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					                                                 Spectrochimica Acta Part A 62 (2005) 213–220




            A spectroscopic study on applicability of spectral analysis for
           simultaneous quantification of l-dopa, benserazide and ascorbic
                           acid in batch and flow systems
                                   Joanna Karpi´ ska∗ , Jerzy Smyk, El˙ bieta Wołyniec
                                               n                      z
                                  Institute of Chemistry, University of Bialystok, ul. Hurtowa 1, 15-399 Białystok, Poland

                                                 Received 7 December 2004; accepted 16 December 2004



Abstract

  The usefulness of derivative spectrophotometry for simultaneous assay of l-dopa, benserazide and ascorbic acid in pharmaceuticals
was studied. The parameters of derivatisation depends on composition of solution in which particular compound was determined. For
quantification of l-dopa in mixtures with benserazide or ascorbic acid the first derivative was used. Its determination in ternary mixture (l-
dopa + benserazide + ascorbic acid) is possible by third derivative spectra. Benserazide was assayed in presence of l-dopa using first derivative
while in ternary mixture by third derivative. Direct determination of ascorbic acid is possible applying first derivative only in presence of
l-dopa. The elaborated derivative spectrophotometric methods were used for assaying of l-dopa and benserazide in their commercial form
“Madopar”. The proposed spectrophotometric derivative method of simultaneous determination of l-dopa and benserazide was combined
with FIA technique.
© 2004 Elsevier B.V. All rights reserved.

Keywords: l-dopa; Benserazide; Ascorbic acid; Derivative spectrophotometry; FIA; Stopped flow mode




1. Introduction                                                                  attractive. They allow simplifying analytical procedure, re-
                                                                                 duction time and cost of individual analysis. Derivative spec-
    Procedures based on analysis of absorption spectra in the                    trophotometry is one of the methods of improving the selec-
UV–vis range are instrumental methods the most often used                        tivity of spectrophotometrical determination. This technique
in analytical laboratories. Their main advantage is usually                      is based on the analysis of derivative spectra. Derivatisation
simple procedure and cheap and easy access to analytical                         of absorption spectra leads to separate of overlapped signals
apparatus. The spectrophotometers are the basic equipments                       and as the consequence the elimination of influence of ac-
of each laboratory. The main disadvantage of spectrophoto-                       companied substances on results of determination.
metric determination is its low selectivity. The spectrum of                         In presented paper the usefulness of spectral analysis for
complex mixture is the sum of individual components. An im-                      simultaneous assay of l-dopa in binary or ternary mixtures
provement in sensitivity and selectivity can be achieved by the                  with ascorbic acid and/or benserazide is discussed. The ob-
use of selective spectrophotometric reagents, by implemen-                       tained results were evaluated in respect to their applicability
tation masking reactions or by separation of an analyte from                     for simultaneous determination of studied compounds in their
a matrix. An introduction of additional operation increases                      binary and ternary mixtures. As derivative method is charac-
a risk of contamination or lost of analyte. Due to mentioned                     terized by versatility and flexibility it can be coupled with oth-
above reasons, the direct methods of analysis seem to be very                    ers analytical techniques, e.g. chromatography or flow anal-
                                                                                 ysis. The elaborated derivative method of determination of
 ∗   Corresponding author. Tel.: +4885 7457809; fax: +4885 7477800.              l-dopa was implemented into flow system. For this purpose
     E-mail address: joasia@uwb.edu.pl (J. Karpi´ ska).
                                                n                                the flow system with stopped flow was used. The worked up

1386-1425/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2004.12.029
214                                           n
                                      J. Karpi´ ska et al. / Spectrochimica Acta Part A 62 (2005) 213–220

