INTRODUCTION:

Parkinson’s Syndrome, also known as Parkinson’s disease, for James Pakinson first described the disease in 1817. Parkinson’s disease (PD) is a progressive, slow-moving, and agerelated neurodegenerative disease that currently affects about 10 million people worldwide. The symptoms of PD are motor dysfunction, depression and decreased olfactory, followed by dyskinesia such as tremor, stiffness, unstable posture and gait disorder. It has negative impacts to the patient‘s life quality and occurred irregularly with the unknown cause. At present, there is no method to reverse or cure Parkinson’s disease, and could only delay the deterioration of the disease.

Istradefylline, chemically 8-[(E)-2-(3,4-dimethoxyphenyl) ethenyl]-1,3-diethyl-7-methyl-2,3,6,7-tetrahydro-1H-purine-2,6-dione having molecular formula C₂₀H₂₄N₄O₄and molecular weight 384.43 g/mol. Istradefylline is a light yellow-green crystalline powder. Istradefylline has a dissociation constant (pKa) of 0.78. The aqueous solubility of istradefylline is ~0.5 μg/mL across the physiological pH range and 0.6 μg/mL in water. Istradefylline is a kind of selective adenosine A_2A receptor antagonists, by the exploitation of Japan Kyowa Hakko Kirin company. Istradefylline tablets (trade name Nouriast, specification 20 mg) obtains Japan PMDA approval listing in March, 2013. Istradefylline was granted FDA approval on 27 August 2019. It was clinically used to improve dyskinesia in the early stages of PD and prolong the half life period of L-dopa and improve the wearing off phenomenon and other side effects produced by L-dopa, further prolong the recurrence time of patients. Istradefylline is considered as a suitable drug in the treatment of Parkinson’s disease¹, unlike standard dopaminergic therapies for Parkinson's, Istradefylline targets adenosine A_2A receptors in the basal ganglia^2,3. This region of the brain is highly involved in motor control. Istradefylline is indicated as an adjunct treatment to levodopa and carbidopa for Parkinson's disease. Literature survey reveals some analytical methods reported related to istradefylline which estimation of istradefylline and carbidopa by LC-MS⁴, Photostability studies by HPLC⁵, related substances and impurity studies of istradefylline⁶. However there is no analytical method reported yet for estimation of istradefylline in dosage form. The spectrophotophotometric methods⁷ have a lack of selectivity and sensitivity, so chromatographic method is a method of choice for simultaneous estimation of istradefyllin. Out of many chromatographic method , HPLC^8-18 is a versatile analytical technique with good sensitivity and selectivity. The aim of this work was to develop an HPLC method for estimation of istradefylline in tablet dosage form.

Fig. 1: Structure of Istradefylline

MATERIALS AND METHODS:

Instrumentation

Chromatographic separation was performed on a Shimadzu LC-20AD HPLC system equipped with a C18 G column (250 × 4.6 mm i.d, 5 μm particle), binary pumps, degasser, variable wave length detector and Rheodyne injector with 20 μl loop volume. ‘LC solution’ software was used to collect and process the data.

Chemicals and Reagents:

Istradefylline pure drug (purity 98.65 %) was purchased online. Marketed formulation Nourianz™ tablets (Kyowa Kirin, Inc) labeled to contain 20 mg of Istradefyllin procured online. HPLC grade water and acetonitrile was from MERCK India Ltd. HPLC grade methanol was from standard reagent Pvt Ltd Hyderabad. Analytical grade orthophosphoric acid was from SD Fine chemicals Mumbai, India. Nylon membrane filters 0.2 µm and 0.45 µm were from PALL life sciences Mumbai, India.

