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  1. 1. Talanta 46 (1998) 39–44 Spectrophotometric methods for the determination of certain catecholamine derivatives in pharmaceutical preparations P. Nagaraja a, *, K.C. Srinivasa Murthy b , K.S. Rangappa a , N.M. Made Gowda c a Department of Studies in Chemistry, Mysore Uni6ersity, Manasagangotri, Mysore 570006, India b Cipla Limited, Virgonagar, Bangalore 560 049, India c Department of Chemistry, Western Illinois Uni6ersity, Macomb, IL 61455, USA Received 28 April 1997; received in revised form 24 July 1997; accepted 29 July 1997 Abstract Two simple, rapid and sensitive spectrophotometric methods for the determination of catecholamine derivatives (pyrocatechol, dopamine, levodopa and methyldopa) are developed. The first method involves the oxidation of o-dihydroxybenzene derivatives by N-bromosuccinimide followed by oxidative coupling with isoniazid leading to the formation of a red-coloured products of maximum absorbance (umax =480–490 nm). The second method is based on the formation of green to blue complex (umax =635–660 nm) between o- dihydroxybenzene derivatives and sodium nitroprusside in the presence of hydroxylamine hydrochloride. All measurements of the two procedures are carried out in an alkaline medium at room temperature. The two methods are successfully applied for the determination of dopamine hydrochloride, levodopa and methyldopa in injections and tablets of pharmaceutical preparation. The common excipients used as additives in pharmaceuticals do not interfere in the proposed methods. The reliability of these methods are established by parallel determination with the reported and official methods. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Pyrocatechol; Dopamine hydrochloride; Levodopa; Methyldopa; Isoniazid; Hydroxylamine hydrochloride; Sodium nitroprusside; N-Bromosuccinimide; Spectrophotometry; Pharmaceuticals 1. Introduction Catecholamine drugs are aromatic vic-diols in which either the 3- or 4-position is unsubstituted and these positions are not sterically blocked. These drugs are now widely used in the treatment of bronchial asthma, hypertension, Parkinson’s disease, myocardial infarction and cardiac surgery. Dopamine, a neurotransmitter, is one of the naturally occurring catecholamines, and its hydrochloride salt is being used in the treatment of acute congestive failure and renal failure [1]. This has stimulated many investigators to work out compendial methods for the determination of catecholamine in authentic and dosage forms. Various methods like spectrofluorimetry [2,3], spectrophotometry [4], ion-exchange column chromatography [5], gas chromatography [6,7]* Corresponding author. 0039-9140/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0039-9140(97)00245-2
  2. 2. P. Nagaraja et al. / Talanta 46 (1998) 39–4440 Table 1 Experimental conditions and optical characteristics Parameters Method BA DPHPCLLDP MDPPCL DPH Blue GreenColour Red Red Red Red 480 660umax (nm) 485 490 480 635 108 3Stability (h) 1024 48 2.4–12.0 5.0–16.0 1.5–10.0Beer’s law (mg ml−1 ) 2.4–17.00.8–5.85 2.8–14.0 7.4×103 7.59×103 Molar absorptivity (l mol−1 cm−1 ) 1.27×104 6.47×103 8.38×103 3.96×103 0.02570.0235Sandell’s sensitivity (mg cm−2 ) 0.05330.0086 0.01450.0293 Regression equationa 0.0200 0.0663Slope (a) 0.1109 0.0390 0.03880.0410 0.011−0.001−0.050Intercept (b) −0.0030.037 −0.032 0.9977 0.9999Correlation coefficient 0.9980 1.0101 0.9958 0.9994 a y=ax+b, where x is the concentration of PCL, DPH, LDP or MDP in mg ml−1 . Table 2 Experimental conditions Volume of NBS (0.05%) or HAH Volume of NaOH (0.01 M) orVolume of INH (0.02%) or SNPMethod Na2CO3 (5%) in ml(0.3%) in ml(0.07%) in ml 1.5a (1.25–2.0)b 1.5a (1.0–2.5)b A 4.0a (2.0–6.0)b 2.5 (1.0–4.0) 2.5 (1.0–3.5)B 0.5 (0.25–1.0) a Used in the proposed procedure. b Range for maximum absorbance and stability. and radioimmunoassay [8,9] have been described in the literature for the determination of do- pamine and dopa from the various biological samples and pharmaceutical preparation. The present work describes the two simple sensitive and accurate spectrophotometric methods for the determination of catecholamine derivatives (pyro- catechol, levodopa, methyldopa and dopamine hydrochloride) using isoniazid (INH) in the pres- ence of N-bromosuccinimide (NBS). The other method determines the pyrocatechol and do- pamine hydrochloride using sodium nitroprusside (SNP) in the presence of hydroxylamine hy- drochloride (HAH). The methods are adopted for the assay of three catecholamine drugs in pure and pharmaceutical formulations. These three drugs are officially listed in USP [10] which de- scribes a nonaqueous titration for the assay of dopamine hydrochloride in raw material and an HPLC technique for injection solutions. A visual titration and UV spectrophotometric methods at 280 nm are prescribed for levodopa and methyl- dopa, respectively. 