J. Sep. Sci. 2007, 30, 1893 – 1898 S. Mennickent et al.

1893

Sigrid Mennickent1 Short Communication

Mario Nail1
Mario Vega2
Marta de Diego1
1
Department of Pharmacy,
Faculty of Pharmacy, University
of Concepci
n, Concepci
n,
Chile
2
Department of Bromatology,
Nutrition and Dietetic, Faculty
of Pharmacy, University of
Concepci
n, Concepci
n, Chile
Quantitative determination of L-DOPA in tablets by
high performance thin layer chromatography
A densitometric high performance thin-layer chromatographic (HPTLC) method was
developed and validated for quantitative analysis of L-DOPA in tablets. Chromato-
graphic separation was achieved on precoated silica gel F 254 HPTLC plates using a
mixture of acetone – chloroform – n-butanol – acetic acid glacial–water
(60:40:40:40:35 v/v/v/v/v) as mobile phase. Quantitative analysis was carried out at a
wavelength of 497 nm. The method was linear between 100 and 500 ng/lL, with a
correlation coefficient of 0.999. The intra-assay variation was between 0.26 and
0.65% and the interassay was between 0.52 and 2.04%. The detection limit was
1.12 ng/lL, and the quantification limit was 3.29 ng/lL. The accuracy ranged from
100.40 to 101.09%, with a CV not higher than 1.40%. The method was successfully
applied to quantify L-DOPA in real pharmaceutical samples, including the compari-
son with HPLC measurements. The method was fast, specific, with a good precision,
and accurate for the quantitative determination of L-DOPA in tablets.
Keywords: Drugs / HPTLC / L-DOPA / Pharmaceutical preparations / Quantitative analysis /
Received: December 18, 2006; revised: March 15, 2007; accepted: March 15, 2007
DOI 10.1002/jssc.200600533

1 Introduction
L-DOPA, the levorotatory isomer of dihydroxyphenylala-
nine, a natural amino acid, is the intermediate precursor
of the neurotransmitter dopamine. Its chemical struc-
ture is shown in Fig. 1. The actions of L-DOPA are mainly
those of dopamine. Unlike dopamine, L-DOPA can readily
enter the Central Nervous System and is used in the treat-
ment of conditions, such as Parkinson’s disease, which
are associated with depletion of dopamine in the brain
[1–4]. L-DOPA is considered by many clinicians as the
drug of choice in the management of idiophatic Parkin-
sonian syndrome [2]. Figure 1. Chemical structure of L-DOPA.
L-DOPA is rapidly decarboxylated in the human body,
so that very little unchanged drug is available to cross
the blood–brain barrier for central conversion into dopa-
mine. Consequently, L-DOPA is usually given together moisture or atmospheric oxygen, it is rapidly oxidized
with a peripheral dopa-decarboxylase inhibitor such as resulting in drug loss and potency reduction [1, 2, 5, 6].
carbidopa or benserazide to increase the proportion of L- Scientific literature reports several methods for the
DOPA that can enter the brain and to reduce its adverse determination of L-DOPA in biological fluids and phar-
effects [1–3]. maceutical preparations, such as spectrophotometry,
It is very important to determine the quantity of L- mostly for pharmaceuticals [7–22], HPLC, for biological
DOPA in its dosage forms because, in the presence of fluids [23–33], electrophoresis [34] and voltammetry [35].
The official method for the quantitative determination
of L-DOPA in tablets is by HPLC [5]. Nevertheless, HPLC
Correspondence: Prof. Sigrid Mennickent, Department of Phar- and other mentioned techniques have often suffered
macy, Faculty of Pharmacy, University of Concepci
n, P.O. Box from diverse disadvantages with regard to cost or selec-
237, Concepci
n, Chile
tivity, with complex sample preparation procedures, and
E-mail: smennick@udec.cl
Fax: +56-41-2207086 long analysis time.

