Journal of Chromatography A 1724 (2024) 464901

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

A high-throughput UPLC-MS-MS Bio-analytical method for the analysis of

veterinary pharmaceutical residues in Chicken Tissues, Application of
efficient-valid-green (EVG) Framework as a Competence Tool
Sarah S. Saleh a, *, Ahmed Samir b, Hayam M. Lotfy c, *, Christine K. Nessim d
a
Analytical Chemistry Department, Faculty of Pharmacy, October University for Modern Sciences and Arts (MSA) 11787 6th October City, Egypt
b
Biochemistry Department, Faculty of Pharmacy, October University for Modern Sciences and Arts (MSA) 11787 6th October City, Egypt
c
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Future University in Egypt, 11835 Cairo, Egypt
d
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ahram Canadian University, 6th of October City 12566, Cairo, Egypt

A R T I C L E I N F O A B S T R A C T

Keywords: Antibacterial medications are receiving the most attention due to hypersensitivity reactions and the emergence of
Response surface methodology bacterial mutants resistant to antibiotics. Treating Animals with uncontrolled amounts of antibiotics will extend
EVG beyond their lives and affect humans. This study aims to determine the concentration of the residues of sulfa­
Maximum residue limit
dimidine, sulfaquinoxaline, diaveridine, and vitamin K3 in the tissues of poultry (muscles and liver) after
AGREEprep
I-optimal
treatment with the combined veterinary formulation. A UPLC-MS-MS method was developed using Poroshell 120
EC–C18 and a mobile phase composed of acetonitrile and distilled water, containing 0.1 % formic acid, in the
ratio of (85:15 v/v) at a flow rate of 0.6 mL/min. Sample extraction solvent was optimized using response surface
methodology (RSM) to be acetonitrile: methanol in the ratio (49.8: 50.2 v/v), and the method was validated
according to the FDA bioanalytical method validation protocol over the range (50–1000 µg/Kg) for sulfaqui­
noxaline and (50–750 µg/Kg) for the other 3 drugs. The greenness of the sample preparation and analytical
method was assessed by applying Analytical Eco-scale (AES) and AGREE coupled with AGREEprep. The
Competence of the study was evaluated via the EVG framework known as Efficiency, validation, and greenness,
to achieve a balance point represented by a radar chart. The method was applied to decide the time required for
poultry products to be safe for human use after administration of the studied drugs. It was found that, after the
administration of the last dose, minimally 7 days are required till the levels of the drugs drop to the maximum
residue limit determined by the FDA/WHO in animal tissues.

1. Introduction poultry products represent around 40 % of global meat production ac­

cording to data published by the Food and Agriculture Organization
Antibacterial drugs are receiving the biggest attention due to mul­ (FAO) [1]. In the year 2021, poultry meat yearly consumption per capita
tiple hazards associated with their uncontrolled usage such as hyper­ in the Middle East region was estimated to reach its maximum in Saudi
sensitivity and evolution of antibiotic-resistant bacterial mutants such as Arabia (41.5 kg), then Libya (38.4 Kg), Egypt (21.3 Kg), and finally
methicillin-resistant Staphylococcus aureus (MRSA), vancomycin- Sudan (1.75 Kg) [2]. This huge percentage and the tremendous increase
resistant Enterococcus (VRE), and multidrug-resistant Tuberculosis in demand required a parallel development in the industry to satisfy the
(MDR-TB). The restrictions regarding the usage of antimicrobials in demand. Thus, the use of antibiotics like tetracycline, bacitracin, sul­
humans play an important role in decreasing the unwanted effects, but phonamides, and others is common in the poultry industry for treat­
the case is not the same when it comes to the usage of antimicrobials in ment, prophylaxis or even growth promotion [3].
animals. The effect of uncontrolled usage of antimicrobials in animals is To control the leakage of antibiotics to humans, Codex Alimentarius
not limited to animals’ lives but reaches humans in different ways. One Commission (CAC), the central part of the Joint FAO/WHO Food Stan­
of these ways is the consumption of poultry products of birds that have dards Program, places food guidelines known as Codex Alimentarius
been treated with antimicrobials. Some estimations reported that [4]. These guidelines determine the maximum residue limit (MRL) of

* Corresponding authors.
E-mail addresses: [email protected], [email protected] (S.S. Saleh), [email protected] (H.M. Lotfy).

https://doi.org/10.1016/j.chroma.2024.464901
Received 6 January 2024; Received in revised form 31 March 2024; Accepted 9 April 2024
Available online 13 April 2024
0021-9673/© 2024 Elsevier B.V. All rights reserved.
S.S. Saleh et al. Journal of Chromatography A 1724 (2024) 464901

