Development and validation of a new stability indicating RP-UFLC method for the estimation of Perampanel in presence of internal standard

 

N S Yamani*, Mukthinuthalapati Mathrusri Annapurna

Department of Pharmaceutical Analysis, Gandhi Institute of Technology and Management (Deemed to be University), GITAM Institute of Pharmacy, Visakhapatnam, Andhra pradesh-530045, India.

 

ABSTRACT:

Perampanel is chemically α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor antagonist. Perampanel is used for the treatment of seizures which involve one part of brain both in adults as well as in children who are above four years. In the present study a new stability indicating reverse phase ultrafast liquid chromatographic method has been proposed for the quantification of Perampanel in presence of an internal standard, Rufinamide using water: acetonitrile as mobile phase. Linearity was observed over the concentration range 0.05-50 μg/mL with linear regression equation y = 1.3839x - 0.0302 (r² = 0.9999). The LOD and LOQ were found to be 0.01451 μg/mL and 0.0437 μg/mL. Perampanel was exposed to different stress conditions such as acidic hydrolysis, alkaline hydrolysis, oxidation and thermal treatment and the proposed method was validated.

 

 

 

INTRODUCTION:

Perampanel (Figure 1) is used for treating seizures (Anti-epileptic drug). It is a non-competitive α- amino 3-hydroxy 5-methyl 4-isooxazole propionic acid receptor agonist^1-3. It is α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor antagonist. It is used for the patients above 12 years for the treatment of partial onset seizures and its exact mechanism is known. Literature survey reveals that many bio analytical methods were developed using LC-MS/MS^4-6 and HPLC^7-9 techniques. The authors of some of these methods used fluorescence detection and for the present study the authors have chosen a reverse phase UFLC method for the estimation of Parampanel in tablets. Forced degradation studies were performed and the method was validated as per ICH guidelines.

 

 

Figure 1: Chemical structure of Parampanel

 

MATERIALS AND METHODS:

Shimadzu Model CBM-20A/20 Alite UFLC system equipped with PDA detector was employed for the study and Agilent C₁₈ column was used. Perampanel API was procured from as gift sample Torrent Pharmaceuticals (India) and it is available as tablets Torpanel (Torrent Pharmaceuticals) (India) (Label claim: 2 mg) and Fycompa (Eisai Pharmaceuticals India Pvt Ltd) (Label claim: 2 mg, 4 mg, 6 mg, 8 mg) and all the chemicals were of AR grade. The internal standard (IS), Rufinamide was procured from Eisai Pharmaceuticals India Pvt Ltd as gift sample. Acetonitrile (HPLC grade), sodium hydroxide, hydrochloric acid and hydrogen peroxide were obtained from Merck (India).

 

Procedure

Preparation of Perampanel drug solution

50 mg of Perampanel was accurately weighed and transferred in to a 50 mL volumetric flask and the volume was made up with HPLC grade acetonitrile (1000 μg/mL) and later the dilutions were made with the mobile phase (water: acetonitrile) (60: 40, v/v) and were filtered through 0.45 μm membrane prior to injection. In similar manner Rufinamide (1000 μg/mL) stock solution was made and 10 μg/mL of solution was added to the volumetric flask just before injecting in to the system throughout the study and the peak areas of Perampanel as well as Rufinamide were observed. The total runtime was 15 minutes.

 

Method validation¹⁰

Linearity, Precision, Accuracy, Robustness

The internals standard, Rufinamide (IS) (10 μg/mL) was added to all the Perampanel drug solutions (0.05-50 μg/mL) and the peak areas of Perampanel as well as Rufinamide were noted. The mean peak area ratio of Perampanel to that of Rufinamide was calculated from the respective chromatograms for each diluted solution and a calibration curve was drawn by taking mean peak area ratio values on the y-axis against the concentration of Perampanel on the x-axis.

 

Intraday and inter-day precision were studied using three different concentrations of Perampanel (15, 20 and 25 μg/mL) on the same day and on three consecutive days respectively in presence of Rufinamide (IS) and thereby the % RSD was calculated. The accuracy of the assay method was evaluated in triplicate at three concentration levels (50, 100 and 150 %) in presence of Rufinamide (IS) and the percentage recoveries were calculated. Robustness study was performed by incorporating small changes in the optimised chromatographic conditions in presence of Rufinamide (IS) and finally the % RSD was calculated.

