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Flow Injection Spectrophotometric Determination of L-Dopa and Carbidopa

in Pharmaceutical Formulations Using a Crude Extract of Sweet Potato Root
[Ipomoea batatas (L.) Lam.] as Enz...

Article in The Analyst · May 1997

DOI: 10.1039/A606852I · Source: PubMed

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Flow Injection Spectrophotometric Determination of
L-Dopa and Carbidopa in Pharmaceutical Formulations
Using a Crude Extract of Sweet Potato Root [Ipomoea
batatas (L.) Lam.] as Enzymatic Source
Orlando Fatibello-Filho* and Iolanda da Cruz Vieira
Departamento de Quı́mica, Grupo de Quı́mica Analı́tica, Centro de Ciências Exatas e de
Tecnologia, Universidade Federal de São Carlos, Caixa Postal 676, CEP 13.560-970, São Carlos,
SP, Brazil
Published on 01 January 1997 on http://pubs.rsc.org | doi:10.1039/A606852I
A flow injection (FI) spectrophotometric method is
proposed for the determination of L-dopa and carbidopa
in pharmaceutical formulations. After selection of the
extraction medium (e.g., buffer-to-tissue ratio, pH, buffer
concentration, protective agents and/or stabilizers) and
storage conditions, crude extract of sweet potato root
[Ipomoea batatas (L.) Lam.] was used as an enzymatic
source of polyphenol oxidase (Tyrosinase; catechol
Downloaded on 15 January 2013
oxidase; EC.1.14.18.1) directly in the carrier. This enzyme
catalyses the oxidation of these catecholamines to the
corresponding dopaquinone. Further, dopaquinone
undergoes a rapid spontaneous auto-oxidation to
leucodopachrome, which is in turn oxidized to
dopachrome; this last compound has a strong absorption
at 480 and 360 nm for L-dopa and carbidopa, respectively.
For the optimum extraction conditions found the enzyme
activity of the crude extract did not vary for at least 5
months when stored at 4 °C and decreased by only 4–5%
during an 8 h working period at 25 °C. The results
obtained for L-dopa and carbidopa by the proposed
enzymatic FI method were in close agreement with the
label values (r1 = 0.9699 and r2 = 0.9999) and also with
those obtained using a pharmacopeial method
(r3 = 0.9675). The throughput was 26 samples h21, and
2.30 ml of crude extract were consumed in each
determination, corresponding to only 72 mg of the
original sweet potato root. The detection limit (three times
the signal blank/slope) was 1.5 31025 and 2.0 3 1025
mol l21 for L-dopa and carbidopa, respectively; the
recovery of L-dopa and carbidopa from three samples
ranged from 98.6 to 106.3% of the added amount.
Keywords: L-Dopa; carbidopa; flow system; polyphenol
oxidase; tyrosinase; catechol oxidase; sweet potato root
[Ipomoea batatas (L.) Lam.]; pharmaceutical formulations
l-Dopa [(2)-3-(3,4-dihydroxyphenyl)-l-alanine] and carbi-
dopa [(2)-l-2-(3,4-dihydroxybenzyl)-2-hydrazinopropionic
acid] are catecholamines with an alkylamine chain attached to a
benzene ring bearing two hydroxyl groups. l-Dopa is an
important neurotransmitter that is used for the treatment of
neural disorders such as Parkinson’s disease. It is effective in
relieving hypokinesia and can also decrease rigidity, oculogyric
crises and tremor.1 After its oral administration, l-dopa is
absorbed through the bowel at the level of the small intestine
and metabolized by a decarboxylation process to dopamine and
then to other metabolites. Both carbidopa and benserazide are
used as inhibitors for the decarboxylase activity.2 Hence, the
development of a method for the selective determination of l-
dopa and carbidopa is important, since they are frequently found
together in some pharmaceutical formulations.
View Article Online / Journal Homepage / Table of Contents for this issue
Analyst, April 1997, Vol. 122 (345–350) 345
Several methods have been proposed for the determination of
these catecholamine drugs in biological specimens and/or
pharmaceutical formulations. The determination of catechol-
amines in biological specimens normally requires the use of
trace analysis techniques, mainly chromatography with flu-
orimetric or electrochemical detection.3 On the other hand,
catecholamines are present in relatively large amounts in
pharmaceutical formulations and much effort has been devoted
to the development of simple, rapid, accurate and precise
analytical methods. HPLC has been widely used for the
determination of l-dopa in brain, plasma, urine, liver, serum,
tissue and biological fluids.4–8 A photokinetic method based on
the strong inhibitory effect of the catecholamines on the
photochemical reaction between Rose Bengal and EDTA has
been proposed.9 A fluorimetric method10 based on the inhibi-
tion by catechlolamines of the photoreduction of phloxin
(tetrachlorotetrabromofluorescein) by EDTA has also been
proposed. A flow injection (FI) determination of epinephrine,
norepinephrine, dopamine and l-dopa by their chemilumino-
genic oxidation with potassium permanganate in acidic medium
in the presence of formaldehyde has been reported.11
Spectrophotometric methods have also been proposed for the
determination of catecholamines in pharmaceutical for-
mulations based on the direct measurement of l-dopa at 280 and
290 nm12 and by first-derivative spectrophotometry at 276
nm.13 With other UV spectrophotometric methods, this com-
pound has been determined in the presence of germanium
dioxide,14 boric acid,15 Na2B4O716 and Na2HPO417 at 292,
239.5, 287 and 292.5 nm, respectively. In addition, catechol-
amines have been determined in the visible region after reaction
with metaperiodate,18 isonicotinic acid hydrazide in alkaline
III
medium,19 Fe and o-phenanthroline in a moderately acidic
medium, PdCl2,21 molybdophosphoric acid,22 ninhydrin,23
20
ammonium metavanadate24and p-aminophenol in the presence
of KIO4 after its oxidation in alkaline medium.25 Nevertheless,
there are few enzymatic methods for determining cate-
cholamines described in the literature26–30 and of these only one
laborious manual spectrophotometric method30 has been pro-
posed. l-Dopa was incubated with mammalian tyrosinase in the
presence of an optimum concentration of ZnII ions for 50–60
min and the melanochrome formed in this reaction was
monitored at 540 nm.
Uchiyama and co-workers31,32 used a cucumber juice
solution (crude extract) containing ascorbate oxidase as a carrier
in an FI system for the determination of l-ascorbic acid31 and
dehydroascorbic acid.32 In another paper,33 they proposed an FI
procedure for the determination of polyphenols using banana
pulp and spinach leaf solution as carriers. The oxygen
consumption in the enzymatic reaction was monitored by an
oxygen electrode and its concentration decrease was inversely
proportional to the phenolic substrate concentration. However,
the former enzymatic source is easily oxidized in air and its

