Anti- Parkinsonian Drug Estimation by RP-HPLC

Aim: The main aim of the current study is to give best and simple method for the estimation of antiparkinsonian drugs named Carbidopa, levodopa and entacapone. Study Design: Simultaneous estimation of Carbidopa, levodopa and entacapone was performed by using Quadrapumped (SHIMADZU Prominance-i, LC-2030C) RP-HPLC equipped with PDA detector. Place and Duration of Study: Chalapathi Drug Testing Laboratory, Chalapathi Institute Of Pharmaceutical Sciences, Lam, Guntur-522034, Andhra Pradesh, India during the period of August 2019 to February 2020. Methodology: The assets of the study can determined as the process of qualification and quantification was done on SHIMADZU Prominance-i, LC-2030C system equipped with Phenomenex ODS (150 x 4.6 mm, 5μm) column and mobile phase was optimized using combination of acetonitrile and 0.1% ortho phosphoric acid in the ration of 50:50 v/v at a flow rate 1.0 ml/min. The wavelength was set as 270nm at ambient temperature by injecting 20μl of solution and the run time was fixed for 5 min. Results: Calibration plot shown best regression over the concentration range of 5-160 μg/ml of Carbidopa, Levodopa and Entacapone standard solutions. The LOD and LOQ were found to be 0.85 and 2.54 μg/ml for Entacapone, 0.24 and 0.71 μg/ml for Levodopa, 0.14 and 0.43 μg/ml for Carbidopa respectively. The accuracy of the proposed method was determined by performing recovery studies and was found to be between 98-102%. The repeatability testing for both sample Original Research Article Nadendla and Abhinandana; JPRI, 33(2): 14-25, 2021; Article no.JPRI.65558 15 and standard solutions was found as %RSD<2.0% which is within the acceptable limits showing that the method is precise as well. The proposed method was successfully applied for the marketed formulations of Carbidopa, Levodopa and Entacapone tablets. In addition the main feature of proposed method is economic and eco-friendly with less retention time around 5.0 min. Conclusion: Including all the optimized method parameters and statistical results given it can be concluded as a new, simple, sensitive, precise and accurate economical analytical method was developed and validated by RP-HPLC for the detection and quantification of Carbidopa, Levodopa and Entacapone which can be applied to the marketed formulation where there are no official compendial methods reported for this particular combination. The high sensitivity (LOD), mobile phase utilized and run time (=5) can be determined as an important features for this proposal.


INTRODUCTION
Parkinson's disease is a progressive disorder of the nervous system that affects movement. Young adults rarely experience Parkinson's disease. It ordinarily begins in middle or late life, and the risk increases with age. People usually develop the disease around their sixties or older. Men are one-and-a-half times more likely to get Parkinson's disease than women. Parkinson's disease is caused by the gradual break down or death of certain nerve cells in the brain. This leads to a reduction in the amount of a chemical called dopamine in the brain. Carbidopa, levodopa and entacapone is the combination of drugs approved by U.S. FDA in June 2003, to treat adults with Parkinson's disease. Levodopa is an immediate precursor to dopamine. Enta capone is a reversible catechol-O-methyl transferase inhibitor which prevents the degradation of levodopa. Carbidopa is a aromatic peripheral L-amino acid decarboxylase inhibitor. Combination of Levodopa, carbidopa & enta capone (Catechols), intended as improved therapy for Parkinson Disease [1-8].

Chemical Resources
Entacapone, levodopa and carbidopa working standards are procured as a gift sample from Aurobindo Pharma Pvt., India. ortho phosphoric acid (OPA), triethyl amine are purchased from LOBA chemical laboratories Pvt. Ltd., HPLC grade water and acetonitrile are purchased from Thermo Fisher Scientific Pvt. Ltd., India.

METHOD VALIDATION
The analytical method validation was done according to ICH Q2 (R1) guidelines of validation of analytical methods for the parameters of specificity, system suitability, linearity, the limit of detection (LOD), the limit of quantification (LOQ), accuracy, precision and robustness were discussed [9][10][11][12].

Specificity
Specificity is the ability of the analytical method to produce a response for the analyte in the presence of other components present in the solution; technically they can be like impurities, degradants or matrix. In this method the specificity is tested for the standard solution and blank and found no interference in the blank injection. Tailing factor and theoretical plates were taken into consideration.

