b Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
Amprolium hydrochloride (AMP) is widely used to prevent and treat coccidiosis in chickens. AMP (1-[(4-amino-2-propyl-5-pyrimidinyl) methyl]-2-methyl pyridinium)  is sometimes formulated with Ethopabate (ETHOP) which is 4-acetamido-2-ethoxybenzoic acid methyl ester. The chemical structures of the two drugs are shown in Scheme 1.
The poultry industry is one of the main agricultural industries in Egypt. The size of the labour force is about 1.5 million permanent workers and about 1 million temporary workers. The industry contributes a large part of the country's supply of animal protein. Poultry meat is popular among egyptian consumers across all income categories, because of its low cost compared to red meat and fish . Anticoccidiosis veterinary drugs are administered orally or are mixed with animal feed and water; however, the unreasonable and extensive use of these drugs can result in high levels of residues in edible animal products like meat, muscle, egg, and liver which contains high reservoir of drugs taken. The US Code of Federal Regulations has established maximum residual limits (MRLs) for AMP is 1 ppm in chicken liver .
Literature survey revealed that AMP is official in British Pharmacopeia . Various analytical methods have been used for the determination of AMP either alone or in combination with other drugs in pharmaceutical formulation or in other matrices such as surface water, eggs, chicken feed, chicken muscles, chicken plasma and chicken liver. These methods include determination in pharmaceutical dosage form with liquid chromatography coupled with ultraviolet (UV) , liquid chromatography mass spectrometry (LC-MS) , thin layer chromatography , spectrophotometric methods , atomic spectrometry  and electrochemical method . AMP was also determined in tissues, biological fluid and feed with liquid chromatography coupled with ultraviolet  or fluorescence ,  detection, spectrofluorimetry , liquid chromatography-mass spectrometry (LC-MS) [14-19], spectrophotometric methods  and capillary electrophoresis . Although the above mentioned techniques offer a high degree of specificity, however, sample preparation, instrumentation limitations, long analysis time. The uses of organic solvents with high costs and harmful to the environment are some of the drawbacks associated with these techniques which challenge their use in routine analysis.
On the contrary, electrochemistry, particularly, ion selective electrodes (ISEs), is an eco-friendly analytical technique characterized by instrumental simplicity, moderate cost, and portability . ISEs have other advantages such as low energy consumption, limited sample pretreatment, rapidity, being non-destructive, adaptability to small sample volumes and on-line monitoring [23, 24]. Such desirable characteristics have made ISEs the center of considerable attention across many disciplines of science, and they have potential applications of clinical, environmental, and food analyses [25, 26].
The molecular recognition and inclusion complexation are of current interest in host-guest and supramolecular chemistry and offer a promising approach to chemical sensing [27, 28]. The use of selective inclusion complexation and complementary ionic or hydrogen bonding are two main strategies for preparing synthetic host molecules, which recognise the structure of guest molecules . Modified cyclodextrins (CDs), either natural or synthetic, are viewed as molecular receptors, as is shown in Scheme 2. In the case of natural CD, cooperative binding with certain guest molecules was mostly attributed to intermolecular hydrogen bonding between the CD molecules, while intermolecular interactions between the host and guest molecules (hydrogen bonds, hydrophobic interactions and Van der Waals forces) contributed to cooperative binding processes when synthetic CDs were used . Although the size and geometry of the guest mainly govern the binding strength, it is possible to influence the host-guest interactions by modifying the three hydroxyl groups on each glucose unit. Indeed, the use of 2-hydroxy propyl β-cyclodextrin enhanced the interaction properties between host and guest molecules . Calixarenes are well-known as selective ligands for various ions through dipole-dipole interactions, as shown in Scheme 3. They can complex with a large variety of cation substrates to form stable host-guest inclusion complexes. This property of calixarenes has been largely exploited for the development of a number of cation selective electrodes [32-34]. The present work evaluates the possibility of using 2-hydroxypropyl β-cyclodextrin and p-tert-butylcalixarene as sensor ionophores in the preparation of AMP selective electrodes Ⅱ and Ⅲ, respectively, using PVC as a polymeric matrix to immobilise the sensors and to attain the formation of highly stable complexes.