346 Analyst, April 1997, Vol. 122
colour changes rapidly from white–yellow to brown after the
peel has been removed. In addition, it is necessary to pre-
oxidize the banana pulp completely in air and store it for 1
month before use. Also, it is difficult to use banana pulp solution
as a carrier solution in an FI procedure owing to the instability
of the baseline. On the other hand, the spinach leaf solution did
not exhibit high enzyme activity immediately and had to be
stored at 4 °C for 24 h in a refrigerator to release the enzyme
polyphenol oxidase from the cells before a centrifugation step in
the presence of 0.3% m/v sodium azide to prevent oxidation.
Even in the presence of this antioxidant, the long-term stability
of the spinach leaf solution at 4 °C is only 7 d.
In this work, an FI spectrophotometric procedure is reported
for determining l-dopa and carbidopa in pharmaceutical
formulations. A crude extract of sweet potato root [Ipomoea
Published on 01 January 1997 on http://pubs.rsc.org | doi:10.1039/A606852I
batatas (L.) Lam.] was used as the enzymatic source of
polyphenol oxidase (PPO; tyrosinase; catechol oxidase;
EC.1.14.18.1 ) directly in the carrier. This enzyme catalyses the
oxidation of these catecholamines to the corresponding dopa-
quinone. Further, dopaquinone is converted to leucodo-
pachrome by a rapid spontaneous auto-oxidation which is in
turn oxidized to dopachrome which presents a strong absorption
at 480 and 360 nm for l-dopa and carbidopa, respectively. The
use of an insoluble poly(vinylpyrrolidone) (PVP) such as
Polyclar SB-100 to remove natural phenolic compounds (e.g.,
Downloaded on 15 January 2013
chlorogenic and isochlorogenic acids) from solution in the
preparation of the crude extract of sweet potato root led to a
considerable increase in the enzyme activity, storage time and
stability of the baseline as well as to a decrease in the time
required to obtain the enzymatic carrier, since no previous
storage is needed to release the enzyme. The enzymatic FI
method proposed here to determine either l-dopa or carbidopa
selectively could be employed as an inexpensive alternative to
those procedures that use pure enzymes and/or chromogenic
reagents.
Experimental
Apparatus
A DuPont Instruments (Newtown, CT, USA) Model RC-5B
centrifuge, provided with a Model SS-34 rotor, was used.
A Hewlett-Packard (Boise, ID, USA) Model 8452A UV–
visible spectrophotometer was used in all spectrophotometric
measurements.
An eight-channel Ismatec (Zurich, Switzerland) Model
7618-40 peristaltic pump supplied with Tygon pump tubing was
used for the propulsion of the fluids. The manifold was
constructed with polyethylene tubing (0.8 mm id). Sample
injection was performed using a laboratory-constructed three-
piece manual commutator34 made of Perspex, with two fixed
side bars and a sliding central bar, which is moved for sampling
and injection. FI spectrophotometric measurements were car-
ried out using a Femto (São Paulo, Brazil) Model 435
spectrophotometer with a glass flow cell (optical path 1.0 cm)
connected to a Cole Parmer (Niles, IL, USA) Model 12020000
two-channel strip-chart recorder. The effect of temperature on
the enzymatic reaction was evaluated using a Tecnal (Pir-
acicaba, Brazil) Model TE184 thermostatically controlled
water-bath.
Reagents and Solutions
All reagents were of analytical-reagent grade and all solutions
were prepared with water from a Millipore (Bedford, MA,
USA) Milli-Q system (Model UV Plus Ultra-Low Organics