2. Experimental 2.1. Apparatus A Jasco Model UVIDEC-610 UV-VIS spec- trophotometer with 1.0-cm matched cells was used for the electronic spectral measurements. 2.2. Reagents Dopamine hydrochloride (Sigma, USA), levodopa (SD Fine, India), methyldopa (SD Fine, India), Pyrocatechol (CDH, India), Isoniazid
  3. 3. P. Nagaraja et al. / Talanta 46 (1998) 39–44 41 Fig. 1. Absorption spectra of: (1) PCL (3 mg ml−1 )+NBS+INH product; (2) LDP (6 mg ml−1 )+NBS+INH product; (3) DPH (7 mg ml−1 )+NBS+INH product; (4) MDP (8 mg ml−1 )+NBS+INH product; (5) NBS+INH reagent blank. Fig. 2. Absorption spectra of: (1) PCL (5 mg ml−1 )+HAH+SNP product; (2) DPH (8.5 mg ml−1 )+HAH+SNP product; (3) HAH+SNP reagent blank. (BDH, Poole, UK), sodium nitroprusside (E- Merck, Germany) were used. All other chemicals used were of analytical reagent grade. Deionised water was used to pre- pare all solutions and in all experiments. 2.3. Solutions Freshly prepared aqueous solutions of the pure drugs and pyrocatechol (PCL) (protected from sun light) (50 mg ml−1 ) were used as the standard
  4. 4. P. Nagaraja et al. / Talanta 46 (1998) 39–4442 Scheme 1 solution for analytical purposes. Dopamine hy- drochloride (DPH), levodopa (LDP), and methyl- dopa (MDP) were standardised by the reported method [11]. Solutions of 0.2% aqueous isoniazid, 0.07% aqueous hydroxylamine hydrochloride, 0.3% aqueous sodium nitroprusside, 0.05% N- bromosuccinimide, 5% sodium carbonate and 0.01 M sodium hydroxide were used. 2.4. General procedure 2.4.1. Method A Aliquots of standard solutions of PCL (20–146 mg), DPH (70–350 mg), LDP (60–300 mg) or MDP (125–400 mg) were transferred to a 25-ml calibrated flask, to which 0.05% NBS, 0.02% INH and 0.01 M sodium hydroxide were added to the catecholamine solution, and the mixture was set aside for 5 min. The contents were diluted to the mark and mixed well. The absorbance at umax was measured against a reagent blank. 2.4.2. Method B Aliquots of standard solutions of PCL (37–250 mg) or DPH (60–425 mg) were transferred to a 25-ml calibrated flask, to which 0.3% SNP, 0.07% HAH and 5% sodium carbonate solution were added, and the mixture was set aside for 5 min. The contents were diluted to the mark and mixed well. The absorbance at umax was measured against a reagent blank. Details of the experimental conditions of the two methods are given in Tables 1 and 2. 2.5. Procedure for the assay of catecholamines in pharmaceutical preparation 2.5.1. Tablets Twenty tablets were weighed and finely pow- dered. A weighed amount of the powder contain- ing 50 mg of LDP or MDP was dissolved in water and filtered. The filtrate was made up to 100 ml and an aliquot of this solution was treated as described above for the determination of LDP or MDP. 2.5.2. Injection DPH injection solutions were appropriately di- luted with water to get the required concentration of the drug, and then the general procedure was followed. The amount of DPH was calculated from a calibration graph. 3. Results and discussion 3.1. Absorption spectra A red-coloured oxidating coupling product with an absorption maximum at 480–490 nm is formed when PCL, DPH, LDP or MDP were allowed to react with NBS in the presence of INH in a sodium hydroxide medium. Green to blue prod- ucts with absorption maxima at 635–660 nm were formed, when PCL or DPH are allowed to react with SNP in the presence of HAH in a sodium carbonate medium. The absorption spectra of
