Introduction

Parkinson’s disease, PD, is one of the most common neurodegenerative disorders in humans leading to progressive deterioration of motor function due to loss of dopamine-producing brain cells [1]. Therapeutics with levodopa (levo-3,4-dihydroxyphenylalanine) will soon be entering its second half-century of use as the most effective symptomatic therapy for PD. Levodopa has provided revolutionary and lifelong benefits for averting disability and improving quality of life, as experienced by millions of PD patients [1]. Since dopamine cannot cross the protective blood–brain barrier, whereas levodopa can, levodopa is widely prescribed to increase dopamine concentration in the treatment of PD [1]. In human, levodopa can normally be prepared via biosynthesis from a nonessential amino acid called tyrosine. However, auto-oxidization of levodopa occurred in the peripheral system, can produce serious side effects, such as paranoia schizophrenia, nausea, vomiting and dyskinesia [1]. Therefore, determination of levodopa is crucial to control its dosage. Several methods have been reported for determination of levodopa in pharmaceutical and biological samples including chromatography [2], spectroscopy [3] and electroanalysis [4,5,6,7,8].

Acetaminophen or paracetamol, the most extensively used medication for pain and fever in both the United States and Europe, is an effective and safe agent, used worldwide for the relief of mild to moderate pain associated with headaches, backaches, arthritis and postoperative pain. It is also used for the reduction of fevers of bacterial or viral origin. Acetaminophen is a non-carcinogenic and an effective substitute for aspirin and used by the patients who are sensitive to aspirin, and it is safe up to therapeutic doses. When administered in suitable doses, acetaminophen has an excellent safety profile, with about 90% of absorption by the organism and subsequently excreted via urine. Overdoses of acetaminophen can lead to the accumulation of toxic metabolites, causing severe and some-times fatal hepatotoxicity and nephrotoxicity [9]. Parkinson’s disease patients chronically use opiates, acetaminophen and drugs treating neuropathic pain such as antidepressants and antiepileptics [9]. Also, it has been found that human absorption of acetaminophen is extremely dependent on gastric emptying. Other drugs that alter gastric emptying can change the pharmacokinetics of acetaminophen. It has been shown that levodopa can influence gastric emptying [10]. Then, simultaneous quantification of these two compounds can provide valuable evidences about the role of them in healthiness. Till now, a variety of methods have been introduced for acetaminophen determination such as, capillary electrophoresis, HPLC, spectroscopy and electrochemical techniques [11,12,13,14,15].

Tyrosine is one of the nonessential amino acids normally produced in human body from phenylalanine. Tyrosine also exists in dairy products, eggs, beans and meats. It has a vital role in production of several important neurotransmitters in brain that help nerve cell communicate. An increased level of tyrosine induces Parkinson’s disease and increased sister chromatid exchange, while the lack of tyrosine may lead to albinism, alkaptonuria and other psychological diseases [16]. In human aqueous humor, tyrosine is the second highest in concentration next to ascorbic acid [16]. Tyrosine is often added to food products and to pharmaceutical formulations. It is critical to mental and physical health as it is needed to produce catecholamines through an iron-containing enzyme called tyrosine hydroxylase [17]. Both tyrosine and levodopa have very similar chemical structure and biological function and linked together by the process taking place in body to convert tyrosine to levodopa to meet its needs. Tyrosine and levodopa are necessary for producing dopamine and some other vital biological compound in our body. Therefore, simultaneous determination of levodopa, acetaminophen and tyrosine is highly demanded.

Electrochemical techniques are among the most appealing choices for biological and pharmaceutical compounds. Electrochemistry often offers analytical techniques which are characterized by instrumental simplicity, moderate cost, high sensitivity, portability and some other unique advantages. Due to similarity between redox reactions that taking place in electrochemical and biological systems, it can be assumed that oxidation-reduction reactions occurring in the body and at the surface of electrodes share similar principles [18,19,20,21,22,23,24].

Graphene, a one-atom-thick monolayer of graphite containing Sp2-bonded carbon atoms with a honeycomb structure, has captured the attention of scientists, researchers and industry worldwide because of some exceptional properties like high electron mobility at room temperature, its large specific surface area, chemically inertness, high thermal conductivity and ballistic transport. However, due to the strong π-π stacking interaction, the reported graphene materials especially those produced by chemical approaches are actually graphene nanoplatelets with a typical thickness of 2–10 graphene layers, which also exhibit similarly fascinating properties as graphene [25]. In the past decade, graphene has been the undisputed champion of the material world and has grabbed appreciable attention for the development of electrochemical sensors and biosensors, supercapacitors and batteries with enhanced performances thanks to its integration with various nanomaterials [26,27,28,29,30,31].

