INTRODUCTION:

Oxcarbazepine is chemically 10,11-Dihydro-10-oxo-5H-dibenz [b, f] azepine-5-carboxamide. Oxcarbazepine (C₁₅H₁₂N₂O₂; 252.27 g/mol) is a white to light orange crystalline powder and slightly soluble in dichloromethane, chloroform, methanol and acetone and practically insoluble in ether, ethanol and water. It is an anti-convulsant as well as a mood-stabilizing drug. It is used for the treatment of anxiety and epilepsy^1-2. Spectrophotometric methods^3-8, voltametry⁹, HPTLC^10-12, GC^13-15, mass spectrophotometry^16-19, micellar chromatography²⁰ and liquid chromatographic methods in biological fluids^21-40 were published in the literature for the determination of Oxcarbazepine. Oxcarbazepine was also assayed in pharmaceutical formulations^41-47and in the present study the authors have proposed a validated isocratic reverse phase stability indicating liquid chromatographic method in capsules.

Figure 1: Chemical structure of Oxcarbazepine

Materials and methods:

Oxcarbazepine is available with brand names Carbamac (Macleods Pharmaceuticals Ltd, India) (Label claim: 300 and 600 mg); Carbox (Micro Labs Ltd, India) (Label claim: 300 mg); Oleptal DT (Torrent), Nictal (IPCA), Vinlep (Sanofi Aventis) with label claim: 150/300/600 mg. HPLC grade Acetonitrile, methanol and Triethylamine were procured from Merck. Oxcarbazepine gift sample was supplied from Macleods Pharmaceuticals Ltd, India.

HPLC instrument and chromatographic conditions

Waters e2695 Separation module with Waters 2489 UV detector with Hypersil BDS C18 column (250 mm x 4.6 mm, 5μ) was chosen or the chromatographic study of Oxcarbazepine and was operated with column temperature 50°C. A mixture of Acetonitrile, methanol and Triethylamine (660:140:200:1) adjusted to pH 6.0 ± 0.05 with the help of ortho phosphoric acid was used as mobile phase with a flow rate of 2.0 ml/min.

Procedure

6.8 gm Potassium dihydrogen phosphate was accurately weighed and dissolved in 1000 ml of water and used as buffer after sonication. A mixture of water and Acetonitrile in the ratio of 660: 340 v/v was used as diluent. Mobile phase consisting of Acetonitrile, methanol and Triethylamine (660:140:200:1) was prepared and the pH of the mixture was adjusted to 6.0 ± 0.05 with ortho phosphoric acid followed by filtration through 0.45μ filter and degassed prior to use.

Stock solution (1000 μg/mL) of Oxcarbazepine was prepared by accurately weighing about 50 mg and dissolved in diluent in a 50 ml volumetric flask and further dilutions were made with diluent. The method was validated by performing different studies – linearity, system precision, method precision, accuracy and robustness. Robustness study was performed by modifying the chromatographic conditions slightly and there by measuring the percentage relative standard deviation of the method developed. 20 μl of all the diluted solutions (5-150 μg/mL) were injected and the peak area (n=3) was noted at the retention time for the linearity study and a calibration curve was plotted (Concentration vs mean peak area). The limit of quantification (LOQ) and limit of detection (LOD) were calculated from the calibration curve response.

Forced degradation studies:

Oxcarbazepine drug solutions were exposed to different stress conditions such as acidic (1 ml, 5N HCl, 60°C, 30 min), alkaline (2 ml, 5N NaOH, 60°C, 30 min), oxidation (5 ml, 30% H₂O₂, 60°C, 30 min), thermal (60°C, 30 min) and photolytic (UV radiation; 254 nm, 24 hours) degradations. The acidic and alkaline stressed samples were neutralised and diluted with the mobile phase for the chromatographic study.

Assay of Oxcarbazepine tablets:

Oxcarbazepine tablets of three different brands were procured from the local pharmacy store, weighed and extracted with diluent and further dilutions were made as per the requirement. These solutions were injected in to the HPLC system and the peak area was noted at the retention time. The quantity of Oxcarbazepine was determined from the linear regression equation developed and the percentage of purity of the three brands was calculated.