different compounds, including pharmaceuticals, which are allowed to 2.3. Standard solutions
be present in food and contribute to the quality, safety, and equity of
global food trading. Stock standard solution of each drug (SDM, SQX, DVR, VK3, and
Coccimix® is a veterinary drug formulation that consists of four SMZ) were prepared in the diluent (methanol: acetonitrile in the ratio of
drugs, two sulfonamide antibiotics, sulfadimidine sodium (SDM) and 50:50 v/v) to prepare concentrations of (500 μg/mL). The standard so­
sulfaquinoxaline sodium (SQX), which inhibit the bacterial synthesis of lutions were found to be stable for one month when kept in the refrig­
folic acid by competing with p-aminobenzoic acid for dihydrofolate erator at 4 ◦ C. To prepare the working solutions (50 µg/mL) for each
synthetase in metabolism, one antiprotozoal namely diaveridine (DVR), drug, appropriate dilutions were done from the stock solutions using the
and vitamin K3 (VK3), also known as menadione. Coccimix is indicated mobile phase.
for both the prevention and treatment of a disease known as coccidiosis,
which is a parasitic infection that may occur as a result of the coccidian 2.4. Poultry tissue samples
protozoa that infects poultry. The chemical structures for the four drugs
are shown in Supplementary material Fig. 1SM. 2.4.1. Sample collection and preparation
Several chromatographic methods have been reported for the In this study, 60 Hubbard-Specific Pathogen Free (HSPF) broiler
determination of different sulfonamides in chicken [5] such as HPLC-UV chickens were used. The broilers were grown to a weight of 750–1000 g
[6–9], HPLC with fluorescence detection [10], UPLC [11,12], and gas in the Animal Health Research Institute (AHRI) - animal house in Dokki,
chromatography [13,14]. Moreover, a few chromatographic methods Egypt. Each animal has been kept in a separate cage under positive
have been reported for the analysis of DVR alone [15,16] or in combi­ pressure and filtered air in the barrier device housed and has free access
nation with other veterinary drugs in chicken [17–19]. For VK3, it has to sterile water and food with provided veterinarian care. All procedures
been quantified in eggs using liquid chromatography tandem mass involving animals were reviewed and approved by the MSA Faculty of
spectrometry [20], and in feed premixes using gas chromatography Pharmacy Research Ethics Committee (REC). The 55 chickens were
[21]. The analysis of the dosage form (Coccimix ®) quaternary mixture divided into 3 groups, 50 broilers as the test group, 2 broilers acted as a
has been reported using spectrophotometry [22] and liquid chroma­ positive control group and the other 3 broilers acted as a negative
tography [23], but both methods could not be applied for the analysis of control group.
the mixture of the four drugs in poultry tissues due to matrix interfer­ When the chickens reached the required weight, the birds in the test
ence and critical residual concentrations that cannot be detected by group were fed the Coccimix formulation, which was provided by
either of these methods. Thus, a new bioanalytical method was needed. Pharma Swede - Egypt, by dissolving 1 gram of the Coccimix formula in
This decline curve study aims to analyze the critical concentration of 1 liter of drinking water for 5 days. After the fifth day of treatment, the
the residues of the studied drugs in the tissues of poultry treated with chickens were slaughtered in groups of 5 broilers every day using a
Coccimix in order to decide the time required to reach the MRL, then the highly sharp knife creating one cut parallel to the jawbone. One broiler
poultry product will be safe for human use. Bioanalytical method was from the control group and four from the test group, were slaughtered
successfully developed using UPLC-MS-MS. The design of experiment over an 11-day post-administration period. Five grams of breast, thigh,
with response surface methodology ensured the development of optimal and liver tissues of each broiler were extracted freshly and directly after
extraction procedure of the studied drug residuals from such a complex slaughtering, then they were stored in 50 g capacity tubes at − 20 ◦ C
matrix. The greenness of the proposed sample preparation and analyt­ until further processing.
ical method was assessed by applying Analytical Eco-scale (AES) and Two additional samples (separated breast, thigh, and liver) were
AGREE couplesd with AGREEprep. The competence of the whole study collected to study how freezing and heating affected the amount of
was evaluated via a three-pillar framework known as Efficiency, vali­ coccimix residues. Each tissue was immersed in boiling water and
dation, and greenness (EVG) framework to achieve a balance point cooked for 15 min to study the heating effect. For the freezing effect,
represented by a radar chart. other tissues were frozen for 14 days at a temperature of − 20 ◦ C. The
negative control group was spiked with fixed doses of the four medi­
2. Materials and methods cations and an internal standard (100 µg/Kg) to show that there were no
traces of any drug in the chicken tissues whereas the positive control
2.1. Chemicals group received no treatment.

