban on the addition of antibiotics Sodium dihydrogen phosphate dihydrate was obtained from
to animal feed in Europe since 2006 Goucem R [6], in the AlfaAesar.
United States and Canada, this practice persists [7]. In
Algeria, antibiotic supplementation is probable and could Millipore Milli-Q-Plus ultrapure water system was used
even be common, in the absence of any analytical control of throughout the study to obtain HPLC grade water used in the
animal feed. preparation of solutions and samples. Oasis HLB extraction
cartridges 6 cm3 /200 mg were purchased from Waters.
For all these reasons, different analytical methods were
set over the world to assess the food contamination rates by Solutions Preparation
drugs residues. European Union (EU) [8] for instance, as well
as the Algerian regulation, later in 2016 [9], have defined A solution of sodium dihydrogen phosphate dehydrate
maximum residue limits (MRLs) for residues of veterinary at 0.2 mol/L was prepared by dissolving 35.56 g into 1000
drugs in food, to protect consumers’ health by ensuring mL of water. To obtain a solution of citric acid at 0.1 mol/L,
food safety. Maximum residue limits (MRLs) are the higher 21g were dissolved into 1000 mL of water.
levels of residue concentrations of pharmacologically active
substances legally permitted in or on food and feed. Thus, A solution containing McIlvaine buffer for extraction, was
MRLs for muscle, liver and kidney tissues were established at obtained by mixing 1000 mL of citric acid (0.1 mol/L) with
100µg/kg, 300 µg/kg and 600µg/kg, respectively. Concerning 625 mL of disodium hydrogen phosphate (0.2 M). Adjust pH
the three tetracyclines (Tetracycline, Oxytetracycline and to 4.0 ± 0.05 with NaOH or HCl as needed. Then, Na2EDTA-
Chlortetracycline), the marker residue is the sum of parent McIlvaine buffer (0.1 mol/L) was prepared by mixing 60.5g
drug and its 4-epimer, whereas doxycycline is the marker of Na2 EDTA. 2H2O with 1625 mL of McIlvaine buffer.
residue [10].
A solution of trichloroacetic acid 20% was obtained by
The objective of our study was assessment of tetracyclines dissolving 20 g in 100 mL of water.
residues contasmination in chicken meat samples of Algiers
slaughterhouses level. This study was intended as the A rinse solution water/methanol (5%) was prepared by
starting point for further risk assessments of drugs residues adding 5mL of methanol to 100mL of water.
contamination of food in Algeria.
An elution solution of 1% methanol/TFA (V/V) was
Materials and Methods prepared by mixing 1mL TFA with 100mL methanol.

Chemicals and Reagents A solution of ammonium acetate 2 mol /L (15.4 g in 100

ml of ultrapure water) was prepared and then diluted to
The antimicrobial standards Tetracycline Hydrochloride, 1/10th in ultrapure water, for the final reconstitution of the
Chlortetracycline Hydrochloride, Demeclocycline residue to qualitative method.
Hydrochloride, 4-Epioxytetracycline, 4-Epichlortetracycline
Hydrochloride, 4-Epitetracycline Hydrochloride were A formic acid solution 0.2 mol/L was prepared for the
purchased from Merck Sigma-Aldrich ; Doxycycline Hyclate final reconstitution of the residue to confirm and quantify

Zamoum R, et al. Assessment of Tetracyclines Residues Contamination in Chicken Meat Samples of Copyright© Zamoum R, et al.
Algiers Slaughterhouses Level. Adv Clin Toxicol 2023, 8(4): 000280.
3 Advances in Clinical Toxicology

tetracyclines in chicken meat. working solution.