RESULTS AND DISCUSSION:

A new stability indicating liquid chromatographic method has been developed for the determination of Oxcarbazepine in pharmaceutical formulations (Tablets). A review of the literature was done and only the liquid chromatographic methods already established were compared with the present method in Table 1. A mixture of acetonitrile, methanol and triethylamine was selected and the pH was adjusted to 6.0 ± 0.05 with the help of ortho phosphoric acid.

Method optimization was done using Waters e2695 Separation module with Waters 2489 UV detector with Hypersil BDS C18 column (250 mm x 4.6 mm, 5μ) and was operated with column temperature 50°C. A mixture of Acetonitrile, methanol and Triethylamine (660:140:200:1) adjusted to pH 6.0 ± 0.05 with the help of ortho phosphoric acid was used as mobile phase with a flow rate of 2.0 ml/min (UV detection 215 nm).

Table 1: Comparison of proposed method of Oxcarbazepine with the previously published methods

Mobile phase (v/v)	Column	Linearity (µg/mL)	Comments	Ref
0.1% NH4OH (pH adjusted to 5.0): Acetonitrile (Gradient mode)	Inertsil ODS3V (250 X 4.6 mm, 5 µ)	2.5-7.5	Related substances (4 Impurities)	41
0.005 M KH₂PO₄: Methanol: Acetonitrile (70:5:25)	Zorbax Eclipse XDB-C18 (250 X 4.6 mm, 5 µ)	1-1000	Stability indicating	42
Acetonitrile, Potassium phosphate monobasic buffer (pH 6.8): Water (36:8:56)	Diamonsil C18	9.96-99.6	Not Stability indicating	43
Acetronitrile: Water (50: 50)	Hiber C18 (250 X 4.6 mm, 5 µ)	0.05-80	Stability indicating	44
Mixed Phosphate buffer: Acetonitrile (55: 45) (pH adjusted to 6.5)	Symmetry C18 (150 X 4.6, 5µ)	7.5-45	Not Stability indicating	45
Water: Methanol: Acetonitrile (64:30:6)	C18	0.05-30 0.05-20 0.15-4	Carbamazepine Oxcarbazepine Eslicarbazepine & metabolites (Human plasma)	46
o-H₃PO₄: Triethylamine (0.14: 0.1): Acetonitrile (60:40) (pH adjusted to 6.5 with o-H₃PO₄ or TEA)	Octylsilyl silica gel C18 (250 X 4.6 mm, 5 µ)	30-70	Stability indicating	47
Buffer: Acetonitrile: methanol: Triethylamine (660:140:200:1) (pH adjusted to 6.0 ± 0.05 o-H₃PO₄)	Hypersil BDS C18 (250 X 4.6 mm, 5 µ)	25-145	Stability indicating (PDA)	Present method

Method validation⁴⁸

Oxcarbazepine has shown linearity 5-150 μg/mL (R² = 0.9999) (Table 2) with linear regression equation y = 47910x + 6439.8 and the LOQ and LOD were found to be 4.5129 μg/mL and 1.4898 μg/mL respectively (Figure 2). Oxcarbazepine was eluted at 6.531 mins as a sharp peak with theoretical plates 7846 and tailing factor 1.06 satisfying the system suitability criteria (Figure 3). The system precision (n=10) and the method precision (n=6) were determined by injecting the drug solution (50 μg/mL) in to the system and the percentage RSD was calculated (Table 3). Accuracy study was performed and the % recovery was calculated from the spiked samples (50 %, 100 % and 150 %) and the % RSD was less than 2.0 (Table 4). The robustness study results also shown that the % RSD (0.19-0.67) was found to be less than 2.0.