Literature survey show that all previously published electrochemical studies have dealt with individual determination of levodopa, acetaminophen and tyrosine, or simultaneous determination of them with other drugs but no studies have been reported on the simultaneous electrocatalytic determination of levodopa, acetaminophen and tyrosine by using modified electrodes. Now we wish report for the first time the preparation and suitability of an ethyl 2-(4-ferrocenyl-[1,2,3]triazol-1-yl) acetate / graphene modified carbon paste electrode (EFTAGCPE) as a new electrode in the electrocatalysis and in addition the determination of levodopa in an aqueous buffer. Then the analytical performance of the modified electrode in quantification of levodopa is evaluated in the presence of acetaminophen and tyrosine.

Experimental

Apparatus and chemicals

The electrochemical measurements were performed with an Autolab potentiostat/galvanostat (PGSTAT 302 N, Eco Chemie, the Netherlands, www.metrohm-autolab.com). The experimental conditions were controlled with General Purpose Electrochemical System (GPES) software. A conventional three electrodes cell was used at 25 ± 1 °C. An Ag/AgCl/KCl (3.0 M) electrode (Azar Electrode, Urmia, Iran, www.azarelectrode.ir), a platinum wire (Azar Electrode, Urmia, Iran, www.azarelectrode.ir), and the EFTAGCPE were used as the reference, auxiliary and working electrodes, respectively. A Metrohm 710 pH meter (www.metrohm.com) was employed for pH measurements.

All solutions were freshly prepared with double distilled water. Levodopa, acetaminophen, tyrosine and all other reagents were of analytical grade and were purchased from Merck chemical company (Darmstadt, Germany, www.merck.com). The buffers were prepared from orthophosphoric acid and its salts in the pH range of 2.0–9.0. Graphene was purchased from Sigma Aldrich Company (www.sigmaaldrich.com).

Synthesis of ethyl 2-(4-ferrocenyl-[1,2,3]triazol-1-yl) acetate

A clean, dry ball milling vessel (15 ml) was charged with 2 stainless steel grinding balls (10 mm diameter), ethyl 2-bromoacetate (0.05 ml, 0.5 mmol), ethynylferrocene (100 mg, 0.5 mmol), sodium azide (32 mg, 0.5 mmol), sodium ascorbate (45 mg, 0.23 mmo), copper acetate (10 mg, 0.05 mmol) and acidic alumina as milling auxiliary (1 g). The vessel was closed, and the milling was started (milling at 500 rpm). After completion of the reaction, as indicated by TLC, ethyl acetate (2 × 15 mL) was added to the resulting solid mixture. Then the reaction mixture was filtered and washed with additional ethyl acetate. The combined filtrate and washings were evaporated under reduced pressure to dryness and purified by layer chromatography on silica gel (n-hexane: ethyl acetate as eluent) to afford the pure product (134.9 mg, 79%).

Mp: 139–142 °C; 1H NMR (400 MHz, DMSO-d6): δδ 1.23 (t, J = 7.2 Hz, 3H), 4.03 (s, 5H), 4.19 (q, J = 7.2 Hz, 2H), 4.31 (t, J = 1.8 Hz, 2H), 4.74 (t, J = 1.8 Hz, 2H), 5.39 (s, 2H), 8.17 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δδ 14.4, 50.8, 61.9, 66.8, 68.8, 69.7, 76.1, 122.4, 145.8, 167.7; MS: m/z (%): 339 (M+, 100), 311 (M+- N2, 3), 224 (M+- C4H7N2O2, 27), 199 (M+- C5H8N3O2, 8), 121 (C5H5Fe+, 65).

Preparation of the electrode

To obtain the best conditions in the preparation of the EFTAGCPEs, we optimized the ratio of EFTA, and graphene. The results of our studied showed that the maximum peak current intensity of levodopa could be obtained at the surface of EFTAGCPE with optimum ratio of EFTA and graphene.

