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. 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. 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. 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. 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. 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.
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