SIMPLE SPECTROPHOTOMETRIC METHODS FOR ESTIMATION OF DRUGS AND PHARMACEUTICALS USING NBS-METHYL ORANGE DYE COUPLE

SIMPLE SPECTROPHOTOMETRIC METHODS FOR ESTIMATION OF DRUGS AND PHARMACEUTICALS USING NBS-METHYL ORANGE DYE COUPLE

B. Vishnuvardhan Reddy, Gopi Mamidi and G. Venkateshwarlu ^*

Department of Chemistry, University College of Science, Osmania University, Hyderabad - 500007, Telangana, India.

ABSTRACT: Simple, sensitive, cost-effective and selective methods are designed and developed for the oxidative indirect spectrophotometric determination of drugs and pharmaceuticals, viz., Argatrobane Monohydrate (ARG), Bortezomib (BOR), Chloroquine Phospate (CHP), Granisetron HCl (GRA), Ibandronate Sodium (IBA) based on their reactivity towards n-bromo succinamide (NBS in excess). The method is based on the oxidation of drugs by n-bromo succinamide (excess) and estimating the amount of un-reacted NBS by Methyl Orange dye at λ_max 508 nm. The calibration curves obeyed Beer’s law over the concentration range of 10-70 µg mL^-1 (ARG), 8-56 µg mL^-1 (BOR), 6-42 µg mL^-1 (CHP), 4-28 µg mL^-1 (GRA) & 5-35 µg mL^-1 (IBA). This method has been applied for the determination of drugs in their pure form as well as in tablet formulations. Statistical analysis of the results using Student’s t-test for accuracy and F-test for precision revealed no significant difference between the proposed methods and the literature method at the 95% confidence level with respect to accuracy and precision. The method has been validated in terms of guidelines of the International Council of Harmonization.

Argatrobane monohydrate, Bortezomib, Chloroquine phosphate, Granisetron HCl, Ibandronate sodium, Estimation, NBS, Methyl orange

INTRODUCTION: Argatrobane Monohydrate: Argatrobane monohydrate, ARG Fig. 1 is chemically [((2R, 4R)-4-methyl-1,2,3,4-tetrahydro-8- quinolinesulfonyl)- L- arginyl- 2- piperidine-carboxylic acid mono hydrate]. It is a direct thrombin inhibitor under clinical development as adjunctive therapy to thrombolytic agents in acute myocardial infarction (AMI). Recent clinical trials have shown argatroban to be especially effective when administered in conjunction with a thrombolytic agent within 6 h of the onset of AMI symptoms.

Biochemical studies have shown that argatroban is a potent and selective thrombin inhibitor; Argatroban is a direct thrombin inhibitor that reversibly binds to the thrombin active site. Argatroban does not require the co-factor antithrombin III for antithrombotic activity. Argatroban exerts its anticoagulant effects by inhibiting thrombin catalyzed or -induced reactions.

FIG. 1: CHEMICAL STRUCTURE OF ARGATROBAN MONOHYDRATE

Argatroban is capable of inhibiting the action of both free and clot-associated thrombin ¹. Several methods are known for the quantitative determination of ARG ^2-8.

Bortezomib: Bortezomib is a novel cytotoxic chemical entity that potently and specifically inhibits the proteolytic activity of the proteasome and thus the degradation of poly-ubiquitinated proteins destined for catalysis by the proteasome. Bortezomib is a modified dipeptidyl boronic acid derived from leucine and phenylalanine. The chemical name for bortezomib, the monomeric boronic acid, is [(1R)-3- methyl-1-[[(2S)-1-oxo-3-phenyl- 2- [(pyrazinyl carbonyl) amino] propyl] amino]butyl]boronic acid, Fig. 2 ⁹.

FIG. 2: CHEMICAL STRUCTURE OF BORTEZOMIB

Because of its physiological significance, it has been quantified by several methods which are enumerated in the recent reference ^10-16. 

Chloroquine Phosphate: Chloroquine phosphate (CQP), chemically known as 7-chloro-4[{4-(diethylamino)-1-methylbutyl} amino] quinoline, is a 4-aminoquinolein antimalarial drug Fig. 3. It is the prototype synthetic antimalarial drug most widely used to treat all types of malaria infections.

