Microsoft Word - 07-D-PJAEC-28012022-422-C-Revised Galley Proof-29- Cross Mark ISSN-1996-918X Pak. J. Anal. Environ. Che m. Vol. 23, No. 2 (2022) 247 – 258 http://doi.org/10.21743/pjaec/2022.12.07 Spectrophotometric Determination of Catecholamine Containing Drugs Using Calcon Dye Ikram K. Othman and Theia’a N. Al-Sabha* Chemistry Department, College of Education for Pure Science, Mosul University, Mosul, Iraq. *Corresponding Author Email: dr_theiaa@yahoo.co.uk Received 28 January 2022, Revised 03 July 2022, Accepted 27 September 2022 -------------------------------------------------------------------------------------------------------------------------------------------- Abstract Calcon dye has been used for spectrophotometric determination of catecholamine-containing drugs, namely, adrenaline, methyldopa and dopamine in their pure forms and pharmaceutical formulations. The method is based on the oxidation of the above drugs with an excess of N- bromosuccini mide (NBS) in an acidic medium. The residual oxidizing agent bleaches the blackish-brown color of calcon measured at 510 nm. The decolorization of the dye is proportional to the residual amount of NBS, which is proportional to the concentration of the drug. Linear calibration graphs were obtained in the concentration range 0.5-16.0, 2.0-40.0 and 1.036.0 μgmL-1 with molar absorptivity values 1.10×104, 3.2×103 and 4.3×103 Lmol-1cm-1 for above drugs, respectively. The method is simple, sensitive, accurate, precise and free from excipients. The developed method was successfully applied to determine the drugs in their pharmaceutical formulations. Keywords: Catecholamine drugs, Calcon, Oxidation, Spectrophotometry -------------------------------------------------------------------------------------------------------------------------------------------- Introduction Catecholamines are a class of monoamines synthesized from tyrosine, which contain a catechol group and a side chain with an amino group in their structure [1]. Catecholamines- containing drugs are widely used to treat different disorders, such as playing a key role in the mechanisms of emotions, learning, memorization, and sleep, as well as in psychomotor activity and neural regulation [2]. Adrenaline (Epinephrine) is a hormone that is involved in regulating visceral functions (such as respiration) [3,4]. It is chemically known as 4-[(1R)-1-hydroxy-2- (methylamino)ethyl]benzene-1,2-diol (I). It is a neurotransmitter for the mammalian central nervous system. Many diseases are associated with a change in catecholamine concentration. It is an active principle of adrenal gland balm and is a drug used to treat nasal congestion, asthma, heart block, hypotension and cardiac arrest. A change in the concentration of adrenaline in biological fluids (blood, urine, cerebrospinal fluid) can serve as a reliable indicator of a violation of homeostasis [5,6]. Methyldopa, is in the family of alpha-2-adrenergic receptor agonists. Chemically it is named S-2-amino-3-(3,4- dihydroxyphenyl)-2-methyl-propanoic acid (II). It is an antihypertensive drug used to lower blood pressure and treat some hypertension and gestational hypertension cases in renal failure and complicated pregnancies [7,8]. Dopamine constitutes approximately 80% of the catecholamine content material Pak. J. Anal. Environ. Che m. Vol. 23, No. 2 (2022) 248 with inside the brain [9]. Chemically known as 2-(3, 4-dihydroxyphenyl) ethylamine (III), It is classified among the heart-stimulating drugs [10], which is a hormone secreted by the adrenal gland. It contributes to the fight or flight response processes [11] and the circulatory system (shock) due to myocardial infarction and trauma [12]. (I) (II) (III) Different techniques have been described for determination of catecholamine drugs such as voltammetry [13-16], chromatography [17-21], amperometry [22], capillary electrophoresis [23,24] and fluorometry [25]. However, these techniques are expensive, need experience and are not available in all laboratories. The spectrophotometric technique is still the preferred method due to its simplicity. Various spectrophotometric methods using different reagents have been reported for the determination of catecholamine drugs. These methods include ion