flow procedure was applied for determination of l-dopa in
its multiple pharmaceutical “Madopar 62.5”.
    l-dopa or ((−)3-(3,4-dihydroxyphenyl)-l-alanine), a nat-
urally occurring amino acid, is the immediate precursor of
the neurotransmitter dopamine. It is used for the treatment
of neural disorders such as Parkinson’s disease which is con-
nected with dopamine shortage in definite regions of brain.
                                                                           Fig. 1. Schematic diagram of the FIA manifold used for the l-dopa deter-
As dopamine is unable to penetrate the blood–brain barrier,                mination. Pp: perictaltic pump; Iv: injection valve; D: detector; R: reaction
it cannot be administered directly. l-dopa, after its oral ad-             coil; S: carrier stream; Ss: sample solution.
ministration is absorbed through the bowel at the level of the
small intestine, achieving maximum concentration in blood                  (Model 5020) (Iv). The flow system was made of PTFE tub-
after about 3 h. It reaches the brain where it is metabolized to           ing of 0.8 mm i.d. The sample was spectrophotometrically
dopamine in decarboxylation process. This process may also                 measured by means of a Hewlett-Packard HP-8452A diode
to some extent occur before l-dopa reaches the brain. l-dopa               array spectrophotometer (D), equipped with a Hellman flow
in therapy of Parkinsonism is administrated in combination                 cell (1 cm light-path and inner volume 18 l).
with inhibitors of aromatic amino acids decarboxylase, e.g.
carbidopa or benserazide to avoid premature decarboxyla-                   2.1.2. Chromatography
tion.                                                                          The chromatographic system (Thermo Separation) con-
    Several instrumental methods like chromatography [1],                  sisted of the 3D detector Spectra System UV 3000, the low-
spectrophotometry [2–5], flow injection technique [6–9] and                 gradient pump P2000, the vacuum membrane degasser SCM
others have been proposed for the determination of l-dopa                  Thermo Separation and the Rheodyne loop injector (20 l),
individually in pharmaceutical preparations.                               was used. ChromQuest Chromatography Data System soft-
    There are a very few methods devoted to the simultaneous               ware version for Windows NT was used for the acquisition
determination of l-dopa and benserazide in binary mixtures                 and storage of data. The measurements at 280 nm were car-
based on derivative spectrophotometry method [10,11] but                   ried out using the reversed-phase analytical column, Lichro-
no papers concerned on the simultaneous determination of l-                spher RP-C18150 × 4.6 (5 l) (Merck, Germany). The mo-
dopa, and benserazide in binary mixtures using flow system.                 bile phase was composed with 0.1 M potassium dihydrogen
    As antioxidants are very often assembled together in phar-             ortophosphate adjusted to pH 3.0 by 1 M orthophosphoric
maceuticals, the aim of the presented work was to discuss the              acid. The flow rate was set at 1.5 ml/min.
usefulness of analysis of derivative spectra for the simultane-
ous determination of l-dopa, benserazide hydrochloride and                 2.2. Reagents
ascorbic acid without the initial separation of them.
                                                                               All reagents used were of analytical grade.
                                                                               l-dopa (LD) – Sigma–Aldrich, USA: the stock solution
2. Experimental                                                            of l-dopa (0.01 M) was prepared by dissolving an appropri-
                                                                           ate amount of compound in 0.01 M hydrochloric acid. The
2.1. Apparatus                                                             standard solution was stored in dark bottle at 4 ◦ C. Working
                                                                           solutions (5 × 10−6 to 5 × 10−4 M) were prepared freshly
   A Hewlett-Packard HP-8452A diode array spectropho-                      every day by an appropriate dilution of stock solution with
tometer coupled to the User Data vision software was used                  double distilled water.
for the acquisition and storage of spectra. The 1 cm quartz                    Benserazide hydrochloride (BEN) – Sigma–Aldrich,
cuvette was applied for spectral analysis. The used diode-                 USA: the stock solution of benserazide hydrochloride
array spectrophotometer was supplied with a series of 328                  (0.01 M) was prepared by dissolving an appropriate amount
individual photodiodes. The spectrophotometer instrumental                 of compound in 0.01 M hydrochloric acid. The standard so-
working conditions were: integration time (1 s), spectral band             lution was stored in dark bottle at 4 ◦ C. Working solutions
width 2 nm and spectrum scan 0.1 s.                                        (5 × 10−6 to 5 × 10−4 M) were obtained by appropriate di-
   Computer Pentium II 333 MHz, 64 MB RAM equipped                         lutions of the standard solution with double distilled water.
in Grams/32 software package Version 5.2 (Galactic, USA)                   The working solutions were prepared freshly every day.
was used for generation of the derivative spectra.                             Ascorbic acid (Vitamin C) (AA) – Polfa, Poland: the stock
                                                                           solution of ascorbic acid (0.01 M) was prepared by dissolving
2.1.1. FIA assembly                                                        an appropriate amount of compound in 0.01 M hydrochloric
   The finally proposed single-channel FIA manifold is                      acid. The standard solution was stored in dark bottle at 4 ◦ C.
shown in Fig. 1. The carrier solution (S) (1 × 10−4 M hy-                  Working solutions (5 × 10−6 to 5 × 10−4 M) were prepared
drochloric acid), was aspirated by the Gilson (model Mini-                 by appropriate dilutions of the standard solution with double
plus 2) peristaltic pump (Pp). The sample solution (Ss) was                distilled water. The working solutions were prepared freshly
injected into the carrier stream via a Rheodyne injection valve            every day.
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                                     J. Karpi´ ska et al. / Spectrochimica Acta Part A 62 (2005) 213–220                                          215

