DEVELOPMENT AND VALIDATION OF STABILITY INDICATING HPTLC METHOD FOR DETERMINATION OF PHENOBARBITONE AS BULK DRUG AND IN PHARMACEUTICAL FORMULATION

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING HPTLC METHOD FOR DETERMINATION OF PHENOBARBITONE AS BULK DRUG AND IN PHARMACEUTICAL FORMULATION

P.R. Mhatre*, S.H. Gatkal, V.V. Chopade and P.D. Chaudhari

Department of Quality Assuranse Techniques, Modern College of Pharmacy Nigdi, Pune-411044, Maharashtra, India

ABSTRACT

A sensitive, selective, precise and stability indicating high performance thin layer chromatographic method of analysis of Phenobarbitone both as a bulk drug and in formulations was developed and validated in pharmaceutical dosage form. The method employed TLC aluminium plates precoated with silica gel 60F-254 as the stationary phase. The solvent system consisted of dichloromethane: ethyl acetate: glacial acetic acid (9.5:0.5:0.1 v/v/v/v). This system was found to give compact spots for Phenobarbitone (Rf value of 0.70). Phenobarbitone was subjected to acid and alkali hydrolysis, oxidation, photodegradation and dry heat treatment also the degraded products were well separated from the pure drug. Densitometric analysis of Phenobarbitone was carried out in the absorbance mode at 244 nm. The linear regression data for the calibration plots showed good linear relationship with r²=/0.998/ in the concentration range of 200-/600 ng/band. The method was validated for precision, accuracy, ruggedness and recovery. The limits of detection and quantitation were 5.82 and 14.60 ng per spot, respectively. The drug undergoes degradation under acidic, alkaline conditions, oxidation and dry heat treatment. Peaks of degraded product were resolved from the standard drug with significantly different Rf values. This indicates that the drug is susceptible to acid hydrolysis/alkaline hydrolysis, oxidation, dry heat degradation. Statistical analysis proves that the method is reproducible and selective for the estimation of the said drug. As the method could effectively separate the drug from its degradation products, it can be employed as a stability indicating one.

Phenobarbitone, HPTLC, Validation, Stability-indicating, Degradation

INTRODUCTION: Barbiturates have been popular hypnotics and sedatives of the last century upto 1960s, but are not used now to promote sleep or to calm patients. They are prototype of CNS depressants. Barbiturates are substituted derivative of barbituric acid ¹.

Chemically phenobarbitone is 5-ethyl-5-phenylbarbituric acid or 5-ethyl-5-phenyl-2,4, 6 (1H, 3H, 5H)   pyrimidinetrione  or  phenobarbituric acid (figure 1).

FIGURE 1: STRUCTURE OF PHENOBARBITONE

Changes in chemic structure of barbituric acid increases lipid solubility speed up the onset of action, and increses the hypnotic potency of the compound ².

Phenobarbital is called a barbiturate that acts by slowing down the activity of the brain. It  has sedative and hypnotic properties, which will help patients to relax before surgery or help to sleep. It also reduces or controls seizures or convulsions, except for absence (petit mal) seizures. Generic Phenobarbital oral elixir is available containing a high amount of alcohol which can increase possible unpleasant   Effects ^{3, 4}. Literature survey further revealed that the analytical method available for determination of drug phenobarbitone by modified conductometric analysis^.5.

MATERIALS AND METHODS: Phenobarbitone sample was obtained from Abbott Healthcare, Mumbai. The solvent used methanol (AR grade), dichloromethane (AR grade), ethyl acetate (AR grade), glacial acetic acid (AR grade), NaOH (AR grade), HCl (AR grade) and H₂O₂ (AR grade). These chemicals were purchased from Merck Chemicals (Mumbai, India).

Equipment: Camag HPTLC system consisting Linomat 5 applicator, camag TLC scanner 3 and WinCATS software V-1.4.4 was used for chromatographic separation. Spotting of samples was done by using Hamilton microliter syringe.

Preparation of Standard stock solution: Standard stock solution of Phenobarbitone was prepared by dissolving 10 mg of drug in 100 ml   of methanol to get concentration 100µg/ml. 2 ml standard stock solution of Phenobarbitone was then diluted in 10 ml methanol to get working standard solution 20 µg/ml (100 ng/5µl). From the stock solution 2, 4, 6, 8 and 10µl were applied on TLC plate, at a distance 10mm from both x-axis and y-axis.