Sulfadimidine sodium (SDM), sulfaquinoxaline sodium (SQX), dia­ 2.4.2. Sample extraction via response surface methodology
veridine (DVR), vitamin K3 (VK3), and the internal standard sulfa­ I-optimal design was used to test the suitability of the extracting
methoxazole (SMX) were kindly gifted by Pharma Swede company, solvent composition using response surface methodology (RSM). Nega­
Egypt; and their purity was tested using the reported method [23] where tive blank samples were thawed at room temperature and mixed thor­
it was found to be 98.2 %, 99.3 %, 99.0 %, 98.4 %, 97.3 %, respectively. oughly with 5 mL of the extracting solvent (methanol: acetonitrile) in
Coccimix® formulation was provided by Pharma Swede company, different ratios ranging from (30:70 v/v) to (70:30 v/v) and homoge­
Egypt. HPLC grades of methanol, acetonitrile, and formic acid were nized again at ambient temperature, then sonicated for diverse time
obtained from (Merck, Germany), and distilled water. intervals ranging from 5 to 10 min on ultrasonic water bath. The samples
were centrifuged for 5 min at 9000 g-force at room temperature.
2.2. Instrumentation Extraction was repeated twice on the residue with sonication and
centrifugation using 5 mL of extracting solvent. After collecting and
UPLC-MS-MS system 6240 (Agilent, USA) equipped with triple filtering the supernatants from the previous procedures through syringe
quadrupole (TQ) mass detector spectrometer, ultra-performance LC bi­ filters 0.45 µm, the volumes were completed by extracting solvent to a
nary pump, 1290 infinity sampler, and Agilent Poroshell 120 EC-C18 volume of 25-mL. The four drugs’ recovery percentages were calculated
(4.6* 50 mm) of particle size 2.7 µm with pore size 100 Å. Electronic to determine the best extraction conditions. The optimal conditions
balance (Vibra, Japan), centrifuge (Centurion Scientific, UK), magnetic were used for the actual chicken samples obtained from the decline
stirrer (Stuart, UK), ultrasonic water bath (Elma, Germany), syringe curve investigation.
filters 0.45 μm, pH meter (Jenway 3510, UK) and Homogenizer (Stuart
SHM1 Model, USA). 2.5. Chromatographic and spectrometric conditions

The mobile phase composing of acetonitrile and distilled water,

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S.S. Saleh et al. Journal of Chromatography A 1724 (2024) 464901

containing 0.1 % formic acid, in the ratio of (85:15 v/v) was employed to previously explained, and UPLC-MS-MS system was used to analyze the
apply an isocratic elution at a flow rate 0.6 mL/min. Prior to application, clear supernatant. The relative peak areas were plotted against the
degassing of mobile phase was done using an ultrasonic bath. The concentrations of spiked SQX in tissues in the range of (50–1000 µg/Kg)
applied method was positive-ion electrospray ionization (ESI+). MS-MS and spiked SDM, DVR, and VK3 in the range of (50–750 µg/Kg) which
detection in multiple reaction monitoring (MRM) mode was done as m/z allowed the construction of the calibration plots and the computation of
279.2 < 186.1 for SDM, m/z 301.2 < 156.1 for SQX, m/z 261.1 < 245.2 the regression equations.
for DVR, m/z 172.1 < 123.1 for VK3, and m/z 254.1 < 156.1 for SMZ.
Processing the data from the mass spectrometer was accomplished using 2.7. Competence profile
the software Mass lynx V 4.1. The FDA’s guidance for industry and bio-
analytical validation was followed throughout the process of method The competency profile of the UPLC-MS/MS method is assessed
validation [24]. against the three main pillars of the EVG framework: a) efficiency
attained by utilizing the Analytical Quality by Design (AQbD) method­
2.6. Construction of calibration plots ology to develop and optimize the working conditions of the planned
method; b) validation using the ICH guidelines was verified to the
Serial dilutions were performed for each of the working standard designed working conditions; and c) greenness evaluated using one or
solutions of the four drugs. 50 μL was spiked into 5 gs of neat muscles more greenness tools which are: Analytical Eco-Scale (AES), Green
(breast and thigh) and liver samples from the negative control group. Analytical procedure Index (GAPI) and Analytical Greenness Metric
Finally, a fixed aliquot of SMZ (IS), equivalent to (100 µg/Kg), was (AGREE) coupled with Analytical Greenness Tool for Sample Prepara­
spiked into the neat tissues as well. The sample was extracted as tion (AGREEprep). Moreover, an equilibrium between the three pillars

Fig. 1. The MS/MS (m/z) of the cited drugs (SDM, SXQ, DVR, and VK3) and internal standard (SMZ) using the adopted mass spectrometric conditions.

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S.S. Saleh et al. Journal of Chromatography A 1724 (2024) 464901