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1894 S. Mennickent et al.
High performance TLC (HPTLC) is a technique carried
out within a short period of time, requires few mobile
phases, and allows for the analysis of a large number of
samples simultaneously. The ability of HPTLC to analyze
many samples in parallel has the advantage over other
techniques because separation of 10 or 20 samples takes
the same time as the separation of 1 sample. Amounts of
the order of nanograms (UV detection) and smaller than
picograms (fluorescence detection) can be detected.
No HPTLC method for the determination of L-DOPA
was found in the literature.
Therefore, the aim of this study was to develop and val-
idate a simple, rapid, sensitive, accurate, precise, and eco-
nomical HPTLC method for the quantitative determina-
tion of L-DOPA in tablets.
2 Experimental
2.1 Materials
2.1.1 Instrumentation
HPTLC system: Spectrodensitometer Scanner 3 (Camag,
Muttenz, Switzerland), equipped with software CATS
1.2.3 (Camag). Band application device: ATS automatic
(Camag); chromatographic chamber (Camag); and HPTLC
plates precoated with silica gel F254 (Merck, Darmstadt,
Germany).
2.1.2 Reagents and chemicals
USP standard of L-DOPA was obtained from Sigma-
Aldrich (St. Louis, MO). Methanol, acetone, chloroform, n-
butanol, glacial acetic acid, and monobasic potassium
phosphate were from Merck. All of the reagents were of
proanalysis quality. Pharmaceutical preparations of L-
DOPA were purchased from a local drugstore.
2.2 Methods
2.2.1 Standard solution preparation
A stock solution of L-DOPA was prepared by dissolving an
appropriate amount in water–methanol (7:3 v/v) to a con-
centration of 1 mg/mL. Working solutions of analyte
(100–500 ng/lL) were prepared by appropriate dilutions
of the stock solution with the same mixture of solvents.
All solutions were protected from light and stored in
refrigerator at 2–48C.
2.2.2 Sample preparation
Pharmaceutical preparations were tablets, nominally
containing 100 mg of L-DOPA and some nonspecific exci-
pients. They were processed as follows [5]: 20 tablets were
weighed and ground into fine powder and an accurately
weighed portion equivalent to 40 mg of L-DOPA was
diluted to 100 mL with water–methanol (7:3 v/v). The sol-
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J. Sep. Sci. 2007, 30, 1893 – 1898
ution was centrifuged and the supernatant was used.
Appropriate dilutions were done for the validation of the
method.
2.2.3 Chromatographic conditions
Chromatography was carried out on silica gel F254
HPTLC plates, previously activated at 1208C for 20 min.
Sample application was done on 4 mm bands using an
automatic ATS device (Camag). For the chromatographic
development, acetone–chloroform–n-butanol–acetic
acid glacial–water (60:40:40:40:35 v/v/v/v/v) was used as
mobile phase. The length of development was 8 cm dur-
ing a period of about 12 min. Vertical development
chambers were used.
Ninhydrine (0.5%) in ethanol was used as a revelator
compound, by the immersion of plates into Deeping
Device (Camag).
Densitometry readings were carried out using a Scan-
ner 3 Camag Spectrodensitometer assisted by a computer
equipped with software WINCATS 1.2.3, and a tungsten
lamp was used as the radiation source. Determination
was performed at a wavelength of 497 nm.
2.2.4 HPLC procedure
As the HPLC is the official method for the determination
of L-DOPA in tablets, the analysis in real pharmaceutical
samples was done by HPTLC and HPLC methods.
By HPLC method, the pharmaceutical preparation was
processed in the same way as by HPTLC (Section 2.2.2) [5].
This procedure was carried out in triplicate. Then, 20 lL
of each test solution and a standard solution of 0.4 mg/
mL was injected into an HPLC system Perkin Elmer series
200 (Norwalk, CT, USA) with an Applied Biosystems
model 785-A programmable absorbance detector (Foster,
CA, USA) and a Perkin Elmer Nelson model 1022 data pro-
cessor. The column was a Lichrospher 100 RP-18,
25 cm64 mm with 5 lm particle size (Merck). The
mobile phase was monobasic potassium phosphate buf-
fer (0.01 M; pH = 2); flow rate, 1 mL/min, and detector
wavelength, 280 nm [5]. The buffer was prepared by dis-
solving 1.36 g of monobasic potassium phosphate
(KH2PO4) in water, and adjusting to pH = 2 with phos-
phoric acid (H3PO4).
2.2.5 Validation
The parameters validated were linearity, detection limit,
quantification limit, precision, accuracy, and selectivity.
Validation was performed in compliance with interna-
tional standards [5, 36–38], using adequate statistical
estimates (RSD, Student’s t-test, ANOVA) [37, 39].
2.2.5.1 Linearity
Linearity was determined by a calibration curve with
