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Document Type : Original Article

Authors

Department of Chemistry, College of Science, University of Baghdad, Baghdad, Iraq

Abstract

A new FI method was examined with many advantages such as simplicity, high sensitivity and accuracy, economical in terms of material consumption, environmentally friendly, and less hazardous to laboratory with the minimum volume of reactants. Flow injection analysis is a process employ for estimation of Oxymetazoline hydrochloride in pharmaceutical forms by spectrophotometric with merging zone technology. This method depends on the oxidation-reduction reaction a cross the oxidizing agent the drug while reducing it to iron(II). The reduced form reacts with the reagent 2,2`-bipyridyl to give a pink complex is measured at λmax (521) nm. Linear calibration curve obeyed Beer's law 10-500 μg.mL-1 with detection limit 6.195 μg.mL-1 for the developed FIA method. The velocity of sampling was 72 sample.h-1, the RSD was 2.716%, and the average recovery was 101%. By using distilled water as a carrier of chemicals with this analytical technique, it is less dangerous and less polluted. The suggested method was effectively used to estimate OXY in medicinal drugs and biological materials. The obtained results were statistically treated and comparable to the official method (British Pharmacopeia).
A new FI method was examined with many advantages such as simplicity, high sensitivity and accuracy, economical in terms of material consumption, environmentally friendly, and less hazardous to laboratory with the minimum volume of reactants. Flow injection analysis is a process employ for estimation of Oxymetazoline hydrochloride in pharmaceutical forms by spectrophotometric with merging zone technology. This method depends on the oxidation-reduction reaction a cross the oxidizing agent the drug while reducing it to iron(II). The reduced form reacts with the reagent 2,2`-bipyridyl to give a pink complex is measured at λmax (521) nm. Linear calibration curve obeyed Beer's law 10-500 μg.mL-1 with detection limit 6.195 μg.mL-1 for the developed FIA method. The velocity of sampling was 72 sample.h-1, the RSD was 2.716%, and the average recovery was 101%. By using distilled water as a carrier of chemicals with this analytical technique, it is less dangerous and less polluted. The suggested method was effectively used to estimate OXY in medicinal drugs and biological materials. The obtained results were statistically treated and comparable to the official method (British Pharmacopeia).

Graphical Abstract

Indirect Sensitive Determination of Oxymetazoline. HCl in Pure Pharmaceutical Drugs and Biological Samples Using a Modified Sensor Unit Via a Green Method of FI/MZ System

Keywords

Main Subjects

Introduction

Oxymetazoline hydrochloride (OXY) is chemically known as 3-[(4,5-Dihydro-1H-imidazol-2-yl) methyl]-6-(1,1-dimethylethyl)-2,4dimethyl-phenol hydrochloride [1]. Figure 1 displays the structural formula of the oxymetazoline hydrochloride molecule. Is a white crystalline powder an imidazole derivative and belongs to a group of drugs similar to the action of the sympathetic system. It has a direct effect on alpha-adrenergic receptor agonists. The α-Adrenergic receptor agonists activate them which lead to the muscle contraction. Smooth vessels and dilated capillaries in the respiratory passages and sinuses [2] and this leads to better breathing by reducing congestion as a result of colds, sinusitis, hay fever, or other allergies in the upper respiratory tracts [3]. It is also used to relieve eye redness due to itching, exposure to dust or pollen, or the use of contact lenses [4]. There are different techniques used to estimate Oxymetazoline.HCl such as high-performance liquid chromatography [5, 6], Fluorophotometric, [7], Spectrophotometric [8], Electrophoresis [9], liquid chromatographic-mass spectrometry [10], chemiluminescence [11] and potentiometry [12]. The suggested method of Flow Injection Analysis/Merging Zones (CFIA/MZ) technique as automatic analytical tool is characterized by high speed and accuracy. It is considered among the methods of green chemistry due to its many advantages including the high sensitivity in estimating with a high repeatability of the result and consuming small quantities of reactant and does not need the expensive and toxic reagents. It also does not require further treatment of the samples and extraction or pre-concentration of the trace concentrations. In addition, the analysis only takes a short time due to the high sampling rate per hour [13-16]. In this manuscript, the indirect determination of Oxymetazoline.HCl through a pink complex formed and measured at wavelength 521 nm by reducing iron(III) to iron(II), and then complicating with a selective organic reagent.

