Int J Pharm Pharm Sci, Vol 9, Issue 10, 224-229Original Article


DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR PHENYTOIN SODIUM AND PHENOBARBITONE IN BULK AND PHARMACEUTICAL DOSAGE FORM

RADHIKA SHAH1*, RAGIN SHAH2

1School of Pharmacy, R. K. University, Bhavnagar Highway, Kasturbadham, Rajkot 360020, Gujarat, India, 2Arihant School of Pharmacy and Bioresearch Institute, Adalaj, Gandhinagar, Gujarat, India
Email: radhika.shah75@gmail.com

Received: 12May 2017 Revised and Accepted: 31 Aug 2017

ABSTRACT

Objective: To develop an accurate, simple, rapid, precise, and linear RP-HPLC method for the simultaneous estimation of phenytoin sodium and phenobarbitone in tablet dosage form and validated as per ICH guidelines.

Methods: The method used was a reverse phase HPLC (RP-HPLC) method using Hypersil BDS C18, (250×4.6 mm, 5 μm) column, mobile phase comprising ofmethanol: phosphate buffer (pH 5.0) (50:50), flow rate of 1.0 ml/min and a detection wavelength of 215 nm using a UV detector.

Results: The retention time for phenytoin sodium and phenobarbitone was found to be 3.97 min and 6.90 min, respectively. The linearity of developed methodwas achieved in the range of 10-30 μg/ml for phenytoin sodium and 3-9 μg/ml for phenobarbitone. The detection (LOD) and quantitation (LOQ) limits were 1.44 and 4.36 µg/ml for phenytoin sodium and 0.40 and 1.35µg/mlfor phenobarbitonerespectively.

Conclusion: A simple, accurate, precise, linear and rapid RP-HPLC method was developed for simultaneous quantitative estimation ofphenytoin sodium and phenobarbitone in bulk and pharmaceutical formulation. The method was validated as per ICH guidelines. Hence, the method holds good for the routine analysis of phenytoin sodium and phenobarbitonein various pharmaceutical industries as well as in academics.

Keywords: Phenytoin Sodium, Phenobarbitone, RP-HPLC, Method Development, Validation

© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open-access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2017v9i10.19917

INTRODUCTION

Phenytoin sodium(fig. 1) is5,5-diphenylimidazolidine-2,4-dione sodium salt. Phenytoin sodiumbelongs to the category of drugs referred to as anticonvulsant and anti-epileptic. Phenytoin is one of the most commonly used antiepileptic medications in clinical practice for generalized seizures.It is used to prevent and control seizures. It works by reducing the spread of seizure activity in the brain.Phenytoin acts on sodium channels on the neuronal cell membrane, limiting the spread of seizure activity and reducing seizure propagation. By promoting sodium efflux from neurons, phenytoin tends to stabilize the threshold against hyperexcitability caused by excessive stimulation or environmental changes capable of reducing membrane sodium gradient. This includes the reduction of post-tetanic potentiation at synapses. Loss of post-tetanic potentiation prevents cortical seizure foci from detonating adjacent cortical areas [1-4].

Fig.1: Chemical structure of phenytoin sodium [1]

Phenobarbitone (fig. 2) is5-ethyl-5-phenyl-2,4,6(1H,3H,5H)-pyrimidinetrione. Phenobarbitone belongs to a class of drugs known as barbiturate anticonvulsants.It works by controlling the abnormal electrical activity in the brain that occurs during a seizure. Phenobarbitone acts on GABA receptors, increasing synaptic inhibition. This has the effect of elevating seizure threshold and reducing the spread of seizure activity from a seizure focus. Phenobarbital may also inhibit calcium channels, resulting in a decrease in excitatory transmitter release. The sedative-hypnotic effects of phenobarbitone are likely the result of its effect on the polysynaptic midbrain reticular formation, which controls CNS arousal[5-7].

Fig.2: Chemical structure of phenobarbitone [5]

Phenytoin and phenobarbitone both depress the motor cortex, raise the seizure threshold and reduce the spread of seizure. Phenytoin stabilises neuronal membrane, inhibiting movement of sodium and calcium ions during the nerve impulse. Phenobarbitone aids GABA mediated inhibition of nerve cells[8, 9].