standard L-DOPA solutions in the range of 100–500 ng/
lL. Each solution was spotted six times. This range
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J. Sep. Sci. 2007, 30, 1893 – 1898
includes the solution concentration of the tablets as
assayed by USP.
2.2.5.2 Detection and quantification limits
In order to determine the detection limit (LOD) and the
quantification limit (LOQ), analyte concentrations corre-
sponding to the lower part of the linear range of the cali-
bration curve were used. Solutions of concentrations 25,
50, and 100 ng/lL were prepared, each of them being
applied ten times. LOD and LOQ were calculated by using
equations [36, 38]: LOD = 3.3r/b; LOQ = 10r/b, where r is
the SD of the response and “b” corresponds to the slope
obtained from the linearity study of the method.
2.2.5.3 Precision
This parameter was determined by the measurement of
instrumental, intra-assay, and intermediate precision.
Instrumental precision was measured for ten spots of
the standard at each concentration.
L-DOPA solutions, from pharmaceutical samples, of
100, 300, and 500 ng/lL were used in intra-assay and
intermediate precision studies.
The intra-assay precision was studied by analyzing
repeatedly, in the same laboratory and on the same day,
five solutions at each concentration, each of which was
independently prepared and each of them being applied
five times [5, 36–38].
For intermediate precision (interassay precision), each
solution was analyzed three times on the same day for
three different days [5, 36–38].
2.2.5.4 Accuracy
The accuracy was determined by addition standard,
applying the method to pharmaceutical preparations to
which known amounts of standard substance corre-
sponding to 80, 100, and 120% of the concentration
expected in the samples were added. The accuracy was
then calculated from the test results as the percentage of
analyte recovered by the assay.
2.2.5.5 Selectivity
The selectivity of the method was evaluated by observing
any interference from carbidopa, a compound present in
most of the L-DOPA pharmaceutical preparations (see
Section 1).
2.2.6 Application of the method
The method was used for the quantitative determination
of L-DOPA in tablets.
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Other Techniques 1895
3 Results and discussion
3.1 Optimization of the method
In order to validate an efficient method for the analysis
of L-DOPA in tablets, preliminary tests were performed
with the objective to select adequate and optimum con-
ditions. Parameters, such as detection wavelength, ideal
mobile phase and their proportions, and concentration
of the standard solutions were exhaustively studied.
Several eluents were tested using different proportions
of solvents, such as methanol, water, ethyl acetate, for-
mic acid, n-butanol, acetone, chloroform, and glacial ace-
tic acid. The best results were obtained with the mobile
phase mentioned by Fernndez et al. [23].
3.2 Validation of the method
The method proved to be linear between 100 and 500 ng/
lL (100, 200, 300, 400, and 500 ng/lL). The calibration
curve had a correlation coefficient of 0.999, indicating a
linear relationship between the concentration of L-DOPA
and peak area over the range investigated. The slope of
the curve was 11.82 and the intercept was 33.78. The
analysis of variance (ANOVA) showed an Fc of 40.00 and
an Ftheoretical (a = 0.05; t = 1.10) of 4.96. H0 (b = 0) was
rejected (b = slope of the curve).
The detection limit (LOD) was 1.12 ng/lL and the quan-
tification limit (LOQ) was 3.29 ng/lL. These are adequate
values for the detection and quantification of L-DOPA in
tablets.
Precision analysis studies showed an instrumental pre-
cision between 0.23 and 0.68%; an intra-assay variation
between 0.26 and 0.65% and an interassay variation
between 0.52 and 2.04% (Table 1). These values are
adequate for the analysis of drugs in concentrations as
low as nanograms [36–38].
The recovery obtained did not differ from the real
value in more than 1.09% and is independent of the con-
centration with a CV less than 1.40% as shown in Table 2.
When L-DOPA was analyzed in the mixture with carbi-
dopa, no interferences were found for any of the com-
Table 1. Precision of the method
CV (%)
Concentration Instrumental Intra-assay Intermediate
(ng/lL) precisiona) precisionb) precisionc)
100 0.45 0.48 2.04
300 0.68 0.65 0.77
500 0.23 0.26 0.52
a) n = 10; analyzed on the same day.
b) n = 5; analyzed on the same day (for each concentration).
c) n = 9; analyzed on three different days (for each concen-
tration).
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1896 S. Mennickent et al. J. Sep. Sci. 2007, 30, 1893 – 1898

