Talanta 46 (1998) 39–44

Spectrophotometric methods for the determination of certaincatecholamine 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, Indiab 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 ofo-dihydroxybenzene derivatives by N-bromosuccinimide followed by oxidative coupling with isoniazid leading to theformation of a red-coloured products of maximum absorbance (lmax=480–490 nm). The second method is based onthe formation of green to blue complex (lmax=635–660 nm) between o- dihydroxybenzene derivatives and sodiumnitroprusside in the presence of hydroxylamine hydrochloride. All measurements of the two procedures are carriedout in an alkaline medium at room temperature. The two methods are successfully applied for the determination ofdopamine hydrochloride, levodopa and methyldopa in injections and tablets of pharmaceutical preparation. Thecommon excipients used as additives in pharmaceuticals do not interfere in the proposed methods. The reliability ofthese methods are established by parallel determination with the reported and official methods. © 1998 ElsevierScience 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 inwhich either the 3- or 4-position is unsubstitutedand these positions are not sterically blocked.These drugs are now widely used in the treatmentof bronchial asthma, hypertension, Parkinson’sdisease, myocardial infarction and cardiac

surgery. Dopamine, a neurotransmitter, is one ofthe naturally occurring catecholamines, and itshydrochloride salt is being used in the treatmentof acute congestive failure and renal failure [1].This has stimulated many investigators to workout compendial methods for the determination ofcatecholamine in authentic and dosage forms.Various methods like spectrofluorimetry [2,3],spectrophotometry [4], ion-exchange columnchromatography [5], gas chromatography [6,7]* Corresponding author.

0039-9140/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved.

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P. Nagaraja et al. / Talanta 46 (1998) 39–4440

Table 1Experimental conditions and optical characteristics

Parameters Method

BA

DPHPCLLDP MDPPCL DPH

Blue GreenColour Red Red Red Red480 660lmax (nm) 485 490 480 635

108 3Stability (h) 1024 482.4–12.0 5.0–16.0 1.5–10.0Beer’s law (mg ml−1) 2.4–17.00.8–5.85 2.8–14.0

7.4×1037.59×103Molar 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 0.01450.0293Regression equationa

0.0200 0.0663Slope (a) 0.1109 0.0390 0.03880.04100.011−0.001−0.050Intercept (b) −0.0030.037 −0.032

0.9977 0.9999Correlation coefficient 0.9980 1.0101 0.9958 0.9994

ay=ax+b, where x is the concentration of PCL, DPH, LDP or MDP in mg ml−1.

Table 2Experimental conditions

Volume of NBS (0.05%) or HAH Volume of NaOH (0.01 M) orVolume of INH (0.02%) or SNPMethodNa2CO3 (5%) in ml(0.3%) in ml(0.07%) in ml

1.5a (1.25–2.0)b 1.5a (1.0–2.5)bA 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)

aUsed in the proposed procedure.bRange for maximum absorbance and stability.

and radioimmunoassay [8,9] have been describedin the literature for the determination of do-pamine and dopa from the various biologicalsamples and pharmaceutical preparation. Thepresent work describes the two simple sensitiveand accurate spectrophotometric methods for thedetermination of catecholamine derivatives (pyro-catechol, levodopa, methyldopa and dopaminehydrochloride) using isoniazid (INH) in the pres-ence of N-bromosuccinimide (NBS). The othermethod determines the pyrocatechol and do-pamine hydrochloride using sodium nitroprusside(SNP) in the presence of hydroxylamine hy-drochloride (HAH). The methods are adopted forthe assay of three catecholamine drugs in pureand pharmaceutical formulations. These threedrugs are officially listed in USP [10] which de-scribes a nonaqueous titration for the assay ofdopamine hydrochloride in raw material and an

HPLC technique for injection solutions. A visualtitration and UV spectrophotometric methods at280 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 wasused for the electronic spectral measurements.

2.2. Reagents

Dopamine hydrochloride (Sigma, USA),levodopa (SD Fine, India), methyldopa (SD Fine,India), Pyrocatechol (CDH, India), Isoniazid

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 analyticalreagent grade. Deionised water was used to pre-pare all solutions and in all experiments.

2.3. Solutions

Freshly prepared aqueous solutions of the puredrugs and pyrocatechol (PCL) (protected fromsun light) (50 mg ml−1) were used as the standard

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 reportedmethod [11]. Solutions of 0.2% aqueous isoniazid,0.07% aqueous hydroxylamine hydrochloride,0.3% aqueous sodium nitroprusside, 0.05% N-bromosuccinimide, 5% sodium carbonate and0.01 M sodium hydroxide were used.

