(2024) 18:151
Hussein et al. BMC Chemistry
https://doi.org/10.1186/s13065-024-01255-7

BMC Chemistry

RESEARCH

Open AccessEco‑conscious potentiometric sensing:a multiwalled carbon nanotube‑based platform
for tulathromycin monitoring in livestock
products
Omnia G. Hussein1*, Hany H. Monir1, Hala E. Zaazaa1 and Maha M. Galal1

Abstract
Tulathromycin (TUL) is a widely used veterinary antibiotic for treating bovine and porcine respiratory infections. Consuming animal-derived food contaminated with this medication may jeopardize human health. This work adopted
the first portable potentiometric platform for direct TUL sensing in pharmaceutical and food products. The sensor
employed a plasticized PVC membrane on a glassy carbon electrode doped with calix[6]arene and multi-walledcarbon nanotubes (MWCNT) in a single solid contact layer for selective binding and signal stability. Characterization
via scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) confirmed the material’s
integrity. The MWCNT-based sensor produced a stable Nernstian response (1.0 × ­10−7 to 1.0 × ­10−3 M) and a limit
of detection (LOD) of 9.76 × ­10–8 M with instantaneous response (8 ± 2 s). IUPAC validation revealed high selectivity
for TUL against interfering ions, minimal drift (0.6 mV/h), and functionality over a broad pH range (2.0–7.0), allowing
direct application to dosage form, spiked milk, and liver samples. Eco-Scale, AGREE, and Whiteness assessment proved
the method’s ecological sustainability, economic viability, and practical feasibility, surpassing traditional approaches.
Keywords Ion-selective electrodes, Tulathromycin, Multiwalled carbon nanotube, Potentiometric sensor, Whiteness,
Livestock products, Veterinary drug residues, Food analysis*Correspondence:
Omnia G. Hussein
omneia.gamal@pharma.cu.edu.eg
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
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regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Hussein et al. BMC Chemistry(2024) 18:151

Page 2 of 16

Graphical Abstract

Introduction
The contamination of animal-derived foods with veterinary drugs, particularly antibiotics, has emerged as amajor concern for human health. The excessive use ofantibiotics and the lack of attention to antimicrobial
withdrawal times can lead to the accumulation of drug
remnants and their toxic metabolites in milk, meat, andother edible tissues [1]. One of the most critical consequences of these residues is the transmission of antibiotic-resistant bacteria, posing a profound threat to publichealth [2].
Tulathromycin ­(C41H79N3O12, TUL) (Fig. S-1) is abroadly utilized, long-lasting macrolide that belongs to
the triamilide subclass. It is approved for treating respiratory infections in swine and cattle and is often administered to goats off-label [3, 4]. TUL is available as asterile aqueous solution, characterized by expansive tissue distribution and a slow excretion half-life of 4–6 days,
resulting in persistent drug concentration in the lungs[5]. Due to minimal metabolism, the unchanged drug
predominates in edible tissues, urine, feces, and bile oftreated animals. Notably, the liver accumulates the highest levels of TUL residues, making it a key matrix forinvestigation [6].
Given its persistence and capacity to accumulate in tissues (LogP 3.8), TUL poses formidable risks to humanhealth and aquatic ecosystems [7, 8]. To ensure foodsafety, the European Union and other nations haveestablished regulations governing TUL’s maximum residue levels (MRL) [9]. Nevertheless, farmers continue toengage in inappropriate practices, such as administering
antibiotics with incorrect doses or durations while overusing them for routine livestock management [10]. Consequently, acceptable thresholds of TUL are exceeded,causing adverse effects on consumers’ well-being, including the potential development of allergies in hypersensitive people and the emergence of antibiotic-resistantpathogens [11]. Therefore, there is an urgent need for
rapid and effective monitoring techniques for suchcompounds.
The analysis of TUL in biological matrices has heavily
relied on chromatographic techniques, particularly liquid
chromatography coupled with tandem mass spectrometry (LC–MS/MS) methods [4, 12–20]. Despite theirhigh sensitivity and specificity, these methods suffer fromsignificant limitations, entailing costly equipment, highenergy consumption, the requirement for well-trained
staff, extended analysis time, and arduous sample purification and preparation steps. On top of that, the need for
expensive and environmentally detrimental organic solvents and the generation of toxic waste can restrict theirpracticality in routine analysis.
In contrast, electrochemical sensors, specifically solidcontact ion-selective electrodes (SC-ISEs), represent a