The EFTAGCPEs were prepared by hand mixing 0.01 g of EFTA (Scheme 1) with 0.89 g graphite powder and 0.1 g graphene with a mortar and pestle. Then, ~ 0.7 mL of paraffin oil was added to the above mixture and mixed for 20 min until a uniformly-wetted paste was obtained. The paste was then packed into the end of a glass tube (ca. 3.4 mm i.d. and 15 cm long). A copper wire inserted into the carbon paste provided the electrical contact. When necessary, a new surface was prepared by pushing an excess of the paste out of the tube and polishing with a weighing paper.

Scheme 1
scheme 1

Chemical structure of EFTA

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For comparison, EFTA modified CPE (EFTACPE) without graphene, graphene carbon paste electrode (GCPE) without EFTA and unmodified CPE in the absence of both EFTA and graphene were also prepared in the same way.

Procedure of real samples preparation

The acetaminophen oral solution was diluted 1000 times with deionized water; then, different volume of the diluted solution was transferred into a 25 mL volumetric flask and diluted to the mark with phosphate buffer (pH 7.0). The acetaminophen content was analyzed using the standard addition method.

Urine samples were stored in a refrigerator immediately after collection. 10 mL of the sample was centrifuged for 15 min at 2000 rpm. The supernatant was filtered out using a 0.45 μm filter. Then, different volume of the solution was transferred into a 25 mL volumetric flask and diluted to the mark with phosphate buffer (pH 7.0). The diluted urine sample was spiked with different amounts of levodopa, acetaminophen and tyrosine.

For preparing the plasma and serum of blood, 10 mL of blood was separated after putting the sample in an incubator at 37 °C for 30 min and centrifuging it. The serum and plasma were separated from each other. After filtering, the samples were diluted with phosphate buffer (pH 7.0) without any further treatment. The diluted samples were spiked with different amounts of levodopa, acetaminophen and tyrosine.

Results and discussion

Electrochemical properties of EFTAGCPE

To the best of our knowledge there is no prior report on the electrochemical properties and, in particular, the electrocatalytic activity of EFTA in an aqueous media. Therefore, EFTAGCPE was prepared and its electrochemical properties were studied in a phosphate buffer (pH 7.0) using cyclic voltammetry. It should be noted that one of the advantages of EFTA as an electrode modifier is its insolubility in aqueous media. Experimental results showed reproducible and well-defined cyclic voltammograms. Anodic and cathodic peak potentials were 0.33 and 0.21 V vs. Ag/AgCl/KCl (3.0 M) respectively. The observed peak separation potential, ΔEp = (Epa − Epc) of 120 mV, was greater than the value of 59/n mV which is expected for a reversible system [32], suggesting that the redox couple of EFTA in EFTAGCPE has a quasi-reversiblebe havior in aqueous medium [32].

In addition, the long-term stability of the EFTAGCPE was tested over a 3-week period. When cyclic voltammograms were recorded after the modified electrode was stored in atmosphere at room temperature, the peak potential for levodopa oxidation was unchanged, and the current signals showed a less than 2.0% decrease relative to the initial response. The antifouling properties of the modified electrode toward levodopa oxidation and its oxidation products were investigated by recording the cyclic voltammograms of the modified electrode before and after use in the presence of levodopa. Cyclic voltammograms were recorded in the presence of levodopa after having cycled the potential 15 times at a scan rate of 10 mVs−1. The peak potentials were unchanged, and the currents decreased by less than 2.1%. Therefore, at the surface of EFTAGCPE, not only did the sensitivity increase, but the fouling effect of the analyte and its oxidation product also decreased.

Electrocatalytic oxidation of levodopa at an EFTAGCPE

The electrochemical behavior of levodopa is dependent on the pH value of the aqueous solution, whereas the electrochemical properties of Fc/Fc+ redox couple are independent on pH. Therefore, pH optimization of the solution seems to be necessary in order to obtain the electrocatalytic oxidation of levodopa. Thus the electrochemical behavior of levodopa was studied in 0.1 M phosphate buffer in different pH values (2.0 < pH < 9.0) at the surface of EFTAGCPE by cyclic voltammetry. It was found that the electrocatalytic oxidation of levodopa at the surface of EFTAGCPE was more favored under neutral conditions than in acidic or basic medium. This appears as a gradual growth in the anodic peak current and a simultaneous decrease in the cathodic peak current in the cyclic voltammograms of EFTAGCPE. Thus, the pH 7.0 was chosen as the optimum pH for electrocatalysis of levodopa oxidation at the surface of EFTAGCPE.