Table. 2. Linearity of Oxcarbazepine

Conc. (µg/ml)	Mean peak area ± SD (% RSD)*
0	0
5	238659 ± 1694.4789 (0.71)
10	479759 ± 1871.0601 (0.39)
25	1199499 ± 7316.9439 (0.61)
40	1918568 ± 16115.9712 (0.84)
50	2398753 ± 19429.8993 (0.81)
60	2871659 ± 12060.9678 (0.42)
75	3590176 ± 24054.1792 (0.67)
100	4790365 ± 34011.5915 (0.71)
120	5710245 ± 45110.9355 (0.79)
150	7236254 ± 34734.0192 (0.48)

*Mean of three replicates

Figure 2: Calibration curve of Oxcarbazepine

Table.3. Precision studies of Oxcarbazepine

S. No.	System Precision	Method Precision
1	2398815	100.1
2	2398521	100.2
3	2397568	99.8
4	2398753	100.2
5	2394236	99.9
6	2387523	100.7
7	2391236	-
8	2394368	-
9	2398569	-
10	2389652	-
Mean	2394924	100.2
SD	4215.96	0.31
RSD	0.18	0.31

Table.4. Accuracy studies of Oxcarbazepine

Spiked level	Amount recovered	% Recovery
50%	49.85	99.7
50%	49.15	98.3
50%	49.36	98.72
100%	98.54	98.54
100%	98.48	98.48
100%	98.41	98.41
150%	148.65	99.1
150%	148.85	99.23
150%	148.19	98.79
Mean		98.81
SD		0.456
RSD		0.4615

Assay of Oxcarbazepine tablets:

Oxcarbazepine tablet formulations (Figure 3) of three different brands were evaluated by using the method optimised and found that the recovery was 99.54% - 99.83% (Table 5). There is no interference of excipients.

Table. 5. Assay of marketed formulations of Abiraterone

Brand Name	Label Claim (mg)	Drug obtained	% Recovery
Brand I	300	298.61	99.54
Brand II	300	299.32	99.77
Brand III	300	299.49	99.83

Forced degradation studies:⁴⁹

Oxcarbazepine drug solutions were exposed to different stress conditions such as acidic, alkaline, thermal photolytic and oxidation. During acidic degradation 26.8 % and during alkaline degradation 44.6 % of the drug was decomposed (Figure 3). In oxidation (2.5%), thermal (0.3 %) and photolytic (1.5%) degradations less than 2% degradation was observed indicating that the drug is highly resistant towards oxidation, thermal and photolytic conditions (Table 6).

Table. 6. Stress degradation studies of Oxcarbazepine

Stress conditions	*Drug recovered (%)	*Drug decomposed (%)	Peaks observed	Theoretical plates	Tailing factor
Oxcarbazepine standard (API)	100.00	-	6.531	7846	1.06
Acidic degradation 1 ml 5N HCl, 60°C, 30 mins	73.20	26.80	6.412	7965	1.01
Alkaline degradation 2 ml 5N NaOH, 60°C, 30 mins	55.40	44.60	6.422	7584	1.02
Oxidative degradation 5 ml 30% H₂O₂, 60°C, 30 mins	97.50	2.50	6.391	8013	1.11
Thermal degradation 60°C, 30 mins	99.70	0.30	6.446	7963	1.08
Photolytic degradation 254 nm, 24 hours	98.50	1.50	6.429	8029	1.02

*Mean of three replicates


Blank	Oxcarbazepine standard (API) (Rt = 6.531 min)

Acidic degradation (Rt = 6.412 min)	Alkaline degradation (Rt = 6.422 min)

Oxidative degradation (Rt = 6.391 min)	Thermal degradation (Rt = 6.446 min)

Photolytic degradation (Rt = 6.429 min)	Oxcarbazepine formulation (Tablet) (Rt = 6.323 min)

Figure 3: Representative Cchromatograms of Oxcarbazepine observed during forced degradation studies

CONCLUSIONS:

The method proposed for the determination of Oxcarbazepine tablets is sensitive, simple and economical. There is no interference of excipients. Oxcarbazepine is very much sensitive towards acidic and alkaline conditions and the suggested method is precise and accurate.

ACKNOWLEDGEMENT:

The authors are grateful to Macleods Pharmaceuticals Ltd, India for providing the gift samples of Oxcarbazepine. The authors have no conflict of interest.

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Received on 16.12.2018 Modified on 18.01.2019

Research J. Pharm. and Tech 2019; 12(2):723-728.

DOI: 10.5958/0974-360X.2019.00128.8