Hussein et al. BMC Chemistry(2024) 18:151

cutting-edge analytical realm with miniaturized, simple,
mobile, eco-friendly, and affordable devices [21]. These
allow for speedy, accurate, and reproducible online monitoring of small volumes of samples with minimal pretreatment steps and thus have garnered attention acrossvarious scientific fields with prospective uses in medicine, environmental monitoring, and food analysis [22–24]. Based on the existing literature, no potentiometricsensor has been proposed for analyzing TUL. While anelectrochemical voltammetric method was reported for
determining TUL in pork samples [25], potentiometricsensing distinguishes itself by its passive operation that
does not involve any external driving forces or redox
reactions, low power, and hardly any sample consumption compared to the voltammetric approach [26, 27].
These attributes hold particular significance when thesample volumes are restricted; and the analyte concentration is low [28].
The optimization of the ion-selective membrane(ISM) begins with creating recognition sites capable of
selectively binding the target ions in what is known as“host–guest chemistry” [29]. This study investigated two
prominent classes of supramolecular ionophores: modified cyclodextrins and calixarenes, renowned for theircapacity to form stable inclusion complexes with theanalyte [30]. Incorporating nanomaterials has also revolutionized the development of electrochemical sensors, offering remarkable advancements in sensitivity,
selectivity, and operational efficiency [31–34]. Carbon
nanotubes, particularly multi-walled carbon nanotubes(MWCNT) and their nanohybrids, have served as thefoundation for highly sophisticated sensing devices capable of detecting trace analytes across diverse domains
such as medicine, food, agriculture, forensic and environmental research [35–39]. The distinctive structure of
MWCNT imparts unique features to traditional carbonaceous electrodes, such as vast active surface area, exceptional electrical conductivity, rapid and efficient chargetransfer with minimal resistance, signal amplificationand stabilization, electrocatalytic properties, reductionof ISM overpotential, in addition to increased durability
with chemical, light and thermal resistance [40]. This has
led to a new generation of miniaturized sensors exhibiting high selectivity and lower detection limits. Thesesensors also eliminate the potential instability and irreproducibility associated with the formation of an aqueous layer, widely acknowledged as the major limitation of
SC-ISEs [40–42]
For the first time, in this work, we aim to develop an
eco-conscious, cost-effective, readily used solid-contact
potentiometric sensor for accurate and sensitive determination of TUL in pharmaceutical formulations andanimal food samples (bovine liver and milk). Central

Page 3 of 16

to our approach is exploring the synergistic potential
of CX-6 and MWCNT within the sensor’s membrane,
aiming to optimize performance and enhance analyticalcapabilities.

Experimental
Instruments

An Adwa pH bench meter (AD1020 pH/mV/ISE/Temperature, Hungary), equipped with a glassy pH electrode(AD1131B, Hungary) and a double-junction Ag/AgCl
reference electrode with an inner filling of 3.0 M KCl
solution and outer filling of 10% ­KNO3 (Thermo Fisher
Scientific, MA, USA), was set up for pH and potential measurements. A working glassy carbon electrode(GCE) (Ø 3 mm, CHI104) was bought from CH Instruments, Inc. (Texas, USA). ­WiseStir® Magnetic Stirrer(DAIHAN Scientific, Seoul, Korea) was also utilized during experiments.
Materials and reagents

Tulathromycin analytical standard was kindly provided
by Pharma Swede (10th of Ramadan City, Egypt) with acertified purity of 97.35%. D­ raxxin® injectable solution,stated to contain 100 mg of TUL per mL, was manufactured by Zoetis Belgium (Batch No. 548396). Deionized double-distilled water and pure analytical grade
chemicals, solvents, and reagents were utilized. Polyvinyl chloride (PVC, high molecular weight), tetrakis(4chlorophenyl)boron
potassium ≥ 98%(KTCPB),
1-nitro-2-(n-octyloxy)benzene 98% (NPOE), dibutyl
sebacate ≥ 97% (DBS), tritolyl phosphate 90% (TTP),
dioctyl phthalate ≥ 99.5% (DOP), dibutyl phthalate 99%(DBP), calix[6]arene 97% (CX6), (2-hydroxypropyl)B-cyclodextrin (average ­Mw ~ 1,460) (β-CD), calix[4]arene-25,26,27,28-tetrol 95% (CX4), 4-tert-butylcalix[8]arene ≥ 90% (tBu-CX8), tetrahydrofuran ≥ 99.9% (THF),
multi-walled carbon nanotube powder (MWCNT)(≥ 98% carbon basis, O.D. × I.D. × L 10 nm ± 1 nm × 4.5
nm ± 0.5nm × 3- ~ 6 μm, TEM) was supplied by SigmaAldrich (Germany); glacial acetic acid 100% and sodiumhydroxide pellets were provided by Merck (Germany),
boric acid 85% from El-Nasr Chemicals Co., Egypt, andorthophosphoric acid (abt. 85% LR) from S.D.Fine-Chem
Ltd., India.
Solutions

A stock standard solution of TUL (1.0 × ­10–2 M) was
prepared by dissolving 80.6 mg of TUL in BrittonRobinson buffer (BRB) with pH (4.0 ± 0.2) into a 10-mL
measuring flask. From this solution, precise aliquots
were withdrawn to prepare a working series of dilutions(1.0 × ­10–9–1.0 × ­10–3 M) utilizing the same buffer. A
stock MWCNT suspension (1.0 mg m
­ L−1) was obtainedHussein et al. BMC Chemistry(2024) 18:151

by sonicating MWCNT in THF for at least 30 min. Prior
to each use, the suspension was sonicated for 10 min toachieve equal dispersion. BRB buffer was made by adding 0.04 M of phosphoric acid and acetic acid to 0.04 M
boric acid, followed by pH adjustment to 4.0 using 1.0 M
NaOH solution.
Fabrication of SC‑ISE sensors