Chromatographic conditions:

The chromatographic system used for the method development and validation consisted of Shimadzu HPLC comprising of LC-20AD binary gradient pump, a variable wavelength programmable SPD-20A detector and an SCL 20A system controller. A Rheodyne injector 7725i fitted with a 20 μL loop was used and data were recorded and evaluated by use of LC solutions software version 5.0. Separation was performed on a Enable C18G (250 × 4.6 mm i.d.,5µ) at ambient temperature. A mixture of 0.1% ortho-phosphoric acid and acetonitrile in the ratio of 30: 70 v/v was found to be the most suitable mobile phase for ideal chromatographic analysis of istradefylline. The solvent mixture was filtered through 0.45 μ membrane filter and sonicated before use. It was pumped through the column at a flow rate of 1.0 mL/min. Injection volume was 20 μL and the column was maintained at room temperature. The column was equilibrated by pumping the mobile phase through the column for at least 30 minutes prior to the injection of the drug solution. The detection of the drug was monitored at 246 nm. The run time was set at 10 min. The optimized chromatographic conditions are shown in Table 1.

Table 1: Optimized chromatographic conditions

Parameters	Conditions
Stationary Phase (Column)	C₁₈ (250 × 4.6 mm i.d.,5µ)
Mobile Phase	Acetonitrile: 0.1% ortho-phosphoric acid (70:30,v/v)
Flow rate (ml/min)	1.0 mL/min
Run time (min)	5 min
Column temperature (°C)	Ambient
Volume of injection loop (μL)	20
Detection wavelength (nm)	246 nm
Retention time (min)	3.125

Fig. 2: Chromatogram of standard solution of Istradefylline

Preparation of mobile phase:

Mobile phase was prepared by mixing 700 mL of HPLC grade acetonitrile with 300 mL of 0.1% ortho-phosphoric acid (prepared by mixing 0.1 ml of ortho-phosphoric acid in 100 ml of HPLC grade water. The mobile phase was sonicated for 10 min and filtered through the 0.45 μm membrane filter.

Preparation of standard stock solutions:

The standard stock solutions of 100 μg/mL of the drug were prepared by dissolving 50 mg of pure drug in the mobile phase in a 50 mL volumetric flask and the volume was made up to the mark. Resulting solutions were further diluted with mobile phase to obtain a final concentration of 100 μg/mL and stored under refrigeration. Aliquots of standard stock solutions were put in a 10 mL volumetric flask and diluted up to the mark with mobile phase. In such a way, the final concentrations of the drug were in the range of 10–90 μg/mL.

Preparation of sample solution:

To determine the content of istradefylline in tablet dosage form (Label claim: 20 mg/tablet) ten tablets were accurately weighed and triturated to fine powder. The powder weight equivalent to 10 mg of istradefylline was taken and dissolved in 50 ml of mobile phase. The resulting solution (2.5 ml) was transferred to a 10 ml volumetric flask and diluted up to the mark with mobile phase. The final solution was filtered through 0.45μ membrane filter using injection filter. A 20 µL of the filtrate was injected into chromatographic system. The peak area of the istradefylline was determined and concentration was found using linear regression equation obtained from calibration curve.

Method Validation:

The developed method was validated as per ICH guidelines¹⁹ by evaluating linearity, accuracy, precision, robustness, ruggedness, detection limit, quantification limit and stability. Coefficients of variation and relative errors of less than 2 % were considered acceptable.

System Suitability Test:

Before performing validation experiments, system suitability test (SST) has to be applied to indicate that HPLC system and method are capable of providing data with admissible quality. SST was performed by investigating capacity factor, tailing factor, theoretical plates number, and also relative standard deviation (RSD) of the peak areas.

Stability:

Stability was assessed by analyzing QC standard solutions after keeping them at room temperature for 48 h. Obtained results were investigated as recovery values and compared to the freshly prepared solutions.

Linearity:

A stock solution of istradefylline of 1000 μg/mL was prepared with mobile phase. From it, various working standard solutions were prepared in the range of 10 to 120 μg/ml and injected into HPLC. It was shown that the selected drug had linearity in the range of 10–90 μg/mL. The calibration plot (peak area of istradefylline versus istradefylline concentration) was generated by replicate analysis (n=9) at all concentration levels and the linear relationship was evaluated using the least square method within Microsoft Excel® program.

Accuracy

The accuracy of the method was carried out using one set of different standard addition methods at different concentration levels, 80 %, 100 % and 120 %, and then comparing the difference between the spiked value (theoretical value) and actual found value.