Figure 1: Structural formula of oxymetazoline hydrochloride molecule

Materials and Methods

The stock solution of Oxy.HCl (1000 μg. mL-1, SDI) was prepared by weighing 0.1g of the pure substance and dissolving it with distilled water to the mark in a 100 mL volumetric vial. The stock solution of the organic reagent 2,2`-BPY (3.2×10-3M) was prepared by dissolving 0.05 g (M.wt=156.18 g/mol, Merck) in 3 mL ethanol, and then it was transferred to a volumetric bottle of 100 mL and the volume was supplemented with distilled water to the mark. The oxidizing agent FeCl3 (3×10-3M) was prepared by dissolving 0.05 g (M.wt=162.204 g/mol, Merck) with distilled water in a volumetric container of 100 mL and the volume was increased for the mark.

Biological specimens (plasma and urine) preparation

Plasma specimens

Blood samples were obtained from people of good health in a glass tube including (EDTA), and then centrifuged for 15 minutes at 3000 rpm. 0.5 ml of plasma was withdrawn, placed in a plastic tube, and stored by freezing until use [17] for plasma specimens attended 100 μg/mL.

Urine specimens

Samples were taken from different healthy individuals. After adding 2 mL of HClO4 acid to precipitate the protein, the samples can then be centrifuged at 3000 rpm and utilized [18] for urine specimens attended 100 μg/mL.

Preparation of dosage forms

Three pharmaceutical drugs were prepared in the form of drops from different companies with a concentration of 50 μg/mL of OXY by withdrawn 1 mL and it was placed in 50 mL volumetric vial, and then diluted with distilled water to the mark.

-Nazordin 0.5 mg of OXY, (SDI)-Iliadin 0.5 mg of OXY, (Merck).

-Rinerge 0.5 mg of OXY, Laboratorios Atral, S.A.

Apparatus and flow injection manifold

The absorbance measurements were carried out in batch by using a double-beam Shimadzu UV- 1800 UV-Visible Spectrophotometer from Japan and a quartz cuvette with an optical lengthwise of 1 cm. The recommended FI system was created as a semi-automated model with a one channel manifold, as depicted in Figure 2, was utilized in the creation of FIA/MZ system (the OXY assessment approach). Distilled water was pumped through injection valve as a carrier stream to transport the reactants through the new FIA system. Peristaltic pump (Shenchen, LabM1) (six-three-way injection valve, homemade) [19] that rotates 90 degree and three Teflon loops (I.d =0.5mm). There were three loaded solutions: sample solution (OXY) in L 1, oxidizing agent solution (FeCl3) in L2, and the reagent solution (BPY) in L3. These chemicals are mixing inside a glass reaction coil 2 mm (I.D). The complex formed (pink) was measured at λmax=521 nm. The modified photometer 301-D+, VIS Spectrophotometer one streak (Japan) was utilized in the developed FIA technique for absorbance and spectral control. To measure the responses represented as mean peak elevation (n=3) (mV) or optical multimeter absorption (DT9205A, OVA, China) for the absorbance mensuration, a Kompensograph C1032 (Siemens) was utilized. A flow cell made of quartz silica (QS, 1 cm) with an internal magnitude of 80 μL was placed inside the detection unit (the modified sensor unit).

Figure 2: The developed CFIA system for determination of OXY.HCl in dosage forms and biological samples

Outcomes and explanations

The initial study for the reaction, 1 mL of OXY (200 μg/mL), 1 mL of FeCl3 (3×10-3 M), and 1 mL of BPY (3.2×10-3 M) were added into a volumetric vial (20 mL) capacity and they were diluted to the mark with distilled water, the highest absorbance of the pink-colored complex was measured at wavelength 521 nm against the blank solution, as illustrated in Figure 3.

The suggested mechanism of the reaction

The indirect spectrophotometric determination of OXY was done through the reduction of iron(III) to Fe(II), and then it was reacted with a selective organic analytical agent (2,2`-bipyridyl) to produce a pink complex [20] that was necessary for the spectroscopic measurement of OXY, as indicated in Scheme 1.

Figure 3: The Absorption spectrum of: (A) pink colored complex against blank solution (20 μg. mL-1) (B) blank solution against distilled water

Scheme 1: The proposed mechanism of oxidation-reduction reaction between OXY with Ferric chloride

The complexation ratio between the reagent with drug was measured by two methods (molar ratio and Jobs method). The ratio [1:3, D: R] was concerned, as exhibited in Figure 4.

