Novel potentiometric methods for the estimation of bisoprolol and alverine in pharmaceutical forms and human serum

Two new potentiometric sensors were created for the quantification of bisoprolol fumarate and alverine citrate in bulk pharmaceutical dosage forms and human serum. Bisoprolol and alverine sensors were manufactured by combining potassium tetrakis (p-chlorophenyl) borate ion pairs to serve as electroactive substances, plasticized poly (vinyl chloride) matrix membranes, and o-nitrophenyl octyl ether. They demonstrated high responses over the concentration ranges of 1.0×10-6 to 1.0×10-2 mol L-1 bisoprolol and alverine with close to Nernstian cationic slopes of 52 and 56 mV decade-1, respectively. The detection limits for bisoprolol and alverine were 2.6×10-6 and 1.75×10-6 mol L-1, respectively. For both medications, the response time was instantaneous (2.0 s). The working pH ranges for bisoprolol and alverine were 4.50-8.50 and 2.00-8.80, respectively. For both sensors, the life cycle was long (3 months). The sensors were used in pharmaceutical dosage types for the assay of bisoprolol and alverine, recording average recoveries of 99.40% and 99.98% respectively and were also successfully used for estimating the two drugs in human serum with an average recovery of 99.60% for both drugs. For all multiple staged interfering materials, the reported latest potentiometric sensor methods displayed high selectivity. The current sensor obtained a high percentage recovery and an excellent relative standard deviation compared with those obtained from previously published methods. * Corresponding author: Dina S. Elshaprawy, Department of Chemistry, Faculty of Science, Zagazig University, Zagazig, Egypt, e-mail: dinaelshaprawy@yahoo.com Khaled Elgendy and Mohamed A.F. Elmosallamy: Department of Chemistry, Faculty of Science, Zagazig University, Zagazig, Egypt Moustafa K. Soltan: Faculty of Pharmacy, Zagazig University, Zagazig, Egypt, Oman College of Health Sciences, Oman Alaa S. Amin: Department of Chemistry, Faculty of Science, Benha University, Benha, Egypt Open Access. © 2021 Khaled Elgendy et al., published by De Gruyter.   This work is licensed under the Creative Commons Attribution 4.0 International License.


Introduction
Potentiometry is an electrochemical method widely studied by researchers due to its many advantages. Thousands of potentiometric sensors have been successfully developed and applied in diverse fields such as medicine, environmental monitoring, agriculture, industry, and pharmaceutical sciences. From a medical point of view, clinical drug research and the determination of drugs in biological samples are extremely significant. Such experiments are performed using various analytical instruments, including electrochemical sensors. With regards to many parameters, potentiometric sensors can out-perform other instruments and are therefore commonly used in research. The use of potentiometric sensors to recognize drug molecules in body fluids has shown promising results in the literature. In this research, we apply potentiometry-based sensors to biological blood serum samples for the determination of drugs and document their performance characteristics [1][2][3][4][5].
High blood pressure is a widespread global health issue and is one of the key causes of cardiovascular disease. Bisoprolol fumarate (BIS), ((E)-but 2enedioicacid;1-(propan-2-ylamino)-3 [4-(2-propanyloxyethoxymethyl) phenoxy] propan-2-ol) (Figure 1a) is used for the treatment of hypertension. It is considered a potent drug with a longhalf life that can be used once daily to reduce the need for multiple doses of antihypertensive drugs. Bisoprolol is well-tolerated, possibly because of the selectivity of its β1-adrenergic receptor. It is a useful alternative to nonselective β-blocker medications such as carvedilol and labetalol in the treatment of hypertension . It can be used alone or in conjunction with other medications to treat hypertension and can also be used in chronic obstructive pulmonary disease (COPD) due to its receptor selectivity (no β2 effect).
Pharmacodynamics and mechanisms of action are a reduction of heart rate (chronotropy) by bisoprolol, contractility (inotropy) and blood pressure alterations. Bisoprolol decreases cardiovascular mortality and allcause mortality in patients with heart failure and reduced cardiac ejection fraction (CEF) has been documented in several clinical trials [6].
Bisoprolol's mechanisms of action in hypertension are thought to be achieved by β1 adrenoceptor antagonism, resulting in lower cardiac output. Bisoprolol is a competitive, cardio-selective β1-adrenergic antagonist that decreases cardiac workload through competitive inhibition of β1-adrenergic receptors by reducing contractility and oxygen requirements [7]. Side effects can include stomach cramps or colic.
Alverine is a smooth muscle (an involuntary muscle found in the gastro-intestinal tract, uterus, etc.) relaxant. In conditions such as irritable bowel syndrome, biliary colic, diverticular disease, and different types of intestinal colic, this prevents muscle spasms that occur in the gut. Diverticular disease results from persistent habitual constipation in which the colonic wall produces small, inflamed pouches. In the case of irritable bowel syndrome, normal intestinal muscle function is lost. Symptoms such as heartburn, stomach pain and bloating, alternating constipation and diarrhea are the product of muscle spasms. Alverine is a smooth muscle relaxant. Smooth muscle is a type of muscle that is not under voluntary control; it is the muscle present in places such as the gut and uterus. [8,9]. It can also be used to alleviate dysmenorrhea.