Mobile Phase Preparation On the other hand, the confirmation and quantification
step was achieved. Indeed, a matrix-matched standard
Mobile phases A and B for qualitative method were calibration curve was obtained by preparing five tubes, each
respectively, 0.1% TFA aqueous solution and 0.1% TFA in containing 2±0.04 g of meat chicken. Increasing volumes
methanol/acetonitrile (70:30). Mobile phases A and B for of the 0.5 mg/mL working solution (10, 20, 30, and 40µL)
quantitative method were respectively, 0.2% ammonium were added to every tube to obtain 0.5, 1, 1.5, and 2 µg/mL
formate with formic acid aqueous solution and 0.2% spiking concentrations. Purified water was added to every
ammonium formate and formic acid in acetonitrile. tube to reach a final volume of 10mL. All samples contained
Demeclocycline as an internal standard.
Standard Stock Solutions and Working Solutions
Extraction Procedure
Standard stock solutions 0.5 mg/mL were prepared in
methanol independently. The prepared stock solutions were The extraction procedure depends on the type of the
stored below –18°C. analysis method:
• For the qualitative method, tubes containing the
An internal standard stock solution was prepared with prepared samples were kept in the dark for 10 minutes.
ultrapure water to obtain 1 μg/mL of working solution, which To 2g amount of homogenized sample, weighed into
was stored at 4°C. Sulfaphenazole was used as an internal 15mL polypropylene centrifuge tube, was added 8mL
standard for the identification of oxytetracycline, tetracycline, of acetonitrile, which was vortex-mixed for 30sec.
chlortetracycline, and doxycycline. Demeclocycline was After hand, the tubes were shaken 10 min (100 tours/
used for the quantification of the four tetracyclines and min) on a rotary shaker. The tubes were centrifuged 5
theirs epimers. Working solutions were prepared using the min at 14000 g at about 4°C, and 6mL of supernatant
stock solution diluted with water. The working solutions were decanted carefully into a clean second centrifuge
were prepared daily. For confirmation and quantification, tube. The supernatant was evaporated and dried under
the working solutions were a mixture of the tetracyclines nitrogen at 50°C. The residue was dissolved and projected
and their epimers, prepared by serial dilutions of the stock to a constant volume of 0.6mL using the ammonium
solution in methanol and were stored in brown glass vials acetate 0.2mol/L. Then the residue was filtered through
at 4°C a 0.45µm filter membrane and analyzed.
• For the confirmation and the quantification method,
Sample Preparation tubes containing the prepared samples were kept in
the dark for 10 minutes. To 2g amount of homogenized
A 100g sample of chicken meat was homogenized with a sample, weighed into 50mL polypropylene centrifuge
Moulinex mixer. Then, 2g were weighted, placed in propylene tube, was added 10mL of Na2 EDTA-McIlvaine buffer,
tubes and stored at 20°C until analysis. which was vortex-mixed for 30sec. The tubes were
then stirred for 10 min at 100 revolutionss/min) on a
The identification of tetracyclines residues in chicken rotary shaker. Subsequently, tubes were centrifuged for
meat samples was performed by preparing a negative control 5 min at 14000 g at +4°C. Afterwards, the supernatant
using a blank sample (free of the interest analytes), and a was collected in a 50mL polypropylene clean tube.
quality control (QC) which was a fortified sample at a half of The extraction was repeated with 10mL of Na2 EDTA-
the MRL value (50µg/kg) for validation method (MRL i.e 100 McIlvaine buffer. The combined supernatant fluid was
µg/kg for samples qualitative analysis purpose). The internal mixed with 2mL of trichloracetic acid before freezing for
standard used at this stage was sulfaphenazole. A volume of 15 to 20 min. Then, tubes were centrifuged at a rotate
200 μL of the 1 μg/mL working solution of internal standard speed of 14000g for 10 min at +4°C, and filtered with fast
was added to all samples before the extraction procedure. filter paper. The supernatant was purified by using the
solid-phase extraction (SPE). The procedure used for the
A 2g homogeneous sample (accurate to 0.04 g) was SPE extraction is shown in Figure 1. After evaporation
placed into a 50mL polypropylene centrifuge tube with of extracts under nitrogen at 50°C, the dry residue was
600µL for fortified samples and 800µL of water for samples reconstituted with 500µL of 20% formate acid, filtered
to be analyzed and the blank sample. Fortified samples through a 0.45µm filter membrane and analyzed.
are obtained by adding 200 μL of 0.5 mg/mL tetracyclines

Zamoum R, et al. Assessment of Tetracyclines Residues Contamination in Chicken Meat Samples of Copyright© Zamoum R, et al.
Algiers Slaughterhouses Level. Adv Clin Toxicol 2023, 8(4): 000280.
4 Advances in Clinical Toxicology

Figure 1: Chlortetracycline keto-enol forms chromatogram.

Liquid Chromatography−Tandem Mass and mobile phase A was then maintained within 5min in the
Spectrometry initial conditions. The resulting total run time was 25 min.
The mobile phase B consisted of 0.1% TFA in methanol/
The analysis was performed on a Perkin Elmer HPLC acetonitrile (70:30).
coupled to an AB Sciex QTrap 3200 triple quadrupole mass
spectrometer (MS/MS). All devices being controlled by The gradient used for the quantitative analysis started
ANALYST 5.1 software. The analytical column was Symmetry with 2 min at 90% of eluent A (0.2% ammonium formate
C18, 150 x 3.9mm, 5 μm of particle size from Waters, and with formic acid aqueous solution) and decreased linearly
heated to 30°C for the tetracyclines identification. Sunfire to 40% within 7 min. This composition was maintained for
C18 100x2.1mm column was used at 3°C for the the 2 min and mobile phase A was then increased linearly to
quantification method. The mobile phase was at a flow rate 90% at 13 min. With a final equilibration time of 4 min in
of 600 μL/min for both methods. The gradient profile for the the initial conditions, the resulting total run time was 17min.
qualitative method started with 5 min at 81% of eluent A The Eluent B contained 0.2% ammonium formate and formic
(0.1% TFA aqueous solution) and decreased linearly to 40% acid in acetonitrile.
at 19min. This composition was increased to 81% at 20min