association using eosin Y and application of cloud point extraction technique [26], charge transfer complexes using bromanil [27], oxidation-reduction using ferric ion [28-30], diazotisation using p- nitroaniline [31], 4-aminoantipyrine in the basic medium [32], complexation reaction using 4-aminoantipyrine and copper ion, ion- pair complex using triiodide ion [33] and using ferrous ion as a complexing agent in alkaline medium [34], oxidative coupling using p-toluidine in the presence of sodium periodide [35], 2, 6-diaminopyridine in the presence of potassium periodate [36] and 3- methyl-2-benzothiazolinone hydrazone hydrochloride monohydrate in the presence of potassium ferricyanide [37]. The aim of this study is to develop an accurate, simple, sensitive and cost-effective method for the spectrophotometric determination of catecholamine containing drugs. The method is based on the oxidation of drugs by N-Bromosuccinimide (NBS) in an acidic medium, and the residual oxidant, which is proportional to the drug concentration, bleach the color of calcon dye and reduction in absorbance is measured at 510 nm, which forms the basis of determination method. Materials and Methods Instruments: UV-Visible spectrophotometer type Shimadzu UV-1650 PC equipped with a 1.0-cm path length silica cell, pH-meter with a combined glass electrode type Philips PW (9421) was used for pH measurements. All calculations in the computing process were performed in Microsoft Excel format. Reagents: Calcon reagent was prepared in a concentration of 500 μgmL-1 by dissolving 0.05 g in distilled water in a 100 mL volumetric flask. Oxidizing agent (NBS) was prepared in a concentration of 5×10 -3 M by dissolving 0.0890 g in 100 mL distilled water. Hydrochloric acid was prepared in a concentration of 1 M by diluting an appropriate volume of conc. HCl. Pak. J. Anal. Environ. Che m. Vol. 23, No. 2 (2022)249 Stock Solutions of Drugs: Stock solutions of adrenaline, methyldopa and dopamine were prepared in a concentration of 100 μgmL -1 by dissolving 0.01 g of each drug in 100 mL distilled water in volumetric flasks. The solutions were kept in the refrigerator. Sample Preparation Aliquots containing 0.5-16.0, 2.0-40.0 and 1.0-36.0 μgmL -1 of adrenaline, methyl dopa and dopamine standard solutions were added respectively, and separated into three series of 10 mL volumetric flasks, followed by adding 1 mL of 1 M HCl and 1.5 mL of 5×10 - 3 M NBS. The solutions were left for 10 min in a water bath adjusted at 30ᵒC for completion of the oxidation process, then 2.6 mL of 130 μgmL -1 calcon was added. The solutions were diluted to the mark, and the absorbance was measured at 510 nm after 20 min for dopamine and 15 min for adrenaline and methyldopa against respective reagent blank. Procedure for Drug Formulations Injection Adrenaline: The content of five ampoules of adrenaline (each ampoule contains 1.0 mg/1 mL adrenaline) or adrenaline darnitsa (each ampoule contains 1.8 mg/1 mL adrenaline) were mixed and then diluted to 50 mL with distilled water to obtain 100 or 180 μgmL -1 respectively. Different volumes of these solutions were taken to obtain concentrations 1.0, 4.0, 6.0 and 10.0 µgmL -1, and they were treated according to the general procedure. The concentration of adrenaline per ampoule was found using the standard calibration curve for the drug in its pure form. Dopamine HCl: The content of three dopadren ampoules (each ampoule contains 200 mg/5 mL of dopamine. HCl). 2 mL were mixed and diluted to 100 mL with distilled water to obtain a solution of 800 µgmL -1 . Then, a solution was prepared by dilution with a concentration of 100 μgmL -1 . Different volumes of the solution were taken to obtain concentrations 4.0, 10.0, 16.0 and 24.0 μgmL-1. They were treated according to the general procedure. The dopamine concentration was found in the ampoule using the calibration curve of the drug in its pure form. Tablet: Ten tablets of aldosam or aldomac (each tablet contains 250 mg methyldopa) were carefully weighed, crushed and mixed well. The weight of the powder equivalent to one tablet was dissolved in 20 mL of distilled water and heated in a water bath for a few minutes to increase the solubility. The solution was allowed to cool and filtered