Water). Sucrose, glucose, fructose, lactose, starch, poly-
(ethylene glycol) 1500, sodium chloride, magnesium stearate
and indigo carmine were purchased from Sigma (St. Louis, MO,
USA). l-Dopa was purchased from BDH (Poole, Dorset, UK)
View Article Online
and carbidopa was kindly provided by PRODOME Chemical
and Pharmaceutical (Campinas, SP, Brazil); 1.0 3 1022 mol l21
stock solutions were prepared daily in 0.1 mol l21 phosphate
buffer of pH 7.0 and standardized by a conventional method.29
Standard solutions from 4.0 3 1024 to 1.0 3 1022 mol l21 were
prepared from the stock solutions in 0.1 mol l21 phosphate
buffer of pH 7.0. Amberlite CG-400 ion-exchange resin from
Aldrich (Milwaukee, WI, USA) was used as received. The
PVPs Polyclar AT, Polyclar SB-100, Polyclar R and Polyclar
K-30 were obtained from GAF (Wayne, NJ, USA) and were
purified essentially as described by McFarlane and Vader,35 i.e.,
the PVPs were boiled for 10 min in 10% v/v HCl and washed
with distilled water until free of chloride ion, then washed with
acetone and dried.
Healthy sweet potato roots [Ipomoea batatas (L.) Lam.]
purchased from a local producer were selected, washed, hand-
peeled, chopped and frozen in liquid nitrogen or in a freezer.
Methods
Preparation of the crude extract
Twenty-five grams of the frozen sweet potato root were
homogenized in a liquefier with 100 ml of 0.1 mol l21
phosphate buffer of pH 7.0, containing 2.5 g of Polyclar SB-100
or other stabilizer/protective agent, for 2 min at 4–6 °C. The
suspension was filtered through four layers of cheesecloth and
centrifuged at 25 000 3 g (18 000 rev min21) for 30 min at 4 °C;
it was stored at this temperature in a refrigerator and utilized as
the enzymatic source in the FI spectrophotometric procedure
after the determination of the PPO activity and total protein.
Measurement of PPO activity
The activity of soluble PPO present in the crude extract was
determined in triplicate by measurement of the absorbance at
410 nm of melanin-like pigments formed in the polymerization
of quinone produced by the reaction between 0.2 ml of
supernatant solution and 2.8 ml of 0.05 mol l21 catechol
solution in 0.1 mol l21 phosphate buffer (pH 7.0) at 25 °C. The
initial rate of the enzyme-catalysed reaction was a linear
function of time for 1.5–2.0 min. One activity unit (U) is defined
as the amount of enzyme that causes an increase of 0.001 A
min21 under the conditions described above.36
Total protein determination
The protein concentration was determined in triplicate by the
method of Lowry et al.37 using bovine serum albumin as
standard.
PPO solution in phosphate buffer
A 120 U PPO solution in 0.1 mol l21 phosphate buffer was
prepared daily by dilution of 33 ml of a 908 U PPO solution in
0.1 mol l21 phosphate buffer in a 250 ml calibrated flask using
the same buffer solution.
FI procedure
Fig. 1 shows a schematic diagram of the spectrophotometric
flow system used. A 120 U PPO solution in 0.1 mol l21
phosphate buffer was used as the carrier (C) at a flow rate of
1.01 ml min21. A solution contained in the sample loop (L, 500
ml) was injected and transported by the carrier after the baseline
had reached a steady-state value. A 400 cm tubular coiled
reactor maintained in a water-bath (R) at 25 °C was placed in
the analytical path in order to provide better reaction conditions.
The coloured zone was measured in the flow-through spec-
trophotometric cell (SC) at 370 nm (carbidopa) or 500 nm (l-
dopa).
 View Article Online