Two sensing membrane cocktails were prepared. Sensor 1 comprised 10.0 mg KTCBP, 10.0 mg of CX6, and190.0 mg PVC mixed with 0.39 mL of NPOE plasticizer,
all in 4.0 mL THF in a glass tube. For sensor 2, 0.36 mL
of MWCNT suspension was introduced to 1.0 mL of thepreviously prepared cocktail and sonicated for 20 min toattain a homogenous suspension, following establishedreports [43, 44].
The GCEs were initially polished using 0.3 µm A
­ l2O3slurry, washed, sonicated in distilled water for 5 min toremove alumina traces, and then dried at room temperature. Fifteen µL of each sensing mixture was drop-casted
on the surface of the polished GCEs and allowed to dry
overnight. All sensors were preconditioned by soaking in
1.0 × ­10–4 M TUL solution for 2 h before calibration.
Potentiometric measurement

For the calibration of TUL sensors, each electrode,coupled with the silver chloride reference electrode, was placed into working standard solutions(1.0 × ­10–9–1.0 × ­10–3 M) and stirred to equilibrate. Uponreaching a steady response, the potentiometer’s reading
was taken within ± 1 mV. After each measurement, themembranes were rinsed with BRB buffer. The obtained
potential (E, mV), which refers to the galvanic potential
difference recorded between the ion-selective GCE electrode and the Ag/AgCl reference electrode, was graphed
in relation to the logarithmic concentration values of
TUL solutions to construct calibration curves.
The regression equation was derived from the linear part of the calibration curve, and the detection limit(LOD) was determined at the crossover point of extrapolated lines at the lower concentration segment of thecurve.
The method’s accuracy was verified by determining five distinct concentrations of the TUL standard;
the method’s precision was validated by repeating themeasurements of three distinct concentrations of the
TUL standard three times in a single day to evaluate therepeatability and on three consecutive days to assess themethod’s intermediate precision.
Sensors’ selectivity

We employed the separate solutions method (SSM) to
examine the selectivity of the two suggested sensors forPage 4 of 16

TUL with the occurrence of interfering ions [45]. The
study investigated common ions and pollutants possible
in targeted matrices, including K­ +, ­Na+, ­Ca2+, and ­Mg2+,
as well as some compounds structurally related to TUL.
The ability of sensing membranes to distinguish between
the primary ion of interest and other ions with the samecharge sign was calculated and expressed as the logarithm of the selectivity coefficient:potlog(K primaryion,interf erent ) =(EI − ETUL )ZA F
2.303RT

where EI and ETUL are the potential readings (mV) in 1
­ 0–3
M solution of interferent species and primary ion (TUL),
respectively, 2.303RT/ZAF corresponds to the slope of thesuggested sensor (mV/decade).
Effect of pH and the dynamic response time

The impact of pH changes within the range (2.0–10.0)
on recorded potential values was examined by gradually introducing tiny portions of 1.0 M NaOH solution to
1.0 × ­10–3 M and 1.0 × ­10–4 M TUL solutions initially prepared at pH = 2.0 and measuring the electromotive force(emf ) at each pH value.
To determine the practical response time, the durationneeded to attain a stable potential reading (± 1 mV) after
the change of TUL concentration was recorded.
Potentiometric determination in pharmaceuticalformulation

A quantity equivalent to 100 mg TUL (1.0 mL) was accurately transferred from D­ raxxin® Injectable solution (100−1
mg. ­mL ) into a 50-mL measuring flask and completedwith BRB buffer (pH 4.0). This solution was then dilutedby the same buffer to achieve final TUL concentrationsof 4.96 × ­10–5 M and 4.96 × ­10–4 M. The optimized sensor
was immersed in the prepared solutions, the emf measurements were recorded, and the regression equation
was employed to find the respective concentration.
Potentiometric determination in spiked milk and liver

Fresh liver and pasteurized milk samples were purchasedfrom a local butcher and supermarket in El Sayeda Zeinab
district of Cairo Governorate. One gram of chopped liver
was accurately weighed, spiked with a known amountof TUL, and then homogenized in 25 mL BRB buffer(pH = 4.0). For milk samples, a volume of 2.5 mL wastaken into 25 mL of BRB buffer without further treatment. The samples were spiked to achieve concentrations ranging from 1.0 × ­10–4 to 1.0 × ­10–6 M, below andabove TUL specified MRL. Direct potentiometric measurements were carried out using the optimized sensor as
previously described while swiftly alternating between
spiked samples and distilled water during the experiment

Hussein et al. BMC Chemistry(2024) 18:151

to prevent biological matrix components from clinging to
the sensing surface. Five replicates of each concentration
were analyzed, and an un-spiked (blank) sample served
as a control.
Calibration graphs were generated by establishing acorrelation between the recorded emf values and thelogarithm of molar concentrations of TUL. The potential
values for each sample were noted, and the found concentrations of TUL were computed using the respective
regression equation.

