ABSTRACT:

Objectives: The objective of present work was to develop and standardize UV-spectrophotometric method
for estimation of Phenytoin in marketed formulation.
Materials and method: UV-spectrophotometric method was developed using 1 N NaOH as solvent. The
developed method was standardized in terms of validation parameters such as sample, sensitive, precise,
linear, accurate, robust, reproducible as per ICH Q2(R1) guidelines. For estimation of phenytoin in
marketed formulation this newly developed method was successfully applied.
Results: Phenytoin exhibits λmax at 218 nm and in the Beer’s range 2 to 10 µg/ml Beer’s law was obeyed.
The limit of detection was found to be 1.709 µg/ml and limit of quantification was found to be 5.081 µg/ml.
Recovery of Phenytoin in marketed formulation was obtained in range of 102-115%. All the precision and
repeatability results were observed within the accurate range less than 2%.

Conclusion: The method was found to be simple, accurate, environment friendly, reproducible and
marketed formulation of Phenytoin can be estimated.

Key words: Phenytoin, Beer’s law, 1 N NaOH, UV-spectrophotometer, Validation.

INTRODUCTION

Phenytoin or 5,5-diphenylimidazolidine-2,4-dione is an anticonvulsant drug which can be useful in the

treatment of epilepsy. [1][2][5] Phenytoin is one of the potential candidates for epilepsy treatment, but it has
low bioavailability due to lower aqueous solubility. [1] Phenytoin is a high yielding chemically synthesized
anticonvulsant drug, which is prescribed to be taken orally (Phenytoin or Phenytoin sodium) or
intravenously (Phenytoin sodium). It is widely used to control tonic-clonic seizures [1][2][3], partial seizures
[1][2][3] and prophylactic seizures [1][2][3] during neurosurgery or post traumatic injury to the head. [1][2] Also

used to contrary digitalis induced arrhythmia. [2][3] Phenytoin is similarly prescribed in a 10% ointment
preparation to endorse curing of ulcers in patients with diabetes. [2] It is an oldest non-sedative antiepilept ic
drug. [3] Phenytoin is composed of five membered hydantoin with two phenyl groups at the fifth position.
Phenytoin has three-member molecule associated with urea and although has pharmacological tool. [4]

Fig. 1: Structure of Phenytoin

Many formulations of Phenytoin are available in the market for example Dilantin, Epsotoin-300, Pheuton-
100, Elion, PhenyCare 100mg, Eptocan-100, Leotoin-100 etc.

Many researchers have been reported few UV spectroscopic methods for estimation of Phenytoin in
marketed formulations. But those methods which have been reported have their own limitations such as use
of costly and hazardous solvents. So, aim of this work was to develop and standardize UV spectroscopic
method for estimation of Phenytoin in marketed formulation.

MATERIALS AND METHODS

Reagents and chemicals:

Benzil, Urea, NaOH, Ethanol, distilled water, conc. HCl, Chloroform, n-Hexane, Ethyl acetate, methanol,
Iodine.

Procedure for synthesis of Phenytoin:

5.25 g (0.025M) of benzil, 1 g (0.025M) of NaOH in 100ml of water, 1.5 g (0.025M) urea in 50 ml of ethanol
were taken in 250 ml of round bottom flask. The round bottom flask was equipped with reflux condenser.
Mixture was heated under reflux on a steam bath for 3 hours. This mixture was allowed to cool at room
temperature and poured the reaction mixture in 100 ml of cold water. The solution was filtered and acidified
the filtrate with concentrated HCl. White precipitates (M.P. 293-2980C) formed, filtered and washed with
water, recrystallized with ethanol. [4]
Thin layer chromatography of synthesized Phenytoin:

In thin layer chromatography, stationary phase is thin adsorbent material as silica gel which was coated on
to a glass slide. The sample was spotted onto one end of the TLC plate and placed vertically into a closed
chamber containing mobile phase. In 1st trial, ethyl acetate : n-hexane (8:2 ratio), in 2nd trial, ethyl acetate :
n-hexane (2:8 ratio) and in 3rd trial, ethyl acetate : methanol (8:2 ratio) was taken as mobile phase. The
mobile phase travels up the plate by capillary forces and sample components migrate varying distances based
on their affinities for the stationary and mobile phases. When the solvent reaches the top of the plate then
the plate was removed from the iodine chamber and dried. The separated components appear as spots on the
plate.