Reagents and apparatus
Many of the reagents used were of high purity and all tests used double-distilled water. Potassium tetrakis (p-chlorophenyl) borate (KTp-ClPB) and o-nitrophenyloctyl ether (o-NPOE) were bought from Fluka (Buchs, Switzerland). BP Chemicals International supplied poly (vinyl chloride) (PVC) (Breon S 110/OP) (Barry, UK). Authentic samples of bisoprolol and alverine (purity 99.90%) were gathered from (SEDCO) Egypt. From local pharmaceutical stores, commercial bisoprolol (Concor® and Bisotens® 10 mg per tablet) and alverine (Alsotrin® 60 mg per capsule) were obtained. KCl, Pb(NO 3 ) 2 , CaCl 2 , NaCl, NiCl 2 , CoCl 2 , CdCl 2 , ZnCl 2 , MnCl 2 , starch, talc, lactose, fructose, ascorbic acid, glucose, heptaminol HCl, atenolol, propranolol HCl, and amlodipine were used as interference materials and purchased from the El-Nasr Company, Egypt. Serum samples were collected from healthy donors. All biological samples were collected in accordance with the applicable laws and institutional guidelines (Profession and Ethics Legislation, Ministry of Health and Population Resolution No 238/2003). The protocol was accepted by the institutional committee of the Faculty of Science (Zagazig University, Egypt).
Potentiometric measurements were carried out using potentiometric sensors built in conjunction with the EIL-Type RJ 23 calomel reference electrode used by some instruments, such as the PTI-15 digital pH meter. For pH measurements, a glass Ag-AgCl combination electrode (Consort, 5210 B BB5) of the Consort P907 mV pH meter was used.

Solutions
The standard stock solutions of 1.0×10 -2 mol L -1 for both drugs were freshly prepared by dissolving (0.044 g) bisoprolol fumarate and (0.473 g) alverine citrate in the least amount of double-distilled water plus 5 mL acetate buffer of pH = 4.6 to keep the drugs in protonated forms, and the volumetric flasks were made up to the 100 mL mark with double distilled water. A series of standard bisoprolol and alverine solutions covering the range from 1.0×10 -6 to 1.0×10 -2 mol L -1 were prepared by suitable dilutions.

Ion-pair preparation
Ion pair preparation was achieved by mixing a 50 mL drug solution of 1.0×10 -2 mol L -1 with a 50 mL solution of 1.0×10 -2 mol L -1 potassium tetrakis (p-chlorophenyl) borate. The precipitate was filtered, washed with double-distilled water and dried at 90°C for 30 min. The resulting powder was used in the plasticized membrane with o-nitrophenyl octyl ether as an electroactive material.

Sensor construction
The constituents of the membrane sensor were: the ionpair, drug -tetrakis (p-chlorophenyl) borate (10.0 mg) (1.85 mass%), (PVC) (170 mg) (31.48 mass%), and o-nitrophenyl octyl ether (o-NPOE) (360 mg) (66.66 mass%). The membrane was casted and the sensor constructed as described previously [34,35]. The sensors were immersed in BIS and ALV solutions of 1.0×10 -2 mol L -1 for one day prior to use and remained in the same solution while not in use. The sensors were calibrated by spiking the normal solution into a 1.0×10 -6 mol L -1 solution of BIS and ALV calibrating solutions with successive liquids. Alternatively, the calibrations were carried out by immersing the sensors in 50-mL beakers containing regular 1.0×10 -6 -1.0×10 -2 mol L -1 BIS and ALV solutions of 25.0 mL aliquots from low to high concentrations. The electromotive force (EMF) was tracked and plotted against the logarithmic drug concentrations. The calibration graph was used for the subsequent measurement of the unknown drug levels.
Potentiometric selectivity coefficients were calculated using the separate solution method [36,37], where the potential of the drug sensor and the reference electrode cell are calculated using two separate drug and interfering material solutions with the same activity (1.0×10 -2 mol L -1 ). The measurement of the potentiometric selectivity coefficient values is based on the equation: where E drug -the measured potential values of BIS and ALV, E B -measured potential value of the interferent, Sthe slope of the calibration plot in mV decade -1 , a drug -the activity of the drug, Z drug and Z B -the charges of the drug and the interferent, respectively.