Precursor Product Time DP CE CXP Screening RT Quantitative RT

Compound
ion ion (mSec) (V) (eV) (V) (min) (min)
445.5 410.3 30 3
Tetracycline 100 30 13,3 9.77
445.5 427.5 30 5
461.3 201.1 30 6
Oxytetracycline 100 30 12,9 9.66
461.3 443.1 50 3
445.4 428.3 30 4
Doxycycline 100 40 18,5 10.4
445.4 201.3 50 3
479.2 444.2 40 5
Chlortetracycline 100 30 13 10.26
479.2 462.2 10 5
Sulfaphénazole (IS) 315.4 156 100 50 30 5 16.5
Demeclocycline (IS) 465 448 100 45 18 3 9.99
DP: Declustering Potential; CE: Collision Energy; CXP: Collision cell exit potential; RT: Retention Time.
Table 1: The MS/MS parameters of the selected antibacterial drugs.

Zamoum R, et al. Assessment of Tetracyclines Residues Contamination in Chicken Meat Samples of Copyright© Zamoum R, et al.
Algiers Slaughterhouses Level. Adv Clin Toxicol 2023, 8(4): 000280.
5 Advances in Clinical Toxicology

The injection volume was 25µL for the two methods. 1 and Equation 2.
Between the injections of standards and of samples
and between injections of each sample, a blank (Water/ T= B + 1, 64 x SDB Equation 1 &
Acetonitrile, V/V) injection was given.
Fm
= M − 1, 64 x SD Equation 2
Mass spectrometric analysis was carried out using
electrospray ionization (ESI) in the positive mode. ESI The means (B and M) and the standard deviations (SDB
parameters were capillary voltage (IS) of 5500 V, entrance and SD) are calculated, from the responses obtained, at
potential (EP) of 10 V and source temperature of 700°C. The the level of the negative controls and the fortified samples
curtain gas was at 20psi and CAD gas was at level medium. respectively.
Nebulizer gas (GS1) and auxillary gas (GS2) were set at 40
psi and 50 psi, respectively. The specific MS/MS parameters According to European Commission Decision 2002/657/
for each target analyte are shown in Table 1. EC [11], the suitable sensitivity of a screening method is
demonstrated when the CCβ is below or equal to the MRL.
Validation Procedure Therefore, the false-compliant rate is below or equal to 5%
at the MRL level. Consequently, CCβ value is validated only
Before the confirmation step, a qualitative method when Fm> T. Otherwise (Fm <T), additional studies, at a
is implemented to monitor the presence or absence of concentration greater than the target concentration, are
tetracyclines residues in a sample and identification of the necessary.
analytes.
Specificity is the percentage of negative results, found
According to European Commission Decision 2002/657/ among the expected negative results (controls). While the
EC [11], the ratio of the chromatographic retention time of sensitivity is the percentage of positive results found among
the analyte to that of the internal standard, i.e. the relative the expected positive results (QC).
retention time of the analyte, should correspond to that of
the calibration solution with a tolerance ± 2.5% for liquid A sensitivity greater than 95% was considered
chromatography (LC). Validated Excel® sheets were used satisfactory. Detection limits (LOD) were calculated from the
to assess qualitative and quantitative criteria. Finally, 21 QC samples according to Equation 3.
stability of the standard solutions was based on the Fougères
Laboratory studies. Thus, the stability of the stock solutions LOD
= Cval × H 3×B / M Equation 3
in methanol is 6 months below -18°C.
LOD and Cval are in μg / kg, H3 × B is the response (peak
Qualitative Method height) corresponding to 3 times the average noise and M is
The validation was conducted according to European the average of the responses of the 21 supplemented samples.
Commission Decision 2002/657/EC [11] and guidelines for The noise is determined graphically and corresponds to the
the validation of screening methods [10]. Detection capability highest noise level, within the 1min MRM detection window.
(CCβ) is assessed by using fortified samples according the
MRL value (100µg/kg), with tetracyclines (Oxytetracycline, Quantitative Method
tetracycline, chlortetracycline, and doxycycline) for a The validation of a quantitative method was achieved
screening purpose. Thus, CCβ is determined as the lowest according to European Commission Decision 2002/657/EC
concentration for which 20 measurements give less than 5% [11]. Therefore, the matrix matched standard calibration
false-negative results [12]. curve was constructed as described in sample preparation
section. The efficiency of the analytical method was
In this study, the threshold value (T-value), the cut-off assessed by investigating the selectivity, matrix effect,
factor (Fm), detection observed limit (LOD), sensitivity and linearity, trueness, repeatability and applicability. The
specificity were studied [10,11]. quantitative criteria assessed were: regression model fit,
trueness, repeatability, within-laboratory reproducibility
For this, two series of samples are prepared, each one and expended measurement uncertainty. The intermediate
including seven samples. The first series will serve as precision and the repeatability were expressed with a relative
“negative controls”, while the second series is supplemented standard deviation (RSD) and trueness was evaluated using
with the analytes of interest (Quality control QC), at the the recovery rate or bias.
target concentration (Cval = 0.5MRL). This pattern was
repeated for three days. This generates 42 results allowing The expended measurement uncertainty was assessed
the calculation of the values T and Fm according to Equation as a combined standard measurement uncertainty (Uc),