   Madopar 62.5 capsules – Hoffmann-La-Roche Ltd.,                        2.3.5. Chromatographic analysis of l-dopa
Basel, Switzerland: each capsule included 50 mg of l-dopa                    The HPLC method of assaying of l-dopa was used as
and 12.5 mg of benserazide hydrochloride.                                 reference method [13]. For this purpose chromatograms
                                                                          of standard solutions of l-dopa in the concentration range
2.3. Procedures                                                           3 × 10−4 to 4 × 10−3 M were recorded at 280 nm. Calibra-
                                                                          tion graph was drawn as function of dependence of peak
2.3.1. Calibration graph of l-dopa                                        area on concentration. The following equation was obtained:
    An aliquot of l-dopa working solution equivalent to                   y = 3 × 109 x + 83837 (R2 = 0.9999).
5 × 10−6 to 5 × 10−4 M was placed into a 50 ml calibration
flask and diluted to the mark with double distilled water. The
absorption spectra were recorded at the wavelength range                  3. Results and discussion
190–390 nm against double distilled water as a blank. Next
the zero-order spectra were transformed into derivative spec-             3.1. Spectral analysis
tra using Savitsky–Golay procedure [12] (for optimal param-
eters of spectra generation depending upon the mixture con-                  Absorption spectra of l-dopa, benserazide hydrochloride,
tent) by Grams program.                                                   ascorbic acid and their mixture are shown in Fig. 2. The
                                                                          spectrum of l-dopa possess two distinct bands of absorption
                                                                          at λ = 204 and 282 nm and shoulder at 224 nm. The broad
2.3.2. Calibration graph of benserazide hydrochloride
                                                                          and intense band between 214 and 228 nm and small one at
   Appropriate aliquots of water solution of benserazide hy-
                                                                          272 nm are observed in spectrum of benserazide. The absorp-
drochloride were placed into 50 ml calibration flasks and di-
                                                                          tion curve of ascorbic acid has only one band at 260 nm.
luted to the mark with double distilled water. The spectra of
                                                                             As it can be seen in Fig. 2, direct determination of each
standard solutions in the range 5 × 10−6 to 5 × 10−4 M were
                                                                          compound in their mixture is impossible with the use of a UV
recorded (190–390 nm) and subjected to the mathematical
                                                                          spectrophotometry due to the strong overlap of the spectra.
treatment using Savitsky–Golay procedure (for optimal pa-
                                                                          The derivative spectrophotometry was used for the separation
rameters of spectra generation depending upon the mixture
                                                                          of the analytes’ signals. For this purpose zero-order spec-
content) by Grams program.
                                                                          tra of l-dopa, benserazide hydrochloride, ascorbic acid and
                                                                          their mixtures (LD + BEN, LD + AA, LD + BEN + AA) were
2.3.3. Calibration graph of ascorbic acid                                 recorded individually and next subjected to the mathematical
    An aliquot of ascorbic acid solution equivalent to 5 × 10−6
to 5 × 10−4 M of was placed into 50 ml calibration flasks
and diluted to the mark with double distilled water. The ab-
sorption spectra at the wavelength range 190–390 nm against
double distilled water as a blank were recorded using 1 cm
quartz cuvette. The derivative spectra were generated using
Savitsky–Golay procedure (for optimal parameters of spec-
tra generation depending upon the mixture content) by Grams
program.