Figure 1 depicts the cyclic voltammogram responses for the electrocatalytic oxidation of 400.0 μM levodopa at unmodified CPE (curve a), GCPE (curve b), EFTACPE (curve d) and EFTAGCPE (curve e).

Fig. 1
figure 1

Cyclic voltammograms of (a) unmodified CPE in 0.1 M phosphate buffer (pH 7.0) containing 0.4 mM levodopa; (b) GCPE in 0.1 M phosphate buffer (pH 7.0) containing 0.4 mM levodopa; (c) EFTAGCPE in 0.1 M phosphate buffer (pH 7.0); (d) EFTACPE in 0.1 M phosphate buffer (pH 7.0) containing 0.4 mM levodopa and (e) EFTAGCPE in 0.1 M phosphate buffer (pH 7.0) containing 0.4 mM levodopa

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As it is seen, while the anodic peak potential for levodopa oxidation at the GCPE, and unmodified CPE are 610 and 660 mV, respectively, the corresponding potential at EFTACPE and EFTAGCPE is ~330 mV. These results indicate that EFTA can act as a good mediator and peak potential for levodopa oxidation at the EFTACPE and EFTAGCPE shift by ~280 and 330 mV toward negative values compared to GCPE and unmodified CPE, respectively. However, EFTAGCPE shows much higher anodic peak current for the oxidation of levodopa compared to EFTACPE, indicating that the combination of graphene and the mediator (EFTA) has significantly improved the performance of the electrode toward levodopa oxidation. In fact, EFTAGCPE in the absence of levodopa exhibited a well-behaved redox reaction (Fig. 1, curve c) in 0.1 M phosphate buffer (pH 7.0). However, there was a drastic increase in the anodic peak current in the presence of 400.0 μM levodopa (curve e).

Based on these results, we propose an EC′ catalytic mechanism, shown in Scheme 2, to describe the electrochemical oxidation of levodopa at EFTAGCPE. In this scheme, levodopa oxidized in the catalytic (C) reaction by the oxidized form of EFTA produced at the electrode surface via an electrochemical (E) reaction.

Scheme 2
scheme 2

Electrocatalytic oxidation of levodopa at the EFTAGCPE

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The effect of scan rate on the electrocatalytic oxidation of levodopa at the EFTAGCPE was investigated by linear sweep voltammetry (LSV) (Fig. 2). As can be seen in Fig. 2, the oxidation peak potential shifted to more positive potentials with increasing scan rate, confirming the kinetic limitation in the electrochemical reaction. Also, a plot of peak height (Ip) vs. the square root of scan rate (ν1/2) was found to be linear in the range of 2–20 mVs−1, suggesting that, at sufficient overpotential, the process is diffusion rather than surface controlled (Fig. 2a). A plot of the scan rate-normalized current (Ip/ν1/2) vs. scan rate (Fig. 2b) exhibits the characteristic shape typical of an EC ́process [32].

Fig. 2
figure 2

Linear sweep voltammograms of EFTAGCPE in 0.1 M phosphate buffer (pH 7.0) containing 100.0 μM levodopa at various scan rates; numbers 1–7 correspond to 2, 4, 8, 10, 12, 16 and 20 mVs−1, respectively. Insets: Variation of (a) anodic peak current vs. ν1/2; (b) normalized current (Ip/ν1/2) vs. ν

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Figure 3c illustrates the Tafel plot for the sharp rising part of the linear sweep voltammogram at the scan rate of 2 mVs−1. If deprotonation of levodopa is a sufficiently fast step, the Tafel plot can be used to estimate the number of electrons involved in the rate determining step. A Tafel slope of 0.1068 V was obtained which is consistent well with the involvement of one electron in the rate determining step of the electrode process [32], assuming a charge transfer coefficient, α of 0.45.