was tested via the EVG approach and represented by its radar chart. solvents was proposed as the extracting solvent. Response surface
methodology (RSM) was applied to study the significant factors and
3. Results and discussion their impacts on the extraction process. RSM includes several designs
such as central composite design [25], Box–Behnken design [26], and
3.1. Method development and optimization optimal designs. The I-optimal design was chosen to optimize the best
composition of extracting solvent and sonication time for the selected
The determination of critical concentrations of various compounds biological tissues. I-optimal designs create a design with minimum in­
in tissues with acceptable accuracy is a bioanalytical challenge. Two tegrated variance [27]. The design proposed 15 experiments to examine
types of stationary phase were tested using the Acquity UPLC HSST3 the potential impact of the two critical material attributes (CMA),
column (100 Å, 1.8 µm, 50 mm X 2.1 mm) and Agilent Poroshell 120 namely the ratio of acetonitrile to methanol (v/v) and sonication time,
EC–C18 column (100 Å, 2.7 µm, 50 mm X 4.6 mm), where better res­ along with their interactions, on the selected critical quality attributes
olution between the four drugs was achieved using the Agilent Poroshell (CQAs) which were the recovery % of the four drugs (SDM %, SQM %,
column. VK3 %, and DVR %) from spiked chicken tissues. Other insignificant
Variable mobile phase compositions were tested where all various CMAs were excluded from the optimization study after screening tests,
ratios of (methanol: water) showed noisy peak of VK3; and by using and were kept at constant levels (ambient temperature, 5 min of ho­
different ratios of (acetonitrile: water) DVR showed tailed peak. The mogenization, and 5 mL of extracting solvent).
optimum elution was achieved using a mobile phase composed of The detailed experiments are listed in Supplementary material
acetonitrile and 0.1 % formic acid aqueous solution in the ratio of (85:15 Table 3SM.
v/v) at a flow rate 0.6 mL/min. The eluted peaks were efficiently Three CQAs (SDM %, SQX %, and VK3 %) showed quadratic models,
resolved, sharp, and symmetric. The multiple reaction monitoring while the CQA (DVR %) showed a linear model. The variables’ coded
(MRM) of the protonated precursor parent ions [M + H]+ was measured values were expressed by:
as shown in Fig. 1. The parameters of the mass spectrometry were listed
SDM% = +39.30A + 76.88B + 69.45AB (1)
in Supplementary material Table 1SM. The recorded retention times
for the drugs of interest were: 0.86±0.02, 0.88±0.01, 0.68±0.02, 0.83 SQX% = +44.56A + 77.34B + 52.19AB (2)
±0.02, and 2.76±0.03 for SDM, SQX, DVR, VK3, and SMZ (IS),
respectively, as shown in Fig. 2. The total runtime of the proposed VK3% = +40.83A + 72.97B + 62.49AB + 5.10AC (3)
method was shortened to 3 min rather than 12 min for the reported
method [23], which is more cost-effective and eco-friendly. DVR% = +77.80A + 51.67B (4)
Where A is the percentage of acetonitrile (ACN %), B is the per­
3.2. I-optimal design for optimizing sample extraction centage of methanol (METH %), and C is sonication time in minutes. The
sign denotes the variable effect, where the positive sign suggests a direct
Solvent extraction is easily applied because it consumes a minimum effect. Reduced models were created for the four CQAs, where the
sample size and organic solvents. Thus, it is favored by analysts if reduced models showed better prediction than the full models (higher
compared to other extraction procedures, such as microwave-assisted values of R2, R2-adjusted and R2-predicted).
and solid-phase extraction (SPE) as it reduces cost, time, and manipu­ In order to test the significance and proper fit of the model, analysis
lation steps. Several solvents including ethyl acetate, hexane, methanol, of variance (ANOVA) was performed at 95 % confidence. All models
and acetonitrile were screened for extracting the four drugs of interest were found to be significant (p < 0.05). The values of adjusted R2 and R2
from the desired tissues after homogenization, where sulfa-drugs (SDM, approached unity (≥0.9) which implies that the proposed models
SQX), VK3, and IS showed the best recoveries using methanol, while explained more than 90 % of the total variances of the four CQAs. The
DVR showed the highest extraction recovery using acetonitrile as shown difference between the predicted R2 and adjusted R2 values were found
in Supplementary material Table 2SM. Thus, a mixture of both

Fig. 2. The chromatograms of the four cited drugs and IS using the adopted chromatographic conditions.

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S.S. Saleh et al. Journal of Chromatography A 1724 (2024) 464901