2.3.4. Analysis of l-dopa and benserazide hydrochloride
in capsules “Madopar 62.5 ”
   The stock solution was prepared by the transfer of the con-
tent of one capsule of preparate (each capsule includes 50 mg
of l-dopa and 12.5 mg of benserazide hydrochloride) into a
100 ml volumetric flask. The powder was dissolved in 25 ml
of 0.01 M hydrochloric acid and filtered. The filtrate was
transferred into 100 ml volumetric flask and fulfilled to the
mark with 0.01 M hydrochloric acid. The working solution
(concentration 1 × 10−4 M versus l-dopa and 1.7 × 10−5 M
versus benserazide hydrochloride) was prepared by the ap-
propriate dilutions of the stock solution with double distilled
water. The zero-order absorption spectra working solution
was recorded at the wavelength range 190–390 nm against
double distillated water as a blank. The derivative spectra
                                                                          Fig. 2. Zero-order spectra of l-dopa (1 × 10−4 M), benserazide hydrochlo-
were generated using Savitsky–Golay procedure by Grams                    ride (4 × 10−4 M), ascorbic acid (5 × 10−5 M) and their mixture in the range
program.                                                                  190–310 nm.
216                                             n
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treatment. Then the analysis of derivative spectra (first to fifth
derivative order) was done for different polynomial degrees
(2–5) and different derivatisation window widths (5–11). As
a result, many zero-crossing interferent points were obtained,
which enable determination of the each individual compound
in presence of others. In order to select the best wavelengths,
the error of derivative value measurement was examined. The
error, caused by the presence of accompanying substances,
was calculated with the use of following formulae:
             nD
               xi,λ
error(%) =   nD
               yi,λ
                      × 100    for binary mixtures,

and
             nD    n
               xi,λ Dzi,λ
error(%) =      nD
                   yi,λ
                            × 100   for ternary mixtures

where n Dxi,λ and n Dzi,λ are the values of interferent’s ampli-
tudes at zero-crossing points λ, n Dyi,λ the value of analyte
amplitude at zero-crossing point λ.
   The parameters of derivatisation for each individual ana-
lyte were selected in the relation to the composition of solu-
tion in which it was analyzed.
   After detailed examinations first, second and third-order
derivative were chosen for compounds under study. The ob-                    Fig. 4. First-order spectra of l-dopa (1 × 10−4 M), ascorbic acid
tained results are shown in Figs. 3–5 and assembled in Table 1.              (5 × 10−5 M) and their mixture in the range 230–310 nm.
There is observed the strong dependence of derivatisation pa-
rameters on composition of analysed mixture. The third-order                 ers compounds. The derivatisation causes fast zeroing of AA
of derivative enables determination of l-dopa or benserazide                 signals, so its assaying is possible using first derivative only
in ternary mixture. The first derivative was used for simul-                  in binary mixture with l-dopa. The performed study pointed
taneous determination of studied catecholamine and benser-                   that determination of l-dopa in presence of benserazide is
azide in presence of each other. The spectral properties of                  possible at two wavelengths 269.1 and 293.2 nm. So, these
ascorbic acid do not allow to remove the interferences of oth-               wavelengths were used for its further analysis in binary mix-
                                                                             tures.




Fig. 3. Second-order spectra of l-dopa (1 × 10−4 M), ascorbic acid           Fig. 5. First-order spectra of l-dopa (1 × 10−4 M), benserazide hydrochlo-
(5 × 10−5 M) and their mixture in the range 260–320 nm.                      ride (4 × 10−4 M) and their mixture in the range 230–310 nm.
                                                     n
                                             J. Karpi´ ska et al. / Spectrochimica Acta Part A 62 (2005) 213–220                                217

Table 1
Mathematic parameters for generation of derivative spectra of the studied compounds
Analyte                            Mixture                              Parameter                                  Optimal value
l-dopa                             Ternary (LD + BEN + AA)              Derivative order                           III
                                                                        Polynomial degree                          5
                                                                          λ: derivatisation window                 Nine experimental points = 18 nm
                                                                        λmax                                       288.3 nm
                                                                        Error of measurement 3 D288.3 value (%)    +0.2
                                   Binary (LD + BEN)                    Derivative order                           I
                                                                        Polynomial degree                          4
                                                                          λ: derivatisation window                 Nine experimental points = 18 nm
                                                                        λmax                                       269.1 nm
                                                                        Error of measurement 1 D269.1 value (%)    +0.14
                                                                        Derivative order                           I
                                                                        Polynomial degree                          5
                                                                          λ: derivatisation window                 11 experimental points = 22 nm
                                                                        λmax                                       293. 2 nm
                                                                        Error of measurement 1 D293.2 value (%)    −1.19
                                   Binary (LD + AA)                     Derivative order                           II
                                                                        Polynomial degree                          5
                                                                          λ: derivatisation window                 Seven experimental points = 14 nm
                                                                        λmax                                       290 nm
                                                                        Error of measurement 2 D290 value (%)      −0.23
Benserazide hydrochloride          Ternary (LD + BEN + AA)              Derivative order                           III
                                                                        Polynomial degree                          5
                                                                          λ: derivatisation window                 Nine experimental points = 18 nm
                                                                        λmax                                       280.2 nm
                                                                        Error of measurement 3 D280.2 value (%)    −0.5
                                   binary (LD + BEN)                    Derivative order                           I
                                                                        Polynomial degree                          5
                                                                          λ: derivatisation window                 Nine experimental points = 18 nm
                                                                        λmax                                       250.8 nm
                                                                        Error of measurement 1 D250.8 value (%)    −0.81
Ascorbic acid                      Binary (LD + AA)                     Derivative order                           I
                                                                        Polynomial degree                          5
                                                                          λ: derivatisation window                 Seven experimental points = 14 nm
                                                                        λmax                                       280 nm
                                                                        Error of measurement 1 D280 value (%)      −7.2