Validation of Analytical Method ^6-8:

Linearity: A stock solution of Phenobarbitone (100μg/ml) was prepared in methanol. Different volumes of stock solution as 2, 3, 4, 5 and 6μl were spotted on TLC plate to obtain concentration of 200, 300, 400, 500 and 600ng/band of Phenobarbitone, respectively. The data of peak area versus drug concentration were treated by linear least-square regression analysis. The response for the drug was found to be linear in the concentration range 200–600ng/band. The calibration curve is shown in fig. 2.

FIG. 2: CALIBRATION CURVE FOR PHENOBARBITONE

Regression Equation: y = 28.13x + 1288

Coefficient of correlation: 0.998.

Precision: The precision of the method was demonstrated by intra-day and inter-day variation studies. In the intraday studies, 3 different concentrations 200, 400 and 600ng/band of standard stock solution were spotted in triplicate and were analyzed. The percentage RSD was calculated.

TABLE 1: INTRA-DAY PRECISION STUDIES FOR PHENOBARBITONE

TABLE 2  : INTER-DAY PRECISION STUDIES FOR PHENOBARBITONE

 Accuracy: To check the accuracy of the method, recovery studies were carried out by over spotting standard drug solution to pre-analyzed sample solution at three different levels 80, 100 and 120 %. Basic concentration of sample chosen was 300 ng/band of Phenobarbitone bulk drug to which 240, 300 and 360 ng/band of Phenobarbitone tablet were added by over spotting. The areas were noted after development of plate. The drug concentrations of Phenobarbitone were calculated by using regression equation. The results obtained are shown in Table 3.

TABLE 3: RECOVERY STUDIES OF PHENOBARBITONE

Limit of Detection (LOD): LOD is calculated from the formula: -

3.3 

DL  =

S

Where, σ = the standard deviation of the response for the lowest conc. in the range, S   = the slope of the calibration curve.

LOD = Phenobarbitone:  5.82  ng/band

Limit of Quantification (LOQ): The quantitation limit (QL) may be expressed as:

10 

QL =

S

LOQ = Phenobarbitone:   14.60  ng/band

Range: Phenobarbitone: 200 – 600 ng/band.

Specificity: The densitogram was studied for interference at the Rf of Phenobarbitone. Lack of interfering peaks in the blank at the Rf of the Phenobarbitone drug was taken as indication of the specificity of the method. The spectra of standard and sample at corresponding Rf matched exactly, indicating absence of other interference at that Rf.

Robustness: Robustness of the method was determined by carrying out the analysis under conditions during which mobile phase ratio, dimensions of chamber were altered and the effects on the Rf values and area were noted. In case of mobile phase ratio, the percentage change in Rf was not more than 0.4 % & the percentage change in area was not more than 0.06 %. In case of Chamber change, percentage change in Rf value was not more than 0.01 % & the percentage change in area was not more than 0.06 %. The method was found to be robust since, the monitored parameters were not significantly affected.

TABLE 4 : SUMMARY OF VALIDATION PARAMETERS

Stress degradation Studies ^9-11:

Densitogram of Phenobarbitone: Methanol was used as a solvent for solution preparation. Stationary phase was aluminium HPTLC plate (20×10cm) precoated with silica gel F₂₅₄. dichloromethane : ethyl acetate : glacial acetic acid  (9.5:0.5:0.1 v/v/v/v) was used. Standard stock solution of Phenobarbitone 5µl (300ng/band) was applied on TLC plate. The retention factor of Phenobarbitone was 0.70 ± 0.05. The typical densitogram of working standard solutions is as shown in fig. 3.

FIG. 3: THE TYPICAL DENSITOGRAM OF WORKING STANDARD (300 ng/band)

Acidic hydrolysis: To 6 ml of stock solution, 1 ml of 2.5 N HCl was added. The volume was made up to 10 ml with methanol (0.3µg/5µl). This mixture then was kept for 3 hours for 30ᵒC. 5μl of resultant solution (300ng/band) was then applied on TLC plate along with respective blank in adjacent track and the densitogram were developed. The results obtained are shown in fig. 4.

FIG. 4: DENSITOGRAM OF PHENOBARBITONE (300ng/band, at Rf= 0.70) AFTER ACID HYDROLYSIS DEGRADATION OF PHENOBARBITONE WAS OBSERVED AT RF 0.76 AND DEGRADED PRODUCTS WAS FOUND AT Rf 0.55, 0.63, 0.66.

Alkaline hydrolysis: To 6 ml of stock solution, 1 ml of 3 N NaOH was added. The volume was made up to 10 ml with methanol (0.3µg/5µl). This mixture then was kept for 3hours for 30ᵒC. 5μl of resultant solution (300ng/band) was then applied on TLC plate along with respective blank in adjacent track and the densitogram were developed. The results obtained are shown in fig. 5.