in a 100 mL volumetric flask, then completed to the mark with distilled water to obtain 2500 µgmL -1 . The solution was diluted to 100 µgmL-1, and 4.0, 10.0, 16.0 and 24.0 µgmL-1 treated according to the general procedure. Results and Discussion Calcon, called Eriochrome Blue Black R or Mordant Black 17. It is chemically known as 2-Hydroxy-1-(2-hydroxy-1- naphthylazo)naphthalene-4-sulfonic acid sodium salt (I V) that was proposed by Hildebrand and Reilley in 1958 to be used as an indicator for calcium ion [38, 39]. (IV) In the preliminary investigation, it was found that calcon dye has maximum absorption at 510 nm. Experimentally, a quantitative oxidation of calcon dye was noticed in the presence of the oxidizing agent NBS in the acidic medium. The absorbance of the dye decreased by increasing the Pak. J. Anal. Environ. Che m. Vol. 23, No. 2 (2022) 250 concentration of the oxidizing agent by bleaching its color. Based on this feature, the possibility of indirect spectrophotometric determination of the catechol amine drugs (adrenaline, methyldopa and sopamine hydrochloride) has been studied. The increasing concentration of drugs leads to a decrease in the concentration of oxidizing agents for bleaching calcon color. It also leads to an increase of the absorbance at 510 nm, which is proportional to the drug concentration (Fig.1). However, the optimum conditions for the quantitative determination of the above drugs have been considered. Figure 1. Absorption spectra of 130 μgmL-1 calcon i n aci dic medi um (A) i n the presence of NBS and 10 μgmL-1 adrenaline (B), 20 μgmL-1 methyldopa (C) and 15 μgmL-1 dopami ne (D) agai nst reagent bl ank (E) Optimization of Calcon Dye Concentration: To select the optimum amount of calcon to be used for the determination of catecholamine drugs, increasing volumes (milliliters) of a dye solution at a concentration of 500 µgmL-1 were added to a 10 mL volumetric flasks containing 1.0 mL of 1 M HCl. The volume was supplemented with distilled water to the mark, and the absorption was measured at 510 nm. It was found that the linear concentration range of calcon dye was 1-130 µgmL -1 . Therefore, the concentration was fixed at 130 µgmL-1 for the estimation of the drug. Optimization of Oxidizing Agent, Concentration and Volume: The effect of the oxidizing agents, including NBS, potassium dichromate, chloramine-T and cerium (IV) sulfate with a concentration of 5×10 -3 M on the bleaching of the dye in the medium of HCl was considered. The results, cited in Fig. 2, indicated that NBS was the most suitable in the oxidation of the dye, as it gave the lowest absorbance; therefore, it was adopted in the present method. The effect of NBS concentration and volume was studied. It was found that 1.5 mL of 5×10 -3 M was the best for this method. Figure 2. Effect of oxidi zi ng agent on the bleachi ng of 130 µgmL-1 cal con Effect of Acid Experimental results demonstrated that the oxidation of calcon dye and the studied drugs by NBS were present in an acidic medium and at room temperature (23°C) for adrenaline but at 30°C for methyldopa and dopamine. Therefore, the effect of different acids of 1 M was examined for determination of the above drugs in the presence of 130 µgmL -1 calcon dye separately. The results in Table 1 indicate that HCl was suitable acid. However, the concentration and volume of HCl were studied. It was found that 1 mL of 1 M gave maximum absorbance, Table 2 & Fig. 3. The conducted procedure was recommended in subsequent experiments. Figure 3. Effect of volume of 1 M HCl on the absorbance of drugs Pak. J. Anal. Environ. Che m. Vol. 23, No. 2 (2022)251 Table 1. Effect of 1 mL of 1 M aci d on the absorbance of drugs. Absorbance CH3COOHHNO3H3PO 4H2SO 4HCl μgmL-1Drug 0.2510.2840.3170.3690.39310 Adrenaline 0.1060.1170.1360.1770.19220 Methyldopa 0.1310.1470.1850.2260.28515 Dopamine. HCl Table 2. Effect of mol ar concentration of HCl on the absorbance of drugs. Mol ari ty of HCl /Absorbance 3.02.52.01.51.00.5 μgmL-1Drug 0.2210.2520.2960.3490.3850.33110Adrenalin 0.0910.1120.1340.1770.1960.15220Methyldopa 0.1270.1610.1910.2330.2850.17415Dopamine. HCl Effect of Time on the Oxidation and Bleaching: This study was conducted to determine the time period required for the oxidation