|Scheme 2. Chemical structure (A) and toroidal shape (B) of the 2-hydroxy propyl β-cyclodextrin molecule|
|Scheme 3. Chemical structure of the p-tert-butylcalixarene (A). Mode of attachment between the p-tert-butylcalixarene and AMP revealing cation–π interactions (B)|
The objective of this study is to develop and optimize simple potentiometric electrodes for rapid, reproducible, selective, sensitive, accurate, and low cost determination of the cationic drug, AMP in pharmaceutical preparations with ethopabate and also in chicken liver.2. Results and discussion
The present work evaluates the possibility of using 2-hydroxypropyl β-cyclodextrin and p-tert-butylcalixarene as sensor ionophores in the preparation of AMP selective electrodes Ⅱ and Ⅲ, respectively, using PVC as a polymeric matrix to immobilise the sensors and to attain the formation of highly stable complexes.2.1. Performance characteristics of AMP sensors
The fact that AMP behaves as a cation suggests that the use of an ISE membranes which exhibit cation exchange capacity. This was achieved by using a lipophilic cationic exchanger; potassium tetrakis (4-chlorophenyl) borate (TpClPB), where the membrane was initially conditioned in 1 × 10-2 mol L-1 AMP for 1 day to replace the original exchangeable counter ion (K+) of the ion exchanger with AMP.
Table 1 shows the results obtained over a period of two months for two different assemblies of each sensor. The addition of ionic strength adjustor ((NH4)2SO4) to different concentrations of AMP solutions prepared for calibration plots shows no significant differences on the resulting accuracy of the proposed sensors. This is in agreement with previous references , which avoid the use of ionic strength adjustor. Typical calibration plots are shown in Fig. 1. The slopes of the calibration plots are 29.2, 29.3 and 30.2 mV/ concentration decade for sensors Ⅰ, Ⅱ and Ⅲ, respectively. Deviation from the ideal Nernstian slope (30 mV) is due to the electrodes responding to the activity of the drug cation rather than its concentration. The sensors displayed constant potential readings for day to day measurements, and the calibration slopes did not change by more than±2 mV/decade over a period of 16, 44 and 56 days for sensors Ⅰ, Ⅱ and Ⅲ, respectively. The detection limits of the three sensors were estimated according to the IUPAC definition .
|Figure 1. Profile of the potential in mV versus log concentrations of AMP in mol L-1 obtained with sensors Ⅰ, Ⅱ and Ⅲ|
In order to examine the selective recognition of AMP by the 2-hydroxy propyl β-cyclodextrin and p-tert-butylcalixarene ionophores in the membrane phase, the performance of an ionophorebased ISEs for AMP (sensors Ⅱ and Ⅲ) were compared with an ionophore-free ion-exchanger TpClPB (sensor Ⅰ) as a control experiment. It was found the positive AMP ion prefers the high donation sites (OH-groups) of 2-hydroxypropyl β-cyclodextrin and calixarene structures rather than the methyl groups. Thus, in the absence of ionophores in sensor Ⅰ, the lowest slope value is found accompanied by the highest selectivity coefficient values. A higher selectivity coefficient value corresponds with more attack by interfering cations on the electrode membrane. The presence of OH-groups only in sensor Ⅱ was not enough to perform the proper chelation, which was demonstrated by a slope of 29.3 mV/decade and the high selectivity coefficient values compared to sensor Ⅲ. The p-tert-butylcalixarene-based sensor Ⅲ shows the best Nernstian slope (30.2 mV/decade) and selectivity coefficient values. The host-guest complex is stabilized via an electrostatic interaction between the cationic AMP and anionic cavity of p-tert butylcalixarene. Moreover, calixarene has a larger internal cavity size (9.5 Å) than 2-hydroxypropyl β-cyclodextrin (6 Å) . This allows the drug to fit well in the calixarene cavity and strongly bond to the calixarene donation sites. The results reveal that, as ionophores, 2-hydroxypropyl β-cyclodextrin and calix-8-arene provide high stability to the complexes formed with cationic drug present in solution; thus, the membrane selectivity and sensitivity are substantially enhanced. The electrochemical performance characteristics of the proposed sensors were systematically evaluated according to IUPAC standards .2.2. Effect of pH, dynamic response time and repeatability of electrode
