Analysis of ternary mixture of antiParkinsonism drugs by capillary electrophoresis in pharmaceutical dosage forms

A simple CZE method was developed for the analysis of ternary anti-Parkinsonism mixture, levodopa, carbidopa and entacapone. The compounds were simply separated and measured using an untreated fused-silica capillary with 75 μm internal diameter and 85 cm total length, with effective length of 70 cm.. Background electrolyte composed of 20 mM phosphate buffer of pH 7.5 was used under an applied voltage of 15 kV. Photodiode array detector (PDA) was used to identify each compound at different wavelength to obtain high sensitivity. The present method was conducted to the analysis of the ternary mixture in pharmaceutical preparations. The analytical results proved the linearity (r2 ≥ 0.9995), accuracy, precision (% RSD < 2).


Introduction
Parkinsonism is a disease affecting central nervous system due to imbalance of dopamine and results in tremor ,movement disorder, bradykinesia , and postural instability. [1,2] . Entacapone is an inhibitor of catechol-O-methyltransferase (COMT). Entacapone is co administered with levodopa and carbidopa which is inhibitor to aromatic amino acid decarboxylase [3] . Figure 1 shows the structural formula and IUPAC names of entacapone, levodopa and carbidopa [4] .
The studied drugs are officials and have compendial methods in the united states pharmacopoeia [4] . Different analytical methods have been published for the simultaneous analysis of entacapone, levodopa and carbidopa. The literatures includes spectrophotometric method [5] , different separation techniques like HPLC and 85 cm total length, 70 cm effective length. Treatment of the capillary was performed with 1.0 M sodium hydroxide, ultra-pure deionized water and running buffer for 60 min , 30 min, 30 min respectively. The previous treatment was repeated in each day for 10 min. To obtain good reproducibility , the capillary was flushed before each run for 3 min by 0.1 M sodium hydroxide, ultra-pure deionized water and for 5 min by the buffer.
The method was conducted using 20 mM phaospahte buffer with pH 7.5 and a potential of +15 kV at 25°C. The samples were hydrodynamically injected at the anodic side with 50 mbar for 6 s.

General procedure and calibration curve
Working solutions of entacapone, levodopa and carbidopa of concentrations within the linearity range were prepared by diluting aliquots of the stock solutions with running buffer. The optimum conditions of analysis were achieved at 25°C using 20 mM phosphate running buffer, pH 7.5 and applied voltage of +15 kV. The analytes were detected at 210, 280 and 380 nm for carbidopa , levodopa and entacapone respectively. The calibration curves were constructed by plotting the peak area versus the analyte concentration.

Analysis of tablets
The extraction was challenging due to the different solubilities of the studied drugs in water and methanol. Levodopa is soluble in water but is not in methanol.
Carbidopa is water and methanol soluble. While entacapone is methanol soluble and sparingly soluble in water. Different ratios of water and methanol were studied to develop the best extraction of the three drugs. The high percentage of extraction was obtained upon using water : methanol (30 : 70, v/v).
Ten Stalevo tablets were individually weighed and finely powdered. In a 100 mL volumetric flask, an amount of the powder equivalent to the average weight of one tablet was placed and extracted by 80 mL of the extracting solvent. The solution was sonicated for 20 min, filtered and dilution to the mark with the same mixture.
Aliquots of the extract were transferred to 10 mL volumetric flasks and diluted with BGE to the mark so that the final concentrations will be in the linear concentration range.

Results
After optimization of the analytical method, it was found that the optimum parameters for the separation and estimation of the three drugs were achieved by running 20 mM phosphate buffer of pH 7.5 and voltage of +15 kV at 25°C . Table 1 summarizes different optimization parameters. Figure 2 shows the electropherogram of the separated compounds. Raw materials and tables of the studied drugs were analysed by the present method and an acceptable results were obtained ( Table   4,6).