Preliminary investigation

The effect of the volume of the reagent and the oxidizing agent with 20 μg. mL-1 OXY was studied, and it was found that the best volume was 10 mL for each of the reagent (3.2×10-3M) and the oxidizing (3×10-3M), which gave the highest absorbance, these volumes were chosen for successive experiments, as demonstrated in Figure 5a and b.

Calibration curve (batch method)

To estimate OXY, a standard curve was created with a linear range (1-30) μg.mL-1, as represented in Figure 6. To test the accuracy and precision of the suggested procedure, two different levels of OXY drug were depended on the perfect conditions discussed in the created technique. The finding in Table 1 revealed that the proposed approach has a good precision and accuracy by noting few errors, relative error values, and high values for recovery.

Calculation of stability constant

Based on the outcome of the mole ratio approach, which revealed the ratio of BPY to OXY (3:1) as described in above subsection, the stability constant was measured for the hypothesized interaction. Two groups of solutions were made; the first included stoichiometric concentrations of OXY and the reagent BPY, and the second contained excess BPY. The complexation ratio between reagent and OXY is proposed by mechanism and stoichiometry (3:1). According to the equation, the reaction between OXY and BPY stability constant is: (K = 1-α/27α4C2) where, C represents the product's molar concentration (M), which is equal to the concentration of OXY, and α (the degree of dissociation) is expressed as: (α = Am-As/Am) where, Am and As represent the absorbance of a solution include reagent BPY increase and at the equivalent concentration, as shown in Table 2, ΔG; Gibbs free energy, R: constant of gases (8.314 J.mol-1. K-1), T: absolute temp. (298.15K).

Indirect spectrophotometric determination of OXY drug via FI system

By using the conventional spectrophotometric technique, the optimal situations for the reaction of OXY with BPY were established. To examine the optimum practical settings and get automated spectra with a method to estimate OXY, the FI/MZ methodology was used. Therefore, utilizing the batch method for OXY estimate, flow injection analysis methodologies were created.

Figure 6: Linear calibration curve for determination of OXY drug using Batch method.

Improve conditions of the developed FIA system

Chemical parameters

The best concentration of the reagent was verified by injecting several concentrations (6.4×10-4 -3.2×10-3 M) by using a homemade injection valve. It was found the best concentration (3.2×10-3 M) produced the best absorbance expressed as peak height in mV (n=3), as shown in Figure 7a. The best concentration of the oxidizing agent was verified by injecting several concentrations (6×10-4-3×10-3 M), and it was found that the best concentration was 3×10-3 M expressed as the peak height in mV (n=3), as indicated in Figure 7b. Figure 7c shows the best addition sequence, (L1=D, L2=O, and L3=R).

Physical variables

In this reaction, the best loops size, reaction coil length, and flow velocity were studied, as shown in Figure 8a, b, and c. The best loops size (117.75-78.50-78.50 μL), R.C length (50 cm) and flow rate 3.1 mL.min-1.

Purge time

The purge time of a discontinuous sample injected with distilled water as a carrier of chemicals was studied by using the best physical and chemical conditions. Different times were used in seconds, such as 5, 10, 15, 20, and the valve was opened. It was found that the best signal at the time of 20 seconds to get a pure and symmetrical response, as illustrated in the Figure 9.

 

Dispersion of OXY zone

The dispersion is a physical phenomenon that occurs in the flow injection technique as a result of the interaction of different solutions with the sample, it is then spread out across the solution. Three ideas underpin the success of FIA analytical technique [21] such as control on the dispersion of the sample zone, reproducible injection time, and reproducible sample injection volume. The reaction's dispersion was 1.3, as indicated in Figure 10 and Table 3. The dispersion was calculated according to the law:

D = Co/C. The peak height without dilution, (the reaction is outside the FI system) is Co, while the peak height with dilution, (the reaction is within the FI system) is C. In the first experiment, the materials are mixed in a beaker, and then the solution was sent through the system as a carrier to obtain a stable response expressed (Co) in the second experiment which the reaction occurs in FI system, according to following sequence, (OXY into L1, FeCl3 in L2 and BPY in L3). Distilled water is used as a carrier in the system. The components are pushed towards the reaction coil, and then towards the detector, resulting in a response represented by (C).

Calibration curve

After studying the ideal conditions for the reaction, a series of OXY solution was prepared from (1-800) µg.mL-1 injected into L1, FeCl3 in L2, and for BPY in L3. Each measurement was repeated 3 times. The response expressed as average peak height in mV (n=3) was plotted against the OXY concentrations (μg.mL-1), the linear range was (10-500) μg.mL-1, as demonstrated in Table 4 and Figure 11.