Results and discussion
In response to the rising demand for feasible analyticalmethods to detect antibiotics in various food products,
we focused on developing a reliable and cost-effective
sensor for the targeted analysis of TUL macrolide in different matrices while considering environmental safety
and sustainability standards.
Initially, we attempted to create a sensing membrane
cocktail with optimal binding affinity for TUL by screening various ion exchangers, plasticizers, and ionophores.
We then exploited the excellent electrochemical conductivity of MWCNT to stabilize the response and improvesensitivity, ensuring compliance with TUL regulations intargeted tissues. The performance metrics of the developed sensors were evaluated based on IUPAC guidelines.
Finally, the optimized sensor was employed to analyze
TUL in dosage form, spiked milk, and liver samples.
Membrane composition and effect of plasticizers

TUL predominantly behaves as a trivalent cation underacidic conditions, Fig. S-1 [46, 47]; therefore, the cationicexchanger KTCBP was paired with an NPOE plasticizer
as the best combination for good baseline potentiometricresponse.
We started by examining the influence of several
plasticizers, namely NPOE, DOP, DBP, DBS, and TTP,on the performance of electrodes. The calibration
charts revealed that the use of NPOE achieved the best
linear response and the highest sensitivity for the membrane, Fig. S-2 NPOE, DBS, and DBP showed betterslopes (20.1, 20.4, and 21.4 mV/decade) than TTP and
DOP (24.0 and 31.1 mV/decade). Meanwhile, NPOE
demonstrated the best correlation coefficient (0.9998)
and lowest LOD (2.38 × ­10–7 M) compared to other plasticizers. These results can be attributed to variations inpolarity/dielectric constants and functional groups of different plasticizers [48].
Ionophore selection

Ionophores are crucial components of ISEs, serving assites for chemical recognition. A strong binding affinity
between the ionophore and target ion forms an inclusion

Page 5 of 16

complex with reduced solvation free energy, facilitating
its transfer from the hydrophilic sample solution acrossthe hydrophobic membrane [49]. This host-and-guest
intermolecular interaction promotes sensor selectivity.
Additionally, the high lipophilicity of the formed complex reduces the leaching of target ions into the aqueous
phase, extending the sensor lifespan [50].
Cyclodextrins and calixarenes are widely recognizedionophores in ISE research. Calixarenes are macrocyclic
molecules with aromatic basket-shaped cavities. These
cavities have the capacity to establish unique interactions with target ions, such as cation-π interactions, π—πinteractions, van der Waals interactions, and hydrogenbonding. The macrocyclic conformation and cavity size
significantly impact host–guest binding and sensing
membrane selectivity [50, 51].
We investigated the recognition capacity of various calixarene derivatives for TUL, namely CX4, CX6,
and tBu-CX8, in addition to β-CD, compared to an
ionophore-free membrane. The sensors’ readings for
TUL were measured against a reference ion, Tetra-npentylammonium bromide ((Pe)4N+)Br− [49]. (Pe)4N+
is a bulky lipophilic ion, incapable of fitting inside thecalixarene molecular cavity, thus helping identify thecalixarene molecule with the highest TUL affinity. The
efficiency of TUL-ionophore binding is maximized
when the emf difference from (Pe)4N+ is minimized. As
illustrated in Fig. 1. CX6-doped ISM achieved the highest binding and the lowest emf differential (247 mV) as
opposed to other ionophores (CX4 291 mV, tBu-CX8 354
mV, and β-CD 346 mV) and the ionophore-free sensor(302 mV). Thus, CX6 was assigned as the ionophore of
choice in later experiments. Fig. S-3 shows a schematicfor the interaction between the TUL ion and the active
sites in the membrane, predicting the occurrence of π—πinteractions and H-bonding between the carbon tail ofthe oxane ring in TUL and the carbon and oxygen centers
in CX-6.
MWCNT incorporation and characterization

Incorporating MWCNT as a transducing layer in ISMs
has been suggested to improve stability, reduce signal drift, and enhance membrane sensitivity [43]. We
adopted a feasible approach, where MWCNT was dispersed into the NPOE plasticized membrane via ultrasonic vibrations without surfactants [44]. We doped thesensing membranes with different ratios of MWCNT(0.21, 0.36, 0.42 mg.mL−1) to investigate the effect on
potential stability and sensitivity. The most promising outcome in terms of slope and linearity range wasachieved at a concentration of 0.36 mg.mL−1, Fig. S-4.
The structural features of MWCNT powder were
examined using FT-IR spectroscopy following its

Hussein et al. BMC Chemistry(2024) 18:151

Page 6 of 16

Fig. 1 The potentiometric response of ISEs containing various ionophores in 1.0 mM TUL solution after subtracting each ISE’s reading to 1.0 mM(Pe)4N+ solution

placement onto potassium bromide (KBr) discs. Figure 2
reveals a characteristic peak at 1636 ­cm−1 that indicatesthe occurrence of the aromatic C=C stretching vibrationin the graphite arrangement of the MWCNT framework.
A five/seven-membered presence at the junction or closure of the MWCNT is responsible for this phenomenon[52]. Bands of symmetric/asymmetric –CH2 methylene
stretching are observed at 2922 and 2852 ­cm−1 [53]. The
spectrum shows strong bands at 3400 ­cm−1, corresponding to the surface–OH stretching vibrations caused byeither atmospheric moisture adsorption or oxidation
during the purification of nanoparticles [54].