3.2. Calibration graphs                                                               As it was mentioned above, the spectral properties of
                                                                                  ascorbic acid do not allow the elimination of the influence
   Using the optimal mathematical parameters of generation                        of l-dopa or/and benserazide hydrochloride on its determi-
derivative spectra, described above, calibration graphs were                      nation. The performed studies pointed that the determination
constructed at selected wavelengths as a function of the con-                     of AA in presence of l-dopa or benserazide is possible using
centration of each compound alone. The obtained equations                         the selected experimental parameters, only when its concen-
of calibration graphs and the statistical evaluation studied                      tration is equal or higher than the second compound.
compounds are assembled in Table 2.                                                   The obtained results of determination of LD in ternary
                                                                                  mixtures showed that its assay in such composed solution is
3.3. Selectivity of derivative method                                             the difficult analytical task (Table 3). The derivatisation do
                                                                                  not remove the spectral influence of two others ingredients.
   The selectivity of determination of each compound in its                       The determination of l-dopa with an error about 5% is pos-
mixtures without preliminary separation was assayed to com-                       sible only when its concentration is not than twice times less
plete the validation of the elaborated methods. For this pur-                     than others accompanied compounds. The determination of
pose a series of solutions included two (LD + BEN, LD + AA)                       l-dopa in binary mixtures (LD + BEN, LD + AA) is charac-
or all of studied compounds (LD + BEN + AA) were pre-                             terized by better selectivity. The influence of ascorbic acid
pared. Determination of one compound was carried out in-                          upon LD determination is very low. The determination of l-
dividually while others were presumed as interference. The                        dopa is possible even in the presence of five times higher con-
obtained results were used for the estimation of the usefulness                   centration of ascorbic acid than studied catecholamine. The
of the elaborated method for analysis.                                            selectivity of l-dopa determination in presence of benser-
218                                                   n
                                              J. Karpi´ ska et al. / Spectrochimica Acta Part A 62 (2005) 213–220

Table 2
Analytical characteristic of elaborated derivative spectrophotometric methods
Analyte       Mixture                               λ (nm)           Linearity range (mol dm−1 )      Equation of calibration curve       Correlation coefficient
LD            Ternary (LD+BEN+AA)                   288.3            5 × 10−6 to  5 × 10−4            y = −5.75 × 10x − 10−4              0.9999
              Binary (LD + BEN)                     269.1            5 × 10−6 t o 5 × 10−4            y = 2.58 × 102 x + 7 × 10−4         0.9997
                                                    293.2            5 × 10−6 t o 5 × 10−4            y = −3.86 × 102 x − 1.1 × 10−3      0.9997
              Binary (LD+AA)                        290              5 × 10−6 t o 5 × 10−4            y = 1.69 × 102 x + 7 × 10−5         0.9998
BEN           Ternary (LD + BEN + AA)               280.2            5 × 10−6 t o 5 × 10−4            y = −2.02 × 10x − 2 × 10−5          0.9998
              Binary (LD + BEN)                     250.8            5 × 10−6 t o 5 × 10−4            y = −1.01 × 102 x − 6 × 10−6        0.9997
AA            Binary (LD + AA)                      280              5 × 10−6 to 6 × 10−5             y = −5.38 × 102 x − 3.9 × 10−3      0.9857