Analysis of variation (ANOVA) and repeatability

An ANOVA test is a way to find out if survey or experiment results are significant. In other words, they help to figure out the calibration curve if it is needed to reject the null hypothesis or accept the alternate hypothesis. For (n2) freedom degrees, the sum of squares of the difference was computed between the values of yi (response) and ŷi (appraiser response), (imply mistake), and call (about regression) [22, 23]. The variance of values yi was determined from the average value by calculating the sum of their squares (due to regression) and to obtain the sum of squares for one degree of freedom (S1)2, and then the (S1)2 was divided on (So)2 to obtain (F), as shown in the Table 5. Fcrit. (4.7472) << F (20.04). As a result, it is possible to conclude that there is a direct relationship between OXY concentrations and the acceptable signal.

The repeatability of the proposed FIA method was good, as presented in Table 6.

Methods validation

The analytical characteristic of a new technique, including the detection limit, correlation coefficient (r), relative standard deviation, and linear range were computed under the optimal conditions [24, 25], as listed in the Table 7. A calibration curve was created by using the fundamental analytical figure of deserts of the suggested method and a set of OXY standard solutions (Figure 11). Under 95 percent confidence intervals for (n-2) freedom degrees, the standard deviation for the residuals (Sy/x), slope (Sb), and intercept (Sa) of the regression line were shown. The excellent reproducibility of the proposed CFIA technique and high repeatability of outcomes were demonstrated to the small subjects compared the batch method. Because the current study was completed quickly (72 samples were analyzed in 1 hour) due to this technique is simpler and easier.

Effect of interferences

The interferences can be identified by excipients such as (glucose, sucrose, lactose, cellulose, and sodium citrate) were studied to check the accuracy of the proposed technique. A sample of pure 50 μg. mL-1 of OXY spiked with half, equal, and double fold excess the concentration of the selected interferences. The acceptable recovery values demonstrated that there were no interferences during the OXY determination by using new CFIA system, as indicated in the Table 8.

Applications and assessment of the suggested method

Three different medicines containing OXY were examined by the proposed method according to the standard addition method, as depicted in Table 9. The statistical comparison [26, 29] between the proposed method with official British Pharmacopeia method [30] by using the student F-test and t-test showed that the calculated F-test values were 0.1125 and 2.3109, t-test values were 1.2569 and 1.0223 less than the theoretical (critical) F-test (19.00) and t-test (2.78) via CFIA/MZ. The FIA technique is applied with successfully used to determine OXY in spiked human plasma and urine samples. The accuracy and precision of 50 μg.mL-1 of OXY were tested. Three times each concentration was examined. Table 10 and 11 shows that the plasma and urine samples have the acceptable reproducibility.

Conclusion

According to literature, survey in the field of flow injection analysis, there is a few researchers who have used this modern technology to estimate the OXY drugs in serum, plasma, and pharmaceutics. Therefore, the research idea was proposed which involves employing a new green method as CFIA technique. These methods can be used to calculate the OXY concentration in μg. mL-1 without the necessity for an earlier separation step, heating, or prepping the specimen. The main advantage of these methods is their wide operating range [31].

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors' contributions

All authors contributed to data analysis, drafting, and revising of the paper and agreed to be responsible for all the aspects of this work.

Conflict of Interest

We have no conflicts of interest to disclose.

ORCID

Shahad L. Hamed

https://orcid.org/0000-0003-4670-7274

HOW TO CITE THIS ARTICLE

Shahad L. Hamed, Bushra B. Qassim. Indirect Sensitive Determination of Oxymetazoline. HCl in Pure Pharmaceutical Drugs and Biological Samples Using a Modified Sensor Unit via a Green Method of FI/MZ System. Chem. Methodol., 2023, 7(1) 92-105