The influence of pH on the potentiometric response of the proposed sensors to AMP was studied by making calibrations at pH values close to both neutral pH range (6.0-8.0) and acidic pH range (2.5-3.5) by using phosphate buffers as shown in Fig. 2. The reason behind studying the pH effect at different values is the presence of two electrically charged groups within the AMP structure. The first is a permanently-charged quaternary ammonium group which is pH-independent ionic group, while the second one is the ionizable amino group located in the pyrimidine ring with pKa value of 5.3. For this reason, the results showed that a significant differences in the corresponding calibration Nernstian slopes in acidic rather than neutral conditions. At acidic conditions, the AMP molecule is doubly charged (divalent ion), where the quaternary ammonium group as well as the amino group which is completely ionized and sensed and hence a Nernstian slope of almost 30mV was obtained. While at neutral conditions (pH 7), the AMP molecule is almost singly charged (quaternary ammonium group only), while only 3% of the amino group is ionized and therefore it was almost sensed as monovalent ion and the corresponding Nernstian slope of about 60mV was obtained (Fig. 2). Dynamic response time is an important factor for analytical applications of ion-selective electrodes. In this study, practical response time was recorded by increasing AMP concentration up to 10-fold. The required time for the sensors to reach values within±1mV of the final equilibrium potential was 35, 15 and 10s for sensors Ⅰ-Ⅲ, respectively. The time traces of the calibration curve of sensor Ⅲ is presented in Fig. 3. Moreover, the repeatabilityof the potential reading for each electrode was examined by subsequent measurement in 1.0 × 10-5 mol L-1 AMP solution immediately after measuring the first set of solutions in 1.0 × 10-4 mol L-1 AMP. The electrode potential for four replicate measurements in 1.0 × 10-5 mol L-1 solution for sensors Ⅰ-Ⅲ, exhibit a standard deviation of 1.31, 1.42 and 1.21, respectively. While the corresponding values in 1.0 × 10-4 mol L-1 solution showed a standard deviation of 1.59, 1.05 and 0.85, respectively. This indicates excellent repeatability of the potential response of the three proposed electrodes.
2.3. Validation of the proposed method
Methods validation has been performed according to ICH guidelines .
The linearity of the proposed methods is evaluated byanalyzing different concentrations of standardsolutions of AMP in triplicates. The values of correlation coefficients are close to unity indicating good linearity, the linear regression equations of the three proposed sensors are:
E=414.2+29.2 log CAMP r=0.9999 (sensor Ⅰ)
E=285.6+29.3 log CAMP r=0.9999 (sensor Ⅱ)
E=313.8+30.2 log CAMP r=0.9999 (sensor Ⅲ)
The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. This is sometimes termed trueness. The accuracy of the results is checked by applying the proposed methods for determination of three different concentrations (10-2, 10-4 and 10-6 mol L-1) for each of pure AMP, respectively. The concentrations are obtained from the corresponding regression equations then the percentage recoveries are calculated (Table 1).
The precision of the results is checked byapplying the proposed methods for determination of three concentrations of pure AMP within their linearity ranges, respectively; where the concentrations are analyzed three times, intra-daily (repeatability), and analysis of three concentrations on three successive days to evaluate the intermediate precision. The concentrations are obtained from the corresponding regression equations then the percentage recoveries are calculated (Table 1).
The Limit of detection (LOD) was determined according to the IUPAC rec-commendations where the concentration of the primary ion at the point of intersection of the extrapolated lines of the Nernstian (high concentration) and nonresponsive (low concentration) segments of the calibration curve can be considered as an "attainable" detection limit under the stated experimental conditions [36, 39].2.4. Sensors selectivity
The effect of interfering substances upon the performance of the sensors was studied. The response of the three sensors in the presence of susceptible excipients, organic and inorganic related substances was assessed and the results of the calculated selectivity coefficients showed that the proposed sensors displayed high selectivity and no significant interference was observed fromthe susceptible interfering species (Table 2). Clearly, the high selectivity of the proposed sensors towards AMP compared to other inorganic cations (Na+ and K+) can be attributed to its higher lipophilicity, where Na+ and K+ did not interfere probably due to the difficulty of the ion exchange of these inorganic hydrophilic ions into the lipophilic membrane. Additionally, it is important to point out that the co-formulated drug, ethopabate, shows a marked non-Nernstian response due to its practical water insolubility and the absence of an electroactive group (quaternary ammonium nitrogen) in its chemical structure. This remarkably high discrimination facilitates the developmentof an AMP-ISE for selective determination of AMP in their combined dosage form, Amprobate® (discussed below).
2.5. Potentiometric determination of AMP in dosage forms (water soluble powder and solution)
The proposed sensors were employed for assaying of AMP in solution. The results prove the applicability of the sensors as demonstrated by the accurate and precise percentage recoveries. Susceptible soluble powder excipients or solution excipients did not show any interference. Thus, the determination of AMP was carried out without prior treatment or extraction and without interference by ETHOP using sensors Ⅰ-Ⅲ (Table 3).