Fig. 2 FT-IR spectrum of pristine MWCNT using KBr pellets in the 4000–400 c­ m−1 range

Hussein et al. BMC Chemistry(2024) 18:151

Scanning electron microscopy (SEM) (Quanta FEG
250) was employed to analyze the surface morphology of
MWCNT and the prepared membranes. Figure 3a shows
networks of intertwined nanofibers of raw MWCNT athigh magnification. Before doping with MWCNT, thepolymeric nature of the unaltered membrane with aneven distribution of pores across the surface is displayedin Fig. 3b. The difference after MWCNT inclusion is evident in Fig. 3c2 (30 kV), where white particles encapsulated within the membrane pores appear brighter due todifferences in conductivity. More surface details of the
MWCNT/ISM are observed in Fig. 3c1 (10 kV), revealing the uniform dispersion of nanoparticles within themembrane matrix. This modification in the geometricalsurface of the electrode might create an expanded electrochemical active area for TUL sensing.
Performance characteristics and validation parametersof the developed sensors

We investigated the analytical capabilities of the two
designed sensors, MWCNT-free ISM (sensor 1) versus
MWCNT/ISM (sensor 2), by graphing the emf measurements with respect to the logarithmic concentrationsof TUL solutions in Fig. 4. Then, the calibration parameters were extracted and compared in Table 1 according
to IUPAC recommendations [55]. The sensors’ analyticalcharacteristics demonstrated a significant performance

Page 7 of 16

enhancement following the incorporation of MWCNT.
Results exhibited a wider linearity range and lower detection limit for sensor 2 (1.0 × ­10–3–1.0 × ­10–7 M, LOD
9.76 × ­10–8 M) over sensor 1 (1.0 × ­10–3–1.0 × ­10–6 M,
LOD 1.73 × ­10–7 M). This suggests a ten-fold enhancement in signal quantification upon the addition of
MWCNT. Lower detection limits may be linked to
the role of MWCNT in hindering the formation of
the aqueous layer that can accumulate TUL ions andcause leaching into more diluted concentrations during
measurement.
The calculated percentage mean recoveries and relative standard deviations are within the acceptable limits,verifying the sensors’ accuracy, repeatability, and precision, with slightly favorable values in the case of sensor 2(MWCNT/ISM).
The MWCNT/ISM sensor also displayed superior features, including greater potential stability, lower drift,
faster response time, and longer shelf life, as discussed inlater sections. All these characteristics would be crucial
for the analytical applications of a novel sensor, particularly in biological matrices.
Dynamic response times and sensors’ lifetimes

The dynamic response time curve reflects both the time
the sensor takes to attain equilibrium and the stabilityof the response. The potential-time curve for MWCNT/

Fig. 3 Surface SEM images of a raw MWCNT, b unmodified ISM, and c1,2. MWCNT/ISM

Hussein et al. BMC Chemistry(2024) 18:151

Page 8 of 16

Fig. 4 The plot of change in potential (mV) as a function of logarithmic molar concentrations of TUL employing sensors 1 and 2

Table 1 The electrochemical validation parameters of the designed GCE sensors
Parameters

Sensor 1 (MWCNT-free ISM)

Sensor 2 (MWCNT/ISM)

Slope (mV/decade)

19.98

20.22

Intercept (mV)

256.46286.14

Correlation Coefficient (r)11Linearity Range (M)1.0 × ­10–6–1.0 × ­10–3

1.0 × ­10–7–1.0 × ­10–3

Working pH Range

2–7

2–7

Response Time (Sec.)

18

8

Lifetime (Weeks)

3

7

Accuracy (Mean ± RSD%)b

99.84 ± 0.700

100.23 ± 0.576

Repeatabilityc (%)

1.037

1.011

Intermediate ­Precisionc (%)

1.619

1.486

Robustnessd (%)

1.264

0.986

LOQ (M)

1.0 × ­10–6

1.0 × ­10–7

LODe (M)1.73 × ­10–7

9.76 × ­10–8

aa

Average of three measurementsbMean ± RSD% of recoveries for five concentrations of TUL in triplicatesc

RSD% values for three concentration levels (1.0 × ­10–4, 1.0 × ­10–5, 1.0 × ­10–6 M) of TUL, each repeated three times intraday for repeatability and interday for threeconsecutive days for intermediate precisiond

RSD% values for previous concentrations under minor pH variationse

LOD (detection limit) was determined by intercepting the extrapolated lines of non-responsive and the Nernstian sections in the potential profile