of the pharmaceutical compounds and the dye with NBS in hydrochloric acid at different times with shaking. The results in Fig. 4 showed that 10 min was sufficient time for the oxidation of drugs before adding a calcon dye, 15 min for bleaching of a dye for adrenaline and methyldopa, and 20 min for dopamine hydrochloride. The reaction remained stable for at least 24 h for all drugs. Figure 4. Effect of ti me on the oxi dation of drugs and bleaching of calcon for (A) adrenali ne, (B) methyldopa and (C) dopamine. HCl Pak. J. Anal. Environ. Che m. Vol. 23, No. 2 (2022) 252 LOD =,LOQ = Effect of Temperature on Oxidation Reaction and Stability: The effect of the temperature and time on the oxidation of catecholamine drugs was studied in the range 23°C (Room temperature; RT) - 50°C using the optimum conditions and concentrations of the reagents as mentioned above. The absorbance of the dye was measured after 5 min intervals at 510 nm. The results showed in Fig. 5, A, B, and C indicated that high absorbance was reached after 30 min for dopamine and 20 min for adrenaline and methyldopa at 30°C and remained constant for more than 3 h. Whereas higher temperature decreases the absorbance. Effect of Addition Sequence: The addition sequence must follow the general procedure; otherwise, a decrease in absorbance was noticed. Linearity and Calibration Graph: Under the optimum conditions mentioned above, standard calibration graphs for adrenaline, methyldopa and dopamine. HCl with calcon dye was created by plotting the absorbance versus concentration Fig. 6. The proposed methods obeyed Beer’s law in the ranges 0.5- 16.0, 2.0-40.0 and 1.0-36.0 µgmL -1 with low intercept and good correlation coefficients for above drugs respectively. The molar absorptivity values were estimated, and they indicated that the method was sensitive (Table 3). Accuracy (average recovery %) and the relative standard deviation (RSD) for the analysis of three replicates of different concentrations for each of the above drugs explain that the method is accurate and precise. The detection limit (LOD) and quantitation limit (LOQ) [40] were calculated by the subsequent equations: Where: σ = standard deviation of the blank and b = slope of the calibration curve. The results, cited in Table 3, are below the lower limit of Beer's law range. Figure 5. Effect of temperature on the absorbance and stability of the oxi di zation of adrenaline (A), methyl dopa (B) and dopami ne (C) Pak. J. Anal. Environ. Che m. Vol. 23, No. 2 (2022)253 Figure 6. Calibration graphs of adrenaline, methyldopa and dopami ne. HCl Table 3. Quantitative parameters and statistical data for assay of adrenali ne, methyl dopa and dopami ne. Average٭ for six determinations Suggested Mechanism: NBS is an oxidizing agent. It behaves as a bromonation agent in the acidic medium for aliphatic and aromatic organic compounds [41-43]. This method assumed that NBS oxidizes the catecholamine drugs (adrenaline, methyldopa and dopamine hydrochloride) in an acidic medium. Then, the unreacted NBS oxidizes the known amount of calcon and bleach its color. The decrease of NBS concentration upon oxidation of known concentration of drugs leads to an increase in the absorbance of the dye at 510 nm, which is linearly proportional to the concentrations of the studied drug compounds. The suggested mechanism is explained in Scheme 1. Analysis of Catecholamine Medicines in Commercial Formulations: The present method has been successfully applied for the determination of catecholamine drugs (Injection and tablet). The results mentioned in Table 4 indicate that the proposed method is accurate and reproducible. Validation of the Method: For the purpose of demonstrating the efficiency of the developed method, its success in estimating catecholamine drugs, and its freedom from the effect of excipients in its pharmaceutical preparations, the standard addition technique has been applied to pharmaceutical Dopami ne. HClMethyldopaAdrenali neParameters 1.0-36.02.0-40.00.5-16.0Linearity range (µgmL-1) 0.43×1040.32×1041.10×104Molar absorptivity (Lmol-1cm-1) 44.4474.0716.67Sandell’s Sensitivity (µgcm-2) Average٭100.0699.83101.42 recovery (%) 0.1410.2340.053LOD ( µgmL-1) 0.4680.7810.176LOQ ( µgmL-1) RSD٭0.350.850.38 