2.6. Statistical comparison of the obtained results with reference
methods The results obtained were compared with those obtained by applying the official method  for AMP determination (Table 3). Statistical analysis of the results between the proposed and official method using t-test and F ratio showed there is no significant difference between them regarding accuracy and precision. It should be noted that the time required for sample analysis was short in case of ISE compared to official non aqueous titration method using 1-naphtholbenzein solution as indicator versus perchloric acid.2.7. Determination of AMP in chicken liver
AMP was successfully determined in spiked chicken liver samples without prior treatment. The results show the applicability of the proposed sensor Ⅲ (based on its superior selectivity and LOD) for the determination of AMP in spiked liver samples as shown in Table 4. The response time of the proposed sensor was instant (within 10s), so the sensors are rapidly transferred back and forth between the liver sample and the de-ionized water between measurements to protect the sensing component from adhering tothe surface of some matrixcomponents. It is concluded that the proposed sensor Ⅲ can be successfully applied in drugresidue detection studies as its LOD was 1.0 × 10-7 mol/L which is below the MRL of AMP=1ppm (3.6 × 10-6 mol L-1). The results obtained were compared with those obtained by applying the reference method  for AMP determination Table 4. Statistical analysis of the results between the proposedand reference method using t-test and F ratio showed that there is no significant difference between them regarding accuracy and precision.
The responses of the fabricated sensors are sufficiently precise, accurate and prove the great selectivity of the sensors for the quantitative determination of AMP inpureform, inpharmaceutical formulations and chicken liver. Moreover, the use of the proposed sensors compromises the great advantage of eliminating any need for drug pretreatment or separation steps. They can therefore be used for routine analysis of AMP in quality control laboratories. In general, the ISEs proposed here offered high simplicity in design and a very low limit of detection as well as being rapid, simple, and inexpensive and could compete with the many sophisticated methods currently available and we can detect veterinary drug residue in chicken liver to achieve safety target of this study.4. Experimental 4.1. Apparatus
Potentiometric measurements were carried out using an Ag/ AgCl double-junction-type external reference electrode (model no. Z113107-1EAPW Aldrich Chemical Co. Steinheim, Germany; 3.0 mol/L KCl saturated with AgCl as an inner filling solution and 10% KNO3 as a bridge electrolyte) and Jenway digital ion analyser (model 3330; Essex, UK). A Jenway pH glass electrode (Essex, UK) was used for pH adjustments. Magnetic stirrer, Bandelin Sonorox, Rx510S (Budapest, Hungary).4.2. Materials
All chemicals and solvents used were of analytical grade and used water was bi-distilled. Polyvinyl chloride (PVC), 2-nitrophenyloctyl ether (2-NPOE), dioctyl phthalate (DOP), potassium tetrakis (4-chlorophenyl) borate (TpClPB), 2-hydroxypropyl-β-cyclodextrin (2-HP βCD), 4-tert-butylcalixarene (tBC8), Britton-Robinson buffer (BRB) (pH 2-12) and tetrahydrofuran (THF), (Aldrich, Germany).
Amprolium hydrochloride (AMP) was supplied by Memphis Company for pharmaceuticals, Giza, Egypt. Its purity was checked and found to be 100.66±0.626% according to the British pharmacopoeia official method  which is a non-aqueous potentiometric titration method. Ethopabate (ETHOP) was supplied by Memphis Company for pharmaceuticals, Giza, Egypt. Its purity was checked and found to be 99.5% according to the British pharmacopoeia official method  which is a liquid chromatographic method.
1. Amprobate water soluble powder B.No. (413006). Each 1000 g is claimed to contain 250 g of AMP, 16 g of ETHOP and 734 g lactose monohydrate manufactured by Memphis Company for pharmaceuticals and chemical industries, Giza, Egypt.
