INTRODUCTION:

Perampanel is an antiepileptic drug used for the treatment of various neurological disorders caused by neuronal hyperexcitation. Chemically it is α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor antagonist. It has been recently approved in the United States and the European Union for the adjunctive treatment of focal seizures and primary generalized tonic-clonic seizures associated with idiopathic generalized epilepsy. The antagonistic action results in the inhibition of neuronal excitation, repetitive neuronal firing, and the stabilization of hyper-excited neural membranes. It is extensively (> 90%) metabolized^1-3via cytochrome P450 CYP3A4/5.Perampanel was assayed in human plasma using LC-MS^4-5, HPLC^6-7 and HPLC with fluorescence detection^8-9but no stability indicating method was available for the determination of Perampanel tablets. In the present study the authors have proposed a simple validated stability indicating RP-UFLC method for the quantification of Parampanel in tablets. The method was validated as per ICH guidelines.

Figure 1: Chemical structure of Parampanel

MATERIALS AND METHODS:

Perampanel API standard (>99%purity) was procured as gift sample from Eisai Pharmaceuticals India Pvt Ltd. Perampanel is available as film coated tablets with brand name Fycompa (Eisai Pharmaceuticals India Pvt Ltd; Label claim: 2, 4, 6, 8, 10, 12 mg). All other chemicals are of AR grade and all solvents are of HPLC grade. Acetonitrile (HPLC grade), sodium hydroxide, hydrochloric acid, hydrogen peroxide and HPLC grade water were obtained from Merck (India).

Instrumentation:

Chromatographic separation was achieved by Shimadzu Model CBM-20A/20 Alite UFLC system (Shimadzu Co., Kyoto, Japan) equipped with SPD M20A prominence photodiode array detector C8 phenomenex column (150 mm × 4.6 mm i.d. 3.5 µm particle size) maintained at room temperature.

Procedure:

Preparation of Perampanel drug solution:

Accurately weighed 25 mg of Perampanel was transferred in to a 25mL volumetric flask and made up to volume with HPLC grade acetonitrile (1000 μg/mL) and afterwards dilutions were made with mobile phase and filtered through 0.45 μm membrane filter prior to injection.

Optimized chromatographic conditions:

A C8 Phenomenex column (150 mm × 4.6 mm i.d.3.5µm particle size) with mobile phase composition %1 acetic acid: acetonitrile: Water (0.1:50: 50) with flow rate 1.0 mL/min (UV detection at 240nm) was found to be more appropriate to satisfy the system suitability parameters and the method was optimised where a sharp drug peak was eluted (Figure 2B) at about 10.412 min (Table 1).

Figure 2A: Representative chromatogram of mobile phase

Figure 2B: Representative chromatogram of Perampanel standard (10 µg/mL)

Method validation:

The method was validated for the following parameters: linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, specificity and robustness (ICH guidelines, 2005)¹⁰.

Figure 2C: Representative chromatogram of Perampanel tablet (10 µg/mL) (Label claim: 10 mg)

Table 1: Optimized chromatographic conditions

Parameter	Optimized chromatographic conditions
Mobile Phase	0.1 % acetic acid and Acetonitrile and water (50: 50 v/v)
Stationary Phase	C8 Phenomenex column (150 mm × 4.6 mm i.d.3.5µm particle size)
Flow Rate	1.0 mL/min
Detection Range	240 nm
Column temp.	(25°±2°C)
Injection Volume	20µL
Detector	SPD M20A prominence photodiode array detector
Elution	Isocratic mode
Total Run Time	15 mins
Retention time	10.412 ± 0.2 mins

Linearity, Precision, Accuracy, Robustness:

A series of solutions (0.05-120 μg/mL) were prepared from the Perampanel stock solution on dilution with the mobile phase and 20 μL of each of these solutions were injected (n=3) in to the UFLC system and the mean peak area was calculated from the chromatograms. A calibration curve was drawn by taking the concentration of Perampanel on the x-axis and the corresponding mean peak area values on the y-axis.

Intraday and inter-day precision were studied using three different concentrations of Perampanel on the same day and on three consecutive days respectively and the % RSD was calculated. The accuracy of the assay method was evaluated in triplicate at three concentration levels (50, 100 and 150 %), and the percentage recoveries were calculated. Standard addition and recovery experiments were conducted to determine the accuracy of the method for the quantification of Perampanel in the drug product and the percentage recovery was calculated.

The robustness of the method was assessed by exposing the drug solution to different analytical conditions purposely changing from the original optimized conditions. The effects so obtained were summarized to calculate the % RSD and must be less than 2.0% specifying that the proposed method was robust.