Fig. 3
figure 3

Linear sweep voltammogram (at 2 mV s−1) of an EFTAGCPE in 0.1 M phosphate buffer (pH 7.0) containing 100.0 μM levodopa. The points are the data used in the Tafel plot. The inset shows the Tafel plot derived from the linear sweep voltammogram

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Chronoamperometric measurements

Chronoamperometric measurements of levodopa at EFTAGCPE were carried out by setting the working electrode potential at 0.4 V (first potential step) and at 0.1 V (second potential step) for the various concentrations of levodopa in phosphate buffer (pH 7.0) (Fig.4). For an electroactive material (levodopa in this case) with a diffusion coefficient of D, the current observed for the electrochemical reaction at the mass transport limited condition is described by the Cottrell eq. [32]. Experimental plots of I vs. t-1/2 were employed, with the best fits for different concentrations of levodopa (Fig. 4a). The slopes of the resulting straight lines were then plotted vs. levodopa concentration (Fig. 4b). From the resulting slope and Cottrell equation the mean value of the D was found to be 2.3 × 10−6 cm2/s.

Fig. 4
figure 4

Chronoamperograms at EFTAGCPE in 0.1 M phosphate buffer (pH 7.0) for different concentration of levodopa. The numbers 1–5 correspond to 0.0, 0.1, 0.3, 0.5 and 0.8 mM of levodopa. Insets: a Plots of I vs. t-1/2 resulting from chronoamperograms 2–5. b Plot of the slope of the straight lines against levodopa concentration. c dependence of Icat/Il on t1/2 derived from the data of chronoamperograms 1–5

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Chronoamperometry can also be employed to evaluate the catalytic rate constant, k, for the reaction between levodopa and the EFTAGCPE according to the method described by Galus [33]:

$$ {\mathrm{I}}_{\mathrm{C}}/{\ \mathrm{I}}_{\mathrm{L}}={\gamma}^{1/2}\left[{\pi}^{1/2}\operatorname{erf}\ \left({\gamma}^{1/2}\right)+ \exp\ \left(-\gamma \right)/{\gamma}^{1/2}\right] $$
(1)

where IC is the catalytic current of levodopa at the EFTAGCPE, IL is the limited current in the absence of levodopa and γ = kCbt is the argument of the error function (Cb is the bulk concentration of levodopa). In cases where γ exceeds the value of 2, the error function is almost equal to 1 and therefore, the above equation can be shortened to:

$$ {\mathrm{I}}_{\mathrm{C}}/{\ \mathrm{I}}_{\mathrm{L}}={\pi}^{1/2}{\gamma}^{1/2}={\pi}^{1/2}{\left({\mathrm{kC}}_{\mathrm{b}}\mathrm{t}\right)}^{1/2} $$
(2)

where t is the time elapsed. The above equation can be used to calculate the rate constant, k, of the catalytic process from the slope of IC/IL vs. t1/2 at a given levodopa concentration. From the values of the slopes (Fig. 4c), the average value of k was found to be 1.2 × 10 3 M−1 s−1.

Calibration plot and limit of detection

Square wave voltammetry method was used to determine the concentration of levodopa (Fig. 5) (Initial potential = 0 V, End potential = 0.6 V, Step potential = 0.001 V, Amplitude = 0.02 V, Frequency = 10 Hz). The plot of peak current vs. levodopa concentration consisted of two linear segments with slopes of 1.082 and 0.222 μA μM−1 cm−2 in the concentration ranges of 0.2 to 30.0 μM and 30.0 to 400.0 μM, respectively. The decrease in sensitivity (slope) of the second linear segment is likely due to kinetic limitation. The detection limit (3σ) of levodopa was found to be 0.07 μM.

Fig. 5
figure 5

Square wave voltammograms of EFTAGCPE in 0.1 M phosphate buffer (pH 7.0) containing different concentrations of levodopa. Numbers 1–13 correspond to 0.2, 1.0, 2.0, 5.0, 10.0, 15.0, 20.0, 30.0, 50.0, 100.0, 200.0, 300.0 and 400.0 μM of levodopa. Insets: a The plots of the electrocatalytic peak current as a function of levodopa concentration in the range of 0.2–30.0 μM and b The plots of the electrocatalytic peak current as a function of levodopa concentration in the range of 30.0–400.0 μM

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In the case of acetaminophen peak currents at the surface of EFTAGCPE were linearly dependent on the acetaminophen concentrations, over the range of 1.0 × 10−6–1.5 × 10−4 M and the detection limit (3σ) was obtained 5.0 × 10−7 M and for tyrosine, peak current at the surface of EFTAGCPE were linearly dependent on the tyrosine concentrations, over the range of 5.0 × 10−6–1.8 × 10−4 M and the detection limit (3σ) was obtained 2.0 × 10−6 M.