https://doi.org/10.22034/CHEMM.2023.361846.1607

URL: http://www.chemmethod.com/article_158717.html

[1]. Maleek M.I., Cytotoxic and inhibitory activity of ceramide on cancer cell lines, Journal of Biomedical Science and Engineering, 2010, 3:892 [Crossref], [Google Scholar], [Publisher] doi:.
[2]. Boxtel C.H., Santoso B., Edwards I.R., Drug benefits and risks. International Textbook of clinical Pharmacology. 2008. [Crossref], [Google Scholar], [Publisher]
[3]. Hegazy M.A.M., Al-Ghobashy M.A., Eltanany B.M., Khattab F.I., Spectral resolution and simultaneous determination of oxymetazoline hydrochloride and sodium cromoglycate by derivative and ratio-based spectrophotometric methods, European Journal of Chemistry, 2015, 6:319 [Crossref], [Google Scholar], [Publisher]
[4]. Aimanant S., Ziemann P.J., Development of spectrophotometric methods for the analysis of functional groups in oxidized organic aerosol, Aerosol Science and Technology, 2013, 47:581 [Crossref], [Google Scholar], [Publisher]
[5]. Palma Sr R.J., Palkowetz Jr, J.M., High Pressure Liquid Chromatographic Assay of Oxymetazoline Hydrochloride, Analytical Letters, 1981, 14:357 [Crossref], [Google Scholar], [Publisher]
[6]. Hoffmann T.J., Thompson R.D., Seifert J.R., Determination of the nasal decongestant, oxymetazoline hydrochloride, in pharmaceutical formulations by HPLC, Drug development and industrial pharmacy, 1989, 15:743 [Crossref], [Google Scholar], [Publisher]
[7]. Saleem M.S., Othman I.K., Indirect Spectrophotometric Determination of Oxymetazoline Hydrochloride in Pharmaceutical Formulation Using Fluorophotometric Method, Ibn AL-Haitham Journal For Pure and Applied Science, 2016, 29 [Crossref], [Google Scholar], [Publisher]
[8]. Al-Ghabsha T.S., Obedagha A.N., Zakaria R.A., Spectrophotometric Determination of Oxymetazoline Hydrochloride in Pure and Pharmaceutical Preparations Using Diazo-coupling Reaction, Journal of Education and Science, 2019, 28:224 [Crossref], [Google Scholar], [Publisher]
[9].  Chen Q., Li P., Yang H., Li B., Zhu J., Peng L., Nonaqueous capillary electrophoresis conditions for the simultaneous separation of eight alpha-adrenergic blocking agents, Analytical and bioanalytical chemistry, 2010, 398:937 [Crossref], [Google Scholar], [Publisher]
[10]. Hayes F.J., Baker T.R., Dobson R.L., Tsueda M.S., Rapid liquid chromatographic-mass spectrometric assay for oxymetazoline in whole rat blood, Journal of Chromatography A, 1995, 692:73 [Crossref], [Google Scholar], [Publisher]
[11]. García-Campaña A.M., Sendra J.M.B., Vargas M.P.B., Baeyens W.R.G., Zhang X., Flow injection analysis of oxymetazoline hydrochloride with inhibited chemiluminescent detection, Analytica chimica acta, 2004, 516:245 [Crossref], [Google Scholar], [Publisher]
[12]. Issa Y.M., Zayed S.I.M., Construction and analytical applications of plastic membrane electrode for oxymetazoline hydrochloride, Analytical sciences, 2004, 20:297 [Crossref], [Google Scholar], [Publisher]
[13]. Hamed L.L., Qassim B.B., Direct and new flow injection method for assay of Iron as ferrous sulfate in pure and dosage forms through the complexation with 2, 2︡ dipyridyl reagent, International Journal of Pharmaceutical Research, 2020, 12:1329 [Crossref], [Google Scholar], [Publisher]
[14]. Qassim B.B., Hamed L.L., Simple green method high throughput flow injection technique for spectrophotometry determination of Fe (III) in iron drugs through the reaction between DPA-4-sulfonat with hydrogen peroxide using a modified detection unit, International Journal of Drug Delivery Technology, 2020, 10:563 [Crossref], [Google Scholar], [Publisher]
[15]. Qassim B.B., Zydan A.A., Sensitive Simultaneous Estimation of Atorvastatin. Ca in Pure and Dosage Forms Via Developed CFIA Using 1, 2 Naphthoquinone-4-Sulfonate as a Suitable Organic Agent, Indian Journal of Forensic Medicine & Toxicology, 2020, 14:2161 [Crossref], [Google Scholar], [Publisher]