 

Assay of Perampanel _{tablet dosage forms}

Two different brands each of twenty tablets were procured from the pharmacy store, powdered and powder equivalent to 25 mg Perampanel was extracted with acetonitrile, sonicated for half an hour and filtered through 0.45 mm membrane filter followed by dilution using mobile phase on daily basis as per the requirement. 20 µL of these two extracted solutions from the two brands were injected in to the UFLC system and the mean peak area ratio was calculated from the respective chromatogram.

 

Forced degradation studies¹¹

Forced degradation studies were performed in presence of Rufinamide (IS) by applying the stress conditions. Acidic hydrolysis (1 ml 0.1N HCl, 75°C/30 min), alkaline hydrolysis (1 ml 0.1N NaOH, 75°C/30 min), oxidation (1 ml 10% H₂O₂, 75°C/30 min)) and thermal treatment (75°C/30 min).  The drug solutions exposed to acidic and alkaline hydrolysis were neutralised before injecting in to the UFLC system. All the solutions were cooled, filtered through Whatman membrane filter No. 45 and then 20 µL of each solution was injected in to the UFLC chromatographic system only after the addition of Rufinamide (10 μg/mL) (IS) just before injection and the mean peak area ratio was calculated from the respective chromatograms obtained and the percentage recovery was computed.

 

RESULTS AND DISCUSSION:

A new stability indicating RP-UFLC method has been developed for the estimation of Perampanel in tablet dosage forms in presence of Rufinamide (IS) using C18 Agilent column and Water: Acetonitrile (60: 40) as mobile phase with 0.9 ml/min flow rate and the method was validated as per ICH guidelines.  Some of the important parameters of the previous literature were discussed and compared with the present proposed method in Table 1. The representative chromatograms obtained with the optimised conditions for the placebo, the internal standard, Rufinamide (Rt 3.751 min) as well as Perampanel API (Rt 10.646) in presence of IS (Rt 3.802 min) were shown in Figure 2A, Figure 2B and Figure 2C.

 

Table 1: Literature survey

Method / Mobile phase (v/v)	Column	Linearity (µg/mL)	Comment	Ref
Bio analytical method (Water/Formic acid): (Acetonitrile/Formic acid)	C18 Hypersil Gold Column	0.0025 - 2.8	LC-MS/MS (Human plasma) (Protein precipitation) D6-Levetiracetam (Internal standard)	4
Bio analytical method	C8 column	0.00025-0.2	LC-MS/MS (Human plasma) (Liquid extraction) (Internal standard)	5
Bio analytical method	RP Column	-	LC-MS/MS Fluorescence detection (Protein precipitation)	6
Bio analytical method Water/acetonitrile (60:40) with 1 ml/litre phosphoric acid / 320 nm	Reverse-phase monolithic column	0.025-1.0	RP-HPLC (Human plasma) (Protein precipitation) Promethazine hydrochloride(Internal standard)	7
Bio analytical method 0.03 M Sodium acetate  (pH 3.7): Acetonitrile (40: 60)	Kinetex PFP column	0.02-1.0	RP-HPLC (Human plasma) Fluorescence detection Mirtazapine (Internal standard)	8
Bio analytical method Acetonitrile: Water: Acetic acid: Sodium acetate (840:560:3:1.8)	YMC Pack Pro C18 column	0.001-0.5	RP-HPLC (Human plasma) (Liquid extraction) Fluorescence detection	9
0.1% Acetic acid: Acetonitrile (50:50)	C8 Phenomenex	0.05-120	RP-UFLC Stability indicating	10
Water: Acetonitrile (60:40)	Agilent C18	0.05-50	RP-UFLC Stability indicating	Present method

 

Method validation

Linearity, precision, accuracy and robustness

Perampanel follows Beer-Lambert’s law over the concentration 0.05-50 μg/mL (% RSD 0.29-0.84) with linear regression equation y = 1.3839x - 0.0302 (r² = 0.9999) (Table 2). The calibration curve was shown in Figure 2 in which the mean peak area ratio values were plotted on the y-axis against the concentration of Perampanel on the x-axis. The LOD and LOQ were found to be 0.01451 μg/mL and 0.0437 μg/mL.

 

Figure 2:  Representative chromatograms of Perampanel in presence of IS

 

Table:2: Linearity of Perampanel in presence of internal standard

*Mean of three replicates

 

Figure 3: Calibration curve

 

Intraday and inter-day precision were studied at three different concentration levels of Perampanel on the same day and on three consecutive days respectively. The % RSD was found to be 0.0138-0.0215 (Intra-day) (Table 3) and 0.27-0.89 (Inter-day) (Table 4) respectively (<2.0 %) demonstrating that the method is precise.