Sensitivity/ Limit of detection (LOD) and Limit of quantification (LOQ):

The limit of detection (LOD) and limit of quantification (LOQ) were based on the standard deviation of the response and the slope of the constructed calibration curve (n=3), as described in ICH guidelines Q2 (R1). Sensitivity of the method was established with respect to LOD and LOQ for Perampanel and calculated by slope method as mentioned below.

Assay of Perampanel _Tablets:

Twenty tablets were procured from the local 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 this solution was injected in to the UFLC system and the mean peak area (n=3) was noted from the respective chromatogram.

Forced degradation studies¹¹:

Forced degradation studies were performed to determine the ability of the drug to withstand its properties in the applied stress conditions. Perampanel was exposed to different stress conditions such as acidic hydrolysis (1 ml 0.1N HCl, 80°C/30 min), basic hydrolysis (1 ml 0.1N NaOH, 80°C/30 min), oxidation (1 ml 10% H₂O₂, 80°C/30 min)) and thermal treatment (80°C/30 min). The drug solutions exposed to acidic and basic hydrolysis were neutralised before injecting in to the UFLC system. All the solutions were cooled, filtered through What man membrane filter No. 45 and then 20 µL of each solution was injected in to the chromatographic system (n=3) and the mean peak area was calculated from the corresponding chromatograms.

RESULTS AND DISCUSSION:

A simple stability indicating reverse phase ultrafast liquid chromatographic method has been developed for the determination of Perampanel API (active pharmaceutical ingredient) and the method was applied to its tablet dosage forms using C8 Phenomenex column (250 mm × 4.60 mm i.d. 5µm particle size) and mobile phase mixture%1 acetic acid: acetonitrile: water (0.1: 50: 50).

Method development and optimization:

Initially trials were made using C8 Phenomenex column with mobile phase composition methanol: water (60: 40 v/v) with flow rate 1.2 mL/min but Perampanel was eluted as a blunt peak and therefore the mobile phase composition was modified as methanol: water (40:60, v/v) with flow rate 0.8 mL/min and tailing was observed (>2). Finally, mobile phase consisting of a mixture of water, acetonitrile and 0.1% acetic acid (50:50:0.1 v/v) was selected with flow rate 1 mL/min (UV detection at 240 nm) (Isocratic mode) for the chromatographic study where Prampanel was eluted at 10.31 ± 0.2 min with theoretical plates more than 2000 and tailing less than 1.5 within a total run time of 15 min.

Method validation:

Linearity, precision, accuracy and robustness:

Perampanel has shown linearity over the concentration range 0.05-120 μg/mL (Table 2) with linear regression equation y=73084x+ 13800 (r²=0.9997) (Figure 3).

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 0.244-1.523 (Intra-day) (Table 3) and 0.26-0.844 (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 and its results of the method are reported in (Table 6). The % RSD was found to be 0.39-0.71 (<2.0 %) with a recovery of 98.87-101.68 % (Table 5).

The robustness of the assay method was established by introducing small changes in the UFLC conditions which included wavelength (241 and 239 nm), percentage of acetonitrile in the mobile phase (52 and 48%) and flow rate (±0.1 ml/min). Robustness of the method was studied using 10 μg/mL of Perampanel. The % RSD was found to be 0.87-1.23which is less than 2.0 indicating that the method is robust (Table 6).

Table. 2. Linearity of Perampanel

Conc. (µg/mL)	*Mean peak area	% RSD
0.05	36636	0.42
1	73259	0.47
2	146523	0.12
5	366257	1.01
10	732515	0.42
20	1465030	1.43
25	1831287	0.84
40	2930060	0.36
60	4395090	0.73
80	5860120	1.66
100	7325150	0.47
120	8790180	0.12

*mean of three replicates

Figure 3: Calibration curve of Perampanel

Table. 3. Intraday precision study of Perampanel

Conc. (µg/mL)	*Mean peak area	*Mean peak area ± SD (% RSD)
10	732515	739551.± 11331.72(1.523)
10	733515
10	752623
20	1465030	1474053± 10178.06(0.698)
20	1472042
20	1485086
30	2197545	2193557 ± 5368.249(0.244)
30	2187453
30	2195672

*mean of three replicates

Table. 4. Interday precision study of Perampanel

Conc. (µg/mL)	*Mean peak area			*Mean ± SD (% RSD)
Conc. (µg/mL)	Day 1	Day 2	Day 3	*Mean ± SD (% RSD)
10	732325	723525	753523	1326446 ±7195.925(0.54)
20	1464037	1482142	1455483	2607978 ± 7027.33(0.26)
30	2397443	2357353	2375661	2376818 ± 20070.07(0.844)