ISM in Fig. S-5 displays an immediate response with satisfying potential stability to alterations in TUL concentration within 3 s at high concentration levels. At lower

concentration levels, the response time increased to 10 s
to stabilize within 1 mV of the steady-state potential. This
results in an average response time of (8 ± 2) seconds forHussein et al. BMC Chemistry(2024) 18:151

sensor 2, in contrast to (18 ± 2) for sensor 1, indicatingthat the inclusion of MWCNT improved both reaction
time and signal stability. Moreover, this instantaneous
response emphasizes the efficacy of ISEs over conventional chromatographic techniques, especially for applications requiring rapid analysis of numerous samples.
Repeated calibrations consistently yielded a reproducible slope of ± 1 mV/decade over a 3-week period for sensor 1, with frequent incidents of membrane cracking andpeeling, in contrast to sensor 2, which maintained stability for seven weeks (Table 1). This proves that the C − Cbonds forming nanotubes in MWCNT impart exceptional strength, flexibility, and resistance to membranerupture. It underscores the role of MWCNT in enhancing the sensor’s durability and broadening its potential
applications.
Effect of soaking time

To explore the impact of preconditioning time on themeasured response, the sensors were immersed in
1.0 × ­10–4 M of TUL for various periods ranging from 1
to 48 h. Interestingly, soaking the sensor for 2 h up to 12
h showed comparable results in contrast to soaking for alonger time, which resulted in an unfavorable decline inslope and detection limit. The observed decline may be
attributed to the leakage of electroactive molecules into

Page 9 of 16

the soaking solution. Therefore, a duration of 2 h was set
to be the optimal soaking time.
Effect of pH

TUL pH-potential profile was evaluated to establish
the optimal operating parameters for the sensors underinvestigation. A steady electric potential prevails over awide pH range of 2.0 to 7.0 for both sensors, as shownin Fig. 5. TUL possesses three primary amine groups that
undergo ionization in acidic conditions, as indicated bytheir dissociation constants ­(pKa of 8.59, 9.62, and 9.90)[47]. This contrasts with an alkaline pH, where the threeamines become less positively charged, explaining thegradual decline in the measured potential with the subsequent increase in pH above 7.5. Therefore, pH 4.0 was
employed during measurements to ensure the complete
ionization of TUL.
Sensors’ selectivity

We assessed the selectivity of both ISE sensors against
potentially interfering ions in biological matrices ofinterest by applying the separate solution method (SSM)[45]. The calculated selectivity coefficients are expressedpot
as (logK T U L.I ), and are presented in Table 2. The values range from -2.99 to -3.83, indicating a high level of
selectivity towards TUL without significant interference

Fig. 5 Potential pH profile of the proposed sensor 1 (MWCNT-free sensor) and sensor 2 (MWCNT/ISE) using 1.0 × ­10–3 M and 1.0 × ­10–4 M TUL
solutionsHussein et al. BMC Chemistry(2024) 18:151

Page 10 of 16

Table 2 The proposed sensor’s logarithmic selectivity
coefficients as determined by the separate solutions method(SSM)
Interferent ­(10–4 M)

pot

logK TUL. I Sensor 1

pot

logK TUL. I
Sensor ­2aNaCl− 2.99− 3.14KCl− 3.00− 3.34− 3.83MgCl2− 3.33CaCl2− 3.19− 3.68Erythromycin− 1.15− 1.25

Clarithromycin− 1.05− 1.45

Ketoprofen− 3.25− 3.20aaverage of three determinations

from inorganic species such as N
­ a+, ­K+, ­Ca+, and ­Mg2+.
The high selectivity for TUL is due to its strong lipophilic properties, which contrasts with inorganic cations
that are highly hydrophilic, thus limiting their permeability and mobility through the hydrophobic membrane[56]. It is also evident that the increased hydrophobicity
after adding MWCNT in sensor 2 contributed to greater
selectivity values relative to the MWCNT-free ISM.
However, the selectivity towards TUL against structurally related molecules such as erythromycin andclarithromycin would be explained differently. Despite
these drugs having comparable lipophilicity to TUL, theobserved selectivity may be attributed to better binding
affinity between the membrane/ionophore active sites

and the TUL molecule. On the other hand, better selectivity values against ketoprofen, a commonly co-administered analgesic drug, can be linked to its acidic nature.
The presence of an anionic center suggests incomplete
ionization at the pH used, thereby reducing its ion
exchange capacity within the membrane. The resulting selectivity offers an advantage when analyzing TUL
in formulations and animal products containing both
medications.
Potential drift and water layer test

The efficiency of SC-ISEs in real-time applications has
been questioned due to potential drifts that compromise repeatability. The build-up of an aqueous layer
between the solid substrate and the ISM has been
deemed a major culprit for such variations in performance [57]. We conducted the water layer test toevaluate the existence of this disruptive film and thelong-term stability of the optimized sensor. The test
involved monitoring potential shifts in 1.0 × ­10–3 M
of TUL solution for one hour, followed by exposure
to a highly concentrated solution of an interferent ion(Diazepam) with similar ionic behavior at the same pH
for another hour before switching back to the primary
ion solution. Figure 6 illustrates the superior stabilityof MWCNT/ISM, highlighting its enhanced resistanceto the influx of interferent ions into any existing aqueous film. Furthermore, the introduction of MWCNT
diminished the drift from 10.6 mV/h in sensor 1 to
0.6 mV/h in sensor 2, as evidenced by the potential