Zamoum R, et al. Assessment of Tetracyclines Residues Contamination in Chicken Meat Samples of Copyright© Zamoum R, et al.
Algiers Slaughterhouses Level. Adv Clin Toxicol 2023, 8(4): 000280.
6 Advances in Clinical Toxicology

which is determined as a root sum of squares of a standard Fougères Laboratory method, where acetonitrile was used
deviation s characterising the precision of the measurement as a solvent for extraction. This is interesting when a multi-
(repeatability, intermediate precision) and an estimate b residues analysis method is to achieve. Indeed, that procedure
accounting for measurement bias error [13]. Therefore, the was used for identification of twenty seven analytes by LC-
equation to calculate Uc is given by Equation 4. MS/MS.

U
= c s 2 + b 2 Equation 4 As widely described in literature, tetracyclines likely
bind to metallic ions, consequently Na2EDTA dihydrate were
The confirmation of the analytes is based on the added specially to improve their extraction from the meat
verification of the concentration calculated on the overall matrix. Thereby, an extraction was made using this salt
curve compared to the decision limit CCα; the relative before clean-up. In that way, the extraction was performed
intensity of the ionic ratio of the two transitions (MRM2 / for the quantitative method.
MRM1); the relative retention time (referred to the IS tr) and
the ratio of signal and background noise (S / N> 3). Optimization of the Analytical Method

Regarding the quantitative approach, the quantitation MS/MS data acquisition was performed in MRM
of the residue marker of tetracyclines required the analysis (Multiple Reaction Monitoring) mode, where two product
of both of the analyte of interest and its metabolite. ions were chosen for each analyte. This was achieved by
In this case, three metabolites were added for this infusing 0.5µg/mL1 of individual standard solutions directly
purpose: 4-epioxytetracycline, 4-epitetracycline and into the MS/MS system. The precursor ions of all analytes
4-epichlortetracycline. Then, seven analytes were optimized were [M + H] +. Precursor and product ions, declustering
to perform a quantitative method. potential (DP), collision energy (CE) and collision cell exit
potential (CXP), retention times (RT) values are shown in
Samples Collection at the Slaughterhouse Level Table 1. The best sensitivity for all drugs was determined to
provide the highest signals for identification, quantification
This is a cross-sectional descriptive study with an and confirmation.
analytical aim. It is based on the evaluation of the residue
levels of tetracyclines in chicken meat. The MS/MS optimization results show the loss of water
or ammonia after fragmentation the analytes studied.
Sampling of chicken at slaughter points in the Algiers This is due to their structure, which is formed by an
region was exhaustively carried out. The chicken were octahydrotetracene-2-carboxamide skeleton [14].
randomly chosen, with a minimum of five animals per
slaughterhouse. Animals’ age was 60 days, according to The selectivity was assessed by examination the
the health certificate issued by veterinarians. In total, 151 chromatograms. We observed that no interference was
chicken meat samples were collected at the slaughterhouse possible with the three analytes, their metabolites and
level in 2017. Samples were collected then analyzed by LC- doxycycline, since the diagnostic ions obtained do not have
MS/MS using our validated method. the same masses. However, doxycycline precursor ion and
tetracycline precursor ion were identical, but the difference
The conformity of samples has been verified in a first in polarity between the two analytes and the reversed
time by comparing the responses obtained for each sample abundance of the product ions made it easier for us to
relative to the QC analyzed the same day. Secondly, the distinguish the two analytes. In fact, doxycycline being the
samples having exceeded the QC signal are analyzed with a least polar is eluted last among the four tetracyclines after
confirmation and quantification method. Indeed, the ratio of chlortetracycline. The latter is preceded by tetracycline.
the surfaces of the sample and of the QC was calculated for Oxytetracycline is the first to be detected.
all the suspects. Only reports that exceeded unity (value of 1)
were retained for confirmation and quantification. Else ways, the analysis of chlortetracycline, by LC-MS/
MS under these conditions, confirms a phenomenon widely
Results and Discussion described in the literature, which is the tautomerization of
chlortetracycline and its epimer. These studies investigated
Optimization of Extraction and Clean-Up the keto-enol forms of the CTC and its epimer 4-epi-CTC in
tissues (Figure 2). The 6-iso-CTC metabolite was also first
Concerning the optimization of the extraction of described in these studies, but only in eggs and not in tissues.
tetracyclines for a qualitative method, a simple and fast All of these forms of CTC are isobaric compounds and cannot
extraction procedure was performed according to the be distinguished by their specific mass [15].