These values are comparable with values reported by other research groups for electrocatalytic oxidation of levodopa, acetaminophen and tyrosine at the surface of chemically modified electrodes (Table 1).

Table 1 An overview on recently reported nanomaterial-based methods for determination of levodopa or acetaminophen or tyrosine
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Simultaneous determination of levodopa, acetaminophen and tyrosine

It is pertinent to note that the use of modified graphene electrode for simultaneous determination of levodopa, acetaminophen and tyrosine have not reported so for and we believe that this work is the first report on the simultaneous determination of levodopa, acetaminophen and tyrosine using EFTAGCPE.

This was performed by simultaneously changing the concentrations of levodopa, acetaminophen and tyrosine, and also at the same time recording the square wave voltammograms. The voltammetric results showed well-defined anodic peaks at potentials of 275, 485 and 745 mV, which are corresponding to the oxidation of levodopa, acetaminophen and tyrosine, respectively, indicating that simultaneous determination of these compounds is feasible at the EFTAGCPE, as shown in Fig. 6 (Initial potential = −0.1 V, End potential = 0.95 V, Step potential = 0.001 V, Amplitude = 0.02 V, Frequency = 10 Hz).

Fig. 6
figure 6

Square wave voltammograms of EFTAGCPE in 0.1 M phosphate buffer (pH 7.0) containing different concentrations of levodopa + acetaminophen + tyrosine in μM, from inner to outer: 1.0 + 1.0 + 5.0, 5.0 + 5.0 + 10.0, 10.0 + 10.0 + 25.0, 20.0 + 25.0 + 40.0, 80.0 + 70.0 + 100.0, 200.0 + 110.0 + 120.0, 300.0 + 125.0 + 160.0 and 350.0 + 150.0 + 180.0 respectively. Insets: a Plot of Ip vs. levodopa concentration in first linear segment, b plot of Ip vs. levodopa concentrations in second linear segment, c plot of Ip vs. acetaminophen concentrations and d plot of Ip vs. tyrosine concentrations

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The sensitivity of the modified electrode towards the oxidation of levodopa was found to be 1.092 μA μM−1 cm−2, which is very close to the value obtained in the absence of acetaminophen and tyrosine (1.082 μA μM−1 cm−2) (Fig. 6). These results are indicative that the oxidation processes of these compounds at the EFTAGCPE are independent and therefore, simultaneous determination of their mixtures is possible without significant interferences.

Interference studies

The influence of various substances as compounds potentially interfering with the determination of levodopa was studied under optimum conditions. The potentially interfering substances were chosen from the group of substances commonly found with levodopa in pharmaceuticals and/or in biological fluids. The tolerance limit was defined as the maximum concentration of the interfering substance that caused an error of less than ±5% in the determination of levodopa. According to the results, L-lysine, glucose, NADH, acetaminophen, uric acid, L-asparagine, L-serine, L-threonine, L-proline, L-histidine, L-glycine, L-tryptophan, L-phenylalanine, lactose, tyrosine, saccarose, fructose, benzoic acid, methanol, ethanol, urea, caffeine, Mg2+, Al3+, NH4 +, F−, SO4 2− and S2− did not show interference in the determination of levodopa, but dopamine, isoproterenol, epinephrine and norepinephrine can cause interference in the determination of levodopa.

Real sample analysis

In order to evaluate the analytical applicability of the method, also it was applied to the determination of levodopa, acetaminophen and tyrosine in acetaminophen oral solution, human blood serum, human blood serum and urine samples. The results for determination of the three species in real samples are given in table 1S. Satisfactory recovery of the experimental results was found for levodopa, acetaminophen and tyrosine. The reproducibility of the method was demonstrated by the mean relative standard deviation (R.S.D.).

Conclusions

In the present study, a modified carbon paste electrode is constructed. The modified electrode was applied for levodopa determinations in the presence of acetaminophen and tyrosine for the first time. Excellent features, like a wide linear range, low detection limit, high reproducibility and repeatability and long time stability proved the successful application of this sensor for the determinations of levodopa, acetaminophen and tryptophan. Real sample analysis was done and good results were obtained.