Fig. 6 Water layer test of the two suggested sensors by recording potential against time in A 1.0 × ­10–3 M TUL, B 1.0 × ­10–2 M Diazepam HClHussein et al. BMC Chemistry(2024) 18:151

Page 11 of 16

Fig. 7 Study of potential drift over time for the two proposed sensors

Table 3 Quantification of TUL in Draxxin® injection using
MWCNT/ISM sensor
Pharmaceuticalformulation

Draxxin® injection
solution(Batch No.548396)
labeled to contain (100mg of TUL/mL)a

Claimed concentration Recovery ± RSD% a
4.96 × ­10–5

99.60 ± 1.429

4.96 × ­10–4

100.51 ± 1.320

Mean ± RSD%

100.05 ± 1.281

average of three determinations

stability observed within the first hour, Fig. 7. We canthus conclude that leveraging the lipophilic nature of
MWCNT successfully hindered the production of an
inner aqueous film, thereby mitigating instabilities andextending the sensor’s lifetime.

Application in biological and non‑biological samples
Potentiometric determination of TUL in Draxxin injection
solution

The MWCNT/ISM sensor was successfully employed toassess TUL in its pharmaceutical formulation (Draxxin®
solution) without prior sample extraction procedures.
Results shown in Table 3 provided good percentage
recovery (100.05 ± 1.28), suggesting that the developed
sensor has the capability to accurately measure TUL inreal samples without being affected by any additives present in the formulation.
Potentiometric determination of TUL in Bovine liver and milk

The applicability of the MWCNT/ISE sensor was demonstrated in bovine food products. Table 4 presents thecomputed values for the quantity of TUL added, TUL
found, and the recovery % for several spiked samples.
Values of %R and %RSD fall within the acceptable analytical range, proving this technique valuable for direct andTable 4 Determination of TUL in bovine products (bovine liver and milk) using MWCNT/ISM sensor
Bovine products

Spiked concentration (µM)

Found concentration (µM)aRecovery (%)aMeanrecovery(%) ± RSD%

Liver

1
10
100

1.01
9.943
100.16101%99.43%
100.16%100.19 ± 5.029

Milk

1
10
100

1.01849.742
101.14101.84%97.41%
101.14%100.13 ± 6.121

aaverage of five determinations

Hussein et al. BMC Chemistry(2024) 18:151

Page 12 of 16

fast monitoring of TUL in milk and liver (within 8 ± 2s)
without prior treatment. This finding is supported by
the fact that the LOD value for sensor II (9.76 × ­10–8 M)
falls below the MRL of TUL at 4.5 ppm (5.58 µM) in thebovine liver [9]. As for milk, which is not permitted toinclude any TUL residues, the method can be utilized to
identify potential instances of TUL misuse and administration during lactation periods.

Meanwhile, the AGREE metric system offers a comprehensive evaluation tool by considering each of the 12
principles of green analytical chemistry (GAC) [59]. Eachprinciple is assessed on a scale from 0 to 1 in a clock-like
diagram, with the total score in the center. As the scoreapproaches 1, the corresponding segment gets greener.
The resulting AGREE pictogram in Table 5, with a scoreof 0.83, underscores the high level of environmental compatibility achieved by the designed sensor.

Greenness assessment

Whiteness assessment

In our commitment to conserving ecological resources
and minimizing environmental impact, we assessed the
proposed method’s greenness using the analytical EcoScale and Analytical GREEnness Calculator (AGREE).
The Semi-quantitative Eco-Scale approach involves giving penalty points to reagents and instrument parametersthat deviate from the optimal green analysis practices inanalytical methods [58]. Then, the sum of these points isdeducted from 100 to give a final score that verifies theprocedure as ideally green (> 75), satisfactory (> 50), or
inadequate (< 50). As presented in Table 5, the proposed
method scored 86, which is deemed excellent for a green
method.

In contrast to the previous methods, the Whitenessapproach not only focuses on the green component of ananalytical method but also emphasizes the interdependence and harmony between analytical, environmental,
and pragmatic facets [60]. This most recent approach
applies the RGB12 algorithm to comprehensively evaluate
the 12 principles of White analytical Chemistry (WAC)[61]. WAC utilizes a triadic color scheme to symbolize
the key characteristics of the assessed methodology: redsignifies analytical performance, green denotes ecologicalsustainability and safety, and blue represents practicality
and affordability. The algorithm calculates scores for eachcolor, yielding an overall score of “whiteness” that reflects

Table 5 Greenness evaluation of the suggested potentiometric approach utilizing Eco-Scale and AGREE tools
1) Analytical Eco-Scale
Eco-scale hazard