Zamoum R, et al. Assessment of Tetracyclines Residues Contamination in Chicken Meat Samples of Copyright© Zamoum R, et al.
Algiers Slaughterhouses Level. Adv Clin Toxicol 2023, 8(4): 000280.
7 Advances in Clinical Toxicology

Figure 2: Quantitative méthod Chromatograms. CTC : Chlortetracycline, DC : Doxycycline, OTC : Oxytetracycline, TC :

Tetracycline, Epi : Epimer, SC : Calibration Standard, LMR : Maximal Residue Limit.

Zamoum R, et al. Assessment of Tetracyclines Residues Contamination in Chicken Meat Samples of Copyright© Zamoum R, et al.
Algiers Slaughterhouses Level. Adv Clin Toxicol 2023, 8(4): 000280.
8 Advances in Clinical Toxicology

Ion pairing reagents can influence the signal of the target was doxycycline at 10.60min. On the other hand, analytes
compound in the electrosprayed liquid [16]. For this purpose, were eluted after their epimers. Tetracyclines and their
heptafluorobutyric acid (HFBA) and pentafluoropropionic metabolites chromatograms acquired from the quantitative
acid (PFPA) were previously used as ion pairing reagents method.
diluted in mobile phase. However, a lack of repeatability of
retention times (more than a minute) was observed. Thus, Validation
we have opted for the use of trifluoroacetic acid, which
allowed us to obtain satisfactory results. Chromatographic The goal of this work was to identify and quantify
and spectrometric conditions were described in Materials tetracyclines residues in raw chicken meat using the LC-
and Methods section. MS/MS method. Each analyte was identified unequivocally
by the presence of tow transitions eluted at the same time.
The qualitative method had a total run time of 25 min and Indeed, in quantitative method, the first chromatographic
all analytes eluted within 18.5 min. The shortest retention peak is used for the quantification and the second for the
time was observed with oxytetracycline (12.84 min), which confirmation. On the other hand, these two peaks allow the
is explained by a highest affinity with the aqueous phase identification and the confirmation as regards the qualitative
and a lowest interaction with the stationary phase. On the method. The identification of sulfonamides and quinolones
other hand, the longest retention time was observed with antimicrobials residues in meat explains the long run time
doxycycline (18.55 min). in this method.

Extracted ion chromatograms from liquid Qualitative Method Validation Results

chromatography−tandem mass spectrometry on chicken As described in Materials and Methods section,
meat samples, obtained from the qualitative method. qualitative validation criteria were evaluated according to
EC Decision 2002/657/EC [11]. Therefore, sensitivity, CCß,
The total time of run was 18 min for the quantitative Fm and T values were assessed as shown in Table 2. The Cval
method. All analytes eluted within 11min. The first analyte corresponds to half of the MRL value of each analyte (50µg/
eluted was oxytetracycline at 9.86min and the last one kg).

MRM1 MRM2
Analytes
Fm T CCß Se LODe Fm T CCß Se LODe
Tetracycline 8852,84 1722,31 ˂Cval 100% 21,87 821,42 214,31 ˂Cval 100% 32,57
Oxytetracycline 9258,50 4790,06 ˂Cval 95% 31,55 2719,86 1398,50 ˂Cval 100% 32,73
Doxycycline 9218,94 5841,37 ˂Cval 95% 13,25 4384,15 400,58 ˂Cval 100% 42,58
Chlortetracycline 10243,23 7600,76 ˂Cval 95% 16,84 6817,74 1373,85 ˂Cval 100% 82,06
MRL =100 µg/kg, Cval = 50 µg/kg, Se: Sensitivity, LODe: estimated LOD (µg/kg).
Table 2: CCß, Fm, T-value and sensitivity of tetracyclines in chicken meat.