The drug is also prescribed to decrease the symptoms of rheumatoid arthritis and to treat systemic and discoid lupus erythematosus in adults ¹⁷. Determination of chloroquine concentrations in biological samples is important for several reasons, such as assessment of patient compliance, evaluation of pharmacokinetic data and prevention of toxic blood concentrations after prolonged use, especially in the case of treatment of rheumatoid arthritis. Reliable analysis methods are also required for quality control of chloroquine preparations.

FIG. 3: CHEMICAL STRUCTURE OF CHLOROQUINE PHOSPHATE

Other analytical methods reported for the assay of CLQ in pharmaceuticals include visible spectrophotometry ^18-21, spectrofluorimetry ²², high-performance liquid chromatography ^23-25.

Granisetron Hydrochloride: Granisetron hydrochloride (as shown in Fig. 4) is an effective and potent antiemetic drug which is used in the treatment of vomiting and nausea resulting from cancer chemotherapy and radiotherapy in adults and children. Granisetron hydrochloride is also effective in the management of postoperative nausea and vomiting due to the anesthetics ²⁶, Chemically, it is endo-N(9-methyl-9- azabicyclo [3.3.1] non- 3- yl)- 1- methyl- 1H- indazole- 3-carboxamide hydrochloride. GTH selectively blocks type 3 serotonin (5-HT3) receptors, is an antinauseant, antiemetic and specific selective serotonin 5-HT3 receptor antagonist Fig. 4. The molecular formula is C₁₈H₂₄N₄O•HCl and its molecular weight is 348.9.

FIG. 4: CHEMICAL STRUCTURE OF GRANISETRON HYDROCHLORIDE

A few analytical methods viz; RP-HPLC ^{27, 28}, HPTLC ²⁹, and spectrophotometry ^{30, 31} have been developed for the determination of phenylephrine hydrochloride. The aim of this study is to develop simple, selective, accurate, precise and sensitive stability-indicating UV method for the determination of Granisetron hydrochloride in bulk and in pharmaceutical dosage forms (tablets) suitable for routine quality control analysis.

Ibandronate Sodium: Ibandronate sodium (IBA) is one of the nitrogen carrying Bisphosphonate. According to IUPAC nomenclature it is 3-(N-methyl-N-pentyl) amino-1-hydroxypropane-1, 1-diphosphonic acid, sodium salt, monohydrate Fig. 5. It prevents osteoclast-conciliate boneresorption ³². It is precious for the cure of hypercalcemia of malignancy ³³, Paget's disease, postmenopausal osteoporosi and corticosteroid-induced osteoporosis metastatic bone disease ³⁴.

FIG. 5: CHEMICAL STRUCTURE OF IBANDRONATE SODIUM

Several methods have been reported for quantitative determination of IBD in UV-Vis spectrophotometry ³⁵, Ion chromatography and ion pair chromatography ^36-37, RP-HPLC method ³⁸ for bulk drug.

MATERIALS:

Instrument: All absorbance measurements were recorded on Shimadzu 140 double beam spectrophotometer as well as on Thermo Nicolet 100 & Elico double beam SL210 UV- Visible spectrophotometers using matched pair of Quartz cells of 10mm path length.

Materials and Reagents: All the reagents used were of analytical reagent grade and distilled water was used throughout the investigation. NBS solution (0.01%) was prepared by dissolving N-bromosuccinimide (Himedia Laboratories Pvt. Ltd, Mumbai) in water with the aid of heat and standardized. The solution was kept in an amber-colored bottle and was diluted with distilled water appropriately to get 70 μg mL^–1 NBS for use in the spectrophotometric method.

A stock solution of Methyl Orange (5 × 10^-4 M) was prepared by dissolving the dye (s. d. Fine Chem. Ltd., Mumbai) in water and filtered using glass wool. The dye solution was diluted to 50 μg mL^–1.

Hydrochloric Acid (1 M): Concentrated hydrochloric acid (S.D. Fine Chem., Mumbai, India; sp. gr. 1.18) was diluted appropriately with water to get 1 M acid.