Analyst, April 1997, Vol. 122 347

Determination of L-dopa and carbidopa in pharmaceutical
formulations
The contents of 20 tablets were well mixed; from the fine
powder an accurately weighed portion was taken and dissolved
in phosphate buffer (pH 7.0; 0.1 mol l21 at 25 °C). Using a
mechanical shaker or an ultrasonic bath, the powder was
completely disintegrated and the solution was clarified by
passing it through No. 1 filter-paper, after which appropiate
dilutions were made.
Results and Discussion
Preparation of the Crude Extract
The activity and total protein extracted varied according to the
Published on 01 January 1997 on http://pubs.rsc.org | doi:10.1039/A606852I
using these compounds. As can be seen, Polyclar SB-100 in the
concentration ratio of 2.5 : 25.0 m/m was the best compound
among those studied. Polyclar SB-100 is a PVP of high
molecular mass which is supplied as a dry, finely divided white
powder. Its remarkable ability to remove phenolic compounds
from solutions39 and its low solubility made it particularly
useful in the preparation of the crude extract, since it can be
easily removed by filtration or by centrifugation. By using
insoluble polymers, several workers29,39–42 have separated
phenolic compounds that form strong H-bonded complexes
(i.e., those with isolated hydroxyl groups) from many crude
extracts, obtaining very active soluble enzymes. The inhibition
of PPO by –SH compounds and other reducing agents is well
documented.38 l-Cysteine used in this work probably inhibited
the enzyme by interaction with the enzyme’s copper, leading to

extraction procedure and medium used. The buffer-to-tissue
ratio was an important factor in the extraction of PPO from
sweet potato root. In this study, the enzyme was extracted using
ratios of 2–6 : 1 v/m and the highest specific activity was
obtained at a ratio of 4 : 1 v/m. It was also found that PPO could
be extracted with a phosphate buffer solution of low molarity
such as 0.05–0.4 mol l21 with the maximum yield achieved at
a concentration of 0.1 mol l21 (Table 1). The effect of buffer pH
on the extraction of PPO was also investigated in the pH range
6.0–8.0. The highest enzymatic activity was reached at pH 7.0,
Downloaded on 15 January 2013
a decrease in the crude extract activity (see Table 2). The same
effect was observed in our previous work29 and also in that of
Lourenço et al.43 in the preparation of crude extracts of yam
(Alocasia marcrohiza) and sweet potato root [Ipomoea batatas
(L.) Lam.], respectively.
Storage Time and Stability of Crude Extract
For the optimum extraction conditions described above (Poly-
clar SB-100), the enzyme activity of the crude extract did not