2. Amprobate solution B.No. (130934). Each 1000 ml contains 250 g of AMP and 16 g of ETHOP manufactured by Dar Al Dawa Veterinary and Agricultural Industrial Company, Giza, Egypt.4.3. Standard solutions
AMP stock standard solution (1 × 10-2 mol L-1): It was prepared by transferring 0.069 g AMP into a 25-mL volumetric flask, dissolving in a sufficient amount of distilled water then the volume was completed to mark with the same solvent. AMP working standard solutions: Different solutions of varying strengths (1 × 10-7 -1 × 10-2 mol L-1) were freshly prepared by serial dilutions from the stock solution using distilled water.4.4. Procedures 4.4.1. Fabrication of membrane sensors
For the preparation of sensor 1, 400 mg NPOE was mixed with 50 mg TpClPB and 190 mg PVC in a 5-cm Petri dish. The mixture was dissolved in 6 mL THF. Fifty milligrams of 2-hydroxy propyl β-cyclodextrin or 50 mg p-tert-butylcalixarene were added to the previous components for the preparation of sensors Ⅱ and Ⅲ, respectively. The Petri dishes were covered with filter paper and left to stand overnight at room temperature to allow solvent evaporation. Master membranes 0.1 mm in thickness were obtained. From each master membrane, a disk (about 8 mm in diameter) was cut using a cork borer and pasted using THF to an interchangeable PVC tip that was clipped into the end of an electrode glass body. The electrodes were then filled with an internal solution of equal volumes of 10-2 mol L-1 AMP and 10-2 mol L-1 KCl. Ag/AgCl wire (1 mm diameter) was used as an internal reference electrode. The sensors were conditioned by soaking in 10-2 mol L-1 aqueous AMP solution for 24 h, and then were stored in the same solution when not in use. The developed electrodes are shown in Scheme 4
|Scheme 4. Potentiometric cell assembly with a conventional, liquid inner contact ion selective membrane electrode as indicator electrode and a double junction reference electrode|
4.4.2. Sensors calibration
The conditioned sensors were calibrated by separately transferring 50 mL aliquots of solutions (10-7 to 10-2 mol L-1) of AMP into a series of 100 mL beakers. The membrane sensors, in conjunction with Ag/AgCl reference electrode, were immersed in the above test solutions and allowed to equilibrate while stirring. The potential was recorded after stabilizing to±1 mV, and the electromotive force was plotted as a function of the negative logarithm of AMP concentration. The above procedure was repeated after adding 2 mL-1 mol L-1 (NH4)2SO4 (ionic strength adjustor) to the measured solutions.4.4.3. Effect of pH
The effect of pH on the response of the investigated electrodes was studied using 10-3 mol L-1 solutions of AMP in BRB with pH ranging from 2 to 10.4.4.4. Sensors selectivity
The potential response of the three proposed sensors in the presence of a number of related substances was studied and the potentiometric selectivity coefficient, the ability of the sensing membrane in discriminating the primary ion against other ion of the same charge sign, log (KPotPrimaryion, interferent)] was calculated to estimate the degree to which a foreign substance would interfere with the response of the electrodes to their primary ion (AMP with sensors Ⅰ-Ⅲ). The selectivity coefficients were calculated by the separate solutions method (SSM) , using the following equation:
-log (Kprimary ion interferentpot)=(E1 -E2)/S
where E1 is the potential measured in 10-3 mol L-1 of 1ry ion solution (AMP solution), E2 the potential measured (mV) in 10-3 mol L-1 of interferent solution and S is the slope of the investigated sensor (mV/decade).4.4.5. Direct potentiometric determination of AMP in Amprobate (water soluble powder and Solution)
An amount equivalent to 0.069 g AMP from Amprobate water soluble powder or Amprobate solution were accurately transferred into a 25 mL volumetric flask and diluted to the mark with distilled water. The concentrations of these solutions are claimed to be 1 × 10-2 mol L-1 of AMP. The prepared electrode (sensors Ⅰ-Ⅲ) in conjunction with the double junction Ag/AgCl reference electrode was immersed in the prepared solutions and the resulting potentials were recorded and then the respective concentrations were calculated from the corresponding regression equation as shown below under linearity 188.8.131.52.6. Direct potentiometric determination of AMP in chicken liver
One gram of chopped and homogenized chicken liver tissue was weighed into 15 mL distilled water, then 1.0 mL of 10-4 mol L-1 AMP was added and the potential was recorded. Another 1.0 mL of 10-4 mol L-1 AMP was added and the potential was recorded again. Measurements were repeated three times and the corresponding concentrations of AMP were recorded using the standard addition equation .
where Cx is the concentration of AMP to be determined, Vx is the volume of the original sample solution, Vs and Cs are respectively the volume and concentration of the standard solution added to the sample to be analyzed, ΔE is the change in potential after the addition of certain volume of standard solution, and S is the slope of the calibration graph. All the samplings, dilutions, and measurements were performed in polyethylene flasks and beakers. The Recovery % was then calculated.
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