Precision:

The precision of the method was ascertained from the peak area obtained by actual determination of six replicates of a fixed amount of the drug (50 μg/mL). The precision of the assay was also determined in terms of intra- and inter-day variation in the peak areas of a set of drug solutions on three different days. The intra- and inter-day variation in the peak area of the drug solution was calculated in terms of relative standard deviation (RSD).

Robustness:

Robustness of the proposed method for istradefylline was carried out by the slight variation in flow rate, analytical wavelength and mobile phase ratio. The percentage recovery and RSD were noted for istradefylline.

Ruggedness:

The test solutions were prepared as per test method and injected under variable conditions. Ruggedness of the method was studied by different analysts.

Detection limit and quantification limit:

The limit of detection (LOD) and limit of quantification (LOQ) were established based on the calibration curve parameters, according to the following formulas:

LOD=3.3SD/slope

LOQ=10SD/slope

or detection limit=3.3σ/s, quantification limit=10σ/s, where σ is the standard deviation of y-intercept of regression line, and s is the slope of the calibration curve.

Specificity:

The specificity of the proposed method was determined against blank and placebo applications. Here mobile phase was used as blank and excipients like starch, lactose, magnesium stearate were used as placebo.

RESULTS AND DISCUSSION:

Method validation

System Suitability Test

After setting the optimum conditions, system suitability parameters for the developed method were determined and compared with recommended limits. To determine the parameters, the study was performed with standard solution of 50 µg/ml concentration and the results were acquired from six injections. System suitability parameters of the method were demonstrated in Table 2. According to the results, all of the system suitability parameters were within the recommended limits and the method was found to be suitable for the analysis.

Table 2: Results of system suitability test (n = 6)

Parameter	Criteria	Result
Capacity factor(k^’)	k^’> 2	3.824
Tailing factor (T)	T < 2	1.156
Theoretical plates (N)	N> 2000	4320
% RSD (peak area)	% RSD ≤ 1	0.74

Stability:

The sample solution stability was analyzed by injecting the same solution at 0, 12, 24, and 48 h. Identical change was not observed in the developed method. Also, results were found within acceptable limits (% RSD < 2), which are summarized in Table 3.

Table 3: Stability data of Istradefylline (standard solutions)

Time (hr)	Assay (%)	% Difference
Initial	100.08	----
After 12 hr	100.02	0.05
After 24 hr	99.87	0.21
After 36 hr	99.16	0.92
After 48 hr	98.32	1.76

Linearity and sensitivity:

Linearity study was performed with calibration standards with 10, 20, 30, 40, 50,60,70, 80 and 90 µg/ml concentrations. The standards were injected in triplicate. Calibration curves were obtained by plotting the peak areas against the given concentrations. The calibration curve was evaluated by the determination coefficient. The determination coefficient (R²) of the calibration curves was 0.9993. Therefore, the calibration curve for istradefylline was found to be linear within the range of 10–90 µg/ml concentrations as shown in Fig.3. The regression equations were calculated from the calibration graphs. The sensitivity of the analytical method was evaluated by determining the limits of detection (LOD) and quantitation (LOQ). The values of LOD and LOQ are given in Table 4. The low values of LOD and LOQ indicates the sensitivity of method.

Table 4: Spectral and statistical data for determination of Istradefyllin by proposed RP-HPLC method.

Parameter	Result
Detection wavelength (nm)	246
Linearity range (µg/ml)	10-90
Coefficient of determination (r²)	0.9993
Regression equation (Y^a)	Y= 123586x-29037
Slope (m)	123586
Intercept (c)	-29037
Limit of detection, LOD (µg/ml)	0.02
Limit of quantitation, LOQ (µg/ml)	0.06

^aY = mx + c, where x is the concentration (µg/ml).

Fig. 3: Calibration curve for Istradeylline

Accuracy:

To study the reliability, the suitability, and the accuracy of the method, recovery experiments were carried out. Known quantities of the pure drug were added to the preanalyzed sample to make samples at the levels of 80 %, 100 %, and 120 %, and were assayed by the proposed method. Accuracy was calculated as the percentage of recovery. The recovery and relative standard deviation for each of the analytes are given Table 5. From the recovery studies it is evidence that the method is highly accurate and can give excellent results.