Melting point:
Melting point of synthesized Phenytoin was checked by filling Phenytoin sample in capillary and seal the
other end of the capillary by holding it on flame. Tied this capillary to the thermometer and placed in Thiel’s
tube which containing liquid paraffin and after uniform heating to Thiel’s tube the sample starts to melt and
this was considered as melting point of sample.
Test for urea:

To check whether the urea present in the synthesized phenytoin or not, because urea which is used for
synthesis of phenytoin may cause toxicity. Therefore the test for urea is needed in our work.

• For that, take 1 g of synthesized phenytoin sample and dissolve in 10 ml of water and then filtered.

• The filtrate was used to perform the test for urea

Test Observation Inference

2ml of above filtrate in a test tube + No brisk effervescence of nitrogen Urea absent
2-3 drops of sodium hypobromite
solution. Mix well.
Infrared spectroscopy (IR): The IR of the synthesized drug was found out using Shimadzu IR affinity-1
instrument. The sample was prepared by using the KBr pellete technique. Dried KBr powder was used in a
ratio of 100 times the weight of pure phenytoin. The phenytoin and KBr mixture were pressed in a die at
10000 to 15000 pounds. The disk was then held in the path of the IR beam of the instrument for
spectroscopic examination. The resulting spectra showed bands at 3450 cm-1 and 1640 cm-1 due to absorbed
moisture. Carbon dioxide and water absorption were removed manually. The IR spectrum was compared
with the reference spectrum of phenytoin[1].

Selection of wavelength:

Different concentrations of Phenytoin were prepared in 1 N NaOH. Phenytoin 10 µg/ml of working standard
solution was scanned in between 200-400 nm and exhibited maximum absorption at 218 nm in UV
spectrophotometer.

Preparation of stock solution:

0.1g of synthesized Phenytoin was weighed accurately, dissolved in 100 ml of 1 N NaOH by sonication.
Transferred 1 ml of the above solution into 100 ml volumetric flask containing 100 ml of 1 N NaOH. This
was considered as standard stock solution having concentration 10 µg/ml. This standard stock solution was
used for making further dilutions.

Preparation of calibration curve:

Serial dilutions were prepared in range of concentration 2-10 µg/ml from stock solution. The 3 sets of
solutions were analyzed and their absorbance were measured at 218 nm. Linearity curve was plotted as
concentration on x-axis and absorbance on y-axis and linearity regression was calculated.

Method development and validation:

1N NaOH was selected as solvent because Phenytoin was soluble in it. The proposed method was validated
according to International Conference of Harmonization (ICH) has mentioned guidelines i.e. Q2(R1).
According to the ICH guidelines developed method was validated in order to prove the suitability of method
using method parameters for the validation of analytical procedures.

Specificity and selectivity:

The method was found to be selective because Phenytoin was showed maximum absorbance at 218 nm and
solvent i.e. 1N NaOH did not show its absorbance at 218 nm, hence this method was found to be specific.

Linearity:
Linearity was examined in a range of 2-10 µg/ml. Accurately weighed 100mg of Phenytoin and was
transferred into a clean and dried 100 ml of volumetric flask and make up the volume to the mark using 1N
NaOH. From the above stock solution pipetted out 1ml and transferred into a 100 ml volumetric flask and
volume was made using solvent 1N NaOH. From this solution further dilutions are made to examine the
linearity.
LOD and LOQ:
Limit of detection is the concentration at which analyte in the test sample was detected. Limit of
quantification is the concentration at which analyte in the test sample is quantified. Following formulae are
used to calculate LOD and LOQ
LOD= 3.3× standard deviation of regression/slope
LOQ= 10× standard deviation of regression/slope

Precision:
Precision was determined by preparing 3 replicates of solution 2 µg/ml, 6 µg/ml, 10 µg/ml of Phenytoin and
its absorbance was measured at 218 nm and % RSD (Relative Standard Deviation) was calculated.
Method precision was determined by performing two tests,
1) Intraday precision
2) Interday precision
For intraday precision three replicates of solution containing concentration 2 µg/ml, 6 µg /ml, 10 µg/ml of
Phenytoin was analyzed and at different time intervals % RSD was calculated.

For interday precision three replicates of solution containing concentration 2 µg/ml, 6 µg /ml, 10 µg/ml of
Phenytoin was analyzed and at different time intervals % RSD was calculated.