A detailed literature survey revealed there are various RP-HPLC methodshave been developed for the determination phenytoin sodium and phenobarbitone in individual and in combination with other drugs[10-20]. However, till date there is no RP-HPLC method has been reported for simultaneous estimation of phenytoin sodium and phenobarbitone in combined dosage form. Hence, the objective of this study was to develop a simple, specific, accurate, precise and sensitive RP-HPLC assay for the determination of phenytoin sodium and phenobarbitone in combined pharmaceutical tablet dosage form. This method was validated in accordance with ICH guidelines and published literature for method development and validation [22-24].

MATERIALS AND METHODS

Pharmaceutical grade phenytoin sodium and phenobarbitone were procured from Intas Pharmaceuticals Ltd., Ahmedabad. The marketed formulation Epilan C contains phenytoin sodium 100 mg and phenobarbitone30 mgwas purchased from the local market. Methanol, orthophosphoric acid, acetonitrile and HPLC grade water were obtained from Merck. All solvents used in this work are HPLC grade. RP-HPLC Shimadzu (LC 20ATVP) model with Spin chrome (LC SOLUTIONS) software was employed in this method. Analytical column used for the separation of analytes isHypersil BDS C18(250 X 4.6 mm, 5 µm) was used for separation of analytes.

Methods

Selection of wavelength

Standard solutions of phenytoin sodium and phenobarbitone were prepared at aconcentration of 10 μg/ml and scanned by UV/Visible spectrophotometer at the range of 200-400 nm.Combined UV spectrums of phenytoin sodium and phenobarbitoneare depicted in fig. 3. The isosbestic point selected for simultaneous estimation was 215 nm (fig. 3).

Fig.3: UV overlaps spectrum of phenytoin sodium and phenobarbitone

Chromatographic conditions

The developed method used a reverse phaseHypersil BDS C18 (250 X 4.6 mm, 5 µm) column, a mobile phase of methanol: phosphate buffer pH 5 adjusted with 0.1 M NaOH (50:50), flow rate of 1.0 ml/min and a detection wavelength of 215 nm using a UV detector.

Preparation of phosphate buffer (0.05M KH2PO4)

Accurately weighed 6.8 g of potassium dihydrogen phosphate(KH2PO4) taken and dissolved in 800 ml of distilled water. Solution pH was found to be 4.7 which was adjusted to pH 5.0 with 0.1 M NaOH. Final volume was made up to 1000 ml with distilled water.

Preparation of mobile phase

A mixture of 50 volumes of HPLC grade methanol and 50 volumes of phosphate buffer was prepared. The mobile phase was sonicated for 10 min to remove gasses.

Preparation of standard solutions

A standard stock solution of phenytoin sodium was prepared by dissolving 20 mg of phenytoin sodium insufficient mobile phase. The solution was then filtered and sonicated for 5 min and diluted to 100 ml with mobile phase. From this solution, 1 ml taken and diluted up to 10 ml with amobile phase containing 20 µg/ml.This is treated as 100% of astandard stock solution of phenytoin sodium. A standard stock solution of phenobarbitone was prepared by dissolving 6 mg of phenobarbitone insufficient mobile phase. The solution was then filtered and sonicated for 5 min and diluted to 100 ml with mobile phase. From this solution, 1 ml taken and diluted up to 10 ml with amobile phase containing6 µg/ml. This is treated as 100% of astandard stock solution of phenobarbitone.

Sample preparation

Twenty tablets were weighed and finely powdered. The powder equivalent to 200 mg phenytoin sodium and 60 mg phenobarbitone was accurately weighed. This powder was transferred to the volumetric flask of 1000 ml capacity and dissolved in 500 ml of mobile phase. The flask was sonicated for 10 minand volume was made up to the mark with the mobile phase. The above solution was filtered through whatmann filter paper (0.45μ).From this solution, 1 ml taken and diluted up to 10 ml with amobile phase containing 20 µg/ml of phenytoin sodium and 6 μg/ml of phenobarbitone.This solution was used for the estimation ofphenytoin sodium and phenobarbitone.

RESULTS AND DISCUSSION

Method development

Different chromatographic conditions were tried for better separation and resolution. Hypersil BDS C18 (250 X 4.6 mm, 5 µm) column was found satisfactory. Peak purity of phenytoin sodium and phenobarbitone was checked using UV detector and 215 nm was considered satisfactory for detecting both the drugs with adequate sensitivity. A number of solvents in the different ratio over a wide range of pH were tried, but either peak shape was broad or resolution was not good. Repeated trials to obtain good, sharp peak with an efficient resolution between two peaks ofphenytoin sodium and phenobarbitone done on a C18 column in isocratic HPLC. The runtime was 9 min in isocratic trial with mobile phase consisting of methanol: phosphate buffer (pH5.0) (50:50) and C18-Hypersil BDS (250×4.6 mm, 5 μm) column, flow rate 1.0 ml/min and detection wavelength 215 nm gave the satisfactory results in terms of retention time, resolution, symmetry and sensitivity. A typical RP-HPLC chromatogram for simultaneous determination ofphenytoin sodium and phenobarbitone from standard preparation was obtained as shown in (fig. 4).