to be less than 0.2. The high magnitudes and fair concord between both 3.3.2. Linearity and range
values confirm better models’ prediction and no over fitting [25,28]. The linear calibration plots were constructed over the range
The adequate precision showed higher values than 4, which implies a (50–1000 µg/Kg) for SQX and (50–750 µg/Kg) for SDM, VK3, and DVR.
high signal-to-noise ratio. Lack-of-fit was calculated to be insignificant. The zero tissues (spiked with IS) were compared to the blank tissues
Good model fitting was confirmed by the fit statistical values listed in (with no IS) to affirm the lack of interference from tissue samples or with
Table 1. the four studied drugs. The results were of deviations were acceptable
The CMPs interactions and their effect on the tested CQAs are shown (±20 % for LLOQ and ±15 % for HQC, MQC and LQC). The regression
in Fig. 3. The impact of (C: sonication time) is negligible on SDM %, SQX parameters are listed in Table 2. The limit of detection was found to be
% and DVR %. Both SDM % and SQX % were affected by the ratio of (A: equal to 14.08, 16.26, 15.01, and 13.00 µg/Kg for SDM, SQX, VK3, and
ACN %) and (B: METH %), where the highest recovery percentages were DVR, respectively.
observed towards adjusting the ratio towards (A:B 30:70 v/v) as shown
in Fig. 3a and Fig. 3b. On the other hand, for DVR %, the highest re­ 3.3.3. Accuracy and precision
covery percentages were observed towards adjusting the ratio towards For the evaluation of accuracy and precision, four concentrations of
(A:B 70:30 v/v) as shown in Fig 3c. The recovery of VK3 (VK3 %) is each of the four drugs (LLOQ, LQC, MQC and HQC) were analyzed intra-
notably impacted by the interaction between (A:B) and (C) where the day and inter-day using six replicates. The intra-day accuracy was
highest recovery percentages were observed towards adjusting the ratio measured for the selected concentrations of each drug and measured
towards (A:B 30:70 v/v) at higher sonication time as shown in Fig 3d. through 24-hour intervals, while the inter-day accuracy was measured
The criteria of optimization were illustrated by maximizing the four through three-day intervals. Accuracy is measured as recovery %, while
assigned CQAs for the best extraction efficiency by setting the ratio precision is measured as RSD % which is calculated as the percentage of
between (A: ACN %) and (B: METH %) to average at higher sonication the ratio between the standard deviation and the mean value. RSD %
time as shown in Supplementary material Fig. 2SM. Upon applying should be ≤ 15. The accuracy and precision values are listed in Table 3.
derringer’s desirability algorithm, the optimum CMPs were advised to
be (A:B 49.8: 50.2 v/v) and 10 min of sonication with a desirability value 3.3.4. Robustness
of (0.706) as shown in Supplementary material Fig. 3SM. The RSM The robustness was checked by performing the chromatographic
validation was done by triplicate determination for the optimized con­ separation under altered conditions such as small changes in the ratio of
ditions, where the actual recovery % of the four drugs are compared to acetonitrile: 0.1 % formic acid (from 85: 15 to 80: 20 and 90: 10, v/v) in
the predicted ones as shown in Fig. 4. Low error values of 1.11 %, 1.2 %, the eluent retention times of the eluted peaks showed minimum alter­
0.83 %, and 0.76 %, were observed for SDM %, SQX %, VK3 %, and DVR ation, but the peak areas were reserved.
%, respectively. The chromatograms of the real samples and blank
samples used in prediction testing are shown in Supplementary ma­ 3.3.5. Matrix effect
terial Fig. 4SM. To evaluate the potential matrix effect, six different blank tissue
samples at both the low-quality control (LQC) and high-quality control
3.3. Method validation (HQC) levels were compared. The objective was to compare the peak
areas in the post-extracted samples with those of tissues spiked with
The proposed method was validated according to the FDA bio­ standard drugs and the internal standard (IS). The results showed that
analytical method validation protocol [24]. The quality control samples the ionization of the four mentioned drugs and the IS in the ion source
of SDM, DVR, and VK3 were prepared in the concentrations (50, 150, remained unaffected by any components present in the co-eluted tissue
350, 600 µg/ Kg), and (50, 200, 500, 800 µg/ Kg) for SQX as LLQC, LQC, matrix. Additionally, the data presented in Table 4 affirm the efficacy of
MQC, and HQC samples. the sample extracting procedure in eliminating any potential interfer­
ence originating from the tissue matrix.
3.3.1. Selectivity
To study selectivity, six blank samples and spiked tissues with the 3.3.6. Extraction recovery
four drugs and the IS (100 µg/Kg) were compared. No intervention was To evaluate the extraction recovery of the four drugs, the peak areas
detected regarding the retention time. The background noises of the in pre-extracted samples were compared to the peak areas in post-
responses of LLOQ and IS were found to be less than 20 % and 5 % extracted samples at both (LQC) and (HQC) levels. This comparison
respectively. was performed using six replicates. The recovered amounts obtained
from this analysis indicated the effectiveness of the extraction proced­
ure. The specific results of this comparison are presented in Table 4.

Table 1
ANOVA and Fit statistics of the I-optimal design.
Source SDM% SQX% VK3% DVR%

F-value p-value F-value p-value F-value p-value F-value p-value

Model 82.64 < 0.0001 87.59 < 0.0001 89.00 < 0.0001 137.39 < 0.0001
Linear mixture ** 127.13 < 0.0001 142.76 < 0.0001 193.71 < 0.0001 137.39 < 0.0001
AB 38.15 < 0.0001 32.41 < 0.0001 64.15 < 0.0001 —- —-
AC —- —- —- —- 6.12 0.0309 —- —-
Lack of Fit 0.99 0.3761* 1.23 0.4826* 1.54 0.3955* 3.86 0.1467*
R2 0.9323 0.9359 0.9604 0.9136
R2 (adjusted) 0.9210 0.9252 0.9496 0.9069
R2 (predicted) 0.9087 0.9084 0.8958 0.8842
Adequate precision 18.517 18.944 23.005 23.0867
Coefficient of variation (C.V) 7.56 6.08 5.38 4.88

*Non-significant.
** The test for the linear mixture terms of the Scheffé polynomial model compares the linear coefficient estimates to each other rather than comparing the coefficients
to zero.

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S.S. Saleh et al. Journal of Chromatography A 1724 (2024) 464901

Fig. 3. 3-D counterplots of I-optimal design factor interaction for CQAs: (a) SDM %, (b) SQX %,(c) VK3 %, and (d) DVR %.

Fig. 4. Mix-process 2D plot of Desirability and Prediction of the four CQAs versus CMAs.