Potentiometric estimation of BIS and ALV in pharmaceutical formulations
Bisoprolol was calculated by taking the equal weight of one tablet of both Concor ® (10 mg/tablet) and Bisotens ® (10 mg/tablet) and then dissolving them in an adequate volume of double-distilled water. The solution was filtered, a 5 mL acetate buffer was applied to the filtrate, and the 100 mL volumetric flask was then made up to the mark with double-distilled water. In order to assess alverine in Alsotrin® (60.0 mg per capsule), 8 hard gelatin capsules were taken and dissolved in a 100 mL volumetric flask, 5 mL of buffer solution was added and then doubledistilled water was added to the mark. The calibration graph method and the standard addition technique were used to evaluate the substance, the latter based on measuring the potential of a definite volume of the unknown sample before and after applying 2.0 mL of the standard solution to the analyte solution, then calculating the unknown concentration.

Estimation of BIS and ALV in human serum
5 mL of blood from healthy donors containing doses of Concor ® (10 mg/tablet) or Alsotrin® (60.0 mg per capsule) was collected in covered test tubes and these tubes were left for approximately 30 min at room temperature to permit the coagulation of the sample. The clot was extracted using centrifugation for approximately 10 min at 1,000-2,000 g [38]. Human serum was transferred by a Pasteur pipette into a sterile polypropylene tube after the centrifugation stage had finished. The samples were stored at 2-8°C. The standard addition technique was applied for assaying the drugs at room temperature.

Results and discussion
In potentiometric sensor methods (potassium tetrakis (p-chlorophenyl) borate (KTp-ClPB) as a newer ion exchange used for the determination of bisoprolol fumarate or alverine citrate. In our study, drug-tetrakis (p-chlorophenyl) borate ion-pairs have been used as electroactive substances in plasticized (PVC) matrix membranes. The membranes were manufactured using casting solutions with the components listed previously. Referring to IUPAC [36], the electrochemical potentiometric sensor was measured, showing that it exhibited near-Nernstian responses over the concentration ranges of 1.0×10 -6 -1.0×10 -2 mol L -1 of drugs with 52 and 56 mV/decade cationic slopes for BIS and ALV, respectively ( Figure 2, Table 1). The detection limits were 2.6×10 -6 and 1.75×10 -6 mol L -1 for BIS and ALV respectively. These values were calculated in conjunction with IUPAC guidelines [27]. A minimum square review of the data gathered over 13 weeks revealed the following relationships:

Response time
Response time is the time required for the sensor to reach a stable reading. Over the measurement duration, the current potentiometric sensors reported stable potential readings within 1.0 mV, and the calibration slope did not differ by more than 1.0 mV decade -1 over the lifetime of the current sensors. The response time values for the two sensors were 2.0 s for the initial 1.0×10 -2 mol L -1 concentration. The duration of useful life of the sensors was 13 weeks.

Influence of pH
In Table 2, two separate solutions of concentrations 1.0×10 -2 and 1.0×10 -3 mol L -1 of BIS and ALV were used to illustrate the effect of pH on the sensors. In order to study the acidic pH range, dilute hydrochloric acid solution was added, and dilute sodium hydroxide solution was used to study the alkaline pH range. The potential readings for 1.0×10 -2 mol L -1 were fairly constant over the pH range of 4.5-8.5 and 2.0-8.8 for BIS and ALV, respectively, and the readings for 1.0×10 -3 mol L -1 of the drugs were fairly constant over the pH range of 5.5-8.5 and 5.7-7 for BIS and ALV, according to the pH potential graphs (Figures 3 and 4). These drugs were fully soluble, dissociated and sensed as singularly charged ions within these ranges. In a more alkaline medium, the potential of both sensors decreased due to the interference of OHions where the drugs started to precipitate. In a more acidic medium, the potential of BIS increased, probably due to the interference of H + ions.

