P. Nagaraja et al. / Talanta 46 (1998) 39–4440
Experimental conditions and optical characteristics
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
Molar absorptivity (l mol−1
0.02570.0235Sandell’s sensitivity (mg cm−2
) 0.05330.0086 0.01450.0293
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
y=ax+b, where x is the concentration of PCL, DPH, LDP or MDP in mg ml−1
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
2.5 (1.0–4.0) 2.5 (1.0–3.5)B 0.5 (0.25–1.0)
Used in the proposed procedure.
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  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-
A Jasco Model UVIDEC-610 UV-VIS spec-
trophotometer with 1.0-cm matched cells was
used for the electronic spectral measurements.
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 analytical
reagent grade. Deionised water was used to pre-
pare all solutions and in all experiments.
Freshly prepared aqueous solutions of the pure
drugs and pyrocatechol (PCL) (protected from
sun light) (50 mg ml−1
) were used as the standard
P. Nagaraja et al. / Talanta 46 (1998) 39–4442
solution for analytical purposes. Dopamine hy-
drochloride (DPH), levodopa (LDP), and methyl-
dopa (MDP) were standardised by the reported
method . 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
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
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
P. Nagaraja et al. / Talanta 46 (1998) 39–44 43
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
Reported methodBP method Proposed method
200/5 ml 98.8591.21 99.0491.10
500 98.7891.10 97.8390.96
99.590.86 — 99.9590.90MDPe
Average of six determinations.
Marketed by TTK Pharma.
Marketed by TRIOKA Parenterals.
Marketed by Wallace.
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
H2O2, chloramine-T and MnO4
were tried in-
stead of NBS and found to be less effective.
However, in acidic medium dichromate and
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  and Na-
garaja et al.  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.
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.
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
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.
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.  and the
ofﬁcial method .
The proposed methods are simple, rapid, precise,
sensitive and economical. The two methods can be
successfully applied as an alternative to the existing
One of the authors (K.C.S.M.) thanks the
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