3.3.7. Stability studies tissues, the recovery percentage ± relative standard deviation (RSD)
Drug stability in biological tissues can be influenced by various was determined for three separate determinations at both the low- and
factors, including storage conditions, chemical properties of the drug, high-quality control (QC) levels. The results presented in Supplemen­
and the drug container. To evaluate the stability of the drugs in chicken tary Material Table 4SM provide evidence of the favorable stability of

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S.S. Saleh et al. Journal of Chromatography A 1724 (2024) 464901

Table 2 study aimed to examine various tissues, including muscle (breast and
Validation parameters for the proposed UPLC-MS-MS method for the four drugs thigh) and liver, for the presence of residues from SDM, SQX, VK3, and
in chicken tissues. DVR.
Parameters SDM SQX DVR VK3 To determine the residues of these drugs, a UPLC-MS-MS method was
Concentration range (µg/Kg) 50–750 50–1000 50–750 50–750
successfully employed on the tissues (breast, thigh, and liver) obtained
Slope 0.1125 0.0196 3.558 27.866 from the initial group of 50 chickens that received the veterinary
Intercept − 3.472 − 0.8898 − 203.22 6197.3 formulation Coccimix®. The objective of the study was to monitor the
correlation coeff "r" 0.9975 0.9983 0.9967 0.9983 levels of these four drugs’ residues over a span of 15 days and identify
SE intercept 0.5046 0.0971 14.02 126.7618
when they reached their maximum residue limits (MRLs). As per the
Limit of detection LOD (µg/Kg) 14.8 16.26 13 15.01
Limit of quantitation LOQ (µg/ 44.85 49.28 39.4 45.49 guidelines set by Codex Alimentarius, the reported MRLs for SDM and
Kg) SQX are 100 µg/kg, while for DVR, it is 50 µg/kg. As for VK3, no MRL has
Mean 101.27 102.94 105.25 97.04 been established, as it is considered safe for consumption at any con­
Standard deviation (±S.D) 3.84 11.73 14.88 10.24 centration. The determination of the cited antibiotics was performed
accurately, without interference from the tissue matrix. Additionally,
the study investigated the impact of heating and freezing on the levels of
Table 3 antibiotic residues.
Intra- and inter-day accuracy and precision for the determination of SDM, SQX, The declines curves were constructed by plotting the residues’ con­
DVR, and VK3 in chicken tissues. centrations obtained from the UPLC-MS-MS analysis on the y-axis, while
Studied QC Level Intra-day, n = 6 Inter-day, n = 6 × 3 the time elapsed after stopping the administration of the formulation
drugs
Accuracy RSD Accuracy RSD
was plotted on the x-axis (the post-administration days of Coccimix).
% % % % The rate of decline in growing tissue was observed. The decline in
concentration of the drugs SDM, SQX, and DVR obeys the third order,
SDM LLQC (50 µg/Kg) 104.22 2.44 108.44 1.66
LQC (150 µg/Kg) 101.44 4.11 104.55 5.78 and can represented via polynomial equations (y2 = axe2 + bx + c),
MQC (350 µg/ 105.55 4.66 105.66 3.55 while the decline in concentration of VK3 obeys first order with a linear
Kg) equation (y = axe ± b). the decline curves for the four drugs are shown
HQC (600 µg/ 103.82 5.69 99.54 5.66
in Fig. 5.
Kg)
SQX LLQC (50 µg/Kg) 101.33 7.55 102.22 3.66
It was found that the four drugs showed the highest initial concen­
LQC (200 µg/Kg) 103.44 4.76 98.55 8.44 tration in breast tissues. For SDM and SQX, liver tissues needed the
MQC (500 µg/ 104.44 5.44 106.4 4.33 longest time compared to other tissues (breast and thigh) for residual
Kg) amounts of SDM, SQX, and DVR to reach their MRLs. SDM reached its
HQC (800 µg/ 106.54 2.47 99.55 5.09
MRL within 3.5 days in the thigh, 3.7 days in the breast, and 4.5 days in
Kg)
DVR LLQC (50 µg/Kg) 102.33 5.66 99.65 4.98 the liver. SQX reached its MRL within 5.6 days in the thigh, 5 days in the
LQC (150 µg/Kg) 105.33 3.11 100.45 2.44 breast, and 6 days in the liver. DVR reached its MRL within 6 days in the
MQC (350 µg/ 100.44 5.32 103.11 3.66 thigh, 6.4 days in the breast, 6.2 days in the liver. For VK3, it was proved
Kg)
that its concentration declines with time in a linear manner. We
HQC (600 µg/ 99.54 4.14 100.44 2.99
Kg)
recommend and advise that the labeled dose of coccimix® (1 g/L water)
VK3 LLQC (50 µg/Kg) 102.44 3.21 105.33 3.44 be used for 5 consecutive days, and that no poultry be slaughtered until
LQC (150 µg/Kg) 99.54 4.21 100.43 6.44 passing 7 days from the last dose of the treatment.
MQC (350 µg/ 102.44 3.44 101.66 2.87
Kg)
HQC (600 µg/ 101.33 5.22 102.44 2.76
3.5. Risk assessment method
Kg)
The amount of a chemical substance that a person can consume each
*Accuracy was expressed as the Recovery % and intra- and inter-day precision
day without experiencing negative health effects is known as the
was expressed as a percentage of relative standard deviation (RSD %).
acceptable daily intake, or ADI [29]. which are based on NOAELs (No
Observed Adverse Effect Level) and related to safety factors that account
Table 4 for population variances. The ADI of SDM and SQX were decided by the
Matrix effect and extraction recovery percentages. Australian Pesticides and Veterinary Medicines Authority to be 0.02 and
0.01 (mg/Kg/bw/day), where bw is the average body weight of a certain
Concentration µg/Kg Matrix effect% Extraction Recovery%
population. DVR and VK3 have no reported ADI.
SQX 50 70.93 % 81.53 %
This study evaluates the safety of animal and poultry products by
800 64.76 % 86.82 %
SDM 50 61.21 % 85.63 % utilizing the mean value of the Index of Food Safety (IFS), in compliance
600 64.70 % 95.41 % with the guidelines and protocols established by the Codex Alimentarius
DVR 50 73.15 % 87.64 % Commission for risk evaluation of contaminants in foods. The food
600 78.21 % 95.50 % safety index (IFS) of both sulfonamides is calculated by the following
Vit K3 50 79.26 % 79.78 %
equation:
600 74.71 % 91.28 %
IFS = Σ (R x F x f ) / (SI x bw)
the four mentioned drugs in chicken tissues, as all the data obtained fell Where R is the level of a sulfonamide residue in a tissue expressed as
within acceptable limits. (μg/kg); F is the daily intake of poultry product per capita expressed as
(kg/person/day); bw is the average body weight, expressed as kg; SI is
3.4. Decline curve study the safe intake is adopted as the ADI for a correction factor (f = 1). The
IFS is calculated for both SDM and SQX at the assigned day for each drug
This study focused specifically on Coccimix®, one of the most used residue to reach MRL in the selected three tissues (breast, thigh and
poultry formulations in Egypt. The chickens under investigation were liver) related to Egypt’s reference values. If the IFS value is less than 1, it
treated with this formulation, administered at the recommended dose indicates that the risks associated with the sulfonamides may not be as
specified by the manufacturer, Pharma Swede Veterinary Company. The harmful to human health as previously thought. Conversely, if the IFS