2.4. General procedure

2.4.1. Method AAliquots of standard solutions of PCL (20–146

mg), DPH (70–350 mg), LDP (60–300 mg) orMDP (125–400 mg) were transferred to a 25-mlcalibrated flask, to which 0.05% NBS, 0.02% INHand 0.01 M sodium hydroxide were added to thecatecholamine solution, and the mixture was setaside for 5 min. The contents were diluted to themark and mixed well. The absorbance at lmax wasmeasured against a reagent blank.

2.4.2. Method BAliquots of standard solutions of PCL (37–250

mg) or DPH (60–425 mg) were transferred to a25-ml calibrated flask, to which 0.3% SNP, 0.07%HAH and 5% sodium carbonate solution wereadded, and the mixture was set aside for 5 min.The contents were diluted to the mark and mixedwell. The absorbance at lmax was measuredagainst a reagent blank.

Details of the experimental conditions of thetwo methods are given in Tables 1 and 2.

2.5. Procedure for the assay of catecholamines inpharmaceutical preparation

2.5.1. TabletsTwenty tablets were weighed and finely pow-

dered. A weighed amount of the powder contain-ing 50 mg of LDP or MDP was dissolved in waterand filtered. The filtrate was made up to 100 mland an aliquot of this solution was treated asdescribed above for the determination of LDP orMDP.

2.5.2. InjectionDPH injection solutions were appropriately di-

luted with water to get the required concentrationof the drug, and then the general procedure wasfollowed. The amount of DPH was calculatedfrom a calibration graph.

3. Results and discussion

3.1. Absorption spectra

A red-coloured oxidating coupling product withan absorption maximum at 480–490 nm is formedwhen PCL, DPH, LDP or MDP were allowed toreact with NBS in the presence of INH in asodium hydroxide medium. Green to blue prod-ucts with absorption maxima at 635–660 nm wereformed, when PCL or DPH are allowed to reactwith SNP in the presence of HAH in a sodiumcarbonate medium. The absorption spectra of

P. Nagaraja et al. / Talanta 46 (1998) 39–44 43

Table 3Determination of catecholamines in pharmaceutical preparations

DPH content per 5 ml of injection or LDP and MDP content per tablet (mg) %Label claim (mg)Drugrecoverya9RSD

Reported methodBP method Proposed method

BA

DPH99.8290.75100.0791.03Injectionb 200/5 ml 98.8591.21 99.0491.1099.9490.6399.290.82 100.0990.99Injecionc 100.290.51200/5 ml

Tablets99.0190.74 —LDPd 500 98.7891.10 97.8390.96

99.590.86 — 99.9590.90MDPe —250

aAverage of six determinations.bMarketed by TTK Pharma.cMarketed by TRIOKA Parenterals.dMarketed by Wallace.eMarketed by Merind Limited.

both red, green and blue products and the reagentblanks 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 werereadily oxidised to o-benzoquinone by NBS. INH,by virtue of its strong electron-donating group,couples with o-benzoquinone in alkaline mediumleading to the formation of oxidative coupledproducts 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 andMnO4

− oxidising agents does not form any colourunder the experimental conditions.

A characteristic green to blue-coloured productis formed when DPH or PCL is allowed to reactwith SNP in the presence of HAH in an alkalinemedium. Use of this method was unsuccessful forthe 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 acharge transfer absorption using SNP as a reagentfor the analysis of phenol. This indicates that theformation of the green to blue colour by theproposed method B may be due to either theformation of indophenol blue or a coordinationcomplex of CT type.

3.3. Stability

The resultant products of the proposed meth-ods were studied at different temperatures. Theresults indicate that the absorbance values remainconstant in the temperature range 5–70°C. Athigher temperatures the absorbance values de-crease, indicating the dissociation of the productson prolonged heating. The coloured productswere stable for 3–48 h at room temperature.

3.4. Interference

An antioxidant, sodium metabisulphate, andsodium chloride that is commonly present in theDPH injection, and also commonly used excipi-ents such as starch, talc, glucose, lactose, dextrose

P. Nagaraja et al. / Talanta 46 (1998) 39–4444

and magnesium stearate, did not interfere, whilevitamin C, adrenaline and noradrenaline werefound to have interfered. In method B, the resultsof interference shows that a 2-fold excess of LDPand MDP do not interfere.

3.5. Application

The applicability of the method to assay ofpharmaceutical preparations was examined. Theresults obtained (Table 3) compared favourably tothose reported by El-Kommos et al. [13] and theofficial method [11].

4. Conclusions

The proposed methods are simple, rapid, precise,sensitive and economical. The two methods can besuccessfully applied as an alternative to the existingmethods.

Acknowledgements

One of the authors (K.C.S.M.) thanks theMysore University for the support of this researchwork.

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