 

The accuracy of the method was proved by the standard addition method and the recovery values were determined. The % recovery of Perampanel was found to be 99.27-99.76% and the % RSD (0.26-0.61) (<2.0 %) of the method has shown that the method is precise (Table 5).

 

The robustness of the assay method was established by introducing small changes in the UFLC conditions which include wavelength (242 and 238 nm), percentage of acetonitrile in the mobile phase (42 and 38%) and flow rate (0.8 and 1.0 ml/min). Robustness of the method was studied using 10 μg/mL of Perampanel. The % RSD was found to be 0.29-0.97 which is less than 2.0 indicating that the method is robust (Table 6).

 

Table 3: Intraday precision study

*Mean of three replicates

 

 

 

 

Table 4: Interday precision study

Conc. (µg/mL)		*Mean peak area ratio			Statistical Analysis *Mean ± SD (% RSD)
PER	RUF	Day 1	Day 2	Day 3
15	10	20.8453	20.8971	20.9126	20.8850 ± 0.0564 (0.27)
20	10	27.0153	27.0215	27.0623	27.0330 ± 0.0892 (0.33)
25	10	34.7335	34.8215	34.7598	34.7716 ± 0.3095 (0.89)

*Mean of three replicates

 

Table 5: Accuracy study

Spiked conc. ((µg/mL)	Formu lation ( µg/mL)	Total Conc. ( µg/mL)	*Mean Conc. (µg/mL) ± SD (% RSD)	% Recovery
5 (50%)	10 10 10	15 15 15	14.89 ± 0.0387 (0.26)	99.27
10 (100%)	10 10 10	20 20 20	19.93 ± 0.0977 (0.49)	99.65
15 (150%)	10 10 10	25 25 25	24.94 ± 0.1521 (0.61)	99.76

*Mean of three replicates

 

Table 6: Robustness study (10 µg/mL)

Parameter	Condition	*Mean peak area ratio (PER/RUF)	Statistical Analysis *Mean ± SD (% RSD)
Flow rate (± 0.1 ml/min)	0.8	14.107	13.9440 ± 0.1353 (0.97)
	0.9	13.904
	1.0	13.821
Mobile phase ratio Water: Acetonitrile (± 2 %)	62:38	13.862	13.8947 ± 0.0584 (0.42)
	60:40	13.904
	58:42	13.918
Detection wavelength (± 2 nm)	238	13.722	13.8133 ± 0.0401 (0.29)
	240	13.904
	242	13.814

*Mean of three replicates

 

_{Assay of} Perampanel _tablets

The proposed RP-UFLC method was applied to Perampanel tablet dosage forms and found the % recovery as 98.91-99.62 (Table 7) and it was also found that there is no interference of excipients. The chromatograms of Perampanel extracted from the tablet dosage forms (Brand I and Brand II) were shown in Figure 2D and Figure 2E.

 

Table 7:  Assay of Perampanel tablets

*Mean of three replicates

 

 

 

Forced degradation studies

Forced degradation were conducted in presence of the internal standard, Rufinamide. During the forced degradation Perampanel has shown some degradants products during alkaline and oxidation degradations. During acidic degradation Perampanel has shown about 10.94% degradation and during thermal degradation 6.90 % degradation was observed. During the alkaline degradation extra peaks were observed at   1.656, 1.856 and 2.568 min conditions and during the oxidative degradation about 20.11% degradation was observed and an extra peak was observed at 1.977 min. All the system suitability parameters were within the acceptable criteria i.e. theoretical plates were greater than 2000, tailing factor was less than 1.5 and the resolution was greater than 2.0. The chromatographic elution of Perampanel and the internal standard, Rufinamide were well separated during the degradation conditions indicating that the method is specific. The representative chromatograms of Perampanel obtained during the forced degradation studies were shown in Figure 4 and the details were shown in Table 8. 

 

Table 8: Forced degradation studies

*Mean of three replicates

 

Figure 4:  Representative chromatograms of Perampanel during stress degradation

 

CONCLUSIONS:

The new stability indicating RP-UFLC method developed for the determination of Perampanel and the method was validated. The proposed method is sensitive, simple and economical and there is no interference of the excipients. During the forced degradation studies it was observed that Perampanel highly resistant towards acidic, alkaline and thermal conditions.

 

ACKNOWLEDGEMENT:

The authors are grateful to Torrent Pharmaceuticals, India for providing the gift samples of Perampanel. The authors have no conflict of interest.

 

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Accepted on 21.11.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(1):279-284.