The specificity of the method was verified by analyzing Quantitative Method Validation Results
twenty samples of chicken breasts from different origins Several criteria were checked for each of the selected
and the specificity was 100% for all analytes because no transitions: specificity, retention time, relative ion intensity
peak was detected in these samples at the retention time tolerances and signal-to-noise ratio, in terms of identification.
corresponding to each analyte.
Specificity was checked not only by examining the
The criterion of the relative retention time was met chromatograms of different matrix control samples at the
for all validation standards. The variability of the relative target retention times but also by injecting different target
retention time of the four analytes was satisfactory with RSD analytes to verify that there was no interference between
values between 0.36 and 1.86%. monitored MRM transitions.

The limit of detection estimated were shown in Table The chromatograms of different matrix control samples
2. The determined LOD was below than 50 µg/kg for both was examined at the target retention times and results were
transitions for all analytes, except for chlortetracycline for satisfactory. Indeed, tetracyclines analysis was specific for
MRM2 for which LOD was out of Cval value. oxytetracycline, chlortetracycline and tetracycline. However,
the specificity was to a lesser extent for Doxycycline.

Zamoum R, et al. Assessment of Tetracyclines Residues Contamination in Chicken Meat Samples of Copyright© Zamoum R, et al.
Algiers Slaughterhouses Level. Adv Clin Toxicol 2023, 8(4): 000280.
9 Advances in Clinical Toxicology

Therefore, the programming of the four samples of water/ (relative ion intensities) were found to be within the
ACN solutions between injections proved to be essential to maximum permitted tolerances for all validation standards
address this contamination. except two, for doxycycline and 4-epioxytetracycline (Table
3).
The ratio between the two identification transitions

VS
Recovery Interday precision
Concen Precision (%) Relative ion intensity (%)
(%) (RSD %)
tration
Compound
(µg/kg) Trueness Repetability Within-lab. Uncertainty
Tole
reproduc Day 1 Day 2 Day 3
(%) (%) (%) rance
ibility (%)
50 µg/kg 92,3 16,5 20
Oxytetracycline 100 µg/ kg 94,3 12,5 14 39,75 27,28 51,20 9,4% 5,5% 4,7% 25%
150 µg/kg 86,8 13 25,6
50 µg/kg 90,8 19 25
4-epioxytetra
100 µg/ kg 104,6 15 31 49,26 62,83 70,64 28,5% 24,8% 18,6% 25%
cycline
150 µg/kg 88,3 21 35
50 µg/kg 103,5 39 48
Doxycycline 100 µg/ kg 134,7 43 76 95,60 152,3 135,34 5,4% 71,3% 5,8% 50%
150 µg/kg 102,6 22 68
50 µg/kg 102,6 21 22
Tetracycline 100 µg/ kg 97,5 14 20 44,31 39,30 60,12 12,2% 14,5% 12,5% 25%
150 µg/kg 76,6 13,5 30
50 µg/kg 103,0 26 26
4-epitetracycline 100 µg/ kg 104,8 15 24 52,00 47,74 67,60 12,2% 10,4% 8,5% 25%
150 µg/kg 85,5 17 34
50 µg/kg 87,5 20 23,5
Chlortetracycline 100 µg/ kg 84,7 16 19,0 46,9 38 62,6 7,3% 6,3% 7,7% 20%
150 µg/kg 81,3 15,0 31
50 µg/kg 110,7 13,5 21
4-epichlortetracycline 100 µg/ kg 99,6 9,5 14 42,16 28 32,78 7,9% 13,3% 11,0% 20%
150 µg/kg 90,0 8 16
Table 3: Trueness, Repeatability and within-Laboratory Reproducibility results.