The pharmaceutical grade drugs were supplied by Arabindo pharmaceuticals and hetero drugs Pvt. Ltd Hyderabad. A stock standard solution of drugs was prepared by dissolving accurately weighed 10 mg of pure drug in water and diluting to 100 mL in a calibrated flask with water. The solution was diluted stepwise to get working concentrations.

Assay Procedure: Aliquots containing 2-70 µg mL^-1 of the drug were transferred into a series of 10 mL standard flasks using a micro burette. To this, 1 mL of NBS was followed by 1 mL of 1M HCl and contents were shaken well. After 30 minutes, 1 mL of methyl orange dye added to the content. Then contents were shaken well and diluted up to the mark. The absorbance of each solution was measured at 508 nm against the corresponding reagent blank.

Calibration curves were constructed for all the drugs by plotting the absorbance versus the concentration of drugs. The absorbance data was collected for six replicate experiments and absorbance to concentration ratio called the relative response was determined. The relative responses between 95% to 105% of average only are considered for construction of the Calibration curves Fig. 6.

FIG. 6: CALIBRATION CURVES OF THE DRUGS

Procedure for Assay of Pure Drug: Sample solutions of each drug in the beer’s law limits were chosen and recovery experiments were performed to check the accuracy and precision. The concentration chosen and recovery are tabulated in Table 2. For this purpose, the standard deviation method also adapted. Excellent recovery and % RSD being less than 2 speaks about the precision and accuracy of the method Table 1.

TABLE 1: DETERMINATION OF ACCURACY AND PRECISION OF THE METHODS ON PURE DRUG SAMPLES

Procedure for Tablets:

Argatrobane Monohydrate:

Argatroban monohydrate injection is a sterile non-pyrogenic, clear, colorless, to a pale yellow isotonic solution. It is available as a single-use polyolefin bag containing 250 mg of argatroban in 250 ml sodium chloride solution (1 mg/ml). Convenient aliquots were taken from this for the determination of argatroban.

Bortezomib: For the analysis of pharmaceutical formulations ten tablets (Velcade, 3.5mg) were weighted, powered and equivalent to 10 mg of Bortezomib was transferred into 100 mL volumetric flask. 60.0 mL of distilled water was added and ultrasonicated for 20 min, then made up to the mark with distilled water. The resulting solution was mixed and filtered through Whatman filter paper no. 42. From the filtrate, the solution was diluted appropriately with distilled water in order to obtain the working concentration of drug used for the analysis.

Chloroquine Phosphate: Ten tablets of (Kamquine 150mg) were weighed accurately and powdered. The powder equivalent to 50 mg of CHP was transferred into a 100 mL volumetric flask, containing a mixture of distilled water (10.0 mL) and HCl (2.0 mL). The flask was shaken for 5 mints and the solution was filtered using Whatman no. 41 filter paper and further diluted with water to obtain a working standard solution.

Granisetron Hydrochloride: About ten to fifteen tablets (Kytril, 2mg) were powdered and equivalent to about 10 mg of Granisetron hydrochloride had been taken into a 100 ml of volumetric flask and dissolved in 40 ml of distilled water by using 0.5M HCl. This solution was t filtered through Whatman filter paper no. 42. The residue was dissolved in 100 mL of distilled water. It was used as a stock sample solution. The aliquot portions of this stock solution were further diluted with distilled water to get the final concentration required for the determination of the drug.

Ibandronate Sodium: Two tablets (Bondria, 150 mg) were weighed accurately and grounded. A quantity equivalent to 20mg of Ibandronate Sodium was weighed and transferred into a 100ml calibrated flask and the volume was finally diluted to the mark with water, mixed well and filtered using a Whatman no. 42 filter paper. It was used as a stock sample solution and was further diluted with water to get a working solution.

RESULTS AND DISCUSSION: Each method developed for quantification of drugs has been validated in terms of precision, accuracy, the limit of detection, the limit of quantification, linearity, selectivity, and ruggedness. Beer’s law limits, slope, intercept, correlation coefficient, Sandell’s sensitivity and regression equations for each drug are tabulated in Table 2. To assess the precision each experiment was repeated at least 6 times and accuracy is estimated in terms of percent recovery and percent RSD. Excellent percent recovery and RSD is less than 2 for each drug demonstrates the accuracy and precision of the methods.