0.02160.02390.0423Intercept 0.02250.01350.0600Slope 0.99700.99630.9968Correlation coefficient (R2) Pak. J. Anal. Environ. Che m. Vol. 23, No. 2 (2022) 254 preparations for the above drugs. The method was summarized by adding increasing amounts of a standard solution of pure drug to a known quantity of the pharmaceutical preparation. By following the described method, the absorption was measured at the wavelength of 510 nm, and the obtained results were included in Table 5, which indicates that the method has good selectivity. Scheme 1. Suggested mechani sm for oxi dation of catechol ami ne drugs and calcon by NBS R= , , 1-Naphthol Pak. J. Anal. Environ. Che m. Vol. 23, No. 2 (2022)255 Table 4. Anal ysi s of the catechol ami ne medicines i n commerci al formul ations. Average recovery (mg) Average recovery (%) Recoverya (%) Drug content found* (mg) Amount present (µgmL-1) Certi fi ed val ue Pharmaceutical preparation 100.501.011 98.040.984 99.811.006 0.99599.52 99.721.0010 1 mg Adrenali ne ampouleb 98.830.991 100.541.014 98.690.996 0.99299.20 98.720.9910 1 mg Adrenali ne ampoulec 100.501.811 99.291.794 100.081.806 1.79999.98 100.051.8010 1.8 mg Adrenali ne ampouled 97.96244.904 99.93249.8310 99.49248.7316 243.35099.34 99.97249.9324 250 mg Al dosam tablete 99.81249.534 100.67251.6710 100.42251.0416 250.285100.14 99.66249.1524 250 mg Al domac tabletf 99.56199.114 100.26200.5210 99.89199.7816 199.2699.63 98.81197.6224 200 mgDopadren ampoul eg aAverage of four determinations Manufactured by bLINCOLN Pharmaceuticals, India, cOSEL-Turkey, dDARNITSA Pharmaceuticals, Firm-Ukraine,eSDI-Iraq,fMACLEODS Pharmaceuticals Ltd-India and g VEM Pharmaceuticals-Turkey. Table 5. Effect of exci pient i nterferences by the standard addi tion procedure. Drug content found (mg) Average recovery (%) Recovery (%) Amount present (µgmL-1) Certi fi ed val uePharmaceutical preparation 97.494 0.98798.78 100.076 1 mgAdrenaline ampoule (India) 97.054 0.98398.36 99.686 1 mgAdrenaline ampoule (Turkey) 96.144 1.7597.21 98.286 1.8 mgAdrenaline ampoule (Ukraine) 97.154 244.8797.95 98.7510 250 mgAldosam tablet (Iraq) 99.004 249.3299.73 100.4710 250 mgAldomac tablet (India) 199.3499.6798.374200 mgDopadren ampoule (Turkey) Pak. J. Anal. Environ. Che m. Vol. 23, No. 2 (2022) 256 Table 6. Comparison between the proposed method and other methods. Literature methods Present method [44] [36] [29] Anal ytical parameter Adrenaline Methyldopa Dopamine Adrenaline Methyldopa Dopamine Reagent Calcon V+5 Diaminopyridine- KIO4 Fe III-K3 Fe(CN)6 Method Oxidation Chelating complex oxidative coupling Prussian blue formation λmax (nm ) 510 488 478 735 Linearity (µgmL-1) 1.0-36.0 2.0-40.0 0.5-16.0 0.5–140 2-24 0.05–6.00 Molar absorptivity Lmol-1cm-1 1.10×104 0.32×104 0.43×104 2.015×103 1.0481 x 104 3.2 x 104 Temp. (Cᵒ) 30 70 25 R.T. RSD 0.35 0.85 0.38 ≤ 0.88 ≤ 0.86 0.65 Applications Injection Tablet Injection Injection Tablet Injection Comparison between the proposed method and other spectrophotometric methods: The suggested method for the determination of catecholamine drugs has been compared with other described spectrophotometric methods in the literature. The current method was distinguished by its sensitivity over some of the spectrophotometric methods listed in Table 6. This method is simple and could be applied to all catecholamine-containing drugs in their pure form and pharmaceutical preparations. Conclusion A new spectrophotometric method has been suggested for the determination of catecholamine-containing drugs. The method is dependent on the oxidation of drugs by NBS and subsequently reacted with calcon dye. The decolorization of the dye, measured at 510 nm, is proportional to the residual amount of NBS, which is proportional to the concentration of the drug. The method is simple, sensitive and accurate. The linearity range for calibration curves were 0.5-16.0, 2.0-40.0 and 1.0-36.0 μgmL-1 with molar absorptivity values 1.10×10 4 , 3.2×10 3 and 4.3×10 3 Lmol -1 cm -1 for adrenaline, methyldopa and dopamine respectively. The method was applied successfully for the determination of the intended drugs in their pharmaceutical formulations as tablets and injections with good recovery values. 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