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S.S. Saleh et al. Journal of Chromatography A 1724 (2024) 464901

Fig. 5. Decline curves study for the four drugs (SDM, SQX, DVR, VK3) in the three studied tissues (breast, thigh, and liver) showing the time in days to reach
MRLs values.

value is greater than 1, it indicates that the risks associated with the 3.6. Freezing and heating effects
sulfonamides are too great to be ignored and that risk control measures
need to be implemented [30]. All the IFS values listed in Supplemen­ It was observed that freezing at − 20 ◦ C for 15 days decreased the
tary material Table 5SM were found to be less than 1 at the 7th day of amounts of SDM, SQX, and DVR by 13.44 %, 15.12 %, and 11.23 % from
administration, thus risk of sulfonamide residues in Egypt could be their initial residue, while VK3 residues decreased by 3.11 % only. The
ignored. least decrease in the concentration of VK3 upon freezing could be
attributed to the nature of its molecules as soluble organic soluble, so
they linked to molecules of protein during freezing and after thawing

Table 5
Summary of the applied greenness tools.
AES

Studied factor Description Sub-total Pps Score Total

Methanol Quantity (2) 12 Σ Pps ¼ 31

Hazardous (3 pictogram) Danger (2) AES:
Acetonitrile Quantity (2) 8 100 -31 ¼ 69
Hazardous (2 pictogram) Danger (2)
Formic acid Quantity (1) 1
Danger (1)
Heating Less than 1 h 2
Consuming energy 1.5 kWh per sample 2
Waste Between (1–10) mL 3
No dealing 3

GAPI AGREE AGREEprep

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S.S. Saleh et al. Journal of Chromatography A 1724 (2024) 464901