Effect of the interferences
By using certain inorganic cations and cationic drugs as interfering products, the sensors were tested for selectivity. The selectivity coefficients ( ) pot , [39,40] were evaluated to show the degree of interference, using a different solution method. The key dependent factor for the selectivity of ion-exchanger complexes-based membrane sensors [41] is the lipophilicity of the interfering ion and also its movements inside the membrane. We reported that the proposed sensor has a high selectivity towards BIS and ALV drugs and no interference from the studied inorganic cations, carbohydrates or structural analogs, and can therefore be used in concentrations equal to or less than that of the drugs for the determination of BIS and ALV drugs in the presence of these interfering species (Table 3).

Method validation
The validation parameters such as precision, specificity, accuracy, and the limit of detection (LOD) were studied for the proposed electrodes. The lower limit of the detection   value is known to be the concentration of drugs at the point of intersection of the extrapolated linear regions of the calibration curve. The value obtained indicates that low drug concentrations can be identified by the proposed sensor ( Table 1). The LOQ is the point of the linear range at which the Nernstian process begins. The LOQ is considered by some scholars to be the lowest point to make useful determinations.

Accuracy and precision
Through preparing solutions containing three different amounts of pure drugs and their dosage types, the consistency of the current process was apparent. Accuracy was estimated by testing solutions on five replicates on the same day and was also evaluated over five days. From the data in Tables 4 and 5, The accuracy demonstrated by the   Table 3 showed the effective use of the proposed sensors in the production of highly reliable BIS and ALV products, as shown by the recovery percentage.

Analytical applications by standard addition method
These sensors have been used successfully to measure the active ingredients in Concor ® (10 mg per tablet), Bisotens ® (10 mg per tablet) and Alsotrin ® (60 mg per capsule), with recoveries of 99.40 ± 0.54%, 99.67 ± 0.20% and 99.42 ± 0.15%, respectively, where n = 5 (Table 6) using the standard addition technique. The results revealed that the precision was ±0.53 and the accuracy was 99.50%. Repeatability CV w was 0.90% and the between-day-variability CV b was 1.2%. The statistical analysis of Student's t-value at 95% confidence limit showed that the calculated values were less than the theoretical ones. This proves that the prepared sensors can be developed for drug analysis. Furthermore, Concor® (10 mg per tablet) and Also-trin® (60 mg per capsule) drugs were successfully estimated in the human serum by using the standard addition technique for two samples of both drugs and the average recoveries were 99.96 ± 0.81% and 98.40 ± 0.69% for BIS and ALV samples, respectively (Table 7).

Comparison study
A comparison of the output characteristics of the proposed sensor was made with methods previously used for BIS and ALV determination. The data in Tables 8 and 9 indicate the superiority of the proposed MIP-based sensor over the other methods as the proposed sensor gave the widest concentration range, relatively lower limit of detection, and lower RSD values than any of the other methods. The RSD and the percentage recovery values reflect the effective use of the proposed sensors for highly precise and accurate drug determination. This is one of the greatest benefits of imprinting technology; the suggested sensors have no interferences and have a high drug selectivity.

Conclusion
New, sensitive, and simple potentiometric sensors have been developed for the determination of bisoprolol and alverine drugs. The approach had the merits of low cost, high precision, quick response time (2.0 s), and the methods for 1.0×10 -2 bisoprolol and alverine also performed in a broad pH range of 4.50-8.50 and 2.00-8.80. In the presence of a wide range of interfering species, there was a high selectivity of the proposed sensors for BIS and ALV drugs and no interference from the studied inorganic cations, carbohydrates, and structural analogs, meaning that the sensor can be used for the determination of BIS and ALV drugs in concentrations of these species equal to or less than that of the drugs. LOD and LOQ values show the fitness of the new technique compared to existing methods. Direct quantification of bisoprolol and alverine in prescription dosage forms and human serum achieved high precision and recovery of up to 99.60%. The alverine sensor was novel and the first to be implemented, and the bisoprolol sensor was an alternative to the previously mentioned potentiometric sensors.
Funding information: Studies were funded by the Faculty of Science, Zagazig University.