Penalty points

Reagents
Tetrahydrofuran

6

BRB buffer

2

Instruments
Energy consumption0(< 0.1 kWh per sample)
Occupational hazard

0

Waste

6

Total penalty points (PP)14

Eco-Scale total score862) AGREE

Hussein et al. BMC Chemistry(2024) 18:151

the degree to which the method aligns with the WAC
principles.
We applied this approach to evaluate the overall sustainability of the proposed sensor in comparison to areported chromatographic method [62]. As depicted inFig. 8, our electro-sensing technique excels in green andblue areas, denoting superior functionality, simplicity,and environmental safety while performing sufficiently inthe red area with sufficient accuracy and sensitivity. This
achievement stems from employing less harmful reagents like BRB buffer instead of the non-green acetonitrile, minimizing energy consumption, and generating
less waste through non-destructive measurements. Furthermore, the instrument is portable, far less expensive,
significantly simpler, and requires much less time andeffort than the HPLC–MS/MS instrumentation. Consequently, our proposed sensor achieves an impressive
whiteness score of 96.5%, surpassing the HPLC–MS/MS
score of 70.2%. We can conclude then that our proposed

Page 13 of 16

technique is a thoroughly balanced analytical methodthat adheres to sustainability standards and is specificallytailored to meet its objectives.
Merits of the novel sensor over conventional methods

To verify the validity of the designed sensor in determining TUL, statistical tests were used to compare theresults with those of the HPLC method reported in theliterature [20]. Table S-1 shows that the computed t- andF-values were lower than the tabulated ones, indicating
no statistically significant difference between the accuracy and precision of the suggested and reported methods. Furthermore, a comparative study was conducted
between the designated sensor and recently publishedwork on TUL analysis. Table 6 highlights the originalityof the newly designed sensor as the first potentiometricdevice capable of quantifying TUL in biological and nonbiological samples. Our method offered precise detection
at micromolar concentrations, surpassing the sensitivity

Fig. 8 Comparison of the Whiteness of the proposed method (SC-ISE) against the reference method (LC–MS/MS) using the RGB12 modelHussein et al. BMC Chemistry(2024) 18:151

Page 14 of 16

Table 6 A comparison between the developed and reported methods for TUL determination
MethodApplication

Linearity range, detection
limitsOperational simplicity andcost

Analysis time Refs.

LC–MS/MS

Bovine muscle, fat, and liver(0.025–100.0) ng/mL, 0.25 μg/
kg

Lengthy sample preparationand extraction procedures(> 70 min), very expensive

7 min

[63]

LC–MS/MSUrine, plasma, and seminal
plasma in bull

(0.1–10), (0.05–5.0), (0.01–1.0)μg/mL for each matrix, respectively, no specified LOD

Lengthy sample preparationand extraction procedures(> 80 min), very expensive> 5 min[12]

HPLC–DAD

Injection solution(1000.0—3000.0), 184.43 µg/mL No extraction procedures,
relatively expensive

10 min[20]

Voltammetric sensor

Swine liver, flesh, sebum(0.006–2.42) µg/mL, 0.001 ng/
mL

Requires extraction procedures NA(> 30 min)[25]

(0.08–806.1), 0.078 µg/mL

No need for extraction procedures, portable and costeffective

This work

Potentiometric sensor Dosage form Bovine liver
and milkof the HPLC/DAD by more than three orders of magnitude. The MWCNT/ISM sensor could detect TUL
directly in as little as 8 s, whereas other methods required
longer run times (around 10 min), not including the timeneeded for instrument preparation and arduous sample
extraction (up to 80 min). Although these approaches
could achieve lower detection limits, their high cost,potential hazards, and laborious nature render theirapplicability in real-time questionable. Alternatively, theproposed sensor is feasible, green, and suitable for in-situ
analysis.

Conclusion
The present work introduces a novel potentiometricsensor for quantifying TUL in various biological andnon-biological matrices. The designed sensor displayed
promising precision, accuracy, speed, and durability.
Integrating carbon nanotubes effectively controlled thewater layer production and stabilized the membrane
response. Beyond that, the MWCNT/ISM sensor exhibited superior performance, featuring a lower limit ofdetection, faster response time, greater selectivity, minor
drift, and longer shelf-life than the MWCNT-free sensor.
The modified sensor could accurately quantify TUL in its
dosage form with minimum interference from coexistingions. This capability extended to the analysis of milk andliver samples, obviating the need for complex extraction
procedures required by previous methods. Based on thegreenness and whiteness profiles analysis, our method
stands out for its convenience, sustainability, and affordability, proving it a reliable option for monitoring TULlevels in various matrices.
Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s13065-​024-​01255-7.

8s

Supplementary file 1.
Acknowledgements
I would like to convey my sincere appreciation to my colleague and mentor,
Dr. Zeinab M. Goda, for devoting her time and energy to deliver valuable guidance and unwavering support throughout this research endeavor.
Author contributions
OGH: Conceptualization, Investigation, Methodology, Validation, Visualization,and Writing – original draft. HHM, HEZ, and MMG: Conceptualization, Supervision, and Writing – review & editing.
Funding
Open access funding provided by The Science, Technology & Innovation
Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank
(EKB). Open access funding was provided by the Science, Technology & Innovation Funding Authority (STDF) in cooperation with the Egyptian Knowledge
Bank (EKB). This study did not receive specific funding from any agency ororganization.
Availability of data and materials
The datasets used and/or analyzed during the current study are available fromthe corresponding author upon reasonable request.

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr
El Aini Street, Cairo 11562, Egypt.
Received: 25 March 2024 Accepted: 23 July 2024

Hussein et al. BMC Chemistry(2024) 18:151

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