The signal-to-noise ratio was always above 3 for both in terms of trueness, precision and expended uncertainty
transitions on each day and for all compounds. are enumerated in Table 3. The quantitative results were
compliant with the requirements of Decision 2002/657/
A simple linear regression model was chosen for EC except for doxycycline for which, even though trueness
all analytes. Calibration curves were satisfactory with results were acceptable, repeatability and reproducibility
determination coefficient (r2) between de 0.91 à 0.98%, RSD was too high. In the aim to achieve better results, the use
where the lowest values were observed for doxycycline. of an isotopically labeled standard apparently was indicated,
not only of doxycycline Gaugain M, et al. [17], but also for
The quantitative performance of the method results the three tetracyclines. This may reduce the matrix effect,

Zamoum R, et al. Assessment of Tetracyclines Residues Contamination in Chicken Meat Samples of Copyright© Zamoum R, et al.
Algiers Slaughterhouses Level. Adv Clin Toxicol 2023, 8(4): 000280.
10 Advances in Clinical Toxicology

which is not accurately corrected by the internal standard twenty-five (16.55%) samples. Then, oxytetracycline was
demeclocycline [17]. identified in thirteen (8.60%) samples, probably due to
its greater use in cattle than in poultry. Apart from that,
As described in Decision 2002/657/EC, the decision doxycycline was detected in fifteen (9.93%) samples. The
limits (CCα) were assessed and were between 107 and 141 withdrawal time for doxycycline in chickens is five days.
on three days, except for doxycycline, 4-epioxytetracycline This predicts its presence in chicken samples in addition to
and chlortetracycline, for which CCα values (for one day), its more pronounced lipophilic character compared to other
were higher than the maximal CCα expected (144µg/kg). tetracyclines. Three samples contained both oxytetracycline
and doxycycline.
Globally, the validation results were in agreement with
an exposure assessment purpose, even though some values Quantitative Assessment
were out of the maximum permitted tolerances in terms of Five among the 25 positive samples were contaminated
quantitative validation criteria. with oxytetracycline or doxycycline at a level higher than the
MRL value (100µg/kg). The surfaces sample/QC ratio was
Assessment of Tetracyclines Residues in between 1.29 and 3.48. Therefore, calibration and validation
Chicken Meat Samples standards were analyzed simultaneously with the unknown
chicken meat. The calibration results in terms of relative
Qualitative Assessment intensity, relative retention time, CCα, signal to noise, are
The analysis of 151 samples revealed the presence of provided in Table 4.
oxytetracycline or doxycycline alone or both of them in

Concentration Level Relative intensity (MRM2/MRM1) Relative retention time (min) S/N (MRM2)
(µg/kg) DC OXT Epi-OXT DC OXT Epi-OXT DC OXT Epi-OXT
0,09 0,42 0,65 1,06 0,96 0,95 205 330 88,3
50
0,09 0,33 0,89 1,04 0,98 0,98 352 171 92,6
0,08 0,34 0,40 1,06 0,97 0,95
100
0,08 0,34 0,51 1,04 0,98 0,98
0,08 0,35 0,43 1,06 0,97 0,95
150
0,09 0,38 0,56 1,04 0,98 0,98
0,09 0,32 0,43 1,07 0,97 0,96
200
0,08 0,34 0,58 1,04 0,98 0,97
Mean 8,5% 35,4% 55,7% 1,05 0,97 0,97
Tolerance 50% 25% 20% 2,5%
Max 12,7% 44,3% 66,8% 1,08 1,00 0,99
Min 4,23% 26,6% 44,5% 1,03 0,95 0,94
CC α (µg/kg) 140 123 134,5
Table 4: Relative intensity, relative retention time, CCα and signal to noise of sample analysis.

Both transitions exist in all samples with a signal-to-noise values.

ratio significantly greater than 3. Three samples contained
86.49, 97.23 and 120.16 µg/kg of sum of oxyetracycline and Conclusion
its metabolite, 4-epioxytetracycline. Even though this residue
contamination is significant considering the MRL value Qualitative and quantitative methods were successfully
(100µg/kg), these values do not exceed the corresponding validated for the detection and quantification tetracyclines
CCα values (130.15, 129.51 and 130.46 µg/kg respectively). by LC-MS/MS. Thus, a simple, cheap, fast, reliable and
In addition, the same finding was observed with doxycycline, sensitive multiresidues analytical method was developed for
which was quantified at 46.8, 91.65 and 124.24 µg/kg with the simultaneous determination of tetracyclines in chicken
decision limits value of 140 g/kg. Consequently, the five muscle samples using simple liquid extraction with LC-MS/
samples were compliant according to the decision limit MS.