Ruggedness:

Ruggedness was performed by using same proposed method with different analyst to check the
reproducibility.

Robustness:
Phenytoin was determined by performing recovery experiment. % mean recovery of sample was determined
by preparing different levels of the sample solutions a 50%, 100%, 150%.
Weighed accurately 100 mg of Phenytoin was transferred into clean 100 ml volumetric flask and volume is
made upto the mark by using 1 N NaOH as Phenytoin is soluble in it. Three replicates of concentration was
made at each level and recovery study was performed.

Analysis of marketed formulation:

For the determination of phenytoin on marketed formulation the validated method was applied. To calculate
the average weight of tablets, weighed accurately 20 tablets then triturated. The amount of drug in sample
was found to be in good range with the label claim of formulation. Percent assay was found to be 95-101%.

Result and Discussion:

Thin Layer chromatography:

To check the purity of compound TLC was performed and its results are shown in fig. 2(a,b)
Trail no.1 Fig. 2(a)

separation of compound by using ethylacetate:n-hexane (2:8) is not clear as shown in fig. 2(a)

Trail.no.2 Fig. 2(b)

Separation of compound by using ethylacetate:Methanol (8:2) is proper as shown in fig. 2(b)

Melting point:

The melting point of phenytoin was done to check purity of compound and was found to be 297°C. So the
synthesized compound was supposed to be a pure.

3. TEST FOR UREA

• Synthesized phenytoin does not show positive test for urea.

 • Therefore the synthesized phenytoin does not contain urea thus, it is safe for the usage.

IR spectroscopy of synthesized phenytoin:

Fig. 7: IR spectrum of synthesized phenytoin

Method Development:

UV-1800 model was used for development of UV-Spectrophotometric method using 1N NaOH as solvent.
Maximum absorbance of phenytoin was found at 218 nm. Details of method developed were presented in
Table:1.

Table1: Developed method parameters

 Sl. No. Parameters Specifications

1 Method Spectrometric

2 Instrument UV

3 Model 1800

4 Make Shimadzu

5 Software UV Probe

6 Synthesized drug Phenytoin

7 Solvent 1N NaOH

8 Scanning wavelength range 400-200nm

9 λmax 218nm

Method Validation:

Developed method was validated in terms of validation parameters such as Specificity, selectivity, linear
range, precision, robustness, ruggedness and reproducibility as per ICH guidelines.

Specificity and Selectivity:

Phenytoin was showed maximum absorbance at 218 nm and 1N NaOH solvent didn’t show absorbance at
218 nm. Hence the method was found to be specific and selective. (Fig:3 and 4)

Fig. 3: UV Spectrum of Solvent

Fig. 4: UV Spectrum of phenytoin

Linearity: linearity was found in range of 2-10µg/ml. The linearity graph is showed in fig. 5. The linearity
and range is showed in Table 2 and the calibration curve is given in fig. 6.

Fig. 5: Linearity graph of phenytoin

Table 2: linearity and range data of phenytoin

Sl. No. Concentration (µg/ml) Absorbance

1 2 0.121
2 4 0.241
3 6 0.361
4 8 0.485
5 10 0.609

r2 0.999
Slope 0.061
LOD 1.709
LOQ 5.081

Fig 6: linearity curve of phenytoin

Precision:
System Precision: As mentioned in the method in order to determine system precision
Three replicates of phenytoin solution i.e. 2 µg/ml, 6 µg/ml,10 µg/ml was prepared in order to determine
system precision as per mentioned in the method and absorbance of each solution was measured at 218nm.
The % RSD was calculated and it was found to be less than 2% (Table 3)
Table 3: System Precision data of phenytoin

Concentration Absorbance* Standard Deviation % Relative Standard

(µg/ml) Deviation
2 0.121 0.001 0.83
6 0.241 0.001 0.41
10 0.361 0.001 0.28
*= average absorbance of six replicates
Intraday precision:

For intraday precision three replicates of phenytoin solution containing concentration 2µg/ml,
6µg/ml,10µg/ml was prepared and analyzed and % RSD was calculated on the same day at different time
intervals and % RSD was found to be less than 2% (Table 4)