TABLE 2: ANALYTICAL AND REGRESSION PARAMETERS OF SPECTROPHOTOMETRIC METHOD

Factors Effecting Absorbance:

Effect of Acid Concentration: To study the effect of acid concentration, different types of acids were examined (H₂SO₄, HCl, and H₃PO₄ and CH₃COOH) to achieve maximum yield of Redox reaction. The results indicated that the hydrochloric acid was the preferable acid with NBS as oxidant. The reaction was performed in a series of 10 mL volumetric flask containing 8.0 μgmL^-1 of the cited drugs, different volumes (0.5-2.5 mL) of 1M HCl and 1 mL of NBS (0.01%) were added. After 5.0 min of heating time at 60 ± 2 °C in a water bath, the solution was cooled for about 3.0 min, 1 mL of methyl orange dye were added, then complete to 10 mL total volume with water.

It was found that the maximum absorbance was obtained at 1 mL of 1M HCl. Above this volume, the absorbance decreased. Therefore, a volume of 1 mL of 1M HCl was used for all measurements.

Effect of Heating Time: In order to obtain the highest and most stable absorbance, the effect of heating time on the oxidation reaction of drugs were catalyzed by heating in a water bath at 60 ± 2 °C for the periods ranging for 5-10 min. the time required to complete the reaction and maximum absorbance were obtained after 5.0 min of heating. After the oxidation process, the solution must be cooled at least for 3.0 min before addition of dye.

Effect of Oxidant Concentration: When a study on the effect of NBS on color development was performed, it was observed that in both cases the absorbance increased with increase in the volume of NBS. It reached maximum when 1 mL of 70 µg mL^-1 NBS   solution was added to a total volume of 10 mL for drugs solutions. The color intensity decreased above the upper limits. Therefore, 1 mL of 70 µg mL^-1 NBS was used for all measurements.

Effect of Dye Concentration: In order to ascertain the linear relationship between the volume of added NBS and the decrease in absorbance of Methyl Orange dye, experiments were performed using 1 mL of 1M HCl with varying volumes of NBS. The decrease in absorbance was found to be linear up to the 1 mL of NBS with the optimum volume of 1.0 mL of methyl orange dye for fixed concentration drug solution. The color was found to be stable up to 24 h.

Application to Formulations: The proposed methods were applied to the determination of drugs in tablets. The results in Table 3 showed that the methods are successful for the determination of drugs and that the recipients in the dosage forms do not interfere.

Statistical analysis of the results using Student’s t-test for accuracy and F-test for precision revealed no significant difference between the proposed methods and the literature method at the 95% confidence level with respect to accuracy and precision Table 4.

TABLE 3: RESULTS OF ASSAY OF TABLETS BY THE PROPOSED METHODS AND STATISTICAL EVALUATION AND RECOVERY EXPERIMENTS BY STANDARD ADDITION METHOD

TABLE 4: STUDENT’S T-TEST AND F-TEST VALUES FOR PHARMACEUTICAL ANALYSIS

Recovery experiment was performed via standard addition technique to ascertain the accuracy and validity of the proposed methods. To a fixed and known amount/concentration of drug in tablet powder, the pure drug was added at three levels (50, 100 and 150% of the level present in the tablet) and the total was found by the proposed methods. Each experiment was repeated six times and the percent recovery of pure drugs added was within the permissible limits showing the absence of interference by the inactive ingredients in the assay.

CONCLUSION: This is simple, rapid, and cost-effective methods for the determination of drugs have been developed and validated. The proposed method is more sensitive and the methods depend on the use of simple and cheap chemicals and techniques but provide sensitivity comparable to that achieved by a sophisticated and expensive technique like HPLC. Thus, they can be used as alternatives for rapid and routine determination of bulk sample and tablets.

ACKNOWLEDGEMENT: The authors are thankful to the Head, Department of chemistry, University College of Science, Osmania University, Hyderabad, Telangana - 500007 India.