System Suitability
System suitability was performed for the standard solution and confirmed the suitability by taking tailing factor, theoretical plates, % RSD and retention time parameters into the consideration [13][14][15][16].

Linearity
The linearity of an validation parameter which confirms the ability of a method (within a given 16 solution; technically they can be like impurities, degradants or matrix. In this method the or the standard solution and blank and found no interference in the blank injection. Tailing factor and theoretical plates System suitability was performed for the standard solution and confirmed the method suitability by taking tailing factor, theoretical plates, % RSD and retention time parameters The linearity of an validation parameter which confirms the ability of a method (within a given range) to obtain test results which will be directly proportional to the concentration of analyte in the sample. By giving different concentrations of sample solutions it is confirmed that the method is linear in 5-160µg/ml range with 0.999 regression value.

Limit of Detection (LOD)
The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. The values were determined by calculating from slope and regression line by following the equation [17][18]. obtain test results which will be directly proportional to the concentration of analyte in the sample. By giving different concentrations of sample solutions it is confirmed that the method 160µg/ml range with 0.999

LOD = 3.3* σ / S
The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. The values were determined by calculating from regression line by following the

Limit of Quantification (LOQ)
LOQ is the parameter which will explain about the detection and quantification of lowest amount. In the method the values of LOQ was determined from the following formula. LOQ = 10* σ / S

Precision
Precision is an analytical procedure expresses the closeness of agreement between a series of measurement obtained from multiple sampling of the same homogeneous sample under prescribed conditions. In the current study the % RSD for the sample solution was found below <2.0 [19].

Accuracy
Accuracy can be defined as the closeness of agreement between accepted reference value and the value found. In this study recovery was calculated by standard weighing method for 50%, 100% and 150%.

Robustness
A robustness method was performed to confirm whether the method is capable of reproducibility during the deliberate changes taken place in the proposed method.

Identification of Wavelength
Approximately 100 mg of Entacapone, Levodopa And Carbidopa is weighed and transferred into 100 ml volumetric flasks individually, to that 70 ml of diluent is added and sonicated to dissolve the compounds, mixed well and made up to the mark with diluent. From those solutions 2 ml is transferred into 3 individual 100 ml volumetric flask, mixed well and made up to the mark with diluent. The prepared solutions are scanned between 200-400 nm to detect the max. All the spectra are overlaid and the isobestic point is identified as 270 nm.

Specificity
By injecting blank solution it is confirmed that there is no inference found in the standard chromatogram by taking tailing factor and theoretical plates into consideration.

System Suitability
Six replicate injections of sample were given for the test of system suitability and found % RSD was within limits (<2.0). Results were given in Table 1.

Precision
The precision of the relative standard deviation of individual area of entacapone and levodopa and carbidopa were found to be within limits.

Intra-day precision
Intraday precision is determined by analyzing same concentration of entacapone, levodopa and carbidopa for six times in the same day.

Inter-day precision
Interday precision is determined by analyzing the same concentration of entacapone, levodopa and carbidopa on different days.

Linearity
For linearity, Six linear concentrations of enta capone, levodopa and carbidopa (5-160μg/ml) were injected in a triplicate manner. A plot of average peak area versus the concentration in μg/ml or mg/ml is made and from this the correlation coefficient, y-intercept (const. of regression) and slope (coefficient of regression) of the regression line were calculated.

Accuracy
Accuracy can be defined as the closeness of agreement between accepted reference value and the value found. In this study recovery was calculated by standard weighing method for 50%, 100% and 150%.

Robustness
Robustness of the method is performed by altering the chromatographic conditions such as pH of the buffer, wavelength, mobile phase composition and observed the variation of the results which should be within the acceptance criteria.

CONCLUSION
There is no official compendial method was reported for the estimation of entacapone, levodopa and carbidopa. Therefore the proposed method which is new, simple, sensitive, precise and accurate economical analytical method can be used for the regular analysis and also can be applied to the commercial formulation. Depending on all the validated parameters it can be confirmed that this method is the best one that can be applied for the estimation of both active pharmaceutical industries and also commercial pharmaceutical labs. The high sensitivity (LOD), mobile phase utilized (ecofriendly) and run time (=5) can be determined as an important features for this proposal.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.