                                                                                        Table 5
Table 3
                                                                                        Determination of benserazide hydrochloride in presence of l-dopa
Determination of l-dopa in ternary mixtures (LD + BEN + AA)
                                                                                        Concentration of        The presence of        CBEN /CLD      Relative
Concentration of     The presence of         CLD /           Relative error (%)         benserazide taken       interferent                           error (%)
l-dopa taken for     interferent (BEN)       CBEN/AA         λ = 288.3 nm               for analysis (M)        (l-dopa) (M)
analysis (M)         and (AA) (M)
                                                                                        5.0 × 10−5              5.0 × 10−5             1               +0.93
5.0 × 10−5           5.0 × 10−5              1               +4.2
                                                                                        1.0 × 10−4              5.0 × 10−5             2               −1.54
1.0 × 10−4           5.0 × 10−5              2               +4.0
                                                                                        2.5 × 10−4              5.0 × 10−5             5               +0.61
2.5 × 10−4           5.0 × 10−5              5               +1.6
                                                                                        5.0 × 10−4              5.0 × 10−5             10              −0.80
5.0 × 10−4           5.0 × 10−5              10              0.0
                                                                                        5.0 × 10−5              1.0 × 10−4             1/2             +5.30
5.0 × 10−5           1.0 × 10−4              1/2             +5.6
                                                                                        5.0 × 10−5              2.5 × 10−4             1/5             +6.70
5.0 × 10−5           2.5 × 10−4              1/5             +11.8
                                                                                        5.0 × 10−5              5.0 × 10−4             1/10            +7.10
5.0 × 10−5           5.0 × 10−4              1/10            +20.8
                                                                                        5.0 × 10−5              1.0 × 10−3             1/20           +25.90
5.0 × 10−5           1.0 × 10−3              1/20            +17.9

                                                                                        achieved less than 6%. As conclusion it can be stated that
azide depends on analytical wavelength at which the mea-                                spectral properties of l-dopa allow to its assay in presence
surements of derivative value are performed. The reading of                             of benserazide and/or ascorbic acid. The determination
derivative at 269.1 nm allows assaying of l-dopa in concen-                             of benserazide is possible only in binary mixtures but its
tration range of benserazide from equal up to twice times                               determination in ternary mixture demands its equal or higher
higher, while at 293.2 nm from equal up to five times higher                             concentration then others ingredients.
than l-dopa. The results of determination of LD in ternary
and binary (LD + BEN) mixtures are shown in Tables 3 and 4.                             3.4. Determination of l-dopa and benserazide contents
    The determination of benserazide hydrochloride in pres-                             in pharmaceutical preparation “Madopar”
ence of catecholamine (LD + BEN) using the elaborated
method was characterized by good selectivity too. The elabo-                               High selectivity of benserazide hydrochloride and l-dopa
rated method allows determination of benserazide in presence                            determination in binary mixtures (LD + BEN) enabled us to
of l-dopa in concentration range from equal concentration to                            put the above mentioned method into practice. Contents of
ten times higher then BEN. The determination results are                                benserazide hydrochloride and l-dopa in the commercially
assembled in Table 5.                                                                   available product “Madopar 62.5 ” were determined. As se-
    Benserazide determination in ternary mixture                                        lectivity of l-dopa determination was the same at 269.1 and
(LD + BEN + AA) is possible only when its concen-                                       293.2 nm, its determination was performed at both wave-
tration is between 10 and 2 times higher than others                                    lengths. The obtained results (Table 6) were compared with
compounds. The error of determination in this case was                                  those obtained by the HPLC official method [13]. The errors

Table 4
Determination of l-dopa in binary mixtures with benserazide
Concentration of l-dopa                  The presence of interferent                  CLD /CBEN               Relative error (%)              Relative error (%)
taken for analysis (M)                   (BEN) (M)                                                            λ = 269.1 nm                    λ = 293.2 nm
5.0 × 10−5                               5.0 × 10−5                                   1                        −1.8                            −0.2
1.0 × 10−4                               5.0 × 10−5                                   2                        −0.7                             0.0
2.5 × 10−4                               5.0 × 10−5                                   5                        +1.2                            +2.4
5.0 × 10−4                               5.0 × 10−5                                   10                       −1.0                            −0.6
5.0 × 10−5                               1.0 × 10−4                                   1/2                      +4.3                            +1.2
5.0 × 10−5                               2.5 × 10−4                                   1/5                     +13.2                            +4.8
5.0 × 10−5                               5.0 × 10−4                                   1/10                    +32.3                           +11.2
5.0 × 10−5                               1.0 × 10−3                                   1/20                    +65.7                           +25.0
                                                  n
                                          J. Karpi´ ska et al. / Spectrochimica Acta Part A 62 (2005) 213–220                                       219