as shown in Table 1. In addition, it is well known38 that the vary for at least 5 months when the extract was stored in a
enzymatic browning tendency of sweet potato is related to refrigerator at 4 °C and decreased by only 4–5% after an 8 h
natural phenolic compounds (natural substrates present in the working period at 25 °C. Cysteine plus Amberlite CG-400 ion-
root), particularly chlorogenic and isochlorogenic acids which exchange resin also provided a good enzymatic stabilization,
comprise about 80% or more of the total phenolic compounds in but not cysteine alone, as can be seen in Fig. 2.
the root. This process and the oxidation by atmospheric oxygen The long storage time achieved and the low background
are responsible for the decrease in the PPO activity in the crude absorbances of the crude extract obtained with Polyclar SB-100
extract. In order to minimize this effect, several protective in comparison with other substances used here and/or sodium
agents and/or stabilizers were investigated such as Polyclar K- azide used by Uchiyama and Suzuki33 shows the advantage of
30, l-cysteine, l-cysteine + Amberlite CG- 400 ion-exchange the medium, preparation method and biological material used in
resin, Amberlite CG-400 ion-exchange resin + EDTA + this work.
Polyclar AT, Amberlite CG-400 ion-exchange resin, Polyclar
AT, Polyclar R and Polyclar SB-100. Table 2 presents the
Reaction of L-Dopa and/or Carbidopa with PPO
activity (U ml21), total protein (mg ml21) and specific activity
(U mg21 of protein) of the crude extracts obtained in triplicate Fig. 3 shows the reaction steps of the catalytic oxidation of l-
dopa by PPO.27,30,44 This enzyme catalyses the oxidation of l-
dopa to dopaquinone. Further, dopaquinone is converted to
leucodopachrome by a rapid spontaneous auto-oxidation which
is in turn oxidized to dopachrome which has a strong absorption
at 480 nm. Dopachrome can slowly be converted to melanin
through a series of reactions catalysed by Zn2+ ions, as shown in
this Fig. 3. An extensive spectrophotometric study of the
reaction of 120 U of PPO from the crude extract of sweet potato
with 5.0 3 1023 mol l21 l-dopa at 25 °C and pH 7.0 (0.1
mol l21 phosphate buffer) was carried out and the absorption
Fig. 1 Schematic diagram of the FI system used for l-dopa and carbidopa spectrum of the dopachrome produced after 3 min is shown in
determinations. The peristaltic pump is not shown and the broken line in the
central bar of the manual injector shows the injection position after
commutation. C, Carrier solution, 120 U of PPO in 0.1 mol l21 phosphate
buffer (pH 7), flowing at 1.01 ml min21; L, sample loop (100 cm, 500 ml); Table 2 Effect of protective agents and/or stabilizers on the extraction of
S, sample or standard solution; R, tubular coiled reactor (400 cm) in a water- PPO from sweet potato root at 25 °C
bath at 25 °C; SC, spectrophotometric cell [l = 500 nm (l-dopa) and 370
nm (carbidopa)]; W, waste. Protective agent Activity/ Total protein/ Specific activity/
and/or stabilizer U ml21 mg ml21 U mg21 protein
Polyclar K-30 1482 3.51 422
Table 1 Effect of phosphate buffer concentration and pH on the extraction l-Cysteine 1605 3.68 436
of PPO from sweet potato root at 25 °C l-Cysteine +
Amberlite CG-400 1570 3.25 483
Phosphate buffer Amberlite 1930 3.80 508
concentration/mol l21 0.05 0.1 0.2 0.3 0.4 CG-400 + EDTA
Specific activity/ + Polyclar A.T.
U mg21 protein 788 910 623 522 339 Amberlite CG-400 2270 3.75 605
pH 6.0 6.5 7.0 7.5 8.0 Polyclar A.T. 2440 3.80 642
Specific activity/ Polyclar R 2795 3.85 726
U mg21 protein 630 747 916 733 656 Polyclar SB-100 2997 3.30 908