Figure 2. Selectivity of the method.

Peak no. 1: L-DOPA; peak no. 2: car-
bidopa.

Table 2. Method accuracy Table 3. Comparison of the determination of L-DOPA in tab-

lets by the proposed method and by HPLC
Added concen- Found concen- Accuracy (%)b) CV (%)
tration (ng/lL) tration (ng/lL)a) Parameter HPTLC HPLC

Intra-assay (n = 9) Labeled claim (mg per tablet) 100.00 100.00

320 322.10  4.20 100.66 1.30 Amount founda) (mg per tablet) 90.99 90.00
400 404.22  3.70 101.06 0.92 RSD (%) 2.40 1.53
480 485.25  1.20 101.09 0.25 F calc.: 11.86; F theoreticalb): 13.61

Interassay (n = 27)
320 321.27  4.50 100.40 1.40 a) Each result is the mean of nine experiments (three solu-
400 402.30  3.80 100.58 0.94 tions, three determinations of each of them).
480 482.83  2.35 100.60 0.49 b) a = 0.05.

a) Each value is the mean  SD.

b) (Found concentration/added concentration)6100.
pound present in the samples (RF L-DOPA = 0.37; RF carbi-
dopa = 0.79) (Fig. 2). The excipients present in the tablets
do not interfere in the analysis. These are eliminated at
the centrifugation step (see Section 2.2.2), as they are not
dissolved by the working solvents.
3.3 Application of the method
Figure 3 shows the application of the method for the
quantitative determination of L-DOPA in tablets. Table 3
compares the results of the analysis of L-DOPA between
proposed (HPTLC) and official (HPLC) method. The
amounts of L-DOPA found in tablets are fairly close to the
labeled amounts for both techniques and between the
accepted limits by USP [5]. The results obtained from the
two methods were statistically compared with each
other at the 95% confidence level with the aid of F-test.
No significant difference was found between both meth-
ods with respect to mean values and SD since the calcu-
lated F-values were less than the corresponding theoreti-
cal one.
4 Concluding remarks
The results indicate that the HPTLC method is useful for
the determination of L-DOPA in tablets. The method has
the required linearity, precision, accuracy, selectivity
and detection and quantification limits needed for the
quantitative determination of L-DOPA in this dosage
form.
The method resulted was fast, simple, efficient, and
easy to perform. HPTLC separation was obtained within
12 min and the method allows for a large number of sam-
ples to be measured simultaneously with a very good
accuracy, sensitivity, and precision. It is very important
especially in quality control. The procedure works with-
out separation steps and involves only centrifugation
because working solvents do not dissolve the excipients.

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J. Sep. Sci. 2007, 30, 1893 – 1898
Figure 3. Quantitative determination of L-DOPA in tablets by proposed HPTLC method. Tracks 1 and 18: standard 150 ng/lL;
tracks 2 and 17: standard 300 ng/lL; tracks 3 and 16: standard 450 ng/lL; tracks 4–15: samples.
The method has an appropriate low LOD and LOQ,
recovery percentage, and precision for quantitative
determination of L-DOPA in tablets, with the advantages
over other techniques as the time and speed of analysis,
and a lower cost of materials and columns (HPLC).
The authors would like to thank the Research Council at the Uni-
versity of Concepci
n (Project DIUC no. 201.074.034-1.0).
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