Table 4: Intraday precision data of phenytoin

Concentration µg/ml Absorption Standard deviation % Relative standard deviation

Abs 1hr 0.123 0.002 1.63

2 Abs 4hr 0.123 0.0015 1.24

 Abs 8hr 0.122 0.0015 1.25

6 Abs 1hr 0.362 0.0015 0.42

Abs 4hr 0.362 0.002 0.55

Abs 8hr 0362 0.0015 0.42

10 Abs 1hr 0.609 0.0015 0.25

Abs 4hr 0.610 0.002 0.33

Abs 8hr 0.610 0.0021 0.34

*= average absorbance of three replicates

Interday precision: Precision for interday three replicates of phenytoin solution containing
concentration2µg/ml, 6µg/ml, 10µg/ml was prepared and analyzed and then %RSD was calculated on three
consecutive days and %RSD was found to be lesser than 2%(Table-5)

Table 5: Interday precision data of phenytoin

Concentration (µg/ml) Standard deviation % Relative standard

Absorbance deviation

2 Day 1 0.122 0.0021 1.7

Day 2 0.127 0.0025 1.97
Day 3 0.124 0.001 0.81
6 Day 1 0.363 0.002 0.55
Day 2 0.367 0.0015 0.42
Day 3 0.365 0.003 0.82
10 Day 1 0.609 0.001 0.16
Day 2 0.610 0.0015 0.25
Day 3 0.609 0.0015 0.25
*= average absorbance of three replicate

RUGGEDNESS:

Ruggedness was determined by performing the same proposed method by different analyst to check
reproducibility. %RSD was obtained less than 2% which indicates that the method developed was rugged.
(Table 6)
*= average absorbance of three replicates
Concentration(µg/ml) Absorbance* Standard % Relative
deviation Standard deviation
Analyst 1 0.121 0.0006 0.48
2
Analyst 2 0.122 0.001 0.82

Analyst 1 0.361 0.001 0.28

6
Analyst 2 0.362 0.0015 0.42
Analyst 1 0.607 0.002 0.33
10 Analyst 2 0.608 0.002 0.33

Robustness: 1N NaOH used as solvent because phenytoin was soluble in it. Maximum absorbance was
found at 218nm. Robustness is performed with the sonication for 5min and by changing the wavelength
217nm, 218nm, 219nm.The % RSD was obtained to be less than 2% (Table 7)

Table 7: Robustness data of phenytoin

Concentration Absorbance Standard deviation %Relative

(µg/ml) deviation

Change in 0.122
wavelengh 217 0.001 0.82
2
Sonication 5 218 0.122 0.001 0.82
min 219 0.122 0.001 0.82
Change in 217 0.361 0.001 0.28
wavelength
6 Sonication in 5 218 0.361 0.001 0.28
min
219 0.361 0.001 0.28

10 Change in 217 0.609 0.001 0.16

wave length
Sonication 218 0.608 0.001 0.16
219 0.608 0.0015 0.25

Accuracy: Accuracy was determined by performing recovery experiments in which determination of %

mean recovery of sample by standardization method at three different levels 50%, 100%, 150%. of the
sample solutions were prepared. The percent recovery was found in the range of 100-106% (Table 8)
Table 8: Recovery data of phenytoin

Total Standard Sample Absorbance Concentrati Sample %

concentra concentratio concentrat (218nm) on concentra Recover
tion n ion (µg/ml) Y=mx + c tion y
(µg/ml) (µg/ml) (µg/ml) difference
standard sample
(µg/ml)

2 1 1 0.121 0.121 2 1 100

(50%)
1 1 0.121 0.122 2.016 1.016 101.6

1 1 0.121 0.123 2.033 1.033 103

4 1 3 0.241 0.250 4.149 3.149 104.96

(100%)
1 3 0.241 0.245 4.066 3.066 102.2

1 3 0.241 0.249 4.132 3.132 104.4

6 1 5 0.361 0.369 6.132 5.132 102.6

(150%)
1 5 0.361 0.371 6.166 5.166 103.3

1 5 0.361 0.375 6.322 5.322 106.4

CONCLUSION:
As per ICH Q2(R1) guideline the present analytical method was validated and it meets specific acceptance
criteria. It is concluded that the analytical method was simple, sensitive, precise, linear, accurate, robust,
reproducible.

ACKNOWLEDGEMENTS:
The authors are thankful to Rani Chennamma College of Pharmacy, Belagavi for providing chemicals and
instruments. The authors are thankful to Tabssum Inamdar for helping during research work.
Abbreviations
µ: microgram, LOD: Limit of detection, LOQ: Limit of quantification, UV: Ultra violet
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