CONFLICT OF INTEREST: Nil

REFERENCES:

1. Ahmad S, Ahsan A and George M: Simultaneous monitoring of argatroban and its major metabolite using an HPLC method: potential clinical applications. Clinical and Applied Thrombosis- Hemostasis 1999; 5(4): 252-8.
2. Xu-Guang G and Zi-dong Z: Content determination of argatroban injection by RP-HPLC. China Phamacy 2013; 33.
3. Da-qing G: Absorbance correction method for simultaneous estimation of Edaravone and Argatroban in synthetic mixture. Central South Pharmacy 2009; 3(1): 134-37.
4. Rhea JM, Snyder ML and Winkler AM: Development of a fast and simple liquid chromatography-tandem mass spectrometry method for the quantitation of argatroban in patient plasma samples. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 2012; 15: 168-72.
5. Escolar G, Bozzo J and Maragall S: Drug today 2006, 42: 223-36.
6. Jin YJ, Mimma T, Raicu V, Park KC and Shimizu K: Combined argatroban and edaravone caused additive neuroprotection against 15 min of forebrain ischemia in gerbils. Neuroscience Research 2002, 43: 75-79.
7. Keiichi Tesseikai, Katsumi: C/o; Patent EP1437137B1.
8. Combination therapy for acute ischemic stroke study group; Edaravone and argatroban stroke therapy study for acute ischemic stroke; clinicaltrials.gov.
9. Richardson PG, Mitsiades C and Schlossman R: Bortezomib in the front-line treatment of multiple myeloma. Expert Review of Anticancer Therapy 2008; 8: 1053-72.
10. Mikhael J and Chang H: Letters in Drug Design & Discovery 2007; 4: 82-86.
11. Srinivasulu K, Naidu MN, Rajasekhar K, Veerender M and Suryanarayana MV: Development and validation of a stability indicating LC method for the assay and related substances determination of a proteasome inhibitor bortezomib. Chromatography Research International 2012; 1-13.
12. Smith MB and March J: March's Advanced Organic Chemistry, John Wiley &Sons, Hoboken, NJ, USA, 6^th edition, 2007.
13. Pitt B, Remme W and Zannad F: Eplerenone, a selective aldosterone blocker, in patients with left ventricular dysfunction after myocardial infarction. ACC Current Journal Review 2003; 12(4): 57-65.
14. Venkataramanna M, Sudhakar Babu K and Anwar sulaiman KC: A validated stability-indicating UF LC method for bortezomib in the presence of degradation products and its process-related impurities. Journal of Chromatography & Separation Techniques 2012; 3(1): 1-6.
15. Utage M and Swamy BMV: Stability indicating HPLC method for estimation of bortezomib for injection 3.5 mg/ vial. International Journal of Pharmaceutical Research Scholars 2013; 2(1): 93-98.
16. Burgess E, Niegowksa J, Tan KW, Kipnes MS, Roniker B and Patrick JL: Antihypertensive effects of eplerenone and enalapril in patients with essential hypertension. American Journal of Hypertension 2002; 15(1): 57-58.
17. Gennaro and Remington AR: The science and practice of pharmacy. 20^th Lippincott Williams and Wilkins, 2000; 1547.
18. Lawal, A, Umar, RA, Abubakar MG and Faruk UZ: Development and validation of UV spectrophotometric and HPLC methods for quantitative determination of chloroquine and amodiaquine in pharmaceutical formulations. International Journal of Chem Tech Research 2012; 4(2): 669676.
19. Onyegbule FA, Adelusi SA and Onyegbule CE: Extractive visible spectrophotometric determination of chloroquine in pharmaceutical dosage forms and biological materials. International Journal of Pharmaceutical Sciences and Research 2011; 2(1): 72-76.
20. Nagaraja P, Shrestha AK, Shivakumar A and Gowda AK: Spectrophotometric determination of chloroquine, pyrimethamine and trimethoprim by ion pair extraction in pharmaceutical formulation and urine. Yaowu Shipin Fenxi 2010; 18(4): 239-48.
21. Khalil SM, Mohamed GG, Zayed MA and Elqudaby HM: Spectrophotometric determination of chloroquine and pyrimethamine through ion-pair formation with molybdenum and thiocyanate. Microchemical Journal 2000; 64(2): 181-86.