*mean of three replicates

Table. 5. Accuracy study of Perampanel

Spiked conc. (µg/mL)	Formulation (µg/mL)	Total conc. (µg/mL)	Average conc. obtained (µg/mL) ± SD (%RSD)*	% Recovery
5 (50%)	10 10 10	15 15 15	14.85± 0.105357 (0.71)	101.68
				99.88
				98.80
10 (100%)	10 10 10	20 20 20	19.74 ± 0.113(0.57)	100.13
				98.87
				101.37
15 (150%)	10 10 10	25 25 25	24.67 ± 0.0971 (0.39)	100.54
				99.49
				100.88

*mean of three replicates

Table. 6. Robustness study of Perampanel

Parameter	Condition	*Peak area	Mean peak area ±SD (% RSD)*
Flow rate (± 0.1 ml/min)	1.0	732515	734141.7± 7194.58(0.98)
	1.1	734223
	0.9	735687
Detection wavelength (± 2 nm)	241	720365	1322115 ± 9014.322(1.23)
	240	732515
	239	745734
Mobile phase composition 0.1% acetic acid: acetonitrile: water(0.1: 50:50) (± 2 %, v/v)	42:58	733241	734469.3± 6389.88(0.87)
	50:50	732515
	48:52	737652

*mean of three replicates

_{Assay of}Perampanel _Tablets:

Perampanel has shown 99.52-98.93% recovery in tablet formulations and the proposed methods can be satisfactorily applied for the marketed formulationsand there is no interference of excipients. The overlay chromatogram of the mobile phase, Parampanel (API) and that of the table formulation were shown in Figure 4.

Figure 4: Overlay chromatogram of A) Perampanel API (10 µg/mL) B) Brand I and C) Brand II (Label claim: 10 mg)

Stress degradation studies:

Perampanel is highly resistant towards all the degradation conditions and only less than 4 % degradation was observed. The system suitability parameters such as theoretical plates were greater than 2000 and the tailing factor was less than 1.5. Perampanel drug peak was well separated in all the degradation conditions indicating that the method is specific. The representative chromatograms, purity plots, overlay chromatogram and that of the 3D chromatograms of Perampanel during these stress degradation studies were shown in Figure 5, Figure 6, Figure 7 and Figure 8 and the system suitability parameters were shown in Table 7.

Figure 5: Typical chromatograms of A) Mobile phase

B) Perampanel standard (10µg/ml)

C) Perampanel during oxidation

D) Perampanel during thermal degradation

E) Perampanel during base degradation

F) Perampanel during acid degradation

Figure 6: Peak purity plots of Perampanel during A) Oxidation

B) Thermal degradation

C) Base degradation

D) Acid degradation

Figure 7: Overlay chromatogram of A) Mobile phase and Perampanel during stress degradation B) Alkaline degradation

C) Thermal degradation D) Oxidation degradation E) Acidic degradation F) Perampanel standard

Representative 3D chromatogram of Perampanel standard (10 µg/mL)

Acidic degradation

Alkaline degradation

Oxidative degradation

Thermal degradation

Figure 8: 3 D chromatograms of Perampanel (A) Acidic degradation (B) Alkaline degradation (C) Oxidative degradation (D) Thermal degradation

Stress condition Medium/temp./duration	Rt (min)	% Recovery	% Drug degradation	Theoretical plates	Tailing factor
Standard drug	10.489	100	-	13402.494	1.124
Acidic hydrolysis 0.1N HCl/ 80°C/ 30 min	10.267 2.323	97.8	2.2	11097.885	1.076
Alkaline hydrolysis 0.1N NaOH/ 80°C/ 30 min	10.10	96.29	3.71	10217.382	0.993
Oxidation 30%H₂O₂/ 80°C/ 30 min	10.237	97.59	2.41	13364.848	1.136
Thermal degradation/80°C/30 min	10.232 2.606	97.54	2.36	13744.937	1.127

1. Greenwood J, Valdes J. Perampanel (Fycompa) a review of clinical efficacy and safety in epilepsy. P. T. 41; 2016: 683-698.

2. Franco V, Crema F, Iudice A, et al. Novel treatment options for epilepsy: focus on Perampanel. Pharmacol Res. 70; 2013: 35-40.

3. Patsalos PN. The clinical pharmacology profile of the new antiepileptic drug Perampanel: a novel noncompetitive AMPA receptor antagonist. Epilepsia. 56; 2015:12-27.