  5. 5. P. Nagaraja et al. / Talanta 46 (1998) 39–44 43 Table 3 Determination of catecholamines in pharmaceutical preparations DPH content per 5 ml of injection or LDP and MDP content per tablet (mg) %Label claim (mg)Drug recoverya 9RSD Reported methodBP method Proposed method BA DPH 99.8290.75100.0791.03Injectionb 200/5 ml 98.8591.21 99.0491.10 99.9490.6399.290.82 100.0990.99Injecionc 100.290.51200/5 ml Tablets 99.0190.74 —LDPd 500 98.7891.10 97.8390.96 99.590.86 — 99.9590.90MDPe —250 a Average of six determinations. b Marketed by TTK Pharma. c Marketed by TRIOKA Parenterals. d Marketed by Wallace. e Marketed by Merind Limited. both red, green and blue products and the reagent blanks are shown in Figs. 1 and 2. The details of optical characteristics are sum- marised in Table 2. 3.2. Reaction sequence Vicinal dihydroxybenzene derivatives were readily oxidised to o-benzoquinone by NBS. INH, by virtue of its strong electron-donating group, couples with o-benzoquinone in alkaline medium leading to the formation of oxidative coupled products as given in the reaction in Scheme 1 [12,13]. Other oxidising agents such as Cr2O7 2− , H2O2, chloramine-T and MnO4 − were tried in- stead of NBS and found to be less effective. However, in acidic medium dichromate and MnO4 − oxidising agents does not form any colour under the experimental conditions. A characteristic green to blue-coloured product is formed when DPH or PCL is allowed to react with SNP in the presence of HAH in an alkaline medium. Use of this method was unsuccessful for the identification of the product in solid form. However, Guptha and co-workers [14] and Na- garaja et al. [15] have proposed the formation of indophenol blue or a coordination complex with a charge transfer absorption using SNP as a reagent for the analysis of phenol. This indicates that the formation of the green to blue colour by the proposed method B may be due to either the formation of indophenol blue or a coordination complex of CT type. 3.3. Stability The resultant products of the proposed meth- ods were studied at different temperatures. The results indicate that the absorbance values remain constant in the temperature range 5–70°C. At higher temperatures the absorbance values de- crease, indicating the dissociation of the products on prolonged heating. The coloured products were stable for 3–48 h at room temperature. 3.4. Interference An antioxidant, sodium metabisulphate, and sodium chloride that is commonly present in the DPH injection, and also commonly used excipi- ents such as starch, talc, glucose, lactose, dextrose
  6. 6. P. Nagaraja et al. / Talanta 46 (1998) 39–4444 and magnesium stearate, did not interfere, while vitamin C, adrenaline and noradrenaline were found to have interfered. In method B, the results of interference shows that a 2-fold excess of LDP and MDP do not interfere. 3.5. Application The applicability of the method to assay of pharmaceutical preparations was examined. The results obtained (Table 3) compared favourably to those reported by El-Kommos et al. [13] and the official method [11]. 4. Conclusions The proposed methods are simple, rapid, precise, sensitive and economical. The two methods can be successfully applied as an alternative to the existing methods. Acknowledgements One of the authors (K.C.S.M.) thanks the Mysore University for the support of this research work. References [1] B.K. George, in: L.S. Goodman, A. Gilman (Eds.), The Pharmacological Basis of Therapeutics, 3rd ed., The Macmillan Company, NY, 1965, p. 427. [2] C.E. Bell, A.R. Somerville, Biochem. J. 98 (1966) 1C. [3] Imai, Kazuhizo, J. Chromatogr. 105 (1975) 135. [4] R.T. Sane, P.M. Deshpande, C.L. Sawant, S.M. Dolas, V.G. Nayak, S.S. Zarapkar, Indian Drugs (and references cited) 24 (1987) 199. [5] Seki, Tokaichizo, Wada, Hiroshi, J. Chromatogr. 114 (1975) 227. [6] P.J. Murphy, T.L. William, D.L. Kau, J. Pharmacol. Exp. Ther. 199 (1976) 423. [7] K. Satoshi, T. Zenzo, Chem. Pharm. Bull. Tokyo 16 (1968) 1091. [8] B.F. Erlanger, Pharmacol. Rev. 25 (1973) 271. [9] L.J. Ricebery, H.V. Vunakis, L. Levin, Anal. Biochem. 60 (1974) 551. [10] US Pharmacopoeia XXI, United Pharmacopoeial Con- vention, Inc., 12601, Twinbrook Parkway, Rockville, MD 20852, 1985, pp. 348, 353. [11] British Pharmacopeia, London, SIN 85 NQ, 1993, pp. 239, 380 and 424. [12] C.S.P. Sastry, V. Gurucharana Das, K. Ekambareswara Rao, Analyst 110 (1985) 395. [13] M.E. EL-Kommos, F.A. Mohamed, A.S. Khedr, J. As- soc. Off. Anal. Chem. 73 (1990) 516. [14] S. Amlathe, S. Upadhyay, V.K. Guptha, Analyst 112 (1987) 1463. [15] P. Nagaraja, J.M. Bhandari, B.N. Achar, Indian J. Chem. 32A (1993) 641. ..