22. Nelson O, Olajire A and Ayodeji A: Rapid spectrofluorimetric determination of chloroquine Phosphate tablets. International Journal of Drug Development & Research 2010; 2(2): 412-17.
23. Rivas-Granizo, P, Jorge SSRC and Ferraz HG: Development of a stability-indicating LC assay method for determination of chloroquine. Chromatographia 2009; 69(2): 137-41.
24. Ali D, Khezeli T and Manafi MH: Determination of anti-malaria agent chloroquine using single drop liquid-liquid-liquid microextraction. Journal of Separation Science 2009; 32(4): 511-16.
25. Samanidou V., Evaggelopoulou EN and Papadoyannis IN: Simultaneous determination of quinine and chloroquine anti-malarial agents in pharmaceuticals and biological fluids by HPLC and fluorescence detection. Journal of Pharmaceutical and Biomedical Anal 2005; 38(1): 21-28.
26. Aggarwal G, Kumar V, Chaudhary and Hema: Design, optimization and characterization of granisetron HCl loaded nano-gel for transdermal delivery. Pharmaceutical Nanotechnology 2017; 5(4): 317-28.
27. Rao SV, Ramu V, Kumar G, Ravi D and Rambabu C: A new isocratic RP-HPLC method development for the assay of granisetron HCl in API and dosage forms. Rasayan Journal of Chemistry 2012; 5(2): 229-33.
28. Gopalakrishnan S, Jeyashree B, Thenmozhi M and Chenthilnathan A: Development and validation of new RP-HPLC method for the estimation of granisetron hydrochloride in bulk and pharmaceutical dosage form. Journal of Chemical and Pharmaceutical Research 2011; 3(3): 470-76.
29. Prabu S, Lakshmana, Selvamani P and Latha S: HPTLC method for quantitative determination of granisetron hydrochloride in bulk drug and in tablets. Latin American Journal of Pharmacy 2010; 29(8): 1455-58.
30. Rao SV, Ramu G, Rao NVNM and Rambabu, C: Development of simple and sensitive visible spectrophotometric methods for the determination of granisetron hydrochloride in pure and pharmaceutical formulations. International Journal of PharmTech Research 2012; 4(4): 1508-12.
31. Kanchana, Angayer S, Aruna, Ajithadas, Niraimathi, Suresh V and Jerad A: From Acta Ciencia Indica. Chemistry 2010; 36(1): 29-31.
32. Muller R, Hannan M, Smith SY and Bauss F: Intermittent Ibandronate preserves bone quality and bone strength in the lumbar spine after 16 months of treatment in the ovariectomized cynomolgus monkey. Journal of Bone Min Rese 2004; 19: 1787-96.
33. Pecherstorfer M, Herrmann Z, Body JJ and Manegold C: Randomized phase II trial comparing different doses of the bisphosphonate ibandronate in the treatment of hyper-calcemia of malignancy. Journal of Clinical Oncology 1996; 14: 268-76.
34. Coleman RE, Purohit OP, Black C and Vinholes JJ: Double-blind, randomised, placebo-controlled, dose-finding study of oral ibandronate in patients with metastatic bone disease; Annals of Oncology. Official Journal of the European Society for Medical Oncology 1999; 10: 311-16.
35. Anonymous, ICH, Q2 (R1): Text and Methodology International conference on Harmonization, Geneva 2005; 1-13.
36. Jiang Y and Xie Z: Determination of Ibandronate and its Degradation Products by Ion-Pair RP LC with Evaporative Light-Scattering Detection. Chromatographia 2005; 62: 257-61.
37. Tsai EW, Ip DP and Brooks MA: Determination of alendronate in pharmaceutical dosage formulations by ion chromatography with conductivity detection. J Chromatography 1992; 596(2): 217-24.
38. Gawad JB and Jain PS: Development and validation of RP- HPLC assay method for determination of ibandronate sodium in tablet dosage form. International Journal of Innovative Pharmaceutical Research 2012; 3(3): 220-25.
    

    ---

    How to cite this article:

    Reddy BV, Mamidi G and Venkateshwarlu G: Simple spectrophotometric methods for estimation of drugs and pharmaceuticals using NBS-methyl orange dye couple. Int J Pharm Sci & Res 2019; 10(9): 4215-22. doi: 10.13040/IJPSR.0975-8232.10(9).4215-22.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---