[16]. Qassim B.B., Zydan A.A., Novel approach of oxidation-reduction reaction with kmno4 for simultaneous determination of simvastatin drug in either pharmaceutics preparation or human urine using homemade FIA-stopped-flow/merging zone technique, BioChem, 2020, 20 [Crossref], [Google Scholar], [Publisher]
[17]. Bothwell T.H., Mallett B., The determination of iron in plasma or serum, Biochemical Journal, 1955, 59:599 [Crossref], [Google Scholar], [Publisher]
[18]. Abdullah H.J., Qassim B.B., Development and Validation CFIA/MZ System as a Green Method for Determination of Thiol Drug (D-PEN), Egyptian Journal of Chemistry, 2022, 65:259 [Crossref], [Google Scholar], [Publisher]
[19]. Qassim B.B., Hamed L.L., A new green method for indirect determination of ferric ions in biological samples using Ascorbic acid as reducing agent via the development of CFIA system, Journal of Physics, 2021, 1999:012145 [Crossref], [Google Scholar], [Publisher]
[20]. Kochi J.K., Mechanisms of Organic Oxidation and Reduction by Metal Complexes: Electron and ligand transfer processes form the basis for redox reactions of radicals and metal species, Science, 1967, 155:415 [Crossref], [Google Scholar], [Publisher]
[21]. Krull I.S., Swartz M., Analytical method development and validation for the academic researcher, Analytical Letters, 1999, 32:1067 [Crossref], [Google Scholar], [Publisher]
[22]. López A., Ferrero F.J., Valledor M., Campo J.C., Reviejo J., Pingarrón J.M., Development of Continuous Flow Analysis System Based on Amperometric Biosensors, IEEE Sensors Journal, 2022, 22:7542 [Crossref], [Google Scholar], [Publisher]
[23]. Ellison S.L., Barwick V.J., Farrant T.J.D., Practical statistics for the analytical scientist: a bench guide, Royal Society of Chemistry, 2009 [Crossref], [Google Scholar], [Publisher]
[24]. Suarez W.T., Madi A.A., Figueiredo-Filho L., Fatibello-Filho O., Flow-injection spectrophotometric system for captopril determination in pharmaceuticals, Journal of the Brazilian Chemical Society, 2007, 18:1215 [Crossref], [Google Scholar], [Publisher]
[25]. Tzanavaras P.D., Themelis D.G., Economou A., Theodoridis G., "Flow and sequential injection manifolds for the spectrophotometric determination of captopril based on its oxidation by Fe (III)." Microchimica Acta, 2003, 142:55 [Crossref], [Google Scholar], [Publisher]
[26]. Yaseen S.M., Qassim B.B., Al-Lami N.O., "Spectrophotometric Determination of Co (II) in Vitamin B12 Using 2-(biphenyl-4-yl)-3-((2-(2, 4-dinitrophenyl) hydrazono) methyl) imidazo [1, 2-a] pyridine as Ligand by Flow Injection–Merging Zone Analysis." Al-Nahrain Journal of Science, 2020, 23:24 [Crossref], [Google Scholar], [Publisher]
[27]. Tawfeeq, A.H., Qassim B.B., "A Novel Method of CFIA/Merging zones technique for assay of Doxycycline in Bulk and Pharmaceutical preparation depending on Azo Dye Formation," Research Journal of Pharmacy and Technology, 2021, 14:67 [Crossref], [Google Scholar], [Publisher]
[28]. Qassim, Bushra B., and Ahmed A. Alwan. "Indirect Way for the Assay of Captopril Drug in Dosage FormsUsing1, 10- Phenanthroline as a Selective Spectrophotometric Agent for Fe (II) Via Homemade CFIA/Merging Zones Technique," Ibn AL-Haitham Journal for Pure and Applied Science, 2018, 294 [Crossref], [Google Scholar], [Publisher]
[29]. Yaseen S.M., Qasim B.B., Al-lame N.O., Spectrophotometric Determination of Cu (+ II) by Complexation with 2-(4-biphenyl) Imidazo [1, 2-] Pyrimidine-3-Hydrazone and Studying Characteristics of prepared complex, Egyptian Journal of Chemistry, 2021, 64:4 [Crossref], [Google Scholar], [Publisher]
[30]. British Pharmacopoeia (2005), version 4, CD-ROM, Her Majesty’s Stationery Office, pp. 1185 [Crossref], [Google Scholar], [Publisher]
[31]. Turkey N.S., Jeber J.N., Flow Injection Analysis with Turbidity Detection for the Quantitative Determination of Mebeverine Hydrochloride in Pharmaceutical Formulations, Baghdad Science Journal, 2022, 19:141 [Crossref], [Google Scholar], [Publisher]