348 Analyst, April 1997, Vol. 122
Fig. 4. The absorption of this chromophore did not increase
significantly ( ≈ 15%) for the time range 2–10 min. In another
study, the addition of 5.0 3 1023 mol l21 Zn2+ ions to the
reaction medium led to an increase in the wavelength of
absorption, which reached 540 nm after 1 h, indicating the
formation of melanochrome.30,44 A similar spectrophotometric
study was conducted for carbidopa and maximum absorption
was obtained at 360 nm. As can be seen from these two spectra
(Fig. 4), it is possible to determine each of these drugs
selectively. Although dopachrome has a maximum absorption
at 480 nm, all l-dopa determinations were performed at 500 nm
in order to eliminate the interference from the carbidopa
reaction product. Taking into account the enzymatic system
characteristics, an FI system was developed, as shown in
Fig. 1.
Published on 01 January 1997 on http://pubs.rsc.org | doi:10.1039/A606852I
FI Parameters and Reaction Conditions
The effect of varying the sample loop length from 50 to 200 cm
(250–1000 ml) on the absorbance signal was initially evaluated.
The best sample loop length was found to be 100 cm (500 ml).
With respect to sensitivity and analytical frequency, the best
compromise was attained using a coiled reactor 400 cm long
and a carrier flow rate of 1.01 ml min21.
The effect of varying the enzyme concentration in the carrier
solution from 30 to 185 U on the analytical signal (absorbance)
Downloaded on 15 January 2013
Fig. 2 Effect of the extraction medium (A, Polyclar SB-100; B, l-cysteine
+ Amberlite CG-400; and C, l-cysteine) on the storage time and/or stability
of the crude extract of sweet potato root.
Fig. 3 Reaction steps of the catalytic oxidation of l-dopa by PPO.
View Article Online
for l-dopa at concentrations of 5.0 3 1024, 1.0 3 1023, 5.0 3
1023 and 1.0 3 1022 mol l21 was also investigated. In the range
of l-dopa solution concentrations studied, the absorbance signal
increased with an increase in the concentration of the enzyme
solution used up to 100–120 U of PPO and then levelled off
between 120 and 185 U. Consequently, a concentration of 120
U was used in this work.
Table 3 shows the effect of pH in the range 5.0–8.0 on the
absorbance of a 5.0 3 1023 mol l21 l-dopa solution under the
conditions specified in the legend of Fig. 1. The optimum pH for
PPO activity was 7.0. The same optimum pH was found by
Lourenço et al.43 in pure and crude extracts of sweet potato. The
tubular coiled reactor was placed in a water-bath and the effect
of temperature was studied between 5 and 50 °C. The enzyme
exhibited the highest activity in the temperature range 15–25 °C
after which a gradual decline in its activity by heat inactivation
was observed between 25 and 50 °C (see Table 3). In a bath
study, heating at 90 °C for 40 min led to inactivation of 45% of
the enzyme compared with its initial activity using l-dopa as
substrate; thus, this shows the high stability of PPO in the
medium used.
Interference and Recovery Studies
The effect of excipient substances frequently found with
catecholamines in pharmaceutical formulations, such as suc-
rose, glucose, fructose, lactose, starch, poly(ethylene glycol),
sodium chloride, magnesium stearate and indigo carmine, was
evaluated using an FI system similar to that shown in Fig. 1. The
ratios of the concentrations of l-dopa or carbidopa to those of
the excipient substances were fixed at 0.1, 1.0 and 10.0. None of
these substances interfered in the proposed FI method.
Recoveries of 98.6 and 106.3% of l-dopa and carbidopa,
respectively, from two pharmaceutical formulation samples (n
= 6) were obtained using the FI spectrophotometric procedure.
In this study, 4.9, 9.9 and 14.8 mg of l-dopa or carbidopa were
added to each sample (Table 4). This is good evidence of the
accuracy of the proposed method. In addition, the relative
standard deviations were 1.28 and 1.33% for solutions contain-
ing 4.0 3 1023 mol l21 l-dopa or carbidopa (n = 12),
respectively; furthermore, the baselines were stable.
Analytical Curves and Applications
The conditions determined above, i.e., a sample loop (L) of 100
cm (500 ml), a reactor length of 400 cm, a carrier (C) flow rate
of 1.01 ml min21, an enzyme concentration of 120 U in 0.1
 View Article Online

Analyst, April 1997, Vol. 122 349

mol l21 phosphate buffer of pH 7.0 and a temperature of 25 °C,
were used for the proposed method. Triplicate signals for nine
l-dopa standard solutions and six consecutive signals for three
pharmaceutical formulation samples and triplicate signals for
Published on 01 January 1997 on http://pubs.rsc.org | doi:10.1039/A606852I
ten carbidopa standard solutions and six consecutive signals for
three samples are shown in Figs. 5(a) and (b), respectively. The
calibration graphs obtained for l-dopa and carbidopa in the
concentration range from 4.0 3 1024 to 1.0 3 1022 mol l21,
using the flow system depicted in Fig. 1, were A = 0.0033 +
64.72C1, r = 0.9993, and A = 0.0087 + 130.28C2, r = 0.9990,
where C1 and C2 are the concentrations of l-dopa and carbidopa
in mol l21, respectively. After an 8 h working period, no
baseline drift was observed and only a slight variation (4–5%)
of the enzyme activity of the crude extract was observed. Table
5 presents the results obtained for these samples using a
pharmacopeial method45 (a spectrophotometric method that
detects both catecholamines at 280 nm) and the proposed
enzymatic FI method and also those declared on the labels. The
results obtained for l-dopa (1) and carbidopa (2) by the

proposed enzymatic FI method are in close agreement with

those reported (r1 = 0.9699 and r2 = 0.9999). Additionally, the
total concentrations (l-dopa + carbidopa) obtained by the FI

Fig. 4 Absorption spectra of the chromophores formed in the enzymatic

Downloaded on 15 January 2013

reaction of PPO with 5.0 3 1023 mol l21 catecholamines: A, carbidopa and
B, l-dopa at 25 °C and pH 7.