even after protein denaturation. On the other hand, placing in hot water environment to close the exclusion gap created by the GAC. However,
for 15 min reduced the residue levels of SDM, SQX, VK3, and DVR by the analytical procedures that are completed before and after sample
60.14 %. 46.33 %, 71.11 % and 72.43 %, respectively. These results preparation are covered by the first and ninth principles, respectively.
reflect the higher safety of poultry products after cooking procedures Most of these ideas have a direct bearing on how samples are prepared.
that includes immersing in boiling water for at least 15 min that make The ten GSP tenets guide the development of more environmentally
their usage safe even before the passage of period of 7 days from last friendly analytical methods, but they do not evaluate the sample’s
dose administration. environmental impact.
Due to this reason, a recently recommended measuring tool called
3.7. Greenness assessment AGREEprep [39] has emerged, which focuses specifically on sample
preparation. It represents the first reported measure that concentrates on
3.7.1. Analytical eco-scale (AES) metric this aspect. Alongside assessing the level of environmental friendliness,
Green chemistry evaluated procedures by considering factors such as AGREEprep evaluation can effectively identify both strengths and
the quantity and type of solvents as well as the quantity of waste weaknesses in the sample preparation process, thereby promoting more
engendered throughout the analytical process [31]. AES greenness tool eco-friendly practices. The scores assigned to each of the ten evaluation
was employed in this study [32]. Using a mathematical formula, this periods in AGREEprep span from 0 to 1, with the highest and lowest
tool calculates a procedure’s score in terms of penalty points. Sub­ scores indicating the best and poorest achievements, respectively. These
tracting the total number of penalty points from 100 yields a result that individual criterion scores are then weighted and combined to generate
indicates the process’s environmental friendliness, where a score ≥ 75 an overall aggregate score, also ranging from 0 to 1, where a score of 1
indicates "excellent" performance, a score ≥ 50 suggests "acceptable," represents optimal performance. As indicated in (Table 5), the proposed
and a score < 50 indicates "inadequate" [33,34]. For the proposed method achieves an AGREEprep score of 0.58.
technique, the results of AES computations are shown in (Table 5). Ac­
cording to the AES evaluation, the approach received a score of 69, 3.8. A competence balance point via evg framework
suggesting that it is an acceptable green method.
The three pillars of the EVG framework are efficiency, validation,
3.7.2. Green analytical procedure index (GAPI) and greenness [40]. An average score is assigned to each pillar made up
With GAPI, a distinct symbol made up of five pentagrams can be used from the five evaluation criteria (A-E) listed under each pillar, (3) being
to evaluate and quantify the environmental impact connected to each the greatest score and (zero) being the lowest score as shown in Sup­
stage of an analytical process. Three color codes employed by GAPI are plementary material Table 7SM. A radar chart can be used to assess
red, yellow and green from the highest to the lowest risk, respectively how well the three pillars are balanced and to make sure that the sug­
[35]. The goal of the GAPI tool is to evaluate analytical processes in line gested approach has been constructed with the greatest adjustment
with Green Analytical Chemistry’s (GAC) in 15 points [36,37]. possible between them to create an eco-friendly, dependable, and
According to the findings presented in Table 5, the proposed tech­ effective process that in turn can be displayed as a competency profile
nique has a GAPI index consisting of 1 green and 9 yellow pentagrams, for the proposed method. Each pillar’s score would fall into one of four
along with 5 red pentagrams. An off-line sample collection was quartiles (Q1, Q2, Q3, or Q4), which are organized descendingly from
described in (point 1), preservation under specific conditions, storage the greatest scores to the lowest. By calculating the mean scores for the
and transportation of the samples is required in (points 2, 3 and 4). three pillars are compared to determine whether the proposed method
Furthermore, an extraction process is required for these samples at the shows a balancing point among the three pillars. Otherwise, recom­
microliter scale in (points 5 and 6). The proposed approach utilizes re­ mendations for modifications are proposed.
agents and solvents, which is demonstrated by the specific solvent used By assessing the proposed method via EVG framework, the method’s
(point 7). In point 8, it is necessary to eliminate the solvent, so additional efficiency pillar fitted the first quartile (Q1), due to applying response
treatment is required. In the suggested approach, the solvent quantity surface methodology (I-optimal) for optimizing the extraction process
(point 9) was carried out using 10–100 mL. Points 10 and 11, which which is considered the rate-limiting step in this method. The I-optimal
discuss flammability and health risks, indicated that the suggested design studied a considerable number of CQAs and CMPs which
approach is associated with a moderate level of risk, as ACN has a NFPA increased the efficiency of the process. In addition, the efficiency was
health hazard value of 2 and both methanol and ACN have a NFPA enhanced by shortening the runtime (< 3 min) and analyzing 4 analytes
flammability hazard value of 3. According to (point 12), the suggested in a single run which minimizes the cost. The validation pillar belongs to
procedure resulted in higher energy consumption. No occupational second quartile (Q2) with an average score of (2), likewise the greenness
hazards are present in (point 13). The waste generation (point 14) is pillar belongs to second quartile (Q2) with an average score of (1.6) as
between 1 and 10 mL of waste. According to point 15, no waste treat­ shown in Table 6. The three pillars’ pertinence to the same or close
ment was performed. quartiles suggests that the method has achieved a balance point of
competence. By applying the EVG competence tool to the reported
3.7.3. AGREE metric methods [22,23], both method fitted in lower quartiles and a poor
The AGREE software [38] features a circular clock-like pictogram balance point was achieved as shown in Table 6. An electronic supple­
showing 12 numerals, each symbolizing a distinct GAC principle. These mental information (ESI) is supplied for constructing the EVG radar
principles are graded on a scale from 0 (red color, concept not met) to 1 chart.
(dark green, concept met). The central score within the AGREE symbol Hence, the proposed method showed advantages over the reported
represents the average numerical value that was obtained from the 12 ones in terms of efficiency due to the application design of experiment in
data points. The color of each numeral indicates its outcome (Supple­ the method’s development and optimization, lower limits of quantita­
mentary material Table 6SM).. The recommended UPLC-MS-MS tion (LOQs) and standard errors (SE) which indicated higher sensitivity
method’s greenness is confirmed by the suggested method’s AGREE and accuracy of the proposed method. In addition to the robustness
score of 0.6, as indicated in Table 5. testing that was carried out for the proposed method only, and that
confirmed its reliability. And finally, all these advantages qualified the
3.7.4. Analytical greenness tool for sample preparation (AGREEprep) proposed method to be applied for the decline curve study in real
Sample preparation was crucial to our investigation; hence it was chicken samples. A suggestion is made to enhance the greenness pillar of
imperative to evaluate this phase. Green Sample Preparation (GSP) was the proposed method by employing less energy with solvent-free or
recently expanded into ten principles to safeguard human health and the greener solvents such eutectic or as ionic liquids for extraction process.

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 S.S. Saleh et al.
Table 6
A comparative study of the proposed vs reported method using the competence tool EVG radar chart.
Proposed method Reported method [22] Reported method [23]
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S.S. Saleh et al. Journal of Chromatography A 1724 (2024) 464901

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