Zamoum R, et al. Assessment of Tetracyclines Residues Contamination in Chicken Meat Samples of Copyright© Zamoum R, et al.
Algiers Slaughterhouses Level. Adv Clin Toxicol 2023, 8(4): 000280.
11 Advances in Clinical Toxicology

In addition, quantification criteria as required in Decision 9. Algerian Ministry of Commerce (2016) Arrête
2002/657/EC were met for oxytetracycline, tetracycline, interministériel du 20 juin 2016 fixant les listes ainsi
chlortetracycline and their metabolites and doxycycline. que les limites maximales de résidus de médicaments
vétérinaires ou de substances pharmacologiquement
The developed method was effectively applied to real actives tolérées dans les denrées alimentaires d’origine
samples, and the results of tetracyclines residues showed animale.
that oxytetracycline and doxycycline were identified in
twenty-five chicken meat samples. The comparison with 10. European Commission (2010) Guidelines for the
the QC sample allowed us to confirm that five samples validation of screening methods for residues of
were contaminated with an amount over the MRL value. veterinary medicines (initial validation and transfer).
Therefore, quantification of oxytetracyclin, its metabolite
11. European Commission (2002) Decision 2002/657/
and doxycycline in five samples revealed that they were
EC of implementing Council Directive 96/23/EC
compliant according the Decision 2002/657/EC. However,
concerning the performance of analytical methods and
additional studies with a larger sample size are necessary to
the interpretation of results. Ecolex.
confirm tetracyclines residues in chicken muscles to assure
food safety. 12. Pikkemaat MG (2009) Microbial screening methods
for detection of antibiotic residues in slaughter
References animals. Analytical and bioanalytical chemistry 395(4):
893-905.
1. Mungroo NA, Neethirajan S (2014) Biosensors for
the Detection of Antibiotics in Poultry Industry—A 13. Gaugain M, Raynaud A, Bourcier S, Verdon E, Hurtaud-
Review. Biosensors 4(4): 472-493. Pessel D (2021) Development of a liquid chromatography-
tandem mass spectrometry method to determine colistin,
2. Klein EY, Van Boeckel TP, Martinez EM, Pant S, Gandra S, bacitracin and virginiamycin M1 at cross-contamination
et al. (2018) Global increase and geographic convergence levels in animal feed. Food additives contaminants Part
in antibiotic consumption between 2000 and A Chemistry analysis control exposure risk assessment
2015. Proceedings of the National Academy of Sciences 38(9): 1481-1494.
of the United States of America 115(15): E3463–E3470.
14. Susakate S, Poapolathep S, Chokejaroenrat C, Tanhan
3. Méheust D, Chevance A, Moulin G (2016) Suivi des P, Hajslova J, et al. (2019) Multiclass analysis of
ventes de médicaments vétérinaires contenant des antimicrobial drugs in shrimp muscle by ultra-high
antibiotiques en France en 2015. Rapport annuel ANSES performance liquid chromatography-tandem mass
pp: 1‑106. spectrometry. Journal of food and drug analysis 27(1):
118-134.
4. Matzov D, Bashan A, Yonath A (2017) A Bright Future for
Antibiotics? Annual review of biochemistry 86: 567-583. 15. Gaugain M, Gautier S, Bourcier S, Jacques AM, Laurentie
M, et al. (2015) 6-Iso-chlortetracycline or keto form of
5. Marshall BM, Levy SB (2011) Food animals and
chlortetracycline? Need for clarification for relevant
antimicrobials: impacts on human health. Clinical
monitoring of chlortetracycline residues in food. Food
microbiology reviews 24(4): 718-733.
additives & contaminants Part A 32(7): 1105-1115.
6. Goucem R (2015) L’antibiothérapie en aviculture et ses
16. Holcapek M, Volná K, Jandera P, Kolárová L, Lemr K,
implications. ENSV d’Alger.
et al. (2004) Effects of ion-pairing reagents on the
7. Agroligne (2017) Usages des antibiotiques en élevage electrospray signal suppression of sulphonated dyes and
Quels risques pour la santé animale et humaine ? intermediates. Journal of mass spectrom 39(1): 43-50
L’essentiel de l’agroalimentaire et l’agriculture - N°104
17. Gaugain M, Fourmond MP, Fuselier R, Verdon E, Roudaut
9: 60.
B, et al. (2020) Control of Antimicrobials in Feed Using
8. European Commission Decision (2009) Commission Liquid Chromatography−Tandem Mass Spectrometry:
Regulation (EU) No 37/2010 of 22 December 2009 Assessment of Cross-Contamination Rates at the Farm
on pharmacologically active substance and their Level. Journal of Agricultural and Food Chemistry
classification, regarding maximum residue limits 68(34): 9033-9042.
in foodstuffs of animal origin. Official Journal of the
European union.

Zamoum R, et al. Assessment of Tetracyclines Residues Contamination in Chicken Meat Samples of Copyright© Zamoum R, et al.
Algiers Slaughterhouses Level. Adv Clin Toxicol 2023, 8(4): 000280.