Table 6
The results of l-dopa and benserazide determination in pharmaceutical preparation “Madopar 62.5 ”
Analyte                                   Found by elaborated and pharmacopoeial method (mg)/n = 5               Relative errora (%)
                                          1D                1D                 BP                                1D                    1D
                                               269.1             293.2                                                269.1                 293.2

l-dopa included in capsule Madopar        50.92 ± 0.75      50.77 ± 0.79       50.56 ± 0.33                      +0.72 ± 1.06          +0.41 ± 1.22
  50 mg of LD
                                                                                1D                                              Relative errorb (%)
                                                                                     250.8

Benserazide included in capsule Madopar                                         13.18 ± 0.15                                    +5.49 ± 1.21
  12.5 mg of BEN
 a   Vs. official method [13].
 b   Vs. declared value.



of l-dopa determination for both analytical wavelengths are                    4.2. Calibration graphs for determination of studied
very little: ±2.17% and ±1.56% for λ = 269.1 nm and for                        compounds in flow system
λ = 293.2 nm, respectively.
   Simultaneously, the benserazide content in capsules                            Using the optimal parameters of derivative spectra gen-
was assayed. The relative error of determination ver-                          eration, selected previously for batch conditions, cali-
sus declared value did not exceed ±7%. Results of                              bration graphs for determination of l-dopa and benser-
benserazide determination in pharmaceutical are shown in                       azide were drawn at analytical wavelengths as a func-
Table 6.                                                                       tion of the concentration of each compound alone. As
                                                                               well as in case of manual method, the determination
                                                                               of l-dopa was performed at two wavelengths: 269.1
4. Combination of derivative spectrophotometric                                and 293.2 nm with equations y = 2.52 × 102 x + 4 × 10−4
determination with a FIA manifold                                              (r2 = 0.9996) and y = 3.53 × 102 x + 10−5 (r2 = 0.9991), re-
                                                                               spectively. The Beer’s law was obeyed in the range 5 × 10−6
   Next the elaborated derivative spectrophotometric method                    to 5 × 10−4 M for both wavelengths. The calibration curve
of determination l-dopa and benserazide hydrochloride was                      for benserazide determination was recorded at 250.8 nm. The
combined with FIA system. The use flow analysis allows                          equation y = −79.3x + 8 × 10−5 (r2 = 0.9995) obeyed Beer’s
increasing the number of analyzed samples with better preci-                   law in the concentration range 5 × 10−6 to 5 × 10−4 M.
sion. For this purpose the single-channel – FIA manifold was
chosen (Fig. 1). Sample solution was injected directly into                    4.3. Application into assay of real samples
carrier stream of 1 × 10−4 M hydrochloric acid and sample
segment was transported trough reaction coil (R) into flow                          In order to determine l-dopa and benserazide hydrochlo-
cell (D). When sample reached the spectrophotometer flow                        ride contents in pharmaceutical preparation “Madopar
cell, the peristaltic pump was stopped and the spectrum was                     62.5 ” using flow system, preliminary examinations were
recorded. Next, it was converted into derivative spectrum us-                  conducted. First of all the selectivity of determination of LD
ing optimal mathematic parameters, selected previously for                     and BEN in binary mixture (LD + BEN) was examined us-
manual determination of l-dopa and benserazide.                                ing a series of solutions at different compositions. Determi-
                                                                               nation of one compound was carried out, while the second
                                                                               was treated as interference. l-dopa determination in binary
4.1. Optimization of flow parameters                                            mixture is characterized by good selectivity. Results of LD
                                                                               determination are assembled in Table 7.
    At the beginning of experiment, the hydrodynamic pa-                           For both wavelengths the presence of benserazide does not
rameters of FIA manifold were optimized. The injected sam-                     influence on LD determination. Better selectivity is obtained
ple segment was transported from injector valve to detector                    at wavelength λ = 269.1 nm using flow system. BEN can be
flow cell. During travel time segment was dissoluted and dis-                   determined with error less than 3.6% only when its concen-
persed. Both phenomena decreased a local concentration of                      tration is 5–10 times higher than l-dopa concentration. So the
analyte and as the result the heights of absorbance peaks.                     direct assay of benserazide in two-component pharmaceuti-
Hydrodynamic parameters which allowed minimizing disso-                        cal “Madopar 62.5 ” is not possible using flow assembly.
lution and dispersion of sample segment were selected. The                     Simultaneously the same samples were analyzed by official
optimized parameters were: flow rate, the length of reaction                    method. Table 8 shows results of l-dopa determination in
coil, sample volume and travel time. After carefully study the                 pharmaceutical preparation obtained by the derivative spec-
flow rate at 6.6 ml/min, length of reaction coil equal 40 cm,                   trophotometric method combined with FIA assembly as well
600 l of sample volume and 6 s of travel time were selected                    as in batch conditions. The error of l-dopa determination
as optimal.                                                                    versus official method is similar for both analytical wave-
220                                              n
                                         J. Karpi´ ska et al. / Spectrochimica Acta Part A 62 (2005) 213–220