Table 6: Precision results

Precision

Results

Concentration

(µg/mL)

% RSD of Peak area

% RSD of Retention Time

Repeatability

0.89

1.21

1.11

0.02

0.08

0.12

Intermediate precision

1.42

0.75

0.67

0.08

0.06

Reproducibility

1.64

0.78

0.85

0.11

0.17

0.09

Precision:

The precision was demonstrated at three levels: repeatability, intermediate precision, and reproducibility (between laboratories’ precision). Each level of precision was investigated by 3 sequential replicate of injections of three concentrations of 40, 50 and 60 µg/mL. The precision was expressed as relative standard deviation (RSD) or coefficient of variation (CV). The results of three levels of precision are shown in Table 6. The developed method was found to be precise as the RSD values for repeatability, intermediate precision and reproducibility studies were <2 %, respectively as recommended by ICH guidelines (ICH Q2 (R1), 2005).

Robustness and ruggedness:

Robustness of the method was studied by deliberate variations of the analytical parameters such as flow rate (1.0±0.1 mL/min), mobile phase composition (± 5 % organic phase) and analytical wavelength (±2 nm). The results are given in Tables 7. The result shown that have the negligible effect on retention time, recoveries and peak area of istradefylline indicating the developed method is robust. Ruggedness of the method was carried out by different analysts. The results are displayed in Table 8. There is no variation in peak areas and retention time of istradefylline from studies carried out by two analysts as indicated by % RSD < 2 gives the method rugged.

Mobile phase stability:

The stability of the mobile phase was evaluated, so the mobile phase was stored at 4–8 °C for 1 week. The aged mobile phase was compared using a freshly prepared one. The mobile phase was stable up to 1 week at 4–8 °C.

Specificity:

Specificity is the ability to unequivocally assess the analyte in the presence of components that may be expected to be present. Typically, these might include impurities, degradants or matrix. Specificity of an analytical method is its ability to accurately and specifically measure the analyte of interest without interference from blank or placebo. The peak purity of Istradefyllin was assessed by comparing the retention times of standard istradefylline and the sample, and good correlation was obtained between the retention time of the standard and sample. Placebo and blank were injected and there were no peaks. There is no interference of blank and placebo on drug peaks hence, the method is specific. The chromatogram for placebo and blank was shown in Fig 4 and 5 respectively.

Sample Analysis:

The developed and validated method was applied for analysis of tablet formulation contain istradefylline. The sample was analyzed in triplicate. Analysis results were evaluated using a calibration curve. The amount of istradefylline in the samples was calculated from calibration curve equation and recovery and RSD values were determined. The results of analysis are given in Table 9. The recoveries were in good agreement with the label claims. The chromatogram obtained were clear as shown in Fig. 6. It was concluded that the method can be applied successfully for the analysis of istradefylline in tablet dosage form.

Fig. 6: Chromatogram of sample solution of istradefyllin

Table 9: Assay results from commercial formulation

Sample

Labelled amount (mg)

Amount obtained* (mg)

Percentage Recovery*±SD

NOURIANZ™ tablets

(Istradefylline)

19.92

99.62 ± 0.92

*Average of five determinations

CONCLUSION:

The proposed method for the estimation of Isrtradefyllin was validated as per the ICH guidelines and it is simple, specific and reliable. Furthermore, this simple and rapid RP-HPLC method can also be used successfully for the determination of istradefylline in pharmaceutical formulations without any interference from the excipient.

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Received on 31.08.2020 Modified on 28.09.2020

Research J. Pharm. and Tech. 2021; 14(9):4767-4772.

DOI: 10.52711/0974-360X.2021.00829

Analyst	Observed value
Analyst	% RSD of area	% RSD of R.T	Tailing factor(T)	Theoretical plates(N)
Analyst I	0.45	0.07	1.15	4286
Analyst II	0.52	0.06	1.15	4311