Method optimization and development
The optimum conditions were achieved by studying the separation parameters like the concentration of the running buffer, buffer pH, the applied voltage and the capillary temperature. The different parameters were studied and optimized to reach high number of theoretical plates (NTP) and resolution (Table 1)

The effect of buffer pH
The pH of the buffer significantly affecting the electromigration of analytes. The pH was optimized to stabilize both the migration velocity and electroosmotic flow of the analytes. The effect of phosphate buffer pH on the separation was investigated over the range of 5.5 -7. Borate buffer was used to study the effect of alkaline pH from 8 to 10. The alkaline pHs from 8 to 10 result in overlap of carbidopa and entacapone. At low pHs, the entacapone peak area decreases. This reduced peak area is due to its low solubility in acidic pH. Peak area increases as the pH increases. The pH selected was 7.5 at which high number of theoretical plates and good resolution were obtained.

The effect of buffer concentration
The effect of buffer concentration on electrophoretic separation is believed to its effect on both the electro osmotic flow (EOF) and the electric current. Different phosphate buffer concentrations (10-30 mM) were studied. As the concentration of the buffer increases, peak broadening obtained as a result of high migration times and electric current (Table 1). A concentration of 20 mM was optimum and used in further progress.

The effect of applied voltage
The effect of applied voltage on the electrophoretic separation was investigated over the range from 10 to 20 kV. The electroosmotic flow (EOF) increases and the migration time decreases as the applied voltage increases A high current and hence joule heating increase when the applied voltage over 20 kV ( Table 1). The optimum applied voltage selected was 15 kV as it gave high resolution.

The effect of injection time
Investigating the pressure and time for hydrodynamic injections was done. A pressure of 50 mbar was found suitable, as it provided the best peak shape.
Pressure values <50 mbar yielded small peaks with very low response, and values >50 mbar yielded broad peaks. After the pressure was selected, the injection time was studied. A 6 s injection time was selected as optimum. Injection time <6 s yielded low peak responses and higher injection time yielded broad peaks. Both pressure and injection times had nearly negligible effect on the migration times of the drugs.

The effect of capillary temperature
The capillary temperature affects the viscosity of the running buffer and electroosmotic flow (EOF) so it has to be controlled. The effect of temperature was investigated over the range 20-30 °C. The best resolution and reduced joule heating were obtained at 25 °C.

Selection of the detection wavelength
Detections were performed at 210 nm, 280 nm and 380 nm for carbidopa , levodopa and entacapone respectively to obtain maximum sensitivity.

Validation
The ICH guidelines [15] were considered for the study of different validation parameters of the proposed method.

Linearity and range
The produced peak area and the concentrations in µg/ mL of the studied drugs were found to be linearly related. The main validation parameters are summarized in Table 2.

Limits of quantitation and detection
The ICH guide lines [15] provide two formula for the calculation of both LOQ and LOD. ( Table 2). The limits of quantitation for the studied drugs were 1.48,3.  Table 3. The accuracy of the proposed methods was also high as indicated by the low value of percent relative error (%Er) Table 3. The proposed method was satisfactory compared with the compendial methods [4] . The results are shown in (Table 3) and also table 4.

Specificity
Each of the studied compounds were specifically analyzed by the proposed CE method. The specificity was proved by injecting a mixture of the working concentration of the studied drugs. The drug peaks were eluted at migration times of 5.6, 6.6 and 6.9 min for levodopa, carbidopa and entacapone respectively fig.2.

Robustness.
The robustness of the method was evaluated by measuring the effect of small deliberate changes in pH, phosphate buffer concentration, and applied voltage on the migration times of the analytes or the peak area. The results revealed that the small changes of the previous have no significant affect on either migration times or peak area. The results are shown in (Table 5).

Application
The studied drugs were simultaneously determined in their multicomponent tablets by the proposed CE method. The measurement of the studied compounds, levodopa, carbidopa and entacapone in Stalevo® tablets were successfully performed with high % recovery good standard deviation as shown in Table 6. The authors declare that they have no competing interest.

Compliance with Ethical Standards:
Availability of data and material: All needed data and material are available

Conflict of Interest:
Author declares that he has no conflict of interest.
Funding: The authors did not receive fund for the work or for the publication  Sy/x = SD of the residuals.     Figure 1 Structural formula of the studied drugs Electropherogram of(a) levodopa,(b) carbidopa and (c)entacapone