Table 7
Selectivity of l-dopa determination of l-dopa in binary mixtures (LD + BEN) using flow system
Concentration of l-dopa              The presence of interferent             CLD /CBEN                  Relative error (%)          Relative error (%)
taken for analysis (M)               (BEN) (M)                                                          λ = 269.1 nm                λ = 293.2 nm
5.0 × 10−5                           5.0 × 10−5                              1                          −3.8                         +1.0
1.0 × 10−4                           5.0 × 10−5                              2                          +6.0                         +5.0
2.5 × 10−4                           5.0 × 10−5                              5                          +4.8                         +0.8
5.0 × 10−4                           5.0 × 10−5                              10                          0.0                         +0.2
5.0 × 10−5                           1.0 × 10−4                              1/2                        −0.2                         +8.2
5.0 × 10−5                           2.5 × 10−4                              1/5                        −1.6                        +12.6
5.0 × 10−5                           5.0 × 10−4                              1/10                       −7.0                        +12.8
5.0 × 10−5                           1.0 × 10−3                              1/20                       +1.4                        +30.0


Table 8
Comparison of results of l-dopa determination in pharmaceutical preparation “Madopar 62.5 ” using elaborated bath, flow and official methods
Taken for analysis (mg)                             Stationary conditions                                      Flow system

                                                    Found (mg) n = 5          Relative   errora   (%)          Found (mg) n = 5     Relative errora (%)
l-dopa capsules Madopar 62.5 50 mg/cap              50.92 ± 0.76              +1.85 ± 1.51                     49.63 ± 0.50         −0.73 ± 0.99
  determined at 269.1 nm
l-dopa capsules Madopar 62.5 50 mg/cap              50.97 ± 0.71              +1.94 ± 1.42                     51.69 ± 1.14         +3.38 ± 2.27
  determined at 293.2 nm
 a    Vs. official method [13].

lengths. As selectivity is better at 269.1 nm, this wavelength                References
is recommended for determination the studied catecholamine
using combination of derivative spectrophotometry with flow                     [1] M.R. Villanueva Camanas, J.M. Sanchis Mallols, J.R. Torres Lapa-
mode.                                                                              sio, G. Ramis-Ramos, Analyst 120 (1995) 1767–1772.
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                                                                                   Gowda, Talanta 46 (1998) 39–44.
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    The results described above prove that the analysis of spec-                   (2001) 533–534.
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                                                                                   627–633.
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of l-dopa and benserazide is impossible. The selected pa-                      [8] L.H. Marcolino-Junior, M.F.S. Teixeira, A.V. Pereira, O. Fatibello-
rameters of derivatisation enable determination of l-dopa in                       Filho, J. Pharm. Biomed. Anal. 25 (2001) 393–398.
                                                                               [9] O. Fatibello-Filho, I. Da Cruz Vieira, Analyst 122 (1997) 345–350.
binary and ternary mixtures in batch and in the flow system
                                                                              [10] B. Uslu, S.A. Ozkan, Anal. Lett. 35 (2002) 303–314.
without separation of compounds. Determination of benser-                     [11] S.T. Hassib, Anal. Lett. 23 (1990) 2195–2214.
azide is possible only for stationary conditions. The proposed                [12] A. Savitzky, M.J.E. Golay, Anal. Chem. 36 (1964) 1627–1642.
methods are simple, fast and precise.                                         [13] British Pharmacopoeia, HMSO, London, 1988 (Addendum 1990).

				
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