Table 3 Effect of the pH of the 0.1 mol l21 phosphate buffer carrier solution
and of the temperature of the tubular coiled reactor (R) bath on the
absorbance value obtained for l-dopa at 500 nm. Experimental conditions
as in Fig. 1

pH 5.0 5.5 6.0 6.5 7.0 7.5 8.0

Absorbance 0.201 0.218 0.263 0.343 0.375 0.351 0.339
Temperature/
°C 5.0 10.0 20.0 25.0 30.0 40.0 50.0
Absorbance 0.326 0.358 0.385 0.394 0.323 0.225 0.145

Table 4 Results of addition–recovery experiments using l-dopa and

carbidopa with three different standard concentrations

l-Dopa/mg* Carbidopa/mg*
Recovery Recovery
Sample Added Found (%) Added Found (%)
4.90 5.01 102.2 4.90 4.83 98.6
Sinemet 9.90 10.03 101.3 9.90 9.92 100.2
14.80 14.70 99.3 14.80 14.73 99.5 Fig. 5 Transient absorbance signals obtained in triplicate for standard
4.90 5.12 104.5 4.90 5.21 106.3 catecholamines solutions: (a), 4.0; 6.0; 8.0; 10.0; 20.0; 40.0; 60.0; 80.0; and
Cronomet 9.90 10.17 102.7 9.90 10.24 103.4 100.0 3 1024 mol l21 l-dopa and three samples (A, sinemet; B, cronomet;
14.80 14.91 100.7 14.80 15.05 101.7 and C, prolopa) and the standard solutions again and (b), 4.0; 6.0; 8.0; 10.0;
* n = 6. 20.0; 40.0; 60.0; 70.0; 80.0; and 100.0 3 1024 mol l21 carbidopa, the same
three samples and the standard solutions again.

Table 5 Determination of l-dopa and carbidopa in pharmaceutical formulations using the pharmacopeial45 and enzymatic FI spectrophotometric
procedures

Label value/mg Pharmacopeia*/mg Enzymatic FI*/mg Relative error (RE) (%)

Sample l-Dopa Carbidopa l-Dopa + Carbidopa l-Dopa Carbidopa RE1† RE2‡ RE3§
Sinemet 250 25 274.5 ± 0.2 251.0 ± 0.1 24.5 ± 0.1 +0.4 22.0 +0.4
Cronomet 200 50 248.0 ± 0.3 211.2 ± 0.2 52.6 ± 0.1 +5.6 +2.5 +6.4
Prolopa 200 0 206.3 ± 0.1 197.7 ± 0.1 0.0 21.2 0.0 +4.2
1 = Enzymatic FI versus l-dopa label value.
* n = 6, confidence level 95%. † RE ‡ RE2 = Enzymatic FI versus carbidopa label
value. § RE3 = Enzymatic FI (l-dopa + carbidopa) versus pharmacopeial value.

350 Analyst, April 1997, Vol. 122
method agreed with those obtained using the pharmacopeial
method (r3 = 0.9675) and within an acceptable range of error.
The detection limits were 1.5 3 1025 and 2.0 3 1025 mol l21
(three times the signal blank/slope), for l-dopa and carbidopa,
respectively, and the throughput was 26 samples h21 with a
relative standard deviation of less than 1.0% (n = 6). Also, 2.30
ml of crude extract were consumed in each determination,
corresponding to only 72 mg of the original sweet potato
root.
Financial support of FAPESP (Processes 91/2637-5,
92/2637-5), PADCT/CNPq (Process 62.0060/91-3), CNPq
(Process 50.1638/91-1), and also the scholarship granted by
CAPES to I.C.V. are gratefully acknowledged.
Published on 01 January 1997 on http://pubs.rsc.org | doi:10.1039/A606852I
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Paper 6/06852I
Received October 10, 1996
Accepted January 6, 1997