Method validation

The developed RP-HPLC method was validated for parameters like system suitability, linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ) and robustness according to ICH guidelines.

System suitability

Standard solutions were prepared as per above-mentioned method and injected into the chromatographic system. The system suitability parameters like theoretical plates, resolution and asymmetric factor were evaluated. The system suitability parameters were tabulated in table 1. All the parameters were found to be within the limits.

Fig.4: Typical chromatogram of standard solution

Table 1: Results of system suitability studies

Parameters Acceptance limits Phenytoin sodium Phenobarbitone
Retention time - 3.973 6.900
Theoretical plates NLT 2000 8092 8274
Tailing factor (T) NMT 2 1.31 1.32
Resolution NLT 2 12.13

#NLT: Not less than. # NMT: Not more than

Linearity

The linearity of the test solutions for the assay method was prepared fromphenytoin sodium and phenobarbitone standard stock solution at five concentration levels from 50% to 150% of standard concentration. The peak area versus concentration data was treated by least-squares linear regression analysis (fig. 5 and 6). The results have shown an excellent correlation between peak areas and concentration within the concentration range of 10–30 μg/ml for phenytoin sodium, 3–9 μg/ml for phenobarbitone (table 2). The correlation coefficients were found to be 0.997 forphenytoin sodium and 0.998 for phenobarbitone, which meet the method validation acceptance criteria and hence the method was said to be linear for both the drugs.

Fig.5: Linearity chart for phenytoin sodium

Fig.6: Linearity chart for phenobarbitone

Table 2: Linearity data for phenytoin sodium and phenobarbitone

% level Phenytoin sodium concentration (µg/ml) Phenytoin sodium peak area Phenobarbitone concentration(µg/ml) Phenobarbitone peak area
50 10 2130.9 3 790.956
75 15 3432.565 4.5 1266.038
100 20 4492.878 6 1632.345
125 25 5610.012 7.5 2066.555
150 30 6595.911 9 2429.197
Correlation coefficient 0.997 0.998
Slope 222.1 271.8

Precision

The precision of the method was verified by precision method studies. It is done by two methods-repeatability and reproducibility (Intraday and inter-day Precision).

Repeatability

The sample solution was prepared at working concentration as per the test method and analysis was performed. The sample solutions of phenytoin sodium and phenobarbitone injected 6 times into the column. The results of repeatability are as tabulated in table 3. The average was taken, and percent relative standard deviation (%RSD) calculated and reported. % RSD values were found within the limits, and the method was found to be precise.

Reproducibility (Intraday and inter-day precision)

Intraday and inter-day precision were carried out using three different concentrations for phenytoin sodium (10, 20 and 30 μg/ml) and phenobarbitone (3, 6, 9 μg/ml) injected thrice into the column. The results of intraday and inter-day precision were tabulated in table 4. The average was taken, and % RSD was calculated and reported. % RSD values were within the limits, and the method was found to be precise.

Table 3: Repeatability data for phenytoin sodium and phenobarbitone

S. No. Phenytoin sodium 20 µg/mln = 6 peak area Phenobarbitone 6 µg/mln = 6peak area
1 4426.225 1631.509
2 4403.994 1623.362
3 4381.889 1615.218
4 4408.186 1624.932
5 4421.056 1629.75
6 4407.577 1616.7
Mean 4408.1545 1623.579
SD 15.4952407 6.634317
%RSD 0.351513104 0.408623

# n: number of injections =6, # SD= standard deviation, # %RSD: % relative standard deviation

Table 4: Intraday and inter-day precision data for phenytoin sodium and phenobarbitone

Drug Conc.(µg/ml) Intra-day precision Intra-day precision
mean±SD(n=3) %RSD mean±SD(n=3) %RSD
Phenytoin Sodium 10 2247.89±9.39 0.418 2248.67±21.57 1.35
20 4433.55±16.63 0.375 4420.20±35.88 1.81
30 6699.39±25.42 0.379 6678.78±71.05 1.06
Phenobarbitone 3 822.29±6.13 0.746 825.80±8.16 1.58
6 1628±4.33 0.266 1624.60±20.92 1.28
9 2462.86±18.39 0.747 2462.64±24.76 1.19

# n: number of injections =3, # SD= standard deviation, # %RSD: % relative standard deviation

Accuracy

The accuracy of the method was determined by recovery studies by the determination of % mean recovery of both the drugs at three different levels (80 %, 100 % and 120%). At each level, three determinations were performed. The percentage recovery and mean percentage recovery were calculated for the drug was shown in table 5. The observed data were within the required range, which indicates good recovery values and hence the accuracy of the method developed.