FIG. 5: DENSITOGRAM OF PHENOBARBITONE (300ng/band, at Rf= 0.70) AFTER A HYDROLYSIS   DEGRADATION OF PHENOBARBITONE WAS OBSERVED AT RF 0.64 AND DEGRADED PRODUCTS WAS FOUND AT Rf 0.80.

Oxidation: 6 ml of stock solution, 2 ml of 6 % H₂O₂ was added. The volume was made up to 10 ml with Methanol (0.3µg/5µl). This mixture then was kept for 3 hours at RT. 5μl of resultant solution (300 ng/band) was applied on TLC plate along with respective blank in adjacent track and the densitogram were developed. The results obtained are shown in fig. 6.

FIG. 6: DENSITOGRAM OF PHENOBARBITONE (300ng/band, at Rf= 0.70) AFTER OXIDATIVE  DEGRADATION OF PHENOBARBITONE WAS OBSERVED AT RF 0.70 AND DEGRADED PRODUCTS WAS FOUND AT Rf 0.63,0.72,0.77.0.82.

Degradation under Dry Heat ¹²: Dry heat studies were performed by keeping drug sample in oven (50⁰ C) for a period of 120hours. 10mg of exposed drug was weighed accurately and transferred to a 100 ml of volumetric flask and dissolved in methanol, the volume was made up with methanol to get conc. of 100µg/ml. 6 ml standard stock solution of Phenobarbitone was then diluted in 10 ml methanol to get working standard solution (0.3µg/5µl). 5μl of resultant solution (300ng/band) was then applied on TLC plate along with respective blank in adjacent track and the densitogram were developed. The results obtained are shown in fig. 7.

FIG. 7: DENSITOGRAM OF PHENOBARBITONE (300ng/band, at Rf= 0.70) AFTER DRY HEAT DEGRADATION OF PHENOBARBITONE WAS OBSERVED AT RF 0.68 AND DEGRADED PRODUCT WAS FOUND AT Rf 0.43.

Photo-degradation studies:

Long UV-Degradation at 366 nm: The photochemical stability of the drug was studied by exposing the drug sample to long UV (366nm) light for 48 hour 10 mg after exposure, accurately weighed 10 mg of drug in 100 ml of methanol to get concentration 100µg/ml. 6 ml standard stock solution of Phenobarbitone was then diluted in 10 ml methanol to get working standard solution (0.3µg/5µl). 5μl of resultant solution (300ng/band) was then applied on TLC plate.

Short UV-Degradation at 254 nm: The photochemical stability of the drug was studied by exposing the drug sample to short UV (254nm) light for 48 hour 10 mg after exposure, accurately weighed 10 mg of drug in 100 ml of methanol to get concentration 100µg/ml. 6  ml standard stock solution of phenobarbitone was then diluted in 10 ml methanol to get working standard solution (0.3µg/5µl).  5μl of resultant solution (300ng/band) was then applied on TLC plate .

TABLE 5 : SUMMARY OF STRESS DEGRADATION STUDIES OF BULK DRUG

RESULTS AND DISCUSSION: HPTLC method was validated as per ICH guidelines. The developed method was found to be linear within the range of 200 – 600ng/band with R²=0.998. The accuracy of method was determined at 80, 100, 120% level. The % recoveries were found to be 99.96%, 98.52%, and 98.15% within the limit of 98% to 102%.

The LOD and LOQ were found to be 5.82 ng/band and 14.60 ng/band indicating the sensitivity of the method. The developed method was found to be precise as the % RSD values for intraday and inter-day were found to be less than 2%. The method was also found to be robustness indicated by the % RSD values which are less than 2 %. The summary of validation parameters of proposed HPTLC method is shown in table 4.

The stress degradation studies showed that Pheno-barbitone undergoes degradation in acid, base, oxidation, dry heat, (31.4%, 22.14% 31.91% 34%). Summary of the results of stress degradation studies of phenobarbitone are shown in table 5.

CONCLUSION: Theproposed methods are precise, specific, accurate, robust and stability-indicating ones. Phenobarbitone can be determined in bulk and pharmaceutical formulation and percentage degradation. ICH guidelines were fallowed throughout the study for method validation and stress testing, and the suggested method can be applied for quality control and routine analysis.

ACKNOWLEDGEMENT: Authors are gretful to the Ayushakti Ayurved Pvt. Ltd., Palghar for providing instrumentation and necessary facilities to carry out the research work.

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    How to cite this article:

    Mhatre PR, Gatkal SH, Chopade VV and Chaudhari PD: Development and Validation of Stability indicating HPTLC method for determination of Phenobarbitone as Bulk Drug and in Pharmaceutical Formulation.Int J Pharm Sci Res. 2013; 4(2); 806-812.

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