Table 5: Results of accuracy

Level (%) Phenytoin sodium% recovery % mean Phenobarbitone% recovery % mean
80 99.12912961 99.27810819 98.69885722 98.88293264
80 100.0278808 99.58154956
80 98.67731416 98.36839113
100 100.5330846 100.8659537 100.8960369 100.9071524
100 101.7390348 101.1372592
100 100.3257419 100.6881612
120 100.7030532 100.3468355 100.9922779 100.1039046
120 99.59680407 99.88472671
120 100.7406491 99.43470906

Robustness

To determine the robustness of the developed method, experimental conditions were deliberately altered, and the system suitability parameters were evaluated. The solutions prepared as per the test method and injected at different variable conditions like flow rate (0.8, 1.2 ml/min.), mobile phase ratio of methanol: phosphate buffer (52:48, 48:52)and pH(4.8, 5.2).At the flow rate of 1.0 ml/min shows, a sharp peak with good resolution and rest of the flow rates were found to be not satisfactory. The method passed all system suitability parameters indicating that the method was robust.

Table 6: Robustness study for phenytoin sodium and phenobarbitone

Parameter Method condition Phenytoin sodium Phenobarbitone
Plate count Tailing Plate count Tailing
Mobile phase (Methanol: phosphate buffer) 52:48 8267 1.38 8012 1.34
48:52 8395 1.35 8145 1.29
pH 4.8 8254 1.34 8426 1.28
5.2 8412 1.31 8247 1.34
Flow Rate 0.8 8342 1.35 8371 1.31
1.2 8246 1.30 8367 1.32

Detection limit and quantification limit

Limit of detection (LOD) which represents the concentration of the analyte at S/N ratio of 3.3 and limit of quantification (LOQ) at which S/N was 10 were determined experimentally for the proposed methods and results were given in table 7. Hence, the detection limits and quantitation limits of the drugs were given S/N ratios of 3.3 and 10 respectively.

Table 7: LOD and LOQ

Drug LOD(μg/ml) LOQ(μg/ml)
Phenytoin sodium 1.44 4.36
Phenobarbitone 0.40 1.22

DISCUSSION

The developed method can be used for routine analysis because the linearity found is nearing 1 that is 0.997 and 0.998 for phenytoin sodium and phenobarbitone respectively which shows the good regression for linearity. Maximum recovery is obtained by this developed method and the mean percentage recovery for each component is nearing 98% to 100%. Therefore this method can be used for the routine analysis and one most important reason is that the developed method does not involve the use of expensive reagents. The method we developed involves chemicals like methanol and buffer, which are easily available. There are various RP-HPLC methods have been reported for the determination phenytoin sodium and phenobarbitone in individual and in combination with other drugs[10-20].However, till date there was no RP-HPLC method has been reported for simultaneous estimation of phenytoin sodium and phenobarbitone in combined dosage form. So this method is first of its kind.

CONCLUSION

The proposed RP-HPLC method was found tobe simple, specific, accurate, precise, robust, rapid and economical. This method gives good resolution between all the two compounds with a short analysis time. The proposed RP-HPLC method can be useful for routine analysis of phenytoin sodium and phenobarbitone in the tablet dosage form.

ACKNOWLEDGEMENT

The authors would like to thank Arihant School of Pharmacy and Bioresearch Institute, Adalaj, Gandhinagar, Gujarat for providing necessary facilities. The authors are also grateful to Intas Pharmaceuticals, Ahmedabad for providing gift samples of phenytoin sodium and phenobarbitione.

AUTHORS' CONTRIBUTION

Principal author: Planned the experimental setup, performed lab work, interpreted data, and wrote the manuscript.

Co-author contribution: Supervised the development of work and helped in the evaluation of the manuscript.

Both authors read and approved the final manuscript.

CONFLICT OF INTERESTS

Authors have no conflict of interest

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